Chapter i

II. AUTOMATIC ANALYTICAL METHODS FOR ENVIRONMENTAL MONITORING AND CONTROLLaboratory exercises

I. ANALYTICAL TECHNIQUES FOR ENVIRONMENTAL MONITORING AND

CONTROL

Laboratory Exercises

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I.1. SPECTROPHOTOMETRIC DETERMINATION OF AMMONIUM BY THE INDOPHENOL BLUE METHOD

This method is applicable to the determination of the ammonium content in waters (e.g. precipitation) within the range 0.04 to 2.0 mg NH4/L.

PrincipleIn an alkaline solution (pH 10.4-11.5) ammonium ions react with hypochlorite

to form monochloramine. In the presence of phenol and an excess of hypochlorite, the monochloramine will form a blue coloured compound, indophenol, when nitroprusside is used as catalyst. The concentration of ammonium is determined spectrophotometrically at 630 mn.

Reagents and InstrumentationAll the chemicals must be of recognized analytical grade. The water used for

dilution and rinsing should be double-distilled or de-ionized and distilled. Phenol (C6H5OH) Sodium nitroprusside (Na2Fe(NO) (CN)5 · 2H2O) Sodium hydroxide (NaOH) Sodium hypochlorite solution (NaOCl) 1M: make a solution containing approx.

3.5% active chlorine (35 g/L) in 0.1 M NaOH Ammonium chloride (NH4Cl) Sodium thiosulphate (Na2S2O3) Reagent A: dissolve 3.5 g phenol and 0.040 g sodium nitroprusside in 100 mL

water. Store the solution refrigerated in the dark. If the colour of the solution turns greenish, it must be discarded, and a fresh solution prepared.

Reagent B: dissolve 1.8 g sodium hydroxide in some water in a 100 mL volumetric flask. Add 4.0 mL 1 M sodium hypochlorite solution, and dilute with water to the mark. Store the solution refrigerated in the dark. If the solution is stored for weeks, the concentration should be checked by titration with a sodium thiosulphate solution.

Standard ammonium solution I, 100 mg NH4+/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.

Standard ammonium solution II, 4 mg NH4+/L: by means of a pipette, transfer

20.0 mL of standard ammonium solution I to a 500 mL volumetric flask. Dilute

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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 µL

CalibrationPreparation of calibration curve:

1. 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.

2. Add to the test tube 250 µL reagent A using a micro pipette, and mix well. Add then 250 µL 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.

3. Cool the solution to room temperature, and transfer it to a 10 mm cell. Measure the absorbance at 630 nm.

4. 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.

5. 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 ProcedureTransfer 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 NH4

+/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 NH4

+/L must be diluted. With suitable equipment the “Indophenol method” can be made automatic.

InterferencesIron (III) may interfere if the concentration is more than 2 mg/L. If the pH-

value 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.

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I.2. DETERMINATION OF CHLORIDE IN WATERS

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.

PrincipleChloride 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 + 2SCN-

SCN- + Fe3+ = Fe(SCN)2+

Reagents and InstrumentationDuring 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(NO3)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 140-200 °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.

Preparation of the Calibration Curve1. Transfer 2.5, 5.0, 7.5, 10, 15, 20 and 25 mL of standard chloride solution II to 50

mL volumetric flasks, and fill up to the mark with water. These solutions contain 0.5, 1.0, 1.5, 2.0, 3.0, 4.0 and 5.0 mg Cl-/L.

2. Transfer 25 mL of the calibration solutions to 100 mL Erlenmeyer flasks. To each flask add with pipettes 5 mL mercury (II) thiocyanate solution and 2 mL iron (III) nitrate solution. Mix well between and after the additions. After 20 minutes,

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measure the absorbance in 50 mm cells at 460 nm. As reference, use 25 mL water mixed well with 5 mL of reagent (2) and 2 mL of reagent (3).Plot the readings against the concentrations and draw the calibration curve.

Analytical ProcedureTransfer 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.

InterferencesBromide and iodide will give the same absorbance as the equivalent amount

of chloride.

References1. 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.

2. Zall, M., Fisher, D., and Gamer, Q. (1956) Photometric determination of chlorides in water. Anal. Chem., 28, 1665-1668.

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I.3. DETERMINATION OF THE SO2 CONCENTRATION IN AIR

IntroductionA serious ecological problem is the atmospheric pollution by sulphur dioxide

which origins are due to the combustion of sulphur compound combustion in engines or boilers. The slow fight against this pollution started recently; in fact legal rules are very restrictive with the concentration of sulphur in fuels.

The present method for sulphur dioxide determination in the air is based on the absorption and oxidation of sulphur dioxide in the sample producing sulphuric acid. Trapping is performed by means of the air bubbling through an hydrogen peroxide dissolution at fixed pH during a certain period (24 h).

To determine the sulphuric acid concentration formed this is put in contact with a well-known amount of barium and thorine salt. This reaction produces barium sulphate precipitating and the residual barium ions will form a complex coloured by reaction with the thorine that can be measured by absorption at 520 nm.

The difference between the amounts initial and final of barium ions which corresponds with the sulphate concentration in the solution gives the amount of sulphur dioxide oxidized in the sample.

Reaction into the bubbling vessel:

SO2 + H2O2 H2SO4

Formation of colour:

Ba2+ + thorine (Ba - thorine) red colour

(Ba-thorine) + H2SO4 BaSO4 + 2H+ + thorine + (Ba residual-thorine) red

TargetTo determine the daily average concentration of SO2 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.

Reagents and InstrumentationGlassware Reagents

Sampling set Water quality Milli-Q

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(manual control station)SpectrophotometerGlass cell, 10 mm light path

H2O2 HClO4 72% H2SO4 98% Ba(ClO4)2 anhydre1 micropipet, 250 L

10 volumetric flasks 100 mL2 pipettes 5 mL Thorine2 pipettes 10 mL Dioxane

Manual station of controlA manual station consists of the following elements:

- Air entrance: conical funnel for sampling of 3-5 cm, made of polyvinyl chloride and placed vertically with the mouth downwards. It should be placed far away from walls and other disturbing elements.

- Measurer the sample volume and control of the flow-rate.- Aspirating pump: the flow-rate should be adjusted to 2 m3 day.- System of filtration: a 5 cm2 area-trap for particles formed by a filtering device

retaining particles over the range 1-5 µm with an efficiency as close as possible to 100%. This filter will be apt for a flow-rate of 2 m3 day.

- Bubbling device with capacity for a volume of 100 mL. - Connectors: polyvinyl chloride tubing with an internal diameter of 8 cm. Avoid

curves and if not possible the strictly necessary must have a radius higher than 5 cm.

All solutions prepared with water quality of milli-Q. Caution: The following reagents must be carefully handled: perchloric acid,

barium perchlorate, dioxane and thorine.1) 27-30 % H2O2 (w/w) .2) 0.1 mol L-1 perchloric acid; take 4.3 mL of 72% perchloric acid and level to 500

mL. 3) 3) 0.01 mol L-1 perchloric acid: 25mL of 0.1 mol L-1 perchloric acid should be

levelled to 250 mL. 4) Trapping solution. Dilute 10 mL of dissolution 1 of 27-30 % H2O2 up to 1000

mL with water quality milli-Q. Adjust pH over the range 4.0 – 4.5 (pH-meter) by dropping the solution 0.1 mol L-1 of perchloric acid. This solution can be conserved during a month in the refrigerator.

5) Ba(ClO4)2 solution. Solve 0.525 g of anhydrous barium perchlorate in a small volume of solution 3 (0.01 mol L-1 perchloric acid). This solution is transferred to volumetric flask of 250 mL and level with the same acid.

6) Solution of Ba(ClO4)2 /dioxane. Diluted 1 mL of the solution 5 (Ba(ClO4)2) with 40 mL of water milli-Q and transfer into a volumetric flask of 1000 mL; then made up with dioxane.

7) Standard solution of sulphate 0.500 mol L-1. Dilute 28 mL of 98% of H2SO4 up to 1000 mL and titrate with NaOH solution (the hydroxide has been titrated vs the

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acid potassium phthalate). 1 mL of this solution sulphate corresponds with 32 mg of SO2.

