)etermination of - C4 Fatty Acids as p-Bromophenacyl Esters Using 31ass-Capillary Gas Chromatography and Electron-Capture Detection

M. Larsson

Department of Analytical and Marine Chemistry, Chafmers University of Technology and University of GSteborg, S412 96 G6teborg, Sweden

C. Roos

National Laboratory of Forensic Chemistry, Department of Toxicology, S-581 85 Link~ping, Sweden

Key Words

Glass-capillary GC Electron-capture detection C~-C4 carboxylic acids p-Bromophenacyl esters Reagent degradation products

Summary

A method for the determination of low relative molecular mass carboxylic acids (C1--C4) in water is reported. The acids are converted to p-bromophenacyl esters prior to a glass-capillary gas chromatographic separation. By utilizing electron-caputre detection the detectability is substantially improved compared to flame-ionization detection. A comparison of three different ways to treat the water samples and to produce the derivatives is made. It is shown that the e, p-dibromoacetophenone reagent decomposes to a small extent which limits the utility of the reagent. Nevertheless a detection limit for formic acid of approximately 2.5mg1-1 is obtained. The method is applied to the determination of formic and acetic acids in a paper kraft water sample.

Introduction

Determination of low relative molecular mass carboxylic acids in aqueous samples is of interest in a number of fields. Carboxylic acids are important compounds in bacteriological systems as bacterial degradation products [1]. In water ~reatment carboxylic acids are produced from humic acids 12] and in steam generation processes, acids formed from trace organics in the feed water act as corrosive agents [3]. Formic and acetic acids are produced during manufacture of wood pulp [4]. The determination of these two acids is difficult and requires separate treatment and method design. Current methods are inconvenient and not appli- cable to trace analysis.

Determination of low concentrations of acids generally necessitates enrichment prior to instrumental analysis. lhe hydrophilic nature of formic and acetic acids com-

plicates isolation by methods commonly employed for organic compounds such as liquid-liquid extraction or adsorption on solids. Therefore alternative techniques have been suggested, such as head-space determination of pre- viously formed methyl esters [5], isolation by the formation of alkali salts [6-8 , 23, 26], of quaternary ammonium salts [9], or by extractive alkylation [10, 11] introduced by Ehrsson for analytical work [ 12], or ion-exchange chroma- tography [4].

In most cases low relative molecular mass carboxylic acids are determined by gas chromatography (GC) as derivatives for two reasons. The flame-ionization detector has an ex- tremely low response to free formic acid and only a slightly higher response to acetic acid. Secondly, the free acids show poor chromatographic performance. However, at higher concentration levels determinations can be made by direct gas chromatographic injections. Columns packed with Porapak N [13], with polypropylene glycol sebacate on Chromosorb W [14] as stationary phases and columns treated with phosphoric acid [15] have been used. New types of glass-capillary columns [16, 17] seem to be an at- tractive choice for direct injections.

Several derivatives for low relative molecular mass acids have been used for GC. Formic acid together with other acids have been resolved from the reagent peak as ethyl [18] and benzyl derivatives [9, 19]. Increased selectivity and sensitivity have been demonstrated with pentafluoro- benzyl derivatives in combination with'electron-capture detection [8, 11]. Potentially useful electron-caputre derivatives such as p-bromophenacyl esters have been used in combination with flame-ionization detection [6, 7, 10]. These esters have tile advantage of also being able to be determined by liquid chromatography (LC) and UV detec- tion [7, 23, 26]. Derivatives suitable for GC and LC have been reviewed [20].

The aim of this work is as follows:

(i) to determine carboxylic acids, including formic acid, at low concentration levels in aqueous samples;

(ii) to separate the acids as p-bromophenacyl esters by glass-capillary gas chromatography utilizing electron- capture detection;

(iii) to compare different methods for the preparation of the derivatives from water samples (cf. Fig. 1).

