6251 DISINFECTION BY-PRODUCTS: HALOACETIC ACIDS AND TRICHLOROPHENOL*

6251 A. Introduction

1. Sources and Significance

The haloacetic acids (HAAs) are1 formed by the chlorination of natural organic (humic and fulvic) matter. Utilities

usingchlorine as a water disinfectant generate haloacetic acids, usually as the second most prevalent group of known

disinfection by-products1

; the primary group formed is usually the trihalomethanes. Toxicological studies indicate that

dichloroacetic acid and trichloroacetic acid are animal carcinogens.2 The USEPA has promulgated a maximum contaminant level for the sum of five haloacetic acids.3

2. References

1. U.S. ENVIRONMENTAL PROTECTION AGENCY & ASSOCIATION OF METROPOLITAN WATER AGENCIES. 1989. Disinfection By-Products in U.S.

Drinking Water. Vol. 1 Report, James M. Montgomery Consulting

Engineers, Pasadena, Calif.

2. ALCEON CORP. 1993. An Overview of Available Information on the

Toxicity of Drinking Water Disinfectants and Their By-Products.

Cambridge, Mass.

3. U.S. ENVIRONMENTAL PROTECTION AGENCY. 1998. Disinfectants and

Disinfection Byproducts Final Rule. Federal Register 63(241):

69390.

6251 B. Micro Liquid-Liquid Extraction Gas Chromatographic Method

This method1 was developed to analyze simultaneously for

monochloroacetic acid (MCAA), monobromoacetic acid (MBAA),

dichloroacetic acid (DCAA), trichloroacetic acid (TCAA), bromochloroacetic acid (BCAA), dibromoacetic acid (DBAA), and

2,4,6-trichlorophenol (TCPh) in treated and untreated drinking

water. Additional haloacetic acids may be present and analyzed

by this method; however, standards for these compounds are not

readily available and are less stable.

1. General Discussion

a. Principle: The sample is extracted with methyl tertiarybutyl ether (MtBE) at an acidic pH to extract the nondissociated

acidic compounds to be determined. A salting agent is added to

increase extraction efficiency. The extracted compounds are

methylated with diazomethane solution to produce methyl ester

or ether derivatives that can be separated chromatographically. A

temperature-programmable gas chromatograph using a fused

silica capillary column and an electron capture detector (ECD) is

used for analysis. Simultaneous analysis and confirmation using

a single injection can be effected by setting up both the analytical

column and the confirmation column to share a common injection port. Alternatively, use separate analytical and confirmation

columns. Alternative detectors may be used if QC criteria can be

met. Aqueous calibration standards are extracted, methylated,

and analyzed in the same manner to compensate for less than

100% extraction and derivatization efficiencies.

b. Interferences: Impurities in extraction solvent and on glassware and other equipment can interfere. Follow specifications

and cleaning procedures carefully to minimize interference. As

shown in Figure 6251:1, the analysis separates peaks of haloacetic acids from those of other common disinfection by-products. Use of two columns is recommended because for waters

with high carbonate contents, false-positive MCAA peaks have

been observed on the column described in 6251B.3h4).

c. Detection levels: Method detection levels (MDL) are given

in Table 6251:I.2 The method has been shown to be useful for

haloacetic acids over a working range of 0.5 to 30 g/L (1.0 to

30 g/L for MCAA) and 0.25 to 15 g/L for TCPh. The

calibration range can be extended, depending on the compound

and detector characteristics.

d. Safety: The toxicity and carcinogenicity of each reagent has

not been defined precisely. Minimize exposure to these chemicals and use them only in a properly operating ventilation hood.

Avoid exposure to DCAA and TCAA because they are carcinogens.3 Avoid contact with the other haloacetic acids and

their solutions.

MNNG (1-methyl-3-nitro-1-nitrosoguanidine) is carcinogenic. Keep in properly labelled plastic containers, containing

activated carbon, with tight-fitting lids and store in a refrigerator

used only for chemical storage. Store spatulas and glassware for

the handling of MNNG in specially labelled plastic containers

and use only for MNNG.

Diazomethane is toxic, carcinogenic, and an explosion hazard.4 Follow special precautions whenever handling this material. Use only in a properly operating fume hood; CAUTION: Do

not breathe vapors. To avoid explosions, do not heat above

90°C and do not use glassware with ground-glass surfaces (e.g.,

* Approved by Standard Methods Committee, 2007.

Joint Task Group: 21st Edition—Sanwat N. Chaudhuri (chair), Russell Chinn,

Patricia Snyder Fair, Yuefeng Xie.

1
ground-glass joints, sleeve bearings) or glass stirrers. Special

glassware for diazomethane generation and handling, as well as

screw-cap volumetric flasks, are available commercially. Always

use a safety shield when generating diazomethane. Always

quench excess diazomethane with silica gel. Do not store diazomethane/ether solutions; they are extremely hazardous and tend

to become contaminated.

Store ether in tightly-closed amber bottles in an explosion-safe

or -proof refrigerator. Store only with compatible chemicals.

Eliminate all sources of ignition; keep away from heat, sparks,

and flames. Handle ether only in a hood and avoid direct physical

contact. Do not breathe vapors. If ether is spilled or leaks,

evacuate area, ventilate, and absorb on vermiculite or similar

material. Wear appropriate OSHA equipment before entering

spill area. Also see Section 1090.

2. Sampling and Storage

See Section 6010B.1.

Preferably collect grab samples in quadruplicate to allow

sufficient volume for replicates and known additions. Flush

sampling tap until water temperature stabilizes and stagnant lines

are cleared. Collect samples in nominal 40- or 60-mL vials

containing approximately 65 mg crystalline NH4Cl (bake overnight at 100°C to eliminate contaminants), which converts free

chlorine to a combined chlorine residual, and sealed with TFEfaced septa and screw caps. To minimize aeration, fill vials so no

air bubbles pass through the sample. Do not rinse with sample

and do not let vial overfill. Seal sample vials with no headspace.

Analyze samples as soon as possible after collection. Store

dechlorinated samples at 4°C, but for no more than 14 d;5,6 check

compound stability in any unknown sample matrix. Sample

extracts can be held in a freezer at 11°C for 21 d.

