Chapter II

ANALYTICAL TECHNIQUES FOR ENVIRONMENTAL MONITORING AND CONTROL

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II.1. INTRODUCTION
Most of the analytical techniques find their applicability in environmental control, from the simplest to the most complicated one. This has increased the difficulty for the creation of this course that attempts to present over a small length and in a concise and clear manner the most important analytical techniques being applied in the mentioned domain. Regardless of the technique for obtaining the analytical data, in order to be relevant and useful in practice, they must be interpreted statistically and because of this at the beginning of this course we introduced a short chapter on basic statistics. In the following chapters a few basic notions regarding the sampling and the preparing of the sample for analysis were presented. This stage is extremely important for the analytical determination, especially when environmental samples are analyzed and, often, proper attention is not paid to it. The fact is known that generally the sampling and sample preparing for analysis generate the largest errors in environmental analysis. After that, the material was structured in two big chapters as follows: analytical techniques used for environmental analyses and methods for the monitoring of the most important pollutants. For the presentation of the analytical techniques a fairly large importance was given to the classical methods like gravimetry and volumetry, these techniques maintaining a certain importance in almost all the environmental control laboratories. Afterwards, the most important instrumental analysis techniques were presented in a concise manner (in our opinion) with applications for environmental control as follows: spectrophotometric methods in UV and VIS, methods based on the atomic emission and absorption, electrochemical methods, chromatographic methods, mass spectrometry and the hyphenated GC-MS technique. Due to their increasing importance, a chapter regarding the immunoanalytical techniques was included. Unfortunately, due to the fact that the size of the work was limited, in the presentation some important instrumental techniques such as like the radiometric and radiochemical analysis techniques were not included, and the description of most of the analytical techniques was done without extensive information. The course ends with the presentation of the monitoring methods for some important environmental pollutants: phenols, nitrogen compounds, heavy metals, pesticides and polychlorinated biphenyl compounds. We hope this presentation includes the major environmental pollutants, even though we are convinced that some important pollutants were not included, this being due to the limited printing space available to us.

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II.2. ELEMENTARY STATISTICS

Mihaela BADEA, Mihaela-Carmen CHEREGI In an analysis, the collection of the data is followed by the data handling. Statistics is necessary to understand the significance of the collected data and therefore to set up limitations on each step of analysis. The design of the experiments (including size of sample required, accuracy of measurements required, and number of analyses needed) is determined by a proper understanding of what the data represent.

II.2.1. ACCURACY AND PRECISION

Accuracy is the degree of agreement between the measured value and the true value. Precision is defined as the degree of agreement between replicate measurements of the same quantity, or in other words precision is how close the shots are to one another. It is impossible to have good accuracy without good precision, but good precision does not guarantee a good accuracy. For example, in the case of a systematic error in the analysis, the error does not affect the precision, but it does affect the accuracy. Since all real analyses are unknown, the higher the degree of precision, the greater the chance of obtaining the true value.

II.2.2. ERRORS AND WAYS OF EXPRESSING ACCURACY

Two main classes of errors can affect the accuracy or precision of a measured quantity. Determinate (systematic) errors are those that are determinable and that presumably can be either avoided or corrected. Some common determinate errors are: instrumental errors (faulty equipment, uncalibrated weights and glassware, etc), operative errors (personal errors in manipulations, mathematical errors in calculations, prejudice in estimating measurements). Indeterminate errors, often called accidental or random errors, represent the experimental uncertainty that occurs in any measurement. These errors are revealed by small differences in successive measurements made by the same analyst under virtually identical conditions, and they cannot be predicted or estimated. Mathematical laws of probability can be applied to understand these errors. The indeterminate errors should follow a normal distribution, or Gaussian curve (Figure II.2.1). is the true value and s represents the standard deviation of a population measurements, and this measure of precision defines the spread of the normal population distribution. It is apparent that there should be few large errors and that there should be an equal number of positive and negative errors.

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3s

2s

+s

+ 2s

+ 3s

Figure II.2.1. Normal distribution for a population of measurements.

Indeterminate errors come from the limited ability of the analyst to control or to make corrections for external conditions, or the inability to recognize the appearance of factors that will result in errors. There are various ways and units in which the accuracy of a measurement can be expressed, but to calculate an error, the true value, xt must be known. The difference between the true value and the measured value, with the regard to the sign, represents the absolute error, e, and is reported in the same units as the measurement.
e = xi xt

If the measured value is the average of several measurements, the error is called the mean error. The absolute or mean error expressed as percentage of the true value is the relative error.

xi xt 100 xt In very accurate, the relative errors are less than 1 %. er =

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II.2.3. MEASURES OF PRECISION

Some basic definitions from statistics need to be introduced to express the idea of precision. A population is any complete set of data, for example a set of n replicate measurements x1, x2, xn. The mean of this population, x is the arithmetic average and defined by the equation:

x=

x
i =1

where n is the number of measurements and xi is each individual measurement. x is sometimes called sample mean to differentiate it from the true or population mean, . The formula for is the same as above, but n must be at least 20 measurements. The most widely useful measure of precision is the standard deviation, symbolized by s or and is used for small populations The standard deviation is a statistical measure of the precision for a series of repetitive measurements. The advantage of using s to quote uncertainty in a result is that it has the same units as the mean value. Under a normal distribution, one standard deviation encompasses 68 % of the measurement and two standard deviation encompasses 96% of the measurement. The standard deviation is calculated with formula:

s=

(x
i =1

x) 2

n 1

The quantity ( xi x ) is called the residual or the deviation from the mean for each measurement. The quantity (n-1) is called the degrees of freedom for the measurement. For a very large population of replicate measurements is used s2 or 2, the square of the standard deviation, which is called the variance, V.

V = s2 =

(x
i =1

x) 2

n 1

The relative standard deviation (RSD) is useful for comparing the uncertainty between different measurements of varying absolute magnitude. The RSD is

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calculated from the standard deviation, s, and is commonly expresses as percentage (%):
RSD (%) = s 100 x

The RSD (%) is also called the coefficient of variance (CV). The confidence limits are another statistical measure of the precision for a series of repetitive measurements. In case of small populations consisting of the usual two to four replicate laboratory measurements, Student developed the statistics necessary to define the confidence limits. He calculated extensive values of a factor t by which the estimated standard evaluation of the mean of small population could be multiplied to obtain confidence limits for the related population of means. The confidence limits are calculated from the standard deviation using the formula:

=x

t s n

Table II.2.1. Values of Students t. Degree of freedom (n-1) t

1 2 3 4 5 6 7 15

90 % 6.31 2.92 2.35 2.13 2.01 1.94 1.90 1.75 1.65

95 % 12.71 4.30 3.18 2.78 2.57 2.45 2.37 2.13 1.96

99 % 63.66 9.93 5.84 4.60 4.03 3.71 3.50 2.95 2.58

The t term is taken from tables calculated by Student for various numbers of degrees of freedom and degrees of confidence. A brief set of values for Students t factor is given in Table II.2.1. The term confidence interval is also used, and represents the span between the confidence limits.

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II.3. SAMPLE AND SAMPLING AND PREPARATION

Mihaela BADEA Because laboratories process a large volume of samples and must maintain a high level of quality, the processes and procedures in the laboratory must be well defined and rigorously followed. Each step must be documented in a permanent record, including the sampling, sample tracking and sample preparation processes. The way the sample is obtained, handled, prepared, etc. is very important in an analytical laboratory. The results of an analysis can only be useful if the sample really does represent what it is intended to represent .

II.3.1. SAMPLE AND SAMPLING

A sample is a small portion of a larger body of material that is obtained for laboratory analysis. If the purpose of such analysis is to present the concentration of a component in the entire body of material, as it usually always is, then this sample must be representative of the entire body. A representative sample is therefore a sample that possesses all the characteristics of a larger bulk system in exactly the same concentration levels as in the system. In other words, it represents the system and whatever concentration level is found for a given component of a sample, that is also then taken to be the concentration level in the system. In situations in which it is likely that the substance to be determined is not homogeneously distributed throughout the entire system, a series of samples could be obtained from different parts of the bulk system and then combined into one sample. This kind of sample is called a composite sample. Another method for solving the problem of non-homogeneity of a bulk system is to take a selective sample. A selective sample is a sample that is obtained from a particular part of the bulk system that is known, or assumed, to have a different composition. For example, the air next to a leaking gas furnace exhaust would have a higher level of carbon monoxide than the air in a room elsewhere in a building. In the case in which the carbon monoxide level in the immediate vicinity of the leak is important, then a selective sample is acquired. If it is thought that a bulk system is homogeneous for a particular component, then a random sample is taken. This would be just one sample taken from one location at random in the bulk system. An example would be when determining the level of active ingredient in a pharmaceutical product stored in boxes with individual bottles in a warehouse. Having no reason to assume a greater concentration level in one bottle or box compared to another, a sample is chosen at random. Other

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designations for samples are bulk sample, primary sample, secondary sample, subsample, laboratory sample, and test sample. These terms are used when a sample of a bulk system is divided, possibly a number of times, before actually being used in an analysis. For example, a water sample from a well may be collected in a large bottle (bulk sample or primary sample), from which a smaller sample is acquired by pouring into a vial to be taken into the laboratory (secondary sample, sub-sample, or laboratory sample), then poured into a beaker (another secondary sample or sub-sample), before a portion is finally carefully measured into a flask (test sample) and diluted to make the sample solution. II.3.1.1. Statistics of Sampling A consideration of statistics is required in a discussion of sampling because of the randomness with which samples are acquired. The sampling is similar to a laboratory analysis the results vary randomly and are affected by random errors that cannot be compensated. One might think that a lab analyst obtains a single sample from a bulk system, analyzes it one time in the laboratory, and reports the answer for this one analysis as the analysis results. If variances in the sampling and lab work are both insignificant, these results may be valid. However, due to possible large variances in both the sampling and the lab work, such a result cannot be considered reliable. The correct procedure is to perform the analysis many times and deal with the variances with statistics. The fact that sampling introduces a second statistics problem means that one must also consider taking a large number of samples and dealing with the results with statistics just as one performs a laboratory analysis a large number of times and deals with those results with statistics. For example, it can be shown that if a measurement system generates data with a standard deviation of 10 ppm and one needs to know an average concentration to 5 ppm with 95% confidence, then one must perform the analysis 16 times. If the sampling variance is high but the lab analysis variance is low, then one must measure 16 samples each one time. If the lab analysis variance is high but the sampling variance is low, then one must measure one sample 16 times. If both the sampling variance and the lab analysis variance are high, then one must measure 16 samples each 16 times. Chemists want to have as low a variance (or standard deviation) as possible for the greatest accuracy. If it is not possible to have a low enough standard deviation to suit the need, then the number of measurements (either the number of samples, the number of lab analyses, or both) must be increased. II.3.1.2. Sample Handling The importance of a high-quality representative sample has already been underlined. How to obtain the sample and what to do with it once it reaches the laboratory are obviously important factors. But the handling of the sample between the

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sampling site and the laboratory is often something that doesnt receive the adequate consideration. The key concept is that the samples integrity must be strictly maintained and preserved. These can be achieved by adding a preservative if required; by refrigerating the sample if is necessary this for the integrity maintaining; by looking up to the specified holding time; by preventing contamination using a particular material for the storage container (e.g., glass vs. plastic) when is necessary, etc. It is very important to document who has handled the sample and what responsibility each handler has at various junctures between the sampling site and the laboratory. In other words, the chain of custody must be maintained and documented. A sample can have a number of custodians along the way to the laboratory. A sample of lake water may be taken by a sampling technician at the site. The sampling technician may give it to a driver who transports it to the analysis site. A shipping/receiving clerk may log in the sample and give it to a subordinate who takes it to the laboratory. Along the way, this sample is in the hands of five different handlers - the sampling technician, the driver, the shipping/receiving clerk, the subordinate, and the laboratory technician. Each should maintain documentation of his/her activity and duties and copies of the chain of custody should be filed.

II.3.3. SAMPLE PREPARATION

Most samples must be prepared before analysis. The preparation process varies depending on the sample matrix, the material to be analyzed, and the analytical method. The most important processes include extraction and cleanup, digestion, leaching, dilution, and filtering. Depending on the sample matrix, other procedures such as grinding and chemical manipulations may be required. II.3.3.1. Sample Extraction The extraction processes are used more often for the organic analytes that are extracted to bring them into the appropriate solvent prior to analysis. The extraction method varies depending on whether the sample is liquid or solid. Extraction techniques for aqueous samples include liquid-liquid (separation funnel or continuous) and solid-phase. For solid samples, the methods include Soxhlett, supercritical fluid, and sonication. Some extraction processes are repeated multiple times (such as three) to improve the efficiency of extraction. Organic pollutants in potable or non-potable waters, soils, sediments, sludge, solid wastes, and other matrices must be brought into an appropriate organic solvent for their injection into the gas or liquid chromatographic column. Such extraction also enables the increase the concentration of analytes in samples by several orders of

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magnitude for their detection at ppb or ppt level. Depending on the nature of the sample matrices, various extraction techniques may be effectively applied for accurate and low level detection of organics. Liquid-Liquid Extraction The aqueous samples are commonly extracted by the liquid-liquid extraction technique. A measured volume of the liquid sample is repeatedly extracted with an immiscible organic solvent. The selection of organic solvent must meet the following criteria: 1. it must be immiscible with water, 2. the organic pollutants should be soluble in the solvent (their solubility must be greater in this solvent than in water) 3. the density of the solvent should be greater than water when the extraction is carried out in a separation funnel or a continuous liquid-liquid extractor; on the other hand, the solvent should be less dense than water, when extraction is performed in a glass vial. Upon mixing the aqueous sample with the solvent, the pollutants dissolve more in the latter because they are more soluble in the solvent. In other words, they partition or distribute in the aqueous and the solvent phases and, at equilibrium, the concentration ratio of the solute in both the phases is constant. The partition or distribution coefficient, P, is equal to the ratio of the concentration of the solute in the solvent to that in the water. Since P is independent of volume ratio, but constant at any given temperature, increasing the volume of the solvent will cause more dissolution of the solute into the solvent. In other words, the greater the amount of solvent the more of the solute would dissolve in it. For any given volume of solvent, repeated extractions using smaller portions in equal amounts will give greater extraction efficiency than a single-step extraction. Certain widely used solvents such as diethyl ether or methylene chloride are highly volatile. Excess pressure build-up may cause rupture of the separation funnel. It is important to vent out the excess pressure, especially after the first shaking of the sample with the solvent. Before extraction, rinse the separation funnel with a few milliliters of the solvent. A glass container that has even a slight crack should not be used for extraction. Solid Phase Extraction Organic substances can be extracted from aqueous samples by solid-liquid (known as solid phase) extraction. The process is simple, fast, and cost effective in comparison to liquid-liquid extraction. In addition, the analysis can be carried out using a smaller volume of sample. By using a suitable capillary column, a detection level comparable to the liquid-liquid extraction column could be readily attained. The method requires a measured volume of the aqueous sample to be passed through a

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cartridge tube packed with a suitable solid adsorbent material. The organic pollutants in the sample are adsorbed onto the solid surface from which they are eluted by a properly selected solvent. The sample is applied at the top of the tube and drawn through the bed by a syringe or vacuum, maintaining a flow rate of 1 to 2 drops/sec. Alternatively, particles with larger pore size may be used to allow fast flow rates for large volume samples. The tube is washed with a non-polar solvent for polar analytes and polar solvent for non-polar analytes. Finally, the analytes are eluted out of column by a suitable solvent. Polar solvents should be used for polar analytes and non-polar solvents for non-polar analytes. The sample extracts may be concentrated further by evaporation of the solvent. The selection of the adsorbent packing material is based on the polarity of pollutants to be analyzed. The non-polar hydrophobic adsorbents retain the non-polar analytes and allow the polar substances to pass through the column. The hydrophilic adsorbents adsorb the polar components, allowing the non-polar materials to pass through. Various stationary phases for solid phase extraction are used, for example: octadecyl (C-18) bonded silica, octyl (C-8) bonded silica, silica, florisil, silica gel, etc. Soxhlet Extraction Semi-volatile and non-volatile organic pollutants from solid samples may be extracted by Soxhlet extraction. The sample is placed in a porous extraction thimble and immersed in the solvent. The extraction comprises a series of batch processes involving distillation and condensation of the solvent along with periodic fill-in and siphoning of the solvent in and out of the extraction chamber. This causes an intimate mixing of the sample with the solvent. Soxhlet extraction using a fluorocarbon solvent is commonly employed to leach out petroleum hydrocarbons from the soil. Other than this use, its application in environmental analysis is limited, because it is slow, taking up several hours to complete. The extraction also requires a relatively large quantity of solvent and usually a pre-concentration step is necessary. Supercritical Fluid Extraction A supercritical fluid is defined as a substance that is above its critical temperature and pressure. It exhibits remarkable liquid-like solvent properties and, therefore, high extraction efficiency. Such common gases as carbon dioxide and nitrous oxide have been successfully employed as supercritical fluids in the extraction of organics from solid matrices. The solid sample is placed in an extraction vessel into which the pressurized supercritical fluid is pumped. The organic analytes dissolve in the supercritical fluid and are swept out of the extraction chamber into a collection vessel. The pressure is released at the valve attached to the collection device where it drops down to atmospheric pressure. The supercritical fluid then returns to its gaseous state and escapes out, leaving the analytes in the collection vessel in an appropriate solvent such as methylene chloride. The extraction efficiency of supercritical fluids may be enhanced by mixing into it a small amount of a co-solvent such as acetone or

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methanol. Supercritical fluid extraction offers certain advantages over other extraction processes: 1. It is relatively a fast process with greater extraction efficiency; 2. Sample concentration steps may be eliminated; 3. Unlike liquid-liquid extraction or Soxhlet extraction, a large amount of organic solvents is not required. II.3.3.2. Sample Cleanup Samples may undergo a cleanup process to improve the analysis process and generate more reliable results. The sample extracts may be purified by one or more of the following techniques: 1. Partitioning between immiscible solvents 2. Adsorption chromatography 3. Gel permeation chromatography 4. Destruction of interfering substances with acids, alkalis, and oxidizing agents 5. Distillation The cleanup procedures presented in table II.3.1. may be applied for different classes of organic substances. Table II.3.1. Cleanup methods for organic extracts. Analyte group Organochlorine pesticides Polychlorinated biphenyls (PCBs) Organophosphoric pesticides Chlorinated herbicides Chlorinated hydrocarbons Polynuclear aromatic hydrocarbons Cyclic ketones Nitrosamines Phenols Phthalate esters GPC Gel permeation chromatography Cleanup method Florisil, GPC, sulfur Florisil, GPC, sulfur, KMnO4 H2SO4 Florisil, GPC Acid-base Florisil, GPC Alumina, silica gel, GPC Florisil, GPC Alumina, florisil, GPC GPC, acid-base, silica gel Alumina, florisil, GPC

Acid-Base Partitioning This is applied to separate acidic or basic organic compounds from neutral organics. The solvent extract is shaken with water that is highly basic. The acidic organics partition into the aqueous layer, whereas the basic and neutral compounds stay in the organic solvent and separate out. After this, the aqueous layer is acidified to a pH below 2, and then extracted with methylene chloride. The organic layer now

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contains the acid fraction. Phenols, chlorophenoxy acid, herbicides, and semi-volatile organic pollutants are cleaned up by the procedure described above. Alumina Column Cleanup Highly porous and granular aluminum oxide - available in three pH ranges (acidic, neutral and basic) - is used in column chromatography. Analytes are separated from the interfering compounds based on their different chemical polarity. The column is packed with alumina, and then covered under anhydrous Na 2SO4. The extract is then loaded on it. A suitable solvent is selected to elute the analytes. The interfering compound is left adsorbed onto the column. The eluate is then concentrated further. Alumina can be prepared in various activity grades by adding water to Grade I (prepared by heating at >400C until no more water is lost). Among the common pollutants, phthalate esters and nitrosamines are separated. Basic alumina (pH 9 to 10) is most active in separating basic and neutral compounds: alkali, alkaloids, steroids, alcohols, and pigments. Certain solvents such as acetone or ethyl acetate cannot be used. This form of alumina can cause polymerization, dehydration, and condensation reactions. The neutral form is less active than the basic grade and is used to separate aldehydes, ketones, esters, and lactones, etc. The acidic form (pH 4 to 5) is used to separate strong acids and acidic pigments. The alumina column cleanup is also used to separate petroleum wastes. Silica Gel Cleanup Silica gel is a form of amorphous silica with weak acidic properties. It is made by treating H2SO4 with sodium silicate when used for cleanup purposes. Interfering compounds of different polarity are absorbed onto and retained on the column. There are two types of silica gel: activated and deactivated. The former is prepared by heating silica gel for several hours at 150C. It is used to separate hydrocarbons. The deactivated form contains 10 to 20% water and is used to separate plasticizers, steroids, terpenoids, alkaloids, glycosides, dyes, lipids, sugar, esters, and alkali metal cations. In environmental analysis, silica gel is used to clean up sample extracts containing single component pesticides, PCBs, polynuclear aromatic hydrocarbons, and phenol derivatives. Methanol and ethanol decrease adsorbent activity. Florisil Column Cleanup Florisil is a form of magnesium silicate with acidic properties. It is used for clean up of sample extracts containing the following types of analytes: nochlorine pesticides, organophosphoric pesticides, phthalate esters, nitrosamines, haloethers, nitroaromatics, and chlorinated hydrocarbons. Florisil is also used to separate aromatic compounds from aliphatic-aromatic mixtures, as well as to separate esters, ketones, glycerides, steroids, alkaloids, and some carbohydrates. It also separates out nitrogen compounds from hydrocarbons.

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Gel-Permeation Cleanup This separation is based on the size of the porous, hydrophobic gels. The pore size must be greater than the pore size of the molecules to be separated. Gelpermeation cleanup (GPC) is used for cleaning sample extracts from synthetic macromolecules, polymers, proteins, lipids, steroids, viruses, natural resins, and other high molecular weight compounds. Methylene chloride is used as the solvent for separation. Elution is carried out using a suitable solvent, and the eluate is concentrated for analysis. Sulfur Cleanup Sulfur is found in many industrial wastes, marine algae, and sediment samples. Sulfur may mask a region of the chromatogram, overlapping with peaks of interest. For example, in pesticides analysis, sulfur can mask over many pesticides such as lindane, aldrin, and heptachlor. Sulfur has the solubility similar to the organochlorine and organophosphoric pesticides and it cannot be separated by Florisil cleanup method. The removal of sulfur is achieved by treating the extract with one of the following three substances: copper, mercury, or tetrabutyl ammonium-sodium sulfite reagent. The sample extract is vigorously shaken with one of the above reagents. The clean extract, free from sulfur, is then separated from the reagent. Permanganate - Sulfuric Acid Cleanup Interfering substances in the sample extract may often be destroyed by treating the extract with a strong oxidizing agent, such as KMnO 4 or a strong acid like conc. H2SO4, or a combination of both. In such a case, the analyte should be chemically stable to these reagents. For example, interfering substances in the sample extract for the analysis of polychlorinated biphenyls can be effectively destroyed by treatment with a small quantity of KMnO 4 - H2SO4 mixture. PCBs are chemically stable under the condition of treatment, and do not react with the acid-permanganate mixture at such a short contact time. II.3.3.3. Digestion Samples analyzed for metals are usually digested. The digestion process uses strong acids and heat to increase the precision and accuracy of the measurement by providing a homogeneous solution for analysis by removing metals adsorbed to particles and breaking down metal complexes. Different digestion techniques are used depending on the analytical method and target accuracy levels. II.3.3.4. Dilution

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Sometimes it is necessary to dilute the sample prior to analysis. The reasons for this may be that the concentration of the analyte is outside the concentration range where the analytical technique is linear, or other substances in the sample may interfere with the analysis (matrix interference). A record of the dilution factor should be kept with the result. Dilution affects the result itself as well as the detection limit for the result. For non-detected results, the reported result based on the detection limit will be increased proportionately to the dilution, and this needs to be considered in interpreting the results. II.3.3.5. Filtering The sample may or may not be filtered, either in the field or in the laboratory. If the sample is not filtered, the resulting measurement is referred to as a total measurement, while if it is filtered it is considered a dissolved result. For filtered samples, the size of the openings in the filter (such as 1 micron) should be included with the result. Commonly, once a sample has been filtered it is preserved. This information should also be noted with the result. REFERENCES

1. Sampling for Analytical Purposes, Pierre Gy, 1998, John Wiley & Sons, Ltd., West
Sussex, England. 2. Standard Methods for the Examination of Water and Wastewater, 20 th Edition, Ed Leonore S. Clesceri, Arnold E. Greenberg, Andrew D. Eaton, 1998, United Book Press, Inc., Baltimore, USA. 3. Handbook of Environmental Analysis. Chemical Pollutants in Air, Water, Soil and Solid Wastes. Pradyot Patnaik, 1997, CRC Press, Inc., Boca Raton, Florida, USA 4. Environmental Chemistry, Seventh Edition, Stanley E. Manahan, 2000, CRC Press, Inc., Boca Raton, Florida, USA.

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II.4. ANALYTICAL TECHNIQUES USED IN ENVIRONMENTAL ANALYSIS

II.4.1. GRAVIMETRIC METHODS Jos MARTNEZ CALATAYUD
Gravimetric analysis, the most classical of all quantitative methods (among the oldest of analytical techniques), relays on some final determination of weight. The compound of interest (analyte) is precipitated in a solid compound of known composition. The mass of the analyte present in the sample is determined from the mass of the precipitate. Since weight can be measured with greater accuracy than any other fundamental property, gravimetric analysis was ( it is yet) one of the most accurate analytical methods. Gravimetric procedures are lengthy and tedious compared against instrumental methods; and, in addition, samples may have to be extensively treated to remove interfering substances. As a result, only a very few gravimetric methods are currently used in environmental analysis. Based on the preparation of the sample before weighing the analyte compound, there are four fundamental types of gravimetric analysis: (a) physical gravimetry, is the most common type used in environmental control engineering. It involves the physical separation and classification of matter based on volatility and particle size (e.g., total suspended solids). (b) thermogravimetry, for the analysis of volatile solids. Changes in the sample mass when heated are recorded. (c) electrogravimetry or electro-deposition which usually involves the electrochemical reduction and simultaneous deposition of metal ions at a cathode. (d) chemical precipitation, the most common in a classical sense. It relies on a chemical reaction to transform the solved analyte in a very low soluble precipitate. Its most important application in the environmental field is with the analysis of sulfate or sulfite.

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Table II.4.1. Gravimetric methods in environmental analysis control Type of gravimetry Physical Analyte Total Solids Suspended Solids Dissolved Solids Oil & Grease Surfactants Thermal Precipitative Volatile Solids Volatile Solids Volatile Suspended Solids Mg 2+ Na + Silica SO4 2II.4.1.1. Physical Gravimetry Solids As the operational definition of Total Solids (TS) content has been adopted through years of use the following all matter that remains as residue upon evaporation and drying at 180 oC for one hour. This is an operational definition because solids in a water or wastewater sample are a diverse collection of dissolved and particulate matter rather a specific chemical compound. According to this operational definition and depending on certain empirical parameters the result may be smaller or greater than the amount of solids present in the sample. An operational criteria serves for a classification of total solids; this definition establishes the following: all solids passing through a filter paper of a certain pore size (e.g., 1.5 microns, Whatman #934AH) are the total dissolved solids (TDS) and those retained on the filter are the total suspended solids (TSS). Some authors prefer to define the total dissolved solids as all matter that is not retained by a #934AH filter, and then, it is not lost by evaporation and drying at 180 o C for one hour. Most of the impurities in potable waters are inorganic salts that resulted in the dissolved state. Thus, the parameters TS (total solids) and TDS (total dissolved solids) have a relevant importance. Sources of drinking water containing high concentrations of inorganic salts are not suitable (more than 1000 mg/L TDS are Procedure Evaporation Filtration Filtration + Evaporation Extraction with C2Cl3F3 + distillation of solvent Extraction into ethylacetate + evaporation Evaporation + 550oC for 15 min Evaporation + 550oC for 15 min Filtration + 550oC for 15 min With Diammonium hydrogen phosphate and final pyrolysis With zinc uranyl acetate Precipitation/ ignition/ volatilization (with HF) With Ba2+

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unacceptable), because such materials are often difficult to remove in the treatmentplants. Waters of this type are even unsuitable for agricultural uses due to the negative effect on plants of the high ionic concentrations. In natural water samples, the TDS (total dissolved solids) usually correlates well with the total hardness (expressed as total [Ca] + [Mg]); which results as an useful parameter to assess either the need for softening and the corrosivity of a water. It is very important to avoid unrepresentative sampling or particle break-up, this should be considered either at the point of sampling and at laboratory subsampling. The former topic means not disturbing the flow pattern of the sample stream, or at least, the disturbance should be minimized. The sampling errors are associated to the sample size. Figure II.4.1 depicts the percent error expected at different ratios intake velocity/stream velocity, and as a function of particle size. This requires that sample intake velocities should be similar to the flow velocities at the point of mixing. These errors are high for big particles. A recommended caution is to face the sampling device intake into the stream flow at 20 degrees.

Figure II.4.1. Relationship Between Sediment Size and Sampling Bias (reproduced from: http://www.ecs.umass.edu/cee/reckhow/courses/572/572bk15/572BK15.html)

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When the presence of settle able solids is suspected and for producing a homogeneous cross-section of settle able solids, the mixing at the point of sampling is sufficient and depth-integrated sampling is not required. The obtaining of a representative sample in the laboratory (laboratory subsampling) is best accomplished by shaking and a quick pouring the aliquot into the receiving beaker (as depicted in Figure II.4.2)

Figure II.4.2. Recovery vs particle size during sub-sampling with different mixing techniques.reproduced from: http://www.ecs.umass.edu/cee/reckhow/courses/572/572 bk15/572BK15.html) For a general discussion on sampling techniques see reference Handbook for Sampling and Sample Preservation of Water and Wastewater (USEPA, 1982). Collection and save of samples must be performed in clean glasses or highdensity polyethylene flasks; and, analysis should be conducted as soon as possible after sampling.

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Some care must be present in sampling and storing the collected samples: e.g. high contents in alkaline salts leach metal ions from the glass; and, air or solved oxygen oxidize iron and manganese ions and then the precipitation of their basic salts occurs. Procedures A. TS (total solids) or Total Residue. Total solids are the final weight ratio of a dried sample (total weight minus tare) versus the sample volume. Platinum is the most inert material and it is preferred over vycor as the material for evaporating dishes. However, due to the high price of platinum, vycor should be used. On the other hand, porcelain is avoided due to the difficulty on obtaining a constant weight. A special attention should be paid to evaporating dishes while hot or in use; to avoid the absorption of moisture or even the collection of dust, they must be safely stored in a desiccator to avoid the collection of dust and absorption of moisture while it is not in use. Procedure: 1. Preheat the evaporating dish (100 mL recommended capacity) for 1 hour at 550 50 oC; then, cool it protected from room dust, in a drying oven or in the open air for a period of 15 - 20 minutes. Keep into a desiccator down to room temperature and weigh. This operation should be repeated up to constant weight. 2. Measure a sample aliquot (about 75 mL or the required volume to obtain 200 mg TS) and then evaporate to dryness at 98 C on the pre-weighed dish in a drying oven at 98 oC. Additional drying for 1 hour at 103 105 oC is highly recommended. The procedure can be developed on a steam bath instead of the oven. 3. Keep the dish in a desiccator to achieve room temperature and weight with a repeating system to obtain constant weight. 4. Estimated precision: 4 mg or 5% and for settled wastewater is possible to reach 1 mg. B. Suspended Solids (or non-filterable residue). Directly measured by drying and weighing of the solids retained during filtration (Whatman #934AH glass fiber filters with a nominal pore size of 1.4 microns). This approach is preferred for most waters than the indirect method of subtracting dissolved solids from total solids. The identified filters should be weighed, heated and dried while remaining in small aluminum pans. Procedure:

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1. 2. 3. 4. 5. 6.

The filter (#934AH glass fibber) should be previously washed by rinsing it three times with 20 mL of distilled water. Maintain suction until the filter is dry and then, dry by heating at 103-105 o C for 1 hour. Keep the filter into a desiccator to reach the room temperature and then weight it. Filter the sample aliquot required yielding 50 - 200 mg of suspended solids and dry for 1 hour at 103-105 oC. Keep into a desiccator to cool and weigh. Repeat the drying and weighing to achieve a constant weigh ( 0.5 mg). Estimated precision 5 mg/L and 20 mg/L for low and large (up to 200 mg/L or more) concentrations, respectively.

C. Dissolved Solids (D S or filterable residue). An operational definition is: the theoretical dissolved solids are all that is not water. These parameters can be directly obtained by the weight of total solids from the filtered sample; or an indirect alternative is subtracting from the total solids the suspended solids. The drying temperature has a relevant importance: (a) At 103-105 C deviation occurs from the physically occluded water due to crystal irregularities and the presence of water of crystallization. This positive bias could happen with sample waters with high contents in calcium, magnesium, chloride or sulfate. The presence of large amounts of residue (over 200 mg) exacerbates the problem of occluded water. (b) A negative bias should be done by the volatilization of CO 2 from the conversion of bicarbonate. (c) At 180 2o C will occur additional volatilization of some inorganic salts (e.g., carbonate, nitrites and chlorides). This high temperature can be recommended for total loss of occluded water. However, some water of crystallization may remain, especially in the presence of sulfates. (d) In general, 103-105 or 180 may be used for waters of low color and low alkalinity. However, the hard sample waters, alkaline waters, or highly colored waters should be treated at the higher temperature. (e) Drying to 180 2 C gives TDS results closest to the calculated ones from a complete chemical analysis. Procedure: 1. The filter (934AH glass fibber) should be previously washed by rinsing it three times with 20 mL of distilled water. 2. Maintain suction until the filter is dry and then, 3. Analyze the filtrate in accordance with the reported TDS procedure. 4. Final drying for 1 hour may be conducted at either 103-105 o C or o 180 2 C.

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D. Oil and Grease (OG). In their determination are also included, in some cases, other organic substances which can be extracted with petroleum gasoline and whose boiling point is over 200 oC approximately, in total concentrations > 1 mg/L. Interferences and previous treatment: Some members of the organic substances from the before mentioned group are waxes, emulsion producers, detergents... Low boiling point hydrocarbons (gasoline) are not included in the determination. Fatty acid salts (soaps) are not determined. If they must be detected in fatty acid form, before the extraction, sulfuric acid is added to the sample until pH is lower than 2. Fatty acids can be determined separately in a second extraction, acidifying the sample only after the first extraction. Emulsions formation can be avoided during the extraction acidifying until pH 1 or adding sodium chloride. All glass materials used are cleaned carefully before the sample taking or after the determination, they are washed with petroleum gasoline and they are dried. Be careful not to have fat in the grindings Sample taking: - The best method is to use a glass bottle with glass cork, filling to the bottle until a certain annular signal (p.e. 500 milliliters). Technique: 1. In case of low impure waters - the complete content of the bottle of the sample taking goes to a movement separation funnel (1000 milliliter), the bottle is washed once or twice with 10 milliliters of petroleum gasoline every time and the dissolvent is added to the water sample. Next, the petroleum gasoline volume is completed to 25 milliliters. Then the sample is shaken intensely for about two minutes. After separating the layers, the aqueous phase is left in a second separation funnel (1000 milliliters) and the organic phase is joined in a separation funnel of 100 milliliters. The extraction is repeated two more times with 15 milliliters of petroleum gasoline every time. The separation funnels used for the extraction are washed again with about 5 or 10 milliliters of petroleum gasoline and the dissolvent and the extract are joined in the separation funnel of 100 milliliters. If the extract contains finely dispersed substances, these can settle by centrifugation for 15 minutes at 3000 RPM. In order to continue treating the extract, is used a vacuum rotating evaporator. The extract, centrifuged if necessary, is filtered through a soft filter paper with the surface free of fat, having been washed itself previously with petroleum gasoline, and the filtrate goes to an evaporation flask of 100 milliliters. The filter is washed twice with 5 or 10 milliliters of petroleum gasoline. After that,

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2.

3.

4.

we fix the flask with a NS clamp (pay attention standardized grinding) to the tube for steam circulation of the vacuum rotating evaporator and suspended it in a water bath, whose temperature is regulated with a thermostat to 201 oC. The apparatus is evacuated, for a medium number of flask revolutions, with a water tube, until the pressure has lowered until about 50 Torricelli and the residual volume of the extract is about 0.5 milliliters. We start a vacuum oil pump and we connect it, changing the position of a key with three steps, with the apparatus. After that, we start the evaporation for about five minutes at about 25 Torricelli (if it is possible to about 2 Torricelli). After disconnecting the vacuum and the rotation, the flask is separated carefully, it is dried outside for 60 minutes in a desiccator on silica-gel, and it is weighted In case of strongly impure waters or mud with large water content - we evaporate to dryness 500 milliliters or less of water sample or mud in a porcelain capsule with 30 g of sea sand in the water bath. The remainder goes quantitatively to a Soxhlet extraction cartridge, which is put in a Soxhlet extraction apparatus. Later, the capsule is washed with petroleum gasoline. The extraction takes place during 3 or 4 hours with petroleum gasoline. Next, the extraction flask is connected to the vacuum rotating evaporator and the dissolvent evaporates until about 20 milliliters remain. The extract is centrifuged, as described in 1., in stages in case it is necessary we filter it and it goes to a calibrated evaporation flask connected to a vacuum rotating evaporator. Calculation - oil and grease total or extract of petroleum gasoline in mg/L = (a*1000)/b a = mg of the extraction remainder (weight) b = milliliter used of the test of water or mud Results indication - cleared values to 1 mg L-1.

Characterization and Determination of Hydrocarbons The great complexity of the oil can be classified according to the three following groups: saturated hydrocarbons or alkanes; aromatic hydrocarbons; heavy products (aromatic compounds with high molecular weight containing heteroatoms such as sulfur, oxygen or nitrogen). After 24 hours on the surface of water, the crude oil practically has lost by evaporation all hydrocarbons with a number of carbon atoms lower than 14, what corresponds, in fact, to a led distillation at 280 C. Apart from evaporation and certain processes of dissolution, petroleum products undergo chemical and bacterial degradations. The oxidation, which can be associated with a polymerization, results in

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a rise in viscosity and density of the recovered product. The residues obtained after boiling at 300 C are less sensitive to these phenomena. A certain number of determination methods were developed, and they can be classified in two main categories: - The first ones include gravimetric or volumetric methods based on the solvent extraction and those based on the density or the optical properties of the elements to determine. This type of not very significant methods applies badly to routine controls because of the evaporation wastages or the interferences whose elements can cause and which, while being extractable, are not hydrocarbons; - The second group are much more significant methods. Infrared spectrometry allows under certain conditions the simultaneous determination of hydrocarbons and phenols but its sensitivity varies with hydrocarbons found. The use of gas chromatography makes it possible to reach a good sensitivity for the determination of the hydrocarbons whose boiling point is located between 150 and 450 C. It also makes it possible to specify the importance of N-alkanes compared to other present hydrocarbons. The determination can be carried out either by injecting water directly in the apparatus, or with a preliminary extraction with a solvent. In this case, we use either the carbon tetrachloride, or pentane. Of course, this method cannot be used for hydrocarbons whose boiling point is higher than the one of the employed solvent, because of the peaks interference on the chromatograms. If we prefer the direct injection of water, we can use a separation pre-column; this method is very appropriate then for the determination of light hydrocarbons. From a practical point of view, it is difficult to carry out homogenous sample taking. Indeed, the hydrocarbons are presented in a surface film form or in droplets; they can also be emulsified in water or adhere to the suspended particles. It is convenient there to carry out sample taking in a turbulent column or using a special apparatus; the sample taking after decantation corresponds in fact to a preconcentration. Sample taking is carried out in a turbulent zone, at a medium depth; and it can be manual or automatic. For the manual sample taking is used a glass bottle with ground or screwed stopper whose capacity is determined by weighing. The automatic sample taking is done by a special apparatus (paddle wheels, scoops, in pressure or depression air, solenoid winnow, etc.) respecting certain obligations. Because of the homogeneity errors, it is advisable to multiply the sample taking and to consider an average value of the obtained results. The possible evaporation or microbial action wastages limits the storage times and imposes very quick extraction after sample taking. In case of a pollution accident, hydrocarbon quantity is important, it is possible to practice an organic solvent extraction, then a residue weighing after solvent evaporation. See greases and oils determination method in wastewater. We can employ carbon tetrachloride, hexane, ether oil or chloroform; recovery percentages can be different according to the used solvent. Solvent evaporation can lead to azeotropic entailed wastage, so this method is

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satisfactory only for hydrocarbons having more than 12 carbon atoms by molecule (P.E. higher than 220 C approximately). If the recovered quantity is sufficient, liquid chromatography allows the saturated or aromatic hydrocarbons determination, and the gas chromatographic method is used to specify the compounds composition. II.4.1.2. Thermogravimetry Thermogravimetry and/or combustion analysis involves the sample treatment at 500 oC or more; this procedure leads to the oxidation and/or volatilization of some of the sample compounds. After this process, two different procedures could be applied: (a) Measuring the loss of sample weight, used for volatile solids analysis in environmental engineering (thermogravimetric procedure). Remember to cool the sample to room temperature before weighing or the method accuracy will suffer severely due to convection currents around the balance pan created by the differences in temperature. A steady increase in apparent weight indicates this problem. (b) (b) Alternatively trapping the evolved gases and weighing the trap is used in many fields for the determination of total carbon and hydrogen in solids (this is known as combustion analysis). Volatile Solids and Fixed Solids After ignition of the sample for 15 minutes at 550 oC, the lost weight of material is called the volatile solids, and fixed solids are all those substances that remain as residue. The operational definition for volatile solids would be: all matter lost upon ignition at 550 oC for 15 minutes, but not lost upon drying at 103-105 oC for 1 hour. The lost volatile portion upon ignition is generally assumed to be equivalent to the organic fraction and the residual portion is considered the inorganic fraction; most of this is calcium carbonate (for samples over the interval from of moderate to high hardness), which decomposes only at temperatures over 800 oC. Temperatures over 600 oC could damage the glass fibber filters by melting and a significant loss of weight can occur. Combining the fractions obtained from ignition and filtration procedures, there are 9 separate groups: namely, TS, total solids; fixed solids; volatile solids; TDS, total dissolved solids; fixed dissolved solids; volatile dissolved solids; TSS, total suspended solids; fixed suspended solids; and, VSS, volatile suspended solids. But usually, only four of these are used: namely, TS, TDS, TSS, and VSS. Negative errors can occur due to the decomposition of certain inorganic compounds; e.g. loss of ammonium salts (NH 4HCO3 NH3 + H2O + CO2) or CO2 release from magnesium carbonate (MgCO 3 MgO + CO2). Positive bias should be due to incomplete oxidation of organic compounds or loss of recalcitrant water of crystallization.

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Additional problems appear in the analysis of sediments, sludge and soils. A special attention should be paid to occluded water, at least 1-hour ignition interval is recommended. Procedure: 1. Vessels, filters, evaporating dish and any other material should be dry and weighed to a constant weigh. 2. Ignite the sample in a furnace for 15 20 min at 550 50 C. Enlarge ignition period up to 1 hour for sludge, soil and sediment samples. 3. Cool in open air for 15 min and then keep it in a desiccator down to room temperature

Figure II.4.3. Combustion (or thermogravimetric) analysis for determination of carbon and hydrogen. The total carbon and hydrogen contents in a solid sample can be determined by combustion in the presence of pure and dry oxygen (Figure II.4.3.). With the aid of a catalyst (a transition metal), the combustion process releases only CO 2 and H2O. Two successive traps, P2O5 and ascarite, serve to retain all water and carbon dioxide released, respectively. P2O5 is an efficient desiccant (it is deliquescent) and the sodium hydroxideimpregnated asbestos (ascarite) will trap all of the carbon dioxide through the chemical reaction, NaOH + CO2 NaHCO3. The third tube is placed for protecting the two former traps from backflow of atmospheric water and carbon dioxide. After the sample combustion both tubes are weighed to determine the increase. This type of analysis is sometimes applied to soils, sediments, dried sludge and extracted aquatic organic matter.

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II.4.1.3. Precipitative Gravimetry Gravimetric Determination of Ionic Contents An ion gravimetric determination requires the conversion of the analyte into an insoluble compound of a known stoichiometry, removed from the mother solution by filtration and weighing. First of all, to obtain a non-filterable precipitate, crystal growth should occur faster than crystal nucleation; if not tiny micro-crystals are formed rather than a few large ones and micro-crystals behave as colloids and pass through the filtering devices. Heating the solutions and adding the precipitant reagent slowly with rapid mixing to decrease the super-saturation degree will help. Co-precipitation, when unwanted ions or molecules are retained (physically trapped) in the precipitate due to: Inclusion, a single substitution in the crystal lattice by an ion of similar size. Occlusion, the physical trapping of impurities from mother liquor within crystal irregularities. Crystal purity increases by re-dissolving the precipitate and then repeat the precipitation; it also means yield losses due to solubility. A. Sulfates, Sulfites and Barium Methods based on the weight of precipitated BaSO4. Sulfate. The historical method of choice for sulfate in waters and wastewaters is the gravimetry with barium (APHA et al., 1985), Ba+2 + SO4-2 = BaSO4 (precipitated) 1. Barium sulfate precipitates quantitatively by adding excess of ion Ba2+ under acidic conditions (5x10-2 mol L-1 in hydrochloric acid), Low pH is required to avoid the co-precipitation of barium carbonates and/or phosphates. 2. The formation of high purity and non-filterable crystals implies to allow reaction to continue for at least 2 hours at temperature over 80 - 90 oC. This process (digestion of the precipitate) minimizes the formation of filterable BaSO 4 crystals from the initial colloidal particles. 3. Filter the precipitate by decantation on a filter paper and the resulting solid mass is washed and dried in an oven for 1 hour at 800 oC. During this process the reducing character of the filter paper can originate barium sulfur, BaSO4 + C BaS + 4 CO. To avoid it, add a drop of sulfuric acid and repeat the calcination. This calcination procedure (1 hour at 800 900 C) should be performed on the porcelain crucible used for this operation. 4. Finally it is weighed at room temperature (keep it into a desiccator). Final weight, total minus tare (crucible).

