1012
ANALYTICAL CHEMISTRY, VOL. 51, NO. 7, JUNE 1979
that n o accurate linear extrapolations can be made. T h e advantages of a two-phase photometric titration over potentiometric titration and visual titrations have been discussed in connection with acid-base titrations ( I ) . In general, the same considerations apply to ion-pair titrations.
CONCLUSIONS Photometric ion-pair titrations performed in a two-phase medium show considerable promise for use in the determination of drugs and ionic surfactants. When dilution corrections are negligible, it is not necessary to know the values of any of the various equilibrium constants which appear in Equations 2 and 3, and 6 and 7 in order to perform accurate titrations. The time required to perform a titration by this method is only slightly longer than that required to perform a photometric titration in a one-phase medium. I t is possible to reverse the present approach by using an organic cation as titrant for an anionic sample species. I t is also possible to monitor the absorbance of the organic phase by placing a hydrophobic silicone-treated paper over the end of the filter probe in place of the normal hydrophilic paper (1). I t would be useful to have available anionic titrants which have higher ion-pair extraction constants, K,, with amine drugs. This would make possible the accurate titration of smaller drug molecules. Preliminary investigations have
shown the utility of some other anionic titrants such as dipicrylamine and laurylsulfate, and work is continuing in this area.
LITERATURE CITED F. F. Cantwell and H. Y. Mohammed, Anal. Chem., 51, 218 (1979). A. Galik, Tabnta, 14, 731 (1967). A. Galik, Talanta, 15, 771 (1968). M. A. Leonard, in "Comprehensive Analytical Chemistry", G. Svehla, Ed., Vol. VIII, Elsevier, New York, 1977, Chap. 3. (5) E. Heinerth, in "Anionic Surfactants--ChemicaI Analysis", J. Cross, Ed., Marcel Dekker, New York, 1977, Chap. 6. (6) D. Hummel "Identificationand Analysis of Surface-Active Agents", Vol. 1, Interscience, New York, 1962. (7) J. T. Cross, in "Cationic Surfactants", E. Jungermann. Ed., Marcel Dekker, New York, 1970, Chap. 13. (8) E. D. Carkhuff and W. F. Boyd, J . Am. fharm. Assoc.. Sci. Ed., 43, 240 (1954). (9) G. N. Thomis and A. 2 . Kotionis, Anal. Chim. Acta, 14, 457 (1956). (10) K. Behrends, Fresenius' 2. Anal. Chem., 250, 241 (1970). (11) K. Behrends, Fresenius' 2. Anal. Chem.. 250, 246 (1970). (12) "United States Pharmacopea" 19th Rev., Mack Publishing Go., Easton, Pa., 1975. (13) K. Gustavii and G. Schill, Acta fharm. Suecica, 3, 241 (1966). (14) G. Schill, in "Ion Exchange and Solvent Extraction",J. A. Marlnsky and Y. Marcus, Ed., Vol. 6. Marcel Dekker, New York, 1974, Chap. 1. (15) G. J. Divatia and J. A. Biles, J . fharm. Sci., 50, 916 (1961). (16) T. D. Doyle and J. Levine, J . Assoc. Off. Anal. Chem.,51, 191 (1968).
(1) (2) (3) (4)
RECEIVED for review November 14, 1978. Accepted February 23, 1979. This work was supported by the National Research Council of Canada and the University of Alberta.
Determination of Tryptophan in Foods and Feedstuffs with a Kinetic Method Hans Steinhart Institute of Nutrition Physiology, Technical University of Munich, 0-8050 Freising- Weihenstephan, Federal Republic of Germany
A fluorometric method for the determination of tryptophan in foods and feedstuffs Is described. The samples under investigation were hydrolyzed with 4 N Ba(OH), and, after preclpitatlon of the Ba2+ ions with H,S04, the pH value was elevated to pH 10.8. I n the fluorescence cuvette, a 20% formaldehyde solution of pH 10.8 was added to the alkaline hydrolysate of the samples. The increase in the fluorescence over a time interval of 60 s was measured. The increase in the fluorescence was linear in the concentration range of 0 to 100 nmol tryptophan/mL solution. This kinetic method is suitable for the determination of tryptophan in foods and feedstuffs. From the proposed reaction mechanism, it follows that indole compounds and guanine disturb the determination.