:hr0matographia Vol. 17 No. 4, April 1983 Originals 185

:209-5893/83/4 0185-06 $ 02.00/0 �9 1983 Friedr. Vieweg & Sohn Verlagsgesellschaft mbH

O Fig. 1

Br C--CH=--Br + RCOON(CsH13}4 Formation of p-bromo- phenacyl esters in or- ganic phase using ex- tractive alkylation of

0 tetrahexylammonium ~ II ion pairs of carboxyl ic

Br C--CH=--OOCR + BrN(C$H13)4 acids.

Experimental

Chemicals

Dichloromethane (p. a., Merck) was washed with dilute KOH and distilled water until neutral. Acetonitrile (p. a., Baker) was used w.ithout any purification. Water was distil- led twice before use. A stock solution of carboxylic acids was made by mixing 100~1 each of formic acetic acid (p.a., Merck), n-Propionic acid (pumm, Kebo) and n-Butyric acid (p.a., Fluka) in 100ml distilled water and made alkaline with 5M KOH. This solution was stable for several months when stored in a refrigerator. Fresh standard solutions were made daily by diluting the stock mixture with water. Most investigations were made at the 10mg1-1 level.

Buffer solutions of various pHs were made by titration of either NaH2PO4, Na2 H PO4 (Merck), K2 H PO4 (Fisher Sci. Company) or boric acid (Merck) with 1 M HC1 or 1 M KOH. KOH pellets (puriss., EKA, Sweden), were washed in methanol prior to use. All buffer solutions were washed with small portions of purified dichloromethane (see above). Tetrabutylammonium hydrogen sulphate, TBA, (Merck, p. a.) and tetrahexylammonium hydrogen sulphate, THA, (Fluka, purum) were dissolved in water and the solutions were neutralised by KOH.

The ~,p-dibromoacetophenone (Aldrich, 98%) was recrystal- lized in ethanol. Reagent solutions were made in either dichloromethane or in acetonitrile to a concentration cor- responding to a molar excess of at least fifty times over the total amount of acid. At the 10mg1-1 level for four acids this means a reagent concentration of 9.4mgrnl -~ sample. Solutions were prepared daily.

For the crown ether catalyzed derivatizations, the reagent solution contained dicyclohexyl- 18-crown-6 (Aldrich,techn.) at a molar concentration one hundredth that of the deriva- tization agent, that is, 0.13mgm1-1 sample.

As a GC internal standard, 4,4'-dibromobiphenyl (Fluka) was added directly to the reagent mixture to a concentra- tion of 10 to 20ggm1-1 sample.

Gas Chromatography

Split and splitless injections were made on a Carlo Erba Fractovap 4160 and a Carlo Erba Fractovap 2101 equipped with an electron-capture detector and a flame-ionization detector respectively.

A Duran glass capillary column (30m x 0.3mm) with a rather thick (0.8/~m) stationary phase layer of OV-73 was used. It was persilylated according to Grob [21] and coated by the static technique [22]. GC conditions are given in Fig. 2 and Fig. 3.

Gas Chromatography - Mass Spectrometry (GC-MS)

For qualitative analysis of the derivatives, a Carlo Erba Fractovap 2101 GC coupled to a Varian Mat 112 mass spectrometer with a spectro system 100 MS computer was used. The chromatograph was equipped with a Grob-type split/splitless injector and the same type of glass capillary column as described under the GC section but with a 0.2/am stationary phase layer. The conditions were as f01- lows: injection temperature 260 ~ temperature program 170~ ~ 3~ -1 . 1/al was injected with a preset split. ting ratio of 1 : 20. Helium was used as carrier gas at a fl0w rate of 40cm s -1 . The total effluent from the gas chroma. tographic column entered the ion source of the mass spectrometer via a non restricted all glass connection. El mass spectra were recorded in the mass range m/e 40400 at a scan rate of 1 s/decade.

Sample Preparation and Derivatization

All sample treatments: freeze drying, derivatization and extraction, were carried out in 5ml test tubes with Teflon- lined screw caps. During freeze drying, these caps were replaced by perforated Teflon films. Derivatization reac- tions were carried out in a thermostatted alumina block (Reacti-Therm, Pierce).

x2048

I

0

Fig. 2

3 is.