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Chloride. It could be gravimetrically determined by precipitation with silver. However, the usually recommended procedure for water and wastewater samples (APHA et al., 1985) is the titration by precipitation with potassium chromate (red precipitate of silver chromate) as titrant. Interferences come from the precipitation of different silver salts with a lot of present anions namely: bromide, iodide, cyanide (high contents of these ions is not usual), and reduced sulfur species like sulfite, sulfide, and thiosulfate. To avoid the precipitation of reduced sulfur ions it is advisable to add hydrogen peroxide to transform into sulfate ions on the other group. Determination of barium as BaSO4. The precipitation is not carried out by slow addition of precipitant to the analyte containing solution. Such a procedure tends to produce conditions of super-saturation and a consequent rapid formation of small, relatively impure solid particles results. This effect cannot be entirely eliminated and is a source of error. The homogeneous precipitation techniques are able to avoid this erroneous effect; the precipitating reagent is generated slowly and homogeneously by slow hydrolysis of sulfamic acid in boiling aqueous solution: NH2SO2OH + H2O NH4HSO4 Determination of barium as oxalate Procedure: Add 10 mL of solution concentrated ammonium chloride to 200 mL of sample and then one drop of 1% heliantine. Adjust the suitable medium by dropping hydrochloric acid up to red color and immediately the required ammonium up to the change into yellow. Filter to separate any precipitate if formed. Add excess of acetic acid (clearly acidic medium) and 25 mL of the sodium oxalate solution. Let it settle for 24 hours at room temperature and protected from dust. Filter on paper (no ashes material) and wash with a lot of boiling water up to no acidic residues can be detected in the filtrate. Dry at 110 C and then calcinate the precipitate with the aid of Pt crucible. Cool to room temperature into a desiccator and the weigh. FIA-gravimetric procedures The best testimony to the high flexibility of FIA for adaptation to all types of procedures is probably the fact that the analytical balance has been used as a detector in FIA manifolds. In fact, classical gravimetric analysis has been implemented in continuous flow-systems (precipitation weighing included). For this purpose, two fixed volumes of sample and reagent are inserted into two channels. A stream of dry air propels both solutions to a merging point, where they react to form a precipitate. The precipitate is retained on a filter inside the flow-cell, which is accommodated on the balance pan. The air stream also functions to dry the retained precipitate on the filter prior to weighing. In this way, constancy in the amount of moisture retained by

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each sample is ensured. After weighing, the precipitate is removed by dissolution with an injection of an appropriate solvent. In this way, a throughput of up to 30 samples/h can be achieved. At the present, there are very few published procedures on the FIA-gravimetric analysis. One devoted to calcium determination is reported (as references O. Jacintho, M.A.Z. Arruda, E.A.G. Zagatto and B.F. Reis, ACA 258, 192, 129 133). According to the authors classical gravimetric analysis, sometimes regarded as obsolete, remains important mainly for accuracy assessment and the evaluation of standards for instrumental calibrations gravimetry is seldom applied to large-scale analysis, because it involves a number of prolonged operations usually to be carried out by skilled analysts.for routine analyses, the favorable characteristics of gravimetry could be better exploited after automation. .. the feasibility of gravimetry in flow analysis is demonstrated with the determination of barium as oxalate . Reactions of precipitation have been proposed in flow systems with the goal of the separation and/or pre-concentration of the analyte or turbidimetric determinations. In the former purpose filtration separation is required and is accomplished by placing the filter at a strategic part of the flow assembly. Separations analyte-interferences can be accomplished through selective dissolution of the retained precipitate. If only selectivity enhancement is needed. When the goal is the preconcentration, the analyte is precipitated, retained in the filter, washed and leaded to the flow-cell of the detector. The detection limit associated was 0.03% (w /v) Ba for a signal-to-noise ratio of three. Unlike the usual gravimetric procedures that are based on calculations on the obtained stoichiometric precipitate, the flow procedure requires calibration graphs, as the drying step was only partial. Linear calibration graphs were obtained over the range 0.40 -1.60% (w /v) (R = 0.999994; n = 5). The linearity of the calibration graph revealed that the retained water was proportional to the amount of precipitate formed, which means the humidity of the crystals was constant. A slight variation under < 5% of the slope of the calibration equations was observed. The analysis of up to 30 samples h -I was possible. According to authors carryover effects were not relevant. B. Silica The gravimetric method for determination of silica in water should be selected according to the form that is present in the sample, as ionic or colloidal state. A method for the total content is reserved for samples presenting high contents (several tens of mg L-1). For small amounts (les than 10 mg L -1) is suitable the spectrophotometric method to the o-phosphate ionic form. To avoid silica adsorption on the glass walls (high silica content samples) use polyethylene. Gravimetric procedure. The silica is precipitated partially de-hydrated by concentrated hydrochloric acid; total loss of water molecules is completed through a

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calcination step. Addition of hydrofluoric acid converts the precipitate into a volatile fluorsaliclic acid and after new calcinations the weigh loss corresponds with the pure silica content. Procedure 1. To 1 2 liters of sample add 10 mL of hydrochloric acid. 2. Dry to residue (Pt capsule is preferred) by means of a water bath 3. Keep the residue for 1 hour at 105 110 C. 4. Add 5 mL of HCl and keep for 5 min protected from dust. After addition of 50 mL of water, warm gently for 1 min and filter. 5. Wash the residue with 0.5 mL l -1 HCl and rinse with pure water to complete elimination of any acidic residue by testing that the washing water is without presence of chloride (AgNO3 reaction). 6. Dry completely the residue, place it into the furnace at 110 C and repeat the above reported procedure by using a second filter. 7. Calcination of the two filters at 1200 C. 8. Add 10 mL of HF and two drops of 50 % sulfuric acid. Dry and calcinate at 1200 C up to constant weigh. 9. Estimated precision 0.2 mg L-1. Any used reagent (pure water included) should be silica exempted and the hydrofluoric acid gives a non-volatile residue. C. Determination of Lithium Only recommended for high contents like (thermo-mineral waters). D. Determination of Magnesium The gravimetric procedure should be applied for contents over 10 mg L -1 and it is performed on the filtrate from the separation of calcium; and then, weighed as magnesium pyrophosphate (MgP2O7). Procedure: 1. Evaporate the acetic filtrate down to 150 mL. 2. Add two drops of HCl and 20 mL of 20 % ammonium phosphate and warm to boil. 3. Add ammonia to about neutral point plus concentrated ammonia to 1/5 volume. If a slow precipitation is observed, rub the beaker walls with the aid of a glass rod. 4. Keep at room temperature for a 12 h interval. 5. Filter by using a free ashes paper and wash with 50 % ammonia. Dry and calcinate with a weighed crucible. 6. Weigh.

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E. Determination of Sodium The gravimetric alternative to the flame photometric estimation of sodium is the procedure based on the weigh of the precipitate obtained with reaction with zinc uranyl acetate. Procedure: 1. Pre-wash and dry into a desiccator (at void) a filter crucible of pore average 10 -20 m. 2. Filter the precipitate and wash (5-fold) with 2 mL of ethylic alcohol at 96 C; previously saturated with zinc and sodium uranyl acetate. Then wash (3 - fold) with 1 mL of ethyl oxide; and, eliminate the ethyl remaining by aspiration. The dry process will end into a desiccator up to constant weigh. 3. The sodium amount (mg L-1) is = M x 14,95 V (being M, mg of precipitate and V, mL of sample. REFERENCES 1. Jean RODIER. LAnalyse de leau-Eaux naturelles, eaux rsiduaires, eau de mer . 7 dition, Dunod, BORDAS, Paris 1984. 2. F.DIENERT, F.WANDENBUCKLE. Sur la dtermination de la silice dans les eaux, Comptes rendus Acad. Des Sciences, 1923,176,1478. 3. F.DIENERT, F.WANDENBUCKLE. Une tude de la slice collodale, Comptes rendus Acad. Des Sciences, 1924,178,564. 4. R.B. FISCHER, T.B.RHINIHAMMER. Rapid precipitation of barium sulphate, Anal. Chem., 25, (1953), 1544. 5. F.P.TREADWELL, M. BOLL. Manuel de chimie analytique. Dunot edit. Paris, II, (1939), 73. 6. F.P.TREADWELL, M. BOLL. Manuel de chimie analytique. Dunot edit. Paris, II, (1939), 67. 7. E.H.TYNER. Anal. Chem., 20, (1948), 76. 8. K.M. BUTLER, E. TUTHILL, An Application of the Uranyl Zinc Acetate Method for the Determination of Sodium in Biological Material. J.Biol.Chem., 1931,93171. 9. J. RANCHET, P. CLEMENT. Contribution lamlioration du dosage des hydrocarbures dans les eaux. Bull. Liaison Labo. P. et C.,1977, 91, sept-oct, p.67. 10.APHA, AWWA, WPCF (1985) Standard Methods for the Examination of Water and Wastewater, APHA, Washington, 16th edition, pp. 32-34, 76-80, 92-100, or 15th edition, pp. 30-32, 70-73, 90-98, or 14th edition, pp. 34-36, 71-75, 89-98. 11.Guy, H.P. & Norman V.W., Techniques of Water-Resources Investigations of the United States Geological Survey, Book 3, Chapter C2, Field Methods for Measurement of Fluvial Sediment. 12.Hem, J.D., Chapt. 4 in Water Analysis, Volume 1, Minear & Keith eds. 13.Hem, J.D., USGS Water Supply Paper #1473: 1st ed. 1959; 2nd ed. 1970.

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14.Rubinson, K.A. (1987) Chemical Analysis, Little, Brown & Co., Publ., Boston, pp. 178-179, 243-245. 15.Sawyer, C.N. & P.L. McCarty (1978) Chemistry for Environmental Engineers, McGraw Hill Publ., pp. 65-69, 284-285, 454-462. 16.Snoeyink, V.L. & Jenkins, D. (1980) Water Chemistry, John Wiley & Sons, Publ., New York, pp. 74-82. 17.USEPA (1982) Handbook for Sampling and Sample Preservation of Water and Wastewater, US EPA, EMSL, September, 1982; EPA-600/4-82-029 [Gov.Docs. EP1.23/5:600/4-82-029]. 18.ISO ICS 13 Environment. Health Protection. Safety (Excludes ISO 14000) en http://webstore.ansi.org/ansidocstore/dept.asp?dept_id=220 19.Gravimetric procedures (from web-site of ISO) are 107 references for metallic samples (silica, aluminum, etc.), soils, etc. a) Ba rule: ISO 548:1981 b) Sulfates: ISO 2480:1972; ISO 2997:1974

II.4.2. VOLUMETRIC METHODS

Jos MARTNEZ CALATAYUD II.4.2.1. Fundamentals of Titrimetry Volumetric or titrimetric analysis is a classic quantitative method which employs an exactly measured volume of a standard solution containing a known concentration of reagent "A"; this reagent A is added step by step (from a burette) to the unknown concentration of the analyte B in the sample; the process proceeds until a perceptible change is produced (chemical indicator). The change, end-point, should be produced as close as possible to the complete reaction A + B in an stoichiometric completion. This theoretical point is known as the equivalencepoint; where the number of added A equivalents to the sample solution equals the number of equivalents of "B" originally present. Alternatively, a non-perceptible change could be also used, a physical indicators in which a physical property (conductivity, absorbance, etc.) is continuously measured. If the A additions to the sample proceeds far away from the equivalence point; further calculations allow to obtain the endpoint. Two basic modalities are normally used: namely, direct titration (as above reported) and back titration. The latter is based on the addition of an intermediate reagent having the excess (not reacted with the analyte) is titrated. Titrimetric methods reach a precision of up to 0.1%. Accuracy is related to the coincidence among end-point (or indicator point) and the equivalence-point.

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Several basic requirements can be reported for a well-defined titrimetry: (a) A quick and quantitative reaction A + B is necessary. (b) A standard titrant solution of accurately known concentration of A to react with the analyte with a well-known and repeatable stoichiometric procedure. (c) An indicator (external or internal) to identify the endpoint. The random and systematic error resulting from the empirical estimation of the endpoint may be estimated by conducting a blank titration. (d) When the chemical for the titrant solution is not available in a kinetically stable form of pure and well-defined composition, the titrant must be standardized. This operation is an independent titration against stable, pure chemical known as a primary standard. (e) Accurate measurements of the sample and added titrant volumes. (f) It should be interesting if possible to have an A + B reaction sufficiently selective to avoid a previous sample treatment to remove interferences. Titrations can be performed manually step by step (or point by point), or automatically, where the titrant A is introduced continuously. At present, in analytical chemistry automation is of paramount importance for the following reasons: (a) convenience and speed, (b) performing analysis without supervision, (c) enable application of elaborate techniques for analyzing the data by computerized systems. For automatic transfer of the titrant to the analyte solution are proposed several instrumental devices: (a) Piston burettes are highly reliable and do not require calibration, (b) Peristaltic pumps highly versatile and reliable. However and due to the changing properties of the flexible tubing they need to be periodically calibrated. (c) Suction-stroke piston pumps or metering pumps are more precise and require less calibration; they enjoy a similar degree of versatility. Other empirical limitation of the pumps is the use of corrosive reagents, highly concentrated mineral acids (not in titrimetry) and organic solvents. There are commercially available acid and solvent resistant tubing. Several advantages of these pumps are the simplicity of operation, simple and reliable control of flow-rate, easily automated, allow to work in a wide range of flow rates (for the titrant transfer; a relevant parameter to be optimized). For titrations in small volumes low flow rates are used (0.1 - 1 mL/min). There are very relevant factors as stirring speed, configuration of the cell, input of titration, etc. Of paramount importance are the kinetics of the volumetric reaction and the response of the indicator system.

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Table II.4.2.Titrimetric methods for environmental water analysis.

Analyte (A) Standardized Chemical Indicator titrant solution (internal) (B) Observed change Neutralization (acid-base) Alkalinity HCl Acidity NaOH Nitrogen (III) H2SO4 Volatile Acids NaOH Precipitation Chloride AgNO3 Chloride Hg2+ nitrate Complex formation Ca2+ EDTA Hardness EDTA methyl red phenolphthalein potassium chromate yellow-precipitate Diphenylcarbazone Eriochrome Blue Black R intense red - deep blue Eriochrome Black T intense red - deep blue pH 12 Ca-EDTA 2Direct titration As Ca and Mg total amounts, expressed as carbonates Winkler Permanganate I3- - S2O32I3- - S2O32DPD Ferrous K2Cr2O7 Sample pretreatment Volumetric reaction

OH- + H+ H+ + OH digest/distillatio Macro-Kjeldahl n NH3 & Acidimetric distillation distillation

Oxidation/Reduction Dissolved Na2S2O3 O2(DO) Ca2+ KMnO4 Chlorine/ClO2 Na2S2O3 SO32Na2S2O3

Chlorine/ClO2 FeSO4 Concentration Fe(NH4)2(SO4)2 of dissolved oxygen, COD

Starch Deep blue colorless (white turbidity) auto Starch Deep blue colorless (white turbidity) Starch Deep blue colorless (white turbidity) DPD ferroin

Mn2+, I(I-) oxalate I(I-) I(I-)

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Complexation Titrations In a complexometric titrations commonly a metal is the analyte and the most usual titrant is the sodium salt of the ethylenediaminetetraacetic acid (Na 2-EDTA). The EDTA molecule is: [HOOCCH2]2NHCH2CH2NH[CH2COOH]2 This is a hexadentate ligand, which binds very strongly to many metals, and its stability is highly dependent of the pH. The complete complexation of the metal is quick as the stability constants are quite large and the formed complexes show a welldefined stoichiometry, 1:1. The end-point can be estimated either by a chelating metal chemical (internal indicator) or an ion-selective electrode sensitive to the analyte. This former and classic method employs an specific dye of the metallochromic type, a chelating ligand which gives a solution change in color in going from the complexed to the uncomplexed indicator-analyte form. To be effective, the indicator-analyte complex at the suitable pH for the procedure must not be affected by other metal ions in the sample and obviously, must bind the metal less strongly than EDTA does. A. Hardness. Definitions and Environmental Significance Hardness means the total concentration of divalent cations in the water sample. These may include two relevant concentrations, Ca (II) and Mg (II), and any other with a negligible concentration, like Sr (II), Ba (II), Fe (II) and Mn (II). Hardness may be classified according to: (a) the constituent anions: namely, calcium-hardness and magnesiumhardness; or, (b) by the counter ions, like carbonate or non-carbonate hardness. The term total hardness means the Ca-hardness plus the Mg-hardness or the sum carbonate hardness plus non-carbonate hardness. The carbonate hardness means the hardness that could be precipitated as carbonates, precipitation that occurs when the sample is heated, evaporated or if the pH was raised. Carbonate hardness could be equal to the alkalinity (in mg L -1 as CaCO3) when the alkalinity is smaller than the total hardness. The non-carbonate hardness is also known as remaining hardness. When hardness is less than alkalinity, the carbonate hardness is similar to the total hardness and non-carbonate hardness is equal to zero. Hardness is commonly minimized in drinking water treatment plants by precipitate softening or cation exchange. To calculate the chemical requirements for this softening, it is necessary to establish both the total hardness, and the calcium hardness.

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Hardness is considered a problem in drinking waters and sometimes in industrial process waters and agricultural waters due to several considerations. Divalent cations will complex with the carboxyl groups in soap and cause its precipitation; inhibiting its action; and in addition, may cause severe hydraulic problems in pipes by precipitating as metal carbonates, especially when the water is heated as it happens in water-heaters and industrial boilers. On the other hand, a controversial point is that hardness has been associated with lower incidences of cardiovascular health problems. Hardness is originated by limestone and related minerals. As calcium is the dominant hardness species and carbonate-bicarbonate is the most prevalent counterions, hardness has historically been expressed as a mass concentration of equivalent calcium carbonate. B. Fundamentals of the Determination Eriochrome black T forms a deep red complex with calcium and magnesium when the indicator is added to the sample with adjusted pH (at about 10); these pH values allow the complex formation due to a partially de-protonated form that is effective as a chelating agent (pKs, are 6.3 and 11.6). In addition, at this pH the color change experienced by the indicator is most easily perceptible by the human operator. More important is the fact that Na 2-EDTA is highly effective at high pH for calcium and magnesium. At this pH many metal ions will hydrolyze and precipitate; due to that, the presence in the solution of an auxiliary complexing agent is need. A complexing agent which competes successfully against hydroxide binding preventing precipitation but with no enough strength to interfere with the complexing reaction of Na2-EDTA or the metallochromic indicator; for total hardness triethanolamine and ammonia are used. Also useful might be citrate or tartrate. The order of reaction is as follows: Ca (II) and Mg (II) present in the sample are complexed by the addition of the indicator; a high excess of both remain in the cationic form. The titration with Na 2-EDTA forms complexes with metallic cations (here Ca and Mg), the order and extent is in according to the stability constant and the status of the ion in solution. Due to that; first the EDTA complexes the Ca (II) present as cation, then the Mg (II) and finally Ca (II) and the Mg (II) (with this order) complexed with the indicator. The indicator passes to a free ionic form and the solution suffers the change of color, from complex Mg (II) - indicator to the free indicator; from intense red to deep blue; the last coloration gives the end-point. To ensure this way, a small amount of Mg EDTA should be added with the buffer. It is also possible to perform the back titration. A known amount of Na 2EDTA is added; this amount is in excess. Then, with a different metallic standard solution, the excess of Na2-EDTA is titrated; bearing in mind that the titrant metals bind less strongly to Na2-EDTA than the analyte.

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Some compounds, binding the metallic cations, can interfere as cyanide, sulfide, and hydroxylamine; these samples require a previous addition of a suitable inhibitor. An inter-laboratory study by using a synthetic sample containing 610 mg L -1 CaCO3 found a relative standard deviation and a relative bias of 2.9% and 0.8%, respectively. Procedure: Place exactly 50 mL of the sample into a 125 mL Erlenmeyer flask and add 1-2 mL of the buffer solution and several drops, 5 6, of the indicator. Finally, titrate with the EDTA solution until all reddish coloration disappears. Maintain constant stirring during this operation. For best results the titration should be completed within 5 minutes. Reagents: (a) To prepare the buffer solution, dissolve 16.9 g ammonium chloride in 143 mL conc. ammonium hydroxide. Add 1.25 g Mg-EDTA and dilute to 250 mL with distilled water. (b) Indicator Solution (approx. 0.011 mol L -1) - Dissolve 0.5 g of Eriochrome Black T (1-(1-hydroxy-2-naphthylazo)-5-nitro-2-naphthol-4-sulfonic acid) in 100 g triethanolamine (2,2',2''-nitrilotriethanol). (c) EDTA Titrant Solution (approx. 0.01mol L-1) - Dissolve 3.723 g Na2-EDTA (sodium ethylenediaminetetraacetate dihydrate) in 1000 mL distilled water. The resulting solution is titrated vs. the standard calcium solution. (d) Standard Calcium Solution (0.005 mmol L-1) - Weigh 0.5005 g anhydrous calcium carbonate (primary standard grade) and carefully add small amounts of 6 M HCl (approx 50% of conc.) until CaCO 3 just dissolves. Add 200 mL distilled water and boil for a few minutes to expel dissolved CO 2. Cool, add a few drops of methyl red indicator and adjust to the "intermediate" orange color by adding 3N NH4OH or 6M HCl as needed. Transfer quantitatively to a 1-liter volumetric flask and dilute to the "mark" with distilled water. C. Calcium Calcium can be determined by atomic absorption spectrophotometry (AAS), by titrimetry, or by a redox method or the most usual, the EDTA-complexometric method. The complexometric procedure is quite similar to the above reported for total hardness. The reader must bear in mind several modifications, to avoid the magnesium analytical activity and make the analysis specific for this calcium-hardness determination. First, magnesium is partially removed by using a higher pH (about 12) which results in precipitation as Mg(OH) 2 and calcium hydroxide does not precipitate (less acidic cation). Second, use a metallochromic indicator that only complexes with calcium as the Eriochrome blue black R. Due to the larger stability constant of the EDTA-Ca over the corresponding magnesium, EDTA binds preferentially with Ca. An inter-laboratory study resulted in

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a standard deviation of 9.2%, and a relative bias of 1.9% for synthetic water samples containing 108 mg/L calcium and 82 mg/L magnesium. Procedure: Place exactly 50 mL of the sample into a 125-ml Erlenmeyer flask: then, add 3 mL of NaOH solution or the required amount if the pH is still below 12. Add solid Eriochrome Blue Black R indicator (0.1-0.2 g). Finally, titrate with the Na2 - EDTA solution with continuous stirring, until the color changes from red to royal blue. Reagents: (a) Standard 0.01 mol L-1 Na2-EDTA Titrant Solution prepared as above. For a routine analysis the accurate weighing and level is enough. (b) 1 mol L-1 sodium hydroxide solution, solve 40 g L-1 of NaOH (c) Eriochrome Blue Black R, grind in a mortar 200 mg powdered dye [sodium-1-(2hydroxyl-1-naphthylazo)-2-naphthol-4-sulfonic acid] together with 100 g NaCl to about 40-50 mesh. (d) Standard calcium solution, weigh 0.5005 g anhydrous calcium carbonate (primary standard grade) and proceeds as above reported Redox Titrations Redox titrations are based on oxidation-reduction reactions between the analyte and the titrant. Common strong oxidants used as titrant include dichromate (Cr2O7-2), iodate (IO3-), iodine (I3-), Cerium (IV) and permanganate (MnO 4 -). Most of the oxidizing (titrant) agents are stable, only permanganate solution should be protected from room light and dust, However, the reducing titrant agents can be affected by oxidation by atmospheric oxygen, and their concentration (titer) must be checked regularly against the corresponding standard. The most used reducing agents are thiosulfate (S2O3-2), arsenite (AsO3-3), ferrocyanide (Fe(CN)6-4), ferrous (Fe+2) and sulfite (SO3-2). Residual Chlorine by the N,N-Diethyl-p-Phenylene Diamine (DPD) Method The N,N-Diethyl-p-phenylene (also known as DPD) reacts with chlorine or tri-iodide to form an intense red coloration due to a free radical; this is titrated (back titration) with ferrous ion producing a colorless solution. DPO reacts with a faster kinetics with chlorine. Reaction with monochloramine is slower depending on its concentration; and in addition, the presence of HgCl2 apparently inhibits this reaction maybe by forming an unreactive complex Hg(II)-monochloramine. After addition of iodide, monochloramine reacts quickly to form tri-iodide, which then develops the red color, by reacting with the DPD. The reaction with dichloramine requires larger amounts of iodide and the reaction is slower. The DPD solutions are kept in an acidified media to avoid the oxidation by atmospheric oxygen, reaction catalyzed by a basic medium. Periodically replacing of

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the solution is recommended (about one month). The neutral buffer for the reaction chlorine or tri-iodide with DPD should be added in the moment of the reaction. The presence of oxidants, like hydrogen peroxide and oxidizing species of manganese and persulfate, interfere seriously by oxidizing iodide. The detection limit is 18 g L-1. Procedure: Place 5 mL of the buffer reagent and the DPD indicator solution and then, add 100 mL sample and mix. (a) For free residual chlorine (FRC): Titrate rapidly with ferrous ammonium sulfate until the red color disappears (volume A). (b) For monochloramine (MCA): Add a small amount of potassium iodide to solution from step (a) and mix. Continue titration until the red color again disappears (volume B). (c). Dichloramine (DCA): Add about 1 g of KI to the solution titrated in step (b) and mix. After two minutes continue titrating until the red color is again discharged (volume C). For high dichloramine concentrations, allow 2 minutes standing time if color driftback indicates incomplete reaction. For low expected amount off dichloramine concentrations can be used half the reported amount of potassium iodide. Reagents: (a) Phosphate buffer solution: Dissolve 24 g anhydrous Na 2HPO4 plus 46 g anhydrous KH2PO4 in pure water. Add to this solution 100 mL distilled water in which 800 mg Na2EDTA have been dissolved, after adding 20 mg HgCl 2 to inhibit biological growth. Fill to the mark (1 L) with water. (b) Dissolve 1 g N,N-diethyl-p-phenylenediamine oxalate in water containing 2 mL conc. H2SO4 and 200 mg Na2-EDTA dihydrate. Make up to 1 liter and the resulting solution must be stored in a brown glass-tight closed bottle. (c) Ferrous ammonium sulfate as titrant: Dissolve 1.106 g Fe(NH 4)2(SO4)2.6H20 in water containing 1/4 mL of conc. H2SO4 and fill up to 1 L. (d) Solid potassium iodide. Ozone (iodometric method) A portion of the gas stream is directed to a gas bubbler filled with 2% KI solution for a well-known period of time. The stoichiometric reaction is: O3 + 2KI + H2O I2 + O2 + 2OH- + 2K+ The iodine formed is then titrated with sodium thiosulfate using starch. Dissolved Oxygen A. Environmental Significance

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Oxygen is a rather insoluble gas, its solubility ranges from 14.6 mg L -1 at 0oC down to about 7 mg L-1 at 35oC. Its solubility also varies with atmospheric pressure and salinity. The paramount importance of the dissolved Oxygen (D.O.) can be summarized with the following points: (a) is required for the survival of aquatic life; (b) is very important in biological treatment processes; (c) the parameter D.O. is used in the assessment of oxidation state in ground-waters and sediments; (d) is used in the assessment of the strength of a wastewater through either the Biochemical Oxygen Demand (BOD) or respirometric studies. As it is required for the survival of aquatic life it is considered an important water quality parameter for natural aquatic systems. The larval stages of certain coldwater fishes are quite sensitive to the oxygen concentration. In addition, discharges of organic wastes may depress the dissolved oxygen concentration; mainly due to the microbial-mediated oxidation of the waste upon discharge of organic compounds. In the assessment of the oxidation state in sediments (collected into aquatic sediments) and deep ground-water samples (collected deeper into the surface) the D.O. often drops. Eventually the concentration level is low enough to allow the anaerobic process Thinking on biological treatment processes and in a general point of view, the D.O. concentrations indicate when aerobic and anaerobic organisms will predominate. Commonly, dissolved oxygen determinations establish the suitability of oxygen transfer systems to aerobic suspended culture operations such as activated sludge. It may also be used to indicate the suitability for the growth of such sensitive organisms such as the nitrifying bacteria. Dissolved oxygen may be determined by the Winkler titrimetry and the Membrane Electrode method. The difference between both methods is that the Winkler method measures dissolved oxygen concentration where the potentiometric electrode method measures the activity. The activity coefficient relates both parameters. B. Fundamentals in the determination of D. O. The oxygen performs a quick oxidation of manganese ions [from Mn (II) to Mn (IV)] at high pH (see the diagrams E pH). At low pHs, oxidizes iodide to triiodide (formed iodine combines with excess of iodide) and the resulting tri-iodide is titrated as usual with sodium thiosulfate with the aid of the starch for better end-point. The titration is performed at pH under 5; smaller pH media produce a relevant decomposition of the thiosulfate. The reaction sequence is: Mn (II) + 2 OH- Mn (OH)2 4 Mn (OH)2 + O2 + 2 H2O 4 Mn (OH)3

117

2 Mn (OH)3 + 3 H2SO4 Mn2(SO4) + 6 H2O Mn2(SO4)3 + 3 I- 2 Mn2+ + I3- + 3 SO42I3- + 2 S2O32- S4O62- + 3 ITo eliminate the interferences from nitrite, the alkali azide iodide reagent also contains sodium azide, NaN3. The azide reacts with it to release nitrogen gas and nitrous oxide, neither of which interferes. NaN3 + H+ HN3 + Na+ HN3 + NO2- + H+ N2 + N2O + H2O The nitrite interferes by raising the apparent concentration of dissolved oxygen according to the following reaction. 2NO2- + 3I- + 4H+ I3- +2 NO + 2H2O Note that the produced nitric oxide can then scavenge any oxygen introduced during titration to produce nitrite and start the cycle. 2 NO + 1/2O2 + H2O 2NO2- + 2H+ Other interferences include reducing agents as ferrous iron, sulfite, thiosulfate and aldehydes. Thiosulfate is not a primary standard reagent due to an ill-defined number of hydration water molecules; its solution should be necessarily standardized against potassium dichromate or potassium iodate. The reaction with dichromate is not as quick as iodate and in addition the green coloration from Cr (III) may interfere with the endpoint. In either case, to an excess of iodide is added a known amount of the primary standard. 2IO3- + 16I- + 12H+ 6I3- + 6H2O Cr2O7-2 + 9I- + 14H+ 2Cr+3 + 3I3- + 7H2O These oxidants treated with an excess of iodide release stoichiometric amounts of tri-iodide, which then reacts with the thiosulfate. Thiosulfate solutions should be preserved from the growth of sulfur bacteria (oxidize the reagent to sulfate) by adding a small amount of NaOH. In addition, high pH will retard downward pH drift from absorption of atmospheric carbon dioxide and minimize the conversion of thiosulfate into sulfate and elemental sulfur. C. Sampling This parameter D. O is obviously very sensitive to sample contact with air; it should be limited to avoid relevant increases in D.O. Specialized sampling techniques

118

are required to minimize the close contact atmospheric air-water sample. Specialized samplers for this parameter are commercially available. D. Analytical Procedure Procedure: To a 300 mL BOD bottle filled with sample add 1 mL of manganous sulfate solution and 1 mL of the mixture alkali-iodide-azide: Cap avoiding the trapping of any air bubble; then shake. If any air bubble has been trapped, add a small amount of distilled water and re-cap. Allow the precipitate to settle to about half the height of the bottle and add 1 mL of concentrated sulfuric acid. Re-cap and mix. Titrate (200 mL of sample) with the 0.025 mol L -1 sodium thiosulfate solution without reaching the colorless end-point. The titration proceeds until a very pale yellow color; stop the titration, add some few drops of the starch solution and continues the titration until the deep blue color disappears. A white turbidity remains. Reagents: Dissolve 400 g MnSO4.2H2O in pure water and dilute to 1 liter. The alkali-iodide-azide reagent is prepared by dissolving 500 g NaOH and 150 g KI in water; then add 10 g NaN3 in 40 mL water and dilute to 1 liter 0.025 mol L-1 Sodium Thiosulfate - Dissolve 6.205 g of Na 2S2O3.5H2O in water and add 0.4 g NaOH; then level to 1 liter. This solution should be standardized with Potassium Iodate or Potassium Dichromate with the aid of starch for a clear endpoint. The starch solution is prepared by dissolving 2g of soluble starch and 0.2g of salicylic acid (recommended as solution preservative) in 100 mL of hot pure water.

(a)

(b) (c)

(a) (b) (c)

(d)

Chemical Oxygen Demand, COD The chemical oxygen demand is expressed as the amount of potassium dichromate reduced by the sample during 2 h of reflux in a medium of boiling, containing 5O% H2SO4 and Ag2SO4 as catalyst. The reaction between dichromate (add an excess) and organic matter is as follows: CxHyOn + R Cr2O72- x CO2 + 2R Cr3+ + [7R+n-2x] H2O

(a) reduction of dichromate: 6e- + 14H+ + Cr2O7-2 2Cr3+ + 7H2O Eo = 1.33 volts

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(b) after oxidation of the organic matter is complete, the excess dichromate is titrated by the reducing agent Fe+2, Fe+2 Fe+3 + eEo = -0.77 volts

The endpoint of this titration of dichromate can be determined colorimetrically by using 1,10-phenanthroline, which forms a colored complex with the resulting Fe (II) ions. At the endpoint the color changes from blue-green to reddish brown (Fe(II) -phenanthroline). The chloride ion interferes by being oxidized to chlorine (6Cl - + 14H+ + -2 Cr2O7 3Cl2 + 2Cr3+ + 7H2O); the presence of Ag 2SO4 (used as catalysts) avoids this precise oxidation and the chloride is subjected to an unpredictable oxidation procedure. Also it can be precipitated as AgCl The addition of HgCl2 serves to remove the free chlorine. The presence of organic impurities can be oxidized by Cr2O7-2 resulting in an excess of Cr 3+; the correction is established through a blank with pure water. REFERENCES

1. http://www.ecs.umass.edu/cee/reckhow/courses/572/572bk16/572BK16.html
2. D. Harvey, Modern Analytical Chemistry, McGRAW Hill, 2000, ISBN 0-07237547. 3. APHA, AWWA, and WPCF, Standard Methods for the Examination of Water and Wastewater, APHA, Washington (16th ed.). 4. H. A. Flaschka, EDTA Titrations, Pergamon Press, New York. 1959. 5. Kramer, J.R. (1982) "Alkalinity and Acidity", Chapter 3 in Water Analysis: Volume 1, Inorganic Species, R.A. Minear & L.H. Keith editors, Academic Press, pp.85135. 6. McQuaker et al. (1983) Environ. Sci. & Technol., 17:431-435. 7. G. G. Christian. Analytical Chemistry, Wiley, New York, 2003. 8. Sawyer, C.N. and P.L. McCarty (1978) Chemistry for Environmental Engineering, 3rd Edition, McGraw-Hill Publ., pp. 24-29, 168-188, 343-376. 9. Schwarzenbach, G & H.A. Flaschka (1969) Complexometric Titrations, Methuen, London. 10.V. L. Snoeyink and D. Jenkins, Water Chemistry, Wiley. 1980. 11.http://www.tau.ac.il/~advanal/TitrimetricMethodsOfAnalysis.htm Titrimetric methods of analysis 12.http://people.morehead-st.edu/fs/r.hunt/360-04vol.doc. Volumetric Methods

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II.4.3. UV-VIS SPECTROMETRY

Andrei Florin DNE II.4.3.1. Nature of Electromagnetic Radiation Electromagnetic radiation is a form of radiant energy which exhibits both wave and particle properties. Some phenomena such as: refraction, reflection and rotation of plane-polarized light are examples of wave properties. On the other hand photoelectric effect suggests particle properties of the electromagnetic radiation. The particle-wave duality explains the behavior and the nature of electromagnetic radiation. Wave properties. As indicated in Figure II.4.4 an electromagnetic wave has an electric component and a magnetic component oscillating in planes perpendicular to each other. Only electric component is active in ordinary energy transfer interactions with matter.
Electric Vector

Wavelength Electric Component Amplitude Direction of Propagation Magnetic Component

Magnetic Vector

Figure II.4.4. An electromagnetic wave. In Figure II.4.3, wavelength, , is the distance between two corresponding point on the wave. Another important property of an electromagnetic wave is its frequency, . The units of frequency are cycles per second or sec -1. The wavelength and frequency are related to the velocity of light by expression:

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 = c/n

(II.4.1)

where c = the velocity of light in a vacuum and n is the refractive index. Particle properties. To describe how electromagnetic radiation interacts with matter, a light beam is considered as a train of photons. The energy of each photon is proportional to the frequency of the radiation and is given by the relationship: E = h = hc/n (II.4.2)

where E = the energy of photon in ergs, = the frequency of the electromagnetic radiation in cycles per second and h = Plancks constant, 6.624 x 10-27 erg.sec. The individual photon energy is the basis for the phenomenon of light absorption. When the photon energy matches an allowed energy transition within the material through which the photon is passing, the energy (the photon) can be absorbed. Photons cannot lose just part of their energy through normal absorption. The photon energy must be equal to an allowed energy transition in the absorbing species. The entire range of radiation is commonly referred to as the electromagnetic spectrum. The ultraviolet and visible part of the spectrum is a very small part of the total range of possible (and detectable) frequencies of electromagnetic radiation (see Figure II.4.5). At longer wavelengths than visible light (about 800 nm) there is the infrared (IR) region, and at frequencies higher than blue light there is the ultraviolet (UV) region (10-400 nm). The UV region is most useful for analytical purpose is 200-400 nm. The 10200 nm region is the vacuum ultraviolet region and need special instruments for measurements.

II.4.3.2. Molecular Absorption of Electromagnetic Radiation

The total energy state of a molecule includes electronic, vibrational and rotational components. All of these energy components are quantified. The absorption of UV or visible radiation generates a transition between electronic levels of the molecule. The energy difference between molecular electronic levels is much greater than that between vibrational states. Thus for each electronic level of a molecule, there are also superimposed vibrational and rotational states as illustrated in Figure II.4.6. Therefore, for an electronic transition we observe a broad band absorption spectrum and that is typical of most absorptions of ultraviolet and visible radiation. The wavelength of the maximum of the broad band correspond to the electronic transition and the width of it to the superimposed vibrational and rotational transitions

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Electromagnetic radiation rays X rays Ultraviolet Visible , nm 10 10 10 Infrared 10 10 10 Microwaves Radiowaves

2

, Hz 3x10
16

3x10

15

3x10 3x10 3x10 3x10

14

13

12

11

Figure II.4.5. UV-Vis domain of the spectrum.

V3 E1 V2 V1 V0 Energy V3 V2 E0 V1 V0 r3 r2 r1 r0 r0 to r3 are rotational energy levels E1 E2 E3 E0 and E1 are electronic energy levels with E0 = ground state electronic level V0 to V3 are vibrational energy levels with V0 = lowest vibrational level in given electronic level

Figure II.4.6. Schematic representation of molecular electronic, vibrational and rotational levels.

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II.4.3.3. Quantitative Law of Radiation Absorption In quantitative absorption studies, a beam of radiation is directed at a sample and the intensity of the radiation that is transmitted is measured. If the photons that strike the sample posses an energy equal to that required to cause a quantized energy change, absorption may occur. Let us consider a monochromatic radiation. Consider the changes in radiation intensity that occurs as monochromatic radiation passes through the absorption cell in Figure II.4.7. We first fill the cell with a blank solution, which normally consists of the solvent plus sample constituents other than the principal absorbing species. With this blank solution in the cell, the transmitted intensity of the radiation ( It) represents the incident intensity of the radiation minus that lost by scattering, reflection and any absorption by the other constituents (normally quite small). We denote this radiation intensity as I0. I (I dl)

I0 dl l

It

Figure II.4.7. Radiation absorption process. Referring to figure II.4.7, let us consider what happens to the radiation as it passes through the sample. Using the differential notation of calculus, -dI represents the decrease in radiation intensity in an infinitesimally small layer, dl, i.e., the amount of radiation absorbed in this layer. The loss in radiation intensity, -dI, is directly proportional to radiation intensity in that point: - dI = k I dl (II.4.3)

where k is a proportionality constant; the negative sign is introduced because the radiation intensity decrease as dl increases. Integrating the equation II.4.3 over the entire cell length, l, gives the loss in radiation intensity due to absorption by the sample.

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It

I0

dI = k I

dl
0

(II.4.4)

Solving, ln I0/It = kl; That is the Lambert law. Very often the transmitted radiation intensity is denoted I (without subscript t) denomination that we shall use in what follows. Converting from natural logarithms to base 10 logarithms (designated by log) we obtain: log I0/I = k' l or I = I010-k'l (II.4.6) It = I0e-kl (II.4.5)

where k = 0.4343 k. Beer (a German physicist) established that if the radiation absorption is due to a dissolved specie, the proportionality constant k is dependent of that concentration (c), so we can write: k = ac (II.4.7)

where a is denoted the specific absorptivity (the concentration is given in grams/litre). If the concentration is given in moles/litre a is replaced by , that is defined as the molar absorptivity (commonly called the molar absorption coefficient). The value of is characteristic of the absorbing molecule or ion in a particular solvent and at a particular wavelength. The value of is independent of concentration and the path length of the radiation. If we replace k' with c in equation (II.4.6) we obtain: A = log I0/I = cl; I = I010-cl (II.4.8)

Equation (II.4.8) has been referred to as the Lambert-Beer law. In equation (II.4.8.) A is the absorbance. The term I/I0 is defined as the transmittance (symbol T), which is the fraction of the incident radiation that is transmitted by the sample. The percent transmittance is defined as 100 x T. Therefore from equation (II.4.8): log T = - cl or -log T = cl = A (II.4.9)

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In the derivation of the Lambert-Beer law it is assumed that: (1) the incident radiation is monochromatic, (2) the absorbing species act independently of each other in the absorption process and (3) the absorption occurs in a volume of uniform cross section. The relationships between absorbance, transmittance and concentration at a given wavelength are illustrated graphically in Figure II.4.8.
T% 100 80 60

A 1 1.0 0.8

0.6 0.4 0.2

40 20

Concentration

Figure II.4.8. Absorbance and transmittance vs. concentration at a given wavelength and cell pathlength. Multiple Component Systems. When systems that contain more than one absorbing component are studied, it is assumed that the species act independently of one another and that their absorbances are additives. In general, if there are n components, the total absorbance expression at any wavelength, , takes the form:
A =

A = l c
n n n n

(II.4.10)

In principle, n absorbance measurements at n different wavelength are required to determine the concentration of n components in a mixture. This provides n independent simultaneous equations in n unknowns.

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Deviations from Lambert-Beers Law Many absorbing systems in dilute solution follow Lambert-Beers law rather closely. For some systems, however, absorbance varies in a non-linear way with respect to concentration. Such behavior is called a deviation from Lambert-Beers law. In order to treat these systems, a curved calibration plot may be prepared with samples of different concentrations. True deviations from Lambert-Beers law occur only in systems where the concentration of the absorbing species is so high that the index of refraction for the absorbed radiation is changed. Apparent deviations from Lambert-Beers law may be due both to limitations of instrumentation and to effect of non-symmetrical chemical equilibrium. Instrumentation Limitations Indeterminate instrumental variations which cause apparent deviations include: (1) stray radiation reaching the detector (reflected within the instrument), (2) sensitivity changes in the detector, and (3) power fluctuations of radiation source and the detector amplification system. Double beam operation tends to cancel out most of the random causes of deviation. Another instrumental cause of deviation is the necessity of working with a band of wavelengths rather than truly monochromatic radiation. Unless the molar absorptivity is invariant within the wavelength band used, the absorbance measured is an average absorbance over the band. Due to the logarithmic nature of absorbance, this is not a true average. The greater the slope of the absorption curve through the wavelength band is, the greater the deviation. Figure II.4.9 demonstrates that the shape of the calibration curve often depends on the bandwidth. Two wavelength bands of equal width are designated 1 and 2. The best wavelength for quantitative analysis is 1 for two reasons. First, at the absorption maximum the change in absorbance with concentration is at a maximum; this yields greater sensitivity and higher accuracy. Second, within this band the molar absorptivity is relatively constant and a linear calibration curve is obtained as in Figure II.4.9.

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1 C 2 A C/2 d (a) d

at 1

at 2

Concentration (b)

Concentration (c)

Figure II.4.9. Effects of finite bandwidth. (a) One component at two concentrations. (b) linear curve at 1. (c) Non-linear calibration curve at 2. Non-Symmetrical Chemical Equilibrium An absorbing species that is involved in non-symmetrical chemical equilibrium may exhibit apparent deviations from Beers law. An example of such system is presented below. Let us consider an aqueous solution of a weak acid HA which has an absorption maximum at 1. The anion of the acid A- is non-absorbing at 1. HA is involved in the equilibrium: HA + H2O = H3 O+ + AFor which we have the acidity constant Ka. The analytical concentration, CHA, includes both forms: CHA = (HA) + (A-). As long as the ratio (HA)/CHA remains constant, Lambert-Beers law is valid when using either (HA) or C HA.. But this ratio

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depends on the pH of the solution: fraction un-dissociated = (HA)/ C HA = (HA) / [(HA) + (A-)] = (HA) / {[(HA) + Ka(HA)]/(H3O+)} = 1/[1 + Ka/(H3O+)]. If (H3O+) >> Ka, the acid exists primarily as HA, and there is no problem. If + (H3 O ) << Ka the acid is highly dissociated with a correspondingly small absorbance. At a constant pH in the intermediate region (buffered solution), the fraction dissociated does not vary with total concentration and absorbance is proportional to CHA. However, in un-buffered solutions the fraction dissociated changes with pH, which in turn is a function of total concentration, C HA. Such a system will show an apparent deviation from Lambert-Beers law if CHA is used as the concentration. II.4.3.4. Quantitative Analysis in the UV-VIS The spectral domain of the UV/VIS is well known because it includes the visible part of the spectrum and is widely used in quantitative analysis. Measurements are based on the Lambert-Beer law. It is not necessary that the compound contain a chromophore as long as derivatization is carried out before measurement to ensure absorption of the light. Through derivatization, it becomes possible to quantify a chemical species that has no significant absorption. The derivatization (a chemical transformation) that has to be specific, total, rapid, reproducible and has to yield a UV-VIS absorbing derivative that is stable in solution. This is the principle of photometric, spectrophotometric or colorimetric analysis. The first two methods of analysis use more or less narrow spectral bands obtained with filters or monochromators. In the case of colorimetric analysis the measurements were carried out with white light without any optical instrument. Visual comparison of the sample color with that of reference solution of known concentration was performed. II.4.3.5. Instrumentation for UV-VIS Spectrometry A UV-VIS spectrophotometer consists of three main components: the source, the dispersive system (combined in a monochromator) and a detector. The sample can be placed in the optical path before or after the dispersive system and the recorded spectra can be treated using a number of different computer algorithms. In figure II.4.10 are presented three mechanisms for transmitting illumination through a sample solution. Light Sources Two light sources are commonly used in the UV-VIS domain: an incandescent lamp made from a tungsten filament housed in a glass envelope is used for the visible portion of the spectrum, above 350 nm; a medium pressure deuterium arc lamp is used for the ultraviolet portion of the spectrum.

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Dispersive system. Light emitted by the source is dispersed by prisms or gratings, which are integrated into an assembly called a monochromator. The monochromator extracts a narrow spectral band of the spectrum. For simple devices as dispersive system is used a wideband, interchangeable color filter.
Sample Window Lens To detector Light source Light source Transparent sample container Lens Optical fibers Waste Lens Window

To detector Light source

Thickness traversed

Sample container

Figure II.4.10. Three common methods of transmitting the probe illumination through a sample solution. Detectors Two types of detectors exist: photomultiplier tubes and semiconductors (e.g. silicon photodiodes and charge transfer devices (CCD/CID)). The detector measures the light signal at a given wavelength. It converts the light intensity selected by the monochromator exit into an electrical signal. Single Beam Spectrophotometers Many routine measurements are conducted at fixed wavelengths using simple photocolorimeters equipped with wideband, interchangeable color filters. An

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analytical blank (containing the solvent and reagents for the analysis, without the sample to be measured) is first placed in the optical path, and then is replaced by the solution to be analyzed. Double Beam Optical Spectrophotometers (Scanning Type) In Figure II.4.11 is presented the scheme of a double beam spectrophotometer. One of the beams passes through the sample while the other passes through the reference.
Blank Mirror Mirror

Rotating mirror (detail) Sample Monochromator Lamp Chopper 1 Chopper 2 Detector Amplifier Display/ Printer

ADC

Computer

Figure II.4.11. Double-beam instrument employing two choppers. Two rotating mirrors, called choppers, which are synchronized with the displacement of the grating, allow the comparison of transmitted light at the detector of the two beams with the same wavelength. Amplification of the modulated signal allows the elimination of the stray light. Double beam spectrophotometers allow differential measurements to be made between the sample and the analytical blank. They are preferable to single beam instruments for measurements in problematic solutions. REFERENCES 1. R.L. Pecsok and L.D. Shields, Modern Methods of Chemical Analysis, John Wiley and Sons Inc., New York, London, 1968. 2. Gary D. Christian, Analytical chemistry (6th edition), Wiley, New York, 2004.

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3. 4. 5. 6. 7. 8. 9.

M. Gore, ed., Spectrophotometry and spectrofluorimetry, 2nd Edition, Oxford University Press, Oxford, 2000. R. Kellner, J, M, Mermet, M. Otto, H.M. Widmer Eds, Analytical Chemistry, Wiley-VCH, Weinheim, 1998. Rouessac, F. and Rouessac, A., Chemical Analysis. Modern Instrumentation Methods and Techniques, John Wiley and Sons, Ltd., Chicester, New York, 2000. A. F. Dne, Metode Instrumentale de Analiz Chimic, Editura tiinific, Bucureti, 1995. Encyclopedia of Analytical Science, Vol. 9, pp. 5297-5353, Academic Press, London, 1995. H.A. Strobel, W.R. Heineman, Chemical Instrumentation: A Systematic Approach, 3rd Ed., Wiley, New York, 1989. H. Willard, H. Merritt, L.Lynne and J.A. Dean, Instrumental Methods of Analysis, 5th Ed., Van Nostrand Company, New York, 1981.

