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Laboratory Exercises
I.1.
SPECTROPHOTOMETRIC DETERMINATION OF
AMMONIUM BY THE INDOPHENOL BLUE METHOD
Phenol (C6H5OH)
Standard
ammonium
solution
I,
100
mg
NH 4+/L:
Ammonium chloride must be dried for one hour at 100 C.
Dissolve 0.2965 g of the dried salt in water in a 1000 mL volumetric flask. Dilute
to the mark with water. The solution is stable for six months when stored in
a refrigerator.
1.
2.
3.
4.
5.
with water to the mark. This standard ammonium solution, and the ammonium
solutions made for preparing the calibration curve, must be freshly made.
Spectrophotometer with optical cell of 10 mm.
Water bath with thermostat, 50 C
Test tubes: 30 mL
Volumetric flasks: 10, 500 and 1000 mL
Pipettes: 1.0, 2.0, 4.0, 5.0, 10.0, 20.0, 25.0, 50.0 mL.
Micropipette: 250 L
Calibration
Preparation of calibration curve:
Transfer to 100 mL volumetric flask 0.0, 1.0, 2.0, 5.0, 10.0, 25.0 and 50.0 mL of
standard ammonium solution II. Dilute to the mark with water. The
concentrations of these solutions are 0.00, 0.04, 0.08, 0.2, 0.4, 1.0 and 2.0 mg
NH4+/L. Transfer 5.0 mL of each of these standard solutions and 5.0 mL of water
to a 30 mL test tube.
Add to the test tube 250 L reagent A using a micro pipette, and mix well. Add
then 250 L reagent B using a micro-pipette and mix well. Cover the opening of
the tube with some inert material. Place the tube in the water bath at 50 C for
two hours.
Cool the solution to room temperature, and transfer it to a 10 mm cell. Measure
the absorbance at 630 nm.
Prepare a calibration curve by plotting the absorbance of each of the standard
solutions against its concentration of ammonium. Prepare a new calibration curve
for each series of samples.
In order to check for ammonium in the reagents, take a photometric reading of
the blank (0.00 mg NH4+/L) against water. The absorbance should not exceed
0.020.
Analytical Procedure
Transfer 5.0 mL of the sample and 5.0 mL of water to a 30 mL test tube.
Proceed according to the instructions presented at points 2 and 3 of above. Convert the
spectrophotometric readings of the sample to mg NH 4+/L by means of the calibration
curve. The concentration may be expressed as mg N/L by multiplying with 0.778.
Samples containing more than 2.0 mg NH 4+/L must be diluted. With suitable
equipment the Indophenol method can be made automatic.
Interferences
Iron (III) may interfere if the concentration is more than 2 mg/L. If the pHvalue of the sample is lower than 3, the sample should be neutralized.
If the sample is turbid, both the sample and the blank should be filtered through a
white band filter.
I.2.
The method can be used for direct determination of the chloride ion content in
water samples within the range 0.05 to 5 mg/L.
Principle
Chloride ions will substitute the thiocyanate ions in undissociated mercury
thiocyanate. The released thiocyanate ions react with ferric ions forming a dark red
iron-thiocyanate complex.
The absorbance is measured at 460 nm.
2 Cl- + Hg(SCN)2 = HgCl2 + 2SCNSCN- + Fe3+ = Fe(SCN)2+
Reagents and Instrumentation
During the analysis, use only chemicals of recognized analytical grade and
only double-distilled or deionized and distilled water.
Perchloric acid (HClO4) 72% ;
Mercury (II) thiocyanate (Hg(SCN) 2);
Iron (III) nitrate nonahydrate (Fe(NO3)3 9H2O);
Sodium chloride (NaCl);
Ethanol (C2H5OH);
Perchloric acid, 1:1. Mix 1 volume 72% perchloric acid with 1 volume of water;
Mercury (II) thiocyanate solution, saturated. Shake 1 g Hg(SCN) 2 with 1000 mL
ethanol. Filter the solution after 24 hours. The solution may be stored in a glass
bottle at room temperature;
Iron (III) nitrate solution, 6%. Dissolve 6 g Fe(NO 3)3 9H2O in 100 mL 1:1
perchloric acid. Filter the solution after 24 hours;
Standard chloride solution I, 1000 mg/L. Dissolve 412.5 mg NaCl dried at 140200 C, in water and fill it up to 250 mL with water;
Standard chloride solution II, 10 mg/L. Dilute 10.0 mL standard chloride solution
I to 100 mL with water.
1.
2.
Analytical Procedure
Transfer 25 mL of the precipitation sample to a 100 mL Erlenmeyer flask.
Proceed as above. Read the chloride content of the sample from the calibration curve.
Interferences
Bromide and iodide will give the same absorbance as the equivalent amount
of chloride.
1.
2.
References
Iwasaki, I., Utsumi, S., and Ozawa, T. (1952) New colorimetric determination of
chloride using mercuric thiocyanate and ferric ion. Bull. Chem. Soc. Japan, 25,
226.
Zall, M., Fisher, D., and Gamer, Q. (1956) Photometric determination of
chlorides in water. Anal. Chem., 28, 1665-1668.
H2SO4
Formation of colour:
Ba2+ + thorine (Ba - thorine) red colour
(Ba-thorine) + H2SO4 BaSO4 + 2H+ + thorine + (Ba residual-thorine) red
Target
To determine the daily average concentration of SO 2 in samples of
atmospheric air.
Handle correctly a manual control station for the air quality.
To be trained on the correct use of materials and processes with possible
chemical risk.
6)
7)
8)
9)
10)
1)
2)
3)
4)
5)
6)
7)
Development of the colorimetric reaction in air samples. Adjust the volume in the
bubbling flash to 100 mL. Take an aliquot of 4.0 mL to be mixed with 10 mL of
barium perchlorate/dioxane (solution 6) and 0.25 mL of thorine solution (solution
10) as reported at the point 2.2 of above.
Measurement of the absorbance. Adjust the wavelength at 520 nm and test the
apparatus with a blank solution adjusting the absorbance at 0.800; place the
standard solutions and record the corresponding absorbance values. Finally, read
the sample solution before ten minutes.
Calculation of the SO2 concentration. Calculate the straight calibration line by
dotting absorbance vs the different concentrations of SO 2. Due to the developed
reaction, the blank displays the maximum absorbance and the slope is negative.
Finally and to calculate the SO 2 concentration in the bubbling device as most
accurate as possible, the range (from the calibration graph) used should be 0 and
6 g mL-1. If the concentration is over the reported range, make the
corresponding dilution.
Results
1) Display the conditions of the sampling process:
- Measurement of the gas counter before to start the sampling (m3)
- Data and hour of the sampling (start)
- Measurement of the gas counter at the sampling end (m3)
- Data and hour of the sampling (end)
2) Display the absorbance values and depict these obtained absorbance values vs the
sulphur dioxide concentration and adjust by regression the obtained curve.
Calculate de air concentration of the pollutant in g m-3.
-
Related Questions
Justify the negative value of the slope of the straight line represented in this lab
exercise.
Which technique should be suitable to obtain information in real time on the
concentration of SO2 in the air?
Main mechanisms of oxidation that can undergo the SO 2 in the atmosphere.
Describe the effects of the SO2 on the different receptors.
Explain the problems derived from pollution due to acid rain", its origins, effects
and possible solutions.
Comments on the particle concentration in suspension and the concentration of
SO2, according to the European legal rules.
10
I.4.
Dioxane or isopropanol
11
12
13
14
15
into a polyethylene bottle previously washed with nitric acid (not to wash nor to rub
the leaves) and conserve them in a dry place until the moment of the analysis.
Add into the sample recipient 20 mL of 0.1 mol L -1 HNO3 and shake
vigorously during a couple of minutes. Transfer the content into a 100 mL glass beaker
and wash the sampling bottle (three times) with 4 mL of 0.1 mol L -1 HNO3 and add
the washing nitric to the beaker containing the solved lead.
Note: Do not throw away the leaves.
1.b) Sampling and sample treatment for analysis of atmospheric lead trapped
on the surface of a particle filter (sample B). With the aid of the sampling device force
sufficient air volume (about 2 m 3) through the filter to obtain the lead amount
deposited on the filter enough to be into de calibration range. Once finished the
sample collection, the filter is carefully translated (use clamps) into a Petri plate. Then
it is transferred to the laboratory, introduced into a bottle and adds 20 mL of 0.1 mol L 1
of HNO3. Proceed as reported in the previous section.
2) Measurement of the area of leaves. In order to determine the total surface
of the leaves on which the lead is deposited, rinse the leaves with distilled water and
dried by pressing between filter paper. Draw its contour on a measurement paper, cut
the paper and weigh. An square piece of paper, 10 cm of side, is also weighed to
calculate the area in cm2 of the total surface of the sample of leaves.
3) Extraction and determination of lead. With each one of the sample solutions
A and B operate as follows: mix into the extraction funnel the content of the beaker, 5
mL of the alkaline complexing solution and 5 mL of the dithizone solution. The
mixture is shaken vigorously during 5 minutes. Place the funnel quiet during 5
minutes to allow the phases (aqueous and organic) separation. The funnel is opened
and the organic phase is passed into a dry test tube. Add an end of spatula of
anhydrous Na2SO4 into the test tube, it is shaken and the organic phase is filtered
through a paper filter, gathering the filtrate on a dry test tube which is immediately
closed to avoid the evaporation of part of the solvent.
Caution: test tubes and the photometer cell must be completely dry: use hot
air if required. Adjust wavelength at 520 nm and put zero absorbance with
dichloromethane.