Tryptophan in foods and feedstuffs is mostly elevated by spectrophotometric methods. The p-DAB (I) and the glyoxylic acid methods ( 2 ) are the most important ones. In many cases, tryptophan is determined either from alkaline protein hydrolysates or from enzymatic partial hydrolysates. T h e spectrophotometric methods are somewhat disadvantageous because they need much work and because it is not yet quite clear whether and to what extent accompanying substances influence the color intensity or whether they falsify the measurements by causing shifts in the absorption maximum. Moreover, spectrophotometric methods are not as sensitive as fluorometric methods. 0003-2700/79/0351-1012$01.OO/O
Guilbault and Froehlich ( 3 , 4 )proposed a method for the determination of tryptophan in serum which uses the fact that the fluorescence changes when tryptophan reacts with formaldehyde. When this method is adapted to hydrolysates of foods and feedstuffs, there are, however, problems. The reason for these difficulties may be the complexity of the basic reactions. I t was the aim of this work to develop a fast and exact fluorometric procedure to determine tryptophan in foods and feedstuffs. For this purpose, a kinetic method was developed which is rather specific for tryptophan. The reaction of tryptophan with formaldehyde in alkaline media is the basic reaction of this method.
PRINCIPLE Fluorescence of T r y p t o p h a n in the P r e s e n c e of Formaldehyde. Tryptophan and other indole compounds react in different ways with formaldehyde. Some of the products of the reaction of formaldehyde with tryptophan are suitable for the quantitative analysis of tryptophan. Norharman, which fluoresces a t the emission wavelength of ,A, = 453 nm when it is excited a t A, = 373 nm ( 5 ) ,is formed from tryptophan when the reaction is brought about under strong acidic conditions in the presence of Fe3+ ions a t a temperature of about 100 "C. Larsson et al. (6, 7)also reported that tryptophan and tryptophan-containing peptides (tryptophan being on the amino end of the chain) result in strongly fluorescing products that are suitable for the quantitative analysis of tryptophan when they are treated with form1979 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 51, NO. 7, JUNE 1979
1013
cmbsion 70
0-
60
Flgure 2. Reaction mechanism of the reaction of tryptophan with formaldehyde at pH 10.8 50
LO
/' /'
30 2
4
6
8
10
12
14
16
18
20mih
L
J
Figure 1. Fluorescence of tryptophan during the reaction with formaldehyde at different pH values (Aex = 289 nm; ,A, = 348 nm)
Figure 3. Formation of radical cations during irradiation of tryptophan
aldehyde and hydrochloric acid. In the present experiments, the fluorescence properties of tryptophan were investigated in the presence of formaldehyde a t different pH values. For this purpose, 1mL of a tryptophan solution in buffers with different pH values was pipetted into a fluorescence cuvette, and 1 mL of a 20% formaldehyde solution of the same p H value was added. The recording of the fluorescence began 15 s after the mixing of the educts by shaking the cuvette. The reactions were conducted in carbonate buffer of pH 10.8, in phosphate buffer of pH 9.0 and 7.2, and in acetate buffer of pH 4.0. The excitation wavelength was 289 nm and the emission was recorded a t 348 nm. The slit widths were 10 nm at the excitation as well as at the emission site. The fluorescence diagrams are shown in Figure 1. The absolute amounts of fluorescence of the samples with different p H values are not comparable because the fluorescence maximum shifted by about 8 nm to the red when it was measured a t pH 10.8 (8). A product with a stronger fluorescence than tryptophan itself was formed when tryptophan reacted with formaldehyde in acetate buffer of pH 4.0. The velocity of the reaction was relatively slow because the increase of the fluorescence was only 28 units in 20 min. When the reaction was conducted in phosphate buffer of pH 7.2, the fluorescence decreased slowly during the first minutes before it started to increase. The