The exact mechanism of this reaction has not yet been determined and this work is being further pursued. Analysis of Mixtures. Various mixtures of Methyl Orange, 4,4'-azodianiline, were prepared and analyzed. A typical chromatogram of the mixture 4,4'-azodianiline and p-phenylazoaniline is shown in Figure 5. The pure sample, p-phenylazoaniline, is reduced to p-phenylenediamine and aniline, and by comparing the peak areas for the pure compound to those from the mixture, the difference in peak areas is due to the quantity of 4,4'-azodianiline in the mixture. The results of this study are given in Table VI. Thus, not only can the total azo compound be determined for a mixture, but also the qualitative and quantitative analysis can be ascertained.
CONCLUSIONS
A method is presented in which within one piece of apparatus, nonvolatile azo, nitro, and sulfonate compounds can be determined. By use of the carbohydrazide reduction and gas chromatographic analysis, one can handle a variety of compounds from the easily reactive azo bond to the stubborn reduction of p-nitroanilines. The analysis is not limited to conditions of the solution reduction using hydrazine. The analysis is rapid and specific, and it enables resolution of mixtures of azo and nitro compounds. Received for review April 13, 1973. Accepted June 29, 1973. This work was supported by National Science Foundation Grant No. G P 28054.
Determination of Bis(chloromethy1) Ether in Air by Gas Chromatography-Mass Spectrometry L. A. Shadoff, G. J. Kallos, and J. S. Woods Analytical Laboratories, The Dow Chemical Company, Midland, Mich.
Bis(chloromethy1) ether (bisCME) may be determined at the one part per billion level in air by concentrating the organics on a retentive substrate (Chromosorb 101) with subsequent analysis by gas chromatography-mass spectrometery. By monitoring CzH40CI+, the most intense ion in the mass spectrum of bisCME, during elution of the trapped organics, an extremely specific analysis is performed. For the signal to be assigned to bisCME three things must occur simultaneously: the mass must be correct, the retention time must be correct, and the observed chlorine isotope ratio must be correct. It has also been shown that bisCME may be quantitatively trapped and retained for extended periods of time and determined within 10Y0 accuracy.
Bis(chloromethy1) ether (bisCME), a possible impurity in the chloromethylating reagent chloromethyl-methyl ether, has recently been reported to be a carcinogen when present a t low concentration in the air (1-5). To ensure that the environment in production facilities utilizing this reagent contains no hazard from bisCME, a n analytical procedure was needed to monitor the atmosphere a t the 1-vppb (volume part per billion) level. A recent report by Collier (6) employs trapping the organics from air onto Porapak Q with analysis of the organics by high resolution mass spectrometry. We present here recovery data for this trapping method and use a method (1) B.
L. Van Duuren, B. M . Goldschrnidt, L. Langseth, et ai., Arch. En-
Health, 16, 472 (1968). Gargus. W. H. Reese. J r . , and H. A . Rutter, Toxicoi. Appi. Pharrnacol.. 15,92 (1969). (3) K. J . Leong, H. N. MacFarland, and W. H . Reese, J r . , Arch. Environ. Health. 22, 663 (1971 ) (4) B. L. Van Duuren, A . Sinak, B. M . Goldschrnidt, C. Katz. and S. Melchionne. J. Nat. Cancer lnst.. 43, 481 (1969) (5) B. L . Van Duuren, Ann, N. Y . Acad. Sci.. 163, 633 (1969). (6) L . Collier, Environ. Sci. Technoi. 6 , 930 (1972). viron (2) J . L .
of analysis not subject to the possible interference encountered using mass spectrometry alone. The analytical method chosen is gas chromatographymass spectrometry (GC-MS). This technique has extremely high specificity for bisCME, high sensitivity, and rapidity of analysis. Specificity is needed because of the diverse substances encountered a t the part-per-billion level in air. Positive identification of bisCME is required to avoid unnecessary evacuation of personnel and possible production shut-down. High sensitivity is required since 1 vppb corresponds to 4.7 ng/l. of air as calculated from the Ideal Gas Law. Rapidity of analysis generates immediate knowledge of hazards.