II.4.4. ATOMIC ABSORPTION AND EMISSION

Mihaela Carmen CHEREGI II.4.4.1. Introduction Atomic absorption and emission spectroscopy, perhaps the oldest instrumental techniques now widely used, are two methods of quantitative analysis that can be used to measure approximately 70 elements (metals, metalloids and non-metals) in environmental samples. This chapter deals with the spectroscopy of atoms. Since atoms cannot rotate or vibrate as molecules do, only electronic transitions can occur when energy is absorbed. Because the transitions are quantized, line spectra are observed. The principle behind these methods of elemental analysis depends on measurements made on an analyte that is transformed into free atoms. The sample solution is heated in the instrument to a temperature of between 2000 or 3000 degrees Celsius to break chemical bonds, liberate the elements and transform them into a gaseous atomic state. Thus, the total concentration of the element is measured without distinguishing the chemical structures present in the cold sample. There are various ways to obtain free atoms and to measure the absorption or emission of radiation by these. The principal techniques described in this chapter are: a. atomic absorption spectrometry (AAS), in which the amount of the radiation absorbed by ground state atoms in a flame or in a small electrical oven (graphite furnace) is measured;

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b. atomic emission spectrometry (AES), in which atoms are excited in a flame, electrical arc, spark, inductively coupled plasma, and the radiation intensity emitted by a fraction of excited atoms that return to their ground-state is measured. Many models of instruments allow measurements to be conducted by these techniques, which rely on different principles, and concentrations in the mg/L (ppm) range or lower can be accessed. II.4.4.2. Atomic Absorption Spectrometry Principles The law that governs the absorption of light by the atoms is Kirchoffs law and it states that incandescent gases can absorb the same radiation that they can emit in certain conditions. The main steps in AAS measurements are: 1. aspiration of sample into the flame (or graphite furnace); 2. conversion of the sample into free atoms and the gaseous atomic state formation (atomic cloud); 3. absorption of the monochromatic radiation by the ground state atoms from the atomic cloud; 4. measurement of the transmitted radiation intensity; 5. extraction of the quantitative information from the registered signal. The monochromatic light is given off by hallow cathode lamp, the cathode made from the same element as that being determined. Therefore, the wavelengths of radiation given off by the source are the same as those absorbed by the atoms in the flame. AAS is identical in principle to absorption spectrophotometry described in a previous chapter and the Lambert-Beers law is followed in this technique, too: A=kC where A is the absorbance , C is the concentration of the element and k is a coefficient unique to each element at a given wavelength. The absorption depends on the number of ground state atoms of atomic cloud in the flame and on the path length in the flame. Both of these variables are difficult to determine, but the path length can be held constant and the concentration of the atomic cloud is directly proportional to the concentration of the analyte in the solution being aspirated. The instrument yields the absorbance by rationing the transmitted intensities in the presence (I) and absence (I0) of the sample: A = - logT = log(I0/I)

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Measurements are made by comparing the unknown to standard solutions. The procedure used involves classical protocols: prepare the calibration curve or standard additions, as long as the range of concentrations stays within the linear conditions of absorbance. The main disadvantage of making measurements in AAS is that a different source is required for each element. Instrumentation Similarly to absorption spectrophotometry, the basic components for AAS are a light source, a cell (the flame), a monochromator and a detector. The optical scheme and the working principle of an atomic absorption spectrometer are presented in Figure II.4.12. In order to simplify the graph, it was consider that the line bandwidth emitted by the source is equal to the line bandwidth absorbed by the atoms. The beam-light emitted by the source ( I0), which must be a very narrow band characteristic of the analyte metal, passes through the atomization device (flame or graphite furnace). In the atomization device, most metallic compounds are decomposed and the metal is reduced to the elemental state, forming a cloud of atoms. The atoms absorb a part of I0 that is proportional to the analyte concentration in the sample. The attenuated beam-light (I) is then focused on the entrance slit of the monochromator, located after the atomization device. The monochromators role is to select a very narrow band of wavelengths and to eliminate extraneous light resulted from the atomization device. The optical path ends at the entrance slit of the detector (photomultiplier tube). The detector measures the ratio I0/I and the logarithm of the ratio is displayed. I0 is the full intensity of the beam-light emitted by the source and perceived by the detector in the absence of the sample, and I is the attenuated intensity of the beam-light perceived by the detector when the sample is present. The various components of an atomic absorption spectrometer are described as follows. A. Sources The key element in AAS is the source, which must emit a sharp-line because the width of the absorption line is very narrow, around 10 -3 nm. A sharp-line source that emits monochromatic and specific wavelengths and used almost exclusively in AAS is the hollow cathode lamp (HCL). The diagrams of a HCL and of the excitation of the cathode atoms are presented in Figure II.4.13. HCL is a glass tube with a borosilicate or quartz window, depending on the wavelengths emitted by the cathode (e.g. quartz is used for lines in the UV region). The tube is filled with an inert gas (argon or neon) at a reduced pressure. Inside of the lamp, a cylindrical hollow cathode made of the element to be determined (a cathode of lead is used for lead determination) and a tungsten or zirconium anode are enclosed. A higher voltage (300V) is applied between the electrodes, causing the inert gas atoms to be ionized at the anode. The positive ions are accelerated toward the negative cathode

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and some of them possess enough energy to strip atoms from the cathode, which form, at the surface, an atomic gas. The atomic gas is excited to higher electronic level by collisions with the high-energy inert gas atoms. When the excited cathode atoms return to their ground state, the characteristic emission spectrum of elements is obtained. The emission steps may be represented as follows:
Ne M (C ) M (G ) * M (G ) + h
+

where M(C) is the element in its metallic state in cathode, M(G)* is the element in its excite atomic state and M(G) is the element in its atomic state.

(a)

(b)

Figure II.4.12. Schematic diagrams of an atomic absorption instrument (a) and of its working principle (b).

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Screen Atomic vapor

Metal

+
Cathode Anode

Ne
*

Quartz window

Ne Ne + Ne Ne +

Excited metal atom

Ne Ne Ne

Electron Inert gas (Ne, Ar)

(a) (b) Figure II.4.13. (a) Schematic diagram of HCL. (b) Diagram of cathode atoms excitation under impact with inert gas ions. These lines are passed through the atomization device, and certain ones are absorbed by the analyte atoms because they possess the right energy to result in the discrete electronic transitions. The most strongly absorbed line is the one corresponding to the electronic transition from the ground state to the first excited state, known as the resonance line. The width of the emitted lines of the cathode-excited atoms depends on the Doppler, Stark (ionization) and Lorentz (pressure) effects but even then it is narrower than the corresponding absorption band of the sample atoms, and therefore, the entire source line-width is absorbed. There are more then 100 types of HCLs made in pure elements. HCLs cannot be used for mercury and sodium (their boiling points are too low), and then classical lamps using metallic vapor are used instead. For certain sample types multi-element hallow cathode lamps are attractive, the cathode is an alloy of several elements and the lines of all the elements are emitted. They may exhibit shorter lifetimes than the single-element lamps, due to the selective volatilization of one of the elements form the cathode and its condensation on the walls of the lamp. In some instances, a brighter source such as microwave-excited or electrodeless discharge lamp composed of the element to be measured, may be preferred. Electrode-less lamps with very intense emission use a radio-frequency emitter to excite the metallic vapor and are especially used for elements such as As, Hg, Se or P. B. Devices for sample atomization Flame atomization. The flame is a mixture of a fuel and an oxidant gas and it has a rectangular base of about 10 cm by 1 mm. It is aligned with the optical axis of the instrument. The sample solution is aspirated by the nebulizer and is introduced in the flame as a fine spray. In the flame, the analyte is transformed in an atomic cloud

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following the processes: solvent evaporation, dissociation of the salt into free ions, ions reduction to gaseous atoms. For sensitive measurements atomization should be as complete as possible. Collisions with high-energy flame gas atoms are principally responsible for atomization. Thus, the hotter the flame, the more effective is the process. In Table II.4.2 are listed some analytically useful flames and their temperature. The most popular flames in AAS are acetyleneair and acetyleneN 2O (for refractory elements). The hydrogen argonentrained air flame is preferred for wavelengths below 200 nm where the acetyleneair flame absorbs a large fraction of the radiation. It is used for arsenic and selenium when they are separated from the sample by volatilization as their hydrides (AsH3, H2Se) and passage of these gases into the flame. Table II.4.2. Recommended flame type and their temperature for analytical determinations by flame AAS. Combustible mixture H2 air H2 O2 CH4 O2 C2H2 air C3H8 air C3H8 O2 C4H10 air C2H2 O2 C2H2 N2O H2 Ar entrained air Tmax ( K) 2300 2950 2950 2450 1998 3173 2200 3400 3200 1850

Flames must also be stable and something must be known about the background levels of chemical species derived from the flame gases as well as those derived from the sample. Not all the parts of a flame are valuable for atomization or for observation of the atomic cloud spectrometrically. Free radicals present in the flame have absorption and emission spectra in the near UV and they can interfere with the measurement of the element. Thus, the observation flame height must be adjusted for some elements. Electrothermal atomization. For the lowest possible detection limit and greater sensitivity the electrothermal or resistive furnace atomizer (Figure II.4.14.) was the second development in the sample module for AAS. It is a flameless device without nebulization and consists of a hollow graphite rod or cup that can hold a precise volume/quantity of the sample (a few L or mg deposited with an automated syringe). This rod of 8 mm inner diameter and 40 mm length is oriented parallel to the

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optical axis and it is surrounded by a double sleeve containing an inert gas to protect it from oxidation and allow circulating water to cool the device.
Transverse hole for introducing sample Hollow graphite rod

Inert gas (Ar) Sample

HCL beam-light

Cooling water

To power source

Figure II.4.14. Schematic diagram of electrothermal atomizer. A small volume of sample (15 L) is placed in the graphite rod through a hole in the top, and then the rod is heated resistively by passing an electrical current. To avoid splashing, the temperature is gradually increased according to a three-step cycle: 1. the sample is dried at a low temperature for a few seconds (~100 to 200 C) 2. pyrolysis at 500 to 1400 C to destroy organic matter that produces smoke and scatters the light source during measurement; the smoke from pyrolysis in flushed out by flowing argon gas, and finally the sample is rapidly thermally atomized at a high temperature up to 3500 C. 3. the absorption of the metal atoms in the hollow portion of the rod is measured and a sharp peak of absorbance versus time is recorded as the light path passes through the atomic cloud. The heating is done in an inert atmosphere to prevent oxidation of the graphite at high temperature and also to prevent the refractory metal oxides. Certain metals such as molybdenum, vanadium, nickel, and barium react with the graphite at high temperature forming carbides and to prevent this pyrolytically coated tubes are used. Electrothermal atomizers have a conversion efficiency of 100 % compared to 0.1 % from flame atomization, therefore the detection limits are often 100 to 1000 times improved over those of flame aspiration, many metals being determined at concentrations of 1 g/L. Other high melting, electrically conducting substances can also be adapted as atomizers. A coil-shaped tungsten filament (W-coil) serves for low-cost, compact and

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portable instrumentation for environmental and clinical analyses. High heating temperatures up to 3000 C can be achieved by using a simple power supply such as a car battery. For over 20 elements that have been determined using W-coil AAS, the detection limits are even better than or within a factor of 10 of those of graphite furnace, while the linear dynamic ranges of concentrations remain unaffected. Chemical vaporization. Other distinctive modes of atomization are the conversion of the compounds of metalloids and many low-activity metals to hydrides. For hydride preparation sodium boronhydride is added to an acidified sample. For instance, arsenic, bismuth, tin, lead, antimony or selenium, which are difficult to reduce in a flame when they are in higher oxidation states, will react with the reducing agent in a separate vessel to form the volatile hydride. The generator vessel must be flushed with Ar or N2 to remove oxygen. When hydride formation is complete, the hydride is carried into a quartz cell placed in the flame by a further flushing, decomposed by heat, and measured by atomic absorption. For mercury, the bound mercury is reduced by a reducing agent such as tin chloride (SnCl2) in acid solution. The elemental mercury is formed instead of hydride and it may be volatilized easily by bubbling an inert gas through the solution and passing the gas through a special cell, which does not need to be put into the flame. This is called cold vapor method, and requires specialized instruments. Because chemical atomization is a selective reaction, these metalloids and low-activity metals are also concentrated by removal from the original matrix. The determination of these elements in the presence of considerable amounts of organic matter often requires the organic material digestion, with a strong oxidizing agent, and the interfering substances masking. Quantitative Determinations in Atomic Absorption Spectrometry AAS is widely applicable as a generally sensitive technique for quantitative determination of elements (about 70). Hollow cathode lamps are available for all and microwave electrode-less lamps for many. AAS is capable of a precision of 2 % and when a double-beam procedure and a background correction are employed, precision can be better then 1 %. Sample preparation. In the majority of cases samples are present in solution and for samples that requires dissolution, acid digestion or solvent extraction may be the first step of analysis. Liquids with high salt content offer problems when the solution is evaporated. Encrustation of salts around the slot of the burner leads to unsteadiness of the sample flow and of fuel and oxidant gases and introduces serious errors. In electrothermal atomization salts rise the smoke and high background correction. A way to remove these difficulties may be the complexation of the analyte in the sample solution and then the extraction of the complexed species. The extraction also offers the advantage of concentrating the analyte, eliminating the interferences, and enchanting the

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sensitivity of the measurements if an organic extractant is used. Some common complexing agents for metals and solvents are: ammonium pyrrolidine dithiocarbamate used with methyl isobutyl ketone; 8-hydroxyquinoline with ethyl acetate or butanol and ethylenediaminotetraacetic acid. Many metals, metalloids and low-activity metals are most sensitively determined by chemical conversion to hydrides. Calibration curves and working conditions. Analytical signal readings are quantified by reference to a working curve, a plot of absorbance versus concentration for each analyte. The standard solutions must be prepared to be nearly identical in composition with the samples as possible in order to minimize the errors. Ultra-pure reagents and solvents should be used, especially in the case of organic solvents. In spectrophotometric measurements the precision is best when the absorbance varies in the range of 0.15 to 1.0 units. If an internal standard is used in a dual-channel instrument, the absorbance range can be extended with a good precision. When the concentration range of an element in a sample exceeds the optimum absorbance values, several options are open: the variation of the optical path length through the flame by a rotating burner; the use of a less sensitive line of the element; the dilution of the sample. Standard addition method. Analyte determination using standard addition method is attractive only if the concentration of the analyte in the sample is very low. The method involves spiking an equal volume of standard solutions, at least three different concentrations, to equal volumes of blank solution and sample aliquots, respectively. The absorbance for all these solutions is measured and then it is plotted versus concentration. The linear curve obtained is extended through the concentration axis and the distance from the point of its intersection to the origin is equal to the concentration of the analyte in the sample. II.4.4.3. Atomic Emission Spectrometry Principles The main steps in AES measurement are: 1. Conversion of a sample to free-atom gas. 2. Excitation of the atoms and their ions to higher electronic states. 3. Measurement of the emission wavelengths and intensities. 4. Extraction of qualitative and quantitative information from these signals. AES is a well-established technique for determining inorganic constituents in various types of samples. Basically, the sample solution is introduced into the excitation device as a fine spray, where the solvent evaporates leaving the dehydrated salt. The salt is dissociated into free gaseous atoms in the ground state and then a certain fraction of these atoms can be raised to an excited electronic state, by the effect of high temperature. These excited atoms return to their ground state emitting photons

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(h) of characteristic wavelength. The intensity of the emitted radiation is proportional to the concentration of analyte in the sample. In AES, one or several specific spectral lines are monitored for each analyte. Hence, the emission of radiation can occur from either excited or ionized atoms, thousands of different spectral lines can be observed. Some of these lines could be more intense than those of the analyte, which can be present at ultra-trace levels, and, for this reason, a high performance monochromator is required. Flame photometric analysis is much simpler and it is not always necessary to have a high-resolution monochromator, a simple interference filter may be sufficed. The monochromator can be replaced by a polychromator a double module in which the exit slit of monochromator has been replaced by a multi-channel detector mounted in the focal plane. It should be noted that the next generation will be represented by the grating spectrometers; in recent years prism spectrometers have become relatively rare. Nowadays, spectrometers able to solve many interferences and problems related to the matrix effects are used, the instrument being programmed to identify and quantify the elements in a real sample. Instrumentation The atomic emission spectrometer consists of three principal components: the device responsible for bringing the sample to a sufficient temperature; the optics including a mono- or polychromator that represent the heart of the apparatus; and a microcomputer that controls the instrument. A. Excitation sources The excitation source is the most critical module because it must volatilize and atomize the sample as uniformly as possible since the concentration of atoms in the atomic cloud should be representative of that in a sample. It should also excite atoms. The excitation sources must fulfill the following criteria: 1. high energy flux; 2. reproducibility in sample introduction and energy transfer; 3. stability of excitation; 4. high sensitivity; 5. ease of use. The main excitation sources are flame; high-voltage arc or spark, inductively coupled plasma and glow discharge. Flame is a lower-energy source and the emission spectrum is much simpler, as there are fewer lines. Only the alkaline and alkaline earth metals are determined by flame emission spectrometry. High-voltage arc and spark are used for solid samples and they are mostly used in semi-quantitative analysis in industry.

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Inductively coupled plasma (ICP) emission spectrometry is used for rapid multi-element determinations in environmental samples. Different from AAS, the chemical interference in ICP-AES is very low. The plasma is an incandescent ionized gas (argon) heated inductively by radiofrequency energy at 450 MHz and 25 kW (Figure II.4.15.). Argon gas, which is ionized by a discharge of a Tesla coil or a pilot spark, flows upward through the quartz tubes, the external tube having at its superior part two water-cooled copper tubes. The copper tubes are connected to the radiofrequency generator creating a variable magnetic field in the flowing gas inside the coil. This induces a circulating eddy current in a gas, which in turn heats it. As this environment becomes more and more conductive, the temperature increases considerable, the temperature reaching 9000 to 10,000 K. The plasma is isolated from the tubes by a gaseous sheath of flowing, non-ionized argon injected through an external tube concentric to the first one. The sample aerosol generated in the nebulizer and spay chamber is introduced into the ICP via a third tube with a diameter of 12 mm. At this high temperature, the sample atomizes, ionizes and excites producing emission spectra. Also, the high temperature of plasma eliminates many chemical interferences present in flame because molecules of compounds formed completely dissociate. The plasma is well suited for refractory elements (boron, phosphorus, uranium, etc.) and difficult to excited elements (zinc, cadmium).

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Figure II.4.15. Schematic diagram of ICP used for AES. The advantages of using an ICP include the possibility to identify and quantify all the elements excepting argon, in concentration from ultra-trace levels to major components; working curves covering 5 decades of concentrations (1 ppb100 ppm) and the detection limits are mostly in ppb range; multi-element analysis can be accomplished in 30 seconds consuming 0.5 mL of sample solution, 30 elements can be determined simultaneously. Spectral interferences from ionatom recombination, spectral line overlaps, molecular bad emission, or stray light can occur that may alter the net signal intensity. These can be avoided by selecting alternate analytical wavelengths and making background corrections. Wavelength Separator This is the device that separates specific emission lines of the analyte using planar, concave or echelle gratings. Predefined elements, characterized by precise spectral lines, are detected by as many photomultiplier tubes as there are secondary slits, each corresponding to an analytical line or measurement channel. A fixed optic arrangement using echelle grating in association with a focusing prism produces a double dispersion of the lines, in both the horizontal (due to the grating) and vertical (due to the prism) dimensions. This order-separating device allows simultaneous detection over the whole spectral range. There are also instruments with dispersion surfaces compatible with two-dimensional sensors. Their sensitivity and spectral response allows simultaneous measurements of lines. Wavelength scanning instruments (monochromator type) have the dispersive system movable in order to focus each wavelength on the fixed exit slit. Several units of this type can be placed in series, constituting double or triple monochromators of high performance. Quantitative Determinations in Emission Absorption Spectrometry AES is described as a very sensitive (detection limits of few ppt) and rapid quantitative elemental analysis. The dynamic range of emission methods is so large that concentration levels are identified by terms major (>10 %), minor (1 %), and trace (<0.01 %). However, each application may be considered as an individual case. Certain instruments better tolerate dominating matrices such as soil or mud, where there are high concentrations of elements such as Si, Fe and Al. The calibration curves are used for quantitative determinations. A well selected set of standards employed to obtain plots of intensity ( Ia) of the analytical lines versus concentration (C):

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Ia = m C where m is the slope of the curve. In every case, a background correction Ib should be made if background is greater than the order of magnitude of acceptable error. A more correct equation for the working curve than may be: Ia I b = m C Accuracy and precision are dependent on the regulation of excitation conditions, including the selection of an appropriate integration time. Many sources fluctuate and times of volatilization and excitation of elements will vary. II.4.4.4. Physical and Chemical Interferences in AAS and AES The effectiveness of flame spectrometric methods for a particular element depends only in part on external variables such as fuel gas pressure and width of the burner slot. It is also determined by processes called interferences or inter-element (matrix) effects.
Table II.4.3. Sources of interference and error associated with flame atomization*.
Type of error Chemical (cation or anion interference) Background interference Cause of error Technique affected Analyte stable compounds formation that atomized with difficulty in flame (AF, EF) 1. Emission by flame gases (EF, less important in AF); emission by analyte (AF) 2. Absorption and light scattering by molecular species in flame (AF) Ionization of analytes (AF, EF) Flame temperature change with solvent composition or high analyte concentration appears; possible encrustation of solids on burner (AF, EF) Emissionn or absorption of another atomic species or its oxide within waveband passed; (EF less important for AF) Method of reducing error Use hotter or fuel-rich flame; avoid certain anions; use solvent extraction or ion exchange to effect prior separation of offending ion; chelate the analyte 1. Change flame or use other analytical line for EF; modulate source emission for AF and selectively detect analytical signal. 2. Correct for background by deuterium arc, Zeeman, and other methods; between 190 and 175 nm use separate flame with Ar shielding. Add easily ionized non-analyte; lower flame temperature Ensure constant solvent composition; wait for steady state; reduce concentrations

Ionization interference Excitation interference

Spectral interference

Use narrower slit or select another intense analytical line in a spectral region free of such interference

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* It is assumed that instrumental variables such as spectral slit width and gas pressure will be set appropriately. AF , atomic absorption flame spectrometry; EF atomic emission flame spectrometry.

The main types of interferences and general ways of combating them are presented in the Table II.4.3. REFERENCES 1. Environmental Chemistry, Seventh Edition 2000, Stanley E. Manahan, Boca Raton: CRC Press LLC. 2. Fundamentals of Environmental Chemistry 2001, Stanley E. Manahan, Boca Raton: CRC Press LLC. 3. Air Pollution Control Technology Handbook 2002, Karl B. Schnelle, Charles A. Brown, CRC Press LLC. 4. Chemical Analysis Modern Instrumental Methods and Techniques 2003, Francis Rouessac, Annick Rouessac, John Wiley & Sons, New York. 5. Analytical Chemistry, Fifth Edition 1994, Gary D. Christian, John Wiley & Sons, New York. 6. The Art and Science of Chemical Analysis 2001, C.G. Enke, John Wiley & Sons, New York. 7. N.H. Bings, A. Bogaerts, J.A.C. Broekaert, Anal Chem., 2002, 74, 2691-2712. 8. X. Hou, K.E. Levine, A. Salido, B.T. Jones, M. Ezer, S. Elwood, J.B. Simeonsson , Anal. Sci., 2001, 17, 175-180. 9. K.W. Jackson, Anal. Chem., 2000, 72, 159R-167R. 10.P.M. Cooke, Anal. Chem., 2000, 72, 169R-188R.

II.4.5. ELECTROCHEMICAL METHODS OF ANALYSIS

Giuseppe PALLESCHI, Alina Stelua LUPU II.4.5.1. Introduction Electroanalytical techniques represent the most effective answer to the increasing worldwide demand of reliable and rapid determinations of the widest variety of analytes in complex matrices. Electroanalytical chemistry can play a very important role in the protection of our environment. In particular, electrochemical sensors and detectors are very attractive for on-site monitoring of priority pollutants, as well as for addressing other environmental needs. Such devices satisfy many of the requirements for on-site environmental analysis. They are inherently sensitive and

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selective towards electroactive species, fast and accurate, compact, portable and inexpensive. The potential benefits of electrochemical monitoring, in an environmental context are: 1. An applied potential in voltammetric sensors can lead to high selectivity and specificity, and hence probing of speciation. Each chemical species as well as each element or oxidation state has an associated potential for oxidation and reduction. Such specificity is not possible with most other analytical techniques. 2. The choice of electrode material can lead to selectivity, particularly in ionselective electrodes. In voltammetric sensors and at some electrode materials, certain species do not react, so that interference problems may be resolved in this way. An obvious example is the high over-potential for hydrogen evolution at mercury electrodes. 3. Modern electrochemical instrumentation, particularly with controlled potential, associated with voltammetric sensors, leads to high sensitivity and low detection limits, since complex applied potential programs can be used together with accumulation of the species to be measured at the electrode surface. 4. There is the possibility of furnishing not only the results but also treated data in real time or close to real time, using computerized control and particularly in flow systems for on-line monitoring. 5. Portable sensors with dedicated instrumentation, possibly battery-powered, that can be used outside the laboratory. 6. Miniaturized sensors, for application in situations where other probes may not be usable. The electroanalytical techniques are concerned with the interplay between electricity and chemistry, namely measurements of electrical quantities, such as current, potential and charge, and their relationship to chemical parameters. The three types of electroanalytical measurements that can be performed offer different degrees of selectivity. II.4.5.2. Conductimetric Methods The concentration of charge is obtained through measurement of solution resistance and is therefore not species-selective. Conductimetric detectors, however, can be useful in situations where it is necessary to ascertain, for example, whether the total ion concentration is below a certain permissible maximum level or for use as an on-line detector after separation of a mixture of ions by ion chromatography. II.4.5.3. Potentiometric Methods

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The equilibrium potential of an indicator electrode is measured against a selected reference electrode using a high-impedance voltmeter, i.e., effectively at zero current. Thus, the current path between the two electrodes can be highly resistive. At an inert redox indicator electrode such as platinum the potential measured is a mixed potential, a function of all species present in solution and their concentrations. In ionselective electrodes, careful choice of the electrode material can give good selectivity to one particular species, in many cases, with only minimal interference from other ions. Detection limits of the order of 10 -7 mol/L of the total concentration of the ion present in a particular oxidation state, although down to 10 -11 mol/L differences in concentration can be measured. Many ions, which include both metals and anions, may be analyzed rapidly and with a good degree of accuracy by ion-selective electrodes. In addition, dissolved gases, such as oxygen, carbon dioxide, ammonia, and oxides of nitrogen, could be analyzed by this technique using a gas sensor electrode. A selective-ion electrode system constitutes the two half-cells, which are a sensing electrode and a reference electrode, a readout meter, and a solution containing the specific ion to be analyzed. The sensing electrode could be a solid state, a liquid membrane, or a gas-sensing electrode, or the most familiar type, a glass electrode. The reference electrode should be either a single junction or a double junction type electrode containing a freely flowing filling solution and should produce a stable potential. A filling solution completes the electrical circuit between the sample and the internal cell. The filling solutions, commonly used in the reference electrodes, are KCl and KNO 3. A filling solution, however, must not contain the ion to be analyzed. When a sensing electrode is immersed in a solution containing the same ion to which it is selective, a potential develops across the surface of its membrane. This potential is measured as voltage and is proportional to the concentration of the ion in the solution. The voltage caused by the sensing electrode is compared against a stable potential produced by a reference electrode. pH Electrode The pH electrode is the most well-known and simplest member of this group and can be used to illustrate the basic principles of ISEs. This is a device for measuring the concentration of hydrogen ions and hence the degree of acidity of a solution - since pH is defined as the negative logarithm of the hydrogen ion concentration; i.e. pH=7 means a concentration of 1x10 -7 moles per liter. (To be more precise, the term concentration should really be replaced by activity or effective concentration. This is an important factor in ISE measurements. The difference between activity and concentration is explained in more detail later, but it may be noted here that in dilute solutions they are essentially the same. Nevertheless, in order to avoid confusion, the more familiar term of concentration will be used in this section.

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The most essential component of a pH electrode is a special, glass membrane sensitive to hydrogen ions, but not to other ionic species. When the electrode is immersed in a test solution containing hydrogen ions the external ions interact with the external side of the membrane and a variation of the membrane potential occurs between the external and internal concentrations. Thus, there is a build up of charge on the inside of the membrane, which is proportional to the number of hydrogen ions in the external solution. Because of the need for equilibrium conditions there is very little current flow and so this potential difference can only be measured relative to a separate and stable reference system which is also in contact with the test solution, but is unaffected by it (see later for a discussion of reference electrodes). A sensitive, high impedance millivoltmeter or digital measuring system must be used to measure this potential difference accurately. The potential difference developed across the membrane is in fact directly proportional to the logarithm of the ionic concentration in the external solution. Thus, in order to determine the pH of an unknown solution, it is only necessary to measure the potential difference in two standard solutions of known pH, construct a straight line calibration graph by plotting millivolts versus pH (= - log [H +]) then read off the unknown pH from the measured voltage. In order to measure the electrode potential developed at the ion-selective membrane the ISE/pH electrode must be immersed in the test solution together with a separate reference system and the two must be connected via a millivolt measuring system. At equilibrium, the electrons added or removed from the solution by the ISE membrane (depending on whether it is cation or anion sensitive) are balanced by an equal and opposite charge at the reference interface. This causes a positive or negative deviation from the original stable reference voltage that is registered on the external measuring system. The relationship between the ionic concentration (activity) and the electrode potential is given by the Nernst equation: E = E0 + (2.303RT/ nF) Log(A) where E = the total potential (in mV) developed between the sensing and reference electrodes; E0 = is a constant which is characteristic of the particular ISE/reference pair. (It is the sum of all the liquid junction potentials in the electrochemical cell); 2.303 = the conversion factor from natural to base 10 logarithm; R = the gas constant (8.314 joules/degree/mole); T = the absolute Temperature; n = the charge on the ion (with sign); F = the Faraday constant (96,500 coulombs); log(A) = the logarithm of the activity of the measured ion. The factor 2.303RT/nF is known as the slope of the electrode (from the straight line plot of E versus log(A) which is the basis of ISE calibration graphs). At constant temperature this should be a constant depending on the valence of the ion

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being measured. Under normal operating conditions, the slope is seen to vary between 50 and 60 mV for mono-valent ions (25 to 30 mV for divalent ions) because of variations in temperature, deviations from "ideal" behavior, and variable ionic conduction across the ion-selective membrane. This means that, when measuring samples, a potential difference of about 55 mV can be expected for every decade change in concentration; i.e. equivalent to 1 pH unit. Differences Between pH and Other Ion-Selective Electrodes i) In contrast to the pH membrane, other ion-selective membranes are not highly selective and sensitive to some of the other ions that may be present in the test solution, thus causing the problem of ionic interference. ii) The calculation of ionic concentration is more dependent on a precise value for the potential difference than is the pH value. For example, it would take an error of more than 5 millivolts to cause a change of 0.1 pH units, but only a 10 -3 V error will cause a 4% error in the calculated concentration of a monovalent ion and an 8% error for a divalent ion. Thus, when measuring other ions, it is essential to take extra precautions to minimize any drift in the value and any errors in the measurement of the electrode potential. iii) Most ISEs have a much lower linear range and higher detection limit than the pH electrode. Many show a curved calibration line in the region 10 -7 to 10-5 mol/L and very few can be used to determine concentrations below 1x10 -7 mol/L. Thus, for low concentration samples, it may be necessary to construct a calibration graph with several points in order to define the curve more precisely in the non-linear range. iv) It is more usual to plot a calibration graph using the ionic concentration with a logarithmic scale on the X-axis rather than the pX factor (analogous to pH) on a linear axis. v) Some ISEs will only work effectively over a narrow pH range. II.4.5.4. Voltammetric Methods Where the current is registered as a function of applied potential, more information and lower detection limits can usually be gained. Several species that react at different applied potentials can be determined almost simultaneously in the same experiment without the need for prior separation. Very low detection limits of down to the 10-12 mol/L level can be reached using state-of-the-art instrumentation and pre-concentration of the analyte on the electrode surface. In many practical sensors or detectors used after separation, e.g., by high-pressure liquid chromatography or capillary electrophoresis, or in detectors in continuous flow, after the voltammetric profile has been investigated, amperometric sensors at fixed potential can be employed. The collective number of voltammetry stands for a number of different electroanalytical methods for the determination and identification of inorganic and

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organic substances by the measurement and interpretation of currents in dependence on the applied voltage to an electrode. In voltammetric measurements, the species of interest are accumulated at an appropriate working electrode by applying a suited voltage during a defined time under constant agitation of the solution in the electrochemical cell. Accumulation may occur by reduction of the species to the element and forming an amalgam with the working electrode, e.g. hanging mercury drop electrode (HMDE), by forming an insoluble compound with the material of the electrode, e.g. HgSe, or by adsorption of very stable complexes of the metal ion to the electrode, e.g., Ni(DMG) 2. After this, agitation is stopped and the voltage at the working electrode is continuously changed to more positive values in order to re-oxidize the reduced species during accumulation (anodic stripping voltammetry, or ASV) or to more negative values to reduce species accumulated in a reducible form (cathodic stripping voltammetry, or CSV). The maximum of the cathodic or anodic current (i P) flowing at distinct voltages is directly representative for the quantity of the species whereas the voltage at the maximum of the current (Up) is indicative of the kind of species. As only the Faraday currents evolved by the electrode reactions of the species to be determined are of interest, all other kinds of interfering currents, above all Faraday currents from accompanying substances (iFR), capacitive currents (iC), and migration currents (iM), must be prevented or suppressed as far as possible. This can be achieved by a suitable sample preparation, a scrupulous choice of the supporting electrolyte, a correct voltage and time for accumulation and applying a convenient mode of current-voltage measurement. In published voltammetric methods, the voltages are mostly given relative to the applied reference electrode. Various monographs on polarography and voltammetry have been published [2,3]. The instrumentation for voltammetric measurements consists of two main parts: the electronics, mostly a polarograph, and the electrochemical cell. The electronics consist of a very stabilized voltage source (potentiostat), devices for the superimposition of different pulses and waves, timer for synchronization, amplifier for the measurement of current in the range of picoamperes to milliamperes, recording units like x/t or x/y recorders, visible display unit (VDU), and printer. In modern instruments, the use of microprocessors and computers has resulted not only in simplification of the voltammetric measurement but also in an increase in the accuracy and precision of the analyses. The electrochemical cell consists of a vessel of appropriate size (from a few L to several mL) containing a mixture of the supporting electrolyte and the solution to be analyzed in which the working electrode, the reference electrode, the auxiliary electrode, a gas inlet tube, and a stirring device are immersed. Working Electrodes (WE) Working electrodes (WEs) are those at which the electrode processes occur. They are usually made of an inert material that is not affected by electrode reactions.

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Different kinds of working electrodes can be used for voltammetric analyses. The choice of the electrode depends on the metal to be determined, on the kind of accumulation to be used, as well as on the voltage range to be applied. A. Hanging Mercury Drop Electrode (HMDE) The HMDE is the most frequently used WE for voltammetric analyses. The surface area of this electrode may be selected from fractions of l mm 2 to several mm2. Its main advantage is the ease of replacing the electrode with high reproducibility and very constant surface area (1-2%), i.e., each measurement can be made with a completely new electrode. The whole surface of the electrode is electroactive. Besides this, mercury has a great over-voltage against hydrogen, i.e., a rather negative voltage can be applied without reduction of H+ to H2. The application of a positive voltage is limited by the dissolution of Hg above all by the formation of insoluble or undissociated compounds of Hg with substances in the electrolyte. Nowadays there are two main types of stationary mercury electrodes. One type consists of a capillary joint to a displacement vessel into which a micrometer gauge rod is screwed. The drop size is determined by the screw thread of the micrometer and the rotation [1]. The other type uses a microvalve (needle valve or flat valve) instead of the micrometer rod to determine the drop size by the time the valve is opened (usually a few msec). B. Glassy Carbon Electrode (GCE) Glassy carbon electrodes (GCEs) are mainly used for anodic oxidations at stationary and rotating disk electrodes above all in electrochemical detectors in highperformance liquid chromatography (HPLC) and as auxiliary electrodes in voltammetric cells [7]. Plane or cylindrical electrodes of very different sizes are disposable. Glassy carbon has many advantages compared with other electrode materials (e.g., noble metals) because its surface is practically gas-tight and cap easily be renewed by polishing. An important feature in electrochemical applications is its very low chemical reactivity. For the determination of most metal ions it is not as versatile as mercury because the application of negative voltages is limited by the lack of over-voltage against hydrogen. The main application of glassy carbon in the determination of metal ions is its use as carrier material for the mercury film electrode. C. Mercury Film Electrode The mercury film electrode (MFE) offers some advantages in the determination of amalgam-forming metals by stripping voltammetry in the concentration range below 0.5 g/Litre [1]. The MFE is formed by electrodeposition of a thin mercury film (10-50 nm thick) on an inert carrier material-mostly glassy carbon. This mercury film is formed in situ during the accumulation of the metal ion to be determined by addition of a certain concentration of a soluble mercury salt into the electrolyte (e.g., l mg Hg(NO 3)2/Litre). Though the MFE is distinguished by its

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high sensitivity, this electrode has some disadvantages. After every determination step the surface of the electrode must be thoroughly cleaned and the reproduction of a further identical mercury film is not guaranteed because by the cleaning the active sites of the carrier material may have changed. The mercury film is seldom uniform and depends also on the composition of the electrolyte. The MFE is somewhat difficult to use. D. Gold Electrode As the surface reactions in the anodic region are much simpler for gold than for platinum, gold electrodes are often preferred to platinum electrodes in electrochemical measurements. The rotating gold electrode has been proven to be very useful in the determination of traces of mercury in biological materials [1]. The surface of gold electrodes is very susceptible to oxidation in the presence of coordinating anions at applied positive voltage. This behavior can be used for cleaning and polishing of the electrode surface. Reference Electrodes (RE) In a voltammetric cell, the voltage applied to a working electrode is measured relative to a reference electrode (RE). The potential of the reference electrode, URE, should be constant and independent of the electrolyte in the cell. It should not be changed by the passage of a small current, i.e., the electrode must be unpolarizable. These types of electrodes normally consist of a metal in contact with its sparingly soluble salt and a solution containing the respective anion in a relatively high concentration. The RE is joined to the electrolyte in the cell by a liquid conducting connection. At the boundary between the liquid junction and the electrolyte within the cell, a junction potential occurs which is included in the voltage of the reference electrode. This diffusion potential may cause interferences if the solution in the reference electrode and the analyte are very different. The REs usually used in potentiometry are also suitable for voltammetry. The most used REs with their voltages (URE) at 25C relative to the normal hydrogen electrode (NHE) are: Hg/Hg2Cl2 (sat. KC1): + 0.244 V Ag/AgCl (sat. KC1): + 0.198 V Ag/AgCl (3 M KC1): + 0.207 V Ag/AgCl (sat. KNO3): + 0.467 V Auxiliary Electrodes (AE) Of the three electrodes arranged in the electrochemical cell, the auxiliary electrode (AE) is the current carrying counter electrode to the working electrode. By its use the RE is protected against the flux of current and by this no polarization causing a shift of its potential occurs. The AE must be made of very good conducting material that is chemically and electrochemically as inert as possible. Rods of

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platinum and glassy carbon have proven to be the electrodes of choice for voltammetric measurements. If Pt wires sealed in glass tubes are used, attention must be given to possibly serious contamination with lead. Pt can only be sealed in soft Pbcontaining glasses that can be leached out by the electrolyte. II.4.5.5. Modes of Current-Voltage Measurements There are different currents governing a voltage-current curve (voltammogram). Only the Faraday current (iP) resulting from the electrode reaction of the species to be determined is of analytical interest. While most interfering currents can be suppressed or eliminated by different provisions, the capacitive current (i C) can only be diminished or eliminated by the application of appropriate measurement methods. This capacitive current arises from the electrical double layer on each electrode, which behaves as a capacitor that is recharged by every change of voltage. The charge required for this appears in the voltammogram as a current that is not specific to the species to be determined. The ratio i C/iF determines to a great part the detection limit of voltammetric methods. Different modes of measurement have been developed to overcome this problem and to enhance sensitivity. Direct Current Mode (DC) Measurements in the direct current (DC) mode are performed by continuously changing the voltage applied to the working electrode (in cathodic or anodic direction) after the accumulation step. At the voltage of the reduction or oxidation of the accumulated species an asymmetrical current peak is obtained. The baseline is a sloping curve. The current at the peak maximum (i P) is representative for the concentration of the species, whereas the corresponding voltage (Up) is indicative for the kind of species. In this measurement mode the i C is not compensated and the evaluation of the voltammogram is rendered difficult by the form of the baseline. This ancient method is seldom applied to the determination of trace metals in biological materials. Normal Pulse Mode (NP) In the normal pulse mode (NP) short square-wave DC pulses (duration 50-60 msec) with continuously increasing amplitudes are superimposed to a constant base voltage at the working electrode. These pulses must be exactly synchronized with the current measurement. After the application of a pulse the i C induced by the pulse voltage decreases exponentially with time whereas the interesting i F occurring at a particular voltage, depending on the kind of the species to be analyzed, decreases with the square root of the duration of the pulse. In order to eliminate the influence of the i C after a delay of 30-40 msec from the start of the pulse the iF is measured during 15-20 msec and stored until the next measurement. These currents may be recorded directly in function of the applied pulse

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amplitude. In this case a curve with a shape analogous to the DC mode is obtained, but the iC is largely suppressed and the sensitivity increased. Mostly, the current of the preceding pulse is subtracted from that of the following pulse and the difference is plotted function of the voltage. A differentiated curve results. In both cases the current maximum (iP) is directly proportional to the concentration of the species and the respective voltage (Up) indicative of its kind. This measurement mode is particularly suitable for solid working electrodes, e.g., in electrochemical detectors for ion chromatography and HPLC. By using an HMDE, the maximum applicable pulse amplitude is limited (U < 500 mV) because by the application of greater pulses the mercury drop may be disturbed and fall off. Differential Pulse Mode (DP) In principle, this method is a combination of the DC and the NP mode. Square-wave DC pulses of small and constant amplitude (U= 5-100 mV) during 4060 msec are superimposed on the continuously changing DC voltage. The application of the pulses and the measurements of the currents must be correctly synchronized. For every pulse the current is measured twice during precisely defined identical intervals (e.g., 20 msec). The first measurement ends just before the start of the pulse, the second with the end of the pulse. The current of the first measurement is subtracted from that of the second and the resulting derivative i/U is plotted in function of the DC voltage ramp. The shape of the curve shows rather sharp peaks on a smooth baseline. As both measurements take place within a short time interval, the ramp DC voltage will be almost the same. With the pulse amplitude being small and the measurement of the current occurring toward the end of the pulse, the respective i C has completely decayed. Any residual iC is eliminated still further by subtracting the two currents. The DC ramp voltage at the current maximum (Up) is indicative of the kind of species; the maximum current (i P) is directly proportional to the concentration of the species. The DP mode provides the most sensitive measurement method in many applications. Because evaluation of the voltammogram is rather simple, this mode of measurement has become the most widely used. Alternating Current Mode (AC) Alternating current (AC) measurement is in principle a DC measurement where a sinusoidal AC voltage with a small constant amplitude (10-100 mV) and a moderate frequency (30-100 Hz) is superimposed on the continuously changing DC voltage ramp. This superimposed AC voltage causes an AC component in the cell current which depends on the applied DC voltage. After filtering out the DC component the AC component is measured selectively. Plotting the AC current as function of the DC voltage gives a peak (not totally symmetrical) in the DC voltage region where the reaction at the working electrode occurs. The Up is indicative to the

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kind of species. According to Matsuda [1], the maximum current i P, representative of the concentration, can be estimated: ip = constant n2 1/2 U C where n = number of electrons involved in the electrode reaction; = frequency of superimposed AC voltage; U = amplitude of superimposed AC voltage; C = concentration of the species. The relationship ip = constant 1/2 can only be used within certain limits. The measured AC current consists of two components: the Faraday AC current (i F) caused by the electrode reaction, which is of interest, and the capacitive AC current (i d resulting from the electrical double layer. Whereas the i F is proportional to 1/2, the iC is directly proportional to . Thus the relation iF/iC decreases with increasing frequency. For quantitative determinations lower frequencies (10-100 Hz) should be used. The phase of the iF is shifted by /4 relative to the applied AC voltage, whereas the interfering iC is shifted by /2. By use of a phase-selective detector the ratio i F/iC and hence the sensitivity of the method can be increased. In the AC mode, above all, species with reversible electrode reactions can be evaluated quantitatively. The i P decreases rapidly with increasing irreversibility. This insensitivity to irreversible reactions can sometimes be an advantage, because many interfering substances (e. g., organic species) react irreversibly at the working electrode. Chronoamperometry Chronoamperometry involves stepping the potential or the working electrode from a value at which no Faraday reaction occurs to a potential at which the surface concentration or the electroactive species is effectively zero. A stationary working electrode and unstirred solution are used. The resulting current-time dependence is monitored. As mass transport under these conditions is solely by diffusion, the current-time curve reflects the change in the concentration gradient in the vicinity of the surface. This involves a gradual expansion of the diffusion layer associated with the depletion of the reactant, and hence decreased slope of the concentration profile as time progresses. Accordingly, the current (at a planar electrode) decays with time, as given by the Cottrell equation: i(t) = nFACD1/2/1/2 t1/2 = kt-1/2 Such an it1/2 constancy is often termed "Cottrell behavior". Deviations from such behavior occur at long times (usually over l00 s) as a result of natural convection effects, or when using microelectrodes with high perimeter-to-area ratios. In the latter case, a time-independent current (proportional to the concentration) is obtained for t > 0.1 s due to a large radial diffusion contribution. Similar considerations apply to spherical electrodes whose current response

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following a potential step contains a time-dependent and time-independent terms. Recall also that for small values of t (t < 50 ms) the chronoamperometric signal contains a background contribution of the charging current. Additional transient background contributions (associated with surface redox reactions) are common to solid-electrode chronoamperometric experiments. Square-Wave Mode (SW) In this method developed by Barker and Jenkins [1], a square-wave (SW) AC voltage of a modulation of small, constant amplitude (U = 5-30 mV) and constant frequency (10-500 Hz) is superimposed on a linearly changing DC voltage ramp. For each changing DC voltage step, either one square-wave cycle or a burst of several cycles is applied. In order to minimize the capacity current (i C arising from the superimposition of the AC voltage, the current measurement is made toward the end of each half cycle of the wave. The lower the frequency, the greater the elimination of the iC due to the greater relaxation time for this current to decrease. The Faraday current (iF), which is of interest, decreases far more slowly during the same time. The DC component is filtered out by the detector and the resulting i F is amplified, rectified and recorded as a function of the DC ramp voltage. Peak-shaped voltammograms are obtained. The DC voltage (Up) at the current maximum (i P) is indicative for the kind of species. As in the AC mode, i P = constant n2 1/2 U C, i.e., for constant instrumental parameters and a distinct species i P is directly proportional to the concentration of the species. In this measurement mode higher frequencies can be used to enhance the sensitivity. Square-wave voltammetry is especially well suited for reversible systems. II.4.5.6. Stripping Voltammetry Voltammetric methods are suitable for the determination of, for example, metal ions in biological materials in concentrations down to the sub-ppb range. For determinations at rather low concentrations (10 -11-10-6 M) mostly stripping methods are used. Stripping methods consist of two basic steps: 1. The pre-electrolysis, in which the species to be determined is electrochemically enriched on a suitable stationary working electrode with constant mixing of the solution during a precisely defined time (t PE: pre-electrolysis time) and a suitable voltage (UPE: pre-electrolysis voltage). UPE = UP (200 to 400) mV. This is normally followed by a resting or "quiescent" period (e.g., 20 sec) without stirring of the solution. 2. The electrolytic determination itself, in which the enriched species on the working electrode is re-dissolved under voltammetric conditions, i.e., without stirring, by altering the electrode DC voltage continuously. This voltage-current curve can be measured with any of the normal voltammetric methods (DC, NP, DP, AC, SW). All these methods give current peaks in function of the electrode voltage. For reversible

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electrode reactions the iP is directly proportional to the concentration of the species in the original solution [6]. Several species can be determined in the same aliquot of the analyte: IP = constant . n3/2 A D1/2 1/2 C where n = number of electrons involved in re-dissolution; A = surface area of the electrode; D = diffusion coefficient; = sweep rate of the continuously changed DC voltage (U/t); C = concentration of the species. Anodic Stripping Voltammetry (ASV) For anodic stripping voltammetry (ASV) mostly HMDE and MFE are used as working electrodes. The enrichment step can occur by three different kinds of reactions: 1. The cation is reduced to the metal that forms an amalgam soluble in the mercury of the working electrode. Mn+ + ne- + Hg M(Hg) 2. The cation is reduced to a metal film on the electrode surface. Mn+ + ne- M/electrode 3. The cation is reduced to a lower oxidation state, which forms a sparingly soluble deposit on the electrode surface. Mm+ + ne- M(m-n)+/electrode The metal is determined by measuring the current-voltage curve with the applying of an anodic (positive) voltage sweep and recording the anodic dissolution current (i.e., re-oxidation). This method is above all suitable for the determination of Bi, Cu, TI, Pb, Sn, Cd, Zn, and Hg, the latter by the use of a gold electrode. This method has been applied to the determination of different metal ions in body fluids, e.g., Pb, Tl, Cu and Hg [6]. Cathodic Stripping Voltammetry (CSV) The enrichment in cathodic stripping voltammetry (CSV) occurs by the formation of sparingly soluble compounds of the species to be determined with ions from the material of the electrode forming a film on the surface of the WE. The commonly used WE is the HMDE. The enrichment voltage (U PE) is such that the material of the WE is oxidized in presence of the substance being analyzed. The more

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insoluble is the compound formed, the more negative will be the voltage at which the oxidation of the electrode material will occur (Nernst equation). Hg0 Hg22+ + 2eHg22+ + 2Cl- Hg2Cl2 The species to be enriched may also be formed by an electrochemical reaction on the surface of the working electrode, e.g., SeO32- + 6e- + 6H+ Se2- + 3H2O Hg0 Hg2+ + 2eHg2+ + Se2- HgSe The determination occurs by measuring the current-voltage curve by applying a cathodic voltage sweep (negative) to the WE. The measured cathodic current arises from the reduction of the cations (mostly Hg 2+ or Hg22+) present in the film of the sparingly soluble substances, e.g., Hg2Cl2 + 2e- HgO + 2CIHgSe + 2e- HgO + Se2The concentration range that can be determined depends on the type and size of the WE. At higher concentrations the film of the sparingly soluble substances adsorbed to the electrode surface becomes too thick and the observed current peaks are broad and may even be split. In some cases the integral of the cathodic current and not the iP is proportional to the concentration in the analyte. For the determination of Se, this element must be in the oxidation state +IV. This method has also been applied to biological material, e.g., for the determination of Se [6]. Adsorptive Stripping Voltammetry (ADSV) In adsorptive stripping voltammetry (ADSV), the accumulation of the species of interest occurs by adsorption of a suitable complex of the species to the surface of the WE. During the accumulation, there is no direct interaction between the species and the material of the WE. Therefore, WEs of different materials (e.g., Hg, Au, Pt, glassy carbon) can be used. The complexes must be very stable, sparingly soluble, and maximally hydrophobic in the applied milieu. Their structure should facilitate the electron transfer between the WE and the complex or the central ion. This relatively new method has considerably enlarged the number of trace elements that can be determined by stripping voltammetry. Thus, metal ions, which do not form amalgams soluble in Hg as needed in ASV, can also be determined with very high sensitivity

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down to the ppt range, e.g., Ni, Co, Zn, Cu, Pt, Mo [6]. In general, the accumulation occurs in two steps, i.e., the complex formation with a suitable ligand (L) and its adsorption to the surface of the WE at a voltage (U PE) during a defined time (tPE) without any Faraday electrode reaction: M + L mL (complex formation) ML + WE MLads/WE As adsorption can take place in some cases without an applied voltage to the WE, the time elapsing between exposing the electrode surface to the solution and the start of the adsorption step should be well defined. Automation of all steps of a determination is to be preferred. The determination may take place by different electrode reactions: 1. The voltage at the WE is continuously changed to more negative values (CSV) and the current resulting from the reduction of the complexed metal ion is measured. iP = constant C and UP is indicative of the kind of metal. MLads/WE + ne- M + L + WE
-U

2. Metal ions that cannot be reduced in the applicable voltage range may be complexed with a ligand that can be reduced (oxidized). MLads/WE + ne- Mn+ + Lred + WE
-U

3. In some cases neither the metal ion nor the ligand undergoes an electrode reaction but, by the adsorbed complex hydrogen is catalytically evolved. The magnitude of the corresponding current is in a limited range directly proportional to the concentration of the adsorbed species. ADSV is in most cases more sensitive and much faster than ASV and CSV. But this method demands a scrupulous sample preparation because many substances, above all surface-active ones, interfere. Electrochemical sensor technology is still limited in scope, and hence cannot solve all environmental monitoring needs. Yet, a vast array of electrochemical sensors has been applied in recent years for monitoring a wide range of inorganic and organic pollutants. We are continuously witnessing the introduction of new electrochemical sensing devices, based on a wide range of chemical or biological recognition materials. In addition, mass production techniques (adapted from the microelectronic industry) enable the fabrication of extremely small and reproducible, and yet inexpensive (disposable), sensing devices. Such devices are being coupled with light and user-friendly microprocessor-based instrumentation.