8) Standard solution of sulphate 0.125 mol L-1. 25 mL from solution 7 (0.5 mol L-1) are taken and diluted with mili-Q water into a volumetric flask 1000 mL. 1 mL of this solution corresponds with 800 g of SO2.

9) Standard sulphate solution 0.00125 mol L-1. Dilute 10 mL of the stock solution (0.125 mol L-1) in a 100 mL volumetric flask with water (1 mL of this solution corresponds to 80 g of SO2).

10) Thorine solution 2.5 g L-1. Dissolve 0.125 g of thorine in 20 mL of solution 9 (sulphate standard 0.00125 mol L-1) and put into a volumetric flask of 50 mL and level it with milli-Q quality water.

Note: This solution should be freshly prepared daily.

Analytical Procedure1) Sampling and sample pre-treatment. Place 100 mL of the trapping solution

(solution 4) into the bubbling flask and start up the sampling equipment, an air volume exactly measured is forced through the bubbling device, so that the sulphur dioxide concentration remains within the calibration linear range. If it is necessary perform the required dilution. Write down the conditions of the sampling

2) Calibration graph.2.1) Sulphate standard solutions. A series of sulphate standard solutions is

prepared by diluting 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0 mL of the 0.00125 mol L-1 stock solution (solution 9) in volumetric flasks of 100 mL and made up with water. The resulting concentrations are, respectively: 0.8, 1.6, 2.4, 3.2, 4.0, 4.8, 5.6, 6.4, 7.2 and 8.0 g mL-1.

2.2) Blank standard solution. Prepare a solution by mixing 4.0 mL of trapping solution (solution 4) with 10 mL of barium perchlorate/dioxane solution (solution 6) and 0.25 mL for thorine solution (solution 10). This blank solution presents the maximum absorbance. The thorine solution aliquot should be added immediately before recording the absorbance. This solution remains unchanged for 30 minutes protected against room light.

3) Development of the colorimetric solution with the aid of the standard sulphate solutions. By using 4.0 mL of each standard sulphate solutions (point 2.2 of above) and by applying the procedure reported for the blank assay (point 2.2 of above). The resulting absorbance should be recorded before ten minutes.

4) 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.

5) 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

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standard solutions and record the corresponding absorbance values. Finally, read the sample solution before ten minutes.

6) Calculation of the SO2 concentration. Calculate the straight calibration line by dotting absorbance vs the different concentrations of SO2. Due to the developed reaction, the blank displays the maximum absorbance and the slope is negative.

7) Finally and to calculate the SO2 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 SO2 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.

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I.4. DETERMINATION OF SULPHATE IN WATERS

This method is applicable to the determination of sulphate in waters within the range 0.05 mg S/L to 4 mg S/L. Samples containing higher concentrations must be diluted prior to the analysis.

PrincipleBa(ClO4)2 is added in excess to precipitate the sulphate as barium sulphate in

an organic solvent. The organic solvent will minimize the solubility product of barium sulphate.

The excess concentration of barium (II) ions in the solution is determined spectrophotometrically at 520 nm through the reaction with thorin (the sodium salt of 4-(ortho-arsenophenyl-azo)-3-hydroxy-2,7-naphtalene-disulphonic acid).

Several organic solvents may be used. The most favourable calibration curve is obtained with dioxane.

Reagents and InstrumentationAll chemicals, except thorin, must be of recognized analytical grade. The

water used for dilution and rinsing must be double distilled or deionized. Sulphuric acid (H2SO4) 0.05 M Perchloric acid, (HClO4) 72 % Barium perchlorate (Ba(ClO4)2), anhydrous Dioxane or isopropanol Thorin (disodium salt) Cation exchange resin, strongly acidic (e.g. Dowex 50 W x 8, 50-100 mesh). 0.1 M perchloric acid (HClO4). 0.01 M perchloric acid (HClO4). Barium perchlorate stock solution 210.0 mg anhydrous barium perchlorate,

(Ba(ClO4)2) , is dissolved in 0.1 M HClO4 to a volume of 100 mL in a volumetric flask.

Barium perchlorate reagent solution 10.0 mL of stock solution is diluted to 1000 mL with dioxane or isopropanol.

Thorin reagent solution 125.0 mg of the disodium salt is dissolved in 5 mL 0.01 M HClO4 and diluted to 50 mL in a volumetric flask. A fresh solution should be prepared each day.

Sulphate standard solution 31.25 mL 0.05 M H2SO4 is diluted to 1000 mL in a volumetric flask. The concentration is equal to 50 mg S/L.

Spectrophotometer for measuring absorbance at 520 nm. Optical glass spectrophotometer cells; 20 mm

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Micro pipette: 250 µL Bulb pipettes: 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 mL Burette: 50 mL Ion exchange columns: 15 cm length, 1 cm diameter Test tubes: 30 mL Volumetric flasks: 50, 100 and 1000 mL

All glassware should be of borosilicate and should be thoroughly rinsed in distilled water before use.

Calibration CurvePrepare a series of standard solutions containing 0, 0.5, 1.0, 1.5 ..... 4 mg S/L

by diluting 0, 1, 2, 3, ..... 8 mL of the sulphate standard solution to 100 mL with water in volumetric flasks. Transfer 4 mL of each of these standard solutions to a test tube. Add 10 mL barium perchlorate reagent solution and 250 µL thorin solution. Use a micropipette for the thorin solution. Mix thoroughly (do not use rubber stoppers!)

Transfer the solutions to optical cells. The spectrophotometer wavelength is set at 520 nm, and 0% transmission is adjusted according to the procedure in the manual of the photometer. Then gain and/or slit width is adjusted to give a reading of 0.80 absorbance units with the blank (0 mg S/L) in the sample compartment. Measure the absorbance of the solutions within 10 minutes after addition of the thorin solution. This is especially important for low concentrations of sulphate and for the blank because the barium-thorin compound may precipitate from the solution.

A calibration graph is constructed from the absorbance readings obtained from the standard solutions. The calibration curve is not linear below 0.5 mg S/L. This is suppressed by adding sulphate in a quantity corresponding to 0.5 mg S/L to all samples and blanks. The detection limit is then 0.05 mg S/L.

Analytical ProcedureCations are removed by treating the sample with a strongly acidic cation

exchange resin.Transfer 4 mL of the pre-treated sample to a test tube and proceed according

to above. Determine the sulphur concentration of the sample from the absorbance reading by means of the calibration curve.

With suitable equipment, the barium perchlorate-thorin method can be made automatic.

InterferencesPhosphate will interfere with this method.

References1. Persson, G.A. (1966) Automatic colorimetric determination of low concentrations of sulphate for measuring sulphur dioxide in ambient air. Air Water Pollut., 10, 845-852.

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I.5. DETERMINATION OF ATMOSPHERIC LEAD (II)

IntroductionThe lead is a relevant atmospheric polluting agent. Its main anthropogenic

source was, until year 2001, the emission of gases from the gasoline used by engine vehicles which used lead tetraethyl as an anti-detonation additive. In addition, it is necessary to consider important lead sources in some places, like battery manufacturers, paintings, insecticides, metal smelting and metal recovery. The atmospheric lead is deposited on the surface of the leaves of vegetables located in the proximities of a highway or on dust particles at the edge of the highways. Its associated persistence to those dust particles will be of several years.

The atmospheric lead concentration occurs at small concentrations, normally associated to particles, which means if we forced a high air volume to pass through a filter; the deposited particles contain lead enough to be detected. In these conditions the liquid-liquid extraction procedure is suitable to isolate and concentrate the recovered analyte.

As empirical application of this procedure, the lead deposited on the surface of the leaves of a plant or tree or in the paper of filter dissolves in nitric acid. To eliminate the interferences of other metals the complexing agent CN- is added.

Dithizone (or diphenylthiocarbazone) reacts with many metallic ions to form chelates with intense coloration, which can be extracted by organic solvents. When a solution of dithizone in dichloromethane is put in contact with an aqueous solution of a metal at the appropriated pH, the metal-dithizonate is formed and hosted into the organic layer. The reagent (dithizone) excess is passed into the aqueous phase by alkalinizing.

