## 6251 DISINFECTION BY-PRODUCTS: HALOACETIC ACIDS AND TRICHLOROPHENOL*

6251 A. Introduction

1. Sources and Significance

The haloacetic acids (HAAs) are 1 formed by the chlorination of natural organic (humic and fulvic) matter. Utilitiesusingchlorine as a water disinfectant generate haloacetic acids, usually as the second most prevalent group of known disinfection by-products 1 ; the primary group formed is usu

ally the trihalomethanes. Toxicological studies indicate thatdichloroacetic acid and trichloroacetic acid are animal carcinogens. 2 The USEPA has promulgated a maximum contami

nant level for the sum of five haloacetic acids. 3 2. References

1. U.S. ENVIRONMENTAL PROTECTION A GENCY & A SSOCIATION OF M ETRO

POLITAN WATER A GENCIES . 1989. Disinfection By-Products in U.S. Drinking Water. Vol. 1 Report, James M. Montgomery Consulting Engineers, Pasadena, Calif. 2. ALCEON C ORP . 1993. An Overview of Available Information on the Toxicity of Drinking Water Disinfectants and Their By-Products. Cambridge, Mass. 3. U.S. E NVIRONMENTAL P ROTECTION A GENCY . 1998. Disinfectants and Disinfection Byproducts Final Rule. Federal Register 63(241): 69390.



## 6251 B. Micro Liquid-Liquid Extraction Gas Chromatographic Method

This method 1 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 tertiary

butyl 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 analyticalcolumn 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. Asshown 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.3 h4).

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 hasnot 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 mate

rial. Use only in a properly operating fume hood; C AUTION : 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.

1ground-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. Alwaysquench 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 NH 4 Cl (bake over

night at  100°C to eliminate contaminants), which converts freechlorine 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 bywashing 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

21 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 fromlaminated 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 63 Ni ECDwith 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 H 2 SO 4 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 , Na2 SO 4 , 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, NH 4 Cl. 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. T ABLE 6251:I. M ETHOD D ETECTION LEVELS AND PRECISION D ATA*

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

31) 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 f 1). 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, H 2 SO 4 , conc. i. Copper (II) sulfate pentahydrate, CuSO 4 5H 2 O. 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. (N OTE : 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  Lof 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 withMtBE. 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 5 e below, but substitute dibromoacetic acid stock solution as the solution added to theouter 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

4ether of the phenol), in a 10-mL volumetric flask and bring tovolume 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 ofmulticomponent 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.5 e , 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 themulticomponent 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 H 2 SO 4 added will reduce pH to less than 0.5 before beginning microextraction. Test a separate 10-mL portion by addin g 1 g CuSO 4 , 4 g granular Na 2 SO 4 [6251B.4 b 1)], and 0.5 mL conc H 2 SO 4 , 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.4 l2) 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 H 2 SO 4 , 3 g CuSO 4 , 12 g baked Na 2 SO 4 [¶4 b 1)], and 3 mL MtBE. Immediately cap and shake briefly by hand to break up any salt clumps.

When using automated extraction, place vials in mechanicalshaker wooden holding block. Shake vials at fast speed (approxT ABLE 6251:II. ANALYTICAL S TANDARDS

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 ofSouthern 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

5imately 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 diazomethane 9 : Using the apparatus in Fig

ure 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: N OTE : 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 Na 2 SO 4 [6251B.4 b 2)] 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 MtBEsolvent 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 extractand 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 forboth 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 eachsample 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 ( A
a ) data for each compound and an internal standard peak DISINFECTION BY-PRODUCTS: HAA (6251)/Micro Liquid-Liquid Extraction GC Method

6area ( A i ) for each level; use these peak areas to calculate relative response for each compound.

Relative response  A a/ A i A calibration curve passing through zero is generated from the plotted points for each compound using the relative responseversus 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 requiresmany 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.