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Fast-responding electrochemical sensors are also being adapted for detection in on-line monitoring or flow-injection systems (as needed for continuous monitoring or field screening applications). Other advances of selective and stable recognition elements, smart sensors and molecular devices, remote electrodes, multi-parameter sensor arrays or micromachining and nanotechnology, are certain to have a major impact on pollution control. Additional efforts should be given to the development of new immobilization procedures (that increase the stability of the biocomponent), to the design of new electrocatalysts (that facilitate the detection of additional priority pollutants), to the replacement of classical mercury electrodes with well-defined solid surfaces, to address the fouling and degradation of electrochemical sensors during use, to the development of immunoassay-based electrochemical sensors and of remote electrodes for unattended operations, and introduction of multi-sensor systems for simultaneous monitoring of several priority contaminants. On-going commercialization efforts, coupled with regulatory acceptance, should lead to the translation of these and future research efforts into large-scale environmental applications. REFERENCES 1. P. T. Kissinger and W. Heineman (eds.), Laboratory Techniques in Electroanalytical Chemistry, Marcel Dekker, New York, 1984. 2. A. M. Bond, Modern Polarographic Methods in Analytical Chemistry, Marcel Dekker, New York, 1980. 3. P. Rach and H. Seiler, Polarography and Voltammetry in Trace Analysis, Hiithig, Heidelberg, 1987. 4. H. Seiler, in Analyses oJ Hazardous Substances in Biological Materials, VoI. 2 (I. Angerer and K. H. Schaller, eds.), VCH, Weinheim, 1988. 5. J. Wang, Analytical Electrochemistry, Wiley-VCH, ISBN 0471-28272-3, 2000. 6. A. Sigel, H.Sigel, Handbook on METALS IN CLINICAL AND ANALYTICAL CHEMISTRY, Marcel Dekker Inc. ISBN 0-8247-9094-4, 1994.

II.4.6. CHROMATOGRAPHY
Monica CATAL ICARDO, Jos MARTNEZ CALATAYUD

II.4.6.1. Introduction

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The concept of chromatographic separation has a long history. Thus, Aristotle's contemporaries used various types of sorbents (earths) to process seawater. More scientific applications were developed much later, by Runge in 1850, Schnbein in 1861, Goppelsrder in 1861 and Day in 1900, and also by Kvita, Albrecht and Engler in the same period. In 1903, the Russian botanist Mikhail Tswett (18721920), who is currently held as the father of chromatography, conducted his pioneering experiment involving the passage of a plant extract through a column filled with a sorbent material (Figure II.4.16). In subsequent years, Tswett refined his invention. Thus, in 1906 he succeeded in isolating chlorophylls and xanthophylls in a plant extract in petroleum ether by passing it through a glass column packed with finely divided calcium carbonate.

Figure II.4.16. Drawing of the original chromatograph from the first Tsweet article. Separation was done in space temporal separation was accomplished at a later time. The isolated species formed colored bands in the column, hence the name of the new technique [from the Greek khroma (colour) and grafein (to write)] according to the much-repeated historical version. To the author, however, the name comes from Chronos, the Greek god of time. Since then, chromatographic techniques have evolved dramatically, hand-in-hand with technological breakthroughs. It was not until about thirty years after Tswett's discovery that the usefulness of adsorption chromatography for separation purposes was envisaged by R. Kuhn and his coworkers, in 1931. A few years later, Sthal and Kirchner developed thin layer chromatography by transposing the original operating principle to thin layers of sorbent.

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This inspired the following historical definition of chromatography: a separation method based on the different velocity at which the components of a sample go through a stationary phase pushed or swept by a mobile phase. Chromatographic separation involves an interaction and the partitioning of the analyte between two immiscible phases for which it exhibits some affinity, namely: the mobile phase and the stationary phase. The stationary phase in a chromatographic column can be solid (packing the column) or liquid (anchored to a solid surface). The mobile phase, also called the eluent, can be a gas, liquid or supercritical fluid that sweeps the sample components as it goes through the stationary phase. The way an analyte distributes itself between the two phases depends on its partition coefficient, which dictates the velocity at which it will travel or migrate across the stationary phase. If the chromatographic process is allowed to proceed for a long enough time, differences in migration velocity between the sample components eventually result in their separation. Those components that are strongly retained by the stationary phase will move slowly in the mobile phase flow, whereas those that are weakly bound to such a phase will move faster. There will thus be a spatial separation: each sample component will move separately from the others. As a result, each sample component will reach a preset point (the detector) after a different time, so there will be both spatial and temporal separation between all. Once each component has been isolated, it can be identified and/or quantified individually. II.4.6.2. Types of Chromatography Chromatographic methods can be classified according to various criteria. One is based on the way the stationary and mobile phases are brought into contact; thus, there is column (three-dimensional) chromatography and planar (two-dimensional) chromatography. In column chromatography, the stationary phase is held in a narrow tube (a column) through which the mobile phase is passed by gravity or under pressure. Columns can be of the packed and open-end types; the former are filled with particles containing the stationary phase (SP) and the latter consist of hollow capillaries the walls of which are coated with the SP. In planar chromatography, the stationary phase is placed on a flat plate and the mobile phase travels across it by gravity or capillarity. Both types of chromatography rely on identical equilibria, however. One other classifying criterion is the type of stationary and mobile phases used. Thus, there is liquid, gas and supercritical fluid chromatography, which use a liquid, a gas and a supercritical fluid, respectively, as the mobile phase. Liquid chromatography can be implemented in a column or on a planar surface; on the other hand, gas and supercritical fluid chromatography can only be performed in a column. A third criterion is the underlying retention mechanism, i.e. the type of

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physicalchemical interaction between the stationary phase and the sample components. Such a mechanism can be essentially of five different types however, the overall phenomenon behind the separation usually involves more than one, namely: (a) Adsorption. This occurs when the stationary phase consists of particles of a solid sorbent the surface of which retains the sample components that will compete for the mobile phase molecules. In this way, the retentionelution cycle is successively completed. (b) Partitioning. This is a liquidliquid extraction process by which the sample components are separated as a function of differences in polarity. The column is packed with a solid support coated with a liquid immiscible with the mobile phase or eluent. The mobile phase is usually non-polar (an organic solvent) and the stationary phase polar. These are the ingredients of so-called normal chromatography as opposed to reversed phase chromatography; the latter uses a polar (aqueous) mobile phase and a non-polar stationary phase. (c) Ion exchange. In ion-exchange chromatography, the stationary phase is a resin with negatively or positively charged covalently bound groups that attract ions of the opposite sign via electrostatic forces. This type of chromatography is dealt with in detail later on. (d) Exclusion. Exclusion chromatography, also known as gel chromatography, relies on a screening effect dependent on the size of ions and molecules. The column is filled with a porous stationary phase or a gel, so the packing particles possess inner channels. The larger molecules in the sample can only pass between the gel particles, whereas the smaller ones can also penetrate the gel via tortuous paths. As a result, the bulkier particles will leave the column before the smaller ones. In addition, adsorption of the analytes onto the gel surface can give rise to partition coefficients greater or less than unity. (e) Affinity. In affinity chromatography, the solute interacts in a specific manner with a functional group in a molecule covalently bound to the stationary phase (immobilized on it). For example, only the protein reacting with an antibody will bind to a column if the antibody is immobilized on the stationary phase. Selectivity here can be modulated by choosing an appropriate group to interact with the analyte and exploiting both chemical bonding and steric effects. II.4.6.3. Classification of Chromatographic Processes The classification can be made according to different criteria, like 1. By the nature of the phases; 2. By the configuration of the separation system; 3. By the interaction between the analyte and stationary phase; and 4. By the composition of the mobile phase (liquid chromatography only). A more complete and general classification scheme is depicted in Figure II.4.17.

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Components of Column Chromatography A column chromatography assembly (Figure II.4.18) essentially includes the following elements: (a) containers for the mobile phase and solvent treatment; (b) a pumping system; (c) a sample insertion system; (d) a column; (e) a detector; and (f) a recorder or a data acquisition, processing and delivery system.

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Column Chromatography
Character mobil phase

Gas
Liquid
GLS partition

Liquid

Character Solid stationary GSC phase adsorption

Planar

Columna CL or HPLC

PC paper

TLC; HPTLC Thin-layer

LSC adsorption

LLC partition

SEC size exclusion /gel permeation

IEC ion exchange

BPC Bonded phase

GPC gel permeation

GGC gel filtration

Figure II.4.17. General classification scheme.

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Solvent/s container Data collection and presentation

Pumping

Injector

Column

Detector

Figure II.4.18. Block diagram of a liquid chromatograph. Whichever the type of analyte interaction is involved, the stationary phase is packed in the column and the sample is inserted at one end and swept by a continuous (or, less often, intermittent) flow of the mobile phase. As noted earlier, because each analyte interacts differently with the two phases, it will travel at a different velocity through the column. If velocity differences between analytes are sufficiently large and the column sufficiently long, the analytes will be separated into more or less sharp bands. An appropriate detector can be placed at the end of the column in analytical chromatography to identify and/or quantify each species ( viz. to record a chromatogram, where each band will show as a peak at a position dependent on the retention time of the analyte concerned); alternatively, each species can be collected in a separate vessel for subsequent measurement with an appropriate detector. The longer the column is, the more efficiently can two species be separated; however, an increased dispersion of the sample components in the bulk mobile phase reduces the separation efficiency of the column. The optimization process should be aimed at improving such efficiency; this entails minimizing band broadening and altering the relative migration rates in order to achieve complete separation of species at the column offset. This requires the prior identification of the variables influencing the analyte migration rates and the factors resulting in band broadening. The most influential experimental variables in addition to the nature of the stationary and mobile phases - and the temperature in gas chromatography - are the flow-rate of the mobile phase and various characteristics of the column including the size of the packing particles, the length and diameter of the column, and the degree of uniformity of the packing - and also the thickness of the liquid film when the stationary phase is liquid. A comprehensive description of the influence of each experimental variable is obviously beyond the scope of this chapter and can be found, together with a definition of the most commonplace chromatographic terms, in any book about chromatography. Rather, this chapter summarizes the general theory of chromatographic separation.

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II.4.6.4. Chromatographic Theory. An Overview The analyte (i.e. the sample component to be isolated) partitions itself between the stationary and mobile phase depending on its specific affinity for each. In 1941, Martin and Synge developed the earliest theoretical description of chromatography, which earned them the Nobel Prize in 1952. The central element in their theoretical development is the upstream partitioning concept and the definition of theoretical plate - by analogy with fractional distillation -, which is the column section where the average concentrations of the analyte in the stationary and mobile phase are consistent with the above-described partition coefficient. XM XS

K =

[X S ] [X M ]

The number of theoretical plates of a column dictates its separation efficiency. The ratio of the column length to the number of theoretical plates it contains is called the plate height.

HETP

Figure II.4.19. Theoretical plate in a chromatographic column. Therefore, the efficiency of a separation relies on the number of theoretical plates of the column. A straightforward description of the procedure used to determine the size (length) of the theoretical plate (and hence the number of plates in a column) based on the dynamics of chromatographic separation is provided below. The size of a theoretical plate is defined as the height equivalent to a theoretical plate (HETP), which is a function of various column-related factors including the following: (a) the effect of mass transfer; (b) the eddy effect; (c) the diffusive effect; and (d) miscellaneous effects including interactions between solutes, the size and uniformity of the packing particles, the thickness of the mobile phase, and

166

variations in the flow-rate of the mobile phase across the column (as a result of gaseous phases being compressible). In Table II.4.4 are presented auxiliary parameters and equations in cromatography. Table II.4.4. Auxiliary parameters and equations in chromatography. Parameter Parameter name Distribution constant (coefficient) Retention time Definition Equilibrium constant AmobileAstationary

K=
tR

cS cM

Time interval from sample insertion to maximum peak appearance tM Dead time Time interval required for a nonretained solute to be transported through the column. Capacity factor Describes the migration velocity of a solute into the column. Theoretically K V t t kA = A S = R M 1<kA<5. (kA<15). Depending on the VM tM T, mobile phase composition and filling in the column. Selectivity Indicating the relative situation of factor two peaks: being B the most retained K k (t ) t = B = B = R B M and A the most quickly eluted K A kA (t R ) A tM >1. Depending from mobile and stationary phases composition. H: plate height H and N indicate the efficiency of the N: number of column. theoretical >N and <H >efficiency 2 plates tR N = 16 W Resolution Indicates the column ability to 2[ (t R ) B (t R ) A ] separate two different solutes. RS = RS 1.5 complete separation W +W
A B

N ( 1) kB RS = 4 1 + k B
CS: solute concentration in the stationary phase;

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CM: solute concentration in the mobile phase; W: base-wide peak. W = 4. Mass Transfer Effect An analyte that was very strongly retained by the stationary phase would stop at the first plate and require a large volume of mobile phase for elution. On the other hand, one not retained at all by the stationary phase would migrate with the eluent front. In between these two extremes, actual analytes are retained to a variable degree and require the use of specific conditions for separation. During the separation process, the analyte passes from the stationary phase to the mobile phase and back many times. The transfer is not instantaneous; rather, it depends on the rate of diffusion of the analyte in the two phases. The diffusion rate in turn depends on the prevailing concentration gradient. Equilibrium, which can never be fully reached in a dynamic system of this type, is favored by high diffusion coefficients, short diffusion distances and large interface areas between the two phases. Thus, if the mobile phase is thick, the solute will have to travel long distances to reach the stationary phase and vice versa. In summary, the analyte will travel farther down the column than one would expect from its partition coefficient alone. As a result, the actual chromatographic peak will be broader than expected. This effect is especially prominent in gaseous phases as a result of the increased transfer coefficient within a gas. The transfer of an analyte from the mobile phase to the stationary phase and back is favored by the following: (a) a high diffusion coefficient; (b) a small distance between the two phases; (c) a large interface area; and (d) a low velocity of the mobile phase. These variables (marked with Cv) contribute to the HETP as follows: Cv = k dg2 v / 2 (1+k)2 Ds where k is the effective partition coefficient, dg the thickness of the stationary phase; v the velocity of the mobile phase, and Ds the diffusion coefficient of the analyte in the stationary phase. Eddy Effect The velocity of the mobile phase is not the same at every point of a theoretical plate. In fact, it is lower near the stationary phase than in the middle of the stream; the travel of the mobile phase through the stationary phase can be compared to that of a mountain stream flowing along an uneven bed splitting into several independent courses at different points.

168

As a result, the velocity at which the analyte travels varies between two extreme values. Thus, some analyte molecules (or ions) will travel very rapidly, while others will be delayed and give rise to a new dispersion factor and hence to broadened chromatographic peaks. The contribution of the eddy effect (A) to the HETP is: A = 2 dp where is a measure of unevenness in the stationary phase packing and dp the average diameter of the support particles.

a)

b)

Figure II.4.20. a) Illustration of the eddy effect; b) Types of particle packing and resulting peaks. As can be seen in Figure II.4.20 minimizing the adverse effects of this phenomenon entails using the thinnest possible a stationary phase. In some cases, column efficiency can be improved by using a stationary phase of low viscosity with an increased diffusion coefficient for the solutes. Diffusive Effect A solute in a mobile phase can travel (diffuse) freely in any direction, not only towards the stationary phase or away from it. Diffusion causes displacements from the regions of increased concentration by effect of a concentration gradient. The resulting dispersion will be higher for solutes with high diffusion coefficients and mobile phases flowing at a high rate. This phenomenon (marked with B/v) has the opposite effects of mass transfer. Its contribution to the HETP is: B/v = 2 Dm / v where is the tortuosity coefficient, Dm the diffusion coefficient of the solute in the mobile phase and v the velocity of the mobile phase (in cm s1). The mass transfer, eddy and diffusive effects in combination do not delay retention of the solute by the stationary phase, so they do not alter the mean velocity of the solute (i.e. the maximum of the chromatographic peak). However, they have a

169

decisive influence on the solute dispersion within the chromatographic system - they result in broader peaks and hence is poorer analyte separation. With provision for these three effects, the HETP for a column can be calculated from: HETP = 2 dp + 2 Dm / v + 8 k dg2 v / 2 (1+k)2 Ds Because v is the sole variable that can be altered by the operator once the chromatographic system has been set up, the previous equation can be simplified to: HETP = A + B / v + Cv A plot of this expression, known as the Van Deemter equation (see Figure II.4.21) clearly reflects the presence of a minimum HETP value that coincides with the optimum value that to be pursued for optimal separation. The operator should thus choose the v value leading to the minimum HETP. In order to expedite separation, however, it may be preferable to use a v value slightly higher than that resulting in the lowest possible HETP.
HETP

C A voptimum

Flow-rate mobile phase (v)

Figure II.4.21. Graphic aspect of the Van Deemter equation. Miscellaneous Effects The three above-described effects are not the only ones influencing chromatographic development. In fact, a few others exist that contribute to peak broadening and thus influence HETP. Thus, (a) Each solute molecule or ion can act independently of the others. In the presence of interactions between one another, solutes may be eluted sooner than expected (e.g. in non-linear chromatography). Such interactions are absent from linear

170

chromatography, so partition coefficients are identical at any point in the chromatographic system. (b) The affinity of the stationary phase for the solutes varies across its surface. Thus, sorbent surfaces can be partially blocked by other polar substances such as water. (c) The thickness of a liquid stationary phase is not uniform throughout the chromatographic system. (d) Because gaseous mobile phases are compressible, their flow-rate changes with the distance traveled through the column. (e) The term Cv encompasses diffusion in one phase only, so it should be replaced with (C + C)v, which includes diffusion in both. For these reasons, the previous equation is incomplete. Some authors have proposed alternative expressions descriptive of a similar variation of HETP with v. The most important parameters and equations in chromatography are presented in Table II.4.4. II.4.6.5. Ion Chromatography Monica CATAL ICARDO, Jose MARTNEZ CALATAYUD Ion chromatography peaked in popularity in the 1960s and 1970s. Although the subsequent inception of a new generation of chromatographic methods has lessened its significance, it continues to be widely used in routine applications. Originally, ion chromatograph relied on two successive processes, namely: (a) the chromatographic separation of ionic compounds by interaction and/or exchange with charged sites on the stationary phase, and ( b) the neutralization or suppression of the large amount of salt species produced in the previous step by formation of non-conducting species (water or carbonic acid) and the enhancement of the intrinsic conductivity of the analytes by conversion into strong acids or bases. The latter step was dictated by the type of detector used (a conductimeter). In Figure II.4.22 is presented the general scheme of an ion chromatograph.

EC MP P I

SC CD

Figure II.4.22. General scheme of an ion chromatograph. MP vessel holding the mobile phase; P mobile phase propulsion system; I sample injection system; EC separation column packed with ion-exchange resin; SC suppressor column; CD conductimetric detector.

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Ion Exchange A. Stationary Phase The mobile phase consists of a resin with a backbone bearing of a specific functional group capable of exchanging ions.

(I)

(II)

The backbone of a typical ion exchanger consists of a styrene-divinylbenzene copolymer. This forms a three-dimensional hydrocarbon structure containing many instances of the following chemical sequence:

This backbone can be easily obtained and is physically and chemically stable under certain conditions. For use as an ion exchanger, it is supplied with ionic groups as follows:

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Depending on the character of the ionic group introduced, the exchanger can be of various types the most prominent of which are acid, cationic and basic (anionic). Table II.4.5 shows selected examples of each type of exchanger. One end of the functional group is covalently bound to the hydrocarbon backbone; the other (counterion), which bears charge of the opposite sign, binds to it via electrostatic forces. This latter portion can be exchanged with other ions present in the mobile phase. Table II.4.5. Types of exchangers. Ionic exchangers Cation exchange Anion exchange Type Strong acidic Weakly acidic Strongly basic Weakly basic Chemical group Sulfonic acid: -SO3H; -CH2CH2SO3H Carboxylic acid: -COOH; -CH2COOH Quaternary amine: -CH2N(CH3)3+OH-; -CH2CH2N(CH2CH3)3+OHAmine: -NH3+OH-; -CH2CH2NH(CH2CH3)2+OH-

B. Ion-exchange Processes A resin bearing sulfonic groups (RSO3H+), for example, will establish the following ionic equilibrium: n R-SO3-H+ + Mn+ (R-SO3-)nMn+ + n H+ solid solution solid solution In an acid medium, all ionic sites in the resin will be occupied by hydrogen ions. However, such H+ ions can be displaced by other ions. As a rule, the affinity of the resin will be maximal for ions possessing a high charge and charge density, and a small size - size here means the actual size of the ion in solution, hydration sphere included. Once the target ions have been bound to and retained by the resin, they can be eluted by using a solution containing an anion with a higher affinity for the resin (a strong acid) in order to displace the equilibrium back to the left. Some lists rank ions for affinity to specific resins. The lists for the most commonplace resins can be used as guidance; the rankings, however, can change slightly depending on the particular experimental conditions. C. Mobile Phase In ion chromatography, the mobile phase is usually an aqueous solution occasionally containing some miscible organic solvent and an ionic species with buffering properties. Any conventional buffer is theoretically useful for this purpose.

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Anion exchangers are used with buffers consisting of positively charged species and the opposite is true for cation exchangers - phosphate buffer can usually be used with both types of exchanger. Eluent power and elution selectivity depend on the particular type and concentration of the species added to the mobile phase. In some applications, the composition of the mobile phase is changed during the chromatographic process; this is known as gradient elution. D. Detectors The ideal detectors for ion chromatography are those based on conductivity, which are universal for charged species and can be highly sensitive in addition to simple, inexpensive, easily miniaturized, robust and long-lasting. Their lack of selectivity poses no problem here as the analytes are previously isolated. However, ensuring that all analytes will be eluted from the column within a reasonable time entails using a high electrolyte concentration; this decreases the sensitivity of the determination because of the conductivity of the eluent concealing that of the sample components. As stated above, this problem can be overcome by using appropriate suppressors behind the ion-exchange column. E. Ion Suppression The earliest suppressors used in ion chromatography were ion-exchange resins that converted the solvent ions into scarcely ionized molecular species without altering the analyte ions. Thus, with HCl as eluent, the suppressor column contained an anionic exchanger in hydroxide form, so the exchange process was as follows: H+(aq) + Cl(aq) + Resin+OH(s) Resin+Cl(s) + H2O For anions, the suppressor was the acid form of a cation-exchange resin and the eluent a carbonate or bicarbonate solution. Suppressor columns must be regenerated on a regular basis (every 810 h) by converting the packing back to the original acid or basic form. More recently, new types of suppressors such as the following have been developed: (a) Hollow fibers of polymeric ion-exchange material that can be regenerated by passing an appropriate solution over their outer surfaces. (b) Micro-membrane suppressors based on ion-exchange membranes that are described later on. (c) Electrolytic membrane-based suppressors, where ion transfer across the membrane is favored by applying an electrical field. (d) Packed-column mini-suppressors, which use two suppressor cartridges connected to a 10-port valve. In its starting position, the eluate is passed through one of the cartridges only. When the regeneration capacity of such a cartridge, which is transparent, is exhausted (viz. when the pH indicator it contains exhibits

174

a color change that signals the need for regeneration), the valve is switched to have the eluent pass through the second cartridge and the first is replaced. (e) Continuously regenerated packed-column suppressors, which rely on the ion reflux principle. This is an ion-exchange technique involving the passage of water over an electrically polarized resin bed and using an electrolytic reaction to produce the eluent and suppressor medium. Applications The quality of environmental water (rain, lake, underground, river) is usually assessed from analyses for inorganic ions such as sulfate, chloride, nitrate, sodium, potassium, ammonium, magnesium and calcium. Monitoring ion contents in water involves the simultaneous separation and determination of anions and cations by ion chromatography. A number of approaches have been explored for this purpose including the use of mixed beds of cation and anion exchangers or two individual columns and as many detectors. A. Determination of anions (CrO42, MoO42, BrO3, SeO32, SeO42, HAsO42 and WO42) and cations (Cu2+, Ni2+, Pb2+ and Cd2+) This is a joint determination of several ions in river water samples. The analytes are all known to be toxic to humans, animals and plants. Toxicological analyses must not only be highly selective, but also allow the speciation of ions as their deleterious effects depend on their specific valence states. This determination involves the chromatographic separation of both metal ions (following chelation with Na2EDTA) and non-metal ions (by suppressed anion-exchange chromatography). The novelty here is that the analysis time is reduced by performing gradient elution under optimal conditions; in this way, each analysis takes less than 20 min. The use of a gradient introduces a gradually increasing competitive advantage in the elution process (i.e. the sweeping of retained ions); however, the use of a conductimetric detector can lead to baseline drift and substantially increased salt concentrations in the eluent as a result. In this particular determination, elution gradients were programmed by using a baseline balancing method. The operational procedure was follows: a volume of 50 L of sample was inserted into the chromatographic system following in-line removal of organic matter by using a Dionex OnGuard-RP cartridge and metal cations were pre-chelated with 0.25 mmol L1 EDTA. The experimental set-up consisted of a Dionex AG9 4 50 mm i.d. column followed by a Dionex AS9 4 250 mm i.d. column packed with a 15 m thick bed of polystyrenedivinylbenzene substrate with a completely aminated anion-exchange latex the backbone of which was polyacrylate-based as binder. The suppressor was of the membrane, sandwiched layer type [viz. an Anion Micro-Membrane Suppressor (AMMS-II)] and the regenerating solution 25 mmol L1 H2SO4. The mobile phase was 3.5 mmol L1 NaHCO3 at pH 9.75.

175

The eluent was passed between two ion-exchange resins over the outer surface of which the regenerating solution was circulated upstream. The limits of detection thus achieved ranged from 2 g L 1 for Ni, Cd, Cu and Cr to 6 for SeO32. B. Determination of Na2+, NH4+, K+, Mg2+, Ca2+, IO3, BrO3, Br, NO3 and Cl in various types of water (river, pond, tap) The process here involved the use of two columns packed with and anion exchanger and a cation exchanger, respectively. The columns were placed one after the other or accommodated in the loops of two injection valves. Figure II.4.23A shows the assembly with the two columns arranged in a serial manner. The sequence in which the two were placed was found to influence the elution profile. A high cation concentration can result in the formation of ion-pairs with the anions; therefore, the cation-exchange column should precede the anionexchange column when the eluent contains many cations. However, the peaks for the cations deteriorate when these are passed through the anion-exchange column, which is not the case with those for the anions. Also, ions such as calcium, iodate, bromate and nitrite cannot be determined under these conditions owing to the resulting peak overlap. These led to the development of the alternative depicted in figure II.4.23B, in which two 6-port valves were used in such a way that the cations separated in the cation-exchange column accommodated in the loop of the first valve were not passed through the anion-exchange column located in the loop of the second valve. When the sample volume (20 L) was injected, the two valves were in their injection positions, so the sample was allowed to reach the loops accommodating the two columns. Cations were retained by the cation-exchange column while anions were passed through it and reach the anion-exchange column. Within 1.45 min, all anions were retained by the latter column, the second valve then being switched to the next position.

176

(A)
FM

Columns cationic and anionic

UV

(B)
FM

V1

V2

UV
Cation exchange Anion exchange column column

Figure II.4.23. Schematic diagrams of the chromatograph configuration. S: sample (Injected volume, 20 L); P: Pump (1 mL min -1); FM: Mobile Phase (H2SO4 1.0 mM + L-hystidine 0.1 mM); UV: UV detector (210 nm); C: conductimetric detector). The anion exchange column was a 50x4.6 mm TSKgel IC-Anion-SW; the cation exchange column was a 150x4.6 mm TSK gel Super IC-Cation. Separating the cations took 10 min, after which the valves were switched and all analytes allowed to reach the detector. For comparison purposes, the detection was done with a spectrophotometer and a conductimeter. Cations were detected by UV-VIS absorption spectrophotometry in an indirect manner and the mobile phase was supplied with an absorbing additive (viz. the aminoacid L-hystidine). The passage of each cation through the detector produced a negative peak. Absorbance measurements were made at 210 nm. The limits of detection thus achieved, in mg L 1, with UV and conductimetric detection, were as follows: Na + (0.092, 0.1), NH4+ (0.059, 0.09), K+ (0.14, 0.2), Mg2+ (0.14, 0.1), Ca2+ (0.4, 0.3), IO3 (0.2, ), BrO3 (0.2, ), Br (0.042, ), NO3 (0.042, ) and Cl (, 7.1). The process took roughly 20 min. The presence of L- hystidine slightly increased the retention times for the cations as it competed with them for active sites. C. Simultaneous determination of inorganic nitrogen, nitrate, nitrite and ammonium in a micro-column (Figure II.4.24) Nitrogen can occur as various chemical species with valences ranging from +5 to 3. Also, the nitrogen cycle encompasses a wide variety of chemical and

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biological processes in the environment. In this example, the analytical method was applied to river water as the continuous release of nitrogen species into rivers increases eutrophication and environmental pollution. A micro-ion chromatographic column was used to speciate inorganic nitrogen and the separation system was coupled to two serially arranged detectors (a spectrophotometer and a spectrofluorimeter). Nitrate and nitrite were detected at 206 nm with the former. On the other hand, ammonium was post-column derivatized with o-phthalaldehyde in the presence of 2-mercaptoethanol to measure the light emitted by the resulting derivative at 470 nm upon excitation at 410 nm.

+ HOCH2CH2SH + NH2R
MEC

OPA

amine

isoindole

The stationary phase was an IC-Anion SW anion-exchange resin packed into a fused-silica tube 100 mm long 0.32 mm i.d. The mobile phase was a 20 mmol L 1 solution of sodium sulfate at pH 5.7. The analytical response was linear over the range 0.020.1 mmoL 1 for the three species and the limit of detection was 1.6, 2.3 and 17 mol L 1 for nitrate, nitrite and ammonium, respectively. The repeatability in retention time and peak height and area at an analyte concentration of 0.1 mmol L 1 was always better than 2%. Each analysis took less than 10 min. Samples were previously passed through a Develosil C30 column to remove hydrophobic substances.

S FM R

C V

UV

T-L

Figure II.4.24. Flow assembly for simultaneous determination of inorganic nitrogen. P: Syringe pump; V: sample injector; C: separation column; T-L: water bath (65 C) and reaction coil (fused-silica capillary tube of 50 m i.d. x 2 m); UV: UV detector; F: fluorescence detector; S: sample; FM: eluent; R: reagent solution; W: waste

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D. Determination of phenols and chlorophenols in water for human consumption The separation of inorganic ions is not the sole application of ion chromatography. In the following example, a total of 14 phenols and chlorophenols were determined as products of the decomposition of the fungicide tecnazene upon irradiation with UVVisible light. Chlorophenols have been widely used for more than fifty years despite their toxicity, persistency and in some cases carcinogenicity. European legislation has set the maximum tolerated total phenol concentration in water for human consumption at 0.5 g L 1 and that for individual phenols at 0.1 g L1. The real samples used were drinking water collected from the public supply network in various places. The experimental set-up (Figure II.4.25) included on-line pre-concentration, ion-exchange chromatographic separation and amperometric detection with a glassy carbon working electrode and an Ag/AgCl reference electrode, using a potential difference of +1.2 V between the two. The elution was done in the gradient mode, using a constant concentration (10 mmol L1) of sulfuric acid and a variable concentration (3665% v/v) of acetonitrile. The pre-concentration was done in an NG1 5 cm 4 mm guard column accommodated in the loop of a 6-port injection valve. A sample volume of 5 mL was passed through the column to retain the target analytes. Then, the valve was switched to the insertion position and eluted substances were passed through the separation column (an NS1 25 cm 4 mm ion-exchange column). Separation took less than 30 min, and column pre-concentration and elution 12 min in all. The limits of detection achieved ranged from 200 to 500 pmol L 1 for all phenols except 2,6-dichlorophenol (1 nmol L1), 3,5-dichlorophenol (5 nmol L1) and 2,3,5,6-tetrachlorophenol (1 nmol L1). The repeatability (n = 5) in both retention times and peak areas was better than 3.2% for concentrations over the range 50250 nmol L1.

AS FM

P
GC

AC

UV

Figure II.4.25. Flow assembly for phenols and polyphenols determination. AS: auto-sampler; P: gradient pump (1 mL min -1); FM: mobile phase; D: amperometric detector; UV: UV reference detector. GC: pre-column; AC: analytical column; W: waste. II.4.6.6. Gas Chromatography (GC)

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Monica CATAL ICARDO, Jos MARTNEZ CALATAYUD Gas chromatography can be directly applied to volatile compounds; however, it is more commonly used with liquid samples the components of which can be readily volatilized at the working temperature by derivatization. The sample should be volatilized immediately upon injection into the chromatographic system. The elution is done using an inert gas as the mobile phase, as this is intended to carry the analyte through the column rather than interact with it unlike most liquid chromatography applications. Gas chromatography can be of two different types depending on the nature of the stationary phase, namely: gas-solid chromatography (GSC) and gas-liquid chromatography (GLC). Gassolid chromatography uses a stationary phase that retains the analytes by adsorption. Its scope of application is highly restricted as a result of it usually giving tailed peaks - a consequence of non-linear adsorption - and of strongly polar molecules being retained almost permanently. For these reasons, it is usually applied to species of low molecular weight only. Gas-liquid chromatography, henceforward called simply gas chromatography, relies on the partitioning of the analyte between a gaseous mobile phase and a stationary phase consisting of a liquid immobilized onto the surface of an inert support. Instrumentation for Gas Chromatography The basic components of a gas chromatograph are depicted in Figure II.4.26.

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Figure II.4.26. Schematic figure of a gas chromatograph. W, waste; D, detector; C, computer; is, injection sample; gc, control of the gas flow-rate; sc, separation column; and, rc, reference column (nor present in any commercially available model). The carrier gas, which must be chemically inert, is usually helium, argon, nitrogen, carbon dioxide or hydrogen. The choice is frequently dictated by the type of detector used. Also, the gas container must be equipped with pressure control and measurement facilities, a flow meter and a molecular sieve (to remove water or other impurities). The sample introduction system should allow the insertion of a sample plug of appropriate size into the system. The most common insertion method involves using a micro-volumetric syringe to inject the liquid or gaseous sample via a silicone rubber septum into a vaporization chamber located at the column top. The chamber is about 50 C below the boiling point for the least volatile component of the sample. The mobile phase then sweeps the sample to the first plate in the chromatographic column, which minimizes dispersion. Injected sample volumes usually range from 10 3 to 20 L. For sub-microliter volumes, a stream splitter is used to circulate the sample through several channels only one of which leads to the column; in this way, only a small fraction of sample reaches the column while the rest is sent to waste. Quantitative work, where reproducible insertion of the sample is crucial, requires the use of rotary valves similar to those employed in HPLC and FIA. Solid samples can also be introduced into the chromatographic system, using glass vials of very thin walls that are broken from the outside once the vials have entered the injection chamber at the top of the column.

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Portable chromatographs for the determination of the typically low concentrations encountered in fieldwork can use sample volumes from 10 L to 1 mL. Gas chromatographic columns can essentially be of the packed or open-end types (see Table II.4.6 and Figure II.4.27). Table II.4.6. Characteristics of the different types of columns. Type of column Classical type Filled capillary Porous layer capillary Open capillary tubular Length/ Diameter 1-10 m/ 2-4 mm 10-50 m/ 1 mm 25-200 m/ 0.1-0.5 mm 5-100 m/ 0.1-0.5 mm d.column Perme V M Loading ation V capacity d.load S Increasing Increasing Diminution 10 3-5 Flow- Throughput rate /sample gas, dispersion mL/min 30-100 0.5-3.0 0.5-3.0 0.5-3.0 Diminution Increasing

a) 2 3

b) 2 1

c)

Figure II.4.27. Front view of different columns: a) Filled classical column; b) Porous layer capillary column; and, c) Open tubular capillary. 1. Tubing; 2. Supporting particle; 3. Stationary phase film; 4. Stationary phase.

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The earliest GC systems used packed columns where the stationary phase was a thin film of liquid coating the surface of an inert, finely divided solid support. A packed column consists of a glass, metal or Teflon tube 250 cm long and 14 mm in inner diameter. The tube is packed with a finely divided, homogeneous solid coated with a thin film (0.051 m) of the stationary phase (a liquid). For easier accommodation into the chromatographic oven, the column is coiled to a diameter of about 15 cm. The efficiency of the column increases markedly with decreasing size of the packing particles; however, this also increases pressure within the system, so particles are rarely smaller than 150 m in diameter. Packed columns afford larger injected volumes than do capillary columns; however, throughput and efficiency are better with unpacked columns of a very small inner diameter (a few tenths of a millimeter). These capillary columns are used with a stationary phase consisting of a uniform film of liquid a few tenths of a micrometer thick that is used to coat the inner walls of the capillary tube. This type of column is also known as open-end column. The materials from which these columns are made, and their coiled configuration, coincide with those of packed columns. Their inner diameters typically range from 250 to 320 m, but can be smaller (200 or even 150 m). The sample volume is usually very small, so the detector must be highly sensitive. The thickness of the stationary phase usually ranges from 5 m for highly volatile species to 0.1 m for less volatile ones. Although packed columns are more inexpensive and easy to use, capillary columns provide higher resolution. One problem with GC in both packed and capillary columns arises from the physical adsorption of polar compounds onto the surface of the stationary phase, which usually contains silicates. Overcoming it entails pre-treating the columns to remove SiOH groups that form on the support surface through hydrolysis by existing moisture. The liquid stationary phase used should be scarcely volatile, thermally stable and chemically inert; also, it should provide capacity and selectivity factor ( k and ) values within appropriate ranges for the analytes. The most suitable stationary phase must be determined on a case-by-case basis; in any case, the material should be similarly polar to the sample components. The column temperature is very important here and should be strictly controlled by using a thermostated oven. The optimum temperature in each case depends on the boiling point of the particular sample and the degree of separation required. Usually, the working temperature is slightly above the mean boiling point (bp) for the sample components. If the bp range spanned by such components is too wide, a temperature program is used instead to change the column temperature during the separation process. Usually, resolution improves with decreasing temperature, at

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the expense of longer elution times; a compromise must therefore usually be made in this respect. As a rule, the retention time doubles with each rise in temperature of 30 C. The chromatographic column is followed by the detector. The types of detectors used are rather different from those employed in liquid chromatography and can be classified as follows in terms of sensitivity: (a) Medium Sensitivity: Thermal Conductivity Detector, TCD or catharometer Gas density balance (to check other detectors, not commercial (b) High sensitivity: (b.1)- non-radioactive ionization: flame Ionization Detector, FID thermo-ionic (b.2)- radioactive ionization: electron capture detector, ECD ionization of argon The most commonplace - even in portable equipment - are the thermal conductivity detector (TCD), the flame ionization detector (FID) and the electron capture detector (ECD). In addition, a mass, IR or NMR detector is frequently used to facilitate the identification of individual components in mixtures. The thermal conductivity detector (TCD, see Figure II.4.28), also called catharometer, was used in the earliest GC applications. It relies on a combination of the thermal and electrical properties of the sample components. This type of detector is very simple, affords wide linear ranges, responds to both inorganic and organic compounds, and is non-destructive. On the other hand, it is scarcely sensitive, so it cannot be used with capillary columns, where sample size is usually very small.

Electric wire Gas outlet Detector hot block Gas inlet

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Figure II.4.28. Detector of thermal conductivity. The flame ionization detector (FID, see Figure II.4.29) is the best for organic compounds and one of the most commonplaces in current commercial instruments.

Electrostatic Field

Ion current

+
High Voltage Electrode

Flame Collector Electrode

Air Sample Fuel

Figure II.4.29. Flame ionization detector. The effluent from the column reaches a burner where it is mixed with hydrogen and air that is electrically ignited. Burning of most organic compounds under these conditions produces ions and electrons that make the flame space conductive. By applying a potential difference between an electrode that can be the burner end itself and the collector electrode, placed above the flame, an electrical current is produced that represents the detector response to a sample component concentration. This type of detector is scarcely sensitive to flow-rate changes in the mobile phase and not responsive to water, CO 2, SO2 or nitrogen oxides, so it is unaffected by the presence of moisture or oxide impurities in the sample; however, it is highly sensitive to the target species. Also, it exhibits a very broad linear response range and low background noise, and is robust and easy to use, but has the disadvantage of its destructive character. The electron capture detector (ECD, see Figure II.4.30) irradiates the effluent column with radiation to alter the electrical conductivity of the gas. The presence of electron-withdrawing organic molecules decreases the current. This type of detector is selective for organic molecules bearing electronegative functional groups (halides, peroxides, quinones and nitro compounds), but is insensitive to amines, alcohols and

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hydrocarbons. It exhibits a high analytical sensitivity and scarcely alters the sample. Its linear response range, however, is somewhat narrow.

Figure II.4.30. Electron capture detector. Automated systems for continuous on-line monitoring collect samples online, condition them and perform their analysis to provide relevant information in real time; this avoids potential contamination of samples during storage and transport. Also, the analytical results can be used as feedback for process control purposes. Conventional gas chromatographs are bulky and heavy. This entails transferring samples from the collection site to the laboratory for analysis, which, as noted earlier, makes it difficult to preserve their integrity. This has prompted the development of portable equipment exploiting recent micro-technological advances. Portable GC instruments are currently available from manufacturers such as Varian, Agilent and PerkinElmer. These instruments, however, only afford the analysis of volatile components (gases), as they cannot provide the temperatures required to convert semi-volatile substances into volatile ones. One instrument that overcomes this restriction is the microFAST GC, which is ten times faster than conventional instruments and affords the determination of environmental pollutants in real time. The instrument uses dual separation columns to ensure that each sample is analyzed individually in a simultaneous manner in each column. Columns in conventional GC instruments are heated by conduction; however, the microFAST GC uses a proprietary system where the heater is inserted between the two columns to heat them not only by conduction, but also according to a temperature program. This heating system is more precise and efficient, and uses less energy than conventional ones. Each column is 13 m long and 100320 m in inner diameter. It consumes little gas (less than 5 mL min1) and also little hydrogen (less than 50 mL min 1), which allows the use of small, light gas containers.

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One of the most critical components is the sample collection unit, which must condition, concentrate and inject samples into the GC column -conventional injection systems are useless with trace level concentrations. Very large injected sample volumes give rather broad bands, detract from resolution and can deteriorate the column, hence the need to pre-concentrate samples. One alternative here is the use of a micro-sorbent trap (a microtrap) for both injection and pre-concentration. The microtrap consists of a sorbent material packed in a capillary tube that is placed in front of the chromatographic column. It acts by trapping organic compounds, which are rapidly released by electrical heating for passage through the separation column. The sample can be circulated in a continuous manner (through an on-line microtrap); alternatively, the microtrap can be serially connected to an injection valve to construct a sequential valve microtrap (SVM). This allows the sample to be injected in a single volume or in several small ones. Applications A. Automatic on-line pre-concentration and gas chromatographic monitoring of four volatile organic compounds by use of a helical sorbent microtrap. A portable set-up consisting of a membrane module, a helical sorbent trap and a gas chromatograph was used for this purpose. For continuous on-line sampling, the analytes were continuously collected and enriched by the membrane-and-trap interface and directly transferred to the chromatographic column by thermal desorption. The membrane, 0.025 mm thick and 163 mm 2 in surface area, was made of poly(dimethylsiloxane) (PDMS) or bisphenol A polycarbonatepoly(dimethylsiloxane) and used to adsorb the sample. Under pressure from the carrier gas, the sample was released from the membrane and transferred to the collector trap. The trap consisted of a helical sorbent accommodated in a silicosteel tube. The analyte was concentrated at room temperature in the trap and then desorbed at fixed intervals by pulse flash electrical heating as a concentrated sample plug for injection into the column (an MXT1 3 m 0.32 mm i.d. silicosteel capillary column coated with a 3 m thick PDMS film), which was placed in an oven at 60 C. Hydrogen at a flow-rate of 5 mL min 1 was used as the carrier gas. Following separation, organic compounds were determined with a flame ionization detector. The limits of detection achieved were in the picogram-permilliliter region and depended on the trap pre-concentration time and on the variables influencing permeation through the membrane. Safe sampling of the helical sorbent microtrap was found to be improved by the helical configuration of the sorbent, which generated a turbulent rotation flow on the surface of the sorbent, and by the use of low carrier gas flow-rates, analyte concentrations and trapping temperatures. The method was tested on diesel engine exhaust. Automobile exhaust gases particularly those from diesel engines - constitute a major source of urban pollution.