4) Calibration graph. Next table depicts the reagents (in this order) to be
mixed for preparing the standard solutions set.
Funnel:
mL of standard solution, 5 mg L-1
mL of 0.1 mol L-1 HNO3
mL of alkaline complexing solution
mL of 25 mg L-1 dithizone
[Pb] (mg L-1) organic layer
1
0
20
5
5
0
2
0.5
20
5
5
0.5
3
1.0
20
5
5
1.0
4
1.5
20
5
5
1.5
5
2.0
20
5
5
2.0
6
2.5
20
5
5
2.5
16
As reported for the sample solutions, the mixture is shaken vigorously during
5 minutes; after 5 minutes (both layers are separated) and the funnel is opened to
deliver the organic phase into a dry test tube. Add an end of spatula of anhydrous
Na2SO4. After filtering the organic phase through a paper filter the resulting organic
solution is kept in a test tube tightly closed to avoid solvent evaporation.
Caution: test tubes and the photometer cell must be completely dry: use hot
air if required. Adjust wavelength at 520 nm and put zero absorbance with
dichloromethane.
Avoid in any moment the contact of the cyanide solution with acidic solutions;
evolution of cianhydric acid is extremely dangerous (smelling to bite almonds).
Do not throw away the residual solutions; it should be placed into the
corresponding deposits.
Related Questions
Propose other techniques of analysis to determine lead in the atmosphere.
Indicate advantages and disadvantages.
List other heavy metals that can be determined jointly with the proposed
techniques.
Make a list of the different experimental variables associated to the analysis,
indicating possible sources of error.
17
I.6.
Principle
The method proposed for the separation and determination of mercury is
based on the selective precipitation of mercury with the reagent Cadion A (4nitrophenyl diazoaminoazobenzene) and the extraction of the formed complex with
toluene. By measuring the absorbance of the extract in toluene and using a calibration
curve the quantity of mercury from the analyzed sample can be determined. The
formation of the mercury complex takes place at a pH of the aqueous phase of 5.5
11. The mercury (II) form with the Cadion a complex with the following structure:
N
NO2
N N
NO2
Hg
N N
18
Calibration Curve
From the solution of 50 g Hg(II)/mL are taken with a micropipette 0.1; 0.2;
0.4; and 0.6 mL which are being introduced in closed cylinders. With a pipette are then
introduced 5 mL of distilled water, washing the walls of the cylinder to carry the
whole quantity of mercury in the aqueous solution. Afterwards are introduced: 1 mL
sodium acetate solution 0.2 M and 1 mL reagent solution 0.015 %; the mixture is
stirred and left to settle for 10 min for the formation of the complex.
After this are introduced 5 mL toluene and the mixture is stirred strongly for 1
min. It is important that after the first seconds, of stirring it is stopped and the plug is
removed to release the toluene vapors, after which the stirring is continued. After the
phase separation (about 10 15 min) a part of the toluene extract is removed with a
dry pipette and it is introduced directly in the spectrophotometer cell. The absorbance
determinations are done against a reference sample prepared in the same manner as
the standards (distilled water, sodium acetate solution, reagent are introduced and the
toluene extraction is executed). In the reference sample no mercury is introduced.
Cells with the optical path of 1 cm and a wavelength of 490 nm will be used.
The absorbance values measured are represented graphically function of the mercury
quantities taken for analysis. In this manner is obtained a calibration curve which, if
the procedure was followed properly, will be a straight line passing through the origin.
Mercury Analysis
The analyzed sample may contain in addition to the mercury ions (II) large
quantities of ions of Cu(II); Co(II); Ni(II); Fe(II, III); Zn(II); Ca(II); Mg(II), etc. From
this sample is received for analysis a certain quantity in a volumetric flask of 100 mL.
The volumetric flask is filled to the mark with distilled water and the mixture is
homogenized. From this sample are then taken samples (two or three) that are
introduced in the cylinders for the extraction. The samples taken for analysis must be
between 0.1 1 mL. In each cylinder are then introduced 1 mL of sodium and
potassium tartrate solution, which will form complexes with some ions, which might
19
precipitate from the reaction media. Subsequently the working procedure for the
calibration curve will be used.
Interpretation of the Experimental Data
The absorbance values measured for the samples taken for analysis allow,
with the aid of the calibration curve, the determination of the quantity of mercury from
these samples. Knowing the quantity of mercury from each sample and also its
percentage from the total volume of the sample taken for analysis, meaning 100 mL,
the quantity of mercury received for analysis can be determined. The result is given in
mg of mercury.
Observations: The toluene is lighter than water, and after stirring, it is
separated in the upper part. The absorbance measurements are always done against a
reference sample that does not contain mercury.
20
I.7.
Introduction
The metallic lead and its compounds are toxic; they can enter the organism by
ingestion, inhaling, or absorption through the skin (the acute lead poisoning is rare due
to the poor absorption). The continuous assimilation of small quantities of lead is by
far the most dangerous. Initially, the lead is weakly bonded to the erythrocytes and
only a small part is eliminated through the urine, most of it being accumulated in the
bones.
From all the organic compounds of lead, the trialkyl compounds have the
strongest neurotoxic effect on mammals. After assimilation, the tetraalkyl compounds
are quickly metabolized to the trialkyl compounds.
In the environment, the main source of organic lead compounds is their use as
antioxidant agents for gasoline. As a result of the laws, in most countries this pollution
source is decreasing. Once entered in the atmosphere, the tetraalkyl compounds of
lead are decomposed photocatalytically, a process that can be very quick in the given
conditions. The decomposition products are lead compounds that are tri- di- and
monoalkylated. The lead is an omnipresent polluting element in all areas of the
biosphere.
Rarely, the natural waters contain a concentration larger than 5 g/L Pb, but
higher concentrations of lead were reported. Another source of lead in the tap water
are the lead pipes through which it circulates.
For the determination of lead can be applied a colorimetric method namely the
dithizone method, atomic absorption spectrometry with flame and respectively
electrothermal atomization (graphite furnace), and the atomic emission spectrometry
with inductively coupled plasma. The atomic absorption spectrometry with flame
presents a relatively not very high detection limit, requiring the applying of an
extraction procedure for the analysis of the drinking water in which low
concentrations of lead are found. The atomic absorption spectrometry with
electrothermal atomization is much more sensitive for low concentrations of lead and
does not require an extraction step. The atomic emission spectrometry with
inductively coupled plasma has a similar sensitivity to the atomic absorption
spectrometry with flame.
21
In this work is presented a procedure for the determination of lead from the air
of a workplace exposed to this chemical pollutant, by the method of atomic absorption
spectrometry with electrothermal atomization.
Reagents and Apparatus
Nitric acid 65 % Suprapur (Aldrich), ammonium dihydrogen phosphate
(Merck), distilled water (conductivity 4 S/cm).
Nitric acid 0.2 %. It is prepared by diluting 3.076 mL HNO 3 Suprapur with
1000 mL distilled water.
Matrix modifier for the graphite furnace are dissolved 0.4 g of ammonium
dihydrogen phosphate in 10 mL of distilled water.
Commercially available lead standard with the concentration of 1000 mg/L
(Aldrich).
Lead standard with the concentration of 10 mg/L: in a volumetric flask of 25
mL of Teflon are diluted 250 L of lead standard with the concentration of 1000
mg/L with 25 mL of Suprapur HNO3 0.2 %.
Lead standard with the concentration of 100 g/L: in a volumetric flask of 25
mL of teflon are diluted 250 L of lead standard with the concentration of 10 mg/L
with 25 mL of super-pure HNO3 0.2 %.
Lead standard with the concentration of 10 g/L: in a volumetric flask of 25
mL teflon are diluted 250 L of lead standard with the concentration of 100 g/L
with 25 mL of Suprapur HNO3 0.2 %.
The lead standards with the concentrations of 2 and 4 g/L are obtained by the
automated dilution made by the autosampler of the atomic absorption
spectrophotometer Perkin Elmer , model Analyst 700 of the lead standard with
the concentration of 10 g/L with nitric acid 0.2 %.
All the lead determinations were done using an atomic absorption
spectrophotometer Perkin Elmer, model Analyst 700, equipped with :
radiation source consisting in a light source emitting the line spectra
characteristic for the element being analyzed (Pb): a lamp with a hollow cathode
(placed on an automated transport system with eight positions for lamps).
correction source for the background: a deuterium lamp.
monochromator- used for the isolation of an absorption line from the
characteristic line spectra for the analyzed element.
solid detector associated with an electronic amplifying system and measuring
equipment.
atomization source represented by the graphite furnace with longitudinal
heating.
The voltage is applied along the graphite tube, parallel with the radiation
beam. The supply source, the electronic systems, and the pneumatic control of the
inert gas for the graphite furnace are incorporated in the spectrometer.
-
22
23
Temperature
(C)
Ramp
(time-s)
Hold
(time-s)
1
2
3
4
5
6
100
140
700
1800
2600
5
15
10
0
1
20
15
20
5
3
Internal
gas flow
(mL/min)
250
250
250
0
250
250
Type of gas
(norm/spl.)
Reading
N
N
N
N
N
N
No
No
No
Yes
No
No
The five steps in the temperature program of the graphite furnace have the
following purpose:
1.2. - drying takes place after the injection of the sample in the furnace.
The sample must be dried at a corresponding low temperature to avoid its
decomposition. For the aqueous solutions, the drying is applied at
temperatures of 100 140 C. The use of a temperature ramp ensures the
increase of the temperature in a well determined interval. In the case of the
graphite tubes with integrated platform (the case of the present work), the
time corresponding to the temperature ramp is shorter, a longer time is
applied for the atomization of the sample from the tube walls. After the
temperature ramp, the furnace is maintained at the selected drying
temperature, until full drying. Because only a few microliters of sample are
used, the time for maintaining at constant temperature is under 1 minute.