overall increase was 14 units within 20 min. The reason for this transient decrease in fluorescence a t the beginning was probably that the overall reaction was still slower than a t p H 4.0. But the fluorescence of the end product was also higher than that of tryptophan. There were different effects in the alkaline range. From the beginning of the measurement on, there was a steep increase in the fluorescence at p H 9.0 which reached its maximum after 45 s and then decreased fast below the level of tryptophan. The increase in the fluorescence lasted longer at pH 10.8 than at pH 9.0, the maximum was reached after 6 min. The decrease of the fluorescence was distinctly slower. Probably, another product was formed a t the alkaline p H values; this product, when irradiated, is assumed to decompose faster than the product formed under acidic conditions. No measurements were made at higher pH values because the fluorescence decreases strongly (9).All measurements were repeated five times: they were reproducible. The mechanism shown in Figure 2 is proposed for the reaction of tryptophan with formaldehyde at alkaline p H values. At pH 10.8, the indole ring is not yet deprotonized because otherwise no fluorescence
of the tryptophan should be detectable. Besides the reaction of tryptophan with formaldehyde there are still photoinduced reactions. Pailthorpe et al. (10) found that tryptophan is one of the most photo-labile amino acids when it is irradiated at 250-500 nm. Detailed explanations of the photoinduced reactions of tryptophan are given by Udenfriend (11, 12). Experiments were conducted with continuous irradiation (IO), flash photolysis (10, 13,14),and laser flash photolysis (15). From these experiments, it may be deduced that an electron is removed from the indole and a radical ion is formed (Figure 3). This radical ion is unstable, and it reacts immediately by forming different primary and secondary reaction products. Until now, nine photolysis products were found (IO),the most important ones are HzOz (16) and kynurenine, respectively N-formylkynurenine (17). The curves of Figure 1 may be caused by an overlap of the two reaction types, namely the reaction of tryptophan with formaldehyde and the photoinduced reactions. The fluorescence spectra of some purine and pyrimidine compounds were measured also a t pH 10.8, taking the same reaction conditions as mentioned above. Uracil, cytosine, and adenine did not fluoresce at these conditions. There was also no fluorescence when a 20% formaldehyde solution was added to these compounds. Guanine, however, fluoresced at A,, = 290 nm and A,, mar = 345 nm. As can be seen from Figure 4a, guanine was stable, in contrast to tryptophan, when it was irradiated with the 140-W xenon high pressure lamp of the fluorometer. When 1 mL of 20% formaldehyde solution of pH 10.8 was added to 1 mL guanine solution (0.25 nmol guanine/mL solution) as described above, the fluorescence decreased, however, rapidly (Figure 4b). The same experiments were conducted with some other indole derivatives, for example with tryptamine and indole-acetic acid. The fluorescence of these substances was similar to that of tryptophan as well, with or without the addition of formaldehyde. Principle of the Method. The principle of the kinetic tryptophan analysis is the measurement of the increase in the fluorescence when tryptophan reacts with formaldehyde a t pH 10.8. It is in principle possible to conduct the reaction at pH 4.0 because the fluorescence also increases at that pH value. There are, however, three reasons to prefer the measurement at pH 10.8. The fluorescence at alkaline pH values is stronger than that at acidic pH values, and the increase in the fluorescence during the reaction of tryptophan with formaldehyde is also higher so that there are greater
1014
ANALYTICAL CHEMISTRY, VOL. 51, NO. 7, JUNE 1979 emission
emission
t
2
4
6
15 min
b'
2
L
6
15 min
(a) Fluorescence of guanine at pH 10.8 (Aex = 290 nm; A,,, = 345 nm). (b) fluorescence at the reaction of guanine with formaldehyde at pH 10.8 Figure 4.