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B. Detection of bis(di-isopropylaminoethyl)disulphide, a degradation product of the nerve agent VX. The GCMS tandem was originally conceived more than thirty years ago for use on planetary aircraft and space tests; however, scientists soon envisaged other possibilities in this hyphenated technique. Thus, field-portable GCMS systems are highly useful for the rapid, reliable identification of analytes. Both vehicle-mounted and hand-carried equipment of this type has been developed in recent years. The most widely used type of mass analyzer is the linear quadrupole, which is straightforward, compact and durable. In this particular determination, a field-portable GCMS system was used for the determination of a chemical warfare agent, viz. O-ethyl S-(2-diisopropylaminoethyl)methylphosphonothiolate (VX). However, VX cannot be determined as such, so it must be previously converted into a detectable degradation product. Sampling was done by solid-phase micro-extraction (SPME), using a 100 mm thick polydimethylsiloxane fiber coating. The sole sample treatment required was the addition of 500 l of 2.5 M NaOH and methanol (in a 1:1 ratio) to effect the catalytic degradation of VX to bis(di-isopropylaminoethyl)disulphide (DES)2. A vial containing a contaminated soil sample and the reagent was heated at 30 C for 30 min, during which SPME headspace sampling took place. Field analyses were performed on an electron impact ionization GCMSEI system mounted on a van. The column was an HP-5MS 30 0.25 mm i.d. model coated with a 0.25 m thick film and the mobile phase H2 at a flow-rate of 1 mL min 1. The oven was heated to 40C at 250 C min1 and the temperature was then held for 2 min. Desorption from the SPME fiber was done in the splitless injection mode for 2 min, followed by injector purge at 50 mL min1. The injection temperature was 270 C. Mass spectra were recorded over the m/z range 35350. The analysis time was less than one hour and the method allowed (DES) 2 concentrations as low as 1 g/g to be detected. II.4.6.7. High Performance Liquid Chromatography (HPLC) Monica CATAL ICARDO, Jos MARTNEZ CALATAYUD Based on the general theoretical principles exposed in introducing chromatography, the contact surface between the mobile phase and stationary phase should be as large as possible. This makes the size of the particles constituting the stationary phase (or its support) especially influential; in fact, the smaller such particles are, the greater will be the number of theoretical plates of the column and

188

the higher its separation efficiency. Packing uniformity is also very important to avoid distorted, poorly resolved chromatographic peaks. The small particle sizes used now do not allow the mobile phase to progress through the column solely by gravity. This entails using an external force such as that provided by pumping at a high pressure. This in turn calls for a stronger chromatographic system consisting of materials capable of withstanding the high pressures to be used. The system is made even more complicated in relation to traditional liquid chromatography by the need to: (a) separate chemically similar components; (b) deal with complex samples by integrating some sample pretreatment operations on-line; (c) improve sensitivity and detection limits by usually post-column derivatization of the resolved components in order to enhance their detection characteristics; and (d) use carefully programmed gradients of the mobile phase. The use of very small particles in order not to allow the mobile phase to travel by gravity was started in 1964 by J. Calvin Giddings; this signaled the inception of liquid chromatography as we know it today. Two years later, S.R. Lipsky constructed the first assembly for what is currently known as high performance liquid chromatography (HPLC, see Figure II.4.31).
Solvent proportioning valve Pulse Injection valve damper Drain valve Precolumn Waste Solid reservoirs Detector Column

Figure II.4.31. Schematic diagram of a HPLC system. Propulsion Unit and Elution Modes (isocratic and gradient-based) The propulsion system is intended to provide the pressure required to force the mobile phase to pass through the column at as controlled and uniform flow-rate as possible (i.e. in the absence of a pulsating flow). The most widely used propulsion system in this context is the piston pump, which can provide high pressures but causes the flow to pulsate every time the piston is loaded or emptied. This shortcoming, however, can be greatly circumvented by using two pumps in opposite phases.

Pump

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Gradient elution requires accurate, programmed control of the mixing of the solvents making up the eluent. This allows such variables as the solvent polarity, ionic strength and pH to be altered during elution. The outlets of several flasks containing the different solvents (or solutions) are connected to a valve allowing their flow-rates to be continuously controlled. In this way, mixing proportions can be changed as required at any time. The solvents used must be highly pure and degassed as bubbling results in distorted or even spurious peaks. The problem can be worsened by the use of mixed solvents (e.g. acetonitrile or methanol in water) as air is less readily soluble in solvent mixtures than it is in individual solvents. This entails the use of a de-bubbling system to reduce the air concentration below saturation levels. Helium can remove up to 80% and evacuation up to 60% of the air initially present in the system. Some commercial equipment includes an on-line vacuum de-bubbler and passes solvents through a porous PTFE membrane prior to use. Many operators, however, choose to use a helium pretreatment followed by evacuation. Sample Insertion The sample volume to be inserted should be accurately known (the operational scale is in the microliter region) and reproducible. Also, sample dispersion during this step should be virtually zero. The most universal sample insertion system is a six-port valve similar to those used in FIA but constructed in stainless steel in order to withstand the high working pressures required. Its operation is depicted in the corresponding sections on flow-analysis and Figure II.4.32.

Mobile phase

To column

Mobile phase

To column

Sample loop

Sample

Waste

Sample

Waste

Figure II.4.32. Scheme of a six-port valve and its operation. Columns HPLC columns vary in length, diameter and content. They should be as chemically inert as possible and capable of withstanding high pressures. Stainless steel tubes 3.9 or 4.6 mm in diameter are quite suitable for HPLC work.

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A column consisting of particles ca. 5 m in diameter tightly packed in a stainless steel tube of 4.6 mm i.d. easily provides 60 00090 000 theoretical plates per linear meter. The use of a 310 cm long pre-column packed containing the same packing as the column is usually advisable. Its importance lies in the facts that ( a) it can act as a filter by retaining solid particles (impurities) in the sample or solvents that might partially block the column and alter its efficiency and selectivity; and ( b) its absence shortens the column lifetime. The pre-column should be regenerated or replaced on a regular basis. A. Mobile Phase The composition of the mobile phase in HPLC varies. Thus, its eluting power can be altered by changing its polarity ( e.g. by using mixed eluents consisting of two or more solvents). In most cases, pure solvents do not allow all sample components to be separated. Also, the eluent composition need not be maintained throughout the chromatographic process. In fact, it can be changed in a continuous manner (the eluent will initially sweep the less strongly retained components and leave the others anchored to the column). As the eluent polarity is increased, competition with the solutes for binding sites in the stationary phase increases. In this way, the previously retained solutes are gradually swept. This operational mode, where the eluent composition is changed in a continuous, programmed manner, is known as gradient elution. B. Stationary Phase. Packing Type (modified surfaces) In its original and still widely prevailing meaning, chromatographic separation is synonymous with adsorption and/or partitioning. The original irregular particles of silica gel and alumina have been subsequently replaced with regular spherical particles that provide more uniform packing and reduce the distortion in the separation zones (see the general theory of chromatography in its introduction). Particles are now typically 510 m in diameter and have pores 60100 in size. Most HPLC work involves partitioning with liquid stationary phases chemically bonded to a support surface. Especially commonplace among the ligands bonded to silica gel are hydrocarbon chains of 8 or 18 carbon atoms (C8 and C18, respectively). Columns modified with octadecyl ligands are often referred to as ODS columns. There are ODS1 and ODS columns, the two types differing in the proportion active residual OH groups, which is variable in ODS1 (it contains a specific number of OH groups) and zero in ODS. However, manufacturers tend to give their columns a trade name not specifying the treatment they have received. The problem with silica gel as a chromatographic support is its narrow operating pH range (37.5) and working temperature. However, its properties can be altered by deactivating residual silanol groups using the end cropping method.

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Some polymer-based packing can work throughout the pH scale and at temperatures up to 150 C. This has substantially expanded the scope of reversedphase chromatography (e.g. with the separation of basic compounds). C. Temperature Control Unlike gas chromatography, liquid chromatography seldom requires temperature control. If needed, a concentric column can be used to circulate water from a thermostated bath to adjust the temperature of the analytical column. Detectors All types of available analytical detectors can be integrated with a chromatographic separation process provided they can be furnished with a flowthrough cell. The relative importance of each type of detector is to a great extent consistent with its use in other batch and continuous analytical modes. There are some exceptions such as the refractive index detector, the universal character of which made it one of the most useful until the gradient elution mode gained widespread acceptance. However, few types of detectors for HPLC are commercially available. There are two broad categories of HPLC detectors, namely: (a) General detectors, which respond to mass- or volume-related properties of the eluent (e.g. the refractive index, absorption in the UVVIS region). (b) Selective detectors, which respond to the presence of functional groups or specific structures in the analyte (e.g. fluorescence and IR detectors). The differential refractometer, also known as the universal detector, measures changes in refractive index in the eluent after sweeping some solute from the sample. There are various refractometer models; most, however, use two channels to carry the pure solvent and the column eluent, and measure the differences in refractive index by comparison. Baseline changes in gradient elution work do not allow chromatographic changes to be accurately detected, especially when the refractive indices of the solute and solvent are similar. The differential refractometer is extremely sensitive to temperature changes, so it requires careful thermostating. It provides detection limits over the range 1100 ppm. A typical model is depicted in the Figure II.4.33.

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Lens

Mask

Mirror

Detector Reference cell Optical zero

Figure II.4.33. Two different models of the flow-refractometer detector: Left- Fresnel type, reference and eluent are flowing through two channels carved into a prism (1. Lamp; 2. Prism; 3. 4. Liquid; 5. Teflon mask; 6. Steel plate; 7. Incident light). Right - Deflection type; a cell is divided in two asymmetric parts for sample and reference (pure eluent), respectively. The presence of a solute into the reference part changes the refraction of light beam. The UV detector often uses a single interference filter, so it can only measure concentrations at a few different wavelengths. The more sophisticated models use a monochromator to select the most suitable wavelength in each situation; this enables qualitative identification by stopping the mobile phase flow. The UV detector is normally more sensitive than the refractive index detector; thus, the former can detect concentrations down to 0.01 ppm or even as low as a few nanograms if an appropriate chromophore is used to derivatize the analytes post-column. Diode array detectors constitute powerful tools for qualitative analysis as they allow spectra to be directly recorded without the need to stop the mobile phase flow. The ability to resolve overlapped spectra by using spectral derivatives or an alternative chemometric technique results in further increased separation power. Fluorescence detectors can be more sensitive and slightly more selective than UV detectors. Finally, amperometric detectors are widely used for the detection of electroactive biochemical substances. Operational Characteristics of HPLC Detectors In this section, detector characteristics are illustrated for the UV detector.

Light source 193

Sample cell

The flow-cell is usually U- or, especially, Z-shaped (see Figure II.4.34); in the latter, the flow circulates upstream, which allows small solid particles (impurities) and gas bubbles to be removed. (L from lamp; D to detector). The detection limit or lowest detectable concentration is usually taken to be twice background noise (the baseline). Based on the BeerLambert law, the absorbance of a solution depends on the concentration of the absorbing substance and the length of the optical path. The latter is usually 10 mm, but can range from 1 to 10 mm.

Figure II.4.34. Scheme of different flow-cells Z-shape (left) and U-shape (right). With a path length of 10 mm and a background noise (minimum reading) of 0.0004 absorbance units, the detection limit will be: DL = (2 background noise) mol L1 = 0.0008 mol L1 Therefore, with a molar absorptivity = 104 L mol1 cm1, DL = 0.0008/104 = 8 108 mol L1 = 80 nmol L-1 This is the concentration the detector will be able to detect after the solute spreads during the separation process. With a dilution factor of 20, the lowest detectable concentration referred to the sample inserted into column will be: DL = 80 nmol L1 20 = 1600 nmol L1 which, for a substance of molecular weight 300, will lead to: DL = 1600 nmol L1 300 g mol1 = 480 000 ng L1

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If the volume of the injected sample is 5 L, then the detection limit for the system will be: DL = 480 000 ng L1 5 106 L = 2,40 ng The lower limit of the linear range of this detector coincides with the detection limit and its upper limit with the deviations from the BeerLambert law that bend the calibration graph. Except for diode array detectors, all solutes are measured over the same wavelength range. The wavelength of choice will be that best suiting the solutes as a whole, but may be far from the optimum values (maxima) for some solutes. The 254 nm line from an Hg lamp provides 90% of the total amount of light it irradiates; this line is suitable for most organic compounds. The choice of eluent is dictated to some extent by the type of detector used; thus, the effluent should not absorb at the chosen wavelength. The foregoing refers to the magnitude of the signal ( i.e. the height of the chromatographic peak). Obviously, its width is also important as it dictates how efficiently neighboring peaks can be resolved. In principle, the solute can spread throughout the chromatographic system. Peak height is defined mathematically, in terms of variance, as 2total = 2injector + 2column+ 2detector+ 2connectors Based on this equation, the void volume of both the detector and the columndetector connection should be as small as possible. With this type of detector, slight temperature changes rarely alter the analytical response, even though they can modify the baseline. Figure II.4.35 reproduces the obtained peaks in a chromatographic system when the void volume of detector is changed.

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Figure II.4.35. Obtained peaks comparing flow-cells: left, control; medium, 0.8 L cell (Chromatronix 210FC1X10T), path length 1 cm; right 0.1 mL cell (common micro cell), path length 1 mm. Post-column Derivatization The ideal detector would be the one exhibiting selectivity for various sample components. Such selectivity can also be introduced via post-column derivatization, which can also provide improved sensitivity and detection limits. Off-line derivatization, whether in the pre-column or post-column mode, is more difficult to automate; also, it is more labor-intensive and time consuming, and particularly in the pre-column mode - subject to a higher risk of sample contamination and of a greater number of its components entering the separation column. Post-column derivatization is done between the separation column and the detector, using a mixing system and a reaction chamber with temperature regulation. The following requirements should be met for efficient operation: (a) the derivatizing reagent should be compatible with the mobile phase; (b) the derivatization reaction should be fast; (c) based on kinetic and viscosity grounds, the working temperature should be relatively high; and (d) increased reaction times and reactor lengths ( i.e. high void volumes) should be avoided as they result in increased peak width. Additional requirements to be met when the post-column derivatization reaction involves heterogeneous phases include the following: (e) the constituents of the reactant bed should be compatible with, and stable in, the mobile phase; (f) the mobile phase should reach neither the reactants nor the support bed;

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(g) the solid-phase reactor should be resistant not only to chemical agents, but also to physical wear under the action of a continuous flow, a high pressure and, in some cases, an also high temperature; (h) any solid-phase derivatization reactions should be quantitative; and (i) the reactor response should be reproducible. As noted earlier, post-column derivatization can be done in a homogeneous phase (i.e. with all reactants in solution) or a heterogeneous phase (by use of a solidphase reactor). The Figure II.4.36 depicts the scheme of a reaction detector based on packedbed reactor.

PM
Isooctane ethanol NaI Isopropanol Acetic acid isopropanol

P PM P

Column

Reactor

Figure II.4.36. Scheme of a reaction detector based on packed-bed reactor. P: pump; PM: Pressure monitor; V: injection valve; D: Spectrophotometer Applications Some manufacturers such as SRI instruments produce equipment for fieldwork that affords both isocratic elution and gradient elution, and can be furnished with a UV or conductimetric detector. Exemple 1 The Figure II.4.37 depicts a schematic diagram of the chromatograph designed by G.I. Baram for the fieldwork.

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According to this author, a liquid chromatography system for use in fieldwork should meet the following requirements: (a) it should be small and lightweight for easy carrying; (b) it should use little energy and not be affected by small oscillations in electrical power; (c) it should operate over wide ranges of temperature and moisture, and be vibration resistant; (d) it should have as short as possible a warm-up time; (e) it should be compatible with the solutions typically used in field work; and (f) it should be flexible enough for application to a variety of analytical problems.

Eluent 1

PT V P1 P2
injection

AS

Eluent 2

column

Figure II.4.37. Schematic diagram of the chromatograph for the field work. V: valve; P: pump; PT: Pressure transducer; AS: auto-sampler; D: Detector; W: Waste. On the other hand, the sample and analytical procedure used should meet the following requirements: (a) the molecular mass of the substances should be less than 500 and their number not exceed 15 (15 is the usual number); (b) the concentrations of the sample components should fall within the range 1 1000 mg L1 (greater concentrations require pre-concentration of the sample); (c) the sample volume should be 10 L or greater; (d) analysis times (sample preparation included) should not exceed 1030 min.

198

The equipment of the Figure II.4.37 consists of two gradient piston pumps with an inout switching valve, a pressure transducer and a mixer. The pump is connected to the column via a needle, which, together with a tightening device, constitutes the stopped-flow injector. First pump drives the sample to the needle. The column is accommodated in a solid-type heater. The set-up is completed by an auto-sampler capable of holding 46 vials and a double-beam UV spectrophotometric detector. The equipment dimensions are 530 mm (L) 200 mm (W) 300 mm (H) and its weight less than 15 kg. It uses a power supply of 100120/200240 V, 100 VA, 5060 Hz. Its performance was tested in various determinations of toxic environmental substances including the following: (a) The determination of 11 phenols by use of a Eurospher 80-5 C18, 64 mm 2 mm i.d. column and gradient elution at 50 C. The eluents used were 36:63.9:0.1 methanolwatertrifluoroacetic acid and methanol. The flow-rate was 0.15 mL min1 and the working pressure 4 MPa. The sample volume was 5 L and the detection wavelength 230 nm. The same column was also used, under different conditions, to separate triazine, carbamate, urea pesticides, and chlorinated aromatic acids and esters. (b) The determination of 16 polynuclear aromatic hydrocarbons by use of a Nucleosil 5-C18 PAH 75 mm 2 mm i.d. column and gradient elution at 40 C. The eluents used were 65:35 methanol-water and 85:15 acetonitrile-water. The flow-rate was 0.12 mL min 1, the working pressure 1.5 MPa and the detection wavelength 250 and 260 nm. The volume of sample (a methanol solution of an hexane extract of snow) was only 2 L. The same type of column, under different conditions, was used to determine 6 phthalate esters. (c) The determination of 8 polynitro explosives with a Eurospher 80-5 C18, 64 mm 2 mm i.d. column. Elution was done at 45 C in the isocratic mode, using a 50:40:10 mixture of methanol, water and 0.1 M tetrabutylammonium phosphate at pH 6.8 as eluent. The flow-rate was 0.28 mL min 1 and the working pressure 5 MPa. The sample volume required was only 3 mL and the detection wavelength 230 nm. Example 2 A commercially available Milikhron-1 portable HPLC system, from Nauchpribor (Oriol, Russian Federation), was used in this determination. The equipment consists of a syringe pump loading up to 2600 L and providing flow-rates of 2600 L min1, working pressures up to 5 MPa and injected volumes of 520 L. It uses a UV detector that operates over the range 190360 nm; it is a single-beam detector furnished with a mirror that allows the light beam to be passed through two cells, one holding the sample and the other a reference solution. The system was used to determine free fatty acids in natural water samples. The presence of these compounds influences phytoplankton growth. The water surface layer (viz. the topmost layer, which is roughly 50100 m thick) provides the

199

most valuable information for this purpose. The method was applied to oil from sea buckthorn berries (Hippophae rhamnoides) and the results were compared with the fatty acid contents in laboratory culture media of Spirulina platensis algae, the two being quite consistent in terms of both composition and concentration. The determination required the derivatization of free fatty acids and their separation on a Separon C18 column (64 mm 2 mm i.d. column, 5 m particle size). Elution was done in the gradient mode, using acetonitrilewater and ethanol water mixtures. The time required to separate the 14 fatty acids, reconditioning included, was 45 min. REFERENCES 1. Gary D. Christian, Analytical chemistry (6th edition), Wiley 2004, ISBN 0-47145162-2 N.Y. 2. Harvey D., Modern Analytical Chemistry, Mc Graw Hill Comp. Inc., 200, ISBN 007-237547-7. 3. Kenneth A. Rubinson and Judith F. Rubinson, Contemporary Instrumental Analysis, Prentice Hall Inc. 2000, ISBN 0-13-790726-5. 4. L. Cloths and K. M. McErlane, J. Pharm. Biomed. Sc., 2003 (31), 407- 412 5. D. Projean, T. M. Tu and J. Ducharme, J. Chrom. B, 2003 (787) 243-253. 6. D. Wittintgton, E. D. Kharash, J. Chrom. B, 2003 (786) 95 - 103. 7. T. C. R. Santos, J. C. Rocha and Dami Barcel, J. Chrom. A, 2000 (879) 3 - 12. 8. V. Kmetec and R. Roskar, J. Pharm. Biomed. Sc. 2003 (32), 1061- 1066. 9. P. Ptacek, J. Macek and J. Klima, J. Chrom. B, 2003 (789) 405 - 410. 10. J. Calvin Giddings, Anal. Chem. 36 (1964) 1980

II.4.7. MASS SPECTROMETRY

Andrei Valentin MEDVEDOVICI II.4.7.1. Definition Mass spectrometry is the branch of science dealing with qualitative and quantitative interpretation of ions produced under controlled conditions by a sample submitted to analysis, by means of a mass spectrometer . II.4.7.2. Principles Mass spectrometry deals with both quantitative and structural information.

200

Basically, the sample is stroked to generate fragments. Against the fragments generated upon impact, only positive (+) or negative (-) ions are further considered. Selected ions are then characterized according to their mass / charge ratio (m/z). Quantitative information is produced by counting the total number of ions (positive or negative) that are produced or by counting the number of ions having a precise m/z being produced during ionization stage. Structural information derives from a hypothetical reformation of the integer from the resulting ions of known masses. Mass spectrometry is thus achieved by means of the following consecutive operations: (1) sample introduction; (2) sample ionization; (3) ion analysis / processing (mainly separation according to their m/z); (4) ion counting / detection; (5) records of the results and interpretation. The basic schema of a mass spectrometer is given in Figure II.4.38. Some applications require that stage (3) be expanded in order to improve the information amount to be taken from the ionization process or to increase detection selectivity. The sequence between brackets in Figure II.4.38 illustrates this alternative. Hence mass spectrometry deals with ions produced by the sample under controlled conditions and these ions are positively or negatively charged, it clearly results that mass spectrometry should be addressed as (+) MS or (-) MS, according to the charge sign of the ions being analyzed. In modern mass spectrometers, analysis of positive ions can be alternatively switched to analysis of negative ions, but in no cases, simultaneous (+) and (-) ion analysis is affordable. It is also worthwhile to note that ion formation, separation and characterization require an environment free of interferences, meaning that a mass spectrometer is working in deep vacuum conditions (according to the constructive characteristics of the mass analyzer, vacuum levels ranges between 10 -5 and 10-12 torr). Principles and practice of mass spectrometry are detailed in general textbooks such as references 1 - 12. II.4.7.3. The Mass Spectrum The mass spectrum is the plot of the individual ion abundance as function of their respective m/z. Ions are produced during the ionization stage of a sample representing a pure compound or a mixture. The ion mass can be expressed in the following manners: (1) the ion average mass representing the sum of the mean masses of the forming atoms; the mean atomic mass is calculated as a balanced average of exact masses of the existing isotopes and their natural occurrence; (2) the ion mono-isotopic mass represents the sum of the exact masses corresponding to the most abundant natural isotope existing for each of the forming atoms; (3) the ion nominal mass represents the sum of the integer masses of the most abundant natural isotope corresponding to each of the forming atoms. Ion masses are expressed in Daltons (Da). z represents the total number of charges existing on the ion, expressed in units of elementary charge (e).

201

Computer Hardware control Data acquisition, computing & interpretation Stage (5)

Sample inlet Stage (1)

Ion source Stage (2)

Mass analyzer Stage (3)

Detector Stage (4)

Ion isolation (precursor ion)

Ion isolation (collisionally induced)

Product ions analysis n

Vacuum System

Figure II.4.38. Basic schema of a mass spectrometer. In Figure II.4.39 a detail from the atrazine mass spectrum is presented (m/z interval ranging from 197 to 220 Da). The profile view assumes a definite resolution of the mass analyzer, while the line view gives the intensities only for integer values of m/z. The ion with the highest abundance in the mass spectrum is referred as the major ion. Normalization of the abundances of the other ions with respect to the major one leads to the relative abundance measurements (R.A.%) placed on the Oy axis. The molecular ion is the ion produced by the molecules of the sample by means of the removal or addition of one/more electrons.
100 90

ATRAZINE
Molecular formula: C8H14ClN5 Average Mw = 215.69 Monoisotopic Mw = 215.09377 Nominal Mw = 215

100 90 80 70 60 50

Ion Relative Abundance ( R.A. %)

80 70 60 50 40 30 20 10 0

Line Spectra

40 30 20 10 0

Profile Spectra

211 212

214 215

216

198 199

200 201

207 208

209 210

202

204 205

206

217 218

213

203

219 220

215 216

197

201

202

204

209

210 211 212

217 218

214

219

198

199 200

203

205 206

207 208

Mass to charge ratio (m/z)

Figure II.4.39. Detail from the atrazine mass spectrum.

213

202

220

II.4.7.4. Sample Introduction Samples can be brought in the ionization area either in a gas phase as well as in a condensed phase (liquid or solid). Samples subjected to MS analysis have either organic or inorganic nature. More often, organic samples are submitted to MS analysis in order to obtain structural information or confirmation, generally from extremely low amounts. If such structural information is required, simultaneous ionization of different molecular species should be avoided. This means that multi component samples should be first separated in individual constituents and then subjected to MS analysis. Nowadays, all separation techniques (thin layer chromatography, gas chromatography, liquid chromatography, supercritical fluid chromatography, capillary electro chromatography, micellar electrokinetic chromatography micelle staking technique, capillary zone electrophoresis) have been successfully coupled to mass spectrometers, directly or by means of especially designed interfaces. For inorganic samples, information deals with identification and assay of atoms and related isotopic occurrence. In such cases, no special features are imposed for sample introduction, but sample ionization methods are more complex (ion bombardment, inductively coupled plasma). II.4.7.5. Ionization Modes in Mass Spectrometry Gas Phase Ionization A. Electron impact (EI) Achieving electron impact ionization means that focused and accelerated electrons collide the molecules of the analyte introduced in the ionization area in a gaseous state. Ionization arises according to the following patterns: (+) mode

M +e

+2e

(-) mode

M M (1) + e p
.* .*

- - *

(5) (6) (7) (8)

+ * M +e M M + e + 2 es (2) p p M

M +.* Ai+ + B j M +.* Ci+ + D j

M (3) M (4)

Ai +Bj

Ci + D j

M = target molecule; A, B, C, D = molecular fragments; p = primary; s = . secondary: * = activated energy state; = impair electron The basic condition for ionization is that the primary electron posses an energy at least equal to the ionization potential of the target analyte ( Ee-p (I.P.)M). An excess of energy is required to generate molecular fragmentation (reactions 3, 4, 7, 8).

203

As the ionization potential of organic molecules falls in the 8 12 eV range, it results that energies of the primary electron higher than 15 eV should provide proper ionization and fragmentation. The choice of a standardized value for Ee-p = 70eV is justified as long as lower energy values lead to a significant variance of the ionization yield. Within a mass spectrometer, three types of ionic species exist: the molecular ions, the fragment ions produced in the ionization area and metastable ions (produced by decomposition of molecular ions after leaving the ion source). The residence of the ions within the source ranges in the 10 -6 5 x 10-6 seconds interval, while the time taken for ions to run through the mass analyzer region is higher (about 10-5 seconds), illustrates the relation between the lifetime of the molecular ion and the resulting mass spectrum (Figure II.4.40). It is obvious that consistent structural information is obtained if the molecular ion and fragment ions are present with significant abundances within the mass spectrum. For chemical species generating molecular ions with extremely short lifetimes, finding a way of reducing ionization energy is mandatory if their observation is required. The reduction of the primary electron energy is not recommended on that purpose as long as ion formation yield and ionization reproducibility are strongly affected.
Very high Fragmentation fragmentation. is important. Loss of the However, signal of the observation of molecular ion the molecular in the mass ion is possible. spectrum. Less Practically no fragmentation. fragmentation. Occurrence of Only the metastable ions molecular ion is probable. is observable Intense signal in the mass of the spectrum. molecular ion in the mass spectrum.

0,1

10 Molecular ion lifetime (s)

100

1000

Figure II.4.40. Correlation between the molecular ion lifetime and characteristics of the mass spectrum. The major advantages of Electron Impact ionization mode are its stability, ease of operation and control of the beam intensity, lack of contamination problems, relative high sensitivity, and generation of library searchable spectra. B. Chemical Ionization (CI)

204

In CI, ionization of the target molecule is made by interactions with ions produced in the source by means of the electron impact on reagent gas molecules. Existence of reagent gas molecules in large excess with respect to the target molecules (10000:1) imposes an increase of the energy of primary electrons (500 eV). CI is known as a soft ionization technique because energy transfer between reagent gas ions and sample molecules do not exceed 5 eV. Ionization products in CI exhibit an enhanced stability due to their even electron state (compared to impair electron state for ions generated through EI). Interactions in CI are summarized below:

(+) mode
+ RH + e + 2 es p RH

(-) mode (9)

. +

RH

+ RH [RH 2 ]

+ R

(10)

H2O + e-p OH- + H

(15)

[RH 2 ] + + M [ MH] + + RH (11) proton transfer [RH 2 ] + + M [ MRH2] + RH + + M M + + RH [RH 2 ]

+

M + OH- H2O + [M H]+ (16)

-

M + OH [MOH] (12) electrophilic addition

(17)

(13) charge exchange

+ MH M

RH = reagent gas; RH2+ = reagent ion; M or MH = target molecule; p = primary; s = secondary: = impair electron
The following reagents gases are commonly used in CI (reagent ions are placed between brackets): H2 (H3+); CH4 (CH5+); C2H6 (C2H7+); H2O (H3O+); CH3OH (CH3OH2+); CH3CN (CH3CNH+); NH3 (NH4+); CH3NH2 (CH3NH3+); H2NCH2CH2NH2 (H2NCH2CH2NH3+). For negative CI, the following reagent gases are commonly used (reagent ions are placed between brackets): NH 3 (NH2-); N2O mixed with CH4; CH4 + He; H2 + He (OH-); N2O or mixed with N2 (O-.); NF3 (F-); CHF3 (F-); O2 (O2-.); CH2Br2 (Br-); CH2Cl2 (Cl-); CHCl3 (Cl-); CF2Cl2 (Cl-). Specific interactions in the ion source are exemplified below on using methane as a reagent gas: CH4 + e-p CH4+ + 2e-s CH5+ + M [MH]+ + CH4

+ RH 3 (14) anion abstraction

205

CH4+ CH3+ + H CH4+ CH2+ + H2 CH4+ + CH4 C2H3+ + H2 + H CH2+ + CH4 CH5+ + CH3 CH3+ + CH4 C2H5+ + H2 C2H3+ + CH4 C3H5+ + H2

C2H5+ + M [MH]+ + C2H4 C3H5+ + M [MH]+ + C3H4 CH5+ + M [MCH5]+ C2H5+ + M [MC2H5]+ C3H5+ + M [MC3H5]+ C2H5+ + M [M - H]++ C2H6

The advantages of CI mode are: soft ionization, multiple checks of the molecular weight possible due to a large variety of ionization processes, universal or selective action depending on the choice of the reagent gas, easy formation of negative ions, allowing structural and thermochemical measurements. Both EI and CI ionization modes are realized in ion sources constructively similar to the schema depicted in Figure II.4.41.
Reagent gas (CI) Magnet Renium Filament Valve Pressure gauge

Accelerating and focusing plate Sample inlet (vapours)

Repeller Electrode

Ionization area

To Mass Analyzer

Vacuum Anode Ion Source Magnet

Primary electrons (70 eV) Screen system for acceleration and focusing product ions to mass analyzer

Figure II.4.41. Basic construction of an ion source designed for EI or CI ionization modes.

206

Thermal electrons produced by the Renium filament are focused and accelerated by means of the plate, the anode, and/or the magnet poles. The electron beam collides with the molecules of the sample (or the molecules of the reagent gas) in the ionization area. Reagent gas line feeding the system includes also a control valve and a pressure gauge for controlling the reagent ions formed within the source. The repeller electrode is used for the elimination of unwanted product ions (the electrode is positively charged for elimination of negative product ions and negatively charged for elimination of positive product ions). The screen system serves to focus and acceleration of product ions towards the mass analyzer (screens are charged to increased voltages of contrary sign with respect of the product ions to be analyzed). The whole ion source is adequately vacuumated by means of turbomolecular or diffusion pumps. C. Field Ionization (FI) In FI mode, molecules of the analyte in gaseous state are brought close to a surface with a high curvature shape (generally tips, whiskers or blades) subjected to intense electric fields (107 108 V x cm-1). These molecules are readily ionized by means of quantum tunneling of valence electrons from the molecule to the metal surface. The techniques should be considered as soft ionization ones, having as a major drawback the lack in sensitivity. Ionization from Condensed Phases A. Liquid Phases Fast Atom Bombardment (FAB). Target molecules are dissolved in viscous, relatively nonvolatile liquid matrix on a porous surface and are subjected to a bombardment of atoms or ions having keV translation energies. The use of Xe atoms or Cs ions generates similar spectra. In the region above the surface of the frit, target molecules are interacting in a similar way as in CI, [M+H] + and [M-H]- ions occur. The most common viscous liquid is glycerol. The method is suitable for polar molecules with Mw 20,000 Da. If high-energy ions are used instead of atoms, the ionization technique is also known as LSIMS (liquid secondary ions mass spectrometry). Atmospheric Pressure Chemical Ionization (APCI). A liquid flow containing the target molecules is pumped through a heated vaporizer. The jet of vapors containing also liquid droplets (continuously evaporating under heat) is oriented toward a discharge zone (typically of corona form, realized between a tip and a disk shaped counter electrode maintained at 1 4kV potential). The electrical discharge ionizes solvent molecules existing in the gas state. A combination of collisions and charge transfer reactions between solvent ions and target molecules leads to their ionization. The generation of protonated or deprotonated molecular ions ([M+H] + or

207

[M-H]-) is thus possible. The ions are extracted toward the mass analyzer via a capillary tube. The set-up is presented in Figure II.4.42.
corona discharge + + Liquid flow N2 flow

+
Heated sheath (up to 600 oC)

Gas phase

Counter electrode

Needle electrode

Extracting capillary

To mass analyzer

Heated N2 curtain Corona discharge Vacuum

Figure II. 4.42. The APCI source (orthogonal design). APCI is used for a wide range polarity of target molecules and is relatively tolerant to nonvolatile salts or buffers existing in the liquid effluent. It is however especially used for nonpolar compounds. The ionization pattern is not greatly influenced by the solution chemistry of analytes in the carrying phase. It is unsuitable for thermally labile compounds and rarely generates multiply charged ions. The forerunner of APCI was thermo spray (TSP). The technique was never particularly routine and has been superseded by APCI. Electrospray ionization (ESI or AP-ESI). A liquid carrier containing target molecules is mixed with a nitrogen flow and forced through a stainless steel capillary maintained at 3-4 kV. A plume of charged liquid droplets is formed (Taylor cone). An oriented counter current heated nitrogen curtain continuously determines the reduction of the volume of droplets with a corresponding increase of the electric field density on the surface until a critical volume is reached (Reyleigh limit). Resulting ions are extracted from droplets and sampled through a capillary tube to the mass analyzer. Figure II.4.43 is illustrating the operating principle of ESI. ESI is the softest ionization technique available. It is especially suited for polar compounds, even with a non-volatile character. Ions are ejected from the charged liquid droplets as [M+H]+ or [M-H]- ions. Ionization yield is highly influenced by the solution chemistry of the analyte in the carrier flow. ESI is intolerant to nonvolatile salts or buffers. It easily generates multiply charged ions, allowing determination of compounds characterized by a high molecular weight.

208

Liquid flow N2 flow

4 kV

Counter electrode

Charged droplets dispersion (Taylor cone)

Heated N2 curtain Extracting capillary to mass analyzer

Original droplet (+ ions predominate)

Vacuum Reyleigh limit

+ -+ + ++ -++ + + + -+ -

Volume reduction

-+ -+ +++ + + + -+-+ + -- -

-++ + -+ +

+ + + + + Explosion

Solvention cluster

+
Ion extraction

Figure II.4.43. Schematic set-up of an AP-ESI ion source. Electrospray ionization is readily subjected to miniaturization. Nanospray and Electrospray Emitter Arrays are increasing sample throughput, realizing at on-chip scale, sample preparation, concentration, and separation. Dual APCI / AP-ESI ionization modes have been also experienced, leading to an extended range for detecting compounds and analysis throughput. Multiphoton ionization (MPI). The target molecules contained in a liquid carrier are nebulized by mixing with a nitrogen flow. Solvent and analyte are then vaporized together in a quartz tube and are brought in the irradiation zone as a homogenous gaseous phase. Direct photo ionization of the target molecules is statistically unfavorable, resulting in lower ionization yields. Introduction in the nitrogen flow through nebulization of a dopant can indirectly enhance on ionization. The dopant is first ionized and the resulting ions are reacting with the analyte, by means of proton or electron transfer interactions. The use of pulsed tunable dye lasers are strongly enhancing on ionization yields. Two stages should be considered: 1. n photons coherently excite sample molecules to a real, intermediate electronic state (a resonance enhanced process); 2. sequential irradiation of this intermediate with m photons leading to final ionization. By increasing the power density of the laser source it is possible to change from soft molecular ion spectrum to ones dominated by fragmentation. C. Solid Phases Matrix Assisted Laser Desorption Ionization (MALDI). The sample initially

209

mixed with a liquid matrix (eg. sinapinic acid), chosen for its ability to absorb and dissipate energy transferred from a laser beam. The molar ratio analyte / matrix is an important operational parameter, readily varied by the analyst. Once prepared, the mixture is transferred to a sample probe. Probe surface and geometry should also be considered as important for the results of the analysis. After application, the sample and the matrix are converted to a condensed (solid) phase by gas evaporation or vacuum drying, resulting in the co-crystallization of the sample with the matrix. Local cluster and size variations occur across the sample deposition site. New instruments incorporate a microscopic movement sample handler, allowing selection of the promising crystals for excitation with the laser beam. The selection of sweet spots is sometimes assisted by a CCD (charge coupled device) camera. The pulsed laser fascicules generate a plume of ejected particles, including ions and neutral, belonging to matrix and analyte, expanding at supersonic speed from the surface of the probe, generally under right angles. UV lasers are usual (N 2 laser at 337 nm or Nd-YAG). IR lasers (CO 2 or Er-YAG) are used only for specialized applications. A laser pulse takes from 1 to 10 ns and impacts an area of about 0.01 mm2. The laser pulse is repeated over a precise interval (commonly 1 Hz frequency). This parameter should be chosen in accordance with the type of mass analyzer that is used. Time of Flight (TOF) and Fourier Transform Ion Cyclotron Resonance (FTICR) mass analyzers are more often coupled to the MALDI source. MALDI is a soft ionization, tending to produce singly charged ions (resulting in very simple spectra). Both (+) and (-) modes are known, although (+) MALDI is the most common. The (-) mode is frequently used for nucleic acid analysis. In the default operational mode, MALDI induces no fragmentation. Biochemical structural studies using MALDI will involve breakdown of the precursor molecule prior to ionization, enzymically or chemically. Applications of MALDI are covering identification of additives in food profiling and characterization of environmental macromolecules (correlation of the analytical data with structure and environmental impact). The MALDI source is presented in Figure II.4.44. Fission Fragment Ionization or Plasma Desorption Ionization (PDI). Sample molecules are deposited on a Nickel foil. Pulses of 252Cf fission fragments pass through the probe. The thermal shock vaporizes mobile impurity ions (H +, Na+, and H-). These ions interact with nonvolatile sample molecules just after the thermal shock, converting them to ions. PDI can be applied for large biomolecules (up to 50,000 Da). Deposition on nitrocellulose yields simple and multiply charged ions as well, with practically no fragmentation. PDI is nowadays superseded by MALDI. Field Deposition (FD). Similar to FI (see C.), requires deposition of sample molecules in a condensed phase on the high curvature surface subjected to intense electric fields.

210

Laser source Sample Matrix

Probe Desolvation Desorption Proton transfer x Probe handler Matrix / Sample co crystallization mass CCD camera H+ + To mass analyzers

Figure II.4.44. The MALDI source. Secondary Ion Mass Spectrometry (SIMS). A beam of ions (either monoatomic or polyatomic, positive or negative, obtained from inert gases or from electropositive / electronegative elements) collides the sample deposited on a metal surface. Secondary ions ejected from the surface are trapped and analyzed. The technique is suitable also for inorganic samples as well as for organic molecules. In the latter case, protonated molecular ions and sodium adducts are readily formed. Surface imaging (static SIMS) as well as 3D mapping (Dynamic SIMS) is possible. The choice of the primary ions will determine the nature of secondary ones, which are transferred to the mass analyzer. Kinetic and chemisorption theories are explaining ejection of the different types of secondary ions. Inductively Coupled Plasma (ICP). Liquid or solid samples of organic or inorganic nature are dispersed in an Argon flow and introduced in an inductively coupled plasma torch. Temperatures around 8000 oK are atomizing sample and ionizing the resulting atoms. Ionization efficiency approaches 100 % for most elements of interest. Ions are sampled through a skimmer directly to the mass analyzer after formation of a supersonic jet in the differentially pumped region behind the plasma extraction cone. II.4.7.6. Mass Analyzers The mass analyzer is the component of mass spectrometer acting on the specific ions generated within the source, to ordinate them according to the m/z values. The basic functioning principle of a mass analyzer is the interaction of the target ions with external electrostatic and magnetic fields of precise geometry.

211

Sector Mass Analyzers Two types of sectors are used for construction of the mass analyzers: magnetic (B) and electrostatic (E) sectors. The electrostatic sector performs only an isokinetic arrangement of ions. When the ions are introduced in a radial electric field, their pathways became stable only if the centrifugal force equalizes the electric force: m v2 z e E = (1) rE d where E is the potential applied between the curved electrodes, d is the distance between curved electrodes, rE is the radius of the path followed by the ion in the electric field, v is the velocity of the ion, m its mass, z its charge and e is the elementary unit of charge. An ion introduced in a magnetic sector moves on a circular path with the radius rB, for which, again, the centrifugal force should equalize the magnetic force.
m v2 = z e B v rB

(2)

It is worthwhile to note that the magnetic field acts as a momentum ( m x v) separator. As the velocity of the ion is the same in both the electrostatic and the magnetic sectors, the relations (1) and (2) should be combined as follows:

v=

z e E rE md

(3) din relaia (1)

z e B rB = m
It results that:

z e E rE md

(4) introducnd rel. (3) n rel. (2)

m e d 2 = B 2 rB z E rE

(5)

The tandem between electrostatic and magnetic sectors acts as a mass analyzer either by scanning the magnetic field B or the electrostatic field E. From relation (5) it seems that m/z is independent with respect to the initial acceleration potential V, given to the ion during the transfer from the source to the mass analyzer. However, if the velocity during the transfer is equal to the velocity in the electrostatic sector, an instrument defined correlation is found between V and E:

212

E rE 2 d

(6)

The former relationships are illustrated in Figure II.4.45 .

S +
E d

B + rB

rE V

Figure II.4.45. Pathways of a positive ion in electrostatic and magnetic sectors. Characteristics: resolution of 10,000 over a mass range up to 15,000 Da; increased resolution of 100,000 over a mass range up to 100,000 Da can be obtained with a cost in terms of sensitivity; scan speeds are limited by the hysteresis and magnet heating; complex construction; needs deep vacuum conditions (10 -10 10-12 torr). The double focusing mass analyzer is used for high resolution measurements (e.g. the EPA method for dioxins imposes such a requirement) and fundamental MS studies. Time of Flight Mass Analyzers (ToF) ToF mass analyzers are based on the measurement of the individual flight times of ions having identical kinetic energy through a specific path (field free drift tube) separating the ion source and the detection area (see Figure II.4.46.A). The kinetic energy of the ions produced within the source is established by means of the acceleration potential V.
m v2 = z e V 2

(7)

The flight time is thus calculated according to the simple relationship:

213

t=

L = v

L m1 / 2 2 z e V

(8)

It is clearly resulting that:

m 2 e V = t2 2 z L

(9)

For given settings of V and L, the time measurement means m/z discrimination. The specific ToF resolution is enhanced by using the reflector design (see Figure II.4.46.B), allowing a better control over the initial kinetic energy spread, and the spatial distribution. The ion introduction in the flight zone should be pulsed, through generation of discrete packets of ions. The flight time of the ions typically falls in the 1 to 100 ns interval. Characteristics: no instrumental parameters limit the upper margin of the m/z interval; high resolution (up to 20,000) when using the reflector design; high sensitivity, especially at high ion sampling frequencies; needs for high performance electronic controlling detection area; suitable for applications requiring both high resolution and sensitivity for high Mw compounds; The Hadamard multiplexing technique is usable together with the ToF principle.

214

L Field free drift tube

Ion Source

Detection Area

Acceleration plate V Field free drift tube Deflection Electrode +

Ion Source

+
+ +

Acceleration plate Detection Area +

L +

Figure II.4.46. Basic functioning of a ToF mass analyzer. The Quadrupole Mass Analyzers (QMA) The functioning of the QMA is based on the interaction between ions and a hyperbolic field generated within 4 rod shaped electrodes arranged in a square array, interconnected two by two at positive and negative potentials consisting in both constant and radio frequency modulated components (see Figure II.4.47). The potential applied to the pairs of electrodes is:

0 = ( - or +) (VDC + VRF cos(2vt))

(10)

The hyperbolic field generated within the electrodes should be written as following:

= (V DC + VRF cos(2vt ))

x2 y2 r02

(11)

If VRF > VDC, low mass ions are lost on the Ox direction (collision with left / right electrodes due to the fluctuation of the RF potential) and heavy ions are lost on the Oy direction (collision with upper / lower electrodes due to their inertia toward the

215

fast altering variable field). This combination of both high and low pass filters yields to a stability window defined by , VDC, VRF and VDC/VRF ratio.
y z

+r
o

+ -

+
+

+
x y

o +

-o

+o

Figure II.4.47. The set-up of a quadrupole mass analyzer. The mass interval for ions having motions on the Ox and Oy directions smaller than ro (the ions stay within the rods) depends on the V DC/VRF ratio. Generally, the VDC/VRF ratio is chosen such that 1 Da window is selected over the entire mass range. To functioning modes are possible: a. is variable, VDC, VRF and VDC/VRF are constant; b. and VDC/VRF are constant, VDC and VRF are variable. Characteristics: upper mass limit falls in the 3,000 - 4,000 Da interval; accepts relatively high pressure regime (10-4 - 10-5 torr), resulting in simple vacuum generating systems; low costs; low resolution; wide applications when coupled to GC and LC separation systems; suitable for structural confirmation of low M w molecules; needs serial coupling of three devices for allowing MS/MS experiments. Ion Trap Mass Analyzers or Quistors (QIT) The fundamental working principles of an ion trap are the same as those described for the linear quadrupole for the single reason that a quistor results from the imaginary bending of a linear quadrupole to a closed loop. That imaginary operation transforms the left / right rod electrodes in hyperbolic calottes, the upper electrode in a ring while the lower one is reduced to a mathematical point (see Figure II.4.48). The hyperbolic surface calottes (named also end-caps) are perforated to allow ion extraction from the source and ion ejection to detection area. Constant potential is applied to end-caps. Ions can be readily formed in the trap or captured from an external source as well.

216

Ring electrode VRF x cos(t) Trapping ions VRF low Ion ejection VRF high

+ Ion Source + Detection Area

r0

Lissajou shaped ion orbits

End-cap electrode VDC

End-cap electrode VDC Damping gas atoms (He)

Figure II.4.48. The quadrupole ion trap (quistor). A radio frequency modulated potential (VRF x cos(t)) is applied to the inner ring electrode. Within this three-dimensional arrangement ions travel on Lissajou shaped paths. For low VRF amplitude, all ions covering a large mass interval are stored together within the ring electrode, on stable orbits, characterized by precise radii and z expansions. Forcing an ion population on stable orbits within the ring produces the risk of an uncontrolled increase of the paths radii, due to electrostatic repulsions (collisions of ions with electrodes become possible). To avoid this, a damping gas (usually He or H2) is feed at 10-3 torr pressure in the trap. Collisions between ions and damping gas atoms reduce ion energies and force them to move closely around the center of the trap. This is also improving the resolution, because of the limitation of the spatial distribution (the field imperfection is minimal in the center of the trap). When all ions are stabilized within the ring, the amplitude V RF of the radio frequency modulated potential starts to be scanned. Increasing the V RF value forces the ions to be ejected from the trap to the detection area (through the holes of the end cap electrode), starting with low masses. An ion trap holds a fixed maximum number of ions and depending on the VRF scanning ramp, it can be filled and emptied sequentially. The filling / emptying frequency determines the sensitivity of the instrument.

