Avoiding pitfalls in the determination of halocarboxylic acids:
the photochemistry of methylationt


F. Javier Rubio,J Edward T. Urbansky* and Matthew L. Magnuson

 United States Environmental Protection Agency, Office of Research and Development,
National Risk Management Research Laboratory,  Water Supply and Water Resources
Division, Cincinnati OH 45268,  USA. E-mail: urbansky.edward@epa.gov;
Fax: +1 513 569 7658; Tel: +1 513 569 7655

Received 24th January 2000, Accepted 13th March 2000
Published on the Web 12th April 2000
 Haloethanoic (haloacetic) acids are formed during chlorination of drinking water and are regulated by the
 Environmental Protection Agency (EPA). These compounds are normally quantified by gas chromatography
 with electron capture detection (GC-ECD) as the methyl esters. EPA Method 552 uses diazomethane (CH^z)
 for this purpose, but has only been validated by EPA for HAA6: chloro-, dichloro-, bromo-, dibromo-,
 bromochloro- and trichloroacetic acids. EPA Method 552.2 was developed and validated for all nine analytes
 (HAA9=HAA6 + dibromochloro-, bromodichloro- and tribromoethanoic acids). Since the promulgation of
 Method 552.2, which uses acidic methanol, a debate has ensued over discrepancies observed by various
 laboratories when using diazomethane instead. In an effort to identify and eliminate potential sources for these
 discrepancies, a comparative study was undertaken for HAA9. Better accuracy and precision were observed for
 all HAA9 species by Method 552.2; recoveries were satisfactory in de-ionized and tap water. Method 552
 remains satisfactory for HAA6. Systematic differences in instrumental response are observed for the two
 methods, but these are precise and may be accounted for using similarly treated standards and analyte-fortified
 (spiked) samples. That notwithstanding, Method 552 (CH2N2) was shown to be unsuitable for dibromochloro-,
 bromodichloro- and tribromoethanoic acids  (HAA9-6). The primary problem appears to be a photoactivated
 reaction between diazomethane and the HAA9-6  analytes;  however, side reactions were found to occur even hr
 the dark. Analyte loss is most pronounced under typical laboratory lighting (white F40 fluorescent
 lamps+sunlight), but it is also observed under Philips gold F40 lamps (As>520 nm), and in the dark.
1  Aim of investigation

Disinfection of water by chlorination can lead to the formation
of haloacetic acids (HAA), which are part of a larger group of
disinfection byproducts (DBPs). Many of these compounds are
suspect carcinogens, and the Environmental Protection Agency
(EPA) has included many DBPs in the Stage 1 Disinfectants/
Disinfection Byproducts  (D/DBP)  Rule.1'* Maximum con-
taminant levels (MCLs)  for five  haloacetic acids (HAAS)
[HAAS is the sum of the concentrations  (in ngmL~')  of
mono-, di-  and  trichloroethanoic acids  and  mono-  and
dibromoethanoic acids (Al,  A2,  A4,  A6, A9).  HAA6=[|
HAA5+A7. HAA9=Table 1 analytes except A3 and A5] are
regulated to annual averages (for four quarterly averages) of
60 ng mL~'. In this document, the HAAs appear also regulated
as HAA6. Dichloroethanoic acid and trichloroethanoic acid
have a maximum  contaminant level goal (MCLG) of 0 and
0.3ngmL~', respectively. The MCLG  of zero for dichloro-
ethanoic acid is based on evidence of carcinogenicity in animals
that indicates probable human carcinogenicity. Public water
systems are also  encouraged to  monitor  bromodichloro-,
dibromochloro- and tribromoethanoic acids and report  the
results as HAA9,  that is, the sum of the nine haloethanoic
acids.3
  The EPA has promulgated three methods for determining
the HAAs: Methods 552,  552.1 and 552.2. In the USA, only