EXPERIMENTAL Sampling Tubes. These are prepared by packing y,-in. 0.d. X 2-in. long stainless steel tubing with 1Yz inches of SO/lOO mesh Chromosorb 101 (Johns-Manville) using silanized glass wool plugs in the ends. These are conditioned a t 200 "C and 10 cm3/min Nz (or He) flow overnight. They are then cooled under flow and capped immediately upon removal. An identification number is scribed on one of the Swagelok (Crawford Fitting Co.) nuts. S o preliminary extraction is necessary as reported for Porapak Q ( 6 ) . Sampling Method. Spot samples may be obtained by attaching the sampling tube to a manual syringe pump made from a 100 cm3 glass tip hypodermic syringe. Continuous sampling at fixed points is accomplished by attaching a rotary vacuum pump to a throttling valve and manometer. Each sampling tube is calibrated such that the pressure drop corresponding to 1 1. per hour is determined. The pressure drop is then adjusted with the throttling valve to establish the flow. We have established the convention t h a t the sample is drawn onto the end of the sampling tube with the numbered Swagelok nut. Gas Chromatography Column. Four feet of Yd-in. or Yg-in. 0.d. stainless steel tubing is rinsed internally with water, acetone, and methylene chloride and air dried. The cleaned tubing is then packed with SO/lOO mesh Chromosorb 101 (Johns-Manville) and conditioned overnight at 200 "C and 10-30 cm3/min Nz (or He) flow. Gas Standards of bisCME in nitrogen may be prepared by partially filling a 5-1. Saran brand (Trademark of The now Chemical Company abroad) plastic film gas bag with nitrogen
ANALYTICAL CHEMISTRY, VOL. 45, NO. 1 4 , DECEMBER 1973
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3
4
5
6
7
time (rnin.1
Figure 1. Signal for bisCME from LKB9000 and injecting the requisite quantity of liquid bisCME (density = 1.32) through the wall. The hole is immediately sealed with adhesive tape and the bag kneaded for 5 minutes. The remainder of the nitrogen is then added and the bag kneaded for another 5 minutes. This procedure prepared part-per-million standards (eg., 0.37 11 of liquid bisCME yields a 21-vppm standard). Partper-billion gas standards may be prepared from the above standards by dilution into another Saran brand plastic film bag. Thorough kneading is essential to the preparation of reproducible standards. Polyethylene bags are not suitable since bisCME appears to permeate the walls. GC-MS Equipment and Operating Conditions. An LKB 9000A mass spectrometer equipped with an Accelerating Voltage Alternator was set as follows: Column temp, 130 "C; flash heater, off; source slit, 0.20; exit slit, 0.15; sensitivity, 200; trap current, 60 PA; source temp, 270 "C; separator temp, 250 "C; and chart speed, 1 in./min. The sampling tubes are attached to the inlet of the analytical column uia a y,-in. Swagelok union with the numbered nut next to the analytical column. The standard LKB injection port is then attached to the other end of the sampling tube and flow reestablished. This operation is performed as rapidly as possible to minimize the time when flow through the column is stopped. The sample tube is wrapped with glass fiber insulated heat tape and heated as rapidly as possible to 150 "C and maintained at this temperature for 4 min. A retention time of 6 f 0.1 min is obtained for bisCME (see Figure 1).The sampling tubes are capped immediately after removal and are ready for re-use. To check the response, the magnet ( m / e ) adjustment, and the accelerating voltage offset, 0.5 cm3 of a 21-vppm bisCME standard in nitrogen may be injected through the standard septum onto the sampling tube and analyzed as above. This is routinely done after every three samples to ensure accuracy. A CEC (now DuPont Analytical Instruments Div.) Model 21491 mass spectrometer equipped with a repetitive scan attachment is also being used in conjunction with a H-P 5750 gas chromatograph via a Watson-Biemann (7) helium separator. The spectrometer was modified by adding a follower amplifier with a long time constant to the output of the electron multiplier to increase the gain and signal to noise ratio. The sampling tube is attached to the gas chromatograph uia an adaptor to the septum nut. A Swagelok y,-in. Tee fitting is installed on the other end of the sampling tube with a silicone rubber septum placed so that standards may be injected onto the sampling tube. The carrier gas inlet to the injection port is disconnected and attached to the remaining branch of the Tee thus establishing flow through the sampling tube and into the analytical column. The sampling tube is wrapped with heat tape and heated to 150 "C for 4 min driving the organic components onto the analytical column which is at ambient temperature. The column is then programmed to 130 "C at 30 "C/min yielding a retention time for bisCME of 8.5 f 0.2 min. The mass spectrometer is set as follows: multiplier, 3; filament, 9.5; chart speed, 0.8 in./sec; and mass and sweep set to cover m / e 79 to 81. ( 7 ) J. T.