3. Apparatus

Preferably dedicate all analytical glassware used in this

method to this procedure.

a. Sample containers and extraction vials,* 40- or 60-mL

screw-cap vials with TFE-faced silicone septa. Clean vials by

washing with detergent, rinsing thoroughly with tap water, rinsing with 1:10 HCl, rinsing again with tap water, and finally

rinsing with reagent water. Heat in an oven at 180° for at least

* Wheaton: Industrial Glassware, Millville, NJ; or equivalent.

Figure 6251:1. Haloacetic acids separation from other commonly produced disinfection by-products on a DB-1701 column. Chromatogram produced by

mixing the methyl esters of each haloacetic acid with other disinfection by-products in MtBE (disinfection by-products tentatively identified).

DISINFECTION BY-PRODUCTS: HAA (6251)/Micro Liquid-Liquid Extraction GC Method

2
1 h. Clean caps and septa by rinsing with acetone, then hexane.

Heat at 80°C for not more than 1 h in a clean, forced-air

convection oven.

b. Microsyringes, 5, 10, 25, 50, 100, 250, 500, and 1000 L.

c. Syringe, 30-mL glass hypodermic, metal luer-lok tip with

8.9-cm- (3.5-in.-) long  17 gauge stainless steel pipetting

needle (alternatively use a 30-mL volumetric pipet). See ¶ a

above for glassware cleaning procedure.

d. Micro volumetric flasks,† TFE-lined screw-cap: 2-mL,

5-mL, and 10-mL. Immediately after use, rinse three times with

methanol. Invert to drain. Let air-dry completely in a ventilation

hood.

e. Mechanical shaker,‡ to automate MtBE extraction. Insert

vials into a wooden holding block (20 vial capacity) made from

laminated plywood with drilled holes to accept vials, dimensioned to fit snugly onto the shaker table.

f. Extract and standard solution storage container, 1.8-mL

clear glass, 7- and 14-mL amber glass screw-cap vials with

TFE-lined silicone septa. For cleaning procedures, see ¶ a above.

g. Transfer pipets, 14.6- and 23-cm (5.75- and 9-in.) disposable glass pasteur pipets. See ¶ a above for glassware cleaning

procedure.

h. Gas chromatograph, temperature-programmable (preferably with multiple ramp capability) with injector. Optimally use

an autosampler for sample injection and a computer data system

for peak integration and quantitation. (A detector base that can

mount two electron capture detectors is ideal.)

1) Gas handling equipment: Use carrier (helium) and makeup

(nitrogen) gases of high purity (99.999%) grade that pass

through indicating calcium sulfate, molecular sieve 5A, activated

charcoal, and an oxygen purifying cartridge. Use two-stage

metal diaphragm high-purity regulators at the compressed gas

sources. Use flow controllers to regulate carrier gas flow. Make

all gas lines 0.3-cm (1/8-in.) copper (or stainless steel) tubing;

rinse with acetone and bake before use.

2) Injector, split/splitless (using straight open bore insert).

3) Analytical column,§ 30 m long  0.25 mm ID, fused silica

capillary column with a 0.25-m film thickness or equivalent.

4) Confirmation column, 30 m long  0.25 mm ID, fused

silica capillary column with a 0.25-m film thickness or a 30 m

long  0.25 mm ID, fused silica capillary column# with a

0.5-m film thickness.

5) Detectors, a constant-current pulse-modulated 63Ni ECD

with standard size cell (use two ECDs for simultaneous confirmation analysis).

i. Salt scoops for sodium sulfate, made from stainless steel

1.3-cm- (0.5-in.-) diam bar stock drilled out to a volume of

1.73 mL so that each level scoopful contains 3 g. Alternatively,

weigh the salt.

j. Pipetting dispensers, adjustable 5- and 2-mL sizes with TFE

transfer lines, that can be mounted on the supplier’s reagent

bottles. Use for dispensing H2SO4 and MtBE. Alternatively use

a 3-mL volumetric pipet and a 5-mL graduated pipet with

manual pipet bulbs.

k. Diazomethane generator: Use millimole-size generator

with “o”-ring joint (Figure 6251:2).** Immediately after use,

rinse inner tube twice with 20% NaOH, then rinse twice with tap

water. Immediately add 1 g silica gel to the outside tube to

quench any residual diazomethane solution, rinse twice with

methanol, and twice with tap water. Rinse both inner and outer

tubes with reagent water three times. Bake at 180°C until dry in

a clean, forced-air convection oven.

Alternatively use the millimole-size diazomethane generator

shown in Figure 6251:3.†† To clean, rinse with reagent water

and then with methanol, invert, and let air-dry.

l. pH-strips, pH indicating strips, 0 to 2.5 range.

4. Reagents

a. Extraction solvent, 99 % MtBE, preservative-free.‡‡

b. Sodium sulfate reagents:

1) Granular sodium sulfate, Na2SO4, reagent grade, suitable

for pesticide analysis. Heat at 400°C overnight in a shallow

stainless steel pan covered with aluminum foil. Store in a 1-L

glass bottle with TFE-lined polypropylene cap.

2) Acidified sodium sulfate: To 100 g anhydrous sodium

sulfate, heated as above and cooled, add diethyl ether to just

cover the solid; make a slurry. Add 0.1 mL conc sulfuric acid

and mix thoroughly. Remove ether under low vacuum. Mix 1 g

acidified sodium sulfate with 5 mL reagent water and check that

pH is less than 4. Store remainder of reagent at 130°C.

c. Methanol, pesticide grade or equivalent.

d. Ammonium chloride, NH4Cl.

e. Reagents for diazomethane generation with the apparatus

shown in Figure 6251:2:

1) Sodium hydroxide solution, NaOH, 20%: Dissolve 200 g

ACS low-carbonate-grade pellets in 800 mL reagent water.

2) 1-methyl-3-nitro-1-nitrosoguanidine, MNNG.§§

f. Reagents for diazomethane generation with apparatus

shown in Figure 6251:3:

† Kontes, or equivalent.

‡ Eberbach, or equivalent.

§ Durabond-1701, J&W Scientific, or equivalent.

 Durabond-5, J&W Scientific, or equivalent.

# Durabond-210, J&W Scientific, or equivalent.

** Aldrich, or equivalent.

†† Paxton Woods Glass, Cincinnati, OH, or equivalent.

