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 deter-
mination 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
ANALYTICAL CHEMISTRY, VOL. 51, NO. 7 , JUNE 1979 t o be analyzed as quickly as possible after sampling. As BCME is a rather low boiling compound, (bp 104 "C) interference from other air components in the gas chromatogram may be expected so the separation and/or detection system has t o be very specific. Mass spectrometry without gas chromatography ( 3 , 4 )does not meet this specificity requirement, while gas chromatography alone necessitates column switching ( 5 ) t o obtain the required specificity. T h e "expensive" GC-MS method (6, 7) using capillary columns and a high resolution mass spectrometer seems t o fulfil the demand of specificity, especially when selected ion monitoring is used, although even in such a system an interfering compound has been reported
1017
Gloss wool
TENAX-
GC
(8).
Determination of BCME after derivatization eliminates most of the disadvantages of the direct analysis, i.e., it puts an end t o the need to use BCME during routine analysis, the specificity requirements can be met more easily by the proper choice of the reagent and the conversion into a stable derivative allows the analysis to be repeatedly performed a t any time after sampling. A serious disadvantage of the derivatization method, however, is the deterioration of the detection limit by sample dilution. Moreover, the derivatization method published by Kallos (9) and Solomon (IO)suffers from two weak points: the formation of several derivatives from BCME due t o the presence of two nucleophiles (for example the methoxy- and 2,4,6-trichlorophenoxy anion in the case of Ref. 9) and the use of an impinger as a sampling device which is impractical under process conditions. Moreover, sucking air through the reagent solution results in the formation of interfering compounds while sampling in a humid atmosphere results in deterioration of the reagent solution. In this paper a gas chromatographic method for the determination of BCME is described which combines the concentration of BCME on a porous polymer (Tenax-GC) with a derivatization with p-phenylphenolate which converts BCME into (Ph)20CH20CH20(Ph)2, thus permitting flame ionization detection on the sub-ppb level. T h e required selectivity and sensitivity are obtained by applying a glass capillary GC column in combination with a solid sample injector. This analytical method has been used successfully for more than one year under different process conditions. EXPERIMENTAL Reagents. Bis(chloromethy1)ether was obtained from Fluka, p-phenylphenol from Merck. The sodium hydride (60% in oil) was obtained from Fluka; before use it was carefully freed from oil by repeated washing with sodium-dried n-hexane (nanograde). n-Hexane was obtained from Mallinckrodt. Dimethylformamide, distilled before use in glass and dried over mol. sieves 4 A, was obtained from Merck, sodium hydroxide was obtained from EKA (Sweden). Tenax-GC, 3 5 4 0 mesh, was provided by ow company; the polymer was extracted with methanol for 14 h in a Soxhlet apparatus before use. Apparatus. A Varian 3740 gas chromatograph equipped with a flame ionization detector was used. A solid sample injector (11) was mounted in the standard 1/4-inch injection system; this injector was supplied by Koppe, Best, The Netherlands. Integration of the GC signals was performed with an SP 4000 system (Spectra Physics). Membrane pumps were used for air sampling (Miniport MV 7918, Verder, Vleuten, The Netherlands). Sampling Trap. Air sampling was performed with Tenax-GC, 3 5 4 0 mesh. The traps consist of Pyrex glass tubing, 18 cm long, 6-mm i.d., filled with 400 mg adsorbent. Before use, the filled traps were washed with 5 mL of DMF and 5 mL of methanol, after which they were conditionned under pure nitrogen for 3 h a t 270 "C. To facilitate the solvent elution procedure, the traps were provided with a ground glass joint which fits into a 10-mL measuring glass (see Figure 1). Derivatization Reagent. The derivatization reagent was prepared by mixing 2.5 g of sodium p-phenylphenoxide with 100
Glass wool
Shallow saw cut
Flgure 1. Tenax adsorption trap
mL of DMF. The sodium salt was prepared by careful addition of 8.6 g of oil-free sodium hydride to a solution of 60 g of pphenylphenol in 700 mL of DMF. The reaction mixture was kept under nitrogen and stirred for 3 h a t room temperature. The sodium salt was isolated by filtration at the pump, washed with dry 1,4-dioxane and n-hexane (nanograde) and dried by suction. The p-phenylphenoxide must be kept under nitrogen during all manipulations. The dried salt was stored in a stoppered flask which was placed in a desiccator filled with P,05. (Caution: Sodium hydride reacts violently with water; see also Ref. 12). Whereas the sodium p-phenylphenoxide will keep for more than a year when stored away from moisture, the reagent slurry in DMF has to be prepared every day to prevent decomposition. Chromatographic Conditions. A 25-m long glass capillary column, i.d. 0.29 mm, was used. The soda lime glass (Schott AR) column was deactivated with benzyltriphenylphosphonium chloride and coated with SE 54 by means of the dynamic mercury drop method (13). A 3.4% solution of SE 54 in toluene gives a film thickness of m when a mercury drop length of 16 cm and a coating 0.3 X velocity of 30 cm/min are used. The column oven