217

Characteristics: energy and spatial distributions of ions produced within the source are non critical for the quistor; the use of low potentials allows a relatively high pressure and therefore, an inexpensive vacuum system is required; low costs; small dimensions; low resolution (typically 1 Da); improved resolution is obtained for specific scanning profiles of VRF, with the proportional reduction of the mass interval or sensitivity; inherent time controlled tandem MS capabilities; suitable for structural confirmation; suitable for easy GC and LC interfacing. Ion Cyclotron Resonance Mass Analyzer (ICR). The construction of an ion cyclotron resonance mass analyzer is presented in Figure II.4.49.
Receiver plate (up) Trapping plate (front) Emitter plate (left) + + VDC B Trapping plate (back) Amplifier + ++
+ +

-V

+ + Ion beam (from source)

+ VDC

Counter plate (right) Receiver plate (bottom)

B + + + +

B +

+ +

Figure II.4.49. The Ion Cyclotron Resonance mass analyzer. A strong magnetic field is used for curving the paths of the ions inside the ICR cavity. The intensity of the magnetic field (B) is so high that ions will finally move on circular orbits within the cavity. A small potential is applied to the trapping (front and back) plates (same sign as the ions) in order to prevent lateral ionic loss. As for the magnetic sector, the following relation remains valid:

218

m v = z e B r The angular velocity of the ion () is given by the relation:

(12)

v = 2 v r

(13)

Transferring relation (16) in relation (15), it clearly results that: m eB = z (14)

For an applied magnetic field of 5 T (Tesla) and a mass interval ranging from 15 to 1,500 Da, radio frequencies of kHz to MHz result for the trapped ions. When a radio frequency i is applied to the emitter plate, ions having the angular velocity i = 2 x x i start to absorb the incoming energy (resonance phenomenon) and continuously increase their orbit radius (from circular, the paths became spiral shaped). Once the trajectory of the resonant packets of ions comes closer to the receiver plates, an image current is induced and consequently detected (if the ions approach the top plate, electrons are attracted to this plate from the ground; when the packets of ions are situated in the proximity of the bottom plate, electrons travel from the upper plate to the opposite one, producing a detectable current). Scanning frequencies i over a given interval results in the observation of the signals for the corresponding m/z ions. A faster method (FT-ICR) involves the excitation of ions by means of a fast sweep of frequencies over a broad range. The receiver plates pick-up signals at different moments of time. The signal at time t i corresponds to all ions reaching the resonant state by absorption of energy from the frequency sweep applied at this specific moment. A Fourier transformation should be applied to the image current variation in time, as the mathematical function is periodic. Functions resulting for each individual cyclotron frequency are then converted to m/z values to produce the conventional mass spectrum. Characteristics: unsurpassed resolution (around 50,000 for an m/z 10,000); high detection efficiency (1 molecule detection); the detection process is not destructive; m/z scale higher than 100 Da (usually higher than 15,000); inherent tandem MS capabilities; high costs due to the cooling systems of the superconducting magnets, high vacuum systems and demanding computer facilities; difficult to couple to atmospheric pressure ion sources; suitable for extremely high resolution measurements, fundamental ion chemistry and stability.

219

II.4.7.7. Ion Detection Systems Ion current intensities resulting from the mass analyzer are ranging in an extremely wide interval (10-9 10-18 A). Ion detection is achieved mainly by means of electron (photon) multipliers. The primary ions hit an electrode to generate secondary electrons. Secondary electrons are thus multiplied through successive acceleration / impact processes between electrodes (dynodes made from high emissive Beryllium Copper alloys) having an increased positive potential (12 to 20 dynodes are usually coupled). A different approach is the continuous channel multiplier or channeltron. The body of the channeltron is built up from a Lead doped glass with high secondary emissive properties and electrical resistivity. A voltage applied between the ends of the conical shaped curved cavity generates a field gradient on the inner walls. If the speed of the incoming ions is less than 1.8 x 10 4 m/s, lower detection sensitivity may be obtained due to a poorer extraction capacity of the secondary electrons. A solution to this problem is to increase the velocity of the ions ejected from the mass analyzer just before detection. In such a post acceleration set-up (PAD) an electrode operated at high potential (up to 30 kV) is placed before the electron multiplier and strongly increases electrostatically the velocity of the incident ions, with a corresponding gain in terms of secondary electron yield. Because photon multipliers are much more robust compared to electron multipliers, some manufacturers prefer to introduce a phosphorescent screen between the post acceleration electrode and the multiplier itself. II.4.7.8. Multiple Sequential MS (Tandem MS) As described already in figure II.4.38, for some specific purposes, it may result a real need in isolation of a specific ion (precursor or parent) and its further fragmentation followed by the analysis of the resulting ionic fragments (product or daughter ions). Theoretically, this loop may be repeated few times consecutively, resulting in (MS)n experiments. The main objective of MS/MS hyphenation is to enhance the selectivity characterizing methods focused on quantitative aims or structural / stability studies for the target analytes. According to the IUPAC Compendium of Chemical Terminology, tandem mass spectrometry represents an arrangement in which ions are subjected to two or more sequential stages of analysis (which may be separated spatially or temporally) according to the quotient mass/charge. MS hyphenation in space requires the use of two ore more mass analyzers coupled serially (one for each ion discrimination process). MS hyphenation in time means the use of a single mass analyzer, while the selection of precursor / product ions

220

is made successively, at different moments with respect to the beginning of the process. Once the precursor ion has been isolated, it is necessary to initiate its dissociation to produce the corresponding fragment ions (products). The following modes for achieving dissociation of precursor ions are used in practice: 1. Collision Induced Dissociation (CID); 2. Surface Induced Dissociation (SID); 3. Infrared Multiphoton Dissociation (IRMPD); 4. Blackbody Infrared Dissociation (BIRD); 5. Sustained Off-Resonance Irradiation (SORI); 6. Electron Capture Dissociation (ECD); 7. Post Source Decay (PSD). Modes 1-2 have wide spread applications, modes 3-6 are characteristic for trapping mass analyzers while the last mode is dedicated to MALDI sources. The most common mode of dissociation of precursor ions is undoubtedly CID. The process requires Helium atoms to collide precursor ions resulting in their further fragmentation. As the energy control upon neutral species (He atoms) is difficult, reproducible CID processes are obtained by means of the controlled acceleration of the precursor ions. As example for a tandem MS instrumentation (spatially developed) the triple quadrupole (QQQ) set-up is further discussed (see Figure II.4.50). The second quadrupole is used only for acceleration of the precursor (parent) ion to achieve CID. Other arrangements designed for spatial tandem MS experiments are: 1. Multiple magnetic sectors; 2. Quadrupole / Time of Flight (Q/TOF); 3. Time of Flight / Time of Flight (TOF/TOF). The typical illustration for in time MS hyphenation is the ion trap (quistor). As ions obtained from the source are all trapped within the ring electrode, ejection of all ions excepting the parent ones is realized without activating detection. Once the precursor ions are trapped, an increased constant potential is applied to the calotte electrodes to produce their acceleration. Collision induced dissociation arises, while VRF is set again at lower values to trap all product (daughter) ions. Scanning V RF amplitude results in ejection of the product (daughter) ions in the increased order of their m/z value, this time with an activated detection system. This procedure can be repeated many times (commercially available systems are allowing up to 13 repetitions), although more than MS 4 experiments are not commonly required. The ICR based instrumentation is also suitable to work under time delayed MS hyphenation. It seems clear that MS hyphenation in time is less expensive with respect to spatially developed tandem instruments, as the use of a single mass analyzer is required.

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Collision Induced Dissociation (CID) He atoms Ion Source + + + + + Acceleration electrode +

+

Precursor ions

+
+

Product ions

Quadrupole 2

Quadrupole 3

Figure II.4.50. Triple quadrupole arrangement for tandem MS. II.4.7.9. Mass Spectrometry Working Modes The possible ways of achieving MS experiments are illustrated in the Figure II.4.51: SINGLE MS STAGE
Ion Source mi/z, i = 1, ,n Mass Analyzer m1/z, m2/z,, mn/z arranged and transmitted mj/z: transmitted mi/z, i j: blocked Detector R.A. = f(m/z) Technique Full Scan Mode Qualitative and quantitative purposes (less sensitive) Selected Ion Monitoring (SIM) Quantitative analysis (sensitivity gain 1,000) Multiple Ion Detection (MID) Quantitative analysis (with increased sensitivity) + structural confirmation

mi/z, i = 1, ,n

R.A. = f(mj/z) R.A. = f(mj/z, mq/z, mk/z); R.A.(mj)/RA(mq) R.A.(mj)/RA(mk) R.A.(mq)/RA(mk)

mi/z, i = 1, ,n

mj/z; mq/z; mk/z: transmitted mi/z, i j, q, k: blocked

MULTIPLE MS STAGES
Ion Source mi/z, i = 1,,n mi/z, i = 1,,n Mass Analyzer 1 mj/z: transmitted mi/z, i j: blocked m1/z, m2/z,, mn/z transmitted and arranged CID mj/z fragmente d to mi/z, i = 1,..,n m1/z, m2/z,, mn/z Mass Analyzer 2 m1/z, m2/z,, mn/z transmitted and arranged mj/z: transmitted mi/z, i j: blocked Detector Technique

R.A. f(mi/z) R.A. f(mj/z)

Product Ion Scan Precursor Ion Scan

Electron Multiplier

222

Ion Source

Mass Analyzer 1

CID fragmente d to mi/z, i = 1,..,n m1/z, m2/z,, mn/z fragmente d to mi/z, i = 1,..,n mj/z fragmente d to mi/z, i = 1,..,n

Mass Analyzer 2

Detector

Technique

mi/z, i = 1,,n

m1/z, m2/z,, mn/z transmitted and arranged

m1/z, m2/z,, mn/z transmitted and arranged

m = ct.

Constant neutral loss/gain scan

mi/z, i = 1,,n

mj/z: transmitted mi/z, i j: blocked

mj/z: transmitted mi/z, i j: blocked

R.A. f(mj/z) R.A. f(mj/z, mq/z, mk/z); R.A.(mj) RA(mq); R.A.(mj) RA(mk); R.A.(mq) RA(mk);

Single Reaction Monitoring (SRM)

= Multiple Reaction Monitoring (MRM)

mi/z, i = 1,,n

mj/z: transmitted mi/z, i j: blocked

mj/z fragmente d to mi/z, i = 1,,n

mj/z; mq/z; mk/z: transmitted mi/z, i j, q, k: blocked

/ / /

Figure II.4.51. Mass spectrometric modes (for single and multiple MS stages). REFERENCES.

1. K.L. Busch, Mass Spectrometry, 17(6S), 26 (2002). 2. E. de Hoffman, V. Stroobant, Mass Spectrometry: Principles and Applications , 2nd 3. 4. 5. 6.
Edition, J. Wiley & Sons, Chichester (2002). Analytical Mass Spectrometry Strategies for Environmental and Related Applications, W.L. Budde Editor, American Chemical Society, Oxford University Press Inc., New York (2001). Advances in Mass Spectrometry , E. Gelpi Editor, J. Wiley & Sons, Chichester (2001). Mass Spectrometry: Analytical Chemistry by Open Learning , 2nd Edition, J. Barker, J.A. David Editors, J. Wiley & Sons, Chichester (1999). C. Herbert, R. Johnstone, Mass Spectrometry Basics, CRC Press, Boca Raton,

223

Florida (2002).

7. R.M. Smith, Understanding Mass Spectra: A Basic Approach , 2nd Edition, Wiley Interscience (1998). 8. R.A. Hites, Handbook of Mass Spectra of Environmental Contaminants, Lewis Publisher, Boca Raton, Florida (1992). 9. F.W. McLafferty, F. Turecek, Interpretation of Mass Spectra, 4th Edition, University Science Books, Sansalito, California (1996). 10. R.E. Marsh, Quadrupole storage Mass Spectrometry, vol. 102, John Wiley & Sons, New York (1989). 11. FT-ICR MS: Analytical Applications of Fourier Transform Ion Cyclotron Resonance Mass Spectrometry, B. Asamoto Ed., VCH, Weiheim (1991). 12. Tandem Mass Spectrometry, F.W. McLafferty Ed., John Wiley & Sons, New York (1983). Useful Net sites. 1. www.asms.org; 2. http://masspec.scripps.edu; 3. www.i-mass.com; 4. http://chipo.chem.uic.edu; 5. www.chem.arizona.edu.

II.4.8. GAS CHROMATOGRAPHY / MASS SPECTROMETRY

Andrei Valentin MEDVEDOVICI II.4.8.1. Definition Gas chromatography / mass spectrometry (GC/MS) is a coupled analytical technique used for the following purposes: a. confirmation and quantitation of volatile or semi volatile analytes in complex mixtures; b. determination of molecular weights and / or elemental composition of volatile / semi volatile unknowns; c. structural determination of volatile / semi volatile unknowns in a mixture by means of spectral matching or spectral interpretation. II.4.8.2. Principles Gas chromatography is a separation technique suitable for volatile / semi volatile compounds based on their differentiate partition between a moving carrier gas (mobile phase) and a stationary phase consisting in a granular solid adsorbent or a viscous liquid phase uniformly distributed on the surface of an inert support. Because interaction between analytes and the mobile phase does not exist practically, the selectivity in gas chromatography only depends on the analyte / stationary phase interaction.

224

Gas chromatography is achieved on both packed (in practice is considered already obsolete) and open tubular capillary columns (present state of the art). Because packed column gas chromatography exhibits less efficiency (generates broader peaks), special requirements are imposed in terms of selectivity to obtain acceptable resolution. This results in a wide variety of stationary phases routinely used. Gas chromatography on open tubular capillary columns is characterized by a tremendous increase of efficiency (extremely narrow peaks are produced) resulting in a moderate need for selectivity. Stationary phases for capillary gas chromatography (CGC) are basically belonging to five classes of polymeric materials, classified according to their increasing polarity (polydimethylsiloxanes, polyphenylmethylsiloxanes, polymethylcyanopropylsiloxanes, polymethyltrifluoropropylsiloxanes and polyethyleneglycols). Mass spectrometry generates both qualitative and quantitative information related to the target compound, by data interpretation of ionic fragments characterized upon their mass to charge (m/z) ratio and specific formation yields. Otherwise speaking, the mass spectrometer acts as a detector of the gas chromatographic system. The bidimensional information routinely extracted from a classic chromatogram (detector response as function of the time period elapsed from sample injection) is expanded by the addition of a third dimension (detector response as function of the m/z value) see Figure II.4.52. Both GC and MS bring something unique to their union. Therefore, it is not surprisingly at all that the coupling of these two techniques was suggested shortly after the GC development period placed in the mid fifties. Complementarities and compatibility features of the techniques are reviewed in the Table II.4.7. 4.8.3. Interfacing MS to GC As resulting from Table II.4.7, the operating pressure of GC and MS instruments is the main parameter introducing major coupling difficulties. When using open tubular capillary columns having internal diameters below 0.32 mm, optimal carrier gas flow through the column (as resulting from the Van Deemter curves) falls in the 0.5 1.5 mL/min interval. In such case the direct coupling between MS and GC is allowed, as long as modern vacuum systems achieve 10 -5 10-8 torr pressure level under continuous gas flow feeding up to 1.5 mL/min. The direct interface requires the introduction of the capillary column exit in the ion source of the MS instrument. To avoid analyte condensation through the bypass zone from the GC oven to the MS ion source, column heating is mandatory, usually at a temperature at least 10 oC higher than the higher column operating temperature over the whole chromatographic run.

225

Table II.4.7. Parallel between characteristics of GC and MS analytical techniques. # 1 2 3 4 5 Characteristic Ability of handling mixtures Ability to provide structural information Ability to accept vapor state samples Ability to consider ng amount of samples Working pressure GC MS

atmospheric (at the deep vacuum column exit)

When packed columns (or megabore open tubular columns) are used, carrier gas flow is much higher (5 40 mL/min). In such situation, an interface (separator) should be inserted between the chromatographic column and the inlet of the MS ion source. The separator has two main functions: 1. reduction of the carrier gas flow to an acceptable value allowing adequate vacuum level in the ion source; 2. increasing concentration of the analyte in the carrier gas flow fraction transferred in the ion source, to improve sensitivity.

Ion Abundance A Mass/Charge m/z Typical Mass Spectrum

Retention Time tR (min) Typical chromatogram

Figure II.4.52. 3D result of a GC / MS analysis.

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The introduction of a separator between GC and MS means already the insertion of a supplementary void volume between column and detection area, resulting in a significant loss in terms of separation efficiency (up to 60%). The separator behavior is characterized by means of separation yield ( ) and concentration factor (N) calculated according to the following relations:

QMS 100 ; QGC

N =

CFGC CFMS

where QMS and QGC are analyte amount reaching the ion source or exiting the chromatographic column, respectively, and CF GC and GFMS are carrier gas flow exiting column or reaching the MS ion source, respectively. The following devices are used as GC / MS interfaces: supersonic molecular jet separator, porous membrane separator (membranes are made from glass, Teflon, silver, silicone, combined silver and silicone or stainless steel) and slit separator. The working principles are illustrated in Figure II.4.53 (A,B and C). The characteristics are summarized in Table II.4.8. Table II.4.8. Technical characteristics of some separators used for GC / MS interfacing. % 50 50 50 50 50 50 50 40 30 Max. accepted operating temperature (oC) 350 350 280 250 350 250 250 250 350

# 1 2 3 4 5 6 7 8 9

Interface Supersonic molecular jet Porous glass tube Porous Teflon tube Silicone membrane Silver membrane (type 1) Silver membrane (type 2) Porous stainless steel membrane Combined silver frit and silicone membrane Slit

N 40 - 86 5 - 20 1-5 105 2 - 100 4 - 24 10 - 108 105 10 60

Flow interval (mL/min) 1 - 60 1 - 60 2 - 30 1 - 50 1-5 4 - 18 7 - 35 1-5 1 - 100

227

L1 d1

L2 d2 d3

From column

To ion source

Carrier gas Analyte V1

d1=0.1 mm; d2=0.25 mm; L1=0.15 mm; L2=0.50 mm; V1=10-3-10-4 torr; V2= 10-4-10-5 torr V2

B
d From column Membrane To ion source From column

To ion source

Figure II.4.53. Basic construction of some separators used for GC / MS interfacing: A. supersonic molecular jet separator; B. membrane separator; C. slit separator. II.4.8.4. Data System for GC / MS Instrumentation A GC / MS experiment produces a huge amount of data. Assuming that a GC peak exhibits a width (6 ) of about 20 s and that a correct peak profile as a Gauss shaped function could be obtained with at least 10 points (see Figure II.4.54), it clearly results that a MS spectrum should be collected every 2 s. As long as a mass spectrum is obtained by sequential scanning on all m/z channels, each channel should be sampled for 8 ms (accepting a spectral resolution of 1 Da and a m/z interval ranging from 0 to 250; note that for routine GC / MS analysis, m/z scales are much broader: up to 800 or even 1000). However, MS instruments are characterized by a defined resolution. The line spectrum appearance more often used in practice is in fact a profile spectrum, meaning that each MS signal has a Gauss shape. Consequently, for accurate reproduction of the Gauss profile, 10 points are needed. Accordingly, one concludes that each m/z channel is sampled with a 800 s rate for generating the MS response. The whole process is illustrated in Figure II.4.55. In commercially available MS detectors, the voltage output from the preamplifier of the electron multiplier is converted from an analog signal to a digital value (using an analog to digital converter) at a rate of 10,000 to 100,000 times per second, meaning that for 1 minute of a chromatographic run, the instrument will

228

produce up to 6,000,000 numbers. Thus, to avoid saturation of bulk storage devices, most data systems find m/z peaks in real time and convert them into mass / intensity pairs. Each spectrum stored on the hard disk of a computing system is associated to a retention time value.

Figure II.4.54. Ability of reproducing Gauss shaped profiles according to the sampling data frequency.

At least 0.8 mseconds for each data

212

(m/z)
1.0e5 8.0e4 Intensity 6.0e4 4.0e4 2.0e4 0 0 50 100 150 200 250 m/z

1.2e5 1.0e5 8.0e4 6.0e4 4.0e4 2.0e4 0 0 1 2

2/250 = 0.008 seconds for each mass channel

20 seconds
3 4 5 6 Time (min.)

At least 1 MS spectra every 2 seconds

Figure II.4.55. Data production algorithm in an MS detector coupled to a GC system.

219 220

213 214

215

216 217 218

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II.4.8.5. Data Interpretation Modes in GC / MS Unlike traditional GC detectors, a MS instrument coupled to a GC system produces a 3D data set. Each point obtained in the xOy plane (abundance as function of retention time) is obtained by addition of all individual intensities existing on the m/z scanned channels over the collected mass spectrum (placed in the yOz plane). Such manner of data interpretation is named TIC (Total Ion Current Chromatograms). On operator request, the data processing software is able to retrieve only narrow intervals or specific m/z values. Chromatograms obtained by considering only the intensities recorded on specific m/z channels or limited m/z intervals are usually referred as EIC (Extracted Ion Chromatograms). For EIC, the sensitivity is enhanced by means of a higher Signal to Noise ratio (S/N) and by the inherent selectivity induced to the processes. However, the assumption that the MS response is a universal one does not apply anymore, because the intensity of chosen specific lines varies for each chemical species. Note that EIC is related to the functioning of the MS detector in the scan mode (see chapter referring to Mass Spectrometry). The Selected Ion Monitoring (SIM) or Multiple Ion Detection (MID) modes are related to the operation of the MS instrument and not to the data processing mode. Chromatograms obtained in the SIM or MID modes are in fact TIC, because each point in the chromatogram represents the intensities addition on all available m/z channels. II.4.8.6. Qualitative Information in GC / MS Qualitative information in GC / MS is obtained only if the MS instrument runs in the scan mode. MID mode is generally associated to spectral confirmation. Four approaches of examining GC / MS data exist: 1. going through the chromatogram and inspecting individually the MS spectra obtained at the apex of each of the chromatographic peaks; 2. having a comparative look on all mass spectra characterizing compounds separated as integrated peaks within the chromatogram; 3. examining MS spectra acquired during peak elution; 4. comparison between the MS spectra considered as characteristic for a given chromatographic peak with a spectra library. The quality of the structural information strongly depends on the quality of the mass spectrum considered as characteristic for an eluted chromatographic peak. Comparing normalized MS spectra acquired during peak elution lead to a spectral purity check tool, useful for evaluation of an eventual chromatographic co elution phenomenon.

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Conditions for getting good characteristic MS spectrum for a chromatographic peak are: a. enough analyte (at least 0.5 ng reaching the ion source); b. m/z scale large enough to observe the molecular ion as well as its intrinsic isotopic profile; c. avoid spectral saturation (a lot of MS signals having 100% relative abundance within the spectrum see Figure II.4.56); d. avoid elimination of low intensity MS signals by means of an inappropriate choice of the intensity threshold (generally equal to or lower than 0.1% relative abundance illustration in Figure II.4.56); e. eliminate background interference by means of background subtraction (make subtraction of the averaged MS spectrum of the baseline close to the considered chromatographic peak). If spectral saturation is observed during peak elution, is advisable to consider only MS scans at the beginning or at the end of the peak. If low intensity averaged spectrum is obtained, it is advisable to consider only scans around peak apex. There are basically two library searching algorithms. The most common one is the 10 peaks algorithm: from the characteristic MS peak spectrum, the software further considers only the 10 most abundant lines separated by at least 14 Da; this is representing the reduced spectrum; library spectra are also represented in this format; match quality is based on the recovery of the 10 lines in the sample reduced spectrum within the reference reduced spectra (line is present or not and line intensity is comparable or not). Another library searching algorithm is the PBM (Probability Based Matching) initiated by F. McLafferty from Cornell University. PBM allows calculation of the relative uniqueness of the spectral lines (higher m/z lines exhibit higher relative uniqueness compared to lower m/z lines). Comparison in PBM considers three factors: m/z values; their relative intensities; their relative uniqueness.

231

100 90

Good characteristic spectrum

200

Saturated spectrum 173

200

215

Relative Abundance (%)

80 70 60 50 40 30 20 27 10 0 180 190 100 110 120 130 150 170 200 140 160 210 100 130 150 160 180 190 110 120 140 170 200 200 Too high threshold 210 20 40 60 70 90 30 50 80 20 30 50 60 80 90 40 70 43 58 173 68 71 96 104 122 132 158 145 27 104 96 122 132 145 215 43 58 71 68 158

100 90

Relative Abundance (%)

80 70 60 50 40 30 20 10 0 160 170 180 190 200 100 110 130 150 120 140 220 210 20 30 40 50 60 70 80 90 43 68 132 158 58 173 215

m/z

Figure II.4.56. Distinguishing good characteristic spectrum from saturated or high threshold computed ones. Does not matter which algorithm is used, the library search can be made as a forward search (is the characteristic spectrum of the peak retrievable within the library? faster approach) or as reverse search (is the library reference spectra fitting on the characteristic spectrum of the chromatographic peak? slower approach). It is worthwhile to note that spectra libraries are commonly containing Electron Impact (EI) mass spectra produced with a 70 eV striking energy, and obtained with Quadrupole Mass Analyzers. Specific instrumental conditions may not be known. Matching for spectra obtained with Ion Trap Mass Analyzers may lead to altered results. Custom made libraries can be obtained on specific ion production and / or mass analysis.

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II.4.8.7. Quantitative Information in GC / MS Quantitation in GC / MS is based on peak area measurement in TIC or EIC chromatograms resulting either from full scan or SIM / MID operating modes of the MS instrument. With SIM / MID operating modes, the signal intensity is taken only on a single m/z channel (or a limited number of m/z channels). This results in important acquisition timesavings. Instead of scanning over the whole m/z range, SIM / MID modes allow data sampling with an increased frequency only on a given m/z channel or narrow channels interval. Enhanced sensitivity is produced due to: 1. increased accuracy for peak integration (peak shape is better reproduced due to a larger number of points generated during peak elution); 2. increased S/N ratio (the addition of multiple measurements amplifies the signal and self-annihilation of the background due to its random character). The overall gain in sensitivity for SIM / MID operating modes ranges in a 100 to 1,000 folds interval. The control software of modern MS instruments allows the selection of different ions during the same chromatographic run. Such achievement leads to the highest sensitivity obtained for all separated compounds in a single chromatographic run. The conversion of the peak area to analyte amount imposes calibration. The two main calibration strategies are based on external and internal standards. The use of an external standard supposes the availability of the target analyte as a reference standard substance. The best internal standard should be chemically very similar to the analyte itself. Consequently, any losses of the analyte during the sample preparation procedure are duplicated by losses of the internal standard. The very best internal standard is an isotopically labeled version of the analyte. Using isotopic labeled internal standard and the SIM operating mode, sensitivity characterizing GC / MS technique falls in the low pg range. II.4.8.8. Applications GC / MS should be considered as the principal primary screening method for pesticide analysis in food, environmental and clinical samples. Due to the broad range of polarity characterizing the analytes, separations are achieved on stationary phases exhibiting different polar character. Consequently, the open tubular columns used with silicone polymer bonded phases are from 100 % methyl silicone or 95 % methyl and 5% phenyl (nonpolar ones) to 50 % methyl and 50 % phenyl, 50 % methyl and 50 % trifluoromethylpropyl, 86 % methyl and 14 % phenylcyanopropyl or 50 % methyl and 50 % phenylcyanopropyl (medium polar or polar ones). The primary choice concerning the stationary phase for screening residues of pesticides is a low polarity one, either 100 % methyl silicone or 95 % methyl and 5 % phenyl.

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Considering the deactivation degree of the column wall in a close relationship with the deposited layer of stationary phase, films thinner than 0.1 m should be avoided for use, especially for separation of labile pesticides. 25 30 m length fused silica open tubular (FSOT) columns, with 0.20 0.25 mm internal diameter and 0.15 0.33 m film thickness should be considered as a basic starting choice. Nitrogen, helium, and hydrogen are carrier gases commonly used in GC. Hydrogen is not suitable for the MS detector, while nitrogen exhibits less flat Van Deemter curves for gas velocities higher than optimal. Thus, helium remains the best choice. As injectors for capillary GC separation of pesticides and related residues, cold on column (OCI), hot splitless injector (HSI) and programmed temperature vaporizer (PTV) are mainly used. Injection of thermally unstable analytes can be controlled via pressure pulses (based on electronic pressure controlled systems EPC), precise temperature control during injection (achieved by PTV) and the presence of suitable chemical additives. As an example, phenylureas lead in conventional hot splitless injectors to extensive and irreproducible formation of isocyanates and amines. The presence of acetic acid, low molecular mass amines, and organic anhydrides during injection determines a minimization of the thermal decomposition effect or reproducible conversion to the corresponding isocyanates. Studies achieved for organophosphoric and carbamate pesticides, in parallel, on PTV splitless, PTV solvent split, pulsed split less and on column injectors lead to the following hierarchical tolerance: OCI < pulsed HIS < PTV splitless < PTV solvent split. Some other phenomena are associated to injection systems. Tralomethrin, for example, is quantitatively transformed to deltamethrin in HSIs. The latter, when vaporized in packed liner fitted PTVs, readily isomerizes, leading to the split of the chromatographic peak in two equally intense peaks, baseline separated, corresponding to cis and trans isomers. Derivatization may also enhance on thermal stability. Phenylurea pesticides are alkylated with iodoethane and sodium hydride to yield thermostable compounds. When using iodoethane instead of iodomethane, differentiation between parent compounds and the N-demethylated metabolites is possible. The use of a retention gap between the injection port and the capillary column has been widely discussed in literature, especially for OCI. The influence of the solvent, the gap geometry, the injection volume, and the analyte concentration on peak efficiency and symmetry do not lead to a general accepted recommendation considering the use of retention gaps. Checking for each particular case should be more advisable. Both electron impact (EI) and chemical ionization (CI) are currently used as ionization techniques in MS detection, as well as the (+) or (-) ion monitoring modes are encountered. The use of the selective ion-monitoring (SIM) feature drastically enhance on sensitivity although structural confirmation is lost. In Table II.4.9, the ion

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fragments frequently monitored by MS for some pesticides separated by GC are enlisted. Table II.4.9. Major m/e signals in the mass spectra of some pesticides separated in gas-chromatography with mass spectrometric detection (ionization by electron impact).
# 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 Pesticide Azinfos Ethyl Chlorfenvifos Chlorpyrifos Chlordane Coumafos Dichlorvos Dicrotofos DDD (p,p) DDT (p,p) Endosulfan Endrin Ethion Ethoprop Fenchlorfos Fenitrothion Heptachlor Lindane Leptophos Merphos Methoprene Methoxychlor Parathion Parathion (methyl) Phenothrin Resmethrin Stirofos Chemical Classification Organothiophosphate Organophosphate Organothiophosphate (pyridine) Cyclodiene Organothiophosphate (heterocyclic) Organophosphate Organophosphate Organochlorine, Bridged biphenyl Organochlorine, Bridged biphenyl Cyclodiene, Organochlorine Cyclodiene Organothiophosphate (aliphatic) Organothiophosphate (aliphatic) Organothiophosphate (phenyl) Organothiophosphate (phenyl) Cyclodiene, Organochlorine Organochlorine Phosphonothioate Phosphonothioate Ester Organochlorine Organothiophosphate (phenyl) Organothiophosphate (phenyl) Pyrethroid ester Pyrethroid ester Organophosphate Major m/z signals in MS 77, 132, 160 267, 323 197, 242, 270, 298 373 226, 362 109, 185 127 235 235 195, 241, 265, 339 263, 281 97, 125, 153, 231, 384 158, 200, 242 125, 285 109, 125, 260, 277 100, 272 109, 181, 219 171, 377 209, 298 73 227 291 109, 125, 263 123, 183 123, 143, 171 109, 329

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REFERENCES

1. F.W. Karasek, R.E. Clement, Basic Gas Chromatography-Mass Spectrometry: 2. 3. 4. 5. 6. 7.

Principles & Techniques, Elsevier Press, Amsterdam (1988). R.A. Hites, Handbook of Mass Spectra Environmental Contaminants , 2nd Ed., FL Lewis Publishers, Boca Raton (1992). F.W. McLafferty, Registry of Mass Spectral Data with Structures , 5th Ed., J. Wiley & Sons, New York (1989). M. Oehme, Practical Introduction to GC-MS Analysis with Quadrupoles, Wiley VCH, Weinheim (1999). M.C. Mc Master, C. McMaster, GC-MS: A practical Users Guide, Wiley VCH, New York (1998). W.M.A. Niessen, Current Practice of Gas Chromatography Mass Spectrometry , Series Chromatographic Science, Vol. 86, Marcel Dekker, New York (2001). NIST / EPA / NIH Mass Spectral Library for WindowsTM, Gaithersburg, MD:NIST Standard Reference Data (1995).

Useful Net sites 1. www.accustandard.com/asi/; 2. http://ull.chemistry.uakron.edu/gcms; 3. http://depts.washington.edu/spectral/massspec/

II.4.9. IMMUNOASSAY
Giuseppe PALLESCHI, Mihaela BADEA Enzyme immunoassay kits are now available for qualitative field-testing or for laboratory screening and semiquantitative analysis of pesticides, herbicides, polychlorinated biphenyls (PCBs), mononuclear and polynuclear aromatic hydrocarbons, pentachlorophenol, nitroorganics, and many other compounds in aqueous and soil samples. Certain analytes may be quantitatively determined as well, with a degree of accuracy comparable to gas chromatography or high performance liquid chromatography determination. The method is rapid and inexpensive. Antibodies are known to show affinity for specific compounds. Antibodies belong to the family of glycoproteins known as immunoglobulins. They are synthesized by animals as response to the presence of a foreign substance. The macromolecules able to develop such an immunological response are called antigens.

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The specificity of an antibody is directed against a particular site on the antigen known as epitope. An antibody (Ab) recognizes specifically the corresponding antigen (Ag) and forms a complex antibody-antigen (Ab-Ag). II.4.9.1. Immunoassay Principle An immunoassay may be defined as a technique based on the reaction between an antigen and an antibody for measuring the concentration of the desired analyte. The classification of the immunoassays is somewhat variable, but generally reflects the assay principle and the nature of the label used to monitor the immunoaffinity reaction. The first major distinction is made between homogeneous and heterogeneous immunoassays. A homogeneous system does not require separation of free and bound antigen; the assay relies upon the alteration of a property (optical, electrochemical, etc) or function of the label on the formation of the antibody-antigen complex A more sensitive approach that is less prone to interference problems is the heterogeneous assay format in which there is a separation step. Within each of these classes there are different ways for designing the assay. Some examples of immunoassay configuration are described below: a) Competitive, solid-phase immunoassay in which antibody is immobilized on a solid phase and the antigen is labeled. In this format a competition between the labeled and unlabelled antigen (the analyte) for the available antibody binding sites takes place. After the binding reaction the amount of label associated with the solid phase will be inversely related to the concentration of analyte. b) Displacement immunoassay in which the antibody is immobilized on a solid phase and the antigen is labeled. At the beginning of the assay all the available binding sites on the immobilized antibodies are occupied by labeled antigen. When the unlabelled antigen (the analyte) is added there is a displacement of the labeled material and, under appropriate condition, the extent of this displacement will be dependent upon the amount of analyte added. c) Sandwich immunoassay is suitable to use only for high molecular weight antigens which posses at least two antigenic sites (epitopes). A capture antibody is attached directly or indirectly to a solid phase. After the binding reaction a second labeled antibody against a different epitope is added in excess and the amount of labeled antibody associated with the solid phase is directly related to the amount of analyte. There are many types of labels used to monitor the antibody-antigen binding reaction including: particles, metals, dyes, radionuclides, enzymes, substrates and cofactors, electrochemically active compounds.

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II.4.9.2. ELISA Technique The enzyme-linked immunosorbent assay (ELISA), introduced for the first time in 1971, remains the most popular immunoassay technique. In ELISA an enzyme is used as label linked either to the antigen or the antibody. This label in conjunction with a suitable substrate produces the assay signal. In the ELISA one of the reagents is bound to a solid phase. Usually, this solid phase has the form of a 96-well microtitre plate. This plate gives the possibility to handle many samples at one time and it facilitates the automation of the assay. As labels, many enzymes may be used: urease, alkaline phosphatase, horseradish peroxidase, -galactosidase, etc. Each of these can be employed with a number of substrates to generate a signal, which usually takes the form of a colored dye. In the last years, ELISA techniques that involve electrochemical measurements and the screen-printed electrode technology were developed. In the classical spectrophotometric ELISA, antibodies are immobilized to the walls of the test tubes, plates, or microwells. Such test tubes and plates are commercially available and supplied in the test kit. A measured amount (between 10 and 50 L) of sample or sample extract is added to one such test tube containing an assay diluent (a phosphate buffer). An equal volume of analyte-enzyme conjugate (commercially available and supplied in the kit) is then added to the test tube. The enzyme conjugate is a solution containing the same analytes labeled with an enzyme. The solution mixture is incubated or allowed to stand for a specific amount of time. During this period, the enzyme conjugate competes with the analyte molecules for a limited number of antibody binding sites in the test tube. After the incubation, the unbound molecules are washed away, leaving behind the bound ones, anchored onto the antibody sites. Then a solution of a chromogenic substrate (i.e., 3,5tetramethylbenzidine if horseradish peroxidase is used as label) is added to the mixture. The enzyme conjugate, bound to antibody sites on the wall, reacts with the chromogenic substrate forming a blue color. The enzyme catalyses the transformation of the substrate into a product that reacts with the chromogen causing a colored product. Each enzyme molecule can rapidly catalyze the conversion of thousands of substrate molecules into product molecules that react with chromogen. In other words, the darker the color, the greater the amount of bound enzyme conjugate or, conversely, the lesser the amount of the analyte in the sample. Thus, the color intensity is directly proportional to enzyme conjugate concentration and therefore, inversely proportional to the analyte concentration in the sample. For qualitative screening, a visual comparison of color with standards can be made. For semiquantitative determination, however, a spectrophotometer should be used to read the absorbance to plot a calibration standard curve. Usually, the color should be read as soon as possible because it becomes unstable after 30 min. The required period for incubation varies from substance to substance but can range from 5

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to 10 min to 1 or 2 hours. In certain analysis, the immunochemical reaction must be stopped after certain time, using different agents (such as 1 N HCl). An alternative assay procedure involves the use of antibody-coupled magnetic particles. Antibodies specific to the analyte of interest are covalently bound to paramagnetic particles rather than test tubes. The particles are suspended in a buffered saline solution with preservative and stabilizers. The enzyme conjugate and the antibody-coupled magnetic particles suspension are then combined with the sample extract. The mixture is incubated. A magnetic field is applied to separate the magnetic particles, which are then washed and treated with a chromogenic reagent. The reaction is then stopped by adding HCl, and the developed color turn is read by a spectrophotometer. Also a sandwich immunoassay format can be used for different applications. These assay methods, including those discussed above, have both advantages and disadvantages when compared with each other. For example, in the sandwich format, an additional incubation period is required for the second antibody, which increases the analysis time. Particulate systems require centrifugation to separate the bound particles. The more common coated-tube method is simple and rapid and does not involve centrifugation. However, its disadvantage is that the surface of the tube or plate limits the number of antibody for the reaction. Also, there may be loss of antibody because of absorption onto the solid surface. An advantage of the magnetic particle method is that the antibody is covalently bound to the solid particles of uniform size (1 m) giving even distribution throughout the reaction mixture. The method, however, requires the use of a magnetic field for separation. Detection limits in the range of low parts per billion (ppb) can be achieved by immunoassay testing for certain parameters in aqueous samples. For soil samples, detection limits of <10 ppm can be achieved for many contaminants. The presence of substances having the same functional groups can interfere in the test, giving a false positive value. For example, 2,3,6-trichlorophenol may interfere in the test for pentachlorophenol. Such interference effect may, however, be reduced by using an antibody that is most selective for the target analyte. II.4.9.3. Immunochemical Sensors (Immunosensors) Immunosensors are analytical devices incorporating an antibody-based biorecognition molecule utilized in conjunction with or integrated within a physicochemical transducer or transducer microsystem and yielding a digital electronic signal, which is proportional to the concentration of a specific analyte or group of analytes. Immunosensors are very attractive due to their high specificity and the signal achieved is related to a single analyte or small number of related compounds. The main advantages of immunosensors over immunoassays are simplification of the analysis procedures, decrease of analysis time, miniaturization of

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equipment and automation. These devices can be classified depending on the analysis place and time, and can include large multi-analyzers, portable bench-top instruments and one-shot disposable sensors. A range of immunosensors for pesticides, herbicides, toxins, endocrine disrupters and microorganisms has been developed for environmental analysis. These devices have been adapted to be used in flow injection analysis, especially for online analysis of waters. Optical Immunosensors Advances in optical fibers and laser technology have contributed to the wide use of this kind of transducers. Optical sensors based on surface plasmon resonance (SPR) such as BIAcore equipment developed by Pharmacia (Uppsala, Sweden) and IAsys using resonant mirror from Affinity Sensors Ltd (Cambridge, UK) brought a significant improvement in optical sensor technology. The BIAcore developed immunosensors for a range of analytes in water and soil extracts. A detection limit of 1 g L -1 for atrazine with this kind of immunosensors was achieved. Electrochemical Immunosensors Electrochemical immunosensors are based on the use of an electroactive label or substrate, usually employing enzyme labeling and amplification techniques. Different types of electrochemical immunosensors have been developed based on potentiometric, capacitive, conductimetric and amperometric detection for environmental analysis. Different amperometric immunosensors have been developed for pesticide detection. Most are based on screen-printing electrodes for one-shot analysis. A screen-printed immunosensor for 2,4-dichlorophenoxyacetic acid (2,4-D) analysis in soil was developed. The sensor is based on an indirect competitive assay with the antigen conjugate adsorbed directly onto the working electrode surface, and enables to detect ppm of 2,4-D in soil extracts. Potentiometric immunosensors are based on the measurement of changes in potential that arise from the reaction of the analyte with the specific receptor. A range of immunosensors has been developed using this type of transducer where the antibody has been immobilized on an ion-selective electrode. Also, sensors based on ion-sensitive field-effective transducers (ISFETs), chemically sensitive field-effective transistor (CHEMFET) and light-addressable potentiometric sensors (LAPS) have been proposed.

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Piezoelectric / Acoustic Immunosensors Piezoelectric / acoustic immunosensors are based on quartz crystal microbalance (QCM) and, respectively source acoustic transducers can also be classified as direct immunosensors when immunoreagents are used as receptors. Piezoelectric sensors have been used for the detection of pollutants such as pesticides. By immobilizing the antibody for target analytes on the crystal, immunosensors for parathion, atrazine and 2,4-D have been developed. REFERENCES 1. Biosensors for Direct Monitoring of Environmental Pollutants in Field, Ed. Dimitrios P. Nikolelis, Ulrich J. Krull, Joseph Wang and Marco Mascini, 1997, Kluwer Academic Publishers, Netherlands 2. Biosensor Principles and Applications, Ed. Loc J. Blum and Pierre R. Coulet, 1991, Marcel Dekker, Inc., New York, USA 3. Rapid Detection Assays for Food and Water, Ed. Stuart A. Clark, K. Clive Thompson, C. William Keevil and Mark S. Smith, 2001, The Royal Society of Chemistry, Cambridge, UK 4. Immunoassay. A Practical Guide. Ed. Brian Law, 1996, Taylor & Francis Ltd., London, UK 5. Biosensors. A Practical Approach. Ed. A.E.G. Cass, 1990, Oxford University Press, New York, USA 6. Rapid and on-line Instrumentation for Food Quality Assurance, Ed. Ibtisam E. Tothill, 2003, CRC Press, Boca Raton, USA

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II.5. METHODS FOR MONITORING IMPORTANT POLLUTANTS

II.5.1. PHENOLS
Jenny EMNEUS

THE

MOST

A number of phenolic compounds are listed in the European Community (EC) Directive 76/464/ EEC concerning dangerous substances discharged into the aquatic environment [1] and in the US-EPA list of priority pollutants [2, 3] due to their toxicity and persistence in the environment. Directive 75 /440/EEC states that maximum levels of phenolic compounds in surface water for drinking purposes should be in the l-10 pg/L range depending on the required treatment [4]. Current official analytical methods, e.g., US-EPA 604 [2] and 625 (acid extractable section) [3] and the recently introduced 8041 [5] are based on liquid-liquid extraction (LLE) followed by gas chromatography using different detection devices such as electron capture or mass spectrometry (MS). However, there is a general trend to change these procedures to liquid solid extraction (LSE) and liquid chromatography (LC) methods to avoid manipulation of large amounts of toxic organic solvents and because the derivatization of phenols is not straightforward. Furthermore, with atmospheric pressure (API) liquid chromatography-mass spectrometry (LC-MS) interfaces structural information similar to chemical ionization can be obtained and the disadvantages of other LC-MS interfacing devices such as thermospray (TSP) have been overcome. Other emerging approaches are the use of solid-phase microextraction (SPME) and the combination of LSE with supercritical fluid chromatography (SFE). In addition, capillary electrophoresis (CE) is a powerful alternative to classical chromatographic techniques for the separation of polar analytes. Biological techniques, mainly immunoassay and biosensors, have grown in the last few years due to the need to develop fast and cost effective technologies suitable for application in the field and as a screening method prior to chromatographic analysis. Several immunoassays are commercially available for the detection of various organic contaminants. Future trends will focus on biosensors because of their faster response and lower cost. An overview of the current analytical methods for the determination of priority phenolic compounds in water and wastewater is presented here and various aspects such as sample extraction, clean up and final determination will be reviewed. The presence of interfering materials in the sample, mainly humic and fulvic acids in river water, as well as other constituents of water, should always be taken into consideration because they will notably affect analytical performance [5, 6].

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Acidification of the sample can help to overcome this drawback and can avoid the deprotonation of the most acidic phenols. However, in this case adsorption of humic material into the sorbent is increased so a large interfering peak appears somewhere in the chromatogram [7]. Various sample handling strategies for the determination of phenolic compounds in water, such as liquid-liquid extraction (LLE), liquid-solid extraction (LSE) and solid-phase micro-extraction (SPME), have been used in order to eliminate the sample matrix influence on the analysis. The analytical approaches for phenols and phenol derivatives determination from environmental samples are almost based on chromatographic techniques (e. g. gas chromatography (GC), liquid chromatography (LC), capillary electrophoresis (CE)), but also biological techniques ( e. g. immunoassay and biosensors). Gas chromatography in conjunction with various detection devices, mainly FID [2], ECD [8] or MS [9], has been reported by several authors. It is currently being used in US-EPA official methods. This approach has the advantage of high sensitivity and selectivity, and the existence of mass spectra libraries for screening of unknown samples, but in general derivatization is required prior to analysis. GC of underivatized phenols using capillary columns with conventional phases is difficult as phenols, and in particular nitrophenols, tend to tail, even when using highly deactivated columns [8]. Various derivatization reagents have been reported, e.g. pentaflouorobenzoyl bromide [2], acetic anhydride [10], heptaflourobutyric anhydride [10] or diazomethane [11]. However, reagents such as pentaflouorobenzoyl chloride are preferred because they introduce electronegative substituents into the molecule, thus non-nitrated and non-halogenated phenols can be detected using ECD although the method fails for nitrophenols. Derivatization using acetic anhydride is more straightforward but dinitrophenols gave problems. The current US-EPA methodology for phenols (method 604) involves derivatization using pentafluorobenzyl bromide prior to GC separation, followed by either FID or ECD although the use of GC-MS for identification purposes is also recommended [2]. When using this methodology LODs of phenols included in US-EPA list range from 0.1 g/L to 13 g/L and from 0.5 g/L to 2.2 g/L with FID and ECD, respectively 0. Mass spectrometry (MS) is by far the most suitable detection device when analyzing unknown samples because mass spectra libraries, which allow the unequivocal identification of phenols, are available. The US-EPA method 625 provides conditions for GC-MS analysis [3]. However, in the last few years it was pointed out that US-EPA official methods for phenols may often lead to incorrect results because derivatization of phenols, especially nitrophenols, is not straightforward. Recently, the US EPA has reported a new protocol (method 8041), which recommends the derivatization to methylated phenols instead of to pentaflouorobenzoyl ether derivatives. However, this method requires the use of diazomethane, which has potential hazards, associated with its use (carcinogenic, explosive).