, /~v ,

5 10 23 retention time (min)

Chromatogram of aqueous standard solution, analyzed by extracti~ alkylation with THA as counter ion. Concentrations: Appr0x 10mgl - t C1-C 4 carboxylic acids. Peaks: p-bromophenacyl este~ of 1 formic acid, 2 acetic acid, ;3 propionic acid, 4 butyric acid, 5 p-bromobenzoic acid. A: e-hydroxy-p-bromo acetophenone, B: c~-chloro-p-bromo acetophenone and C: e, p-dibromoacetophen0ne

GC conditions: Split injection (1 : 80) of 1/al organic phase contain ing 20ng p.1-1 4,4'-dibromobiphenyl internal standard. Injecti0~ temperature: 260 ~ . Temp. program: 180 ~ to 250 ~ at 5~ -~ Hydrogen carrier gas flow rate: 40cms -1. Detector: ECD, temp 275 ~ Make up flow; Argon + 5% Methane, 30mlmin -1 . Column 30 m i,d. 0.3mm. Stationary phase: OV-73, 0.8#m.

186 Chromatographia Vol. 17 No. 4, April 1983 Originals

x128

0

2

3

4

i i i

5 10 15

retention time (rain)

Fig. 3

Chrornatogram of aqueous standard solution containing approx. 10mg1-1 C 1 - C 4 carboxylic acids.

GC conditions: Splitless injection (20s) of 2/~1 organic phase with 10ng#1-1 internal standard. Temp. program: 40 ~ to 150 ~ in 3 minutes, then 8 ~ per minute to 260 ~ Detector: FID. Notations as Fig. 2.

Use of potassium salts

lml aqueous sample was adjusted to pH 8 by addition of 20% methanolic KOH and freeze dried. Derivatives were prepared by adding l ml acetonitrile containing a, p-di- bromoacetophenone (9.4mgm1-1), dicyclohexyl-18-crown- 6-ether (0.13mgrn1-1) and internal standard (10/agm1-1) and heating for 15min at 80 ~ [7, 23].

Use of quaternary ammonium salts

lml aqueous sample was adjusted to pH 7 by addition of 100/11 0.4M phosphate buffer and either 100/11 0.1M TBA or 0.1M THA was added. After freeze drying, 1 ml dichloro- methane containing 9.4mgml -~ a, p-dibromoacetophenone and internal standard was added and the sample was heated at different temperatures for various times (see Results).

Extractive alkylation

lml aqueous sample was adjusted to pH 7 as above and 100tal one of the tetraalkylammonium solutions was ad- ded. The anions of the acids were extracted as ion-pairs into 1 ml dichloromethane containing a, p-dibromoa- cetophenone (9.4mgml - t ) and internal standard. After extracting the sample for 5min, the mixture was heated at different temperatures for up to 12hours. Extraction and derivatization were thus accomplished in one step.

Ident i f ica t ion of p -Bromobenzo ic Ac id

Extractive alkylation of p-bromobenzoic acid (puriss, Fluka) was at pH 9.6, at 45 ~ for 30min. For identifica- tion, spiked samples of varying concentrations were run. Blank samples were analyzed at pH 9.6 using extractive alkylation and THA as counter ion at different concentra- tions of a, p-dibromoacetophenone. Reaction temperatures of 45 ~ and 100 ~ were used for 30 and 60 min respectively.

The GC temperature program was: 180 ~ isothermally for 4min, then 15~ -1 to 260 ~

R e s u l t s a n d D i s c u s s i o n

Gas C h r o m a t o g r a p h y

Gas chromatograms resulting from extractive alkylation at pH 7, with THA as counter ion, of a standard water solu- tion are shown in Fig. 2 and Fig. 3. In Fig. 2, split injection with electron-capture detection was employed, and splitless injection with flame-ionization detection is shown in Fig. 3. The standard water solution contained acids at the following concentrations: formic acid 12.2mg1-1 ,acetic acid 10mgl -I , propionic acid 9.9mg1-1, butyric acid 9.6mgl -~. Gas chromatographic conditions and injected amounts are given in the figure captions.

