TOPIC 2: THE ANALYTICAL PROCESS

SSCK 1203 2/20122013

STEPS IN AN ANALYSIS
The analytical process consists of the following unit operations

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

Problem definition Method selection Sampling Sample Preparation Eliminating interferences Measurement Reporting

(1) Problem Definition

Questions to be answered by chemical measurements (What information is needed?) Problem - what needs to be found? Information - What is the sample, who will use it, How will it be used? Qualitative or quantitaive - How sensitive must the method be, what is the degree of accuracy/precision, how are interferences eliminated Budget and time constrain How much is the cost, when is the information required?

(2) Method Selection

The analytical method to be used depends on the following Sample type, size and preparation required Skill and training of analyst Tools/instruments available Selectivity, precision, sensitivity required Cost (budget) and speed Time required and target deadlines Availability of methods in the chemical literature (Books, journals, manuals, etc) Availability of standard methods

(3) Sampling
Sampling: The process to get a representative and homogeneous sample - Representative - content of analytical sample reflects content of bulk sample - Homogeneous - the analytical sample has the same content throughout

Sample collection depends on - The type, size, homogeneity of the bulk sample - The physical state of the sample (solid, liquid, gas) - The chemical state of the material to be assayed (Preserve sample so that the identity and concentration of the analyte to be analyzed is not destroyed or altered) Apply statistics and error determined

Sampling Steps
(1) Identify the population from which the sample is to be obtained (2) Collect a gross sample that is truly representative of the population being sampled. (3) Reduce the gross sample to a laboratory sample that is suitable for analysis
Sampling methods commonly used Grab Sample - Portion of sample removed from the target population Composite Sample - Several grab samples combined to form a single sample In-situ Sampling - Sampling done within the population without physically removing the sample

Methods of sampling, liquids and gases are given in standard reference books eg: ASTM (American Society for Testing and Material) APHA (American Public Health Association) AOAC (Association of Official Analytical Chemists International) Homogeneous parent samples Simple grab sample approach taken at random and assumed representative Heterogeneous parent samples - Several samples have to be taken

SAMPLING SOLIDS
Solid materials are heterogenous making sampling difficult Gross composition - special sampling techniques will be required to obtain a representative sample

The larger the particle size, the larger the gross sample should be It is best to take 1/50 to 1/100 of the total bulk

Cone and Quarter Method

The gross sample must be reduced in size to obtain a laboratory sample A commonly used sampling method is the cone and quarter method The sample is crushed and mixed to form a conical pile The pile is flattened and cut into equal quarters, and two opposite quarters are collected at random The quartering process is repeated until the desired sample size is obtained

SAMPLING LIQUIDS
- Liquid samples are homogeneous - easier to sample - The gross sample can be relatively small - Sampling techniques will depend on the types of liquid
Examples 1. Small quantities of non homogeneous liquid sample is shaken and sampled immediately 2. Large volume of liquids are sampled after a transfer or during discharge or if in a pipe, sampled after passing through a pump when they have undergone thorough mixing 3. Large stationary liquids are sampled at different depths using a thief sampler The separate aliquots of liquids can be analyzed individually or can be combined into one gross sample (composite sample) 4. For biological fluids, the timing of sampling is very important

SAMPLING GASES
Gases tend to be homogeneous
A large volume of sample is required because of their low density Examples
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Air analysis:
Air Sampling Filters

Use a `Hi-Vol sampler that contain filters to collect particulates

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Liquid displacement method:

The sample must be slightly soluble in the liquid and does not react with the liquid
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Breath sample:
Air Sampling Pump

Dust Sampler

The subject blows into an evacuated bag

Sample storage and preservation

Samples are preserved to prevent: Decomposition of biological samples (by bacterial action) or heat and light labile samples - Refrigerate after collection until the time of analysis, protect from light, seal or store under vacuum or nitrogen Precipitation of metals from water samples - Acidify by adding 10% HNO3 immediately upon collection Loss of water from hygroscopic material Loss of volatile analytes from water samples

Sample Preservation Techniques

Method
Use appropriate sampling container

Description
Teflon (PTFE) - for ionic analyte Glass container - for organic analyte Antioxidants, Antibacterials To avoid thermal degradation To stabilize or immobilize the analyte For metal analyte

Addition of chemical stabilizers Freezing the sample Adsorption on a solid phase Addition of HNO3 (pH < 2)

Sample storage Conditions

Sample Volatile sample Thermally labile sample Storage Conditions Keep in sealed containers and store in the refrigerator or freezer

Liquid samples

Keep in cool, dark area (away from sunlight)