‡‡ Omnisolv, manufactured by EM Science, Gibbstown, NJ, or equivalent.

§§ Aldrich or equivalent.

TABLE 6251:I. METHOD DETECTION LEVELS AND PRECISION DATA*

Compound

Added

Conc.

g/L

Found

Conc.

g/L

Standard

Deviation

g/L

Relative

Standard

Deviation

%

Method

Detection

Level

g/L

Monochloroacetic acid 0.50 0.54 0.026 4.8 0.082

Monobromoacetic acid 0.50 0.80 0.028 3.4 0.087

Dichloroacetic acid 0.50 0.5 0.017 3.5 0.054

Trichloroacetic acid 0.50 0.5 0.017 3.4 0.054

Bromochloroacetic

acid 0.50 0.49 0.015 3.1 0.04

Dibromoacetic acid 0.50 0.47 0.021 4.4 0.065

2,4,6-Trichlorophenol 0.25 0.27 0.011 4.1 0.034

* Based on the analysis of seven portions of reagent water with known additions.2

DISINFECTION BY-PRODUCTS: HAA (6251)/Micro Liquid-Liquid Extraction GC Method

3
1) Diethylene glycol monoethyl ether. 

2) N-methyl-N-nitroso-p-toluene sulfonamide.##

3) Ethyl ether, absolute.

4) N-methyl-N-nitroso-p-toluene sulfonamide solution: Mix

10 g reagent f 2) in 100 mL 1:1 (v:v) solution of ethyl ether and

reagent f1). Solution is stable for 1 month when stored at 4°C in

an amber-colored bottle with a TFE-lined screw cap.

5) Potassium hydroxide solution, KOH: Dissolve 37 g in

100 mL reagent water.

g. Silica gel, 35/60 mesh activated at 180°C and stored in a

desiccator.

h. Sulfuric acid, H2SO4, conc.

i. Copper (II) sulfate pentahydrate, CuSO4 5H2O.

j. Standard material, see Table 6251:II for source and physical

information.

1) Individual haloacetic acid standard stock solutions: Prepare individual haloacetic acid and the trichlorophenol stock

solutions as follows: Weigh 0.150 g of each acid. Dilute each

standard in MtBE to 10 mL in a screw-top volumetric flask.

(NOTE: Do not use methanol for dilution, because spontaneous

methylation of the haloacetic acid may occur in methanolic

solution.8 Transfer each to a separate clean 14-mL amber vial

and store in a freezer at 11°C. Stock standards are usable for

6 months.

2) Multicomponent haloacetic acid additive solution: Prepare

a six-component additive solution using individual haloacetic

acid stock solutions. Dilute 16.7 L of each stock standard into

a 10-mL volumetric flask containing 9 mL MtBE, but use 8.4 L

of 2,4,6-trichlorophenol solution. After adding all stock solutions, dilute to volume with MtBE. This gives 25 g/mL of each

HAA and 12.5 g/mL for 2,4,6-trichlorophenol. The additive

solution is usable for 3 months when stored at less than 10°C.

Alternatively, prepare known-addition solution monthly in methanol and store at 4°C.

Measure microliter volumes with a gastight syringe using the

solvent flush delivery technique. Do solvent flush with a 25-L

syringe by first drawing up 2.5 L of solvent and then drawing

the syringe plunger to the 5-L mark with air. From the 5-L

mark measure amount of stock solution desired and then deliver

the entire contents to the volumetric flask.

3) Individual haloester standard stock solutions: Prepare individual methyl ester stock solutions as follows: Weigh 0.1 

(molecular weight of ester/molecular weight of acid) g of each

methyl ester in a 10-mL volumetric flask and dilute to mark with

MtBE. Prepare methyl ester for dibromoacetic acid by derivatizing 1 mL of a 20 000-g/mL acid solution with 100 L

methanol (follow derivatization steps in 5e below, but substitute

dibromoacetic acid stock solution as the solution added to the

outer tube for collection of the diazomethane). After derivatizing, transfer ester quantitatively to a 2-mL volumetric flask with

a TFE-lined screw cap and dilute to mark with MtBE. Stock

standards are usable for 6 months when stored at less than

10°C.

4) Multicomponent haloester additive solution: Prepare a multicomponent additive solution by diluting 10 L of each haloester stock standard, but use 5 L of 2,4,6-trichloroanisole (methyl

  Carbitol (Aldrich), or equivalent.

## Diazald (Aldrich), or equivalent.

Figure 6251:2. Easy-to-use diazomethane generator apparatus for preparing small amounts of diazomethane in methyl tertiarybutyl ether (MtBE).

Figure 6251:3. Easy-to-use alternative diazomethane generator for preparing small amounts of diazomethane in MtBE.

DISINFECTION BY-PRODUCTS: HAA (6251)/Micro Liquid-Liquid Extraction GC Method

4
ether of the phenol), in a 10-mL volumetric flask and bring to

volume with MtBE. This will yield a mixture containing approximately 10 g/mL each, except for 2,4,6-trichloroanisole, which

will be approximately 5 g/mL. Additive solution is usable for

3 months when stored at less than 10°C.

5) Direct injection haloester standards: Prepare direct injection standards using 10 g/mL multicomponent haloester additive solution, a 30-g/mL internal standard additive solution [see

¶ k2) below], and a 10-g/mL methanol solution of methyl-2,3-

dibromopropionate [surrogate ester, see ¶ l4) below]. Prepare

direct injection standards by diluting appropriate volumes of

multicomponent haloester additive mix, internal standard additive solution, and surrogate ester solution with enough MtBE to

give a final volume of 1.0 mL.

k. Internal standard, 1,2,3-trichloropropane (IS-TCP), 98%

pure (alternatively use 1,2-dibromopropane (IS-DBP).

1) Internal standard stock solutions: Weigh 50 mg into a

10-mL volumetric flask and bring to volume with methanol. This

will yield a 5000-g/mL stock solution. Stock standards are

usable for 6 months when stored at less than 10°C.

2) Internal standard additive solution, 30 g/mL: Deliver

60 L internal standard stock solution into a 10-mL volumetric

flask and dilute to volume with methanol. Divide evenly among

six 1.8-mL vials and store at 11°C. Additive solution is usable

for 3 months.