The maximum of absorption of the lead-dithizone complex into the organic phase is at 520 nm.

TargetsTo determine the concentration of the lead associated to particles.To illustrate the lead pollution on different vegetal organisms due to engine

gasoline. The use of an easily available methodology based on the liquid-liquid

extraction combined with the visible photometry. An empirical test of the non-aqueous solvents and their most usual

applications.

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Reagents and InstrumentationA) Reagents The glassware should be previously washed with 0.01 moles L -1 nitric

acid to eliminate any possible residue of lead. Glassware and apparatus Reagents

100 mL polyethylene flasks (2) and latex gloves Standard solution 1000 mg/L of Pb(II)

50 mL volumetric cylinder (1) Dithizone 100 mL beaker (1) HNO3

Sampling set DichloromethaneGlass fibber filter 25 % NH3 (aq) Petri plates. Na2SO3

Analytical balance KCNScissors Na2SO4 8 metallic supports8 metallic circles8, 250 mL glass funnels 1 pipettes 5 mL 2, 5 mL pipettesFilter paper 8 conical flasks test tubes (8) 8 tubes with cork of spiral of 10 mL Spectrophotometer (VISIBLE)

B) Solutions to be prepared1) 5 mg L-1 Pb(II) solution. The standard solution is prepared by adding 2.5 mL from the stock solution containing 1000 mg L-1 and levelling to 1 L with 0.1 mol L-1

HNO3. The stock solution, 1000 mg L-1 Pb(II), is prepared by solving 1.5980 g Pb(NO3)2 (reagent analysis) with 0.1 mol L-1 HNO3 and then levelling up to 1000 mL with the same solvent.2) Alkaline complexing solution. For their preparation 750 mL of 25% NH3 (aq) are mixed with 1.0 g of KCN and 1.5 g of Na2SO3 and is levelled to 1 L with pure water. 3) Solution of dithizone (diphenylthiocarbazone) 25 mg/L. 0.025 g of dithizone in 1 L of dichloromethane.

Caution: 2 and 3 solutions must be prepared into the safety funnel.

Analytical Procedure1.a) Sampling and sample pre-treatment of the lead on plant leaves (sample

A). Pick carefully 8 medium leaves near a highway or zone with dense traffic, which

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do not show visible earth particles nor other visible polluting agents. Put the leaves 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 m3) 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: 1 2 3 4 5 6mL of standard solution, 5 mg L-1 0 0.5 1.0 1.5 2.0 2.5mL of 0.1 mol L-1 HNO3 20 20 20 20 20 20mL of alkaline complexing solution 5 5 5 5 5 5mL of 25 mg L-1 dithizone 5 5 5 5 5 5[Pb] (mg L-1) organic layer 0 0.5 1.0 1.5 2.0 2.5

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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 QuestionsPropose 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.

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I.6. SEPARATION BY EXTRACTION ANDSPECTROPHOTOMETRIC DETERMINATION OF MERCURY FROM WASTE WATERS

PrincipleThe method proposed for the separation and determination of mercury is

based on the selective precipitation of mercury with the reagent Cadion A (4-nitrophenyl 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 N NN

N NO2

NNNN

NNO2

Hg

With the given working conditions the reaction is highly selective. No interferences appear from the ions of Cu(II), Au(III), Be(II), Sn(II), Cd(II), Tl (I, III), UO2(II), Pb(II), Mn(II), Fe(II,III), Bi(III), Cr(III), Co(II), Ni(II), Rh(III), Pd(II), Pt(IV), PO4

3-, MoO42- and others. The ion Ag(I) interferes forming a red complex with

the reagent. The ion Cl- does not interfere up to a ratio Cl-/Hg(II) of 50. The ions of Br-, I-, SCN-, S2- and EDTA interfere forming stable combinations with the mercury (II). For the domain 0.1–4 g Hg(II)/mL is observed a linear dependence of the absorbance function of the mercury concentration. The value of the molar absorptivity is of 40000 L mol-1 cm-1. Because the formation of the mercury-cadion complex takes place in a neutral environment, to the reaction media is added a solution of sodium and potassium tartrate, which through the complexes formed will prevent the precipitation of some ions.

The reagent has the absorption maximum at 402 nm, and the mercury complex at 427 nm. The absorbance measurements, for the determination of mercury were done at 490 nm, a wavelength at which the absorbance of the reagent is small.

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Reagents and Instrumentation- Standard solution of mercury chloride with the concentration of 50 g Hg(II)/mL

in distilled water;- Solution of Cadion A (4-nitrophenyl diazoaminoazobenzene) 0.015 % in methanol;- Solution of sodium acetate 0.2 M;- Solution of sodium and potassium tartrate 20 % in distilled water;- Cylinders or test tubes with the volume of 10 – 50 mL provided with plug,

micropipette of 1000 μL; pipettes of 1, 2 and 5 mL, volumetric calibrated flask of 100 mL;

- The absorbance determinations are made with a spectrometer for molecular absorption in UV-VIS.

Calibration CurveFrom 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 AnalysisThe 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

17


precipitate from the reaction media. Subsequently the working procedure for the calibration curve will be used.

Interpretation of the Experimental DataThe 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.

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I.7. DETERMINATION OF LEAD BY ATOMIC ABSORPTION SPECTROMETRY WITH ELECTROTHERMAL ATOMIZATION (ET-AAS)

Maria - Cristina BRATU

IntroductionThe 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 µg/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.

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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 µS/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 µL 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 µg/L: in a volumetric flask of 25 mL of teflon are diluted 250 µL 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 µg/L: in a volumetric flask of 25 mL teflon are diluted 250 µL of lead standard with the concentration of 100 µg/L with 25 mL of Suprapur HNO3 0.2 %.

- The lead standards with the concentrations of 2 and 4 µg/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 µg/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.

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The graphite tube is maintained in position in the furnace by two graphite contacts that ensure the electrical contact for the heating of the tube. The graphite tube is manufactured from pyrolytic graphite and presents an integrated platform L’vov that determines a thermal equilibrium between the sample and the atmosphere inside the tube during the atomization step.

To prevent the oxidation of the graphite tube at high temperatures and to purge from the tube the vapors and fumes, is used an inert gas (argon) both inside the tube and around its outer shell. The cooling of the graphite tube is done by coupling it to a cold water source, an optimal temperature and flow rate of the water could achieve the cooling of the furnace in 20 seconds.- the autosampler – contains all the mechanical and electrical components required for the intake of a correct volume of solution from a selected container, its injection in the graphite tube and the washing of the injection needle.

The use of the autosampler for the injection of the solutions in the graphite tube leads to a considerable improvement of the analytical results compared to the manual pipette measuring.

The measured volume is between 1 – 99 µL. The autosampler also contains a tray with 88 positions, in which are placed

the polypropylene vials used for the reference solutions, the matrix modifiers solutions, the sample solutions and the diluent solutions. The position of each vial is numbered on the tray. - computer – with specific software “AA WinLab”, that controls the spectrophotometer.

Working ProcedureThe air compressor which controls the pneumatic operation of the

spectrometer is started. The argon cylinder is opened and then the graphite furnace cooling circuit is opened. The atomic absorption spectrophotometer and the computer are connected to the network. The specific software for this instrument is loaded, “AA Win Lab”. After an automated equipment verification test, from the method library, is selected the lead determination method based on the use of the graphite furnace with a matrix modifier (ammonium dihydrogen phosphate). The hollow cathode lamp and the deuterium lamp (for the background correction) are switched on – and let to heat up for half an hour.

In the method editor are established: the quantity of sample/standard injected (25 µL), the quantity of matrix modifier added (5µL) and its position on the autosampler tray, position of the diluent (HNO3 0.2 %), number of replicates, type of calibration curve (linear/nonlinear), the concentrations of the standards used for the calibration and the position of the most concentrated standard from which by automated dilution the other standards are prepared and the program of the graphite furnace.

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The Temperature Program of the Graphite Furnace25 µL of blank/standard/sample are introduced in the atomization unit and the

temperature is increased gradually to remove the solvent and interferents as well as possible before the atomization. In the table below is presented the temperature program of the graphite furnace optimised for the determination of lead.