2342
Watson and K. Biemann, Anal. Chem.. 36, 1135 (1964)
Table I. Response for m / e 79 for Various bisCME Standards Standard 1 PI 0.25 crn3 0.5 c m 3
0 . 5 I. 0.1 cm3 0.5 c m 3 1 crn3
0.5 cmJ
10 ng/jd in CH& 21 vpprn in N2 21 vpprn in N2 followed by 1 I. air 10 vpprn in N2 5 7 vpprn in N2 5 7 vpprn in N2 57 vpprn in N2 21 vpprn in N2 after 3 days storage
Equivalent from 1 I . , vppb
2.1 5.3
10.5 5 5.7 28.5 57 10.5
Response, mm 120 f 1 0 280 f 20 570 310 320 2100 4100
f 20 f 20 f 20 f 200 f 120
560 f 20
DISCUSSION The specificity of GC-MS for bisCME is achieved in three ways. Gas chromatographic separation from other substances present in the air yields a retention time characteristic for bisCME. However gas chromatography alone is not sufficiently specific on a single analysis to positively identify the signal as bisCME in a complex mixture. Use of a mass spectrometer detector yields the other two degrees of specificity. The mass spectrum of bisCME shown in Figure 2 has the most intense ion a t m / e 79 and 81 corresponding to loss of a chlorine atom from the molecular ion. If the mass spectrometer is set to continuously monitor these ions during chromatographic elution of an air sample, three things must simultaneously occur for the signal to be assigned to bisCME: The retention time must be correct; response must occur for both m / e 79 and m/e 81; and the relative signals for the ions must be 3 : l due to the chlorine isotopic abundance. Furthermore, even if other substances elute from the column a t the same time as bisCME, a signal will most likely not be recorded for them a t m / e 79 and 81. This reduces somewhat the separation requirements of the gas chromatographic column and produces a signal which may be uniquely assigned to bisCME with a high degree of confidence. The sensitivity of the GC-MS system was determined by analysis of a 10 ng/gl solutinn of bisCME in methylene chloride. This allows an accurately known weight of bisCME to be delivered onto the column. One microliter of this solution produced a 120-mm deflection for m l e 79 (recorder sensitivity = 2.5 mm/mV). This signal is equivalent to 2.1 vppb of bisCME in a 1-1. air sample (Table I), o r conversely a 1-1. air sample is required to achieve 1vppb sensitivity.