Add 20 L of internal standard additive solution to each 2 mL

extract, yielding internal standard of 300 ng/mL.

l. Surrogate, (DBPA-SUR) 2,3-dibromopropionic acid, 99%

pure or 2,3,5,6-tetrafluorobenzoic acid (TFBA-SUR).

1) Surrogate stock solution, 20 000 g/mL: Weigh 0.2000 g

SUR acid into a 10-mL screw-cap volumetric flask and dilute to

mark with MtBE. Stock solutions are usable for 6 months when

stored at less than 10°C.

2) Surrogate additive solution:

a) DBPA-SUR, 10 g/mL: Deliver 5 L DBPA-SUR stock

solution into a 10-mL volumetric flask and dilute to volume with

methanol. Divide evenly among six 1.8-mL vials and store at

11°C. Additive solutions are usable for 3 months.

Add 30 L DBPA-SUR additive solution to each 30-mL

sample portion, yielding DBPA-SUR 10 g/L.

b) TFBA-SUR, 20 g/mL: Deliver 300 L TFBA-SUR stock

solution into 1-L volumetric flask and dilute to volume with

reagent water. Add 0.5 mL TFBA-SUR additive solution to each

30-mL sample, yielding 100 g/L.

3) Esterified surrogate stock solution, 10 000 g/mL: Derivatize 1 mL SUR stock solution and 100 L methanol, according

to derivatization steps in 6251B.5e, but substitute SUR stock

solution and 100 L methanol for MtBE. After derivatizing,

transfer quantitatively to a 2-mL volumetric flask with a TFElined screw cap and dilute to mark with MtBE. Stock solutions

are usable for 6 months when stored at less than 10°C.

4) Surrogate ester additive solution, 10 g/mL: Deliver 10 L

SUR ester stock solution into a 10-mL volumetric flask and

dilute to volume with methanol. Additive solutions are usable for

3 months when stored at less than 10°C.

Add 10 L DBPA-SUR or 100 L TFBA-SUR ester additive

solution to each 1 mL of direct injection standard yielding

100 ng/mL DBPA-SUR or 1000 ng/mL TFBA-SUR.

m. Reagent water: See Section 1080.

n. Calibration standards: Prepare aqueous calibration standards in reagent water by injecting a measured amount of the

multicomponent haloacetic acid solution directly into water using the solvent flush technique. Prepare five different concentration levels from 0.5 to 30 g/L for the HAAs and 0.25 to

15 g/L for 2,4,6-trichlorophenol in 40-mL TFE-lined screw-top

bottles containing 30 mL reagent water. Extract these standards and

process the same way as samples, using the procedure given below.

5. Procedure

a. Sample preparation: Remove samples and standards from

storage and let equilibrate to room temperature. Each time a new

sample matrix is analyzed check that the amount of H2SO4 added

will reduce pH to less than 0.5 before beginning microextraction.

Test a separate 10-mL portion by adding 1 g CuSO4, 4 g granular

Na2SO4 [6251B.4b1)], and 0.5 mL conc H2SO4, mix until salt

dissolves, then test using pH indicating strips.

b. Microextraction: Transfer 30 mL from the sample container

to a 40- or 60-mL vial with TFE-faced septum and screw cap.

Add surrogate additive solution as indicated in 6251B.4l2) to

each sample, including standards and blanks. Add haloacetic

acid additive solution at this step for known additions.

Take one vial at a time and add the following in sequence:

1.5 mL conc H2SO4, 3 g CuSO4, 12 g baked Na2SO4 [¶4b1)],

and 3 mL MtBE. Immediately cap and shake briefly by hand to

break up any salt clumps.

When using automated extraction, place vials in mechanical

shaker wooden holding block. Shake vials at fast speed (approxTABLE 6251:II. ANALYTICAL STANDARDS

Compound*

Purity

%

Molecular

Weight

Boiling

Point7

°C @ mm†

MCAA

MBAA

DCAA

TCAA

BCAA

DBAA

TCPh

IS-DBP

IS-TCP§

SUR-DBPA

SUR-TFBA

99

99

99

98

97

99

95

95

99

99

99

94.5

138.95

128.94

163.39

173.39

217.86

197.45

201.9

147.43

231.88

194.09

183

208

194

198

215‡

195 @ 250

246

140–142

156

160 @ 20

—

MeCA

MeBA

MeDCA

MeTCA

MeBCAA

MeDBA

TCAn

99

98

99

99

98

—

99

108.52

152.98

142.97

177.42

187.42

—

211.48

130 @ 740

144

143

152–153

155

182

132 @ 28

* Sources: BCAA, Radian Corp., Austin, TX; DBAA, Fluka Chemika-BioChemika, Switzerland; TCPh obtainable from Chem Service, Inc., Westchester,

PA; MeDBA and SUR ester, derivatized acid at Metropolitan Water District of

Southern California laboratory; other compounds from Aldrich Chemical Company, Inc., Milwaukee, WI.

† °C at reduced pressure in mm Hg.

‡ Decomposes.

§ Ensure that TCP is not a contaminant when it is used as IS.

DISINFECTION BY-PRODUCTS: HAA (6251)/Micro Liquid-Liquid Extraction GC Method

5
imately 300 cycles/min) for 9 min; alternatively shake manually

for 2 min until salt is dissolved.

Remove vials, place upright, and let stand for at least 3 min

until the phases separate.

c. Preparation of diazomethane9

: Using the apparatus in Figure 6251:2, add approximately 130 mg MNNG to the inside tube

of the generator. Add 0.5 mL reagent water to the MNNG and

secure cap and septum. Add 2 mL MtBE to the outside tube.

Place butyl o-ring in glass joint, place inside tube firmly on top

of O-ring, and clamp securely with a screw-type pinch clamp.

Place generator in an ice water bath containing enough ice to

keep diazomethane MtBE solution at 0°C until used.

Add 600 L 20% NaOH (1 drop/5 s) using a 1-mL gastight

syringe (22-gauge needle) through the generator septum (check

that the syringe needle is on the opposite side of the vapor exit

hole). Let derivatization continue for 30 min after adding NaOH;

use product as soon as possible. Add more ice if necessary to

maintain temperature.