•(This  paper is the  work product  of United States government
employees engaged in their official duties. As such, it is in the public
domain and exempt from copyright restrictions © US government.
JOn leave from Department de Ingenieria Quimica  y  Energetica,
Universidad de Extremadura, Avda. De Elvas s/n,  06071 Badajoz,
Spain. E-mail: fjrubio@unex.es.
EPA-approved methods may be used for regulatory compli-
ance monitoring. Method 552 uses liquid-liquid microextrac-
tion and diazomethane (CH2N2) as derivatizing agent. Method
552.1  uses a solid phase extraction and subsequent esterifica-
tion of the acids with acidic methanol. Method 552.2 uses
liquid-liquid extraction followed by methylation with acidic
methanol  (H2SO4).  Because of  the hazardous nature  of
diazomethane (used in  Method 552), there is  a desire to
eliminate this chemical from standard analyses. This was one of
the driving forces behind the development of Method 552.2,
although longer reaction times are needed. Nonetheless, many
laboratories (including  our  own) have  continued to  use
diazomethane to determine the HAAs.4
  The matter at hand can be expressed as one question: can
either of these two methods be used to quantify HAA9
accurately and precisely? Xie et al.5 compared the efficiencies of
Methods 552 and 552.2, but only for determining HAA6. They
obtained similar results using both methods. EPA's  National
Exposure Research Laboratory (NERL), formerly the Envir-
onmental Monitoring Systems Laboratory  (EMSL), is respon-
sible for issuing approved methods. EMSL validated Method
552 for HAA6 only; nevertheless, some investigators have used
it to determine HAA9. In other words, EPA never approved
Methods  552  or  552.1  for quantifying  bromodichloro-,
dichlorobromo- or tribromoethanoic acids in drinking water.
On the other hand, NERL  did validate Method 552.2 for all
nine compounds.
  Multi-laboratory analyses of split samples from unpublished
EPA DBP formation studies have shown discordant  results—
especially for bromodichloro-,  dibromochloro-  and tribro-
moethanoic acids (HAA9-6)—using the two methods (CH2N2
and H+/MeOH). Disagreement in the results for HAA9-6
analytes was so severe as to render the results meaningless for
248    J. Environ. Monit., 2000, 2, 248-252

                                This journal is © The Royal Society of Chemistry 2000
                                 DOI: 10.1039/b000674m

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those DBFs. It was unclear whether the differences were due to
mistakes made by the analysts, weaknesses in the method(s) or
some peculiarity of the samples. Therefore, two objectives were
established:  (1) evaluate Method 552 (microextraction)  for
HAA9 (actually only HAA9-6; EMSL already did HAA6) and
(2) determine a reason for discrepancies for results obtained by
the different laboratories using the two methods.
2  Experimental

2.1 Reagents
Sulfuric acid for acidification was of ACS reagent grade. The
extraction  solvent,  /ert-butyl  methyl  ether  (MTBE),  was
obtained from J. T. Baker (Phillipsburg, NJ, USA). Silica gel
(35-60 mesh,  Aldrich, Milwaukee, WI, USA) and  sodium
sulfate (Mallinckrodt, Phillipsburg, NJ, USA) were heated at
400 °C for 4h to remove organic contaminants. JV-MethyWV-
nitroso-j5-toluenesulfonamide (Fluka, Buchs,  Switzerland or
Aldrich), diethyl ether (Fisher Scientific, Fair Lawn, NJ, USA)
and carbitol [2-(2-ethoxyethoxy)ethanol] (Aldrich), .were used
in the  generation of diazomethane as per Method 552. High
purity  water was obtained by polishing house reverse osmosis/
UV-irradiated water through a Barnstead (Dubuque, IA, USA)
Easy Pure  system equipped with ion  exchange and  organic
removal cartridges.
  Standards for the nine haloacetic acids in MTBE (EPA-552.2
acids   calibration mix-ICR) were  obtained  from Supelco
(Bellefonte, PA, USA).  The composition  of the  standards
and the concentrations are given in Table 1. This was diluted
1:8 (12.5%) v/v in pesticide residue analysis grade methanol
(Burdick & Jackson, Muskegon, MI, USA). For the  sake of
brevity, all  concentrations for a mixture of the  HAA9 analytes
henceforth  will be expressed relative to tribromoethanoic acid,
analyte 11  in Table 1.
2.2 Standard calibration graphs