The optimised temperature program for lead determination

Step #

Temperature (ºC)

Ramp(time-s)

Hold (time-s)

Internal gas flow

(mL/min)

Type of gas (norm/spl.) Reading

1 100 5 20 250 N No2 140 15 15 250 N No3 700 10 20 250 N No4 1800 0 5 0 N Yes5 2600 1 3 250 N No6 250 N 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 ºC. 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 dissociation of the volatilized molecular species. The atomization temperature is a

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characteristic of the analyzed element. For the atomization is desired the fastest possible temperature increase. That is why the time corresponding to the temperature ramp will be set to the minimum values that will ensure the highest temperature increase rate. At the beginning of this step, is activated the “reading” operation of the spectrometer by measuring the absorbance of the light radiation.

5. cleaning – after the atomization, the graphite furnace can be heated to higher temperatures, so that any sample residue remaining in the furnace will be burned.

Generating of the Calibration Curve “Zero” point of the instrument is read by measuring the absorbance

corresponding to the calibration blank (HNO3 0.2 %). The value of the absorbance corresponding to the calibration blank will be automatically subtracted from the absorbances corresponding to the standards and the samples.

Both for reading of calibration blank and samples is required the addition to the automated preparation of samples of 5 µL of ammonium dihydrogen phosphate (placed in the position 86 of the autosampler) as a matrix modifier.

For the generating of the calibration curve are used lead standards with the concentrations of 2, 4, 10 µg/L. The lead standards with the concentrations of 2 and respectively 4 µg/L are obtained by the automated dilution made by the autosampler of the spectrophotometer of 5 µL and respectively 10 µL of lead standard with the concentration of 10 µg/L (placed in the position 87 of the autosampler) with 20 µL and respectively 15 µL of nitric acid 0.2 % (the position of the diluent in the autosampler – 88).

The absorbances corresponding to the three lead standards are read, with two replicates each at 283.3 nm. The calibration curve is generated automatically by the software of the instrument, in a nonlinear type. The values corresponding to the correlation coefficient and the slope corresponding to the calibration curve are calculated automatically and displayed on the screen of the computer connected to the analytical instrument.

Determination of the Lead Concentration from the Environment of a Workplace

The lead concentration from an environment exposed to this pollutant is determined: inside the linotype workshop from typography.

In order to determine the lead concentration at a respiratory level, the lead is collected from three air samples, at three different moments, using for this, a sampling pump for gaseous samples “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:

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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 µL 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/m3 of air is applied the formula:

(2)

where Vdil. = 50 mL.; Replacing the equation (1) in the relationship (2), is obtained:

(3)

where D = flow rate (L/min); t = time (min).Thus, for the determination of the lead concentration from the collected air

samples are considered the lead concentration obtained on the calibration curve (c- expressed in µg/L), the volume to which the sample absorbent solution was diluted (Vdil. = 50 mL), the sampling flow rate of the air (D- expressed in L/min ) and the sampling time of the air in the absorbent solution (t- expressed in min).

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I.8. DETERMINATION OF MERCURY BY COLD VAPOURATOMIC ABSORPTION SPECTROMETRY

Maria - Cristina BRATU

IntroductionThe 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 µg/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 ProcedureReagents and apparatus

- Tin chloride (Fluka), hydrochloric acid (Baker), potassium permanganate (Spolek), sulfuric acid (Fluka), hydrochloric hydroxylamine (Reactivul); distilled water (conductivity 4 µS/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 %.

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- 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 µL 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 µg/L - prepared by dilution of 500 µL 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.

- Standards of mercury of concentrations of 2 µg/L, 5 µg/L, 10 µg/L used for the calibration of the system AAS-MHS 10 – prepared by adding in 10 mL of absorbent solution for mercury vapours of: 200 µL, 500 µL respectively 1000 µL of mercury standard of the concentration 100 µg/L. 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 µg/L are measured at the wavelength of 253.7 nm .

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The mercury standards of concentrations 2, 5, 10 µg/L are prepared right before the measurements, by introducing in the reaction vessel of the hydride generator MHS –10, in 10 mL of absorbent solution for mercury vapours, of 200 µL, 500 µL and respectively 1000 µL of mercury standard with the concentration 100 µg/L.

Operation of the MHS-10 System Coupled in Series with the Atomic Absorption Spectrophotometer:

In the following figure is presented the scheme of the system MHS-10.

The scheme of the system MHS –10.

When the system MHS-10 is in standby, an argon flow circulates continuously from the input point of the valve (P) towards its output point, (A) and from there through the flow restrictor F3 (that determines a nominal value of the flow of 650 mL/min).

When the reaction vessel containing the mercury standard is connected to the system (concentrations 2, 5, and respectively 10 µg/L), a flow of inert gas circulates through the tube (e) towards the quartz cell, removing the air from the system. A continuous flow of inert gas passes through the restrictor F1 (with a nominal value of

27


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 F3 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 SnCl2 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 µg/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 “MHS- 10” 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/m3 of air is applied the formula:

(2)

where V.sol. abs.= 50 mL.

Replacing the equation (1) in the relationship (2), is obtained:

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(3)

where D = flow rate (L/min); t = time (min)Thus, to determine the concentration of mercury from the collected air

samples is taken into consideration the concentration of mercury obtained from the calibration curve (c- expressed in µg/L), the volume of the absorbent solution (V abs.sol

= 50 mL), the air sampling flow rate (D - expressed in L/min ) and the air sampling time in the absorbent solution (t- expressed in min).

Working Procedure for the Determination of the Mercury Concentration from a Fish Sample

A sample of cod fillet acquired from a supermarket is analyzed. For this, a representative quantity of sample is chopped up, with the aid of a plastic knife. From this well homogenized sample, are weighed about 0.7 g of fish (the exact quantity is recorded). Two samples are performing in parallel, and to a third sample is added a known quantity of mercury standard. The calibration curve is generated using blank solution and mercury standards processed by the same mineralization procedure as the fish samples

Procedure for the Mineralization of the Fish Samples, the Blank Solution and the Mercury Standard (100 µg/L Hg)

In two Erlenmeyer flasks with plug are introduced approximately 0.7 g of fish, 5 mL concentrated H2SO4. The vessels are closed and placed in the water bath set at 70 ºC, for one hour. Then, the flasks are cooled on an ice bath, adding in this position 50 mL of KMnO4 6 %. The flasks are then introduced in the water bath set at 55 ºC, for two hours. Then, the flasks are let to cool at room temperature and are added 15 mL of hydroxylamine hydrochloride 20 %. From the resulted solutions is determined the mercury concentration with the aid of the AAS-MHS -10 system, analyzing two replicates of 10 mL for each solution.

In the case of the blank solution used for the generating of the calibration curve and for the preparing of the calibration standards with the concentrations 2, 5, and respectively 10 µg/L are introduced in the Erlenmeyer flasks with plug 5 mL of concentrated H2SO4, the rest of the procedure being identical as for the mineralization of the fish samples.

The calibration curve is generated using the blank solution and the standards that are processed before the analysis in the same manner as the samples. The mercury standard of concentration 10 mg/L is prepared by dilution of 500 µL commercially available mercury standard with the concentration of 1000 mg/L with 50 mL of a mixture of nitric acid without Hg 1.5 % and sulfuric acid without Hg 1.5 % in a volumetric flask of 50 mL (in the flask are added five drops of KMnO 4 6% for the

29


stabilization of the mercury). The mercury standard with the concentration of 100 µg/L is obtained by introducing in an Erlenmeyer flask with plug, of 700 µL 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 µL 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 µg/L.

For generating the calibration curve, 200 µL, 500 µL, and respectively 1000 µL of mercury standard with the concentration of 100 µg/L mineralized are introduced in 10 mL of mineralized blank solution put in the reaction vessel of the system MHS-10, in order to obtain the calibration standards with the concentrations 2, 5, 10 µg/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 µg/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 SamplesTo calculate the mercury concentration from the fish samples, expressed in

mg/kg sample, the following formula is applied:

(4)

where Vfinal sol. = 70 mL.