If more diazomethane is needed, prepare two or more batches

and combine before use.

Alternatively use the apparatus in Figure 6251:3 to prepare

diazomethane. Add enough ethyl ether to tube 1 to cover the first

impinger. Add 10 mL MtBE to 15-mL collection vial. Set

nitrogen flow at 5 to 10 mL/min. Add 4 mL sulfonamide solution

and 3 mL 37% KOH solution to the second impinger. Connect

tubing as shown and let nitrogen flow purge diazomethane from

the reaction vessel into the collection vial for 30 min. Cap vial

when collection is complete and hold at 0°C. When stored at 0°C

diazomethane solution may be used over a period of 48 h.

d. Separation and concentration: NOTE: Ensure that all items

that come into contact with the sample prior to methylation have

been washed with a dilute solution of sulfuric acid.

The drying step included here may be used if excess diazomethane is required to maintain the persistent yellow color of the

sample (¶ e below). It is not necessary in every case and may be

used at the discretion of the analyst. Plug a small disposable

pipet with a small amount of acid-washed glass wool. Add

approximately 1 g acidified Na2SO4 [6251B.4b2)] to the pipet

and pass exactly 2 mL of the top MtBE sample extract through

the salt, being careful not to transfer any of the aqueous phase.

Rinse the salt in the pipet with two 250-L volumes of solvent

and collect together in a receiver ampule (a 2-mL volumetric

flask with TFE-lined screw cap) for subsequent concentration

and methylation.

Concentrate MtBE extract to approximately 1.7 mL using a

moderate stream of nitrogen blowing on the surface of the

extract.

e. Derivatization: Add 20 L of 30 g/mL internal standard

additive solution to each concentrated extract. (The internal

standard is added at this time to minimize manipulation in the

presence of diazomethane.) Cool in an explosion-safe freezer or

in an ice bath for 7 min and add diazomethane (cooling extracts

is unnecessary if diazomethane is generated by apparatus shown

in Figure 6251:3).

Uncap one volumetric flask and add 250 L of cold diazomethane/MtBE solution. Cap immediately with TFE-lined screw

cap; mix gently by inverting once. Repeat for remaining extracts.

A persistent yellow color after addition of diazomethane indicates that an excess is available for esterification. If necessary

add more diazomethane solution.

Hold for 15 min at 4°C in an explosion-safe or explosionproof refrigerator. Alternatively, keep extracts in an ice bath

(cooling is unnecessary if diazomethane was generated by the

apparatus shown in Figure 6251:3).

After holding 15 min, place extracts in a hood and let stand

another 15 min until they reach room temperature. Dilute to

mark with MtBE and invert flask to mix. If using an autosampler,

transfer each extract evenly between two labeled autosampler

vials containing approximately 0.01 g silica gel with a 23-cm

(9-in.) disposable pasteur pipet to quench excess diazomethane.

Keep each extract in contact with diazomethane for approximately the same amount of time before quenching. Store extra

autosampler vial in freezer at 10°C as a backup extract. Alternatively, add silica gel to volumetric flask after derivatization is

complete.

f. Gas chromatography: Typical operating conditions for the

chromatograph are as follows:

Injector temperature 160°C; split valve opened at 0.5 min.

Temperature program: 37°C for 21 min, rising 11°C/min to

136°C, holding 3 min at 136°C, rising 20°C/min to 236°C,

holding 3 min at 236°C.

Detector temperature: 300°C.

Carrier gas flow: 30 cm/s at 37°C.

Makeup gas flow: 23 mL/min.

At the beginning of each analytical run, inject two MtBE

solvent blanks to condition the GC and to verify that interferences are absent. A 2-L extract is injected in splitless mode.

Always inject same sample volume and use sample dilution to

obtain response in the calibration range. If levels greater than the

highest standard are obtained, reanalyze diluted sample extract

and readjust internal standard concentration. Calculate concentration only for those compounds that were at levels higher than

the calibration curve; for other compounds use values obtained

from the undiluted sample extract. See Figure 6251:4 for a

chromatogram of an extracted and derivatized 30-g/L standard

on the analytical column. See Table 6251:III for retention times.

A direct standard may be injected after the MtBE solvent blanks

to verify continued system performance.

g. Calibration: Use five levels of calibration standards to

define the quantitation range. The lowest standard should be near

the limit of quantitation (LOQ) (see Table 6251:IV) for each

compound. Use other standards to bracket the expected range of

sample concentrations; do not exceed linear range of detector.

Prepare standards by adding haloacetic acids and trichlorophenol

to reagent water and then extract with the same solvent and

derivatize with the same batch of diazomethane as used for the

samples. Use the same extraction/esterification procedure for

both standards and samples to correct for recovery characteristics. Analyze calibration standards under the same chromatographic conditions as samples.

Analyze calibration standard at one or more levels with each

sample set to verify the working calibration curve. If the calibration standard is within 15% of the expected value, a new

five-level calibration curve is not needed.

6. Calculation

A 2-L injection of each calibration level will provide peak

area (Aa) data for each compound and an internal standard peak

DISINFECTION BY-PRODUCTS: HAA (6251)/Micro Liquid-Liquid Extraction GC Method

6
area (Ai

) for each level; use these peak areas to calculate relative

response for each compound.

Relative response  Aa/Ai

A calibration curve passing through zero is generated from the

plotted points for each compound using the relative response

versus standard concentration. Use the internal standard quantitation method to determine unknown concentrations by a linear,

quadratic, or point-to-point curve fit.

7. Quality Control

The quality control practices considered to be an integral part

of each method are summarized in Table 6020:I.

a. Quality control program: Because sample preparation requires

many manipulations, chances for errors are increased. Consequently, at least follow minimum quality control requirements to

monitor and maintain method performance. Include method blanks,

an initial demonstration of laboratory capability and detection levFigure 6251:4. Chromatogram produced by reagent water with known additions: 30 g/L extracted standard on DB-1701 column.