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The absence of derivatization requirements and the possibility of on-line coupling with LSE without sophisticated instrumental requirements has made LC with C18 or C8 columns the most commonly applied separation technique for the analysis of phenolic compounds. UV detectors are the most common detectors and detection is usually carried out at 280 nm, except for nitrophenols and pentachlorophenol, which show a better signal at 310 nm [6, 7]. Diode array detectors are recommended because even though little decrease in sensitivity occurs, spectral libraries can be used for confirmation purposes [12]. With on-line LSE using Lichrolut EN, LODs below 0.7 g/L were obtained for all phenols using UV detection [6] but values below 0.25 pg/L using off-line LSE were found. However, an important decrease in LODs was obtained when using EC detection instead of UV [13] and values in the low pg/L range for most of the compounds studied were obtained using on-line LSE [14]. The high sensitivity of EC allows a reduction in the sample volume, and when processing only 10 mL of water LODs of 0.02 g/L were obtained [14]. However, UV shows better performance with regard to signal stability although, because of the lower sensitivity, higher sample volumes are required for the LSE step, thus increasing the risk of exceeding the breakthrough volume of the more polar phenols. In summary, EC detection is a good alternative for monitoring relatively clean samples, for instance drinking water, where electrode fouling is not a critical problem. For the analysis of complex samples such as river or wastewater, the more robust UV will be preferred, although EC may be required to detect the low breakthrough volume analytes. Recently coulometric array detectors were introduced. Unlike common electrochemical detectors in which electrodes typically react to 10% or less of the injected sample, coulometric sensors convert 100% of the analyte because oxidation of phenols occurs in a high porosity electrode. In their most sophisticated approach, a serial array of electrodes at increasing potentials provides 3-dimensional chromatograms (similarly to diode array detectors) where the analyte voltammogram facilitates peak identification. An increase in sensitivity of up to three orders of magnitude can be obtained compared to diode array detectors, and LODs ranging from 0.03 ng/L to 0.38 ng/L were obtained when combined with LSE [15]. They cannot be used for multi-screening purposes because the gradient elution is not suitable. Several authors have reported the monitoring of phenolic compounds using direct [16] or indirect [17] fluorescence detection. LODs below the ng/L level were obtained but derivatization to dansyl derivatives was necessary and a tubular flowthrough reactor was required for indirect detection. Different interfaces are currently available for LC-MS experiments. The thermospray interface (TSP) provides a good response in the negative ion mode for the listed phenols with the exception of phenol, 4-methylphenol and 2,4dimethylphenol because current buffers cannot deprotonate them even when working at a high buffer concentration level [12, 14, 16]. The particle beam interface was reported for pentachlorophenol analysis but its use in the environmental field is prevented because it gives lower sensitivity compared with other available devices

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[18]. The main advantage of atmospheric pressure interfaces (API interfaces) are the resulting higher sensitivity (especially when using atmospheric pressure chemical ionization, APCI), robustness, and ease of use. Also, by raising the extraction voltage structural information can be obtained via CID using a simple quadrupole instrument (2,4-dinitrophenol determination). A few papers have dealt with the analysis of phenols using the ionspray (ISP) interface [12, 14. 18]. Since an acidic pH is normally required for the chromatographic analysis of phenolic compounds, post-column addition of buffer is necessary in order to generate ions in solution when per-forming LC-ISP-MS experiments. By far the most interesting feature of the ISP interface is the detection of phenol, 4-methylphenol and 2,4-dimethylphenol, although methanol percentages above 85% are required [19]. In the last few years the application of CE, mainly capillary zone electrophoresis (CZE), has grown in the environmental field mainly because it is fast, has high resolution, and is suitable in the analysis of polar and ionic compounds as well as the more nonpolar ones. Moreover, it offers the possibility of carrying out sample enrichment in the capillary using isotachophoresis and shows compatibility with mass spectrometer devices such ISP. The major drawback of CE is its low loading capacity, which is in the range of few nanoliters. Several reports dealing with the separation of phenols have been published, most of them dedicated to demonstrate the techniques high-resolution applications to specific pollutants. Only a few of the reports refer to the determination of pollutants in environmental matrices. CZE has been applied at basic pH using electrolytes such as sodium borate, cyclohexylaminoetansulfonic acid, and phosphate buffer [20, 21]. In most cases UV detection is used. Typical LODs when CZE is combined with off-line LSE range from 0.3 pg/L to 1 g/L [21], although they can be improved by using EC or fluorescence detection [22]. Deprotonated humic and fulvic acids can be effectively separated from other contaminants due to their different migration kinetics, thus allowing the quantification of the most polar phenols such as phenol or cathecol. However, the basic pH requirement for CZE can be a limitation because it can induce hydrolysis of some analytes such as nitrophenols, or the polymerization of catechol. For this reason, some authors prefer the use of micellar electrokinetic chromatography (MECC) 2 [23]. Even though this approach is just emerging, it is really promising because it has the advantage that trace enrichment is carried out on the top of the capillary and the LODs can be lowered, thus overcoming their major drawback. Interest in developing fast, portable and cost-effective field analytical methods that are capable of effectively screening for the presence of target analytes in environmental samples has grown in the last few years. The EPAs Office of Research and Development is leading efforts to develop this promise and to extend the range of analytes, which can be monitored [24]. Standard operating protocols are written in formats such as those found in EPA solid waste (SW-846) protocols [25]. Biosensors and the more established immunoassays (ELISA) are really promising in this area.

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They take advantage of the high specificity of biological recognition to selectively monitor target analytes in complex samples. One of the commercially available immunoassay for phenolic compounds is for pentachlorophenol. Cross reactivity studies showed the good specificity of the method; only 2,4,5,6-tetrachlorophenol and 2,3,4,6-tetarchlorophenol gave significant values (54.1% and 15.1%, respectively) and furthermore matrix interferences did not affect immunoassay performance [26]. The method compares favorably with GC-MS and HPLC measurements in water (~0.980) and soil (0.996) and LODs of 60 ng /L are obtained [26]. Other authors have reported the development of immunoassays for the determination of 4-nitropheno1 and 2,4-dinitrophenol in urine and water samples, in the latter case with LODs of 7 g /L [27]. Similarly to immunoassay techniques, biosensors take advantage of the intrinsic specificity enzyme-substrate reactions for selectively monitoring pollutants in environmental samples. Any biosensing device can be divided into three parts: the biological recognition unit, the signal transducer and the detection system, which depends on the chosen transducer. The detection system is usually based on amperometric principle because of its higher sensitivity and selectivity, which characterizes the method. Hence, an electrical transducer and a potentiostat for detection are used. Phenol oxidizes (tyrosinases and laccases) or peroxidases are the enzymes currently used for this propose [28]. Most of the experiments are done with tyrosinases, which show a response mainly for catechol, phenol, 4-chlorophenol and 4-methylphenol [29]. Co-immobilization of laccase can increase the number of detected phenols (e. g. hydroquinone and 2-amino-4-chlorophenol)[30]. Peroxidase enzyme (horseradish peroxidase) can be also applied for a large range of phenols [31]. REFERENCES (1) Vincent, G.; Angeletti, G.; Bjorseth, A.: Kluwer, 1991. (2) 604, E. m.; Environmental Protection Agency, 1984, pp 58-66. (3) 625, E. m.; Environmental Protection Agency, 1984, pp 153-174. (4) Hennion, M. C.; Pichon, V.; Barclo, D. TrAC 1994, 13, 361. (5) 8041, E. m.: Washington D.C., 1995, pp 1-28. (6) Puig, D.; Barclo, D. J. Chromatogr. A 1996, 733, 371. (7) Puig, D.; Barclo, D. Chromatogr. 1995, 40, 435. (8) Mubmann, P.; Levsen, K.; Radeck, W. Fresenius J. Anal. Chem. 1994, 348, 654. (9) Durhan, E.; Lukasewycz, M.; Baker, S. J. Chromatogr. 1993, 629, 767. (10) Mubmann, P.; Preib, A.; Levsen, K.; Wunsch, G.; Efer, J.; Engewald, W. Wom Wasser 1992, 79, 145. (11) Nick, K.; Scholer, H. F. Fresenius L. Anal. Chem. 1992, 343, 304. (12) Ruana, J.; Urbe, I.; Borrull, F. J. Chromatogr. A 1993, 655, 217. (13) Galcern, M. T.; Juregui, O. Anal. Chim. Acta 1995, 304, 75.

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(14) Puig, D.; Barclo, D. Anal. Chim. Acta 1995, 311, 63. (15) Achilli, G.; Cellerino, G. P.; Eril, G. M.; Bird, S. J. Chromatogr. A 1995, 697, 357. (16) Ruiter, C.; Bohle, J. F.; de Jong, G. J.; Brikman, U. A. T.; Frei, R. W. Anal. Chem. 1988, 60, 666. (17) Lamprecht, G.; Huber, J. F. K. J. Chromatogr. A. 1994, 667, 47. (18) Capiello, A.; Famiglini, G.; Palma, P.; Berloni, A.; Bruner, F. Environ. Sci. Technol. 1995, 29, 2295. (19) Puig, D.; Barclo, D. TrAC 1996, 15, 362-375. (20) Bachmann, K.; Gottlicher, B.; Haag, Y.; Hensel, W. J. Anal. Chem. 1994, 350, 716. (21) Aguilar, M.; Farran, A.; Marti, V. Sci. Total Environ. 1993, 132, 133. (22) Gaitonde, C. D.; Pathak, P. V. J. Chromatogr. A 1994, 663, 229. (23) Praus, P.; Dombek, V. Anal. Chim. Acta 1993, 283, 917. (24) Rogers, K. R.; Williams, L. R. TrAC 1995, 14, 289. (25) 4010A, E. m.; EPA: Washington, D. C., 1995, pp 1-17. (26) Hottenstein, C. S.; Jourdan, S. W.; Hayes, M. C.; Rubio, F. M.; Herzog, D. P.; Lawruk, T. S. Environ. Sci. Technol. 1995, 29, 2754. (27) Li, Q. X.; Zhao, M. S.; Gee, S. G.; Kurth, M.; Seiber, J. N.; Hammock, B. D. J. Agric. Food Chem. 1991, 39, 1685. (28) Varga, G. M.; Emnus, J.; Ruzgas, T. Anal. Chem. 1995, 14, 319. (29) Lutz, M.; Burestedt, E.; Emnus, J.; Jarskog, H.; Gobhadi, S.; Gorton, L.; Varga, G. M. Anal. Chim. Acta 1995, 305, 8. (30) Yaropolov, A. I.; Kharybin, A. N.; Emnus, J.; Varga, G. M.; Gorton, L. Anal. Chim. Acta 2000. (31) Wang, J.; Lin, Y. Anal. Chim. Acta 1993, 271, 53.

II.5.2. NITROGEN (Nitrate, Nitrite, Ammonia)

Tomas ALEXANDERSSON II.5.2.1. Nitrogen Nitrogen is an important eutrophic element that can exist in various forms. The most common inorganic forms of molecules containing nitrogen are nitrite ( NO2), nitrate (NO3 ) and ammonia (NH3). Nitrogen can also be a part of an organic compound and is then referred to as organic nitrogen. In the environment microorganisms have the ability to convert one form of nitrogen to another form. The determination of the inorganic forms is usually made on filtered samples whereas it could be valuable to determine organic nitrogen for both filtered and total samples.

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II.5.2.2. Ammonia Ammonia is a gas at room temperature and it is soluble in water. Dissolved ammonia acts as a base with ammonium as the conjugating acid. The equilibrium between the acid/base pair is determined by the pH of the water solution. Usually when an analysis of a water solution is expressed in units of ammonia, the term ammonia refers to the sum of ammonia and ammonium. Ammonia can be analyzed by several different methods, e.g. gas sensitive electrode and wet chemistry. If a measurement is made with an ion selective electrode the sample is mixed with a strong base, such as sodium hydroxide. The raise in pH will convert all ammonium to ammonia, which diffuses through the hydrophobic gas-permeable membrane of the electrode into the internal solution. The incoming ammonia will change the pH in the internal solution and this change is detected by measuring the potential between a pH-electrode and a chloride-reference electrode immersed into the internal solution. By calibrating the electrode, the instrument gives a direct reading of the ammonia concentration. With an electrode it is possible to do determinations directly on turbid and colored samples. Samples with a high concentration of ions may affect the measurement. It is also possible to convert ammonia into a colored compound and measure the color with a spectrophotometer. In the phenate method, ammonia reacts with hypochlorite and phenol with nitroprusside as a catalyst to an intensively blue compound, indophenol. The developed color is determined by absorption at 640 nm. There is also another method, the modified Berthelot method, where ammonia is converted to 5-aminosalicylate. After oxidation and oxidative coupling a green complex is formed, which is determined by absorption at 660 nm. In the standard method EN 11732 for determination of ammonia in a flow injection system the sample is injected into a continuous carrier stream and mixed with an alkaline solution. The ammonium in the sample is transformed to ammonia and is separated in a diffusion cell. There the solution passes a hydrophobic gaspermeable membrane and the ammonia diffuses into a continuous flowing indicator solution. The indicator changes its color due to the change in pH caused by the diffusing ammonia. This color change is measured in a spectrophotometer and is proportional to the ammonia concentration in the sample. II.5.2.3. Nitrite and Nitrate Both nitrite and nitrate are easily soluble in water. The result after an analysis of a water solution is usually expressed in units of nitrite or nitrate nitrogen. Determination of nitrite can be done with wet chemistry methods whereas determination of nitrate also can be done with ion selective electrode and UV spectrophotometric method.

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In the determination of nitrite a reddish purple azo-compound is produced by reaction of diazotized sulfanilamid and N-(1-naphtyl)-ethylenediaminedihydrochloride (NED dihydrochloride) at a pH between 2.0 and 2.5. The amount of color is then determined by measuring the absorbance at 543 nm. This method could also be used for the determination of nitrate if the sample is pretreated in glass column filled with cadmium granules treated with copper sulfate. When the sample passes through the column the nitrate is reduced into nitrite, which then is determined according to the previously described procedure. Use of the cadmium column will determine the samples content of the sum of nitrite and nitrate. In order to get a measure of only nitrate a correction for the amount of nitrite must be done. The nitrate could also be determined with an ion selective electrode. When it is immersed into a solution, a potential develops across an inert membrane that holds a liquid ion exchanger. By calibrating the electrode it is possible to determine the nitrate in the range from 0.1 mg/L to 1400 mg/L. Several different anions such as nitrite, cyanide, sulfide, and bromide may interfere with the measurement. It is also important to have the same pH and ion strength in both sample and calibration solutions, since the method depends rather on the nitrate activity than on the quantity of nitrate. Many on-line instruments utilize the fact that nitrate is absorbing light at 220 nm. It is a very rapid and convenient method since there is no need for reagent solutions and pumps and tubes for sample transport. There is though a possibility of interference from organic matter, which also could absorb at this wavelength. The normal procedure is to make an additional measurement at 275 nm where nitrate does not absorb and use this for correction of the amount of organic matter. This will of course introduce a small uncertainty in the result, which therefore may be regarded more as a tool for process control than an accurate analytical determination. However, if the content of organic matter in the sample is low or constant the accuracy of the method is improved.

II.5.3. CYANIDES
Giuseppe PALLESCHI, Mihaela BADEA Cyanide, a deadly poisonous substance, exists in water as HCN, a weak acid, Ka of 6 x 10-10. The cyanide ion has a strong affinity for many metal ions, forming relatively less-toxic ferrocyanide, [Fe(CN) 6]4-, with iron(II), for example. Volatile HCN is very toxic and has been used in gas chamber executions in the U.S. Cyanide is widely used in industry, especially for metal cleaning and electroplating. It is also one of the main gas and coke scrubber effluent pollutants from gas works and coke ovens. Cyanide is widely used in certain mineral-processing operations. Cyanides are metal salts or complexes that contain the cyanide ion (CN ). These cyanides could be subdivided into two categories: (1) simple cyanides such as

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NaCN, NH4CN, or Ca(CN)2 containing one metal ion (usually an alkaline or alkalineearth metal or ammonium ion) in its formula unit, and (2) complex cyanides such as K4Ce(CN)6 or NaAg(CN)2 containing two different metals in their formula unit, usually one is an alkali metal and the other a heavy metal. The complex cyanide dissociates to metal and polycyanide ions. The latter may further dissociate to CN which forms HCN. The degree and rate of dissociation of complex cyanides depend on several factors, including the nature of the metal, pH of the solution, and dilution. The cyanide ion and HCN are highly toxic to human beings, animals, and aquatic life. The toxicity of CN- is less than that of HCN, but in case of the water pollution, this is not important because most of the free cyanide exists as HCN, as the pH of most waters is substantially lower than the pK a of HCN. Analytical distinction between HCN and other cyanide species present as complex cyanides is possible [1]. The degree of dissociation of the various metallocyanide complexes at equilibrium increases at decreased concentration and low pH. The zinc- and cadmium-cyanide complexes are dissociated almost totally in very dilute solutions, which result in acute toxicity to fish at any ordinary pH. In equally dilute solutions there is much less dissociation for the nickel-cyanide complex and the more stable cyanide complexes formed with copper(I) and silver. The iron-cyanide complex ions are very stable and are not toxic. However, these complexes are subject to extensive and rapid photolysis, yielding toxic HCN, when diluted solutions are exposed to direct sunlight. For total cyanide determination, after removal of the interfering substances, the metal cyanide is converted to HCN gas, which is distilled and adsorbed in a sodium hydroxide solution. Then, the absorption liquid is analyzed using one the following methods: Silver nitrate titrimetric method Colorimetric method Ion-selective electrode method Ion chromatography II.5.3.1. Samples Pretreatment The nature of the preliminary treatment varies according to the interfering substances present. Sulfides, fatty acids, and oxidizing agents need to be removed by special procedures. Most other interfering substances are removed by distillation. Oxidizing agents (such chlorine) decompose most of the cyanides during storage and manipulation. For avoiding this, NaAsO 2 or Na2S2O3 should be added if the presence of the oxidizing agents is identified. Sulfides will distil over with cyanides and, therefore, affect colorimetric, titrimetric and electrochemical procedures. If presence of S 2- is identified lead acetate or lead carbonate is added. The fatty acids are removed by extraction with iso-octane, hexane or CHCl 3.

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The carbonate in high concentration may affect the distillation process by causing a violent release of carbon dioxide with excessive foaming when acid is added before distillation. Such samples should be preserved using calcium hydroxide. II.5.3.2. Silver Nitrate Titrimetric Method Cyanide reacts with silver nitrate as shown below forming the soluble cyanide complex, Ag(CN)2- . 2 CN- + AgNO3 Ag(CN)2- + NO3-

When all the CN ions in the sample are complexed by Ag + ions, any further addition of a few drops of titrant, AgNO 3, produces a distinct color with an indicator that can determine the end point of the titration. Thus, in the presence of a silversensitive indicator, p-dimethylaminobenzilidenerhodanine, Ag+ ions at first combine preferentially with CN. When no more free CN is left, the little excess of Ag + added reacts with the indicator, turning the color of the solution from yellow to salmon. Cyanide concentrations higher than 1 mg/L can be determined by titrimetry. Alternately I may be used as an indicator. Titrate to first appearance of turbidity (due to the formation of AgI). The titration method is suitable for cyanide concentration above 1 mg/L. II.5.3.3. Colorimetric Method Cyanide is converted to cyanogen chloride. CNCl, by treatment with chloramine-T at pH < 8 without hydrolyzing to cyanate, CNO . On addition of pyridine-barbituric acid or pyridine-pyrazolone reagent, CNCl, reacts with pyridine to form an intermediate nitrile, which hydrolyzes to glutaconaldehyde. The latter reacts with barbituric acid or pyralozone to give a blue color (absorbance reading at 578 nm)- the intensity of which is proportional to the concentration of cyanide in the sample. The colorimetric method can be used to determine CN - concentration in the range of 1 5 g/L. II.5.3.4. Ion-Selective Electrode Method The CN- in the alkaline distillate from the preliminary treatment procedures can be determined potentiometrically by using a CN - selective electrode in combination with a double-junction calomel reference electrode and a pH meter having a resolution of 1 mV. This method can be used to determine CN- in the concentration range of 10-5 to 10-2 M CN-.

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II.5.3.5. Cyanide in Solid Samples The determination of soluble cyanides requires sample leaching with distilled water until the solubility equilibrium is established. Cyanide analysis is then performed on the leachate. Low cyanide concentration in the leachate may indicate the presence of soluble metal cyanides. High levels of cyanide indicate soluble cyanide in the solid sample. II.5.3.6. Cyanide in Aerosol and Gas Samples Hydrogen cyanide and cyanide salts in aerosol and gas may be analyzed by NIOSH Method 7904 (NIOSH, 1984). Between 10 and 180 L of air at a flow rate of 0.5 to 1 L/min is passed through a filter-bubbler assembly of a 0.8-m cellulose ester membrane and 10 mL of 0.1 N KOH solution. While cyanide particulates retain over the filter membrane, HCN is trapped over the KOH solution in the bubbler. The membrane filter is then placed in 25 mL of 0.1 N KOH solution for 30 min to extract the cyanide particulates deposited on it. The KOH extract and the bubbler KOH solution are analyzed for cyanide by selective-ion electrode technique using KCN standards. REFERENCES 1. S.J. Broderius, Determination of Hydrocyanic Acid and Free Cyanide in Aqueous Solution. Anal. Chem. 53 (1981) 1472 2. In Situ Monitoring of Aquatic Systems. Chemical Analysis and Speciation . Ed. Jacques Buffle and George Horvai, 2000, John Wiley & Sons, Ltd., West Sussex, England 3. Standard Methods for the Examination of Water and Wastewater , 20th Edition, Ed Leonore S. Clesceri, Arnold E. Greenberg, Andrew D. Eaton, 1998, United Book Press, Inc., Baltimore, USA

II.5.4. HEAVY METALS

Giuseppe PALLESCHI, Mihaela BADEA, Andrei Florin DNE Although many of the metals are toxic, only some metals are major environmental pollutants, because of their widespread use. The European Pollutant Emission Register has classified seven metals as priority pollutants: cadmium, chromium, copper, lead, mercury, nickel and zinc.

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The metal industry and metal ore industry or sintering installations, installations for the production of ferrous or non-ferrous metals are the most important pollutant sources for cadmium (62 %), chromium (57 %), copper (63 %), lead (83 %), mercury (30 %) and zinc (84 %). The pollution with nickel is due to the mineral oils and gas refinery (41 %) and combustion installations (33 %). The effect of metals in the environment (water, air, soil) ranges from beneficial through troublesome to dangerously toxic. Some metals are essential to plant and animal growth while others may have toxic effect. The benefits versus toxicity of some metals depend on their concentration in the environment. Metals in general can be analyzed by Colorimetry and Atomic Absorption Spectrometry (AAS) or Atomic Emission Spectrometry (AES). In addition, some metals may be determined by other methods, including ionselective electrodes, voltammetry, ion chromatography, electrophoresis, neutron activation analysis, redox titration, and gravimetry. The choice of method depends on the precision and sensitivity required. Atomic absorption or emission spectrometry is often chosen, because it is rapid, convenient, and gives the low detection levels as required in the environmental analysis. Although colorimetry methods can give accurate results, they are time consuming and a detection limit below 10 g/L is difficult to achieve for most metals. Preliminary treatment is often required to present the metals to the analytical methodology in an appropriate form. II.5.4.1. Sampling and Treatment Metals can be classified as: - dissolved metals metals that in an un-acidified sample pass through in a 0.45 m membrane filter; - suspended metals metals that in an un-acidified sample are retained by a 0.45 m membrane filter; - total metals the concentration of metals determined in an unfiltered sample after vigorous digestion, or the sum of the concentrations of metals in the dissolved and suspended fractions; - acid-extractable metals the concentration of metals in solution after treatment of an unfiltered sample with hot dilute mineral acid. Before collecting a sample, decide what fraction is to be analyzed (dissolved, suspended, total or acid-extractable). This decision will determine in part whether the sample is acidified with or without filtration and the type of digestion required. Important errors may be introduced during sampling and storage because of contamination from the sampling device, failure to remove residues of previous samples from the sample container, and loss of metals by adsorption and /or precipitation in the sample container.

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The best sample containers are made of quartz or Teflon, but these materials are too expensive. The preferred containers are made of polypropylene or polyethylene. It is strongly recommended to use containers and filters that have been acid rinsed. Samples must be acidified immediately by sampling with concentrated nitric acid to pH<2. For dissolved metals, the samples are filtrated before preserving. After acidifying the sample, the samples are stored in a refrigerator at approximately 4 C to prevent change in volume due to evaporation. In these conditions, samples with several mg/L are stable up to 6 months (except mercury, for which the limit is five weeks). For metal concentrations of g/L, the samples should be analyzed as soon as possible after sampling. Also, for mercury analysis the samples may be preserved by adding 2 mL/L 20 % K2Cr2O7 solution (prepared in HNO3). II.5.4.2. Sample Digestion Aqueous and non-aqueous samples must be digested with an acid before their analysis by atomic absorption or atomic emission spectrophotometry. The metals and their salts present in the sample are converted into their nitrates due to the fact that the nitrates of all metals are soluble in water. Therefore, concentrated nitric acid by itself or in conjunction with hydrochloric acid, sulfuric acid, perchloric acid, or hydrofluoric acid is used in sample digestion for the determination of total metals. Nitric acid alone is, however, adequate for digestion of most metals. The acid digestion is performed using a small volume (5 to 10 mL) of nitric acid alone or in conjunction with one of the previously mentioned acids on a hot plate. Alternatively, a laboratory-grade microwave unit, specifically designed for hot acid digestion, can be used. When the sample is boiled with acid, the latter should not be allowed to dry. The acid extract after boiling and cooling is diluted with water to a measured final volume for analysis. II.5.4.3. Atomic Absorption Spectrometry for Heavy Metals Determination An atomic absorption spectrophotometer consists primarily of a light source to emit the line spectrum of an element (i.e., the element to be analyzed), a heat source to vaporize the sample and dissociate the metal salts into atoms, a monochromator or a filter to isolate the characteristic absorption wavelength, and a photoelectric detector associated with a microprocessor and a digital readout device for measuring the absorbance due to the metal at its corresponding concentration. The light source usually is a hollow cathode lamp or an electrode-less discharge lamp composed of the element to be measured. The heat source is an air-acetylene or air-nitrous oxide flame or a graphite furnace.

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In flame atomic absorption spectrometry, the heat source is a flame. The sample is aspirated into the flame and atomized. The light beam is directed through the flame. The metal atoms absorb energy at their own characteristic wavelength (Table II.5.1). The energy absorbed is proportional to the concentration of the element in the sample. Table II.5.1. Recommended Wavelength, Flame Type, and Technique for Flame Atomic Absorption Analysis. Metal Cadmium Chromium Cobalt Copper Iron Lead Manganese Wavelength (nm) 228.8 357.9 240.7 324.7 248.3 283.3; 217.0 279.5 Flame Air - acetylene Air - acetylene Air - acetylene Air - acetylene Air - acetylene Air - acetylene Air - acetylene N2O - acetylene Air - acetylene N2O - acetylene Air - acetylene N2O - acetylene Air - acetylene Technique Direct aspiration; Chelation extraction Direct aspiration; Chelation extraction Direct aspiration; Chelation extraction Direct aspiration; Chelation extraction Direct aspiration; Chelation extraction Direct aspiration; Chelation extraction Direct aspiration; Chelation extraction Direct aspiration Direct aspiration; Chelation extraction Direct aspiration Direct aspiration Direct aspiration Direct aspiration; Chelation extraction

Molybdenum 313.3 Nickel 232.0 Titanium Tin Vanadium Zinc 365.3 224.6 318.4 213.9

An atomic absorption spectrometer equipped with a graphite furnace or an electrically heated atomizer instead of the standard burner head gives better sensitivity and much lower detection limit than what is obtained with the flame technique. The principle of this technique is the same as for the flame method. A small volume of sample is aspirated into a graphite tube, which is heated in several stages. First, the sample is dried by low current heating. Then, it is charred at an intermediate temperature to destroy the organic matters and volatilize the compounds. Finally, it is heated to incandescence by a high current in an inert atmosphere to atomize the

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element. Atoms in their ground state absorb monochromatic radiation from the source. The intensity of the transmitted light is measured by a photoelectric detector. The intensity is inversely proportional to the number of ground state atoms in the optical path, i.e., the greater the quantity of ground state atoms in the optical path, the greater the absorbance (in other words, the less the amount of light transmitted through). The primary advantage of the graphite furnace technique over the conventional flame method is that the former requires a smaller volume of sample and the detection limit is much lower. Many metals can be determined at a concentration of 1 g/L (1 ppb). A disadvantage of the graphite furnace technique, however, is that because of high sensitivity, interference due to other substances present in the sample can cause a problem. Such interference may arise from molecular absorption or from chemical or matrix effects. This can be reduced or eliminated by correcting for background absorbance and by adding a matrix modifier into the sample. Some common matrix modifiers are: Mg(NO3)2, (NH4)2SO4, (NH4)2S2O8, NH4NO3, ascorbic acid, oxalic acid. Certain metals such as molybdenum, vanadium, nickel, barium, and silicon react with graphite at high temperatures, thus forming carbides. Such chemical interaction may be prevented by using pyrolytically coated tubes. To prevent the formation of metallic oxides and minimize the oxidation of furnace tubes, argon should be used as a purge gas. Chelation-Extraction Method Many metals at low concentrations can be determined by chelation-extraction technique. These metals include cadmium, chromium, cobalt, copper, iron, lead, manganese, nickel, silver, and zinc. A chelating agent such as ammonium pyrrolidine dithiocarbamate (APDC) reacts with the metal, forming the metal chelate, which is then extracted with methyl isobutyl ketone (MIBK). A 100-mL aliquot of aqueous sample is acidified to pH 2 to 3 and mixed with 1 mL APDC solution (4% strength). The chelate is extracted with MIBK by shaking the solution vigorously with the solvent for 1 min. The extract is aspirated directly into the air acetylene flame. The calibration standards of the metal are similarly chelated and extracted in the same manner and the absorbances are plotted against concentrations. APDC chelates of certain metals such as manganese are not very stable at room temperature. Therefore, the analysis should be commenced immediately after the extraction. If an emulsion formation occurs at the interface of water and MIBK, use anhydrous Na2SO4. The chelation-extraction method determines the chromium metal in hexavalent state. In order to determine the total chromium, the metal must be oxidized with KMnO4 under boiling and the excess KMnO4 is destroyed by hydroxylamine hydrochloride prior to chelation and extraction. Low concentrations of aluminum and

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beryllium can be determined by chelating with 8-hydroxyquinoline and extracting the chelates into MIBK and aspirating into a N2O-acetylene flame. Hydride Generation Method Arsenic and selenium can be determined using the hydride generation method. These metals in HCl medium can be converted to their hydrides by treatment with sodium borohydride. The hydrides formed are purged by nitrogen or argon into the atomizer for conversion into the gas-phase atoms. The reaction with NaBH4 is rapid when the metals are in their lower oxidation states as As(III) and Se(IV), respectively. Sample digestion with nitric acid, however, oxidizes these metals to their higher oxidation states, producing As(V) and Se(VI). These metals are reduced to As(III) and Se(IV) by boiling with 6 N HCl for 15 min. The digested sample is then further acidified with conc. HCl, treated with sodium iodide (for As determination only), and heated. Add to this solution 0.5 mL NaBH 4 solution (5% in 0.1 N NaOH, prepared fresh daily) and stir. The hydride generated (arsine or selenium hydride) is purged with the carrier gas such as argon and transported into the atomizer. Standard solutions of these metals are treated with NaBH4 in the same manner for the preparation of the standard calibration curve. The presence of other substances in the samples causes little interference because hydrides are selectively formed and are removed from the solution. The commercially available continuous hydride generator units make the operation simpler than the manual method outlined above. Cold Vapor Method for Mercury Determination Cold vapor atomic absorption spectrophotometric method is applicable only for the mercury analysis. The principle of this method is described below. After acid digestion with nitric acid, mercury and its salts are converted into mercury nitrate. Treatment with stannous chloride reduces mercury into its elemental form, which it volatilizes to vapors. Under aeration the vapors of mercury are carried by air into the absorption cell. The absorbance is measured at the wavelength 253.7 nm. Prior to reduction, any interference from sulfide and chloride are removed by oxidizing the extract with potassium permanganate. Free chlorine produced from chloride is removed by treatment with hydroxylamine sulfate reagent and by sweeping the sample gently with air. After the sample is acid digested with conc. H 2SO4 and HNO3, the extract is treated with two strong oxidizing agents: KMnO 4 and potassium persulfate (K2S2O8). After adding 15 mL of KMnO4 solution (5%) to the acid extract, let the solution stand for 15 min. To this solution, add about 10 mL of 5% K 2S2O8 solution. Heat the mixture to boiling for 2 h in a water bath. The excess of KMnO 4 is destroyed by adding NaCl-hydroxylamine sulfate solution (12% concentration of each, or a 10% hydroxylamine hydrochloride solution instead of (NH2OH)2 H2SO4).

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The calibration standards are made from a soluble mercury salt, such as, mercuric chloride. The standard solutions are analyzed first prior to the sample, following acid digestion, oxidation, and reduction, as described above. A standard calibration curve is constructed by plotting absorbance vs. concentrations of Hg (or mg Hg). The concentration of Hg in the sample is then determined by comparing the absorbance with that in the calibration curve. II.5.4.4. Specific Methods for Determination the Most Important Heavy Metals Pollutants Mercury A particular characteristic of mercury is that it exists in the environment in a number of different chemical and physical forms each with different behavior in terms of transport and environmental effects. Extensive research efforts have been put into the identification and quantification of these species over the last decades. In the atmosphere, the main three forms of Hg are: elemental Hg vapor (Hg 0), Reactive Gaseous Mercury (RGM) and Total Particulate Mercury (TPM). Of these three forms, only Hg0 has been tentatively identified with spectroscopic methods while the other two are operationally defined species, i.e. their chemical and physical structure cannot be exactly identified by experimental methods but are instead characterized by their properties and capability to be collected by different sampling equipment. RGM is defined as a water- soluble mercury species with sufficiently high vapor pressure to exist in the gas phase. The reactive term refers to the capability of stannous chloride to reduce these species in aqueous solutions without pre-treatment. The most likely candidate for RGM species is HgCl 2. TPM consists of mercury bound or strongly adsorbed to atmospheric particulate matter. Another species of particular interest is methylmercury (MeHg) due to the high capacity of this species to bioaccumulate in aquatic foodchains and to its high toxicity. Ambient concentrations of mercury in air may range between 1.0 to 3.6 ng m -3 for elemental mercury, from 1 to 50 pg m -3 for RGM and TPM whereas MeHg has been found in the range of 1 to 20 pg m -3. Mercury concentrations in precipitation samples depends on a number of factors primarily related to emission sources type, location of the monitoring station and meteorological conditions (i.e., frequency and intensity of precipitation events), however, typical concentrations observed in different European sites were in the range of 5 to 80 ng L -1 for total mercury, 5 to 50 pg m-3 for RGM and TPM, whereas MeHg levels were between 0.005 and 0.5 ng L -1. The sampling and analysis of atmospheric Hg is often made as Total Gaseous Mercury (TGM) which is an operationally defined fraction that includes species passing through a 0.45 mm filter or some other simple filtration devices such as quartz wool plugs and which are collected on gold. TGM is mainly composed of elemental

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Hg vapor with minor fractions of other volatile species such as HgCl 2, CH3HgCl or (CH3)2Hg. In the last few years, new automated and manual methods have been developed to measure TGM, RGM and PM. These developments make it possible to determine both urban and background concentrations of RGM, PM and TGM. A. Automated Methods for TGM The Sir Galahad II System is used to determine the mercury concentrations as TGM. The Sir Galahad II is based on the Millennium Merlin fluorescence detector. The air sample is pumped at a known flow rate through a filter and over the Amasil trap. Any mercury (Hg0) or mercury compound present forms an amalgam with the gold, thereby becoming trapped. The mercury can be reliably trapped even at elevated temperatures. On completion of the sampling phase the trap is flushed with argon. This removes any trace of the sample gas that could cause quenching and replaces it with argon, which allows maximum sensitivity. A rapid heating cycle is then activated converting all forms of mercury to the vapor, which enters into the detector where it produces a transient peak. The detection level is below 0.1 picograms. The Tekran Gas Phase Mercury Analyzers (Model 2537A) is suitable for TGM measurements. The pre-filtered sample air stream is passed through gold cartridges where the mercury is collected. The mercury is then thermally desorbed and detected in an integrated Atomic Fluorescence Spectrophotometry (AFS). The instrument utilizes two gold cartridges in parallel, with alternating operation modes (sampling and desorbing/analyzing) on a predefined time base of 10 min. With a sampling flow rate of 1.5 L min-1 a detection limit of 0.15 ng m -3 is achieved. A 47mm diameter Teflon pre-filter protects the sampling cartridges against contamination by particulate matter. The Gardis instrument is based on gold amalgamation and Atomic Absorption Spectrometry (AAS) detection. The Gardis instrument operates with ambient air as carrier gas and does not require Argon or Helium for detection. The sampling is run at about 1 L min -1 with sampling times of 10 minutes. Under these conditions, a detection limit of about 0.1 ng m-3 is achieved. A 25 mm diameter PTFE membrane is used to protect the analyzer gas inlet from contamination caused by aerosol particles.

B. Manual Methods for TGM

The manual methods are based on gold (or silver) trap amalgamation. The samples are manually analyzed using thermal desorption and cold-vapor atomic fluorescence spectrometry (CVAFS) detection. Samples are collected on 10-cm long traps consisting of a 6-mm diameter quartz glass tube containing a mixture of small pieces (1-2 mm) of gold wire and quartz glass grains. Alternatively, the adsorbing material may consist of gold-coated quartz glass grains. The airflow is normally > 0.5 L min-1. With 24 h sampling time the detection limit is typically 0.01 ng m -3.

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C. RGM Measurements The mist chamber (MC) technique has been developed by Lindberg and Stratton (1995). Air is drawn through a Pyrex glass chamber of 100 mL total volume containing 30 mL diluted HCl solution. Part of the MC-solution is dispersed as a fine aerosol, by a nebulizer inside the chamber. A hydrophobic filter at the top of the MC separates the droplets from the air and allows the liquid to drain back into the chamber. Mercury(II) species adsorbed in the MC solution are analyzed after reduction to elemental mercury by SnCl 2 using the CVAFS detection. The air sampling flow rate is 10-15 L min-1 and the detection limit for a 6 h sample is 1 pg m-3 Tubular denuders consist of 6 mm quartz tubes coated with KCl. During sampling, the denuders are electrically heated to approximately 45 oC to avoid water vapor condensation. The sampling flow rate is of 1 L min -1. Analysis is made using thermal desorption and CVAFS detection. The denuders are heated to 450 oC and purged with N2. The mercury released from the denuder is collected on a gold trap, which then is analyzed using CVAFS. The detection limit is of 5 pg m -3 for a 24 hintegrated sample. The annular denuders for sampling of RGM consist of a 15 mm outer diameter quartz tube with an inner, enclosed 8 mm tube. Air is pulled through the space between the two tubes. Both the inner surface of the outer tube and the outer surface of the inner tube are coated with KCl. The RGM is quantitatively collected in the annular denuder at a sampling flow rate of 5 - 10 L min -1. In the analysis step the denuder is heated to 500 C converting the adsorbed RGM to elemental mercury vapor, which is pre-concentrated on a gold trap. The gold trap is then analyzed using the normal desorption and CVAFS detection procedure. The detection limit for a measurement with 2 h sampling time is under 2-3 pg m -3. The annular denuders are suitable for automated applications. Lead In air sampling, high-volume samplers are preferable for accuracy (when it is necessary), but the low-volume technique is also useful for obtaining extensive data. As in all sampling for suspended particulate matter, the accuracy of volume meters should be checked periodically. The size of the pores of filters for collecting leadcontaining particles should be small, possibly less than 0.2 m for glass-fiber filters. Liquid scrubbers containing iodine monochloride and solid scrubbers with activated carbon, cristobalite, or iodine crystals may be used for sampling organic lead compounds in air, in the range of about 1 g m-3 or less up to 10 g m-3. Techniques for sampling water are less complex than for air. The major question is whether or not the water should be filtered before analysis since it is known that lead occurs in water both in the particulate fraction and in solution. For most purposes at least, it is reasonable to sample water without any fractionation of the material collected.

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However, in some cases it may be necessary to determine the biological availability for absorption of the various forms of lead that occur in water, and in soil. The latter is a dust source and may be a food contamination source as well. The preparation of soil and soil dust samples for lead analysis usually involves drying (at 100 C), homogenization by grinding, and sieving. The analytical methods currently in use for the estimation of lead content are of two general types, destructive and non-destructive. In the former, the sample is first oxidized to destroy all organic matter. The ash is then usually dissolved in an aqueous medium, either for further preparative steps or for direct instrumental analysis. Nondestructive methods are more recent and still too complicated for routine studies. They include X-ray fluorescence analysis and fast neutron activation. In selecting methods, consideration must be given to the cost of the equipment and the time involved in performing the analyses. The oldest and best known of the general methods currently in wide use are those based on the formation of the red complex that lead forms with dithizone (diphenylthiocarbazone). Numerous specific procedures have been developed based on the spectrophotometric determination of lead dithizonate. Perhaps no method of instrumental analysis for lead has enjoyed such a rapid acceptance in recent years as atomic absorption spectroscopy ( see II.4.4.2). Electroanalytical methods have also been found useful for lead determinations. These include polarography and, more recently, anodic stripping voltammetry. The polarographic method found wide application until more effective masking procedures were developed to increase the specificity of the dithizone method. Anodic stripping voltammetry is gaining in popularity for lead analysis. Two non-destructive methods for lead analysis have been under investigation in the last two decades. These are neutron activation and X-ray fluorescence. The first of these is not likely to find wide application for lead analysis because of the cost and the need for access to a fast neutron source. Its advantage is that the concentration of many elements can be determined simultaneously. X-ray fluorescence is also theoretically capable of detecting, nondestructively, all elements in a substance. A major obstacle to the wide application of this method is the profound matrix effect of the substances being analyzed. Another problem is the backscatter from the exciting source. Cadmium The most popular method for cadmium determination remains the atomic absorption spectrometry (see II.4.4.2). Cadmium can be determined, also by different types of electro-chemical methods such as classic polarographic methods or anodic stripping voltammetry and cadmium-selective electrodes. The basic principle behind the electrochemical methods is the change in the electrochemical potentials formed when electrons are transferred from one metal to another. A dropping mercury electrode is placed in a solution where

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the metal concentration is to be determined. By changing the charge of the electrode, different metals will be reduced and form an amalgam (a solid solution of metal atoms and mercury) with the mercury electrode. Polarographic waves can thus be recorded. Different metals can be determined simultaneously in a liquid sample, since they form amalgams at different charges. Anodic stripping voltammetry is based on the reverse process, i.e. the release of metals that have already been reduced and are bound to the mercury electrode. During oxidation and release from the amalgam, a peak current can be recorded at a potential that is characteristic for the particular metal. Anodic stripping voltammetry is one of the most sensitive methods for cadmium determination available. The most crucial aspects are complete destruction of all organic materials and the transfer of cadmium ions from the sample into a non-contaminated electrolyte. The method is especially suitable for water analysis, where no sample treatment is necessary. Specific cadmium-selective electrodes are commercially available, but their sensitivity is insufficient for cadmium measurement in most biological materials. Furthermore, the electrodes are not ion specific, and problems can easily arise from various contaminants in the solution used. Cadmium has a number of stable isotopes. Irradiation with neutrons yields new radioactive cadmium isotopes, which can be quantitatively measured on the basis of their specific energy and half-life. The irradiated sample is usually digested before the radioactivity is measured. Sometimes, it may be necessary to concentrate cadmium by chemical methods and to separate the cadmium ions from other isotopes that have an energy spectrum overlapping the one for cadmium before measurement can be carried out. Non-radioactive cadmium can also be added after irradiation to enable measurement of the recovery after digestion and various concentration steps. The detection limit for neutron activation analysis is low, of the order of 0.1-1 mg L -1 sample. However, the method is expensive since the samples have to be irradiated in a reactor, and so it is not normally used for screening. Irradiation with protons, proton-induced X-ray emission (PIXE), can also be used for activation analysis of cadmium. Several elements are measured at the same time. The main advantage of the method is its ability to detect and quantify cadmium in very small volume samples. Chromium Chromium occurs in each of the oxidation states from -2 to +6, but only the 0 (elemental), +2, +3, and +6 states are common. Divalent chromium is unstable in most compounds, as it is easily oxidized to the trivalent form by air. Only the trivalent and hexavalent oxidation states are important for human health. Therefore, they must always be examined separately. Although a compound CrF 6 is well known, the stable forms of hexavalent chromium are almost always bound to oxygen (e.g., CrO 4-2, Cr2O72 ). The trivalent form exists in coordination compounds, but never as the free ion. As a rule, its coordination number is 6, the complexes being generally octahedral.

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As chromium is present in low concentrations, care must be taken to avoid contamination. The collection of dust from air samples may introduce contamination from the chromium in the filters. Water samples may extract chromium from containers. Finally, reagents used in sample dissolution, separation, chelation, acid digestion, and other reactions, may contribute significant amounts of chromium. Thus, it is necessary to control for these influences by simultaneously performing a blank analysis, i.e., by carrying out the whole analysis, including sampling, preparation, and digestion, using all reagents. The sensitivity of instrumental analysis for the determination of chromium does not present any problems for concentrations in the mg/kg range, and a number of techniques can furnish satisfactory precision and accuracy. On the other hand, the sensitivity of the instrumentation for the determination of chromium in the ng or g/kg range is severely limited, and no one method is entirely satisfactory. Atomic absorption spectrometry and inductively coupled plasma emission spectrometry are the most used techniques for determination of the chromium content of water and sewage. For total Cr determination the X-ray fluorescence and neutron activation can be used. Gas chromatography with spectrometric detection can be applied only if Cr is chelated and extracted, with a detection limit of 1 ng. 30 ng/L Cr(III) can be measured using chemiluminescence detection. Copper The most important oxidation state in natural, aqueous environments is copper(II). Any copper(I) present is quickly oxidized by any oxidizing reagent present, or in a disproportionation reaction, unless it is stabilized by complex formation. The copper(II) ion binds preferentially via oxygen to inorganic ligands such as H2O, OH-, CO32-, SO42-, etc. and to organic ligands via phenolic and carboxylic groups. Thus, almost all of the copper in natural samples is complexed with organic compounds. Many cupric compounds and complexes are soluble in water and have a characteristic aqua-blue-green color. The trivalent form of copper is found in only a few compounds and is a strong oxidizing agent. In environmental and mineral environments, the divalent oxidation state readily adsorbs to a variety of hydrated metal oxides including those of iron, aluminum and manganese. Gravimetric and colorimetric methods were the earliest procedures used for the measurement of copper. Gravimetric methods are non-specific and may precipitate other cations including zinc, cadmium, cobalt and nickel. Useful spectrophotometric reagents for copper include cuprizone (biscyclohexanoneoxalydihydrazone), bathrocuproinedisulfonic acid (2,9-dimethyl4,7-diphenyl-1,10-phenanthrolinedisulfonic acid), bathocuproine (dimethyl-4,7diphenyl-1,10-phenanthroline) and more recently 1-(2-pyridylazo)-2-naphthol, BPKQH (benzyl 2-pyridyl ketone 2-quinolylhydrazone) and 2,2'-bicinchoninic acid.

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The bathocuproine method can achieve a limit of detection of 2 g Cu/Litre in water samples. Although colorimetric methods can suffer from lack of specificity, they are nevertheless useful, especially in laboratories where more sophisticated instrumentation is not available. Beyond a spectrophotometer and an analytical balance, no specialized equipment is required. In addition, the methods are, in general, simple, inexpensive, easily taught and rapidly carried out. Atomic absorption spectrophotometric (AAS) methods are the most widely used for the determination of copper in various matrices. A dramatic increase in sensitivity over that obtained by flame AAS is obtained with graphite furnace AAS. Increasingly more common is the use of emission methods in which the sample is introduced into a high temperature inductively coupled argon plasma (ICP) where the element is rapidly vaporized and ionized. The element is detected and quantified by atomic emission spectroscopy (ICP-AES). A further increase in sensitivity is obtained through the coupling of the ICP to a mass spectrometer (ICP-MS). The attraction of the ICP methods is the ability to do multielemental analysis, which is the obvious advantage over other spectroscopic techniques. The ICP-MS technique has the additional advantage that isotopic information can be obtained, which is especially useful if stable isotopes of copper are used. An isotope dilution ICP-MS method reported precision of less than 0.15 % for copper and cadmium in zinc ore and for copper and molybdenum in domestic sludge. Many X-ray fluorescence (XRF) methods, which are non-destructive techniques, have been published for the determination of trace elements including copper. XRF has been used for a long time as a rapid and convenient method for trace element determination although its sensitivity is somewhat lower than anodic stripping voltammetry. Field instruments are available for scans of contaminated sites to estimate the metal in the surface layer of the soil. Ion-selective electrode and potentiometric methods have been used for copper speciation in soil and in seawater. Voltammetric / potentiometric analyses offer sensitivity in the parts per billion (g/kg) range for copper. An attraction of potentiometric methods is their ability to help in the speciation of copper and limited multielement detection. Cathodic stripping voltammetry (CSV) is an extremely sensitive method for copper in both seawater and fresh water, with a limit of detection of 0.005 g/L. Nickel Nickel usually has an oxidation state of two, but also occurs as relatively stable tri- and tetravalent ions. Nickel forms complexes (chelates) that are insoluble in water, but soluble in organic solvents. These compounds are often very stable and play an important role in trace analysis. For example, nickel dimethylglyoxime is the compound that makes possible the separation of nickel from cobalt, which is similar in its chemical and analytical behavior. Prior to the determination of nickel in

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environmental materials, the organic constituents must be oxidized or removed to avoid interference during analysis. As nickel concentrations are often low in relation to analytical detection limits, pre-concentration steps are introduced, which may also separate nickel from substances interfering with analysis. Techniques very frequently employed include chelate extraction with dithiocarbamates, dimethylglyoxime, furildioxime, or 8hydroxyquinoline into organic non-polar solvents. Another pre-concentration technique, prior to analysis of nickel in fresh and seawater, is the use of chelating ionexchanged resins, e.g., Chelex 100 . A less time consuming method for the preconcentration of nickel in seawater involves complexation of the trace metals by 8hydroxyquinoline followed by adsorption on C 18 chemically bonded silica gel. A greater enrichment factor, smaller sample volume, and removal of interfering humic substances can be achieved by pre-concentrating nickel and other trace metals in natural waters on XAD-7 regions (cross-linked polymer of methylmethacrylate) in a two-step procedure at two different pH values. The two most commonly used analytical methods for nickel are atomic absorption spectroscopy and voltammetry. The introduction of a Zeeman-compensated system in electrothermal atomic absorption spectroscopy (EAAS) improved the background compensation and permitted a more rapid and direct determination of nickel levels with considerably lower detection limits (0.09 - 0.5 g/L), which was suitable for routine use. The progresses in voltammetry have made this method the most sensitive. The differential pulse voltammetry (DPV) may be used after prior interfacial accumulation by an adsorption layer of nickel-dimethylglyoxime chelate at the hanging mercury drop electrode (HMDE). The measurement of nickel concentrations as low as 1 ng/L was possible using this method. Though it requires time-consuming sample digestion procedures, voltammetry is more sensitive, more rapid, and less costly than EAAS. An isotope dilution gas chromatography-mass spectrometric method permits the detection of nickel in environmental samples at the ng/L level. The method depends on the preparation of a thermally stable and volatile chelate (chelating agents: sodium diethyldithiocarbonate or lithium bis(trifluoroethyl) dithiocarbamate) followed by on-column injection into a gas chromatographic column and electron ionization of the eluted chelate in the mass spectrometer. The analysis for nickel in natural water is frequently performed by EAAS following pre-concentration. Large concentration factors (200:1) provide detection limits as low as 10 ng/L in seawater analysis. Inductively-coupled plasma atomic emission spectroscopy (ICP-AES) has gained importance in simultaneous multi-element determination.

