HOW IS AN ENVIRONMENTAL SAMPLE PREPARED TO DETERMINE TRACE CYANIDE? WHAT ABOUT PHENOLS?

308



Trace Environmental Quantitative Analysis, Second Edition



In

Cooling water



Screw clamp

Out



To low vacuum source

Inlet tube



Gas scrubber



Condenser



Distilling flask



Heater



FIGURE 3.40 Glass distillation apparatus to prepare wastewater samples to quantitatively

determine cyanide and phenolics.



used to quantitate cyanide. Figure 3.40 depicts a schematic for a two-holed distillation and gas scrubber apparatus used to isolate and recover both cyanide and

phenols from wastewater samples. The inlet tube, along with the connection to a

low-vacuum source, is used to provide purge air. As HCN gas is removed from the

distilling flask, it is trapped in the gas scrubber.

Simple distillation is used to prepare the wastewater sample for the determination

of total phenols. An acidified aqueous sample is merely simple distilled with the

aqueous distillate trapped into a scrubber containing dilute NaOH, as was the case

for cyanide. The contents of the scrubber consist of a dilute solution containing

sodium phenolate. This solution is subject to whatever determinative technique is

applicable to measure trace total phenolics.



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Sample Preparation Techniques



309



105. COULD WE PREPARE A SAMPLE TO DETECT CYANIDE

BY DRIVING HCN INTO THE HEADSPACE?

Yes, indeed. A sample, be it water, soil (enviro-chemical), or whole blood (envirohealth), is acidified, headspace sampled, and injected via a gas-tight syringe into a

dedicated gas chromatograph with a nitrogen-phosphorous detector (NPD).154 A

porous-layer open tabular column is used to provide gas–solid chromatographic

retention and separation of HCN (a fixed gas). The high sensitivity afforded by the

NPD enables the analyst to measure down to low ppb concentration levels of cyanide

in a human specimen. Since static HS is an inherent part of the gas chromatograph,

the IDL cannot be independently determined from the MDL, as discussed earlier in

this chapter. The sketch below shows a sealed HS vial for this application:

Crimp top seal



Headspace



Human specimen



CN− + H+



HCN(sample)



HCN(sample)



KH



HCN(g)



A sketch of what a GC-NPD gas chromatogram would look like after the

acidified specimen (spiked with acetonitrile as the internal standard) is shown below:



NPD

Response



HCN



0



CH3CN



Time after HS sampled and gas-tight syringe injected



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Trace Environmental Quantitative Analysis, Second Edition



Peak Area (HCN) /

Peak Area (CH3CN)



A calibration plot using acetonitrile as the internal standard might resemble the

sketch shown below:



Conc (HCN) / Conc(CH3CN)



Prior to acidifying the sample, ascorbic acid is added to whole blood to minimize

the loss of cyanide ion due to conversion to thiocyanate ion.



106. WHAT IS CHEMICAL DERIVATIZATION AND

WHY IS IT IMPORTANT TO TEQA?

Most priority pollutants (enviro-chemical) or persistent organic pollutants (envirohealth) can be directly injected into a gas chromatograph owing to their physicochemical properties of being relatively nonpolar, semivolatile, and thermally stable

in the hot-injection port of the GC. However, those organic compounds with heteroatom functional groups are polar, nonvolatile, and sometimes thermally labile.

Consider Figure 4.1, where the degree of analyte volatility is plotted against the

degree of analyte polarity. Polar, nonvolatile analytes are converted to less polar

ones, which become semivolatile derivatives. These derivatized organic compounds

fall into the realm of GC and are said to be amenable to analysis by GC. Derivatives

can also be prepared from analytes that yield a more sensitive means of detection

for GC and are of particular importance to HPLC. This author’s first encounter with

the need to make a chemical derivative involved the three chlorophenoxy acid

herbicides (CPHs) — 2,4-D, 2,4,5-T, and 2,4,5-TP (Silvex) — in drinking water.