During the drying process, the internal gas flow is maintained at its
maximum value (250-300 mL/min) for the purging from the tube of the
vaporized solvent.
3. pyrolisis is used for the volatilization of the organic and inorganic
compounds of the matrix selectively from the sample, leaving the analyzed element in
a less complex matrix for analysis. During this step, the temperature is increased as
much as possible to volatilize the matrix components, but up to the analyte
decomposition temperature. The internal gas flow is maintained at 250-300 mL/min
for the removal of the volatilized matrix components.
4. atomization is used for the production of the atom population
corresponding to the element to be analyzed, that will allow the measuring of the
atomic absorption. In this step, the temperature is increased up to the point of
24
25
bubble Gilibrator 2, for each sample recording the exact value of the
measured flow rate, to determine the volume of the collected air:
Volumeair (Vair) = Flow (D)* time(t)
(1)
For the collection of the air samples is used the sampling vessels, in which
are introduced 10 mL of nitric acid 0.2%, acid in which is bubbled a
volume V of air, retaining in the absorbent solution the lead.
Each of the three absorbent solutions (nitric acid 0.2 %) in which the lead was
retained is quantitative transferred in a volumetric flask of 50 mL, which is filled to
the mark with nitric acid 0.2 %. These solutions are introduced in three vials of the
autosampler of the spectrometer and two replicates for each one are analyzed. 25 L
of solution will be analyzed using the atomic absorption spectrophotometer Analyst
700, and the absorbances for each replicate are interpolated on the calibration curve,
thus determining the corresponding concentrations.
To calculate the lead concentration expressed as mg/m 3 of air is applied the
formula:
c Pb ( mg / m 3 )
Vdil * c
Vair ( L) *1000
(2)
c Pb ( mg / m 3 )
Vdil * c
D * t * 1000
(3)
26
I.8.
Introduction
The toxic metals are not biodegradable, having the tendency to accumulate in
the vital organs of humans where they act for a long period of time. The pollution of
the environment with toxic metals is due to the industrial efluents, as well as to the
discharge of wastewater from different sources.
The mercury is a trace element, which in small quantities can produce severe
toxic effects. In the case of humans that are not exposed to mercury through their jobs,
the most probable source for this element is the diet. The reported quantity of mercury
in foodstuff is relatively low, approximately 0.02 g/g, but a large variability exists
due to the type of product, its geographical origin, and the industrial and agricultural
techniques from that area. The mercury accumulates throughout the food chain,
especially in the aquatic environment. The organic and inorganic compounds of
mercury can be present in the natural water and can be concentrated in different
organisms, like fish. That is why the fish can have a higher content of mercury as
compared with other foodstuff, but it is difficult to report a mean concentration of this
element, because it depends on species, age, size and water quality in the residence
area.
From all the methods used for the determination of mercury, the cold vapour
atomic absorption spectrometry (CVAAS) has become probably the most used
technique of analysis being characterised by a special sensitivity and selectivity.
In this work is described a method of analysis based on the cold vapour
atomic absorption spectrometry (CVAAS) used for the determination of mercury from
a working environment exposed to this chemical pollutant, and from a fish sample.
Experimental Procedure
Reagents and apparatus
Tin chloride (Fluka), hydrochloric acid (Baker), potassium permanganate (Spolek),
sulfuric acid (Fluka), hydrochloric hydroxylamine (Reactivul); distilled water
(conductivity 4 S/cm).
Nitric acid 65% puriss. p.a. - Hg < 0,0000005 % (Fluka), sulfuric acid 98% puriss.
p.a. Hg < 0,0000005 % (Fluka).
Acid mixture: nitric acid puriss. p.a 1.5 % and sulfuric acid puriss. p.a 1.5 %.
Absorbent solution for the mercury vapours: 2.4 x 10-2 % KMnO4 in H2SO4 4 %.
27
SnCl2 5 % in HCl 10 % .
Potassium permanganate solution 6%.
Hydroxylamine hydrochloride solution 20 %.
Standard solution of mercury of concentration 1000 mg/L (Aldrich).
Standard solution of mercury of concentration 10 mg/L - prepared by dilution of
500 L of standard solution of mercury of concentration 1000 mg/L with 50 mL of
absorbent solution for mercury vapours made in a volumetric flask of 50 mL.
Standard of mercury of concentration 100 g/L - prepared by dilution of 500 L
de standard of mercury of concentration 10 mg/L with 50 mL of absorbent
solution for mercury vapours made in a volumetric flask of 50 mL.
All the mercury determinations by atomic absorption are made using an atomic
absorption spectrophotometer Perkin Elmer model Analyst 700 coupled with a
manual hydride generation system model MHS-10.
Working Procedure for the Determination of the Mercury Concentration
from a Working Environment
The mercury concentration from an environment exposed to the pollutant is
determined: the enclosure of a metrology laboratory. Among the activities carried out
in this laboratory is the repair of thermometers.
In order to determine the concentration of mercury in air, three air samples
are collected, at three different moments, using for this a gas sampling
pump Ametek. The three air samples are collected with a flow rate of
about 1 L/min, for ten minutes. The sampling flow rate is measured with a
flow rate calibrator with soap bubble Gilibrator 2, for each sample
recording the exact value of the measured flow rate, to determine the
volume of the collected air:
Volumeair (Vair) = Flow (D)* time(t)
(1)
For the collection of the air samples is used the sampling vessel with frit of
100 mL volume, in which are introduced 50 mL of absorbent solution for mercury
vapours, solution in which is bubbled a volume V of air, retaining in the absorbent
solution the mercury.
Before the performing of a set of measurements, is required the calibration of
the AAS-MHS-10 system. For the calibration, the absorbances of the mercury
standards with the concentrations 2, 5, 10 g/L are measured at the wavelength of
253.7 nm .
28
29
the flow of 25 mL/min) and the line (b) towards the immersion tube. The gas from the
immersion tube and the conical shape of the reaction vessel ensure a good
homogenization of the solution. The third flow of inert gas circulates through the
restrictor F2 (with a nominal value of the flow of 400 mL/min) and the line (a) towards
the reaction vessel.
The removal of air from the system with the aid of the argon flow is done for
50 seconds. Then, to perform the analysis, the piston of the device is kept pressed, and
the flow of inert gas that circulates through F 3 is cut off. A pressure is applied between
the output (B) of the valve, on the line (c) towards the reducing agent tank. Thus the
reducing agent is pushed through the tube (d) towards the immersion tube, through
which is added to the standard/sample solution. As reducing agent is used a solution of
SnCl2 5 % prepared in HCl 10 % which is added to the solution to be analyzed for 26
seconds. The ions of Hg2+ from the solution to be analyzed are reduced by the SnCl 2 to
elemental state mercury, the resulted mercury vapours being transported by the argon
to the windowless quartz cell (placed on a metallic support mounted above the burner
of the atomic absorption spectrophotometer Analyst 700), where they absorb the
radiation from the mercury hollow cathode lamp of the spectrophotometer. The
spectrophotometer Analyst 700 is controlled by computer, and after the addition of
the reducing agent to the solution to be analyzed, is started the measuring of the
absorbance for 60 seconds. On the display of the computer the absorbance
corresponding to the mercury vapours is recorded as peak with the height proportional
to the concentration of the element from the analyzed sample.
Afterwards the absorbances corresponding to the maximum of the peaks for
the mercury standards with the concentrations 2, 5, and respectively 10 g/L are read,
with two replicates for each standard, and then the calibration curve is generated.
The mercury concentration is determined from each sample of absorbent
solution in which the mercury was retained.
For this purpose, from the 50 mL of absorbent solution, 10 mL of solution are
analyzed with two replicates, by using the manual hydride generation system MHS10 coupled with the atomic absorption spectrophotometer Analyst 700, and the
absorbances corresponding to each replicate are interpolated on the calibration curve,
thus determining the corresponding concentrations.
To calculate the mercury concentration expressed in mg/m 3 of air is applied
the formula:
c Hg (mg / m 3 )
Vabs.sol . * c
Vair ( L) *1000
(2)
30
c Hg ( mg / m 3 )
Vabs.sol . * c
D * t * 1000
(3)
31
stabilization of the mercury). The mercury standard with the concentration of 100
g/L is obtained by introducing in an Erlenmeyer flask with plug, of 700 L mercury
standard with the concentration of 10 mg/L and 5 mL concentrated H 2SO4, following
the same mineralization procedure as for the fish samples. With all the reagents added
in the mineralization process, the 700 L of mercury standard with the concentration
of 10 mg/L will be diluted with 70 mL of reagent mixture, obtaining the mercury
standard with the concentration of 100 g/L.
For generating the calibration curve, 200 L, 500 L, and respectively 1000
L of mercury standard with the concentration of 100 g/L mineralized are introduced
in 10 mL of mineralized blank solution put in the reaction vessel of the system MHS10, in order to obtain the calibration standards with the concentrations 2, 5, 10 g/L
Hg. After the connection of the reaction vessel with each individual standard, to the
system MHS 10, argon is bubbled in the reaction vessel for 50 seconds to remove the
air from the system. Afterwards the reducing agent is added in the reaction vessel
(solution of SnCl2 5 % prepared in HCl 10 %), for 26 seconds. Under the action of the
reducing agent, the Hg2+ ions are reduced to elemental Hg, the resulted mercury
vapours being transported by the argon to the windowless quartz cell where they
absorb the radiation from the mercury hollow cathode lamp of the spectrophotometer
Analyst 700. The absorbance corresponding to each standard is read with two
replicates. To read the zero mercury point of the instrument in order to remove the
influence of the Hg contained in the reagents, before the reading of the absorbances
corresponding to the mercury standards with the concentrations of 2, 5, 10 g/L Hg,
in the reaction vessel of the device are added 10 mL of mineralized blank solution,
measuring, after the addition of the reducing agent, its absorbance. Subsequently this
value is automatically subtracted from the absorbances corresponding to the mercury
standards and the samples.