was operated isothermally at a temperature which results in a retention time for the BCME derivative of about 20 min. A new capillary column requires about 230 "C which gradually diminishes to 215 OC over the period of 3 months. To increase the life of the capillary column, the oven temperature was set to 100 "C whenever it was not used. The temperature of the sample injection zone was 260 "C; that of the detector, 270 "C. The linear flow rate of the carrier gas, nitrogen, was 20 cm/s. To the FID, a nitrogen make-up flow of 30 mL/min was supplied. The solid sample injector was slightly modified; the rubber septum was replaced by a Teflon stopcock to prevent small septum particles from getting into the hot injector zone or column during the injection. Analytical Procedure. Air, 10-30 L is sucked through the Tenax trap at a flow rate between 0.1-1.2 L/min depending on the expected concentration and the time interval of interest. When air with a high humidity is sampled, the trapped BCME has to be derivatized as soon as possible. The derivatization slurry, 2 mL is pipetted into a 10-mL measuring glass. The trap is placed on this glass and eluted with about 2 X 1.5 mL DMF. The residual solvent in the trap is blown into the reagent with pressurized gas or a pipetting balloon. The trap is removed and the flask stoppered. After 15 minutes at room temperature, the liquid volume in the measuring glass is made
ANALYTICAL CHEMISTRY, VOL. 51, NO. 7 , JUNE 1979
1018
'\
recovery
50
01
ref 2
L air
io io io i o i o 7c i c ,io Flgure 2. Breakthrough volume of BCME (sample: 290 ng) on Tenax-GC (500 mg, 35-60 mesh, air flow 1 L/min) ib
t
I
x
BCME recovery , IO,.\
\
50,
L O % Ret Humidity 8 0 % .. .
'\
1I
20 minutes Figure 4.
Calibration with external standard, injected amount 0.54 ng
BPPME 04 0
1
-days
3
4
5
I
Influence of humidity and time interval between sampling and derivatization on BCME recovery Figure 3.
up to 10 mL with 2 M sodium hydroxide solution. The reaction mixture is cooled to room temperature and 1 mL of n-hexane is added. After vigorous shaking and phase separation, about 0.7 mL of the hexane solution is removed from the reaction mixture and pipetted into a screw cap reaction vial of 1 mL. Then 1 to 5 X 1 pL of the hexane solution is applied to the glass needle of the solid sample injector and injected after evaporation of the solvent and other volatile compounds. The BCME content is calculated from the peak areas of sample and external calibrations with known amounts of BPPME.
RESULTS AND DISCUSSION As shown by Pellizzari (2), water adsorbed on a porous polymer hydrolyzes trapped BCME to a considerable extent. Therefore Tenax-GC, a polymer with a very low water retention (14), was used as adsorbent. T h e "break-through" volume of the trap was determined by measuring the BCME recovery from spiked traps as a function of the dry air volume sucked through the adsorbent. As shown in Figure 2, a t least 80 L of air/g Tenax can be trapped without the BCME recovery being affected. No significant change in adsorption efficiency was observed after the regeneration of used traps (washing with DMF and methanol, followed by a heat treatment at 270 "C for 3 h under pure nitrogen). While the trap efficiency is not affected by air humidity (2), the BCME recovery from the trap depends on the humidity of the air and the time between sampling and derivatization as shown in Figure 3 (see also Ref. 2). When air with a high humidity must be sampled, accurate results can be obtained only when the derivatization has been performed within a few hours after sampling, as shown in this figure. The derivatization procedure was checked by adding known amounts of BCME to the derivatization reagent. The reaction of BCME with the p-phenylphenoxy anion in DMF was shown to be complete within 15 min a t 20 "C. Owing to the choice of solvent only the bis derivative is formed. The p-phenylphenoxy anion is used as a nucleophile to obtain a high boiling
Table I. Influence of BCME Amount on the Recovery BCME spiked on Tenax trap in ng
BCME found (as BPPME)
27
84
in% of the amount added
44
78
108
86
168 24 5 313 580
80 04 83 80
-u a82.1 t 2.9
derivative, thus preventing interferences during the GC analysis with most of the common air pollutants in industrial plants. The high carbon content of the BPPME allows the use of the stable and reliable flame ionization detector. The conversion of BCME into B P P M E is 96 f 5% for amounts of 10 ng up to 600 ng BCME. The response factor of the derivative (BPPME) was determined with BPPME prepared in a large-scale experiment. Standard solutions of B P P M E in n-hexane turned out to be stable for more than one year, thus permitting an easy and safe external standard calibration for routine GC analyses and making the use of BCME superfluous. A chromatogram of a calibration is shown in Figure 4. The efficiency of the total analytical procedure was investigated by adding known amounts of BCME to Tenax traps which were analyzed by the standard procedure immediately after 20 L of dry nitrogen had been sucked through them. The results obtained for different BCME levels are reported in Table I. An average recovery of 82.1 f 2.9% was obtained. The loss of about 18% may be due to irreversible adsorption or hydrolysis of the BCME by the adsorbent or solvent. The same recovery was observed even when 30 L of air of 100% humidity was sampled (Le., 82%).