TABLE 6251:III. RETENTION TIMES

Compound

Retention Time

min

DB-1701

Column

DB-5

Column

DB-210

Column

Methyl chloroacetate (MeCA) 11.11 5.80 10.97

Methyl bromoacetate (MeBA) 18.80 9.23 13.03

Methyl dichloroacetate

(MeDCA) 20.53 10.11 12.72

Methyl trichloroacetate

(MeTCA) 24.78 18.94 14.37

Methyl bromochloroacetate 26.23 19.19 15.11

Methyl dibromoacetate

(MeDBA) 28.64 25.51 16.83

2, 4, 6-Trichloroanisole

(TCAn)  34.7 33.74 22.08

1,2-Dibromopropane (internal

standard) 15.97 10.78 —

1,2,3-Trichloropropane

(internal standard) 25.87 18.43 13.87

Methyl-2,3-dibromopropionate

(surrogate ester) 30.74 28.60 —

Methyl-2,3,6,7- tetrafluorobenzoicate (surrogate) 29.19 26.84 —

TABLE 6251:IV. RECOMMENDED QUANTITATION LIMITS

Compound

Recommended

Quantitation

Limit

g/L

Monochloroacetic acid (MCAA)

Monobromoacetic acid (MBAA)

Dichloroacetic acid (DCAA)

Trichloroacetic acid (TCAA)

Bromochloroacetic acid (BCAA)

Dibromoacetic acid (DBAA)

2,4,6-Trichlorophenol (TCPh)

1.0

0.5

0.6

0.6

0.8

0.6

0.4

DISINFECTION BY-PRODUCTS: HAA (6251)/Micro Liquid-Liquid Extraction GC Method

7
els, assessment of the internal standard recovery, determination of

surrogate compound recoveries, evaluation of calibration data and

curves, sample matrix additions, and precision of replicate sample

analysis. Additional quality control measures may be used.

b. Method blanks: Process a method blank (30 mL reagent

water) with each set of samples. If the blank produces any peak

within the retention time window of a compound that would

prevent its determination, seek out and eliminate the source of

contamination and reanalyze samples.

c. Initial demonstration of capability: To demonstrate an adequate level of performance, conduct the following operations before

analyzing samples and whenever any major analytical change, such

as new analyst or switch in type of column, is made.

1) Accuracy as percent recovery—Establish a calibration

curve as in 6251B.5g and select a representative additive concentration (5 g/L is convenient) for each target compound.

Using a syringe, add the appropriate amount of stock standard

mix to each of a minimum of seven 30-mL portions of reagent

water and analyze.

Calculate average percent recovery (P) and standard deviation of

the recovery (Sr). Compare results to the single-laboratory recovery

and precision data in Table 6251:V. Compare precision at similar

concentrations, that is P  30% of the additive level. Acceptable

mean recovery values are within the interval P  30%. For compounds not meeting this criterion, repeat with another seven samples until satisfactory performance has been demonstrated.

2) Absolute recovery—Use direct injection haloester standards, 6251B.4j5), to check absolute recoveries of extracted and

derivatized haloacetic acids. Calculate absolute recoveries by

comparing the ratio (area for compound/area for internal standard) for samples in ¶ 1) above with the area ratio for direct

injection standard at a tenfold concentration to account for the

extraction (expressed as the haloacetic acid). Typical absolute

recoveries are given in Table 6251:VI; acceptable absolute recoveries are within 30%. Recoveries outside this range may

indicate insufficient shaking or poor methylation, possibly due to

water in the extracts. Correct problem and produce acceptable

absolute recoveries before analyzing samples.

Because of rapid advances in chromatography, columns and

conditions may be modified to improve separation or to lower

cost. Repeat procedure in 7c for each modification.

d. Internal standard assessment: The internal standard corrects for any deviation in sample volume injected. A sample

injection is acceptable if the area counts of the internal standard

peak do not vary more than 20% from the daily calibration

standard(s) IS response.

Reinject an extract exceeding the 20% range. If re-analysis

does not produce acceptable results, re-extract and re-analyze. If

the reextracted sample results are not acceptable or if samples

have exceeded holding time, resample and reanalyze or record

results as suspect and out-of-control.

e. Evaluating surrogate recovery: The surrogate is added

directly to all samples before acidification and extraction. If the

surrogate area is low or absent, it is likely that there has been a

derivatization problem (e.g., water in extract) or extraction problem (e.g., water insufficiently acidified).

An extract is acceptable if the area counts of the surrogate

standard recovery are 30% from the surrogate standard recovery for the daily calibration standard(s).

When surrogate recovery is not acceptable, check the following: locate possible errors in calculations or procedure, degradation of standard solution, contamination sources, and instrument

performance. If these steps do not reveal the problem, re-analyze

the extract. If re-analysis does not produce acceptable results,

reextract and re-analyze samples. If the reextracted sample results are not acceptable or if samples have exceeded holding

time, record results as suspect and out-of-control.

f. Extracted standard calibration: Quantitation is done by internal

standard referencing with relative areas. Produce a minimum fivelevel extracted standard calibration curve for sample quantitation.

If the response for any compound falls outside the predicted response

by more than 15% from a previous calibration, make a new calibration

standard and analyze it until an acceptable curve is obtained.

Analyze calibration standards with each sample set after an

acceptable five-level calibration curve is generated. If the continuing calibration standards are not within 15%, check for errors or

degradation of standards and construct a new calibration curve.

g. Matrix additions: Add each target compound into one sample

per sample set (a sample set is all samples extracted within a 24-h

period) or 10% of the samples, whichever is greater.

The added concentration should be near to or greater than the

background. Take care, particularly with dichloroacetic acid, to

ensure that the addition plus background concentration does not

exceed calibration range (extract dilution may be needed).

Analyze one sample portion to determine the background

concentration (B) of each compound. Add working standard mix

to a second sample portion and analyze to determine the concentration of each compound (A). Calculate percent recovery (Pi

)

TABLE 6251:V. ADDITIVE RECOVERY IN REAGENT WATER*

Compound

Conc.

Added

g/L

Mean

Conc.

Recovered

g/L

Standard

Deviation

g/L

Relative

Standard

Deviation

%

Mean

Recovery

%

Monochloroacetic acid

Monobromoacetic acid

Dichloroacetic acid

Trichloroacetic acid

Dibromoacetic acid

2,4,6-Trichlorophenol

5.00

5.00

5.00

5.00

5.00

2.50

4.90

4.95

4.95

5.06

4.98

2.51

0.19

0.13

0.15

0.16

0.16

0.075

3.88

2.67

3.11

3.06

3.11

2.99

98.0

99.0

99.0

101

99.6

100

* Based on the analysis of seven portions of reagent water with known additions in a single laboratory.