(4) Sample Preparation

Sample preparation (aka sample pretreatment or treatment) is a step in chemical analysis where the sample is brought into the correct size form for analysis General Principles Should not lose any analyte Bring the analyte into the best chemical form for assay method used Remove interferences Should not add any new interferents Dilute or concentrate the sample to bring analyte concentration into the best range for the method

PREPARING A LAB SAMPLE

Replicate samples must always be performed unless the quantity of the analyte or other factors prohibit
(Replicates are samples of the same size carried through the analytical procedure at the same time and the same way)

Ways of converting samples to useful forms Grind solids to a suitable size to obtain a homogeneous sample Dry* the samples to get rid of adsorbed water Weigh dried samples (store in a desiccator) Dissolve in solution (aka dissolution)

SAMPLE DRYING METHODS

Sample

Drying Conditions Heat at 110oC Depends on sample (removes adsorbed vapors) Heat at <100oC

Inorganic sample Common organic sample Biological sample

Hygroscopic sample
Oxidizable sample Heat sensitive sample

Dry in vacuum desiccator

Dry in vacuum desiccator or under nitrogen

Freeze dry

PREPARING SOLUTION (Sample dissolution)

Most analyses are performed on solutions A solvent is chosen that dissolves the whole sample without decomposing the analyte Types of sample dissolution (1) Destructive - those that totally destroy the sample matrix (2) Nondestructive or partially destructive - mild or non evasive dissolution - Destructive dissolution can be used only when the analyte is inorganic or can be converted to an inorganic derivative for measurement - Non destructive dissolution if the analyte to be measured is an organic substance

Errors in Preparing Solutions

Several sources of error are encountered in the sample dissolution step Incomplete dissolution of the analyte Losses of analyte by the volatilization (evaporation) Introduction of analyte (external) as a solvent contamination Contamination from the reaction of the solvent with vessel walls

DISSOLUTION PROCEDURES
Inorganic Solids Simple Dissolution - Dissolve sample in water Wet Digestion (aka acid treatment) - Heat with mineral acids in open/closed container Fusion Technique - Heat with acid or flux until molten state

Organic Solids Dry Ashing - Oxidize by slow heating in oxygen at very high temperature (in furnace) Wet Digestion - Heat with mineral acids in an open or closed container

Acid Treatment Of Inorganic Solids

(In open containers: eg in a beaker on a hot plate) Advantage: low cost Disadvantage: Loss of analyte by volatilization HCl : Carbonates, phosphates, oxides H2SO4 : Organic material at 300C HNO3 : Any metals not dissolve by HCl HClO4 : Steel HF : Silica Aqua Regia (HCl+HNO3 3:1) for unstable inorganic HNO3+HCl+HF (5:15:3) for alloys

Grades of acids Very High Purity Chemicals eg Ultra-Pure (NBS) Analytical Reagents eg Certified ARTM (Fisher) TM (Sigma) Chemically Pure (CP) eg CP TM (Sigma) Practical Grade eg Purified Commercial or Technical Grade

Microwave Digestion System

Rapid, efficient drying and acid decomposition of samples by using a microwave digestion system
Advantages - Sample contained within the digestion vessel - Highly efficient and rapid (5-10 min) - Mostly any sample can be digested - Volatile elements are retained in reaction vessel - Easy to automate: a computer controls the pressure and the temperature Disadvantages - Inability to add reagents during the digestion - Limited amount of sample (typically 1 g or less) - Safety concerns due to the use of high pressures and corrosive reagents

Fusion Techniques
Inorganic samples mixed with large excess of alkali metal salts in a crucible and heated until the substance fuse together in a molten state, the melt is allowed to cool at room temp and dissolved in dilute acid or water (Used when acids fail to dissolve sample, eg. silica, mineral oxides, steel)

Types of flux Base flux (Na carbonates, hydroxides, borate for alkaline metal) Acid flux (pyrosulfates, boric oxide, fluoride acids) Oxidizing Flux (Sodium peroxide or nitrate/alkaline metal + Sodium Carbonate) Disadvantages Contamination by flux material High salt content may complicate analysis High temperature - loss of analyte through evaporation Sample container may react with flux material

Removing Organic Material Before Inorganic Analysis

For inorganic analytes such as trace metals contained in organic materials (animal and plant tissue, biological fluids, etc), apply either dry ashing or wet digestion: - Dry ashing involves slow combustion at 400-7000C, which leaves behind the inorganic residue, soluble in dilute acid - Wet digestion is heating organic material with oxidizing acids (HNO3/H2SO4 mixture), (Inorganic residue left behind is soluble)

 Biological fluids must be free of proteins: - Remove proteins by either dry ashing or wet digestion, or - Precipitate protein using specific reagents and filter/centrifuge to yield protein free filtrate For organic analytes - AVOID oxidizing methods (NO heating in strong acids) - Extract the analyte from sample, or use dialysis or dissolve sample in appropriate solvent