differences; this is very favorable for the quantitative determination. Finally, tryptophan is more stable in the alkaline than in the acidic environment. The difference of the fluorescence is linear over the scope of interest between 0 and 100 nmol tryptophan/mL as will be shown in this work (see below). Since some substances, for instance indole derivatives or guanine, disturb the analysis, the method is not suitable for samples which contain greater amounts of these substances. Because these substances are rarely present in greater amounts in foods and feedstuffs, the method is, in most cases, suitable to assay tryptophan in these samples after alkaline hydrolysis. As can be concluded from the results above, an exact adjustment of the p H value is very important to obtain valuable results. T h e absolute extent of fluorescence of the samples is not important because only the time-dependent change of the fluorescence is used as a criterion in the method. The method is not suitable for samples that contain only small amounts of tryptophan because the change of the emission within the time interval may become too small.
EXPERIMENTAL The method was used for the determination of the tryptophan contents in different foods and feedstuffs. The samples were defatted by ether extraction, lyophilized, and then frozen. The dry matter of the lyophilized samples was determined by heating the samples for 4 h at 105 O C . The hydrolysis of the samples with 4 N Ba(OH)*was conducted according to the method previously described (9). About 300 mg of each sample were hydrolyzed. The pH value of the hydrolysates varied between 9.5 and 10.5. Twenty mL of each hydrolysate in a 50-mL flask were brought up to pH 10.8 with 4 N NaOH by using a pH-stat apparatus. Subsequently the solution was made to 50 mL by adding a 0.3 M carbonate buffer. After that, the hydrolysate was ready for measurement. A 35% stabilized formaldehyde solution (Merck, West Germany, No. 4003) was diluted with a 0.3 M carbonate buffer of pH 10.8 and some drops of 4 N NaOH. The fluorescence measurements were conducted with a Farrand Spectrofluorometer 801. The exitation wavelength A, was 289 nm and the emission wavelength A,, was 356 nm. The slit widths were 10 nm, both at the excitation part and at the emission part of the fluorometer. The fluorescence could be read from a digital multimeter (Philips PM 2421) and it was plotted vs. time. One mL of the hydrolysate was pipetted into a fluorescence cuvette (1cm) with a Eppendorf pipet, subsequently 1 mL of the formaldehyde solution was added, the cuvette was shaken and then brought into the sample chamber of the spectrofluorometer. The irradiation of the reaction mixture in the sample chamber was interrupted at the beginning of the reaction by using a blind. The blind was removed 30 s after the beginning of the reaction
hydrolysis with L N Ba(OH),
1
precipitation of Ba2'with H,SOL ,washing
1
bring up the pH of the hydrolysates to 108 with NaOH and carbonate buffer
5
I m l hydrolysate+lml 20% formaldehyde solution pH108
1
shaking for 30 sec
I
irradiation for 15sec
fluorescence cuvettes in the sample chamber
I
lStmeasurement of the fluorescence
60 sec
Zdmeasurement of the fluorescence Figure 5.
Schematic presentation of the procedure
(that is after the addition of the formaldehyde solution). During the following 15 s, the spectrofluorometer was allowed to stabilize. The fluorescence was plotted for the first time at exactly 45 s, and for the second time at exactly 105 s after the start of the reaction. The differences of the fluorescence could be calculated between t = 45 s and t = 105 s. Three hydrolysates were made from each sample. The measurements were repeated three times and the mean values of the fluorescence increases were taken for the determination of tryptophan. Figure 5 shows a scheme of the procedure. The calibration curves were made with freshly prepared tryptophan solutions in carbonate buffer of pH 10.8. The stock solution contained 100 nmol tryptophan/mL. A dilution series was made with 50, 25, 10, and 5 nmol tryptophanlml. The measurements were conducted as described above. Each point on the calibration curve is the mean of three determinations. The comparative measurements were made with the norharman method described by Denckla and Dewey (5) modified by Steinhart and Kirchgessner (18).