Thus, for the determination of the mercury concentration from the fish samples is considered the concentration of mercury obtained from the calibration

30


curve (c- expressed in µg/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 µL 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:

(5)

where: Conc. det. = concentration determined, obtained after applying equation (4); Conc. calc. = concentration corresponding to the fish sample +5 µg/L (added mercury standard).

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I.9. DETERMINATION OF pH IN PRECIPITATION. POTENTIOMETRIC METHOD

PrincipleThe 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 Bates-Guggenheim convention).

In precipitation samples, the ionic strength will typically be in the region 10 -3

to 10-5. 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.

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However, it is strongly recommended to check the electrode system at regular intervals, by measuring the “apparent pH” of a solution with low ionic strength with known pH or hydrogen ion concentration. The pH readings should be within 0.02 or 0.05 pH-units of the “theoretical” result. If this is not the case, or if the reading is unstable during stirring of the solution, the reference electrode should be replaced. New glass electrodes should be tested against at least two buffers to see that the response is Nernstian.

The reference electrode should preferably be stored in dilute potassium chloride solution (0.1M).

Reagents and InstrumentationThe National Institute of Standardized Technology (NIST) solutions with

known pH.1. 0.05 M potassium hydrogen phthalate (C6H4(COOH)(COOK),

pH = 4.00 at 20 °C pH = 4.01 at 25 °C. Dissolve 10.12 g potassium hydrogen phthalate, C6H4 (COOH) (COOK), dried at 120 °C, in 1000 mL distilled water.

2. 0.025 M potassium dihydrogen phosphate (KH2PO4) and 0.025 M disodium hydrogen phosphate (Na2HPO4) pH = 6.88 at 20 °C pH = 6.86 at 25 °C . Dissolve 3.39 g potassium dihydrogen phosphate, KH2PO4, and 53 g disodium hydrogen phosphate, Na2HPO4, dried at 120° C, in 1000 mL distilled water. Instead of the anhydrous disodium hydrogen phosphate, 4.43 g of undried dihydrate, Na2HPO4 · 2 H2O, may be used.

Commercial available buffer solutions may also be used, but should be checked against the primary standard buffers described above. The buffers should be kept in the dark in well closed bottles of borosilicate or polyethylene.

pH-meter with the possibility of reading to the nearest 0.02 pH-units or preferably to the nearest 0.01 pH-unit.

A glass electrode and a reference electrode must be used with the pH-meter. The reference electrode should be suitable for measurement in low-ionic strength solutions and preferably be of the calomel type filled with saturated potassium chloride. Other reference electrodes or combination electrodes may be used, but all electrodes should be checked for acceptable performance.

Magnetic stirrer, with teflon coated stirring bar.Beakers used for the test solution should be made of borosilicate glass or

polyethylene.

Calibration CurveCalibrate the pH-meter according to the instruction manual for the instrument

using one, or preferably two, buffer solutions. The temperature of the buffer solutions must be known. The calibration should be checked after each set of samples.

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Analytical ProcedureMeasure 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 PairThe behaviour of the reference electrode is the main source of errors in pH-

measurements, 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.

References1. 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.

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I.10. DETECTION OF PESTICIDE USING PRUSSIAN BLUE-SCREEN PRINTED BIOSENSORS

IntroductionThis 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 CH3COOH + HO(CH2)2N+(CH3)3Cl- (1)

AChE is irreversibly inhibited by organophosphorous and carbamate pesticides and a comparison of the activity of AChE before and after exposure to environmental samples can provide an evaluation of the pollution level. The measurement of enzymatic activity can be accomplished by electrochemical [6, 7, 9], chemiluminescent [10] or spectrophotometric methods [11, 12]. In recent years much attention has been devoted to the design of integrate electrochemical biosensors for the detection of compounds inhibiting the activity of cholinesterases. The amperometric detection of pesticide is more accurate and sensible then potentiometric methods, based on measurement of the acetic acid concentration increase. A bienzimatic amperometric system has been developed using the AChE together with the choline

35


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 PartPrinciple of the methodThe 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-

The enzymatic reaction produces thiocholine, which is oxidized at PB modified screen-printed electrodes. PB-modified SPEs covered with an appropriate enzymatic membrane are used in an electrochemical drop system.

Reagents- Glutaraldehyde 25% aqueous solution (v/v) (Sigma) - Nafion from Fluka, - Acetylcholinesterase (AChE) (EC 3.1.1.7, from electric eel, 244 U/mg) (Sigma) - Potassium haxacyanoferrate (III) chloride (Carlo Erba) - Iron (III) chloride (Fluka)- Paraoxon (diethyl-p-nitrophenilphosphate) (Sigma)

As working buffer solution a potassium phosphate buffer saline (0.05M phosphate buffer + 0.1M KCl, pH 7.4) is used.

All chemicals from commercial source were of analytical grade.ApparatusAmperometric measurements are carried out using a PalmSens

Electrochemical Sensor Interface, Palm Instruments BV, Netherlands.

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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 depositionPB 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 100°C to obtain a more stable and active layer of PB.

The PB modified electrodes were stored dry at room temperature in dark. Enzyme immobilizationThe 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 4°C.

Procedure for Pesticide MeasurementThe 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.

4) After that, the biosensor is washed with buffer, and the 2nd step is repeated 37


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 ReactivationThis 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.

38


References1. FAO, Agriculture towards 2010, in: C 93/94 Document of 27th 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, 645-6549. Solè 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, 37-

42.11. Pogačnik 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.

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I.11. DETERMINATION OF STRONG AND WEAK ACIDS INPRECIPITATION. COULOMETRIC TITRATION METHOD

IntroductionThis 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.

PrincipleIn 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- + ½H2

The EMF of a glass-calomel electrode pair is read at intervals and the results are used to construct a Gran’s 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 InstrumentationReagentsDuring 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)

40


- A 4.5 V dry battery with an adjustable series resistance and a mA meter is sufficient for measurements, but “coulometers” are available commercially (e.g. Metrohm).

- Titration vessel, 100 mL. This should have a suitable lid with holes to serve as support for the electrodes and the nitrogen inlet, and be supplied with a thermostat jacket.

- Thermostat (25 °C ± 1 °C) - Sensing electrodes. An ordinary glass electrode (pH range 0-10) and a calomel

reference electrode, or a combined electrode. - Working electrodes. The platinum electrode (2 x 2 cm2) is made of bright platinum

(sheet or net). The silver electrode is made from 99.9% pure silver, 1.0 mm diameter wire, about 30 cm long and coiled to a suitable dimension.

- Pipette: 50 mL - Micro pipettes: 0.5, 1.0 mL- Volumetric flask: 1000 mL

Analytical ProcedureTurn on all instruments, and allow heating for ½ hour. Adjust the pH-meter to

pH = 4.00, using the buffer solution. Transfer 50 mL of the sample into the thermostated titration vessel, and add 1 mL of solution I. Start nitrogen purging and adjust flow to give continuous agitation of the solution. The bubbles should not disturb the solution between the sensing and the working electrodes. Measure the pH of the solution. If the pH of the sample is above 5.6 it may be necessary to add more than 1 mL of solution I.

Wait until pH reading is constant. Switch pH-meter to read millivolts (range 0-240 mV with glass electrode positive) and start the electrolysis current.Read the glass electrode potential vs the calomel electrode every 20 seconds and continue until the potential changes sign (at pH ca. 8). Stop the electrolysis.

Plot Gran’s function, Y, at 25°C (see the following table) against electrolysis time (in seconds). The plot intercepts the abscissa at the equivalence point, t e (F = Faradays constant, R = the universal gas constant, T = absolute temperature).

41


Gran’s function

EmV y EmV y EmV y EmV y

1 1.04 41 4.93 81 23.4 121 111

2 1.08 42 5.13 82 24.4 122 115

3 1.14 43 5.33 83 25.3 123 120

4 1.17 44 5.55 84 26.3 124 125

5 1.22 45 5.77 85 27.4 125 130

6 1.26 46 5.98 86 28.4 126 135

7 1.31 47 6.22 87 29.6 127 140

8 1.36 48 6.47 88 30.7 128 146

9 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

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EmV y EmV y EmV y EmV y

25 2.65 65 12.5 105 59.7 145 283

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 ResultsThe concentration of strong acid in the sample is calculated from the formula:

or

wherei = electrolysis current in ampereste = electrolysis time at equivalence point (seconds)

43


F = Faradays constant (coulombs/mol)Vo = initial sample volume (litres)NH2SO4 = normality of added sulphuric acidVH2SO4 = volume of added sulphuric acid (litres)Notes: Borosilicate glass can be used for storage of samples.