ANALYTICAL CHEMISTRY, VOL. 45, NO. 14, DECEMBER 1973
1
loo
P-
L 0
20
40
CI
60 m / r (amu)
100
I20
Figure 2. Mass spectrum of bisCME
I
Table II. Mass Spectrum of Chloromethyl-Methyl Ether, CICH20CH3
I
II I
I
L
I
.C t i m
II
i;k
I
,
I I
I
\
I'
\
I
\
I I
I
I
b)
-4 I
Figure 3. Signal for bisCME from 21-491 spectrometer
Since this large volume of air may not be directly injected onto a gas chromatographic column, a technique for concentrating the organic components is required. Various trace atmospheric impurities have been determined by trapping on a retentive substrate with subsequent vaporization onto a gas chromatograph (6, 8, 9). One liter of air may be pulled through such a sampling tube to trap the organic components in the air. The sampling tube is then capped and transported to the GC-MS, attached between the injection port and the analytical column, and rapidly heated to desorb the organics which are analyzed for bisCME. The sampling tube must quantitatively absorb the bisCME from the air a t the 1-vppb level. I t must be capable of holding the bisCME for extended periods of time without loss or decomposition since there may be consid(8) J. Novak, V. VaBak, and J. Janak, Anal. Chem., 37,660 (1965). (9) T. A. Bellar, M . F. Brown. and J. E. Sigsby. Jr., Anal. Chem., 35, 1924 (1963).
m/e
Rel. abundance
mle
Rel. abundance
13 14 15 16
17
0.64 2.87 30.27 0.60 0.17
44 45 46 47 48 49
26 27 28 29 30 31 32 33
0.03 0.09 2.37 32.15 4.48 2.01 1.03 0.04
50 51 52 53
1.10 100.00 2.38 0.97 1.37 20.31 1.54 7.99 0.36 0.49
62 63
0.01 0.51
65
0.19
35 36 37 38 39 40 41 42 43
1.54 1.32 0.72 0.43 0.09 0.06 0.07 0.21 0.46
77 78 79 80 81 82 83
0.03 0.09 0.14 0.97 0.14 0.33 0.02
erable time between collection and analysis, and it must quantitatively deliver all the bisCME when heated. To evaluate these criteria, gas standards of bisCME in nitrogen were prepared a t the 57-vppm, 21-vppm, and 10vppb levels. T o test for retentionand subsequent delivery of bisCME, a 0.25-cm3 sample of the 21-vppm standard was injected onto the sample tube a t ambient temperature and desorbed by heating to yield 280 f 20 mm of deflection for m l e 79 (Table I) in good agreement with the response for bisCME in solution. This confirms the ability to trap and deliver bisCME quantitatively a t the nanogram level. To determine whether 1 1. of air would sweep the bisCME completely through the short sampling tube, 0.5 cm3 of the 21-vppm standard was placed on the tube followed by 1 1. of laboratory room air. The response was 570 f 20 mm (Table I) indicating not only that the bisCME is not swept off by 1 1. of air but that atmospheric moisture has no significant effect. Similarly, 500 cm3 of the 10vppb standard yielded 310 f 20 mm deflection for m l e 79 as expected (Table I). This experiment also indicated the presence of benzene in the air of the laboratory as evidenced by a response for m l e 79 but not m l e 81 a t an earlier retention time than the bisCME as shown in Figure l. This is due to the I3C isotope contribution to the C6H6f ion from benzene and does not constitute any significant interference in the analysis for bisCME. The linearity of response was observed by injections of aliquots of the 57-vppm standard as shown in Table I.