RESULTS AND DISCUSSION The calibration curves made at p H 4.0 (acetate buffer) and a t p H 10.8 (carbonate buffer) were both linear in the range from 0 to 100 nmol tryptophan/mL. But the differences in the fluorescence were about 25% smaller a t p H 4.0 than a t pH 10.8. The results are summarized in Table I. The tryptophan values of most of the samples which were analyzed with the kinetic method were similar t o those which were determined with the norharman method. I t is not possible to decide from the data collected by Friedman and Finley (19,
ANALYTICAL CHEMISTRY, VOL. 51, NO. 7, JUNE 1979
1015
Table I . Tryptophan Contents of Some Foods and Feedstuffsa
dry matter
fat in dry matter, %
sample weight for hydrolysis, mg
norharman method, % trp in fat free fresh dry matter substance
kinetic method % trp in fat free fresh dry matter substance
feedstuffs field bean casein feather meal fish meal oat bran oat flakes alkane yeast skim milk powder corn IC1 protein soybean meal manioc meal
92.08 93.30 94.64 93.67 94.48 92.16 94.83 93.88 91.99 94.29 94.33 92.09
1.14 0 3.51 3.26 2.96 6.7 3 0 0.03 4.31 0.42 1.34 0.67
297 298 29 9 300 300 298 299 29 9 298 297 300 297
0.37 1.67 0.94 1.01 0.14 0.35 0.81 0.71 0.11 1.16 0.98 0.08
0.32 1.55 0.85 0.9 1 0.13 0.29 0.77 0.67 0.09 1.12 0.91 0.07
0.36 1.65 0.96 1.07
0.32 1.54 0.87 0.97
0.40 0.28 0.73 0.11 0.39 1.01 0.07
0.34 0.27 0.69 0.09 0.37 0.95 0.06
9.89 7.91 7.62 12.80
2.36 4.61 4.72 2.87
300 296 301 299
0.12 0.15 0.40 0.30
0.01 0.01 0.03 0.04
0.09 0.11 0.38 0.35
0.01 0.01 0.03 0.04
92.46 60.55 96.05
1.15 0.45 4.15
299 300 300
0.18 0.13 0.11
0.17 0.08 0.10
0.18 0.11 0.09
0.17 0.07 0.08
26.61 53.38 26.99 22.98 25.13 29.63 24.39 28.92 26.24 29.01 30.19 26.68 29.94
39.70 38.58 11.73 9.73 10.61 6.63 23.03 9.28 7.38 6.76 36.21 13.37 32.47
301 299 315 330 315 300 300 299 300 300 301 298 300
1.46 1.19 1.27 1.19 1.39 1.15 0.98 0.95 1.15 0.67 1.15 1.06 1.07
0.24 0.38 0.30 0.25 0.31 0.32 0.18 0.25 0.28 0.18 0.22 0.24 0.22
1.45 1.20 1.31 1.11 1.36 1.17 1.24 1.20 1.39 0.70 1.35 0.99 1.20
0.19 0.39 0.30 0.23 0.31 0.32 0.23 0.39 0.32 0.16 0.26 0.22 0.24
26.98 25.55 41.77 44.20
14.60 1.08 37.86 35.13
309 314 316 328
0.95 1.02 1.00 0.82
0.22 0.25 0.29 0.23
1.11 1.13 1.15 0.81
0.25 0.29 0.30 0.23
-
-
vegetables herb tomato spinach cauli flow er plant products noodles bread zein animal products eggs cheese beef belly beef kidney pig tenderloin pig liver pig heart roe belly + neck hind leg rabbit liver broiler broiler liver broiler heart fish trout codfish tunny sardine a
The sample weights and the
%
tryptophan values are average values from three hydrolysis.