The absorbances corresponding to the mercury from the fish samples are
interpolated on the calibration curve and the mercury concentration from 10 mL of
analyzed solution is determined.
Calculation of the Mercury Concentration from the Fish Samples
To calculate the mercury concentration from the fish samples, expressed in
mg/kg sample, the following formula is applied:
c Hg (mg / kg )
V finalsol . (mL) * c
m sample ( g ) * 1000
(4)
32
curve (c- expressed in g/L), the volume of the final solution (after the addition of all
the reagents before the reading of the samples) and the quantity of fish sample
analyzed (expressed in grams).
Applying the Method of Standard Additions for the Verification of
Interferences
In an Erlenmeyer flask with plug, are introduced approximately 0.7 g of fish,
35 L mercury standard with the concentration of 10 mg/L and 5 mL of concentrated
H2SO4. The same procedure for the mineralization as described above is used. Two
replicates are then analysed of 10 mL from the solution resulted after the
mineralization of the sample.
The recovery yield is calculated (%), with the formula:
Recovery(%)
Conc. det.
* 100
Conc. calc.
(5)
where: Conc. det. = concentration determined, obtained after applying equation (4);
Conc. calc. = concentration corresponding to the fish sample +5 g/L (added mercury
standard).
33
I.9.
DETERMINATION OF pH IN PRECIPITATION.
POTENTIOMETRIC METHOD
Principle
The method is based on the determination of the potential difference between
an electrode pair consisting of a glass electrode sensitive to the difference in the
hydrogen ion activity in the sample solution and the internal filling solution, and a
reference electrode, which is supposed to have a constant potential independent of the
composition of the immersing solution. The measured potential difference is compared
with the potential obtained when both electrodes are immersed in a solution or buffer
with known pH or hydrogen ion concentration. The pH is defined by the formula:
pH(sample) = pH(reference) + [(E(sample) E(reference)] F/RT1n10
where E are the electrode potentials, R is the universal gas constant, T the absolute
temperature and F is the Faraday constant.
This is an operationally defined pH. Buffers of known pH are specified by the
National Institute of Standardized Technology (NIST). The primary standard and the
most widely used buffer for pH-meter calibration is 0.05 M potassium hydrogen
phthalate, which has a pH of 4.00 at 20 C, and a hydrogen ion activity of 10 -4 M. This
latter hydrogen ion activity is based on theoretical calculations (the BatesGuggenheim convention).
In precipitation samples, the ionic strength will typically be in the region 10 -3
-5
to 10 . The activity coefficient for monovalent cations such as the hydrogen ion will
therefore be in the range 0.95-0.99. This corresponds to <0.02 pH-units difference
between pH and log[H+]. Much more critical is the assumption of a constant
reference electrode potential when going from a relatively concentrated potassium
hydrogen phthalate solution to extremely dilute precipitations samples. The problem
arises because of the inherent possibility of building up a liquid junction potential
between the internal solution of the reference electrode, and the sample solution. This
liquid junction potential may be larger if the ionic strength difference between the two
solutions is large. It is reduced by making the boundary between the concentrated
filling solution and the sample as sharp as possible. Various designs of pH cells
meeting this criterion have been proposed. Tests of commercial electrodes against
dilute acid solutions and low ionic strength buffers with known pH or hydrogen ion
concentrations have shown, however, that this problem has largely been overcome
with modern pH instrumentation and electrode systems.
34
35
Analytical Procedure
Measure the pH-value of the sample according to the instruction manual for
the instrument. The solution may be stirred, but not vigorously. The temperature of the
sample solution must be the same as the temperature of the buffer solution used for
calibration.
Rinse the electrodes thoroughly with distilled water between each
measurement, and wipe off the excess water with a soft paper.
Store the electrodes in 0.1 M KCl-solution or according to the manufacturers
recommendations. The reference electrode should not be stored in distilled water!
Performance Test of the Electrode Pair
The behaviour of the reference electrode is the main source of errors in pHmeasurements, especially in low ionic strength solutions. In order to check the
performance of the reference electrode, control measurements should be made on
solutions of dilute acids or dilute buffers to verify that correct values are obtained for
solutions of lower ionic strengths. A solution which should give a pH ~4.00 could be
used for the test. A 10-4M HC1-solution should give a pH of 3.99 0.05.
Electrode pairs should also show minimal differences between measurements
made in stirred and unstirred low ionic strength solutions.
Usually the liquid junction between the solution and the saturated KCl-solution in the
reference electrode is formed in a porous plut of ceramic fibre. Slow stirring removes
the concentrated KCl-solution which slowly runs out through this capillary.
If the stirring is too vigorous, the ionic medium in the plug itself may be
diluted. This will increase the liquid junction potential, and should be avoided. The
liquid junction potential may also increase if the porous plug is clogged up by
impurities.
References
1. Bates, R.G. (1965) Determination of pH, theory and practice. New York, Wiley.
2. Linnet, N. (1970) pH measurements in theory and practice. Copenhagen,
Radiometer.
3. Westcott, C.C. (1978) pH measurement. New York, Acad. Press.
4. Davison, W. and Woof, C. (1985) Performance tests for the measurement of pH
with glass electrodes in low ionic strength solutions including natural waters. Anal.
Chem., 57, 2567-2570.
36
I.10.
Introduction
This laboratory exercise presents a new method based on Prussian Blue
modified screen-printed electrodes for screening of organophosphorous and
carbamate pesticides in water.
Pesticides are among the most important environmental pollutants because of
their high toxicity and their significant presence in the environment. The use of
pesticides in agriculture has progressively increased since World War II with a
concomitant increase in world food production. In this context, industrial emission of
pesticides during their production, and more importantly the presence of residues of
these chemicals and their metabolites in food, water and soil, has become a problem
for society at large [1]. Among the many methods reported for pesticide detection,
chromatographic methods such as High Performance Liquid Chromatography (HPLC)
and Gas Chromatography (GC) are used as reference methods [2,3]. Thus enzymatic
methods have been adopted as an alternative to classical methods (GC, HPLC) for
faster and simpler detection of some environmental pollutants [4, 5].
The use of cholinesterase enzymes for inhibition-based determination of
pollutants has shown great promise for environmental screening analysis [6-8].
Cholinesterase occur in nerve tissue and red blood cells and play an important
physiological role as it is responsible for the hydrolysis of acetylthiocoline, a
neurotransmitter operating in the colinergic synapses. The enzymatic hydrolysis of
acetylcholine in the presence of acetylcholinesterase (AChE) proceeds as follow:
AChE
CH3COO(CH2)2N+(CH3)3Cl- + H2O
37
oxidase (ChO); the hydrolyzed choline is oxidized by ChO and the electroactive
compound (H2O2) is quantified amperometrically.
ChO
HO(CH2)2N+(CH3)3Cl- + O2
HOOC(CH2)2N+(CH3)3Cl- + H2O2
(2)
The biosensor proposed in this work is based on the modified Prussian Blue
(PB) transducers coupled with AChE, as enzyme, and acetylthiocholine as substrate.
The thiocholine produced by the enzymatic reaction is measured using screen-printed
electrodes (SPEs) modified with PB. The measurement is carried out in drop, at + 200
mV vs Ag/AgCl.
In a previous work [13] it was demonstrated for the first time the
electrocatalytic effect of PB towards some thiols.
Experimental Part
Principle of the method
The promising advantages of PB as catalyst and the screen printing
technology has been combined to assemble sensors with improved characteristics for
the amperometric determination of thiocholine.
The biosensors for pesticide determination are based on the following
reactions:
AChE
CH3COS(CH2)2N+(CH3)3Cl- + H2O
CH3COOH + HS(CH2)2N+(CH3)3Cl-
38
Electrodes preparation
Screen-printed electrodes used in this experimental work are home-made with
a 245 DEK (Weymouth, England) screen-printing machine. Graphite-based ink
(Electrodag 421) form Acheson Italiana (Milan, Italy) was used to print the working
electrode. The substrate is a flexible polyester film (Autostat HT5) obtained from
Autotype Italia (Milan, Italy). The electrodes are produced in foils of 20. The diameter
of the working electrode is 0.3 cm resulting in an apparent geometric area of 0.07
cm2, the counter in graphite-based ink and the reference in silver chloride.
Prussian Blue deposition
PB modification of SPEs was accomplished by placing a drop (10 L of total
volume) of precursor solutions onto the working electrode area. This is a mixture
obtained adding 5 L of 0.1 mol L-1 ferric chloride in 10 mmol L-1 HCl to 5 L of 0.1
mol L-1 K3[Fe (CN) 6] in 10 mmol L-1 HCl. The drop was carefully placed exclusively
on the working electrode area, in order to avoid the formation of PB on the reference
and counter electrodes that could notably increase the internal resistance of the
system. After 10 minutes the electrodes were rinsed with 3 mL of 10 mmol L -1 HCl.
The electrodes were then left 90 minutes in the oven at 100C to obtain a more stable
and active layer of PB.
The PB modified electrodes were stored dry at room temperature in dark.