  EPA Method 552 microextraction (CHkNJ.  Aliquots of the
diluted commercial solution were injected into 30 mL portions
of high purity water using gas-tight microliter syringes to
prepare  standards  of  concentrations  varying  from  2 to
ISngmL"1.  Standards  were prepared in 60 mL borosilicate
glass vials equipped with PTFE-Iined septa and screw-caps.
Subsequently,  a  30 uL  aliquot  of  surrogate  solution
[20ngmL~'    2-bromopentanoic   acid   (Aldrich)   and
140ugmL~'  2,3,5,6-tetrafluorobenzoic  acid  (Aldrich) in
methanol] was added.  Once  the vials  had  been  prepared,
they were extracted for 45min in a mechanical shaker and
subsequently esterified with diazomethane, proceeding exactly
as specified.6
  EPA  Method  552.2  (H+/MeOH). Standard  solutions of
concentrations  2-15ngmL~1  were  prepared  by  injecting
volumes of the  12.5%  v/v diluted  commercial solution into
40.0 mL portions of high  purity  water. All the steps for
extracting and methylation with  methanol-H2SO4  are as
described in the method.7 The extraction time was 45 min,
the same as in Method  552.

2.3 Recovery of analytes from fortified tap water
Two different waters were used to verify recovery: house de-
ionized  (DI) water  and Tri-Township water  (TTW). EPA's
house de-ionized water is Cincinnati tap water which has been
subjected to reverse osmosis and UV irradiation. TTW was
collected from  a  residential  faucet  in  Logan Township,
Dearborn County, IN, USA. TTW is a chlorinated potable
water system, using  groundwater which presumably has  some
surface  water  infiltration from the Whitewater  River. After
10 min of high flow to flush the pipes, water was collected in a
high density polyethylene bottle  and used within  24 h of
collection.
  Replicates of each water were fortified with the 12.5% v/v
diluted commercial standard: 12 uL for Method 552, and 16 (iL
for Method 552.2, to reach a final concentration of 5 ng mL~'
(relative to tribromoethanoic acid). After capping and mixing,
samples were subjected to the respective method. Unspiked
samples were used to determine the background levels of all
analytes.

2.4 Effect of light on Method 552 microextraction

Discordance in results obtained for the trihaloethanoic acids
(see below)  required a  more thorough  investigation of the
diazomethane  methylation chemistry under the  influence of
light. Triplicate  standard solutions of SngmL"1 (relative to
All) were prepared as described above. All the vials were
treated following Method 5526  up to the point of methylation.
Once the extracts had been transferred to a 2.00 mL volumetric
flask, a 250 uL  aliquot of fresh diazomethane  solution was
added to each flask. The solutions were methylated for 25 min
under  variable  illumination: (1)  normal  laboratory  (white
fluorescent)  light, (2) gold light F40/GO (Philips Lighting,
Valencia, CA, USA) and (3) no light, i.e., in the dark. A 75 mg
portion of  silica gel was  then added  to each extract to
decompose the remaining diazomethane, and the samples were
analyzed by GC-ECD.
   In an effort to find evidence for  the side reactions inferred
from the results described above,  solutions of tribromoetha-
noic  acid  (Aldrich)  of  30ugmL~'  were  prepared in
MTBE. Aliquots of 2.0 mL were  methylated  by bubbling
diazomethane directly into the vials until the appearance of the
yellow tint characteristic of diazomethane.8 The  samples were
then  methylated for  different periods (between  10  and
Table 1 Composition of the EPA Method 552.2 commercial standard used in this study
No.
           Halocarboxylic acid analyte
                                          Formula
  CAS registry no."
Concentration/ng mL~
Al
A2
A3
A4
AS
A6
A7
AS
A9
A10
All
Chlorocthanoic
Bromoethanoic
2,2-Dichloropropanoicc
Dichloroethanoic
2-Bromopropanoic
Trichloroethanoic
Bromochloroethanoic
Bromodichloroethanoic
Dibromoethanoic
Dibromochloroetbanoic
Tribromoethanoic
CICHiCCMl
BrCB>CO2H
CH3C1,CCO,H
CkCHCOiH
CH3CHBrCO,H
C13CCO-,H
BrClCHCO2H
BrCl2CC02H
Br-,CHCO,H
Br,CICCO,H
Br3CCCWf
[79-11-8]
[79-08-3]
[75-99-0]
[79-43-6]
[598-72-1]
[76-03-9]
[5589-96-8]
[71133-14-7]
[631-64-1]
[5278-95-5]
[75-96-7]
300
200
200
300
100
100
200
200
100
200
100
5.30
8.58
9.01
9.41
9.89
13.01
15.04
17.58
18.03
19.58
20.89
'Registry numbers are for the acids, not the dissociated anions that would normally be encountered in water samples. HAA9 excludes analytes
3 and 5. 'Retention times are for the methyl esters and were established using a methyl ester blend standard purchased from Supelco. Times
are for the DB-1701 column. This acid is also known as dalapon.
                                                                             J. Environ. Monit., 2000, 2, 248-252    249

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 360 min), with half of them in the dark and the other half under
 white light. These samples were then analyzed by GC-MS.