Comparison of the two chromatograms show that the formic acid derivative is resolved from the reagent peak after split injection while it is obscured by the reagent peak with splitless injection. Secondly, the derivatives show very good electron-capture response but the detection limits are set by artefact peaks and background levels (discussed below) rather than detector response. Thirdly, with flame- ionization detection the large peak of trihexylamine causes interference in the middle of the chromatogram. This compound is the result of the degradation of tetrahexylam- monium bromide in the hot injector [24] and is not detected by the electron-capture detector under the conditions used. Analogous to the formation of trihexylamine (see above), tributylamine is formed from the decomposition of tetra- butylammonium bromide, which elutes in the early part of the chromatogram and does not interfere with the bromo- phenacyl esters.

For the above reasons, split injection with electron-capture detection is preferred to splitless injection with flame- ionization detection. Potentially, the resolution of the formic acid derivative from the reagent peak may be improved by selection of a stationary phase with a dif- ferent selectivity. A glass-capillary column with OV-1 as stationary phase and a fused silica column, coated with Carbowax 20M, gave a lower resolution as did a fused- silica, methyl-tolyl (50%) silicone column [25 ].

With electron-capture detection, the approximate detection limits were estimated as being five times the background levels. The following detection limits for the carboxylic acids in water were thus calculated: formic acid 2.5mg1-1 , acetic acid 1 mgl -~ , propionic and butyric acids 0.2mgl -~ . The background levels are discussed below.

Chromatographia Vol. 17 No. 4, April 1983 Originals 187

Using extractive alkylation with THA as a counter ion, linearity was established from the detection limit up to approximately 50mgl -~ where the linear range of the electron-capture detector was exceeded. Determination of higher concentrations is possible by simply diluting the organic extract. Work showing linearity for flame-ionization detection of C2-C4 p-bromophenacyl esters has been presented by L'Emeillat et al. [10].

Gas Chromatography - Mass Spectrometry

The ,derivatives formed were identified by GC-MS. The mass spectrum of the formic acid derivative is presented in Fig. 4. Mass spectra of the other derivatives have already been published by L'Emeillat et al. [ 10]. Background peaks appearing in the chromatograms shown in Fig. 2 and Fig. 3 could also be identified (see Fig. 2). Two of these peaks were a-hydroxy-p-bromo acetophenone and c~-chloro-p- bromo acetophenone. The latter is also formed during analysis of samples in the presence of chloride ions [26,27]. The trialkylamines discussed above were also identified.

Derivatization Procedures

Use of potassium salts

The derivatization procedure, using freeze drying to isolate the potassium salts of the acids, was found to be non- reproducible at the 10mgl -~ level. The most severe problem was the fluctuating and sometimes high background levels of formic and acetic acids (1-10mg1-1).

However, occasionally a relative standard deviation (rsd) for all acids of 4 - 1 0 percent (n = 5) could be obtained. An ac- curate pH setting and a complete drying of the salt was probably critical, since these parameters affect the break- down of the reagent (see below). The titration of each sample with KOH was time consuming. Chauhan and Darbre [8] have observed a high content of formic and acetic acids in NaOH and preferred a sodium hydrogen carbonate buffer for the formation of sodium salts of carboxylic acids. We tested a procedure with a potassium phosphate buffer but this did not improve the reproducibil- ity.

100

13 30

|11 130

�9 I.L �9 |

230

M% 242

U

Use of quaternary ammonium salts

The maximum yield of derivatives was obtained quickly, after 5min at 45 ~ for the tetrabutylammonium salts as well as for the tetrahexylammonium salts. The derivatives were stable at this temperature for several hours. Fewer artefacts were observed in the chromatogram, compared to samples analyzed by the extractive alkylation procedure. The precision was poor (rsd 25%, n = 5 ) a t the lOmg1-1 level. The background of acetic acid was lower than in the previ. ously discussed procedure, but the formic acid level was about equal. Chromatographic interferences of trialkyl- amines are discussed above.