Enzyme immobilization
The AChE was immobilized onto the PB-modified electrode surface by the
cross-linking method. For this 4 L mixture of glutaraldehyde (1 % in water), Nafion
(5 % in alcohol), BSA (3% in water) and 0.01 U AChE were left to dry (about 30 min)
onto the electrode area. After preparation the biosensors are kept in buffer solution at
4C.
Procedure for Pesticide Measurement
The detection of pesticide is carried out measuring the enzymatic activity
before and after exposure the enzyme to the sample. The enzymatic activity
measurement before the exposure to the sample is carried out as follow:
1) 50 L of buffer are placed on the electrode and then the + 200 mV potential
is applied and the current recorded (about 5 nA). After measurement the buffer
solution is removed from the biosensor surface.
2) 50 L of working buffer solution containing 3 mM thiocholine are added
on the electrode and the steady-state current is recorded (about 80 nA).
The current due to the oxidation of thiocholine is the difference between the
recorded current in the step 2 minus the recorded current in step 1. This value is the
intensity of current of enzyme uninhibited (I0).
3) The biosensor is washed with buffer solution and incubated for 30 minutes
in 5 mL sample, under stirring. During the incubation step the AChE is inhibited if in
the sample pesticides are present.
39
4) After that, the biosensor is washed with buffer, and the 2 nd step is repeated
and the residual enzymatic activity curren is measured.
The current due to the oxidation of thiocholine, produced after the enzyme
inhibition, is the difference between the current recorded in step 4 minus the current in
the step 1 ( I1).
Percentage inhibition was calculated according to the formula:
I%= [(I0-I1)/I0]100
Biosensor Reactivation
This kind of biosensors are disposable, but it is possible to measure the
pesticide more times using the same electrode. In fact it is possible to use the same
biosensor reactivating the enzyme with pyridine-2-aldoxime methiodide (2-PAM).
After the measurements, the biosensor is washed with distillated water and the
inhibited enzyme is regenerated for 30 sec with a drop 5 mM 2-PAM prepared in
buffer solution. Then, the biosensor is washed and another drop of buffer solution
containing substrate is added to measure the enzymatic activity again. This step
permits to use one biosensor more times.
For measuring an unknown concentration of pesticide present in a water is
necessary to perform all the 4 steps and calculate the percentage inhibition,
respectively. The pesticide concentration is calculated using the calibration curve
which should be performed before the sample analysis.
What do you have to do?
1. Calibration curve. For the calibration curve is necessary to measure and calculate
the inhibition resulted for the next concentrations of paraoxon: 0, 10, 20, 30, 40
and 50 ppb. The paraoxon solutions will be prepared in distilled water and the
steps 1-4 presented at the procedure will be followed for each concentration point.
The degree of inhibition due to the amount of paraoxon will be graphically
represented and the parameters of the linear equation will be calculated.
2. Sample analyses and calculation of the pesticide concentration. Two samples of
drinking water spiked with paraoxon will be analyzed for pesticide concentration
determination. The resulted enzymatic inhibition obtained for each sample will be
introduced in the equation previously obtained and the pesticide concentration will
be calculated.
40
References
1. FAO, Agriculture towards 2010, in: C 93/94 Document of 27 th Session of the FAO
Conference, Rome, 1993.
2. Standard methods for examination of water and wastewater 20th ed.; American
Public Health Association; Washington,1998; pp 6/85-6/90.
3. Liska I.; Slobodnik J. J.Cromatogr.A 1996, 733, 235-258.
4. Preininger C.; Wolfbeis O.S. Biosens. Bioelectron. 1996, 11, 981-990.
5. Bagirova, N.A.; Shekhovtsova, T. N.; van Huystee, R. B. Talanta 2001,
6. Bernabei M.; Chiavarini S.; Cremisini C.; Palleschi G. Biosens. Bioelectron. 1993,
8, 265-271.
7. Cremisini, C.; Di Sario, S.; Mela, J.; Pilloton, R.; Palleschi, G. Anal. Chim. Acta
1995, 311, 273-280.
8. Hart A.L.; Collier W.A.; Janssen D. Biosens. Bioelectron. 1997, 12, 645-654
9. Sol S.; Merkoci A.; Alegret S. Crit. Rev. in Anal. Chem. 7 2003, 33(2), 9-126.
10. Danet A.F.; Badea M.; Aboul-Enein H.Y. Biopolymers (Biospectr.) 2000, 57, 3742.
11. Poganik L.; Franko M. Biosens. Bioelectron. 1999, 14, 569-578.
12. Ellman, G.L.; Courtney K.D.; Andres V; Featherstone R.M. Biochem. Pharmacol.
1961, 7, 88-95.
13. Ricci, F.; Arduini, F.; Amine, A.; Moscone, D.; Palleschi, G. J. Electroanal. Chem.
2004, 563, 229-237.
14. Ricci, F.; Amine, A.; Tuta C.; Ciucu A., Lucarelli F.; Palleschi. Anal. Chim. Acta
2003, 485, 111-120.
41
I.11.
Introduction
This method is applicable to determination of strong acids in precipitation
samples within the concentration range 10 -5 to 10-3 M. The lower concentration limit
is close to the concentrations at background sites without alkaline mineral dust.
Principle
In the coulometric titration method (Liberti et al., 1972), the acid is titrated at
constant current with hydroxyl ions liberated at a platinum electrode, a silver-silver
bromide electrode serving as the counter electrode. The overall reaction is:
Br- + Ag + H2O AgBr + OH- + H2
The EMF of a glass-calomel electrode pair is read at intervals and the results
are used to construct a Grans plot (Gran, 1952; Rosotti and Rosotti, 1965), which
gives the endpoint of the titration by extrapolation of the straight part of the curve.
The only necessary modification is the addition of a constant, known amount
of acid to the sample before the titration, in order to facilitate the titration of weakly
acidic or alkaline samples without interference from carbon dioxide.
Reagents and Instrumentation
Reagents
During analysis, use only reagents of recognized analytical grade. The water
used for dilution and rinsing must be double-distilled or de-ionized and distilled.
Nitrogen gas (N2) 99.9%
Potassium bromide (KBr)
Sulphuric acid (H2SO4) 0.05M
Buffer solution pH = 4.00
Solution I: 1 M KBr and 2.5 10-3 M H2SO4 (transfer 120.0 g KBr and
exactly 50 mL of 0.05M H2SO4 to a 1000 mL volumetric flask. Fill up to the mark
with water.).
Instrumentation
- Expanded-scale pH-meter (Radiometer PHM 26 or an instrument with similar
specifications).
- Constant current source (2-10 mA adjustable)
42
43
Grans function
EmV
EmV
EmV
EmV
1.04
41
4.93
81
23.4
121
111
1.08
42
5.13
82
24.4
122
115
1.14
43
5.33
83
25.3
123
120
1.17
44
5.55
84
26.3
124
125
1.22
45
5.77
85
27.4
125
130
1.26
46
5.98
86
28.4
126
135
1.31
47
6.22
87
29.6
127
140
1.36
48
6.47
88
30.7
128
146
1.42
49
6.75
89
32.0
129
152
10
1.48
50
7.00
90
33.3
130
158
11
1.54
51
7.28
91
34.6
131
164
12
1.60
52
7.57
92
36.0
132
171
13
1.66
53
7.87
93
37.4
133
177
14
1.73
54
8.19
94
38.8
134
185
15
1.80
55
8.51
95
40.4
135
192
16
1.90
56
8.85
96
42.0
136
199
17
1.94
57
9.20
97
43.6
137
207
18
2.02
58
9.57
98
45.3
138
216
19
2.10
59
9.94
99
47.2
139
224
20
2.18
60
10.3
100
49.1
140
233
21
2.26
61
10.7
101
51.0
141
242
22
2.36
62
11.1
102
53.1
142
252
23
2.45
63
11.6
103
55.2
143
262
24
2.54
64
12.1
104
57.4
144
272
25
2.65
65
12.5
105
59.7
145
283
44
EmV
EmV
EmV
EmV
26
2.75
66
13.0
106
61.9
146
294
27
2.86
67
13.5
107
64.4
147
306
28
2.97
68
14.1
108
67.0
148
318
29
3.09
69
14.6
109
69.7
149
331
30
3.21
70
15.2
110
72.4
150
344
31
3.34
71
15.8
111
75.3
151
351
32
3.48
72
16.5
112
78.3
152
371
34
3.61
74
17.1
113
81.5
153
386
34
3.75
74
17.8
114
84.7
154
402
35
3.90
75
18.5
115
88.1
155
418
36
4.06
76
19.3
116
91.6
156
434
37
4.23
77
20.0
117
95.1
157
452
38
4.39
78
20.8
118
98.9
158
470
39
4.56
79
21.7
119
103
159
489
40
4.74
80
22.5
120
106
160
507
Expression of Results
The concentration of strong acid in the sample is calculated from the formula:
or
where
i = electrolysis current in amperes
te = electrolysis time at equivalence point (seconds)
45
46
47
48
Reagents
Pentane
Standards for GC:
Benzene (standard)
Toluene (standard)
Xylene (standard)
Preparation of Solutions
Stock solution A. In a 25 mL flask are introduced 20 mL of pentane; cover
tightly and weigh it. Next, add 500 L of benzene, it is covered
immediately and weighed again.
500 L of toluene are added
immediately. Remove the flask from the balance and level it with pentane.
Standard solution B. In a tube introduce 9 mL of pentane and add 1 mL of
the stock solution A.
Note: cover immediately the flash and work always into a fume hood.
Procedures
1) Sampling and sample pre-treatment. By means of the sampling
equipment, it is forced to flow through the adsorption cartridge an air
volume exactly measured (10 L), so that the benzene concentration,
toluene and xylene are within the calibrated straight linear range. If it is
necessary perform the suitable adjustements.