 2.5 Instrumental methods

   GC-ECD analysts. Extracts were analyzed on a Tremetrics
 540 instrument (Austin, TX, USA), equipped with a Tremetrics
 774 autoinjector. Volumes of 2.0 uL were injected on to a
 J&W (Folsom, CA, USA) DB-1701 column (30 mx 322 urn
 id x 0.25 urn  film thickness)  at  a constant  He  pressure of
 101 kPa (15 Ib in"2). The inlet temperature was 220 °C and the
 detector temperature was 300 °C.  The temperature program
 was as follows: hold at 40 °C for 10 min; ramp at 5 °C min"1 to
 65 °C; ramp at 10°C min"1 to 85 °C; and ramp at 20°Cmin"1
 to 210 °C. Analytes, chemical formulae, CAS registry numbers
 and chromatographic retention times are given in Table 1.

  GC-MS  analysis. GC-MS was  used to analyze samples
 exposed to diazomethane for side reactions (i e., reactions other
 than  methylation).  Samples  were injected into a Hewlett-
 Packard (Palo Alto,  CA,  USA) GC-5890 Series  H gas
 chromatograph  interfaced  to  an MS-5971  mass  selective
 detector. Volumes  of  1.0 uL were  injected  on to a J&W
 Scientific  DB-5  column  (30mx250 urn  idx0.25um  film
 thickness). The inlet temperature was 275 °C and the detector
 temperature  was  280 °C. The temperature program  was as
 follows: hold at 32°C for 10 min; ramp at 4 °C min"1 to 50 °C;
 ramp at 4°C min"1 to 70 °C; hold for 15 min;  and ramp at
 15°Cmin~' to265°C.
3 Results and discussion

3.1 Basic data treatment

Chromatographic relatives  peak areas (^anaiyteMinijitd.) were
plotted against  concentration  (expressed in ngmL"1) for
each  analyte.  Slopes and  .y-intercepts were determined  by
unweighted least-squares linear regression. Slopes, ^-intercepts
and correlation coefficients  are reported in Table 2. Standard
errors in the slopes and jc-intercepts were computed using a
commercial spreadsheet  package  (MicroCal Origin,  v.2.8,
1993).
  In  order to evaluate  Methods  552  and  552.2 for the
quantification  of HAA9 analytes, two groups of compounds
must be considered separately: HAA6 and HAA9-6 (A8, A10,
All). With HAA6, very high correlation coefficients (0.988-
0.999) were obtained using either method, consistent with what
was already determined by NERL.
  Brominated  trihaloethanoic  acids  (A8, A10 and   All)
subjected to Method 552 produced low correlation coefficients
(between 0.762 and 0.867). The results were substantially better
using Method  552.2 (0.960 ^r2^ 0.977).  These results corro-
borate the  validity of Method  552.2 for HAAS, and suggest
                       that Method 552 suffers from a lack ofruggedness for the three
                       brominated trihaloethanoic acids (A8, A10 and All).


                       3.2 Method precision comparison

                       Before determining the calibration  curves  for all  the nine
                       haloacetic acids (HAA9), two sets of nine replicates of HAA9
                       (5.00 ngmL"1, relative to All)  were prepared in high purity
                       water in order to evaluate the reproducibility of the method.
                       One set was analyzed by Method 552 and the other set was
                       subjected to Method 552.2.
                         As expected, the Method 552.2 (H+/MeOH) results are very
                       reproducible for HAA9 analytes. Similar results were observed
                       for the mono- and  dihalocarboxylic acids subjected to EPA
                       Method 552 (CH2N2). As an  example representative of these
                       groups, the results obtained for chloroethanoic acid are shown
                       in Fig. l(e). However, Fig. l(a)-(e) show that  the reproduci-
                       bility was very poor for the brominated trihaloethanoic acids.
                       There is an interesting relationship between the responses of the
                       different analytes. Specifically, the brominated trihaloethanoic
                       acids follow the same trend within a sample as is immediately
                       evident in Fig. l(a)-(c)- The reproducibility for trichloroetha-
                       noic acid is  much  better than  that  for  the brominated
                       trihaloethanoic acids, suggesting that brominated  species are
                       intrinsically less stable (or kinetically more reactive) during the
                       analytical procedure. The precision of replicate analyses is  in
                       fact so bad that we might infer that Method 552 cannot be
                       applied to A8, A10 and All with any certainty.