Extractive alkylation

In order to minimize the hydrolysis of the reagent, mild conditions for the reactions were desirable. For this reason, extractive alkylation was performed at a neutral pH. A slow extractive alkylation was obtained when the TBA ion was used. Formic acid, the most hydrophilic of the four acids, was derivatized with the slowest rate when it was extracted by this less lipophilic counter ion (Fig. 5A).

A maximum yield of derivatives was obtained after 5 rain of extraction when THA was used as a counter ion, except for formic acid, which needed heating at 45 ~ for 30rain (Fig. 5B). The rsd (n = 5) at the 10mg1-1 level was 5.5~ (formic acid), 3.4% (acetic acid), 2.9% (propionic acid) and 1.9% (n-butyric acid) for aqueous standard solutions. The derivatives were stable for several hours at 45 ~ Tn- hexylamine was formed in the injector by degradation of tetrahexyl ammonium bromide [24] and elutes between the acetic and propionic acid derivatives. Upon heating at 80 ~ the derivatization yield was not improved. Due to the shorter reaction time, the THA ion was preferred as counter ion for the extractive alkylation. Typical background levels of formic acid were 0.5-0.6mg1-1 and for acetic acid ca. 0.2mg1-1 . The recovery has been determined by L'Emeillat et al. [10] as 70% for acetic, propionic and butyric acids.

Degradation of (~, p-Dibromoacetophenone

The relatively high background of formic acid was examined in detail and is a decomposition product from the reagent itself. As mentioned above, in chromatograms from blank samples not only the pure reagent was found, but als0 ~- hydroxy-p-bromo acetophenone. This compound is easily oxidized to p-bromophenylglyoxal. According to a method by Girsavicius [28] for the determination of phenylglyoxal. hydrogen peroxide and dilute alkali can be used for complete oxidation of the glyoxal to benzoic acid and formic acid. Analogously, the products formed by further oxidation of p-bromophenylglyoxal should be p-bromobenzoic acid and formic acid (cf. Fig. 6).

The breakdown pathway suggested is supported by the presence of the p-bromophenacyl ester of p-bromobenz0ic

Fig. 4

EI mass spectrum of p-bromophenacyl formate.

188 Chromatographia Vol. 17 No. 4, April 1983 Originals

Atbi~a[ units

Arbitrary units

5-

. . . . . . formic acid

butyric acid

�9 45~ / / ~ / i

�9 80 ~

/ . / " // , J pl / /

14~176 �9 . . . . . . . . . -e l ; I m l ~'~ K 100 200 12h

Reaction time ( min )

Fig. 5A

Reaction rate for extractive alkylation with tetrabutylam- monium ion (0.01 M) of formic acid and butyr ic acid to p- bromophenacyl esters. Reaction temperatures: 45 ~ and 80 ~

4.

3-

2-

1-

- ~_-:.--_--.-__-__-_____-_______:__

--m

' t $o ' ~$o ' ~ t~h Reaction time (rain)

Fig. 5B

Reaction rate for extractive alkvlation with tetrahexylam- monium ion (0.01 M) of formic and butyr ic acids. Symbols as Fig. 5A.

0 0

oxJ o o o

_ @ l l ox / ~ - ~ II II Br C--OH+HCOOH �9 B r - - - ~ ( I ) " - - C - - C - - H

p-bromobenzoic formic acid p-bromophenylglyoxal acid

Fig. 6

Degradation of e, p-dibromoacetophenone to formic and p-bromo- benzoic acids.