ANALYTICAL CHEMISTRY, VOL. 51, NO. 7, JUNE 1979
1019
lower limit of detection of 0.1 ppb can be achieved, especially when the sample amount is increased from 20 to 30 L. Application of this BCME analysis method under several process conditions has shown that a good accuracy, sensitivity, and selectivity can be obtained with relatively simple equipment. Whenever strong interferences are present with the same retention time as BPPME, it is possible to prepare a derivative with a differently substituted phenol. In our work, we have never been obliged to do this. An increase of sensitivity and selectivity can be obtained by applying single ion mass spectrometry to the B P P M E derivative (M+-,m / e 382: int. 25%; m / e 352: int. 80%; m l e 183: int. 100%). By using this method, the analysis costs increase rapidly, but in comparison with the direct BCME GC-MS methods all the advantages of the derivatization mentioned before are preserved.
LITERATURE CITED
I
20 minutes
I
I
20 minutes
Flgure 5. Chromatograms of air sampled inside a chemical plant: (a) air sample volume 20 L, sampling rate 1 L/min, injected volume 2 pL, BPPME signal corresponds with 0.25 ppb (v/v) BCME; (b) ibid., BPPME signal corresponds with 0.8 ppb (v/v) BCME
T h e variation coefficient of the analytical method, determined under process conditions, was found to be about 20% ( n = 14) for a BCME level of 0.3 ppb (v/v) and about 6% ( n = 12) for 1.0 ppb (v/v). Typical chromatograms obtained in a chemical plant are shown in Figure 5 . From this figure it will be clear that a
(1) Nelson, N.: Ecotoxicol. Environ. Safety 1977, 1 , 289. (2) Pellizzari, E. D.; Bunch, J. E.: Burkley, R. E.; McRae, J. Anal. Lett. 1976, 9 , 45. (3) Collier, L. Environ. Sci. Techno/. 1972, 6 , 930. Sonnabend, L. F. A m . Ind. Hyg. Assoc. (4) Tou, L. C.; Westover, L. 6.: J . 1975, 36. 374. ( 5 ) Frankel, L. S.; Black, R. F. Anal. Chem. 1976, 4 8 , 732. (6) Evans, K . P.; Mathias, A.; Melior, N.: Sillvester, R.; Williams, A. E. Anal. Chem. 1975, 47, 821. (7) Shadoff, L. A,: Kallos, G. J.: Woods, J. S. Anal. Chem. 1973, 45, 2341. (8) Schuiting, F. L.: Wlls, E . R. J. Anal. Chem. 1977, 49, 2366. (9) Salomon, R. A . ; Kailos, G. J. Anal. Chem. 1975, 4 7 , 955. (10) Salomon. R. U S Patent 3 944 389. (11) Berg, P. M. J. v.d.: Cox, Th. P. H. Chromatographia 1972, 5 , 301. (12) "Reagents for Organic Synthesis": John Wiiey & Sons: New York, 1967: p 1075. (13) Schomburg, G.; Husmann, H. Chromatographia 1975, 8 , 517. (14) Janak, J.: RuzickovB, J.: NovBk, J. J . Chromatogr. 1974, 99, 689.
RECEIVED for review November 29,1978. Accepted February 26, 1979.
Solubility of 4-Methyl-2-pentanone in Aqueous Phase of Various Salt Concentrations Hiromitsu Kanai, Veronica Inouye, Reginald Goo, and Helen Wakatsuki Chemistry Section, Laboratories Br., Hawaii State D e p a ~ m e n tof Health, Honolulu, Hawaii, 968 73
The solubility of MIBK in water of various salt concentrations was studied by GLC. Although there is an approximately 30% decrease in the volume of MIBK dissolved in typical distilled deionized and seawater samples, this volume effect on the lead analyte signals was not observed in atomic absorption spectrometry.
4-Methyl-2-pentanone (MIBK) has been widely used as the extracting solvent in the complexation-extraction preconcentration technique using ammonium pyrollidine dithiocarbamate (APDC) as the ligand in the atomic absorption spectrophotometric (AAS) analysis of heavy metals in water and wastewater (1). Although it has favorable combustion characteristics, its partial solubility in aqueous phase has been often stated as its drawback. 0003-2700/79/0351-1019$01 .OO/O
Several articles have been published on this subject. Brooks and his co-workers ( 2 ) studied the amount of MIBK that dissolves in seawater whose salinity was 31%~).Munro ( 3 ) determined the MIBK solubility in acid solutions. Recently, Everson and Parker ( 4 , 5 )have done extensive studies on this subject. They showed how MIBK solubility in the aqueous phase varies as a function of ammonium citrate concentrations, and through radioactivity studies showed that lead is mostly in the organic phase and its concentration in the aqueous phase is not significant. Although these articles have characterized many properties of the APDC-MIBK complexation-extraction technique in AAS analysis, there are a few questions which are pertinent to an environmental laboratory such as ours conducting heavy metal analysis of water samples whose salinity varies widely. Instead of performing analysis in highly buffered solution such as the ammonium citrate and model condition, it would be C 1979 American Chemical Society