DISINFECTION BY-PRODUCTS: HAA (6251)/Micro Liquid-Liquid Extraction GC Method

8
as 100 (A B)/T, where T is the known concentration of the

material added to the sample.

Compare percent recovery (Pi

) for each compound with established QC acceptance criteria. Establish QC criteria by initially analyzing seven samples with additions and calculating the

average percent recovery (P) and the standard deviation of the

percent recovery (Sr).

Calculate QC acceptance criteria as follows:

Upper control limit (UCL)  P  3Sr

Lower control limit (LCL)  P 3Sr

The data generated during the initial demonstration of capability, ¶ c1) above, may be used to set the initial upper and lower

control limits.

Monitor all data from dosed samples. Compound recoveries

must fall within established control limits. After 10 new recovery

measurements, recalculate P and Sr using all the data, and

construct new control limits. When the total number of data

points reaches 20, update control limits by calculating P and Sr

using only the most recent 20 data points.

Compare percent recovery (Pi

) for each compound with the

QC acceptance criteria established by the control limits. If recovery of any compound falls outside the designated range,

performance is judged to be out of control. Seek source of

problem immediately and resolve before continuing the analysis.

However, if recovery of a compound meets calibration, blank, internal standard, surrogate, and replicate quality control, laboratory performance is in control, and the recovery problem is matrix-related. Label

result for that compound in the sample as suspect/matrix.

h. Replicate analysis: Analyze sample duplicates to monitor

precision. Analyze duplicates on at least 10% of all samples

randomly selected.

Determine control limits by calculating the range as a function

of the relative standard deviation. The range, R, is the absolute

difference of the duplicate values, X1 and X2, as follows:

R  X1 X2

The normalized range (Rn) is calculated by dividing the range

by the average of the duplicate values (Xm):

Rn  R

Xm

Xm  X1  X2

2

Calculate mean normalized range (Rm) for 20 pairs of duplicate data points initially and 20 pairs of points quarterly:

Rm  ¥Rn

n

where:

n  number of duplicate pairs.

Variance  S2  ¥(Rn Rm)

2

n 1

The standard deviation (s) is the square root of the variance.

Upper and lower control limits are Rm  3s and zero, respectively. Acceptable duplicates fall within the control limits. The

upper warning limit is Rm  2s. If an Rn value is outside the

warning limit, a potential problem is indicated and investigated

before the analysis is out of control. Recalculate control limits

quarterly using the most recent 20 points not including any data

points that are out of control. Recalculate control limits when

any major analytical changes are made and after at least 20

points have been collected.

Analyze any problem and correct it. If the duplicate is not

acceptable, reextract only for those compounds out of control. If

the duplicates are still unacceptable or the sample holding time

has been exceeded, resample and reanalyze. If this is not possible

record results as suspect and out of control. Do not use such data

in range calculations.

i. Additional quality control: Each quarter, analyze QC check

standards from an external source. Independent confirmation

may include interlaboratory split sampling for comparison. Analyze performance evaluation samples, preferably from USEPA

or appropriate state agency, at least once a year. Results for each

target compound must be within established acceptance limits.

Shipping blanks are containers filled with reagent water containing appropriate amount of NH4Cl (see 6251B.2), shipped to

all sample locations with sample bottles, and returned with the

samples. They are used to assess contamination during sampling

and transit. Analyze a shipping blank with each sample set. If the

shipping blank contains reportable levels, compare with the

laboratory reagent blank. If contamination is not detected in the

laboratory reagent blank, the sampling or transportation practices

TABLE 6251:VI. ABSOLUTE RECOVERY DATA FOR REAGENT WATER WITH KNOWN ADDITIONS

Compound

Conc.

Added

g/L

Mean

Conc.

Recovered

g/L

Standard

Deviation

g/L

Relative

Standard

Deviation

%

Mean

Recovery

%

Monochloroacetic acid

Monobromoacetic acid

Dichloroacetic acid

Trichloroacetic acid

Bromochloroacetic acid

Dibromoacetic acid

2,4,6-Trichlorophenol

1.00

1.00

1.00

1.00

0.50

1.00

0.50

0.789

0.706

1.10

0.927

0.49

1.16

0.523

0.047

0.034

0.048

0.051

0.015

0.032

0.030

5.92

4.76

4.38

5.49

3.07

2.75

5.89

78.9

70.6

110

92.7

98

116

105

DISINFECTION BY-PRODUCTS: HAA (6251)/Micro Liquid-Liquid Extraction GC Method

9
may have caused the contamination. Discard all samples in the

set and resample.

Direct ester standards (concentration based on corresponding

acid) may be injected at the start of each set to verify the

sensitivity, chromatography, and retention times on the gas chromatograph.

Make an instrument check of the entire analytical system daily

using data gathered from analyses of reagent blanks, standards,

and replicate samples.

8. Precision and Bias

Single-laboratory method detection levels (MDL) and extracted recovery data from reagent water are presented in Table

6251:I. Data for absolute recoveries in reagent water are given in

Table 6251:VI. Laboratory data from two different laboratories

showing duplicate precision and matrix additions recoveries are

presented in Tables 6251:VII and 6251:VIII.

The precision and bias of analyses performed under the Information Collection Rule10 (ICR) using this method are presented

in Tables 6251:IX and 6251:X, respectively. Precision was determined as relative percent difference for duplicate analyses

[(Rn)(100)] and was calculated only when both analyses in the

duplicate pair showed concentrations at or greater than the ICR

minimum reporting level (MRL). Bias was evaluated as percent

recovery for cases in which the fortified amount was at least half

the backgound concentration. The fortifying concentrations

ranged from the ICR MRL (2 g/L for MCAA, 1 g/L for all

other HAAs) to 40 g/L.

9. References

1. CHINN, R. & S.W. KRASNER. 1989. A simplified technique for the

measurement of halogenated organic acids in drinking water by

electron capture gas chromatography. Presented at the American

Chemical Soc. Pacific Conf. on Chemistry and Spectroscopy, Pasadena, Calif., Oct. 18 –21, 1989.