The glassware must be treated with hot dilute acid and thoroughly soaked in distilled water prior to use. 12 hours with 10% hydrochloric acid at 90°C followed by 24 hours soaking in distilled water is considered adequate. Otherwise, alkali metals from the glass will diffuse into the samples.

References1. Gran, G. (1952) Determination of the equivalence point in potentiometric titrations.

Part II. Analyst, 77, 661-671.2. Liberti, A., Possanzini, M. and Vicedomini, M. (1972) The determination of the

non-volatile acidity of rain water by a coulometric procedure. Analyst, 97, 352-356.3. Rosotti, F.J.C. and Rosotti, H.J. (1965) Potentiometric titrations using Gran’s plots.

Chem. Educ., 42, 375-378.

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I.12. DETERMINATION OF BENZENE, TOLUENE AND XILENE IN AIR BY GASS CHROMATOGRAFY

IntroductionThe aromatic hydrocarbons have an extraordinary industrial importance; they

are raw material for more than 60% of the tonnage of plastics, elastomers and synthetic fibbers and also for dyes, insecticides, medicines, etc. In addition they are also present in fuels obtained from the petroleum. Due to that we can find them as atmospheric polluting agents in very diverse concentrations. The most relevant are benzene, toluene and different xylenes (mixed isomers), followed by the naphthalene and the anthracene.

The main sources of the benzene are the slight fractions of the soft coal tar and the extracted aromatic fractions from gasoline. The toluene is obtained from the same sources that the benzene and can be separated from it and from xylene by successive distillations. More of 50% of the produced toluene becomes benzene by hydro-dealkylation. A part of the fractions with high contents of toluene is added to gasoline to increase the octane degrees. These reasons lead to find benzene, toluene and xylene in the atmosphere like polluting agents. Normally they are analyzed jointly by gas chromatography and the total amount is expressed as BTX (μg of benzene, toluene and xylene m-3).

Gas chromatography (GC) is a technique of separation very used to solve problems in diverse fields, among them the environmental analysis. At the moment it is used routinely as a control technique presenting very good resolution for volatile organic compounds.The theoretical principles of the separation by means of GC do not differ basically from those of the chromatography in general. In the GC the mobile phase is a gas, whereas the stationary phase can be a solid adsorbent or a liquid retained on a solid support (packed column) or impregnating the walls of a capillary column (open column).

To obtain a good separation and good resolution chromatograms it is necessary: a perfect temperature control of the column, the control the flow-rate of the mobile phase and a suitable election of the mobile and stationary phases, as well as of the detection system.

TargetsTo be trained in the correct use of standards and instrumental techniques of

high performance.To be familiar with the gas chromatograph and its performance.Identify and determination of benzene, toluene and xylene in air samples.

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Reagents and ApparatusGlassware and apparatus Reagents

Sampling set PentaneAdsorbent cartridges Standards for GC:Gas chromatograph Benzene (standard)Capillary column non polar Toluene (standard)Detector FID Xylene (standard)Software for data process Analytical balance 19 mL tubes provided with stopcock 25 mL volumetric flasks1, 2, 3 and 4 mL pipettes Microsyringe, 10 µL

Preparation of SolutionsStock solution A. In a 25 mL flask are introduced 20 mL of pentane; cover

tightly and weigh it. Next, add 500 µL of benzene, it is covered immediately and weighed again. 500 µL 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.

Procedures1) 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 µm.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: 1µLColumn: HP5 type phenylmethylsilicone (30m x 0.32 mm x 0.25 µm)

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Gas carrier : Helium, flow-rate 1.5 mL min-1

Injector temperature 230 ºCDetector temperature 250 ºCProgrammed Column temperature: Isotherm at 35 °C during 5 min.From 35 °C to 140 °C at 15 °C min-1

Isotherm at 140 °C; 2 minFinal temperature: 35 °C

5.1.) Identification of benzene, toluene and xylene. A suitable solution of benzene and then of toluene are injected to assign the retention times which allow identify such compounds either in the calibration graph and samples.

5.2.) Standard injections. The different standard solutions of benzene, toluene and xylene are injected in the same conditions obtaining the corresponding chromatograms and correct integration of the areas of the peaks from these compounds.

5.3.) Sample Injection. 1 µL of the extract is also injected in the same conditions obtaining the corresponding chromatogram and the integration areas of the benzene, toluene and xylene peaks which will be identified by its retention time.

Results1) Identify the retention times (tR) for the three compounds.2) Calculate the concentration in µg L-1 of benzene, toluene and xylene using

all dots in the calibration graph.

A (stock solution) Write:Volume of benzene (µL) 500 mg of weighed benzene µg L -1 of benzene Volume of toluene 500 mg of weighed toluene µg L-1 de tolueneVolumen de xylene 500 mg of xylene (exact weight) µg L-1 of xylene

Standard n 1 2 3 4 5Volume of B solution (in mL)µg L -1 of benzeneµg L -1 of tolueneµg L-1 of xylene

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Write in the tables the area of the peaks in the calibration graphBenzene g/LÁrea

Toluene g/LÁrea

Xylene g/LÁrea

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 µg m -3.

Related Questions1) Explain the problems relative to the contamination due to organic compounds, its

origins, effects and possible solutions.2) List the different sampling schemes for organic compounds.3) Draw a scheme of the gas chromatograph.4) What effect has the temperature of the column on the separation of these

compounds?

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I. 13. DETERMINATION OF THE VOLATILE ORGANIC COMPOUNDS FROM THE ATMOSPHERE USING A PORTABLE GAS CHROMATOGRAPH

Maria-Cristina BRATU

IntroductionThe monitoring of the volatile organic compounds (VOC) in the

environmental samples is done either by the collecting of air samples and their subsequent analysis in the laboratory, or by determination at the sampling site using a portable gas chromatograph – (procedure described in this work).

The analysis of the volatile organic compounds from the atmosphere is done, usually, either by their adsorption on cartridges with specific fillings, followed by thermal desorption and their analysis with the aid of a chromatograph provided with a detector MS, FID, or ECD, or by collecting air samples in metallic vessels followed by their pressurization with an inert gas and the later gas chromatographic analysis of the mixture.

Both analysis procedures require the intervention of the human operator increasing the possibility of appearance of systematic errors that are reflected in the precision of the analysis.

The use of a portable gas chromatograph provided either with a FID, PID or ECD does not require the intervention of the operator in the sampling and analysis steps, greatly simplifying the working procedure and avoiding the errors inherent to the intervention of the operator in the sample preparing step.

Reagents and Equipment Carrier gas (nitrogen of chromatographic purity, min 99.999%). Cylinder for the calibration of the portable GC: “SCOTTY” cylinder of 48 L and

300 psi (BTEX Gas Mix), consisting of six analytes with the concentration in nitrogen of 10 ppm each, as follows: benzene, ethyl benzene, toluene, m-xylene, o-xylene, p-xylene.

The determinations of volatile organic compounds were done using the portable gas chromatograph “Photovac”, model Voyager, with the following configuration: two thermostated injection systems (an automated gas injection valve, a gas injection port using a gas syringe), a column oven that ensures the thermostating of four capillary columns mounted at the factory inside it (a “blank” column for the analysis of the total quantity of volatile organic compounds, a column for the analysis of the light volatile organic compounds, a column for the analysis of the medium

50


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.

The scheme of the portable gas chromatograph ”Photovac” model Voyager

Working procedureBefore 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

51


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-ethyl-ketone, 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.

52


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, p-xylene 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

53


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.

54


I.14. SEPARATION AND DETERMINATION OF TRIHALOMETHANES FROM WATERS BY GASCHROMATOGRAPHY

IntroductionThe 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 ProcedurePreparing the filling of the chromatographic columnThe stationary phase used is the squalan (5,6,10,15,19,23-hexamethylen

tetracosan), having the boiling point at 375 ºC and the density of 0.805 g/cm3.