SEPARATION AND PRECONCENTRATION

Analyte must be separated from the matrix to: Eliminate interferences Provide suitable selectivity preconcentrate analyte for more sensitive or accurate measurement Separation methods: Precipitation Chromatography Distillation Addition of masking agents

- Extraction - Dialysis - Electrophoresis

(5) Eliminating Interferences

Interferences are substances that prevent direct measurement of the analyte STANDARD ADDITION The method is often referred to as spiking the sample (by adding a known amount of analyte into the sample) The method is used to eliminate interference for analytes in a complex matrix (eg blood, sediment, human serum, etc)

(6) Measurement
Measurement is often the simplest stage of the analytical process Use reagents of high purity (reagent grade) A blank measurement must be performed for trace analysis Analytical measurements: (1) Classical and (2) instrumental The physical or chemical property proportional to the analyte concentration is measured Calibration - Measuring suitable standards to determine the relationship between analyte quantity and the physical/chemical property being measured

(7) Calculating Results and Reporting

(1) Determine the concentration of the analyte in the analytical sample solution (2) Use results to calculate the amount of analyte in the original (bulk) sample Evaluate the results
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Requires appropriate use of statistics Must be reasonable, reliable and related to the problem as originally stated

Data presentation must be understood and conclusions clearly shown Report results with limitation/accuracy information The report must be verified by a professional chemist or charted chemist

METHOD VALIDATION
Method validation is the process to confirm that the analytical procedure employed for a specific test is suitable for its intended use
Methods need to be validated or revalidated

1. before their introduction into routine use 2. whenever the conditions change for a validated method eg.

instrument with different characteristics 3. whenever the method is changed, and the change is outside the original scope of the method

Validation Parameters

Specificity (Selectivity)

Linearity
Range Accuracy Precision - Repeatability - Intermediate Precision - Reproducibility

Detection Limit (LOD) Quantitation Limit (LOQ) Robustness (Ruggedness) System Suitability Testing

Specificity
The ability to assess the analyte in the presence of components which are expected to be present (eg. matrix, impurities, degradants, etc) - It is not always possible to demonstrate that an analytical procedure is specific for a particular analyte (complete discrimination) - In this case a combination of two or more analytical procedures is recommended to achieve the necessary level of discrimination

Linearity
The range of concentrations of analyte for which the procedure provides results that are in directly proportional to the concentration or amount of analyte in the sample Ways of determining linearity - Use of calibration (standard) curve (Concentrations determination at the linear sections of the graph) - Triplicate (3) measurements at least - Regression, R2 > 0.998

Accuracy
Expression of the closeness of agreement between the accepted value (conventional true value or accepted reference value) and the value obtained by the method Accuracy can be determined in 3 ways - Recovery studies - use the procedure on a pure Standard Reference Material (SRM) and calculate % recovery (Problem method if <90%) - Compare results using 2 or more independent methods (one accurate and validated) - Analyze spiked blank matrix with varying known amounts of a standard

Precision
The closeness of agreement between a series of measurements from multiple sampling Often expressed as standard deviation or relative standard deviation (RSD) Repeatability expresses the precision under the same operating conditions over a short interval of time Intermediate Precision expresses within laboratories variations (different days, different analysts, different equipment, etc) Reproducibility expresses the precision between laboratories (for collaborative studies, and usually applied to standardization of methodology)

Range

The interval between the upper and lower concentrations of analyte in the sample for an analytical procedure that has a suitable level of precision, accuracy and linearity

Limit Of Detection (LOD)

LOD is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated - Evaluated as signal-to-noise ratio ie 3 times standard deviation of the noise (S/N = 3)

Limit Of Quantitation (LOQ)

The lowest amount of analyte in a sample that can be quantitatively determined with suitable precision and accuracy - For assays of low levels of compounds in sample matrices (eg impurities and/or degradation products) Evaluated as signal-to-noise ratio ie 10 times standard deviation of the noise (S/N=10)

Robustness

A measure of the methods capacity to remain unaffected by small, but deliberate variations in method parameters (Provides an indication of its reliability during normal usage)

System Suitability Testing

Tests parameters to be established for a particular procedure depending on the type of procedure being validated (An integral part of many analytical procedures) Tests are based on the concept that the equipment, electronics, analytical operations and samples to be analyzed constitute an integral system that can be evaluated as such

Calibration and Maintenance

Sensors must be calibrated eg. Time, temperature, pressure, humidity, weight Controllers must be qualified, calibrated and maintained at appropriate intervals Environmental requirements for the computerized system must be met