2. U.S. ENVIRONMENTAL PROTECTION AGENCY. 1984. Definition and procedure for the determination of the method detection limit. 40 CFR

Part 136, Appendix B. Federal Register 49, No. 209.

3. ALCEON CORP. 1993. An Overview of Available Information on the

Toxicity of Drinking Water Disinfectants and Their By-Products.

Cambridge, Mass.

4. DE BOER, T.J. & H.J. BACKER. 1963. Organic Synthesis, Collective,

Vol. IV. John Wiley & Sons, New York, N.Y.

TABLE 6251:VII. SAMPLE DUPLICATE DATA FROM TWO LABORATORIES

Laboratory Compound

Number

of Pairs

of

Replicates

Average

Difference

Between

Duplicates

%

Standard

Deviation of

Difference

Between

Duplicates

%

A MCAA

MBAA

DCAA

TCAA

DBAA

BCAA

5

3

7

6

5

11

7.6

1.9

1.5

1.4

6.0

1.8

10.6

1.5

0.8

1.0

6.0

1.3

B MCAA

MBAA

DCAA

TCAA

DBAA

BCAA

10

3

11

11

5

10

16.7

8.9

8.5

5.5

5.4

5.3

14.8

8.4

10.6

3.6

4.0

4.3

TABLE 6251:VIII. FIELD SAMPLE RECOVERY WITH KNOWN ADDITIONS TO

DRINKING WATER, IN TWO LABORATORIES

Laboratory Compound

Added

Conc.

g/L

Number

of

Samples

Mean

Recovery

%

Relative

Standard

Deviation

%

A MCAA

MBAA

DCAA

TCAA

BCAA

DBAA

TCPh

5.0

5.0

5.0

5.0

10.0

5.0

2.5

7

7

7

7

14

7

7

99

101

96

100

96

102

100

4

4

4

3

5

5

6

B MCAA

DCAA

TCAA

MBAA

BCAA

DBAA

TCPh

5.0

4.0

4.0

5.0

2.0

4.0

0.4

13

14

14

14

14

14

14

101

103

103

97

106

102

104

8

7

6

8

8

7

15

TABLE 6251:IX. RELATIVE PERCENT DIFFERENCE (RPD) DETERMINATIONS FROM DUPLICATE SAMPLES

Haloacetic Acid*

MRL

/L N

RPD in Given Percentile Median Sample

Concentration

10th 25th Median 75th 90th gL

HAA5 775 0.0 0.83 2.9 6.4 12 21

Monochloroacetic acid 2.0 232 0.0 1.6 4.9 12. 28 3.2

Dichloroacetic acid 1.0 774 0.0 0.0 2.9 6.3 11 10

Trichloroacetic acid 1.0 716 0.0 0.0 2.4 6.1 10 9.8

Monobromoacetic acid 1.0 98 0.0 0.0 6.9 15. 27 1.4

Dibromoacetic acid 1.0 294 0.0 0.0 3.6 7.4 11 2.4

HAA6 771 0.07 0.86 2.8 6.3 11 24

Bromochloroacetic acid 1.0 656 0.0 0.0 3.2 6.7 12 3.4

* HAA5 data are the sum of the individual concentrations of MCAA  DCAA  TCAA  MBAA  DBAA; HAA6 data are the sum of HAA5  BCAA.

DISINFECTION BY-PRODUCTS: HAA (6251)/Micro Liquid-Liquid Extraction GC Method

10
5. U.S. ENVIRONMENTAL PROTECTION AGENCY. 1996. ICR Sampling

Manual. EPA 814-B-96-001, U.S. Environmental Protection

Agency, Cincinnati, Ohio.

6. MUNCH, D.J., J.W. MUNCH & A.M. PAWLECKI. 1995. Method 552.2,

Determination of Haloacetic Acids and Dalapon in Drinking Water

by Liquid-Liquid Extraction, Derivatization and Gas Chromatography with Electron Capture Detection. U.S. Environmental Protection Agency, Cincinnati, Ohio.

7. LIDE, D.R., ed. 1991. CRC Handbook of Chemistry and Physics,

71st ed. CRC Press, Boca Raton, Fla.

8. XIE, Y., D.A. RECKOW & R.V. RAJAN. 1993. Spontaneous methylation of haloacetic acids in methanolic stock solutions. Environ. Sci.

Technol. 27:1232.

9. FALES, H.M. T.M. JAOUNI & J.F. BABASHAK. 1973. Simple device for

preparing ethereal diazomethane without resorting to codistillation.

Anal. Chem. 45:13.

10. U.S. ENVIRONMENTAL PROTECTION AGENCY. 1996. Monitoring requirements for public drinking water supplies: Cryptosporidium,

Giardia, viruses, disinfection byproducts, water treatment plant data

and other information requirements. Final rule. Federal Register

61(94):24354.

10. Bibliography

U.S. ENVIRONMENTAL PROTECTION AGENCY. 1990. Methods for the Determination of Organic Compounds in Drinking Water. Supplement 1.

EPA-600/4-90-020.

BARTH, R.C. & P.S. FAIR. 1992. Comparison of the microextraction

procedure and Method 552 for the analysis of HAAS and chlorophenols. J. Amer. Water Works Assoc. 84:94.

TABLE 6251:X. PERCENT RECOVERY DETERMINATIONS FROM FORTIFIED SAMPLES

Haloacetic Acid* N

Percent Recovery in Given Percentile Median Sample

Concentration

10th 25th Median 75th 90th gL

HAA5 811 93 98 103 108 120 16.

Monochloroacetic acid 937 88 96 105 116 138 2.0

Dichloroacetic acid 806 89 95 100 106 116 7.2

Trichloroacetic acid 793 90 97 102 110 120 4.9

Monobromoacetic acid 953 91 99 105 112 140 0.0

Dibromoacetic acid 925 92 98 103 114 127 0.0

HAA6 804 93 99 103 108 118 19

Bromochloroacetic acid 873 90 98 102 108 120 1.8

* HAA5 data are the sum of the individual concentrations of MCAA  DCAA  TCAA  MBAA  DBAA; HAA6 data are the sum of HAA5  BCAA.

DISINFECTION BY-PRODUCTS: HAA (6251)/Micro Liquid-Liquid Extraction GC Method

11