ANALYTICAL CHEMISTRY, VOL. 45, NO. 14, DECEMBER 1973
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The accuracy of the analysis is approximately &lo% of the amount reported, as determined from eight standards run on eight different sampling tubes’over a 16-hour period. Furthermore three-day storage at ambient temperature had no deleterious effects on the recovery (see Table I). The signal obtained from the 21-491 spectrometer is illustrated in Figure 3. Since the spectrometer is repeatedly sweeping m l e 79 to 81, the output is a series of mass peaks forming a n envelope as shown in Figure 3a. Closer examination of each sweep shows that the nominal mass peaks a t mle 79 and 81 are composed of several peaks themselves (Figure 3b). These are due to different formula ions having the same nominal mass. For example mle 79 is composed of three peaks. The low mass peak is Br+ (78.9183), the middle is the desired C~H4035C1+(78.9951) and the high mass is C513CH6+ (79.0503) and &H7+ (79.0548). The resolution required to separate these pairs of ions is 1027, 1431, and 17600. With a spectrometer resolution of 1250 (10% valley), three separate peaks are observed for these ions. With this spectrometer resolution, even greater specificity for bisCME is obtained since only ions with the proper formula are observed. The analysis with either spectrometer is quite rapid. Since only mle 79 and 81 are recorded, only those substances which have those ions in their mass spectrum will
yield a signal. Thus a sample may be analyzed even though the column has not eluted all the components from previous samples. In actual practice, a sample may be run every 15 minutes allowing around-the-clock monitoring of air with samples taken once an hour. An analytical method for bisCME has recently been reported (6) using a similar concentration method and direct analysis by high resolution mass spectrometry. This depends solely on a n identification based on the mass (or formula) of mle 79. A serious interference may be encountered if other substances are present which yield the ion CzH40Cl+. This is a very likely occurrence since chloromethyl-methyl ether has this ion in its mass spectrum (see Table 11) and would be expected to be present in large excess. A physical separation is required to avoid the interference from this and similar substances. The analytical system consisting of a sampling tube containing a retentive substrate for collection of air samples and storage for subsequent GC-MS analysis for bisCME a t the part-per-billion level has proven to meet all the requirements for a specific, sensitive, reliable analysis with a fast turn-around time. Received for review November 2 , 1972. Accepted July 12, 1973.
Mass Chromatographic Analysis of Volatiles Alan C. Lanser, J. 0 . Ernst, W. F. Kwolek, and H. J. Dutton Northern Regional Research Laboratory, Peoria, ///. 6 1604
A gas chromatographic method for determining molecular weights called mass chromatography has particular interest for the lipid chemist. The equipment differs from the familiar dual compensating gas chromatograph in using different carrier gases in each column and in employing the “forgotten ideal” gas density balances as detectors in independent mode. Molecular weights are calculated from detector responses for the same component eluted from identical columns with different carrier gases. An analysis of errors, precision, and accuracy of the method is given. A typical example of application in lipid chemistry illustrates the complementary roles of mass chromatography and mass spectroscopy in compound identification.
Mass chromatography is old in principle ( I , 2 ) but new in application. Renewed interest in this procedure is due largely to the availability of instrumentation which facilitates its conduct (3, 4 ) . Mass chromatography differs from familiar dual compensating column gas chromatography (1) A. Liberti, L. Conti, and V. Crescenzi, Atti Accad. Naz. i i n c e i Rend.. 20, 623 (1956). (2) A. Liberti, L. Conti. and V. Crescenzi, Nature (London). 178, 1067 (1956). (3) D. G . Paul and G. E. Umbreit, Res./Deve/op..21, 18 (1970). ( 4 ) C. E. Bennett, L. W. DiCave, Jr., D. G . Paul, J. A . Wegener, and L. J. Levase, Amer. Lab., May 1971. 2344
in two important aspects: different carrier gases (cgl and C ~ Z )are used in each column; each column effluent is monitored by a gas density balance, the ”forgotten” absolute detector ( 0 , 6 ) , in independent inoncompensating) mode. The response of each detector to an unknown is a function of its concentration and the difference between the molecular weights of the unknown and the carrier gas. Two equations can be written involving the amount and molecular weight of the unknown (MWx) corresponding to the observed detector responses for each column (R,,, and RCg2).These two equations in two unknowns can then be solved algebraically ( 3 , 3 ) :
To determine the instrument constant K , known molecular weights (MW,,d) and measured detector responses (R1 and Rz) are substituted in Equation 1, which has been rearranged to give:
Mass chromatography or molecular weight chromatography is of particular interest to lipid chemists because ( 5 ) A. J. P. Martin and A. T. James, Biochem. J . . 63,138 (1956). (6) C. E. Bennett, L. W. DiCave, Jr., and D. G . Paul, Abstract of paper,
“Gas Density-The Forgotten Ideal Detector,” Pittsburgh Conference in Analyticai Chemistry and Applied Spectroscopy, Cleveland, Ohio, March 1972.
ANALYTICAL CHEMISTRY, VOL. 4 5 , NO. 14, DECEMBER 1973