                       3.3 Method recovery comparison

                       Values of background levels of HAAs were determined from
                       unspiked tap water (TTW) samples and these were subtracted
                       prior to comparison. No background levels were observed for
                       unspiked DI water. Table 3 shows the results for the Method
                       552 (microextraction) and Method 552.2.
                         In DI water, both methods show acceptable recoveries for
                       HAA9: 88-114% for Method 552 and 94-127% for Method
                       552.2. Nonetheless, Method 552.2 gives better precision for the
                       brominated trihalo species, ;.a,  BrCl2CCO2H, Br2ClCCO2H
                       and Br3CCO2H. In TTW water, good results were obtained for
                       HAA6 using  either method.  However, Method 552.2 gave
                       better recovery  and precision  than  Method  552  for the
                       brominated trihalocarboxylic acids. Recoveries were as follows
                       (H+/MeOH  vs.  CHzNj): BrCl2CCO2H  (116  vs.  130%),
                       Br2ClCCO2H  (106 vs.  129%) and Br3CCO2H (148 vs. 168%).
                       Although  we obtained  a recovery  for tribromoethanoate
                       outside of the ±30%  range given by Pawlecki-Vonderheide
                       et a/.,9 our recovery is based on a spiked concentration of
                       5.0 ng mL"1 and not 10 ng mL"1 as  was used in developing
                       Method  552.2. A more  thorough evaluation of the matrix
                       might indicate a systematic bias for this analyte (especially near
                       the detection limit), but was beyond the scope of this work.
Table 2 GC-ECD response for HAA9 laboratory standards analyzed according to EPA Method 552 (microextraction) and Method 552.2°

                   Method 552 (microextraction)                            Method 552.2
Analyte
                   Slope x 10"
^-Intercept x 10"
                                                                       Slope x 10"
                                                                                         .y-InterceptxlO"
CICH2C02H
BrCH2CO2H
C12CHCO2H
C13CCO2H
BrClCHCO2H
BrCl,CCO->H
Br2CHCO2H
Br2ClCCO2H
Br3CCO2H
30±2 30±40 0.988
380±17 30+300 0.992
490±20 400 + 600 0.991
1400 + 70 -200 + 600 0.995
1080±40 -200+700 0.993
1400±200 -2200±1700 0.867
1360±50 -300+400 0995
490 + 80 -1800 + 1400 0.83!
260±50 -500±400 0.762
241 ±4°
3600±100
5020 + 70
10000±500
11000+316
16000 + 1200
13 000 ±500
4900+400
1800+180
-130±110
-3100 + 1600
-1700±1600
-8500±4000
-10000±5000
-19000±10000
-8500+4000
-13000+7000
-2700+1500
"Results for the slopes and ^-intercepts cannot be directly compared because different concentrations in the internal standard were
two methods. All peak areas were normalized to the internal standard response.
0.999
0.997
0.999
0.989
0.997
0.977
0.993
0.973
0.960
used in the
250    J. Environ. Monit., 2000, 2, 248-252