EPA Methods 515.1 (drinking water) and 8150 (solid waste) require that CPHs and

other organic acids be converted to methyl esters. Earlier, boron trifluoride–methanol

(BF3-MeOH) was used to convert carboxylic acids to their corresponding methyl

esters (with mixed results from this author’s experience), while more recent methods

favor the more vigorous in situ generation of diazomethane gas. EPA Method 8151A

also considers that pentafluorobenzyl (PFB) esters of CPHs and other “chlorinated

acids of environmental interest” can be made and chromatographed using a GC-ECD.

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311



The PFB moiety in the derivatized ester of the CPH makes the ester extremely

sensitive to detection via GC-ECD.

Let us take a broad view of chemical derivatization in analytical chemistry. The

flowchart below summarizes how most commercially available derivatization

reagents are categorized:



Silylation is the conversion of active hydrogen in a functional group to a trimethyl

silyl (TMS) derivative. This was the first means to chemically convert carboxylic

acids, alcohols, thiols, and primary and secondary amines to TMS esters. TMS esters

are most appropriate where GC-MS is the principal determinative technique. Acylation is the conversion of active hydrogen, as is found in alcohols, phenols, thiols,

and amines, into esters, thioesters, and amides by reacting organic compounds that

contain these functional groups with fluorinated acid anhydrides. Heptafluorobutyrylimidazole and N-methyl-N-bis(trifluoroacetamide) are particularly effective in

converting primary amines to fluorinated amides. Introduction of a perfluoroacyl

moiety in the derivative leads to a significant increase in analyte sensitivity when

using GC-ECD as the determinative technique. Alkylation is the conversion of active

hydrogen by an alkyl or benzyl group to an ester or ether, depending upon whether

the functional group in the organic compound is a carboxylic acid or alcohol or

phenol, respectively. Diazomethane via in situ generation, BF3-MeOH, dimethyl

formamide–dialkyl acetals, and pentafluorobenzyl bromide are commonly used

derivatizing reagents. Enantiomeric purity analysis reagents form diastereomers

when reacted with optically active analytes. Diastereomers are easily separated by

GC. Commercially available reagents include (–)methyl chloroformate that reacts

with enantio-enriched alcohols and N-TFA-L-prolyl chloride that couples with

amines to form diastereomers. Chromotags are derivatizing reagents that add an

ultraviolet-absorbing chromophore to an aliphatic carboxylic acid that converts the

aliphatic acid to a UV-absorbing derivative to enhance sensitivity in HPLC-UV.

Fluorotags convert a minimally fluorescent analyte to a highly fluorescent derivative,

and hence enhance sensitivity in HPLC-FL. The reaction of aliphatic carboxylic

acids with p-bromophenacyl bromide in the presence of 18-crown-6 under alkaline

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conditions to form a strong ultraviolet-absorbing ester, and the conversion of aliphatic carboxylic acids to highly fluorescent 4-bromomethyl-7-methoxycoumarin

represent common uses of chromotags and fluorotags.155

Analytes are usually isolated and recovered via any of the extraction and cleanup

techniques described in this chapter. The extractant or eluent is evaporated to either

dryness or close to dryness in order to concentrate the analyte. The derivatizing

reagent, catalysts, acids, or bases, and any other reagents are then added. Heat is

applied if necessary to increase the reaction rate. The derivatized analyte is extracted

from the product mix and further cleaned up, excess derivatizing reagent is removed

if possible, and then the extract is injected into the appropriate chromatographic

determinative technique. It is important that the excess derivatizing reagent be

chromatographically separated from the derivative(s) to enable quantitative analysis.

Let us digress a bit to some specific examples of the use of chemical derivatization

to accomplish the goals of TEQA.



107. HOW DO YOU MAKE A PFB DERIVATIVE

OF SOME BUTYRIC ACIDS?

This author once attempted to prepare PFB esters of n-butyric, i-butyric, and 2-methyl

butyric acids.156,157 Here is what you need to do:

TO



PREPARE THE REAGENTS:



30% potassium carbonate: Dissolve 7.5 g of K2CO3 (anhydrous) in ∼20 mL of distilled

deionized water (DDI). Transfer to a 25-mL volumetric flask and adjust to mark with

DDI. Transfer to storage vial and label as “30% K2CO3(aq).”