T ABLE 6251:III. RETENTION T IMES

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 — T ABLE 6251:IV. RECOMMENDED Q UANTITATION 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

7els, 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.5 g 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 (S
r ). 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.4 j5), to check absolute recoveries of extracted and derivatized haloacetic acids. Calculate absolute recoveries bycomparing 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 absoluterecoveries 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 7 c 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 aderivatization 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 internalstandard 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 anacceptable 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 mixto a second sample portion and analyze to determine the concentration of each compound ( A ). Calculate percent recovery (P i ) T ABLE 6251:V. A DDITIVE 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

8as 100 ( A

B ) /T

, where T is the known concentration of the material added to the sample.

Compare percent recovery (P i ) for each compound with es

tablished 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 ( S
r).

Calculate QC acceptance criteria as follows:

Upper control limit (UCL)  P  3 S r Lower control limit (LCL)  P

3 S rThe data generated during the initial demonstration of capability, ¶ c 1) 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 S
r 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 S
r using only the most recent 20 data points.

Compare percent recovery (P i ) for each compound with theQC 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, X 1 and X 2 , as follows:

R   X 1 X 2 

The normalized range (R
n ) is calculated by dividing the range by the average of the duplicate values (X
m ):

R n  R
Xm Xm  X 1  X 2 2
Calculate mean normalized range (R
m ) for 20 pairs of dupli

cate data points initially and 20 pairs of points quarterly:

R m  ¥R n

n
where:

n  number of duplicate pairs.

Variance  S 2  ¥ (R n R m ) 2

n
1

The standard deviation ( s ) is the square root of the variance. Upper and lower control limits are R
m  3 s and zero, respec

tively. Acceptable duplicates fall within the control limits. The upper warning limit is R
m  2 s . If an R
n 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 confirmationmay 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 NH 4 Cl (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 T ABLE 6251:VI. A BSOLUTE RECOVERY D ATA FOR REAGENT WATER WITH KNOWN A DDITIONS

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

9may 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 thesensitivity, 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 Rule 10 (ICR) using this method are presentedin 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. C HINN , 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 AmericanChemical Soc. Pacific Conf. on Chemistry and Spectroscopy, Pasadena, Calif., Oct. 18 –21, 1989. 2. U.S. ENVIRONMENTAL PROTECTION A GENCY . 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 C ORP . 1993. An Overview of Available Information on the Toxicity of Drinking Water Disinfectants and Their By-Products. Cambridge, Mass. 4. D E B OER , T.J. & H.J. B ACKER . 1963. Organic Synthesis, Collective, Vol. IV. John Wiley & Sons, New York, N.Y. T ABLE 6251:VII. S AMPLE D UPLICATE D ATA 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

T ABLE 6251:VIII. FIELD S AMPLE RECOVERY WITH KNOWN A DDITIONS TO D RINKING 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

T ABLE 6251:IX. RELATIVE PERCENT D IFFERENCE (RPD) D ETERMINATIONS FROM D UPLICATE S AMPLES

Haloacetic Acid* MRL


/L N RPD in Given Percentile Median Sample Concentration


gL 10th 25th Median 75th 90th

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

105. U.S. ENVIRONMENTAL PROTECTION A GENCY . 1996. ICR Sampling Manual. EPA 814-B-96-001, U.S. Environmental Protection Agency, Cincinnati, Ohio. 6. M UNCH , D.J., J.W. M UNCH & A.M. PAWLECKI . 1995. Method 552.2, Determination of Haloacetic Acids and Dalapon in Drinking Waterby 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. X IE , 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. J AOUNI & J.F. B ABASHAK . 1973. Simple device for preparing ethereal diazomethane without resorting to codistillation. Anal. Chem. 45:13. 10. U.S. ENVIRONMENTAL PROTECTION A GENCY . 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. B ARTH , 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. T ABLE 6251:X. PERCENT RECOVERY D ETERMINATIONS FROM F ORTIFIED S AMPLES

Haloacetic Acid* N Percent Recovery in Given Percentile Median Sample Concentration


gL 10th 25th Median 75th 90th

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