acid in all samples analyzed. This compound is strongly retained and elutes approximately 13 min after the internal standard (Fig. 2). It was identified by running a blank sample to which p-bromobenzoic acid had been added. A direct relationship was obtained in blank samples between the added amount of reagent and the amounts found of both formic acid and p-bromobenzoic acid which is shown in Fig. 7. The concentrations of these two compounds increased when the temperature was raised during the ex- tractive alkylation. The curve of formic acid is not extended further because it was not possible to separate the formic acid derivative from the reagent peak at a reagent concentra- tion of 100mgrnl -~ . Investigations were carried out at pH 9.6 to extract more effectively the less acidic p-bromo- benzoic acid. Therefore quantitative results could not be

Arbitrar units

~ p-bromobenzoic acid 0.5 �9 45*

A A 1oo ~ i// .~.i

, _]T._~__ma. I I I | 510 i i i i i

lOO Reagent concentration (mgm1-1 }

Fig. 7

Content of p-bromophenacyl esters f rom formic and p-bromoben- zoic acids in blank samples versus concentration of ~, p-dibromo- acetophenone used. Extractive alkylat ion of THA ion pairs at pH 9.6. Reaction conditions: 45 ~ for 30min. or 100 ~ for 60min.

At lOOmgm1-1, the formic acid derivative was overlapped by the reagent peak.

directly transferred to the conditions outlined in the proposed method. However, the amount ofp-bromobenzoic acid in blank samples was estimated as 1.4mg1-1. On a molar basis this corresponds to a background level of 0.3mgl -~ for formic acid which is about the same level as the background found. To minimize the reagent degrada- tion problem, fresh solutions should be prepared daily. For the samples analyzed in the present paper, the back- ground levels were reproducible. Nevertheless, care should be taken when new types of samples are to be analyzed. For the analysis of samples of strongly oxidizing character, a standard addition method should be used in order to examine whether the background level of formic acid is enhanced or not.

The reagent breakdown is of course a severe limitation on the utility of the reagent for the determination of low levels of formic acid.

Application

In pulp bleaching, several factors may affect the product quality. To characterize the degradation products from wood carbohydrates, formic and acetic acids were deter- mined in the kraft pulping process [4].

Extractive alkylation of THA ion pairs at pH 7 was used for the analysis of a paper kraft sample. The formic acid concentration was determined as 54mg1-1 and acetic acid as 11 mg1-1 (Fig. 8).

Conclusions

The high response of the electron capture detector to the p-bromophenacyl esters of the C~-Ca carboxylic acids could not be utilized to its full potential, but nevertheless electron-capture detection is preferred to flame-ionization detection. By the use of a capillary column with a thick stationary phase the formic acid derivative isresolved from the reagent peak.

Chromatographia Vol. 17 No. 4, April 1 9 8 3 Originals 189

x 4 0 9 6

f J

o 5

retention time (min)

i.s.I

x 5 1 2

i

10

Fig. 8

Chromatogram of paper kraft sample, analyzed by extractive alkylation at pH 7 with THA as counter ion. Concentrations: Formic acid 54mg1-1, acetic acid 11 mg1-1 . Internal standard: 10mg1-1. EC detection. GC conditions and notations as in Fig. 2.

The detect ion limit for formic acid is set by the decom- posit ion of the reagent which decomposes to a small extent to yield formic and p-bromobenzoic acids.

Of the methods studied for water sample t reatment , ex- tractive alkylation with THA as counter ion is preferred with respect to simplicity, background levels and time of analysis.

Acknowledgements

We wish to thank D. Dyrssen for criticism of the manuscript and G. Eklund for constructive discussions. We acknowledge T. Ohlsson for discussions concerning the reagent degrada- tion and E. Fogelqvist for the mass spectrum of the formic acid derivative.

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G. King (Eds.). Heyden, London 1977. 1211 K. Grob, G. Grob, K. G r o b J r . , H R C & C C 2 , 6 7 7 (1979). [221 J. Bouche, M. Verzele, J. Gas Chromatogr. 6,501 (1968). [231 H. D. Durst, M. Milano, E. J. Kikta, Jr., S. A. Connelly, E~

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Received: Dec. 30, 1982 Accepted: Jan. 11, 1983 C

190 Chromatographia Vol. 17 No. 4, Apr i l 1983 Originals