1% PFBB: Dissolve 0.25 g of PFBB in ∼20 mL of acetone. Transfer to a 25-mL

volumetric flask and adjust to mark with acetone. Transfer contents to storage vial and

label as “1% PFBB(acetone).”

1000 ppm each carboxylic acid: Weigh ∼0.010 g of each acid into a 10-mL volumetric

and already half filled with DDI. Label as “1000 ppm each acid.”



TO



SYNTHESIZE AND EXTRACT THE



PFB



ESTER:



Into a 22-mL headspace vial with crimp top, place 200 µL of the 100 ppm acid, 200

µL of 1% PFBB, 50 µL of 30% K2CO3, and 4 µL acetone. Shake vigorously and allow

the contents of the vial to stand at room temperature for 3 h. Add enough DDI to reach

the neck of the headspace vial. Add 2 mL of pesticide-grade iso-octane. Transfer 1.0 mL

of extract to a 2-mL GC vial and inject 1 µL of extract into a gas chromatograph

incorporating an electron-capture detector (GC-ECD). For a 30 m × 0.32 mm DB-5

(J&W Scientific) capillary column, the following temperature program adequately

separates the PFB esters of C3, C4, and C5 carboxylic acids. Start at 100˚C and hold

for 3 min, then raise the temperature at a rate of 8˚C/min to 150˚C, and then hold for

0.5 min. Under these conditions, we found that propionic acid elutes at 3.099 min,

n-butyric at 3.65 min, and valeric at 6.09 min (principles of programmed temperature

GC will be considered in Chapter 4). Figure 3.41 shows two chromatograms in a

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Sample Preparation Techniques



YP15005.RAW



800



313



700

600



40 ppb iBuOOH, nBuOOH, 2MeBuOOH

in 20 mL aqueous phase

C-GC-ECD



500



30 m × .25 mm DB-1 (J&W scientific)



400

300



5.56



100



VI ON



200

6.28



6.97



0

4



6



8



YP15001.RAW



2

800

700



10



12



14



Blank, PFBB, acetone



600

500

400

300



100



VI ON



200



0

2



4



6



8



10



12



14



FIGURE 3.41 Two chromatograms for the derivation of i-butyric, n-butyric, and 2-methyl

butyric as their PFB esters.

stacked arrangement for the derivatization of i-butyric, n-butyric, and 2-methyl butyric

as their PFB esters. A blank (lower chromatogram) and a spiked blank (upper chromatogram) reveal the presence of these PFB esters. Note that a 40 ppb concentration

level can easily be reached. After these butyric acids are converted to their respective

PFB butyrates, not only are polar acids converted to nonpolar esters, but also significant

increases in analyte sensitivity (using a GC-ECD as stated earlier) are realized. Let us

consider a second illustration of chemical derivatization, this time for HPLC.

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Trace Environmental Quantitative Analysis, Second Edition



108. HOW DO YOU PREPARE A P-BROMOPHENACYL

ESTER OF N-BUTYRIC ACID AS A CHROMOTAG

AND CONDUCT A QUANTITATIVE ANALYSIS?

The following procedure answers this question:

PREPARATION



OF MIXED ALKYLATING REAGENT:



Weigh 0.47 g of p-bromophenacyl bromide (2,4-dibromoacetophenone) and 0.045 g

of 18-crown-6 per every 10 mL using acetone. Dissolve both reagents in enough acetone

prior to adjusting to a final volume.



PREPARATION



OF FATTY ACID STOCK REFERENCE STANDARD:



Prepare an approximately 10,000 ppm stock solution of n-butyric acid (n-BuOOH) in

water by weighing out approximately 0.1 g of the acid and dissolving in a beaker filled

with approximately 5 to 8 mL of water. Dissolve, then transfer to a 10-mL volumetric

flask and adjust to the calibration mark with DDI.



PREPARATION



OF



1M



AQUEOUS



KHCO3:



Prepare a 1 M solution containing potassium bicarbonate dissolved in DDI by dissolving

approximately 10 g of KHCO3 in enough to reach 100 mL. Transfer to storage bottle.