2) Extraction. In the analytical laboratory the content of the sampling tube
is put in contact with 100 mL of pentane to extract the organic compounds.
3) Filtration. The extract is filtered through a nylon filter of 0.45 m.
4) Calibration. Standard solutions of benzene, toluene and xylene. Using
covered tubes, a series of standard solutions of benzene, toluene and
xylene is prepared by diluting 0.5, 1.0, 2.0, 3.0 and 4.0 mL of solution B in
pentane up to a 10 mL.
Note: cover immediately the flash and work always into a fume hood.
5) Analysis. The chromatographic analysis will be performed according to
following conditions:
Injection volume: 1L
49
Write:
500
500
500
50
g L-1 of xylene
Write in the tables the area of the peaks in the calibration graph
Benzene g/L
rea
Toluene g/L
rea
Xylene g/L
rea
3) Draw the calibration lines. Represent the areas obtained in the
chromatograms vs. the concentration values of benzene, toluene and xylene
and applied the corresponding linear regression analysis.
4) Obtain from the calibration lines the corresponding concentrations of
benzene, toluene and xylene in the simple solutions.
Identify the presence of these three compounds by jeans of the retention times.
Obtain the correct peak areas from the chromatogram; then calculate the
concentrations in g m -3.
1)
2)
3)
4)
Related Questions
Explain the problems relative to the contamination due to organic compounds, its
origins, effects and possible solutions.
List the different sampling schemes for organic compounds.
Draw a scheme of the gas chromatograph.
What effect has the temperature of the column on the separation of these
compounds?
51
52
53
volatile organic compounds and a column for the analysis of the heavy volatile organic
compounds), two gas chromatographic detectors connected in parallel with
simultaneous data acquisition (PID- photoionization detector and ECD- electron
capture detector) and the onboard system for data processing/storage. The portable gas
chromatograph is provided with an electronic flow rate controller for the carrier gas
and connection interface for the external PC for the control of the gas chromatograph,
transfer of the acquired data and their re-processing with the chromatographic
software SiteChart.
In the figure below is represented the scheme of the portable gas
chromatograph Photovac model Voyager in which are indicated all its component
parts.
Working procedure
Before starting the operation with the portable gas chromatograph its battery
must be charged.
The internal cylinder of the chromatograph is loaded with carrier gas (nitrogen
with the purity 99.999%) by connecting the input port of the instrument through a
pressure regulator, to a nitrogen cylinder of 200 atm. The filling with carrier gas of the
54
internal cylinder of the chromatograph ensures its autonomy for 7 hours, during this
time on site gas chromatographic measurements can be done.
After the charging with carrier gas, the chromatograph is started by pushing
the On/Off button, being required an interval of about half an hour for its stabilization
and reaching of the optimal working parameters, moment indicated by the green color
of the state led of the gas chromatograph (Ready status indicator).
Before the starting of the determination, a sampling location recognition
naming is entered, using the keyboard of the chromatograph. For this, the following
scheme is followed:
I..
is entered ENTER MENU using one of the four fixed keys located on
the left side of the display (it regards the up and down positioning
order, the keys On/Off, Start/Stop, Enter Menu, Exit) then, using the
keys below the display, is pressed the key below SETUP, then the key
below TAG. In this moment is entered the naming of the sampling
location for the later recognition of chromatogram recorded at that
site.
Also, are chosen the column and the detector, with the aid of which the
analysis will be made. For light compounds (for example: acetone, methyl-ethylketone, chloroform) is chosen the column C, and for the medium ones (benzene,
toluene, xylenes, methyl-isobuthyl-ketone, trichlorethylene) is selected the column B.
The analysis time for the column C is 15 minutes, and for the column B is half an
hour. With regard to the detectors, the data acquisition can take place simultaneously
on both detectors, or only on the photoionization detector (in the case of the
compounds having the ionization potential lower than 10.6 eV) or only on the electron
capture detector (in the case of compounds containing atoms of chlorine or bromine).
For the selection of the column, is entered ENTER MENU - SETUP CONFIG
COLUMN and with the aid of the key Enter Menu is selected or deselected a
chromatographic column.
After the establishing of these parameters, the air sample to be analyzed is
sampling by selecting the key START/STOP. For 20 seconds an air sample will be
drawn in with the aid of a pump (during sampling the indicator led of the sampling
stage, named Sampling status indicator- will have a red color). After the 20 seconds
sample sampling interval, the sample is transported by the carrier gas from the entry
point in the chromatograph, through the previously selected separation column
towards the detector.
Each component of the sample will be retained with a different time inside the
column and will be eluated from the column with a characteristic retention time. If the
response of the detector is reported to the dead time, this response will be presented as
a series of chromatographic peaks separated by time, with one peak for each
component of the sample. The retention time serves for the identification of the
components of the analyzed air sample, and the areas of the chromatographic peaks
are used for the quantitative analysis.
55
The qualitative and quantitative analysis made with the Voyager instrument,
after the analysis of an air sample is based on the retention times and the areas of the
peaks corresponding to the previously analyzed standards. The chromatograph
presents in its database a library of compounds, with a standard of each interesting
compound being stored in its memory by: retention time, peak area and concentration.
The ratio peak area/concentration represents the sensitivity for a certain compound
(expressed in mVS/ppm), and this information is saved in a part of the memory of the
Voyager instrument known as the library which will be automatically updated during
the calibration of the instrument.
In the case of each analyzed sample, the retention times corresponding to the
recorded chromatographic peaks are compared automatically to the retention times of
the compounds from the chromatograph library. If an overlapping within the
established limits of the peak recognition window is observed, than that peak is
identified as one of the compounds from the library. For the calculation of the
concentration corresponding to the identified peak, its area is divided to the sensitivity
of the corresponding standard from the library (the sensitivity- ratio
area/concentration). The retention times and the ratios area/concentration
corresponding to the standards of the compounds from the library are updated every
time the Voyager chromatograph is calibrated.
For the calibration of the portable gas chromatograph was chosen the option
of connecting it through a flow regulator, to a pressurized cylinder that contains a
mixture of calibration gases (in the case of this article was used for the calibration a
cylinder BTEX containing benzene, ethyl benzene, toluene, m-xylene, o-xylene, pxylene with a concentration of 10 ppm each).
The selection of a longer suction interval for the calibration gas is required, to
allow the purging of the whole route between the gas chromatograph and the
calibration cylinder before the start of the calibration. In order to calibrate the
chromatograph, the calibration cylinder is opened and the valve of the flow regulator
is slowly opened. During the suction of the calibration gas through the pump, the
indicator of the flow regulator will be maintained at a balance value found between the
maximum area (red colored area) and the minimum one of the flow, this allowing the
ensuring of a proper flow for the calibration gas. After the introduction of the
calibration gas in the chromatograph, the calibration cylinder is closed and then the
flow regulator is closed.
When the chromatograph has to be calibrated, the desired column is selected,
set as follows: ENTER MENU-SETUP-CONFIG-COLUMN. By pressing in this
moment the key ENTER MENU is activated the column A or B or C on which the
analysis will be carried on, the possibility of selecting one detector or both detectors in
the same time being available (the PID detector or /and ECD). In this moment are
selected the PID or / and the ECD libraries depending on the chosen detectors.
Following the scheme ENTER MENU LIBRARY LIST and using the left-right
arrows is selected the library corresponding to the selected column. For the activation
56
or the deactivation of a compound, is flagged the name of the compound and are
pressed the keys below EDIT CALCMPD. If a compound is not selected, it is
automatically removed from the library. For the entering of the concentration of a
certain compound, are pressed the keys below EDIT CALCONC. By using the
arrow keys are entered the concentrations of the compounds from the calibration gas.
The calibration gas must contain all the compounds that were activated in the selected
library. By pressing the key START/STOP is started the analysis, then are pressed the
keys below DISPLAY GC CMPD to view the list of the compounds, and at the
finishing of the analysis it will be checked if all the calibration compounds are present
in the list. By pressing ENTER MENU LIBRARY- CALL is accepted the last
executed analysis as being proper for the calibration of the portable gas
chromatograph.
57
I.14.
Introduction
The trihalomethanes (CHCl3, CHCl2Br, CHClBr2, CHBr3) are formed in water
during the chlorination process. Several formation mechanisms are possible, from
which we mention the one starting from polyphenolic degradation products of the
fulvic acid from the composition of the natural humus substances.
The concentration of trihalomethanes in water is monitored due to the role
attributed to them in the carcinogenesis processes, the maximum allowed
concentration being 100 g/L (and respectively 30 g/L CHCl3).
Working Procedure
Preparing the filling of the chromatographic column
The stationary phase used is the squalan (5,6,10,15,19,23-hexamethylen
tetracosan), having the boiling point at 375 C and the density of 0.805 g/cm 3.
CH3
CH3
CH (CH2)3
CH3
CH3
CH3
CH3
CH3
CH (CH2)3 CH (CH2)4 CH (CH2)3 CH (CH2)3 CH
CH3
58
O
Si O Si
H2O, H+
OH
OH
Si O Si
2 Cl
CH3
Si Cl
CH3
Cl
Cl
CH3 Si CH3 CH3 Si CH3
Si
O
Si
59
Chromatogram obtained at the separation of the trihalomethanes from waters. 1extraction solvent (pentane); 2- CHCl3; 3-CHCl2Br; 4-CHClBr2; CHBr3.
60
The peaks areas are appreciated from the chromatogram of the reference
sample. The following values are obtained:
The area of a peak is obtained by multiplying the height of the peak with its
width at 0.607 from the height.