CHCH3

CH3

(CH2)3 CHCH3

(CH2)3 CHCH3

(CH2)4 CHCH3

(CH2)3 CHCH3

(CH2)3 CHCH3

CH3

The inert substrate is Chromosorb W, AW-DMCS with granule dimensions 80/100 mesh. The Chromosorb W is a diatomite earth, that contains silicic acid in the hydrated amorphous phase, with a porous structure, thermally treated at 900 ºC with a small quantity of Na2CO3 (3 %) and has a specific surface of 1 – 3.5 m2/g. The AW index designates the previous acid washing of the commercially delivered product. The index DMCS designates the modification of the surface with dimethylchlorosilane as in the scheme from below.

The concentration of the stationary phase on the mix support is of 10 %.

55


For the preparation of 10 g of filling the following procedure is employed:- 1 g squalan is dissolved in 150 mL n-hexane of chromatographic purity - 9.000 g of Chromosorb W, AW-DMCS are weighed with a precision of ±1 mg,- the squalan solution is poured over the Chromosorb and the mixture is

homogenized with the aid of a magnetic stirrer for 15 minutes- the n-hexane is slowly evaporated, preferably in a distillation installation with

rotating still (the heating is done to at the most 85-95 ºC)- the filling prepared in this manner is left for 24 hours in a Petri dish.

A chromatographic column from glass with the internal diameter of 3.5 mm and the length of 1.4 m is used. The filling of the column is done in the following manner:- one of the ends of the column is blocked with a glass wadding plug- that end of the column is connected through a rubber hose to a vacuum pump - through a funnel of proper dimensions the filling is introduced in the column in

small portions. After each addition, the walls of the column are tapped with a wooden rod to obtain a uniform setting of the filling.

Parameters for the Separation- Mobile phase: N2

- Stationary phase: 10 % squalan on Chemosorb W, AW-DMCS- Column: internal diameter 3.5 mm, length 1.4 m- N2 pressure through the detector: 0.3 atm- Entry pressure of the mobile phase in the column: 0.8 atm- Detector: flame ionization type- Temperature of the column: 70 ºC- Temperature of the detector: 250 ºC- Temperature of the injector: 120 ºC- Volume of the injected sample: 3 L- Standards used: CHCl3, CHBr3, CHClBr2 of chromatographic purity.

56

Si O SiO

H2O, H+O SiSi

OH OHSiCH3

CH3

ClCl2+

SiSi CH3CH3

Cl

SiSiCH3 CH3

Cl

O


Method for the Preparing of the Samples and Standardsa) The method for the preparing of the standards is:- In a volumetric calibrated flask of 50 mL, with an impervious plug, are weighed at

the analytical balance quantities of standard substance between 20 and 90 mg. The flask is filled to the mark with methanol. The source solution obtained in this manner kept at + 4 ºC can be used for two weeks.

- Working reference solution obtained by the dilution 1/25 of the source solution with methanol. In this manner are obtained concentrations of trihalomethanes at the order of tens of g/L.

- In a vessel with the capacity of 10 mL, manufactured from polypropylene and having the lid provided with a silicone rubber septum, are introduced 4 mL of distilled water, 5 L of working reference solution (with the aid of a microsyringe) and 1 mL pentane of chromatographic purity.

- The vessel is stirred for 4 minutes.- The phases are allowed to separate.- With the aid of a microsyringe, a part of the organic layer is removed and injected

in the column.b) Method for the separation of the trihalomethanes from waters:- 4 mL of sample are introduced in the previously mentioned extraction vessel. 1

mL of pentane of chromatographic purity is added and the extraction is carried out; 3 L are removed from the organic layer and are injected in the column.

- If the sample contains free chlorine a few granules of sodium thiosulfate are added, and then the trihalomethanes are extracted in pentane.

Processing the ResultsThe aspect of a chromatogram is presented in the next figure:

Chromatogram obtained at the separation of the trihalomethanes from waters. 1-extraction solvent (pentane); 2- CHCl3; 3-CHCl2Br; 4-CHClBr2; CHBr3.

57

A (UV 220 nm)

timp (min)0 5 10 15

1

2

3

4

5A (UV 220 nm)

time (min)0 5 10 15

1

2

3

4

5A (UV 220 nm)

timp (min)0 5 10 15

1

2

3

4

5A (UV 220 nm)

time (min)0 5 10 15

1

2

3

4

5


The peaks areas are appreciated from the chromatogram of the reference sample. The following values are obtained:

The peaks areas are appreciated from the chromatogram of the unknown 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:

where: THM – trihalomethane– concentration of the reference in the respective trihalomethane

58


I.15. DETERMINATION OF THE PHENOLIC COMPOUNDS IN WATER BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Maria-Cristina BRATU

IntroductionThe 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 µm; the pore diameters are: 110-175 Å).

- Standard of phenols: EPA 604-M Phenols Mix (Supelco)- formed from a mixture prepared in methanol of 11 analytes, at different concentrations: phenol (500 µg/mL); 4-nitrophenol (2500 µg/mL); 2,4-dinitrophenol (1500 µg/mL); 2-chlorophenol (500 µg/mL); 2-nitrophenol (500 µg/mL); 2,4-dimethylphenol (500 µg/mL); 4,6-dinitro-o-cresol (2500 µg/mL); 2,4-dichlorophenol (500 µg/mL); 4-chloro-m-cresol (2500 µg/mL); 2,4,6-trichlorophenol (1500 µg/mL); pentachlorophenol (2500 µg/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

59


methanol, with the volume of 1 mL, 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) 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 ProcedureSample preparationThe 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 analysisIn 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 μm, the length of the column being 83 mm and the diameter 4.6 mm.

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.

60


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 µm. The same elution with linear gradient of the mobile phase is used (A: methanol/1% acetic acid; B: H2O/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 ºC, 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 µL from each diluted phenolic compound solution corresponding to 0.6 µg, 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.

61


Taking the calibration curveThe calibration curve is traced in four points. For this, the phenol mixture

standard EPA 604-M “Supelco” is diluted to a ratio of 1:25 (v/v), 1:50 (v/v), 1:100 (v/v), respectively 1:125 (v/v). The concentrations corresponding to the phenolic compounds mixture from the four obtained solutions, expressed in mg/L are presented in table A. The quantities of phenol expressed in µg from the standard solutions prepared for the tracing of the calibration curve are presented in table B.

In four vessels of the autosampler of the chromatograph are introduced approximately 2 mL from each of the four solutions, from which will be automatically injected 60 µL.

Table A. Concentrations (expressed in mg/L) of the phenolic compounds used for the tracing of the calibration curve.

Phenolic comp.in the standard EPA 604-M

Conc. of phenolic comp. in the standard EPA 604-M (Supelco)

Conc. of phenolic comp. in the standard EPA 604-M (Supelco) diluted 1:25with methanol




Phenol 500 mg/L 20 mg/L 10 mg/L 5 mg/L 4 mg/L4-Nitrophenol 2500 mg/L 100 mg/L 50 mg/L 25 mg/L 20 mg/L2,4-Dinitrophenol

1500 mg/L 60 mg/L 30 mg/L 15 mg/L 12 mg/L

2-Chlorophenol 500 mg/L 20 mg/L 10 mg/L 5 mg/L 4 mg/L2-Nitrophenol 500 mg/L 20 mg/L 10 mg/L 5 mg/L 4 mg/L2,4-Dimetilphenol


4,6-Dinitro-o-cresol


2,4-Dichlorophenol


4-Chloro-m-cresol


2,4,6-Trichlorophenol


Pentachlorophenol


62


Table B. Amounts of phenols expressed in µg from standard solutions prepared for the calibration graph (injected volume = 60 µL)

Phenolic comp.in the standard

EPA 604-M

Conc. of phenolic

comp. in the standard

EPA 604-M (Supelco)