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       w
                   Bromodlchloroothanolc acid
        1Z34S3769
  I3
                    Dibromochforoethanolc acid
                                     'a-
  .IS
  x '•
  i
  §05
                      Tribromoethanolc acid
  V
  I*
       W
Triehloropthanolcacld
                       Chloroethanoteacld
                         4     s     6    r-
                         Sample number
Fig. 1 Bar graphs representing the variability in peak areas for nine
replicates of brominated trihalocarboxylic acids, which are adversely
affected when methylated using diazomethane: (a) bromodichloroetha-
noic acid, (b) dibromochloroethanoic acid, (c) tribromoethanoic acid
and (d) trichloroethanoic acid; (e) Chloroethanoic acid is included for
comparison because it is amenable to methylation with diazomethane.
                   »DARK
                   •WKTTE LIGHT
             10
                      110
                               210
                            Time/mln
                                                 410
Fig. 2 Effect of light on recovery of (30 ugrnL"') tribromoethanoic
acid  (delected as  the  methyl  ester)  in  the  presence  of excess
diazomethane (as in EPA Method 552). Graph shows the  GC-MS
peak area in the dark (•) and under normal laboratory lighting (•).
                                          Table 3 Comparison  of analyte recovery (%) in laboratory fortified
                                          samples of Tri-township Water tap water (TTW) and de-ionized water
                                          (DI)"
Method 552 (microextraction) Method 552.2
Analyte
CICH-.CO-.H*
BrCH,CO,H
CUCHCOjH
Cl3CCOoH
BrClCHCO,H
BrCUCCOiH
Br,CHCOJ€
BnClCCO^H
Br3CCO,H
TTW (%)
94+1
100±1
97+1
91 + 1
96±I
130±11
101±1
129±14
168±15
DI (%)
88±1
84+1
91 + 1
88±1
92±I
111±I1
92±1
114±14
114±15
TTW (%)
110±3
114 + 2
J05±l
102+3
103±1
116 + 4
103±2
96±5
148±2C
DI (%)
97±1
94±1
98 + 2
108 + 6
101±2
125 + 8
103 + 3
125±10
127 + 12
"TTW is a local chlorinated potable water system (see text for addi-
tional details). Our facility's  house de-ionized water is  treated  by
reverse   osmosis.   ''Chloroethanoic    acid   concentration  was
5.00 ng mL~'. Other concentrations varied according to the ratios in
the commercial standard as specified in Table 1. For example, bro-
moethanoic acid concentration was  3.33 ngmL"'. The  recovery
obtained for BrsCCOiH is outside the ±30% recovery range speci-
fied in  Method 552.* Presumably, this represents some matrix effect
that would require further characterization if the  objective were to
obtain  an accurate  value for this analyte. However, our objective
here was to verify that a reproducible result could  be obtained for a
fortified sample, and that was  in fact accomplished.
                                                                Table 4  Effect of light exposure during diazomethane methylation
                                                                (Method 552) on the performance of trihaloethanoic acid determina-
                                                                tion"
Analyte
C1CH,CO,H
C13CCO,H
BrCUCCO^H
Br-CICCOJH
Br3CC02H
Peak area*
No light
29.7+0.3
464±6
460 ±30
332±26
100±10
xlO"3
White light
36.0±0.2
346 ±2
114±15
73±11
11±2

Gold light
29.8 ±0.3
485 ±2
550 + 20
415±24
1I9±8
                                          "Brominated trihaloethanoic acids experience the most severe effect.
                                          Chloroethanoic acid is  shown for comparison as it experiences a
                                          minimal effect from exposure to light during methylation with diazo-
                                          methane. 'Values are averages for triplicate samples (5.00ngmL""')
                                          in de-ionized water. Reported uncertainties are the estimated stan-
                                          dard deviations of the means.
                                          3.4 Effect of light on Method 552 (CH2N2) performance for
                                          trihaloethanoic acids

                                          The average peak areas with their standard errors obtained in
                                          the analysis of the triplicate samples in the presence of different
                                          kinds of light are shown in Table 4. As expected, the measured
                                          peak areas demonstrated that the determination of the mono-
                                          and dihaloethanoic acids was unaffected by light. Table 4 gives
                                          the results for CICH2CO2H as representative of the mono- and
                                          dihaloethanoic acids. When BrCl2CCO2H, Br2ClCCO2H and
                                          Br3CCO2H are methylated with diazomethane under labora-
                                          tory (white) lighting, the results are much lower than when the
                                          methylation is carried out under gold light  or in  the dark.
                                          Apparently, the light most responsible for photoactivation has
                                          a wavelength below 520 nm because the results in the dark are
                                          similar to those under gold light. Accordingly, a judicious
                                          choice of laboratory illumination can improve performance of
                                          Method 552 for determining HAA9.