The unknown concentrations are found from the formulas:
A eTHM ...................c eTHM
x
A THM
.................... x
x
A THM
c eTHM
A eTHM
61
I.15.
Introduction
The phenol and substituted phenols represent common by-products in many
industrial processes. At concentrations of the order of ppb, in environmental
conditions depending on temperature and pH, these compounds may persist for several
days or weeks. It was demonstrated that the phenols are toxic for most aquatic
organisms and, even in low concentrations, they have a bad influence on the taste and
smell of water. Because of this, in the case where different types of waters will be used
as water sources for human consumption, is imposed the contamination check for
these organic pollutants. In the European Union, the maximum allowed concentration
for different phenols in the drinking water is extremely small. In this situation, is
imposed the use of an analytical method that will ensure a high selectivity and
sensitivity.
In this work is presented a method for the determination of phenols from
water by using a pre-concentration procedure by selective extraction of these
compounds from water on adsorbent cartridges and determination of the extracted
phenols from the cartridge by high performance liquid chromatography with UV
detection.
Reagents and Apparatus
- Acetonitrile of HPLC grade (Riedel de Haen); water of HPLC grade (Fluka),
methanol of HPLC grade (Fluka), acetic acid (Aldrich).
- Cartridge SPE for pre-concentration: Supelclean ENVI-Chrom P (Supelco)copolymer styrene-divinyl benzene (the particle size is: 80-160 m; the pore
diameters are: 110-175 ).
- Standard of phenols: EPA 604-M Phenols Mix (Supelco)- formed from a mixture
prepared in methanol of 11 analytes, at different concentrations: phenol (500
g/mL); 4-nitrophenol (2500 g/mL); 2,4-dinitrophenol (1500 g/mL); 2chlorophenol (500 g/mL); 2-nitrophenol (500 g/mL); 2,4-dimethylphenol (500
g/mL); 4,6-dinitro-o-cresol (2500 g/mL); 2,4-dichlorophenol (500 g/mL); 4chloro-m-cresol (2500 g/mL); 2,4,6-trichlorophenol (1500 g/mL);
pentachlorophenol (2500 g/mL).
- Standard of phenols: EPA 604-S Phenols Mix (Supelco) - formed from 11
individual phenol solutions, with the concentration of 500 mg/L prepared in
62
methanol, with the volume of 1 mL, phenol; 4-nitrophenol; 2,4-dinitrophenol; 2chlorophenol; 2-nitrophenol; 2,4-dimethylphenol; 4,6-dinitro-o-cresol; 2,4dichlorophenol; 4-chloro-m-cresol; 2,4,6-trichlorophenol; pentachlorophenol) and
a vial with a mixture of all the above mentioned compounds.
- Solid phase extraction system type Vacuum Manifold Supelco with 12 positions.
- Liquid trap type Supelco SPE Vacuum Pump Trap Kit.
- Vacuum pump Heidolph.
The determinations were made using a high performance liquid
chromatograph Perkin Elmer series 200, equipped with:
- quaternary pump; vacuum degasser for solvents;
- auto-sampler (for the automated injection in the chromatographic column of a
previously selected quantity of sample and for the automated washing of the
syringe);
- column oven for thermostating the chromatographic column;
- UV-VIS detector type Diode Array with the possibility of simultaneous spectra
acquisition;
- computer (software).
Working Procedure
Sample preparation
The water sample is acidified to a pH=2 to prevent the ionization of the
phenol compounds.
The preconcentration of the sample on a SPE cartridge
The cartridge is activated by passing through it 6 mL of methanol at a flow rate of
1 mL/min.
The cartridge is conditioned by passing through it 6 mL of acidified water (pH=2)
at a flow rate of 1 mL/min.
Through the cartridge are passed at a flow rate of 2.5 mL/min, 100 mL of sample
placed in an additional vessel.
The elution from the cartridge is done with 2 mL of solvent mixture methanol/
acetic acid 1%, at a flow rate of 1 mL/min.
After this the eluate is analyzed chromatographically in order to separate the
components and determine them.
Chromatographic analysis
In the vessels (vials) of the automated sample changer (auto-sampler) are
introduced the standards and samples to be analyzed (about 2 mL)..
The system HPLC Perkin Elmer series 200 will be used.
A column with reversed phase Brownlee type Pecosphere C 18, is used,
having the pore diameter of 3 m, the length of the column being 83 mm and the
diameter 4.6 mm.
63
An elution with linear gradient of the mobile phase will be used: from 35 %
acetonitrile/water (pH=2.2) up to 100 % acetonitrile, over an interval of 4 minutes.
The mobile phase flows through the chromatographic column with a flow rate
of 2.5 mL/min. The chromatographic analysis done in these conditions takes five
minutes. The elution order from the chromatographic column is the following: phenol;
4-nitrophenol; 2-chlorophenol; 2,4-dinitrophenol; 2-nitrophenol; 2,4-dimethylphenol;
4-chloro-m-cresol; 2,4-dichlorophenol; 4,6-dinitro-o-cresol; 2,4,6-trichlorophenol;
pentachlorophenol.
In the case of water samples with a more complex composition, for a better
separation of the phenolic compounds, can be used another chromatographic column:
type Supelcosil LC-8, with the dimensions 15 cm length x 4.6 mm. i.d. with the
particle diameter of 5 m. The same elution with linear gradient of the mobile phase
is used (A: methanol/1% acetic acid; B: H 2O/1 % acetic acid) as follows: 5-100 % A +
95 0% B in 30 minutes. The mobile phase flows through the chromatographic
column with a flow rate of 1 mL/min. The analysis time to separate the 11 phenolic
compounds is 11 minutes, the elution order from the chromatographic column is the
following: phenol; 4-nitrophenol; 2,4-dinitrophenol; 2-chlorophenol; 2-nitrophenol;
2,4-dimethylphenol; 4,6-dinitro-o-cresol; 2,4-dichlorophenol; 4-chloro-m-cresol;
2,4,6-trichlorophenol; pentachlorophenol.
In both situations, the temperature of the column is set at 30 C, the
measurement being done with a UV-VIS detector type Dyode Array with simultaneous
spectral acquisition, the phenolic compounds being simultaneously monitored at two
wavelengths: 254 nm and 280 nm.
In order to establish the retention time corresponding to each phenolic
compound from the mixture is used the phenol standard: EPA 604-S Phenols Kit
(Supelco) - formed from 11 individual phenol solutions, with the concentration of 500
mg/L. Each phenolic compound is diluted 1:50 with methanol. By injection in the
chromatographic system of 60 L from each diluted phenolic compound solution
corresponding to 0.6 g, is determined the corresponding retention time for each
compound. As a result, the identification of the phenolic compounds separated from
the analyzed sample will be done automatically through the software of the
chromatograph, by comparing with the retention times of the standards previously
introduced in the HPLC system.
The quantitative analysis of the phenolic compounds is done by the
interpolation of the area of the chromatographic peak corresponding for each separate
compound, on the calibration curve obtained using mixtures of different
concentrations of standard solutions of phenolic compounds prepared in methanol
(v/v).
To ensure the reproducibility of the retention times corresponding to the
phenolic compounds, and for a high precision of the determinations, before the start of
the chromatographic analyses, is required an interval for the stabilization of the
chromatographic system of at least one hour.
64
Conc. of
phenolic
comp. in the
standard
EPA 604-M
(Supelco)
500 mg/L
2500 mg/L
1500 mg/L
Conc. of
phenolic
comp. in the
standard
EPA 604-M
(Supelco)
diluted 1:25
with
methanol
20 mg/L
100 mg/L
60 mg/L
Conc. of
phenolic
comp. in the
standard
EPA 604-M
(Supelco)
diluted 1:50
with
methanol
10 mg/L
50 mg/L
30 mg/L
Conc. of
phenolic
comp. in the
standard
EPA 604-M
(Supelco)
diluted 1:100
with
methanol
5 mg/L
25 mg/L
15 mg/L
Conc. of
phenolic
comp. in the
standard
EPA 604-M
(Supelco)
diluted 1:125
with
methanol
4 mg/L
20 mg/L
12 mg/L
Phenol
4-Nitrophenol
2,4Dinitrophenol
2-Chlorophenol
2-Nitrophenol
2,4Dimetilphenol
4,6-Dinitro-ocresol
2,4Dichlorophenol
4-Chloro-mcresol
2,4,6Trichlorophenol
Pentachlorophe
nol
500 mg/L
500 mg/L
500 mg/L
20 mg/L
20 mg/L
20 mg/L
10 mg/L
10 mg/L
10 mg/L
5 mg/L
5 mg/L
5 mg/L
4 mg/L
4 mg/L
4 mg/L
2500 mg/L
100 mg/L
50 mg/L
25 mg/L
20 mg/L
500 mg/L
20 mg/L
10 mg/L
5 mg/L
4 mg/L
2500 mg/L
100 mg/L
50 mg/L
25 mg/L
20 mg/L
1500 mg/L
60 mg/L
30 mg/L
15 mg/L
12 mg/L
2500 mg/L
100 mg/L
50 mg/L
25 mg/L
20 mg/L
65
Table B. Amounts of phenols expressed in g from standard solutions prepared for the
calibration graph (injected volume = 60 L)
Phenolic comp.