Conc. of phenolic


EPA 604-M (Supelco)

diluted 1:25with

methanol

Conc. of phenolic


EPA 604-M (Supelco)

diluted 1:50with

methanol

Conc. of phenolic


EPA 604-M (Supelco)

diluted 1:100with

methanol

Conc. of phenolic


EPA 604-M (Supelco)

diluted 1:125with

methanolPhenol 500 mg/L 1.2 µg 0.6 µg 0.3 µg 0.24 µg4-Nitrophenol 2500 mg/L 6.0 µg 3.0 µg 1.5 µg 1.2 µg2,4-Dinitrophenol

1500 mg/L 3.6 µg 1.8 µg 0.9 µg 0.72 µg

2-Chlorophenol 500 mg/L 1.2 µg 0.6 µg 0.3 µg 0.24 µg2-Nitrophenol 500 mg/L 1.2 µg 0.6 µg 0.3 µg 0.24 µg2,4-Dimetilphenol

500 mg/L 1.2 µg 0.6 µg 0.3 µg 0.24 µg

4,6-Dinitro-o-cresol

2500 mg/L 6.0 µg 3.0 µg 1.5 µg 1.2 µg

2,4-Dichlorophenol

500 mg/L 1.2 µg 0.6 µg 0.3 µg 0.24 µg

4-Chloro-m-cresol

2500 mg/L 6.0 µg 3.0 µg 1.5 µg 1.2 µg

2,4,6-Trichlorophenol

1500 mg/L 3.6 µg 1.8 µg 0.9 µg 0.72 µg

Pentachlorophenol

2500 mg/L 6.0 µg 3.0 µg 1.5 µg 1.2 µg

After the obtaining of the chromatograms corresponding to each injected phenol mixture, the apparatus identifies the compound based on the residence time and automatically integrates the areas corresponding to each maximum. The four chromatograms obtained are considered calibration standards, and again in an automated mode the software of the apparatus generates the calibration curves for each compound at the four concentrations. Different types of calibration curves may be obtained. The most popular (also chosen in our case) is the I st order line with the intersection of the origin. If the “calibration curve” needs to be extended to higher concentration values it is recommended the use of a IInd order equation given that the maximum linearity deviation will not exceed 25 %.

It can be noticed that by the adsorption on the cartridge of 100 mL of sample contaminated with phenolic compounds, followed by the elution with 2 mL of mixture methanol/acetic acid 1%, most phenolic compounds can be easily determined at concentrations below the value of 0.1 ppb using the UV detection.

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I.16. DETERMINATION OF ALDEHYDES AND KETONES IN AMBIENT AIR

Aldehydes and ketones sampled as 2.4-dinitrophenylhydrazones in impregnated tubes can be analysed in extracts with high performance liquid chromatography (HPLC).

Reagents and InstrumentationReagents

- Acetonitrile, HPLC-quality, Rathburn Chemicals Ltd., No. RH 1016. - Methanol, Merck. - Tetrahydrofuran, Merck. - Water, quartz distilled and ion exchanged from a Millipore “MilliQ” water

purification system. - Potassium iodide, p.a, Fluka. - Sulfuric acid, Merck - Ethanol. - Carbonylcompounds needed. - 2,4-Dinitrophenylhydrazine, Fluka.

InstrumentationThe following instruments and equipment may be used:

- A Hewlett-Packard 1050 modular system, consisting of a 79852A quaternary solvent supply system, a G1306A diode array detector, and an autosampler.

- Nova-Pak C18, 4 µm particles (No. 86344, Waters Associates), 150 mm * 3.9 mm i.d.).

- Waters in-line Precolumn Filter, - Syringes and volumetric glassware.

Analytical ProcedureFill 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.

10 µL of a sample or standard solution is separated by using a quaternary mixture of methanol/water/acetonitrile/tetrahydrofuran. The following table shows the gradient profile which is used.

64


Quaternary gradient which separates the carbonylhydrazones of interest (EMEP) at a flow rate of 0.8 mL/min.

Time % 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-2-one, pentanal, hexanal, benzenecarbaldehyde, ethandial, oxopropanal.

BlanksEach 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 µg/m3 of the carbonyl compound in a sample volume of 750 litres.

Preparation of Hydrazones1. 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.

2. A volume corresponding to 500 mg of the carbonylcompound is transferred to 20 mL ethanol.

3. The carbonylsolution (step 2) is than transferred to the DNPH solution (step 1) with stirring. Let the solution stand for 15 minutes to complete the reaction.

65


4. Filter the solution in step 3, and recrystallize the hydrazone from aqueous ethanol. (Hydrazones from unsaturated/aromatic carbonyls should be recrystallized from aqueous acetonitrile.)

5. Dry the hydrazone and do a purity test (HPLC-UV).

CalibrationPrepare 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 µg/mL to 2 µg/mL is suitable for most analyses).

QuantificationThe concentration of carbonylcompounds in the air sample expressed as g/m3 ,

is given by:

C - Concentration of the carbonyl compound in the air sample [µg/m3]c - Concentration of the carbonyl compound in the standard [µg/mL]H(s) - Peak height/area of the carbonyl compound in the standard [counts]H(p) - Peak height/area of the carbonyl compound in the sample [counts]k - Conversion factor (e.g. from hydrazon to carbonyl) methanal:0.1429,

ethanal:0.1964, propenal:0.2373 etc.V - Sample volume [m3]v - Volume of the prepared sample [mL]

InterferencesFailure to remove ozone by the ozone scrubber will result in serious under-

estimating of some carbonylhydrazones.

References1. Vairavamurthy, A., Roberts, J.M. and Newman, L. (1992) Methods for

determination of low molecular weight carbonyl compounds in the atmosphere: a review. Atmos. Environ., 26A, 1965-1993.

2. Slemr, J. (1991) Determination of volatile carbonyl compounds in clean air. Fresenius J. Anal. Chem., 340, 672-677.

3. Dye, C. and Oehme, M. (1992). Comments concerning the HPLC separation of acrolein from other C3 carbonyl compounds as 2,4-dinitrophenylhydrazones: a proposal for improvement. J. High Res. Chrom., 15, 5-8.

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I.17. DETERMINATION OF ALACHLOR USING DIRECT ELISA

IntroductionThis 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 H2O2 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).

Sample Collection, Preservation and StorageCollect all samples in 40 mL bottles into which 3 mg of sodium thiosulfate

crystals have been added to the empty bottles just prior to shipping to the sampling site. When sampling from a water tap, open the tap and allow the system to flush until the water temperature has stabilized (generally one to two minutes). Adjust the flow to about 500 mL/min and collect the sample from the flowing stream. Keep sample sealed from collection time until analysis. When sampling from a body of water, fill

67


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 4°C 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 PreparationAlachlor 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 µg/sample, further dilutions are needed to meet the target range of the assay. Multiple dilutions may be done if the concentration range is unknown.

AnalysisExperiment 1: Optimisation of tracer and antibody concentration (“Bi-

dimensional” ELISA)1. The plate is covered with 100 µL 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 µL / well).

3. 100 µL 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.

68


Optimisation of the antibody concentration.

4. The residual solution is removed, and the plates are washed and dried as described in step 2.

5. 200 µL 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 µL of 4M H2SO4 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.

Experiment 2: Analyte calibration and analysis of real samples1. The plate is covered as described in step 1, experiment 1, except that only the

optimum concentration of antibody is used. Incubation over night at 4C.2. The residual solution is removed and the plate is washed as described in step 2,

experiment 1.3. The analyte calibration (i.e., from 1 ppm to 0 ppm alachlor) is performed on the

“long” dimension of the plate in different sample matrices, as following.

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ptim

isat

ion

of tr

acer

con

cent

ratio

n


The tracer is added to the optimised concentration so that the final volume in each well is 100 µL. The incubation is performed for 5 hours.

4. The residual solution is removed and the plate is washed as described in step 2, experiment 1.

5. 200 µL 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 µL of 4 M H2SO4 and incubate for 25-30 min.

6. The absorbance is read at 450 nm.

Report1 Explain how you choose the "best" antibody and tracer dilution and why you did

in that way?2 Estimate IC50, the dynamic range and the detection limit for alachlor determination

using dilution curve in buffer and sample.3 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).

4 Estimate the concentration of alachlor in the unknown sample.

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PBST

PBST

PBST

Sample undil.

Sample, 1/10

Sample, 1/100

Sample, 1/1000

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Chapter i