Table 11. Reproducibility of the Egg Tryptophan Values Measured with the Kinetic Methoda sample weight, mg 1. hydrolysis 2. hydrolysis 3. hydrolysis
300.37 300.63 301.13
F = fluorescence; t , = 1st value; t ,
1st measurement F t, F t, AF
F t,
F t2
AF
F t,
F t2
AF
448 489 483
475 486 482
51 2 526 522
37 40 40
476 486 483
514 525 523
38 39 40
=
486 528 521
38 39 38
2nd measurement
3rd measurement
2nd value.
supplement) whether the values in Table I agree well with the data reported in the literature because the variation of these data is wide. Some tryptophan values, especially those from animal products and fish were, however, higher when they were determined with the kinetic method rather than with the norharman method. Some reflections suggest, however, that the norharman values might be too low. This method requires more steps than the kinetic method so that pipetting and dilution errors may not be excluded. Above all, the synthesis of norharman
is conducted under strong acidic conditions in the presence of oxidizing substances (FeC13,air) at high temperatures. One hour is needed until norharman is quantitatively formed from tryptophan. Because tryptophan is sensitive to oxidizing substances and to heat, it is possible that part of the tryptophan is destroyed or reacts with other substances of the hydrolysate, so that it may not be fully recovered with the norharman method. It is also possible that the sensitivity to oxidation is higher in the heterogenous mixture of the calibration reactions.
1016
ANALYTICAL CHEMISTRY, VOL. 51, NO. 7, JUNE 1979
The hydrolysates were at once brought up to p H 10.8 when the kinetic method was used. At this p H value, tryptophan is not responsive to oxidation. The reaction time was only 105 s. The fluorescence background, which is constant during the reaction, did not disturb the determination. As was mentioned above, indole compounds disturbed the determination of tryptophan with both the kinetic method and the norharman method. The nucleic bases which may be present in the alkaline hydrolysates were examined with regard to their fluorescence properties under the conditions used. The change of the fluorescence during the reaction could be recognized only when guanine was investigated. The fluorescence, however, did not increase but it decreased strongly (see Figure 4). This is probably an explanation for the different tryptophan values of alkane yeast and IC1 protein compared with the other proteins. The values of these samples were lower when the kinetic method was used because these substances had higher guanine contents of dry weight (approximately 1%) than other samples. It must be shown in further work whether there are still other substances which have a similar negative effect as guanine. On the other hand, there are some major advantages to the kinetic method when compared with other methods. The kinetic method is time-saving compared with colorimetric or fluorometric methods because tryptophan may be determined directly from the alkaline hydrolysates. There are also fewer preparatory steps so that the danger of tryptophan losses is smaller. The sensitivity of the method is relatively high because fluorometric methods are in general more sensitive than the often used colorimetric procedures. Tryptophan may be measured very readily a t a concentration of 5 nmol/mL (=1 fig/mL). The limit of detection is 2 nmol tryptophan/mL (~0.4 pg/mL). At lower concentrations, the differences in the fluorescence often become so small that superpositions of the noise of the apparatus may not be excluded. Amplification of the signal does not prevent this effect. When the kinetic method is used to determine tryptophan from alkaline hydrolysates, it is important to pay particular attention to two points so that reproducible results can be obtained. The fluorescence properties depend strongly on the p H conditions in the reaction mixture as can be seen from the experiments mentioned above. Therefore, an exact ad-
justment of the pH value, preferably with a pH-stat apparatus, is indispensable. Moreover, the exact timing of the reaction is the other important factor for obtaining optimal results. This is valid for all determinations based on kinetic experiments. Concerning the reproducibility of the method, some results from the tryptophan analysis of eggs are summarized in Table 11. The selection of the samples was a t random. It can be seen from this table that the deviations were no higher than 5% in each hydrolysate. The fluorescence properties a t the reaction of tryptophan with formaldehyde depend strongly on the reaction conditions because the overall reaction is very complex. This may be the explanation for having difficulties with the method of Guilbault and Froehlich ( 3 , 4 )when tryptophan is measured in hydrolysates of foods and feedstuffs.