TO



PREPARE THE POTASSIUM SALT OF BUTYRIC ACID (N-BUOOK):



To 5 mL of the stock fatty acid reference standard, in a 50-mL beaker, add enough

1 M KOH solution to adjust the pH to 7 to 8. This is best accomplished by filling a

buret with the 1 M KHCO3 solution and titrating to the desired pH. Adjust the acid

solution to a precise final volume and record. Transfer to a storage vial and label with

a new concentration for the fatty acid.



PREPARATION



OF WORKING CALIBRATION STANDARDS:



Create a series of working calibration solutions with the same final volume according

to the following table. Use a 22-mL headspace vial with crimp top:



Standard No.



Alk Rgt (mL)



Acetone (mL)



µ

RCOOK (µL)



V(total) Adjusted with DDI



0 (blank)

1

2

3

4



1

1

1

1

1



3

3

3

3

3



0

10

50

100

500



5

5

5

5

5



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Sample Preparation Techniques



315



DERIVATIZATION:

Place the 22-mL headspace vial or equivalent into a heater block set at 80˚C and heat

for 30 min. Alternatively, the contents of the vial may be evaporated to dryness and

the residue reconstituted in the HPLC-compatible solvent.



DETERMINATION



OF THE ESTER VIA



HPLC:



Inject 5 µL of the content of the GC vial into an HPLC. Use a gradient elution reversedphase approach as previously developed.



Finally, we consider the use of a fluorescence reagent to convert a carboxylic

acid to a highly fluorescent derivative.



109. WHAT IS THE SAMPLE PREP APPROACH TO PLACING

A FLUOROTAG ON A CARBOXYLIC ACID?

Scheme 3.10 is a flowchart that outlines the sample prep approach for isolating and

recovering perfluorocarboxylic acids from liver homogenate, followed by the preparation of a highly fluorescent derivative using 3-bromoacetyl-7-methoxycoumarin.158 The fact that methoxycoumarins can be used as fluorotags for carboxylic

acids has been known for some time.159,160 In this case, shown in Scheme 3.10, the

perfluorocarboxylate anion is ion pair extracted into 1:1 ethyl acetate:hexane using

a tetrabutyl ammonium cation under alkaline conditions following bath sonication.

The extract is evaporated to just dryness and acetonitrile (a polar solvent) is added,

followed by the 3-bromoacetyl-7-methoxycoumarin (BrAMC) reagent. The derivatived perfluorocarboxylic acid is subsequently injected into an RP-HPLC-FL, as

noted in Scheme 3.10. HPLC-FL as a determinative technique will be introduced in

Chapter 4. There are other derivatizing reagents that do not quite fit into the categories described earlier. We will encounter other derivatization concepts as we

proceed through Chapter 4.



110. WHAT CAN WE CONCLUDE ABOUT SAMPLE PREP?

An attempt was made to introduce most of the recently developed sample prep

techniques as well as provide for the underlying principles of established techniques.

The link between true enviro-chemical quantitative analysis and true enviro-health

quantitative analysis was attempted from the sample prep perspective. Hopefully,

the reader comes away with a deeper appreciation of how samples and specimens

are prepared so that these materials can be more properly introduced to the various

determinative techniques introduced in the next chapter.

One of the unique features of solvent extraction, particularly for metal ions, is the large

variation in distribution ratios and separation factors made possible by controlling the

chemical parameters of the system.

—Henry Freiser

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Trace Environmental Quantitative Analysis, Second Edition



To 50 mg liver homogenate, add 1 mL

water, 1 mL 2M Na2CO3, and 1 mL

0.5M TBA



To prepare TBA:

dissolve TBAHSO4 in

DDI, adjust pH to 10

with 2M NaOH



Sonicate in water bath

for 10 min



Extract w 3 × 4 mL aliquots of EtOAcHexane 1:1 via shaking for 15 min



This approach

should be adaptable

to urine and serum

samples.



Evaporate to just dryness using a

N2 stream; 200 uL ACN

Add: 2 mL

of 0.2% BrAMC

in acetone

Derivatize by heating at 70°C for 25 min;

cool on ice at −30°C for at least 2 hours

to precipitate excess BrAMC



Store sample at this

point if necessary



Filter mixture through

glass wool



Inject 10 μL into a HPLC-FL with λex@366nm and λem@419nm



SCHEME 3.10



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