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Electron microscopy and X-ray microanalysis can be used for the determination of nickel in single dust particles, such as welding fumes and grinding dusts.

REFERENCES 1. In Situ Monitoring of Aquatic Systems. Chemical Analysis and Speciation . Ed. Jacques Buffle and George Horvai, 2000, John Wiley & Sons, Ltd., West Sussex, England 2. Handbook of Metals in Clinical and Analytical Chemistry , Ed. Hans G. Seiler, Astrid Sigel and Helmut Sigel, 1994, Marcel Dekker. Inc., New York, USA 3. Sampling for Analytical Purposes, Pierre Gy, 1998, John Wiley & Sons, Ltd., West Sussex, England 4. The Chemical Analysis of Water. General Principles and Techniques. 2 nd Edition. D.T.E. Hunt and A.L.Wilson, 1988, Alden Press, Oxford, Great Britain 5. Standard Methods for the Examination of Water and Wastewater , 20th Edition, Ed Leonore S. Clesceri, Arnold E. Greenberg, Andrew D. Eaton, 1998, United Book Press, Inc., Baltimore, USA

II.5.5. PESTICIDES
Andrei Valentin MEDVEDOVICI II.5.5.1. Monitoring of Pesticides Definitions Pesticides are substances, mixtures of substances or living (micro) organisms intended to prevent, repel, mitigate, and destroy any pest. Pests are representing living organisms occurring in places where they are not wanted, resulting in damages to humans, animals or crops. A pesticide residue represents any compound resulting from the physical, chemical or biochemical (metabolization in living organisms) degradation of a pesticide, resulting in the enhancement, conservation, or reduction of the initial toxic potential of the parent compound.

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Exceptions - the followings are not regulated as pesticides, despite their apparent fit to the previous given general definitions: a. drugs used to control human or animal diseases; b. fertilizers and nutrients (not considered plant growth regulators); c. biological control agents (e.g. beneficial predators that eat insect pest); d. products containing low risk ingredients (e.g. garlic or mint oil). Classification Pesticides are classified according the following criteria: a. the target pest they are intended to destroy (Acaricides, Algicides, Antifouling agents, Avicides, Bactericides, Fungicides, Herbicides, Insecticides, Molluscicides, Nematicides, Ovicides, Rodenticides, Viruscides); b. their mode of action (Antifeedant, Attractant, Chemosterilant, Defoliant, Desiccant, Fumigant, Growth regulator, Mating disrupters, Plant incorporated protectant, Repellent, Safener, Synergist); c. their nature (Inorganics, Organics, Biochemicals, Microbials); d. type of formulation in which a pesticide is included (Granulars, Dusts, Baits, Coatings, Aerosols, Slow release formulations); e. their impact on health (according to World Health Organization: Extremely, Highly, Moderately and Slightly Hazardous; according to International Agency for Research on Cancer : Carcinogenic, Probably carcinogenic, Possibly carcinogenic, Not classified, Probably not carcinogenic). Glossary of Terms Pesticide sources are either non-point sources or point sources. The non-point sources are represented by a) accidental release on storage; b) accidental drift during application; c) surface run-off from land at application points; d) leaching through soil. The point sources are represented by a) direct application points; b) wastes from manufacturing sites; c) spills during mixing, loading and transportation. The transport of pesticides in the environment can be achieved according to the following processes i) direct fallout from application points; ii) adsorption on carriers; iii) overland and/or subsurface flow (erosion); iv) atmospheric deposition (dry or wet). Pesticide fate: most of the lipophilic pesticides tend to undergo enzymatic reactions that make them more water-soluble and reactive by the attachment of polar functional groups such as OH. Most of these reactions are microsomal mixedfunction oxidase reactions catalyzed by the cytochrome P-450 enzyme system associated with the endoplasmatic reticulum of the cell and occurring most abundantly in the liver of vertebrates. The final products are then involved in conjugation reactions with endogenous conjugating agents (glucuronide, glutathione, sulfate, acetyl) resulting in conjugated species, which have a higher polarity, greater water solubility and thus are more easily eliminated. Pesticide persistence: the period of time during which a pesticide remains active at the application area describes its persistence. Its carryover effect describes the presence of the pesticide at the application area once its mission has been

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accomplished. The half life represents a measure of the pesticide persistence and is quantified as the amount of time taken by a pesticide to decompose by 50% from the applied chemical to an inactive form. Decomposition products may exhibit higher, reduced or no pesticidal activity compared to the parent compound, and may exhibit higher, reduced, or no risk effects on environment and human health. Pesticide resistance: developed resistance of the target to commonly used pesticides over a period of years by means of natural selection of the occurring biotypes. The mechanisms for resistance depend on the pesticides mode of action (as example, photosynthesis inhibition induced by triazines can be avoided by some weed biotypes by developing slight changes in the chloroplast protein structure). Sampling. Sample Preservation and Storage The sampling action should be oriented towards representativity and homogeneity. Sampled quantities should be in accordance with the analytical method further considered and the inherent sensitivity required. Usually, enough is taken for at least the application of a second full analysis procedure. Traditionally, sampling containers are made on glass. Plastic containers (polyethylene, polypropylene or polycarbonate) are often preferred for their robustness. Laboratory tests should be performed prior to the collecting action, in order to demonstrate the suitability of the material of the sampling container. For some particular compounds, adsorption on polymeric materials or even glass may be significant (captan adsorbs readily on glass, while organo-phosphorus compounds, but not glyphosate, are adsorbed on polyethylene). Mainly for water quality monitoring (environmental analysis) samples may be subjected to concentration at the point of collection. Adsorption cartridges or discs are obtained on the collection site and transported to the laboratory for analysis. Preserving sample integrity depends on the stability of the target analytes in a specific matrix. Freezing samples immediately after collection should provide preservation for thermally unstable extremely volatile compounds. Specific chemical addition of stability enhancing compounds is used occasionally (controlling the pH of the media or blocking active sites of the target analytes by means of derivatization). Contamination and cross contamination risks should be attentively considered (conditions for transportation and manipulation of empty sampling containers and collected samples, plasticizers contamination from container walls, etc.). Sample Preparation As showed in Figure II.5.1, sample preparation methods applied to pesticide analysis are of extreme variety. Depending on the type of sample to be analyzed, its size and corresponding concentration levels of the target compounds, more or less complicated procedures are used. More often, two or three successive sample preparation techniques need to be applied to a sample, in order to achieve analyte concentration and to eliminate efficiently the matrix interferences.

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Sample preparation methods used for pesticide isolation arranged according to the sample type are summarized in Figure II.5.2. A. Solid liquid Extraction For practical reasons, classical procedures are continuously upgraded to get improvements in terms of reliability and performances. Such an approach relates with the conventional Soxhlet extraction assisted by focused microwaves (FMSE). The main factors affecting extraction efficiency are microwave power, irradiation time, extractant volume and number of cycles. The result, in terms of extracted mass and repeatability is similar to the classical method, while important reduction of the process duration and saving of extractant are obtained. Organochlorine pesticides (OCPs) and related residues were successfully isolated from sunflower seeds by means of FMSE. Comparison with the ISO 659-1988 reference extraction method proved a better efficiency of FMSE. B. Liquid liquid Extraction (LLE) LLE is the classical method used for pesticide isolation, especially from water and biological fluid samples. Ethyl acetate, dichloromethane, and their mixtures are among the preferred extraction solvents for phenylureas, triazoles, amides, carbamates, benzimidazoles and chlorotriazines. The extraction efficiency is modified by adjustment of pH and ionic strength in the aqueous phase. In situ derivatization of the target analytes is also used as an effective tool (ex. chlorophenoxy acidic herbicides are derivatized with dimethyl sulphate prior to their extraction in n-hexane). The classical way of performing LLE is the separation funnel extraction. Some continuous LLE extractors or steam distillators are also available. C. Solid Phase Extraction (SPE) SPE is used for isolation of the target compounds from liquid media by means of adsorption on a granular solid bed (cartridge) or on a porous solid membrane (disk). After matrix removal, analytes are desorbed from the solid material using a specific solvent or mixture of solvents. The extract is concentrated by solvent thermal vaporization or gas flush. Direct desorption of the analytes into the LC or SFC columns by the mobile phase are achievable using an on-line set-up. When using desorption solvents non-miscible with the solvent of the initial matrix, special attention should be paid to cartridge / disk drying (this operational step is required between sample loading/clean-up and analyte desorption). When using on-line procedures, it is important to estimate the desorption kinetics of the trapped analytes. Contrarily, serious problems related to analyte focusing and chromatographic efficiency loss may arise. Four types of adsorbents are usually shared by SPE applications to pesticides. Hydrophobic modified silica materials (C18, C8, C2, C1) are extensively used for a large variety of samples (biological samples such as serum and urine for atrazine,

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simazine, prometryne, ametryn, sulfonyl ureas or environmental samples such as different types of waters for alachlor, aldicarb, methiocarb - with concomitant hydrolysis, medium polar, neutral and alkaline herbicides, phenyl ureas).

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Sampling Sampling storage Sample preparation Rough isolation Cleanup Target analyte(s) isolation Concentration Derivatization (precolumn) Separation method Derivatization (post column) Detection Data analysis Quantitation Structural confirmation

Accelerated Solvent Extraction (ASE), Focused Microwave Soxhlet Extraction (FMSE), Immuno Afinity Clean-up (ImCU), Liquid-Liquid Extraction (LLE), Low Temperature Lipid Precipitation (LTLP), Matrix solid phase dispersion (MSPD), Microwave Assisted Extraction (MAE), Nanofiltration (NF), Pressurized Fluid Extraction (PFE), Single Drop Microextraction (SDME), Solid Phase Extraction (SPE), Solid Phase Microextraction (SPME), Steam Distilation (SD), Stir Bar Sorptive Extraction (SBSE), Supercritical Fluid Extraction (SFE), Subcritical Fluid extraction (ScFE), Suported Liquid Membrane Extraction (SLME), Ultra-Sonication (US), Size Exclusion Chromatography (SEC), Liquid Chromatography fraction collection (LC). Gas Chromatography (including Fast Temperature Programming and Low Pressure)(GC), Liquid Chromatography (LC) (including capillary LC and capillary electrochromatography CEC), Supercritical Fluid Chromatography (SFC), Thin Layer Chromatography (TLC), Micellar Electrokinetic Chromatography (MEKC), Capillary Electrophoresis (CE).

Atomic Emission Detector (AED), Electrochemical Detection (ELCD), Electron Capture Detection (ECD), Evaporative Light Scatering Detector (ELSD, including condensation nucleation CN), Fluorescence Detector (FLD, including Laser induced fluorescence), Inductively Coupled Plasma Mass Spectrometry (ICP-MS), Mass Spectrometry (MSD), MS/MS, MS(n), Thermoionic Detector (NPD), Spectrometric Detection (UV, Vis, DAD)

Figure II.5.1. General analytical process for pesticide-containing samples.

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SAMPLE PREPARATION METHODS for Pesticides AIR SAMPLES 1. 2. 3. LIQUID SAMPLES Liquid Liquid Extraction (LLE) Size Excluzion Chromatography (SEC) Supported Liquid Membarenes (SLM) or Semi-permeable Membrane devices (SPMDs) 4. Stir Bar Sorptive Extraction (SBSE) 5. Solid Phase Microextraction (SPME) 6. Solid Phase Extraction (SPE) a. on line; b. off line; c. on discs; d. on cartridges I. matrix oriented (clean up) on shielded materials on ion exchangers II. analyte oriented (concentration) on silicagel hydrophobic modified materials on polymeric materials on porous & non-porous graphitized carbon blacks on molecular imprints on immunosorbents on ion exchangers III. mixed (SPE2) SOLID SAMPLES

Vapours 1. Supported Liquid Membarenes (SLM) or Semi-permeable Membrane devices (SPMDs) 2. Gum Phase Extraction (GPE) on Open Tubular Traps

1. 2. 3. 4. 5. 6. 7. 8.

Aerosols 1. Filter retention; 2. Gum Phase Extraction (GPE) on Open Tubular Traps

Soxhlet Extraction (SE) and Focussed Microwave (FMSE) Pressurized Fluid Extraction (PFE) Microwave Assisted Solvent Extraction (MASE) Supercritical Fluid Extraction (SFE) Soil Column Extraction (SCE) Head-space Solid Phase Micro-extraction (HS-SPME) Supported Liquid Membarenes (SLM) or Semi-permeable Membrane devices (SPMDs) Matrix Solid Phase Dispersion (MSPD)

Solid airborne particulates 1. Filter retention; 2. Laser desorption / ionization/MS;

Figure II.5.2. Sample preparation methods for pesticides according to sample type.

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Polymeric materials (styrene divinyl benzene based copolymers such as PLRP and Lichrolut EN) as well as vinyl pyrollidone divinyl benzene copolymers (commercially named OASIS) are mainly in current use. Triazines, ureas and amidic herbicides are better isolated on the more polar OASIS materials while other compounds including carbamates, thiocarbamates, aryloxypropionic acids, aryloxyphenoxypropionic acids and related metabolites are well isolated on styrene divinyl benzene (SDVB) hydrophobic materials. Recently, a new polymeric material, polyaniline, was introduced for extraction of chlorophenolic pesticides in water samples. Non-porous and porous graphitized carbon black solid beds (NPGC and PGC, respectively) are also used for herbicide isolation from water samples. Chlorophenoxy herbicides (requiring on-line derivatization with tetraalkylammonium salts), acetanilides, neutral diphenyl ethers, triazines, phenyl ureas, triazoles, benzimidazoles, phenoxyalkanoic acids and quats (paraquat, diquat, difenzoquat) are readily isolated on graphitized carbon beds. Few applications on pesticide isolation relate to the use of ion exchanging resins. The extraction of chlorophenoxy acidic herbicides residues from green bean samples (2,4-D; MCPA; 2,4-DP; 2,4-DB; MCPB; 2,4,5-TP and benzoic acid derivative of Dicamba) was achieved on hydroxymethylmethacrylate MFE polymer containing quaternary ammonium functional groups. Strong cation exchangers are moreover used for sample clean-up (matrix elimination). Glufosinate and related metabolites in hard water samples and chlormequat in pear, juices, and cereals require matrix clean-up on strong cation exchangers. Two new directions evolved in the last few years related to the SPE of herbicides: the synthesis of immunosorbents and polymeric molecular imprints. Extraction on immunoaffinity sorbents (ISs) is based upon the molecular recognition using antibodies. The extraction and clean-up of complex biological and environmental aqueous samples are achieved in a single step even for large volumes. The entrapment of esterases in a ceramic SiO 2 sol-gel matrix leads to isolation of organophosphates and carbamates. Anti-atrazine monoclonal antibodies and antidinitrophenyl polyclonal antiserum were also immobilized on the same purpose. Immunosorbents (IMS) produced by covalent immobilization of the antibody generated against the target analyte on inert materials (silica gel or activated agarose gel), were successfully used for isolation of analytes from complex matrices. Thifluzamide in peanut samples and imazalil in citrus fruits were rapidly isolated using IMS. Molecular imprints are synthetic polymeric phases on which selective receptors complementary to the target analytes have been generated during preparation. Ethylene glycol dimethylacrylate methacrylic acid copolymers can be imprinted with trialkylmelamines or dibuthylmelamine in order to generate templates selective to atrazine. Surface functionalization with molecular imprinted polymers was obtained also for porous polypropylene films. Desmetryn served for producing its own

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template. 2,4,5 trichlorophenoxy acetic acid was used as template in a system of 4vinylpyridine as monomer, ethylendimethacrylate as reticulation agent and methanol/water mixture as porogen. Some other materials were especially developed for specific matrix elimination. Restricted access or shielded materials eliminate selectively macromolecular compounds from pesticide containing samples (e.g. soil extracts, serum, plasma). Basically, the hydrophilic character of the external particles surface and the lipophilic character of the inner pore surfaces in these materials are responsible on their intrinsic capabilities of discrimination, combining size exclusion and polarity based interaction mechanisms. Owing to the increasing complexity of the sample matrices, sequential coupling of two SPE processes (SPE 2) is sometimes required. Generally, one SPE procedure is addressed to the isolation of the target compounds, while the other acts as a matrix remover (clean-up). D. Solid Phase Microextraction (SPME) SPME consists in the adsorption of the target compounds on a thin polymeric film deposed on the surface of a capillary fiber. The mass transfer can be achieved either from liquid media in direct contact with the extracting coated fiber as well as from gaseous environments. Volatile or semi-volatile pesticides existing in solid samples can be easily transferred in the gas phase on heating in closed vials, followed by trapping of the resulting vapors in the coated fiber (procedure is known as Head Space HD/SPME). The nature of the polymeric film as well as its thickness should be related to the polarity characteristics of the extracted compounds. Chloroanilines, sulphamides, phtalimides and oxazolidones (Dichloran, Chlorothalonil, Vinclozolin, Dichlofluanid, Captan, Folpet and Captofol) were extracted on polyacrylate, polydimethyl siloxane (PDMS), carbowax divinylbenzene and PDMS divinylbenzene films having thickness between 30 and 100 m. Recoveries of the analytes ranged between 70 and 124% when determined in sea water samples. Parameters as sample pH, ionic strength, organic additives, stirring rates, and contact duration have to be individually considered. Organochlorine herbicides were monitored in landfill leachates using PDMS coated fibers while ureas (Chlorsulfuron, Fluometuron, Isoproturon, Linuron, Metobromuron and Monouron) are better isolated on polyacrylate fibers. Trapped compounds can be further thermally desorbed directly into GC inlets, solvent desorbed directly to LC columns or on-line / off-line desorbed by supercritical state carbon dioxide for SFC separations. E. Pressurized Fluid Extraction (PFE); Supercritical Fluid Extraction (SFE); Assisted Solvent Extraction (ASE) Combining properties of supercritical fluids and/or extraction solvents with pressure/temperature/density effects and automatically controlled static/dynamic setup results in an increased flexibility and efficiency of the previously cited methods.

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However, two practices related to SFE / PFE / ASE processes should be mentioned. The first one relates to the fixation effects in the extraction thimble. Such practice was discovered during development of SFE procedures for pesticide determination in fruit juices (diatomaceous earth was solidifying liquid samples). Addition of silica onto solid vegetal materials (such as tobacco leaves) proved to induce a major matrix removal effect during analysis of OCPs. All pesticides enlisted in the 608 EPA method were 100% recovered by means of such a practice at a concentration level of 10 pg x L-1. The other practice relates to in-situ derivatization of target analytes during SFE / PFE / ASE procedures. Pesticides containing carboxyl or phenol groups were readily derivatized simultaneously in PFE by using acetic anhydride for acetylation, N-O-bis (timethylsilyl) trifluoroacetamide (BSTFA) for silylation and borontrifluoride / methanol, phenyltrimethyl ammonium hydroxide and trimethylsulfonium hydroxide for methylation. In-situ degradation of the target compounds in order to generate class markers was also practiced for non-persistent pesticide analysis in biological samples. 3,4 dichloroaniline and 3,5 dichloroaniline as markers for the exposure to fungicides vinclozolin, procymidone, iprodione, chlozolinate and herbicides diuron, linuron, neburon and propanil, respectively, are generated by the in situ hydrolysis simultaneously carried out with steam distillation (SD) removal , followed by liquidliquid extraction (LLE), derivatization with heptafluorobutyric anhydride (HFBA) and gas chromatography / electron capture detection (GC-ECD). F. Matrix Solid Phase Dispersion (MSPD) MSPD is a sample preparation process dating from 1989, realizing simultaneous disruption and extraction of solid or semi solid samples. The method is applied for the isolation of pesticides in tissues (animal or human origin), food, fruits, and vegetables. As an example, Atrazine, Cyanazine, Metribuzin and Simazine were studied to determine their toxicity in aquatic media (including correlations with environmental temperatures and dissolved oxygen content) by monitoring their concentrations in catfish muscle and liver using MSPD as selective extraction step. G. Gum Phase Extraction (GPE) GPE is an application of the sorption phenomena on polymeric materials used during the sampling process at temperatures above their glass transition point (T g). In such conditions, polymeric materials no longer behave as pure solids but enter a gumlike or even liquid-like state with properties similar to those of organic solvents (considering diffusion and distribution constants). The commonly used sorbent is polydimethylsiloxane (PDMS). Open tubular traps are more commonly coated in 1 - 3 m lengths of fused silica columns with 0.3 - 0.5 mm inner diameters and 10 - 15 m film thickness. Air samples should be sucked through the coated tubing. Trapped analytes are thermally desorbed into the gas chromatographic column. Extra column cryo focusing may be necessary.

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H. Stir Bar Sorptive Extraction (SBSE) Stir bar sorptive extraction (SBSE) is based on the same principle as GPE but the polymer covers the external surface of a magnetic bar stirrer. The rod is inserted within the liquid sample and stirred for a defined period of time. The same effect is obtained if PDMS gum is packed into a cartridge and the liquid sample is pushed through using HPLC pumps. Analytes are thermally desorbed directly in the gas chromatographic column or back extracted into an appropriate solvent, with subsequent solvent removal for concentration, re-dissolving and injection to a LC column. Good recoveries were obtained for triazines analyzed in water samples when isolation was made as stated previously (ca. 75% for Simazine, Atrazine, Propazine and Sebuthylazine, ca. 80% for Terbutryn and Prometryn, ca. 110% for Terbuthylazine). Cyanazine, Metribuzin, Desisopropylatrazine and Desethylatrazine were poorly recovered (< 7%). GC-MS was used for separation and detection. I. Supported Liquid Membranes (SLM) Micro porous membrane liquid-liquid extraction (MMLLE) of lipophilic pesticides in biological fluids combines the size exclusion properties of the membrane, reducing lipid co-extraction, with the stirring capabilities of the sample phase together with the permanent pumping of the organic phase. Hollow fibber membranes are also used for pre-concentration of nitrophenolic pesticides in water samples. The mechanism is based upon the pH difference inside and outside the hollow fibber, while the organic solvent was immobilized into the pores of the membrane. Such liquid-liquid-liquid micro extraction device (LLLME) is suitable for interfacing even with micro column liquid chromatography. Supported liquid membrane extraction (SLME) emerges also as a fast and efficient sample preparation alternative solution for pesticides. Aromatic aminophosphonate isolation from water samples based on SLME allowed identification and study of the operational parameters (pH and ionic strength of the aqueous phase, composition of the membrane phase and concentration of analytes) as well as the structure - extraction efficiency relationship. J. Size Exclusion Chromatography (SEC) SEC is generally required as an isolation procedure for pesticides in fat matrices. High molecular mass compounds (e.g. triglycerides, sterols) are excluded from the stationary phase material, while the light fraction is eluted later and is collected for subsequent clean-up, separation or analysis. A practical example is given by the determination of Thiofensulfuron methyl and Tribenuron methyl in cottonseeds and cotton gin trash. Separation Techniques A. Thin Layer Chromatography

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More often, Thin Layer Chromatography (TLC) is associated to the screening of pesticides, especially from water samples. The Automated Multiple Development (AMD) feature highly increases separation capacity and versatility of the method. The AMD approach uses more than 25 different development steps (1 - 3 mm migration distance each) with intermediate drying steps, the mobile phase composition, or nature changing continuously from step to step. Both mobile phase change and drying are made in an automated way, AMD-TLC equipments being commercially available. In fact, the entire procedure represents the application of three or even more different step composition gradients during a single run over TLC silica plates having thickness higher than 100 m. Because of intermediary drying steps, each new development generates focusing of the spots, finally leading to extremely sharp lines. Sample throughput of the method should be considered as satisfactory since 12 to 24 sample applications are feasible on a single TLC plate. Associating densitometry detection, quantitative determinations are allowed, even considering the possibility to produce UV-VIS reflectance spectra of the separated spots. Scratching spots from plates followed by analyte extraction from the solid material in appropriate solvents and subsequent introduction in a mass spectrometer make possible structural confirmation and sensitive quantitation. Standardization of AMD-TLC, as a DIN method for pesticide determination in ground and drinking water, dates since 1993. B. Gas Chromatography (GC) GC is a powerful technique for pesticide separation. Hot splitless injection (HSI) should be considered a rugged technique suitable for most pesticides. Flash vaporization is carried out at ca. 220 oC for most analytes. Some phenyl ureas, carbamates and organophosphoric compounds are susceptible to thermal degradation with HSI. On-column injection (OCI) or programmed temperature vaporization (PTV) are offering powerful alternatives. Silanized glass wool inserts also enhance the decomposition of labile compounds. Some applications of Large Volume Injection (LVI) on PTV are also cited in the literature for phenoxyacetic, trichlorophenoxy acetic and phenoxy propionic herbicides. Electronic Pressure Control (EPC) with pulse programming reduces decomposition of carbamates on injection. Of the GC methods for pesticides reported in literature, more than 90% use capillary columns (25 to 30 m length, 0.25 to 0.32 mm inner diameter and 0.15 to 0.30 film thickness). Medium polarity stationary phases are widely used (SE 52, 54, OV17, DB 5, DB7 or equivalent). Triazines and some phenyl ureas are separated also on nonpolar polydimethylsiloxanes (OV 1 or equivalent). Organophosphorus herbicides and chlorotriazines are separated with increased selectivity on SPB-35 or polyethylene glycols (Carbowax 20 M, Supelcowax RSL 300 or equivalent). The temperature gradient is the key for tuning selectivity in GC. Starting program temperatures for triazines and phenyl ureas are lower (40-60 oC) while higher values are used for phenoxycarboxylic esters (80-85 oC) and organophosphoric derivatives (100 oC). Temperature gradients of 15-30 oC min-1 are generally used for

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less complex samples, up to 260-290 oC and a single ramp. Two different steps are required for complex mixtures or crowded matrices (first gradient range in the interval 10-25 oC min-1, the second one between 3-6 oC min-1). Halogen containing pesticides are detected at the pg level with the ECD ( 63Ni). Low limits are also obtained in some cases with selective NPD or ELCD detection (1 ng). Mass spectrometric detection is widely used due to its universal character as well as for its intrinsic sensitivity and selectivity. Both EI and CI (using methane or isobutane as reagent gas) are used for ionization. C. Liquid Chromatography (LC) The main reasons for failure of GC methods for pesticide analysis are the thermal instability, low volatility, and high polarity characteristic of some target analytes. There are a relatively reduced number of classes of pesticides not suitable for GC separation and therefore requiring the use of liquid chromatography (LC). Among them, phenylurea, carbamates, dinitrophenols, benzimidazoles, azoles, benzoylureas, some organophosphorous, pyrethroid and quaternary ammonium derivatives should be cited. In LC, the separation mechanism dedicated to pesticide separations is undoubtedly the reversed phase one (RP). However, ion exchange (IE) and ion pair (IP) mechanisms are also used, especially for quaternary ammonium derivatives (quats). In RP applications, polar end capped octadecyl silicagel stationary phases are especially recommended for separations requiring high content or even 100 % aqueous containing mobile phases (e.g. separation of water un-extractible organophosphoric pesticides as acephate, methamidophos, monocrotophos, omethoate, oxydemeton-methyl and vamidothion). More often, the addition to mobile phases of concentrated buffers or strong acids is needed, in order to stabilize the analytes in a single form. Tautomeric equilibrium, competitive to chromatographic partition, is strongly affecting peak shape and symmetry. Sometimes, the inherent selectivity of LC affords separation of the tautomeric structures, leading to serious peak splitting, mainly at low concentration of the target compounds. The normal phase (NP) mechanism is used only for achieving enantioselective separations. Enantiomers of the organophosphoric pesticides crotoxyphos, dialifor, fonofos, fenamiphos, fensulfothion, isofenphos, malathion, methamidophos, profenophos, crufomate, prothiophos and trichloronate were successfully separated on polysaccharide based chiral stationary phases (ChiralPak AD, AS, OD and OJ) under NP conditions. Micro LC columns are used for a better compatibility with specific sample preparation methods (e.g. on column focusing) or interfaces for MSD. The use of micro columns also enables temperature gradient as an additional selectivity / efficiency tuning factor. Applications on triazines use temperature gradients ranging from sub ambient conditions up to 70 oC. The detection systems used for LC analysis of pesticides are: UV spectrometric detection (especially diode array detection - DAD - allowing peak

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confirmation by means of spectra comparison), fluorescence detection (FLD), electrochemical detection (ED), evaporative light scattering detection (ELSD) and MSD. Their characteristic sensitivities can be considered to increase in the following order: ELSD < UV (DAD) < ED ~ FLD < MSD. The particle condensation nucleation practices should also enhance ELSD sensitivity. Against the cited detection systems only ELS and MS are universal detectors. UV or FL detection of the target compounds is sometimes not achievable, due to the lack of suitable chromophoric / fluorophoric molecular sites. This could be overcome by derivatization. Two post column derivatization modes are noticeable for pesticide detection. One relates with the fluorescent detection of carbamates based on their reaction with orto-phtalaldehyde (OPA), the other one deals with the photo irradiation of benzoylurea insecticides (namely diflubenzuron, triflumuron, hexaflumuron, lufenuron and flufenoxuron) or pyrethroids (namely phenpropathrin, cyfluthrin, deltamethrin, fenvalerate, acrinathrin, fluvalinate and bifenthrin) with production of highly fluorescent photo degradation products. MS is undoubtedly the solution of the near future for LC detection. Improvements made on interfacing devices together with a continuous and sensible diminution of instrumentation costs promote MS as a universal / selective tunable detection system. Atmospheric pressure electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) are the most robust and popular devices for interfacing MS to LC systems. Although ESI and APCI are more often used, other LC / MS interfaces produce reliable results in pesticide applications: thermospray (TSI), particle beam (PBI) and matrix assisted post source decay LASER desorption / ionization (CID-PSD-MALDI). Multi dimensional techniques (MS / MS or (MS) n) enable to find out the right ratio between selectivity and sensitivity of the detection process. D. Supercritical Fluid Chromatography (SFC) Packed Column Supercritical Fluid Chromatography (P-SFC) offers an interesting alternative for pesticide separations based on the normal phase partition mechanism, even when using nonpolar modified stationary phases. Reproducible retention, high selectivity, and good peak symmetry are obtained on applying both density / pressure and modifier gradients. On-line hyphenation of SFC with SPE or SPME (desorption is made by the supercritical mobile phase) achieves high extraction yields and sample throughput. Interfacing with MSD (both ESI and APCI) does not require any modification of the commercialized interface designs but only an additional solvent flow, generally introduced before the automated pressure relief valve (nozzle) of a downstream configuration. A similar set-up based on the introduction of an additional suitable solvent flow through the nozzle leads to a versatile SFC semi-preparative or even preparative fraction collection / isolation alternative, allowing fast (automated) off-line bi-dimensional separation techniques. Extremely selective, reproducible and fast SFC applications relate to chiral separation

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of pesticide racemates on chemically modified polysaccharides or antibiotic modified stationary phases. E. Micellar Electrokinetic and Capillary Electro Chromatography Micellar Electrokinetic Chromatography (MEKC) and Capillary Electrochromatography (CEC) are powerful techniques achieving efficient pesticide and pesticide residue separations, although accepted standardized methods are still missing. The N-methylcarbamates, carbendazin, imazalil, methylthiophanate, prochloraz, procimidone, thiabendazole, triadimefon, metribuzin, lenacil, ethofumesate, atrazine, terbuthrin, isoproturon, chlorotoluron, linuron, desethyl atrazine, 2-hydroxyatrazine, desethyl 2-hydroxyatrazine, 3-chloro-4-methylphenyl urea are only some of the pesticides being reported to be separated by means of MEKC and CEC. On-line coupling of MEKC with ESI-MS was recently found as achievable using partial filling (PF) or reverse migrating micelles (RMM) techniques. Quantitative migration toxicity relationship for phenoxy acid herbicides has been obtained with MEKC on using micellar Brij 35 based migration media. On-line stacking procedure carried out on injection acts as a pre concentration step and together with SPE for sample preparation shifts detection limits in the low g x L-1 range, even on use of the classical on column diode array detection. Immunochemical Assays Immunoassays (IAs) are based upon the selective interaction between a specific antibody (Ab) and a hapten (H or antigen - Ag). Pesticides are not ordinarily antigenic; consequently they have to be conjugated to a carrier molecule (usually a protein) in order to induce an immune response. The result of the binding reaction between the Ab and H can be measured by means of enzymatic methods (EIAs), radiometric methods (RIAs), fluorescence methods (FIAs, including the polarized PFIA approach) or chemiluminescence methods (CLIAs). Some of the immunoassays designed for pesticides are given in Table II.5.2, together with the corresponding detection limits. Bio- and Immuno Sensors Bio and immunosensors are systems based on measurement of the results generated by means of the binding to a specific receptor of the target analyte. Main advantages related to bio and immuno sensors lies to the high sample throughput, any or limited sample preparation procedures, in situ measurement capabilities and selectivity. Drawbacks are related to inherent matrix interferences (mainly on biosensors), limited shelf life, difficulties for coupling receptors to supports, identification of sensitive transducers and a relative delay in generating the response. The measuring principle of a biosensor designed for pesticide determination is mainly enzyme inhibition. Cyanobacteria, thylakoid membranes, protoplasts,

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immobilized enzymes and labeled enzymes are receptor components. The analyte blocks the enzyme, enzyme systems or electron transport systems of intact cells. In an immunosensor, the antigen antibody selective interaction is monitored. In order to enhance on sensitivity, the indirect monitoring of the Ag Ab interaction should be preferred, using competitive tracers or markers. Determination of alachlor, as an example, can be achieved with increased sensitivity by using liposomeencapsulated markers in a competitive binding reaction instead of enzymes. The conjugation of a lipid to a pesticide allows its incorporation into a liposome structure, leading to competitive liposome immuno assay based sensor. The use of piezoelectric crystals as physical sensors is a continuously developing research direction. Other physical sensors are optics, such as surface plasmon resonance (SPR), interferometric or grating couplers. Bio and immuno sensors are complementary to high-resolution techniques and represent a stimulating border research area between chemical analysis and microelectronics. Table II.5.2. Immunochemical assays for some pesticides.
No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Pesticide Aldrin Atrazine Benomyl Bioallethrin Captan Carbofuran Chlorothalonil Chlorpyrifos 2,4 - D Dichlorprop Dieldrin Heptachlor Hexazinone Imazamethabenz Picloram Pyrimiphos methyl Simazine 2,4,5 - T Thiabendazole Triadimefon Test format RIA CLIA PFIA RIA RIA EIA EIA EIA EIA RIA PFIA PFIA RIA EIA EIA EIA RIA EIA PFIA RIA EIA EIA Ag Type p p p p p p p p p p m p p m p p p p p p m p Detection limit g L-1) 0.7(ng)* 0.025 0.100 1.250 0.03 1.000 0.060 0.070 0.050 5.000 0.600 10 .000 0.08 (ng)* 10.000 0.220 0.500 50.000 30.000 3.000 1.000 0.500 2.000 Sample type Biological fluids Environmental Environmental Food, crops Food Soil Water Soil, water Biological fluids Food Soil Cereal grains Water, urine Wheat grains, flour Surface water Liver Food

m monoclonal antibody; p polyclonal antibody; * - referred as absolute quantity.

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REFERENCES

1. R.B. Geerdink, W.M.A. Niessen and U.A.Th. Brinkman, Trace level

determination of pesticides in water by means of liquid and gas chromatography. Journal of Chromatography A 970: 65-93 (2002). 2. H. Kataoka, New trends in sample preparation for clinical and pharmaceutical analysis. Trends in Analytical Chemistry 22: 232-244 (2003). 3. L.H. Keith (ed) Compilation of EPAs Sampling and Analysis Methods, 2 nd Edition. Boca Raton: CRC Press (1996). 4. C.D.S. Tomlin (ed) The Pesticide Manual, 12th Edition. British Crop Protection Council Publications, Hampshire, UK (2000). 5. E. Mallat and D. Barcel, Immunosensors for pesticide determination in natural waters. Trends in Analytical Chemistry 20: 124-132 (2001). 6. D.B. Barr, Analytical methods for biological monitoring of exposure to pesticides: a review. Journal of Chromatography A 778: 5-29 (2002). 7. V. Camel, Microwave-assisted solvent extraction of environmental samples. Trends in Analytical Chemistry 194: 229-248 (2000). 8. L.J. Krutz, S.A. Senseman and A.S. Sciumbato, Solid-phase micro extraction for herbicide determination in environmental samples. Journal of Chromatography A 999: 103-121 (2003). 9. P.D. Patel, (Bio)sensors for measurement of analytes implicated in food safety: a review. Trends in Analytical Chemistry 212: 96-115 (2002). 10. L. Ramos, E.M. Kriestenson and U.A.Th. Brinkman, Current use of pressurized liquid extraction and sub critical water extraction in environmental analysis. Journal of Chromatography A 975: 3-29 (2002). 11. H.J. Stan (ed), Analysis of Pesticides in Ground and Surface Water . Berlin: Springer Verlag (1995).

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II.5.6. POLYCHLORINATED BIPHENYLS (PCBs)

Giuseppe PALLESCHI, Mihaela BADEA Polychlorinated biphenyls (PCBs) are a group of theoretically 209 different compounds of which 150160 are found in nature. It should be noted that the range of PCBs are very different substances, both with regard to chemical-physical properties and with regard to their biological activity. A distinction is commonly made between the generally more carcinogenic dioxin-like PCBs (non- and mono-ortho chlorinated) and the more immunotoxic bulky PCBs (chlorinated in the ortho positions). PCBs are not found naturally, but are now found all over earth due to their persistence and relative volatility. PCBs are lipid soluble, have very low water solubility and are predominantly associated with particles in natural waters. Due to their extreme mobility and ability to bioaccumulate and magnify in marine food webs, long-lived animals at high trophic levels appear to be most at risk from PCBs. These substances have high boiling points, exhibiting high chemical and thermal stability, and flame resistance. World production of PCBs is minor at the present. PCBs are severely restricted in some countries, including EU countries and banned in others. Since 1929 at least 1 million tones have been produced. More than half is still in the environment or in products. PCBs exist in mixtures composed by several components that produce multiple peaks when a chromatographic method is used. An oven temperature in the range of 200C and detector and injector temperatures around 300C and 250C, respectively, should give good separation, sharpness of peaks, and fast analysis time. Electron capture detector (ECD) is the most commonly used detector for trace level analysis of PCBs by gas chromatography (GC), exhibiting a response to an amount below 0.1 ng PCBs. Thus, on a capillary column, a detection limit (l.o.d.) in the range of 5 g/L can be achieved. With proper sample concentration steps, detection levels several-fold lower to l.o.d. may be obtained. Other halogen-specific detectors such as Hall electrolytic conductivity detector can also be used to analyze PCBs. Because PCBs produce multiple peaks, extra care should be taken to identify the genuine peaks from any other contaminants, such as phthalate esters, sulfur or chlorinated pesticides, and herbicides, to avoid any false positive inference. The following steps should be taken for the qualitative determination: 1. All the major peaks of the reference Aroclor standard must be present in the GC chromatogram of the unknown sample extract.

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2. The retention times of the sample peaks must closely match with that of the standard(s). One or more internal standards (such as dibutyl chlorendate, tetrachloro-m-xylene or p-chlorobiphenyl) should be added into the sample extract, as well as Aroclor standards, to monitor any response time shifts. 3. When the chromatogram of the unknown sample has a number of peaks (some of which show matching with an Aroclor standard), then the ratios of the areas or heights of two or three major peaks of the unknown should be compared with the corresponding peak ratios in the standard at the same retention times. For example, if a sample is found to contain any specific Aroclor, measure the area or height ratio of largest to second largest peak in the sample extract and compare the same to that in the standard. The comparison of peak ratios, however, should not always be strictly followed, as it can lead to erroneous rejection. For example, because some of the same chlorobiphenyl components are common to most Aroclors, the presence of two or more Aroclors in the sample can alter the peakratio pattern. Similarly, the kinetics of oxidative or microbial degradation of chlorinated biphenyls can be different for each compound in the PCB mixture. 4. Finally, the presence of Aroclor found in the sample on the primary column must be re-determined and confirmed on an alternate column or by GC/MS using selective ion monitoring mode. II.5.6.1. Quantitation Although GC/MS is the most reliable technique for qualitative determination of PCBs, its quantitative estimation in environmental samples is more accurately done from the analysis by GC-ECD. The area counts or the heights of all major PCB peaks in the sample are first added up and then compared against the total area or the heights of the same number of peaks at the same retention times in the standard. A singlepoint calibration can be used if the peak areas of the PCBs are close to that of the standard. Often, many environmental samples show the presence of only some but not all characteristic PCB peaks. II.5.6.2. Sample Extraction and Cleanup Aqueous samples are extracted with methylene chloride by liquid-liquid extraction. The extract is concentrated and then exchanged to hexane. Soils, sediments, and solid wastes are extracted by sonication or Soxhlet extraction. Samples should be spiked with one or more surrogate standard solution to determine the accuracy of analysis. Some of the internal standards mentioned above may also be used as surrogates. If only the PCBs are to be analyzed, hexane instead of methylene chloride may be used throughout. Oil samples may be subjected to waste dilution, i.e.,

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diluted with hexane or isooctane and injected onto the GC column for determination by ECD or HECD. Sample cleanup often becomes necessary to remove interfering substances such as phthalate esters and many chlorinated compounds frequently found in wastewaters, sludges, and solid wastes. Most interfering contaminants can be removed from the solvent extract by gel permeation chromatography or by florisil cleanup. In addition to these cleanup methods, the sample extract should be further cleaned up by shaking 1 mL extract with an equal volume of KMnO 4 - H2SO4 (KMnO4 in 1:1 H2SO4). Most organics at trace levels are oxidized under these conditions forming carbon dioxide, water, and other gaseous products, leaving behind the PCBs in the hexane phase. The hexane phase is then washed with water, and the moisture in hexane is removed by anhydrous Na2SO4. If sulfur is known or suspected to be present in the sample, an aliquot of the cleaned extract after KMnO 4 treatment may be subjected to sulfur cleanup either by using mercury or copper powder.

II.5.6.3. Alternative Analytical Methods PCBs at high concentrations can be measured by GC-FID, NMR, and HPLC. Concentrations over 100 ppm can be determined by HPLC by UV detection at 254 nm. A normal phase HPLC technique with column switching can separate PCBs from chlorinated pesticides. PCBs in soils and wastewaters can be rapidly screened on site or in the laboratory by immunoassay technique. Immunoassay test kits are now commercially available from many suppliers. The samples can be tested at the calibration levels of 1 to 50 ppm. The kit primarily contains antibodycoated test tubes or magnetic particles, assay diluent, PCB-enzyme conjugate, a color-forming substance, and a solution to quench the reaction. The method does not distinguish accurately one Aroclor from another. PCBs can be measured semiquantitatively by comparing the optical density of the color formed in the sample against a set of calibration standards using a spectrophotometer. REFERENCES

1. Standard Methods for the Examination of Water and Wastewater , 20th Edition, Ed
Leonore S. Clesceri, Arnold E. Greenberg, Andrew D. Eaton, 1998, United Book Press, Inc., Baltimore, USA 2. Gas Chromatographic Environmental Analysis. Principles, Techniques, Instrumentation, Fabrizio Bruner, 1993, VCH Publishers, Inc.New York, USA

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II.5.7.

BIOLOGICAL OXYGEN DEMAND, BOD

Tomas ALEXANDERSSON

BOD is an acronym for biological oxygen demand, which is an empirical method for determination of how much oxygen is consumed during biological degradation of compounds present in a water sample. The method is used for determination of the impact a wastewater stream will have on a recipient and to evaluate a wastewater plants BOD-removal efficiency. Both oxygen consumed by biological degradation of organic matter and oxygen consumed by oxidation of inorganic material such as sulfide and ferrous iron are measured in the method. First the water sample is diluted to a suitable concentration since normally the BOD concentration is larger than the available oxygen concentration in an airsaturated sample. Nutrients such as nitrogen, phosphate and trace minerals are added to the diluted sample and after adjustment of pH to a value between 6.5 and 7.5 the solution is inoculated with a seed. The seed usually originates from a municipal biological treatment plant. The solution is transferred to three incubation bottles. One bottle is used for determination of the initial dissolved oxygen (DO) concentration. The other two bottles are incubated in a dark space with constant temperature (20C 1C). The time that the bottles are incubated is somewhat different in different countries. Normal incubation periods are either 5 or 7 days. When the appropriate time has elapsed the remaining oxygen concentration is measured in the two bottles. The difference between the initial value and final value corrected for seed consumption is a measure on the samples BOD. When using a seed from a municipal treatment plant with nitrification, any oxygen consumption due to the oxidation of ammonia will also be included in the BOD-value. To prevent this from happening an inhibitor could be added that will make nitrification impossible. Common inhibitors are allylthiourea (ATU) and 2chloro-6-(trichloromethyl) pyridine. There are several criteria that have to be fulfilled before a BOD-analysis can be regarded as valid. First of all should the DO-consumption for unseeded dilution water not exceed 0.2 mg/L. This could otherwise indicate a too poor quality on the dilution water. There is also a criterion for the oxygen consumption for the seeded dilution water, which should not exceed 1.0 mg/L. The final DO-concentration in the bottles should not be lower than 1 mg/L and there should be an oxygen consumption of at least 2 mg/L. If the final value is below the limit the test has to be repeated with a more diluted sample. The consumption could be too low if the sample is too diluted or if it is toxic to the seed.

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The method and procedure could be tested by including a control consisting of a mixture of 150 mg/L glucose and 150 mg/L glutamic acid. In an inter laboratory study it was found that the average BOD 5 value for this control mixture was 198 mg/L with a standard deviation of 30.5 mg/L.

II.5.8. CHEMICAL OXYGEN DEMAND, COD

Tomas ALEXANDERSSON Chemical oxygen demand (COD) is a method for determination of the amount of organic matter that could be oxidized by a strong chemical oxidant and is expressed in oxygen equivalents. The oxidation is done in a boiling mixture of potassium dichromate (K2Cr2O7) and sulfuric acid. Since halides are interfering with the measurement a complexing agent consisting of mercury sulfate (HgSO 4) is usually added. Formerly was the reflux method with titration used for COD determination but today there are several manufacturer that offers commercial tubes filled with reagents and the reading is done with a spectrophotometer. The analysis is rather simple with theses test kits. The procedure is as follows: Shake the test tube in order to stir up the solid matter in the reagent. Add the specified volume of the sample to the tube. Seal the tube and clean the outside of the tube carefully. Boil the sample at the specified temperature and for specified time, which usually means 2 hours at 148 C. Take out the hot tube and invert it two times. Let the tube cool to room temperature, clean the outside of the tube and evaluate in a spectrophotometer. For evaluation of the method a solution of potassium hydrogen phthalate could be used. In a inter laboratory study with a 200 mg COD/L solution the achieved standard deviation was 13 mg/L.

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