                                          3.5 Diazomethane side reactions investigated by GC-MS

                                          Fig. 2  illustrates the effect that exposure to white light has on
                                          the formation of the analyte peak, methyl tribromoethanoate.
                                          A  reasonably stable response  is  observed  in  the aliquots
                                          methylated  in  the  dark,  but progressive  degradation  of
                                                                                  /. Environ. Monit., 2000, 2, 248-252     251

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          120000
           80000'
           40000
                NL.||.,)J	I..I.1	,	l.Jj.lL
                 5.00    15.00   25.00   35.00   45.00
                                          Br3CCO2H
           60000
           SOOOO
           100001
AU.
:.00    15.i
                               BrjCCOjH
        ,00   25.00   35.00   45.00
Fig, 3 GC-MS ohromatogram for the methylation of tribromoethanoic
acid (30 ug mL"1) with diazomethane after 120 min (a) in the dark and
(b)  under  laboratory (white)  lighting.  Side reactions interfere in
quantifying this analyte. Several products were identified by compar-
ison with library spectra (see text for details).
Br3CCO2H occurs in the samples methylated under white light,
confirming the destructive effect that white light exerts on the
determination of the trihaloethanoic acids with diazomethane.
  Fig. 3  shows the GC-MS chromatogram after 120 min of
methylation in the dark and under white light. Fig. 3(a)  shows
only  one  peak  at  38.62 min  (corresponding  to methyl
tribromoethanoate);  hence,  there is  no  appreciable side
reaction  after 120 min of methylation in the dark. However,
Fig. 3(b) shows several  other sizable peaks in addition  to the
methyl tribromoethanoate peak. It is clear that reactions other
than methylation take place when Br3CCO2H and CH2N2 are
combined  in  white light.  Among the peaks  identified by
comparison with library mass spectra were the side reaction
products methyl 2-methylpropanoate, methyl  2-bromopro-
penoate  and methyl  ethanoate.  The  presence  of  these
compounds suggests  that a halogen  atom  abstraction takes
place. Such behavior for CH2N2 is unsurprising, but it has not
been  reported for  the trihaloethanoic acids previously.8'10
Diazomethane can be photolyzed to carbene [eqn. (1)],  which
could abstract halogen atoms.

                    CH2N2->:CH2+N2                 (1)

Carbene and carbenoids can undergo a wide range of reactions,
                                             including  insertions,  abstractions  and  alkens  formation.
                                             Mechanistically, these are unlike the decarboxylation (hydro-
                                             lysis) observed for tribromoethanoic acid with acidic metha-
                                             nol/
                                             4  Conclusion

                                             A comparison of the results obtained using Methods 552 and
                                             552.2 for the determination of HAA9 indicates  that better
                                             precision and accuracy are obtained with Method 552.2 (acidic
                                             methanol). EPA Method 552  can still be used to measure
                                             HAA6 concentrations, but it is not suitable for determining the
                                             additional analytes that make up HAA9. Of particular interest
                                             are the heretofore unreported side reactions of diazomethane
                                             with the brominated trihaloethanoic acids (i.e., BrCl2CCO2H,
                                             Br2ClCCO2H  and Br3CCO2H), especially when exposed to
                                             white light. The results improve when the analysis is performed
                                             under gold light or in the dark; nevertheless, taking precautions
                                             to  limit exposure   to light  does  not  make  the  method
                                             satisfactory   for the  brominated  trihaloethanoates.  The
                                             mechanism of reaction was not studied but can be presumed
                                             to  be either  insertion of a methylene  group or halogen
                                             abstraction due to  the carbenoid behavior  of CH2N2. The
                                             utility of diazomethane in the quantification of trihaloethanoic
                                             acids is therefore reduced on  account of its propensity for
                                             assorted  side reactions with  some  analytes. Satisfactory
                                             recoveries are achieved for all HAA9 analytes using Method
                                             552.2, and it should be used whenever HAA9 determination is
                                             required.
                                                              Acknowledgements

                                                              F.J.R. thanks the Junta de Extremadura for sponsoring his
                                                              visit to the USA and the EPA for its kind hospitality.
                                             References

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                                              2  Environmental Protection Agency, Fed.  Regist., 1998, 63(241),
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                                              4  E. T. Urbansky, D. M. Freeman and F. J. Rubio, J. Environ.
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