LITERATURE CITED Spies, J . R.; Chambers, D . C. Anal. Chem. 1948, 20,30-39. Opienska-Blauth, J.; Charezinski, M.; Berbec, H.Anal. Biochem. 1963, 6 ,69-76. Guilbault. G. G.; Froehlich, P. M. Clin. Chem. ( Winston-Salem, N . C . ) 1973, 19, 1112-1113. Guilbault. G. G.; Froehilch, P. M. Clin. Chem. ( Winston-Salem. N . C . ) 1974, 20,812-815. Denckia, W. D.;Dewey, H. K. J . Lab. Ciin. Med. 1967, 69,160-169. Larsson. L . 4 : Sundler, F.; Hakanson, R. J. Histochem. Cytochem. 1975, 23.873-881. Larsson. L.-I.: Sundler. F.: Hakanson. R. J . Chromatour. 1976. 177. 355-363. Steinhart, H. Landwirtsch. Forsch. 1979, 32,63-73. Steinhart, H. 2. Tierphysioi., Tierernahr. u . Fuftermittelkd. 1978. 4 1 . 48-56. Pailthorpe. M. T.: Bonjour. J . P.; Nicholls, C. H. Photochem. Photobiol. 1973, 17, 209-223. Udenfriend, S. "Fluorescence Assay in Biology and Medicine", Academic Press: New York, 1962; Vol. 1. Udenfriend,S. "Fluorescence Assay in Biology and Medicine", Academic Press: New York, 1969: Vol. 2. Pailthorpe, M. T.; Nicholls, C. H.photochem. photobiol. 1972, 15,465-477. Templer, H.; Thistlethwaite, P. J . Photochem. Phot06iol. 1978, 23,79-85. Bent, D. V.: Hayon, E. J . A m . Chem. Soc. 1975, 97, 2612-2619. McCormick. J. P.; Fischer, J . R . ; Pachlatko, J. P. Science 1976, 791. 468-469. Wairant, P.; Santus, R. Photochem. Photobiol. 1973, 19. 41 1-417. Steinhart. H.: Kirchaessner. M. 2.T?emhysbl.. Tieremcihr. U. FuitermMekd. . . 1978. 4 1 , 18-29: Friedman, M.; Finley, J. W. J . Agric. FoodChem. 1971, 19,626-631.
-
RECEIVED for review October 20, 1978. Accepted February 5, 1979. This work was supported by Deutsche Forschungsgemeinschaft.
Determination of Bis(chloromethy1)ether in Air Leo G. J. v.d. Ven and Arnold Venema" Akzo Research b. v., Corporate Research Deparlment A m h e m , P. 0. Box 60, The Netherlands
A gas chromatographic method is described for the determination of bis(chloromethy1)ether (BCME) in air. The BCME is converted into bis( p-phenylphenoxymethyl) ether (BPPME) after enrichment on a Tenax-GC trap. The chromatographic system consists of a solid sample injector, a glass capillary column, and a flame ionization detector. BCME levels down to 0.1 ppb (v/v) were detected in the atmosphere of Industrial plants.
Although several analytical methods for the determination of low levels of bis(chloromethy1)ether (BCME) in air have 0003-2700/79/0351-1016501.00/0
been published, there still is a need for a method of analysis that is simple, reliable and not too expensive. Two different analytical approaches can be distinguished in the published methods: (1)direct determination of BCME or (2) determination after conversion of BCME into a derivative. Almost all direct BCME determinations use a concentration step on a porous polymer prior to the analysis; after heat desorption, the BCME content is measured by gas chromatography, mass spectrometry, or a combination of the two. A serious drawback of all the direct analysis methods is the necessity to work with the carcinogenic BCME ( I ) , for instance in calibration solutions. Moreover, on account of the instability of BCME to hydrolysis ( Z ) , the trapped sample has Q 1979 American Chemical Society