in the standard
EPA 604-M
Conc. of
phenolic
comp. in the
standard
EPA 604-M
(Supelco)
500 mg/L
2500 mg/L
1500 mg/L
Conc. of
phenolic
comp. in the
standard
EPA 604-M
(Supelco)
diluted 1:25
with
methanol
1.2 g
6.0 g
3.6 g
Conc. of
phenolic
comp. in the
standard
EPA 604-M
(Supelco)
diluted 1:50
with
methanol
0.6 g
3.0 g
1.8 g
Conc. of
phenolic
comp. in the
standard
EPA 604-M
(Supelco)
diluted 1:100
with
methanol
0.3 g
1.5 g
0.9 g
Conc. of
phenolic
comp. in the
standard
EPA 604-M
(Supelco)
diluted 1:125
with
methanol
0.24 g
1.2 g
0.72 g
Phenol
4-Nitrophenol
2,4Dinitrophenol
2-Chlorophenol
2-Nitrophenol
2,4Dimetilphenol
4,6-Dinitro-ocresol
2,4Dichlorophenol
4-Chloro-mcresol
2,4,6Trichlorophenol
Pentachlorophe
nol
500 mg/L
500 mg/L
500 mg/L
1.2 g
1.2 g
1.2 g
0.6 g
0.6 g
0.6 g
0.3 g
0.3 g
0.3 g
0.24 g
0.24 g
0.24 g
2500 mg/L
6.0 g
3.0 g
1.5 g
1.2 g
500 mg/L
1.2 g
0.6 g
0.3 g
0.24 g
2500 mg/L
6.0 g
3.0 g
1.5 g
1.2 g
1500 mg/L
3.6 g
1.8 g
0.9 g
0.72 g
2500 mg/L
6.0 g
3.0 g
1.5 g
1.2 g
66
I.16.
Analytical Procedure
Fill a 5 mL syringe with acetonitrile. (Collect the sample extract in a 3 mL
narrow neck flask). Eluate the derivatives by slowly (approximate 1.5 mL/min)
pushing acetonitrile through the cartridge. Stop the elution when the 3 mL mark is
reached. Transfer approximately 0.5 mL of the sample solution to a 2 mL autosampler
vial and seal the vial. The sample is now ready for HPLC analysis.
67
% Tetrahydrofuran
% Acetonitrile
% Water
% Methanol
0.0
18.0
22.0
60.0
0.0
0.5
18.0
22.0
60.0
0.0
20.0
8.4
37.4
54.2
0.0
24.0
0.0
0.0
34.0
66.0
40.0
0.0
0.0
15.0
85.0
41.0
0.0
0.0
15.0
85.0
45.0
0.0
100.0
0.0
0.0
48.0
18.0
22.0
60.0
0.0
The detection and quantification is carried out at 369 nm (band width 22 nm)
using 474 nm (band width 50 nm) as the reference wavelength. The detection and
quantification of dicarbonyls is carried out at 440 nm (band width 22 nm) using
337 nm (band width 50 nm).
Following carbonylcompounds should be measured: methanal, ethanal,
propenal, propanal, propanone, 2-methyl-propenal, butanal, 2-butanone, 3-buten-2one, pentanal, hexanal, benzenecarbaldehyde, ethandial, oxopropanal.
Blanks
Each day analyses of carbonylcompounds are performed, a laboratory blank
should be prepared. Periodically field blanks should be obtained once every week. The
blank levels of methanal, ethanal, and propanone will probably change with cartridge
batch number and the batch number of acetonitrile. The blank level should not
exceed 0.05 g/m3 of the carbonyl compound in a sample volume of 750 litres.
1.
Preparation of Hydrazones
Dissolve 400 mg of 2,4-dinitrophenylhydrazine in 2 mL 96% sulphuric acid. This
solution is then added, with stirring, to 13 mL 75% ethanol. Any undissolved
solid is removed by aid of a Pasteur pipette.
68
2.
3.
4.
5.
Calibration
Prepare a stock solution from each carbonylhydrazone by dissolving
approximately 5 mg (+/- 1%) in 100 mL acetonitrile. (These stock solutions will be
ready for use.) Calibration solutions are prepared by dilution of the stock solutions
(1 g/mL to 2 g/mL is suitable for most analyses).
Quantification
The concentration of carbonylcompounds in the air sample expressed as g/m 3 ,
is given by:
69
70
I.17.
Introduction
This method provides a quantitative evaluation of alachlor from water
utilizing ELISA (enzyme linked immunosorbent assay) technology. Alachlor is a
Group I compound, with adverse acute health effects from exposure to low
concentrations. The assay is simple and adaptable enough that it may be used on site
in the field as a screening method. Direct ELISA involves usually immobilization of
antibody on the well surface by simple adsorption, followed of addition of analyte
(alachlor) and labeled analyte derivative (alachlor derivative labeled with peroxidase
(HRP)), which will start to compete for antibody (affinity purified anti-alachlor,
developed in sheep) active sites. Using a substrate mixture specific for tracer marker
(in our case the substrate, e. g. 3,3,5,5-Tetrametylbenzidine and H 2O2 in 30 mL
acetate buffer 50 mM, pH 5.2 is specific for HRP), the reacted analyte concentration
can be evaluated indirectly by determining the concentration of bound fraction of
tracer. Spectrometrical detection (450 nm) allows to have a quantitative determination
of alachlor by direct ELISA. The method is applied for alachlor determination from
water samples (e. g. drinking water, surface water, mixed domestic and industrial
wastewaters, groundwater, reagent waters).
2-[(2,6-diethyl-phenyl)(methoxymethyl)amino]-2-oxoethane sulfonic acid
(ESA) cross reacts with alachlor in the ELISA. ESA is not detected by standard HPLC
or GC. Alachlor related acetanilides may also interfere with the determination of
alachlor, such as acetochlor, metolachlor, and metalaxyl. In order to prevent eventual
errors in analysis, the cross reactivity of used antibody for possible interference has to
be evaluated.
-
Instruments
Micro-plate with 96 wells (NUNC, Brand Products).
Universal micro-plate reader (EL 800, Bio-tek Instruments INC.).
Automatic multi-finger pipette (8 positions).
71
the sample container with water from a representative area. Sampling equipment,
including automatic samplers, must not use plastic tubing, plastic gaskets, or any parts
that may leach interfering analytes into the sample. Automatic samplers that composite
samples over time should use refrigerated glass sample containers.
Residual chlorine in the sample should be reduced by adding 50 mg/L of
sodium sulfite (this may be added as a solid with stirring or shaking until dissolved, or
as a prepared solution).
Adjust the sample to pH 2 by adding 6 N HCl. It may require up to 4 mL to
accomplish this. It is very important that the sample be dechlorinated before adding
the acid to lower the pH of the sample. Adding sodium sulfite and HCl to the sample
bottles prior to shipping the bottles to the sampling site is not permitted. HCl should
be added at the sampling site to retard any microbiological degradation of method
analytes. Samples must be iced or refrigerated at 4C from the time of collection until
extraction. Preservation study results show that the analyte is stable for 14 days in
samples that are preserved it. Refrigerated sample extracts may be stored up to 30
days.
Sample Preparation
Alachlor is extracted and concentrated from the sample by SPE (solid phase
extraction) using solid phase extraction disk, 25 mm, SDB-xc (3M Empore TM, or
equivalent). Remove the SDB disk from the sampler and place in a glass bottle with an
ID large enough to allow the filter to lie flat. Add 2.0 mL of pesticide free methanol to
the SDB disk, cap, and shake on a platform orbital shaker for 15 minutes. Transfer the
methanol solution to a 4 mL sample vial with a PTFE-lined cap. Using a positive
displacement pipette or syringe, remove an aliquot of sample and dilute it a minimum
of 1:100 with deionized water. Wrap remainder of the samples with parafilm and store
refrigerated.
Note: If concentrations are higher than 0.5 g/sample, further dilutions are
needed to meet the target range of the assay. Multiple dilutions may be done if the
concentration range is unknown.
Analysis
Experiment 1: Optimisation of tracer and antibody concentration (Bidimensional ELISA)
1. The plate is covered with 100 L of antibody solution (i.e., in the range 1000 - 0
mg/L in 50 mM carbonate buffer, pH 9.6, on the column 1 to 12 by successive
dilution), and the adsorption is allowed to proceed over night at 4C.
2. After the residual solution is removed, the plate is washed with PBST (approx.
200 L / well).
3. 100 L of different concentrations of tracer (i.e., in the range 1/100 - 0 tracer
dilution in PBST) are added on the second dimension (e.g., row A to H) by
successive dilution. The incubation is allowed to proceed for 5 hours.
72
4.
The residual solution is removed, and the plates are washed and dried as
described in step 2.
5. 200 L of the substrate solution is added in each well. The reaction is allowed to
proceed for 10 minutes, when it is stopped by addition of 50 L of 4M H 2SO4 and
incubate again for another 25-30 min.
6. The absorbance is read at 450 nm.
The optimum concentrations of tracer and antibody are chosen as the ones that
give 40 70 % binding.
1.
2.
3.
73
The tracer is added to the optimised concentration so that the final volume in each
well is 100 L. The incubation is performed for 5 hours.
4.
5.
6.
1
2
3
4
The residual solution is removed and the plate is washed as described in step 2,
experiment 1.
200 L of the substrate solution is added in each well. The reaction is allowed to
proceed for 10 min, when it is stopped by addition of 50 L of 4 M H 2SO4 and
incubate for 25-30 min.
The absorbance is read at 450 nm.
Report
Explain how you choose the "best" antibody and tracer dilution and why you did
in that way?
Estimate IC50, the dynamic range and the detection limit for alachlor determination
using dilution curve in buffer and sample.
Approximate the matrix influence in each case (i.e., by calculating the ratio
between IC50 signal of calibration in buffer and signal corresponding to the same
point in the case of urine dilution).
Estimate the concentration of alachlor in the unknown sample.
74