chromatographic procedures are sensitive enough to analyze directly even extremely dilute solutions. We checked the linearity of our systems by making analyses a t several solute concentrations. In most cases we injected an amount of compound necessary to give a reasonable response, and then we injected exactly half that amount. Linearity was considered satisfactory if the second response was half the first. Decomposition or reaction may be recognized by changes in p-value over a period of time. If a compound decomposes during gas chromatography, results cannot be considered reliable. The determination of p-values does not usually depend upon the injection technique simply because the analyst uses the same syringe, the same solution (except for amount of solute), the same mode of injection, and the analyses are carried out in rapid succession. In previous reports we have demonstrated with pesticides the integrity of the p-value in the presence of comparatively large amounts of extraneous materials (3), discussed and illustrated the effect of adding water to systems (S), determined from p-values amounts extracted into upper and lower layers in multiple extractions and in single
worth considering for finding an efficient solvent system to accomplish a given separation.
extractions with unequal phase volumes (4,and presented apparatus for rapid extraction of one liquid phase with another (4). Although widely differing p-values of two substances qualitatively indicate that they may be separated easily by extraction processes, in countercurrent distribution (6, 8) the efficiency of separating two solutes depends on K,/Kb, the ratio of their partition coefficients (called 6 or separation factor, which is formulated as always greater than l ) , and is maximal when K,.Kb is near one. Selection of a solvent system to give the highest 6 value and manipulation of the system to adjust K.'& to a value near one has been discussed (8). Unequal phase volumes may be used (4). p-Values may be changed to partition coefficients ( K ) or 4 by the following equations :
LITERATURE CITED
(1) Ackman, R. G., J . Am. Oil Chemists' floc. 40, 564 (1963). (2) Beroza, M., Bowman, M. C., ANAL. CHEM.37, 291 (1965). (3) Beroza, M., Bowman, M. C., J . Assoc. Ofi. Agr. Chemists 48, 358 (196.5) \ - - - - I .
(4) Beroza, M., IBowman, M. C., ANAL. CHEM.38, 837 (1966). (5) Bowman, M. C., Beroza, M., J . Assoc. Ofic. Agr. Chemists 48, 943 (1965). (6) Craig, L. C., J . Biol. Chem. 155, 519 (1944). (7) Haenni, E. O., Howard, J. W., Joe, F. L., Jr., J . Assoc. O$lc. Agr. Chemists 45, 67 (1962). (8) King. T. P.. Craig. L. C.. "Methods of Biochemical Andysis," 'Vol. X, D. Glick. ed.. DD. 201-28. Interscience. New Pork; 1'962. (9) Scholfield, J. A., Jones, E. P., Butterfield, R. O . , Dutton. H. J.. ANAL. CHEM.35. 1588 (1963). (10) Scholfikld, J. A., 'Nowakowska, J., Dutton, H. J., J . Am. Oil Chemists' SOC. 37, 27 (1960). ~
The values of K for each p-value at intervals of 0.01 have also been given in a previous publication [Table I11 of ref. (3)1. The ease of determining pvalues for compounds that can be analyzed by gas chromatography makes this approach
RECEIVEDfor review March 30, 1966. Accepted August 9, 1966. Mention of proprietary products is for identification only and does not necessarily imply endorsement of these products by the U. S. Depart men t of Agri CUIt ure .
Mass Spectrometric Determination of IUnresolved Components in Gas Chromatographic Effluents CHARLES C. SWEELEY,' WILLIAM H. ELLIOTT,* IAN FRIES, and RAGNAR RYHAGE Kemiska Instifutionen, Karolinska Institutet, Stockholm, Sweden
b A mass spectrometric method is proposed for determining the composition of unresolved or partially resolved mixtures in gas chromatographic effluents. Using a combined gas chromatograph-mass spectrometer equipped with molecule separators, simultaneous recordings are made of the changing intensities of two selected m/e values during elution of the mixture from the chromatographic column. Peaks for the two ions are observed with a single collector, one oscillographic recorder and at constant magnetic field strength by rapid switching of accelerating voltage with a time-actuated relay and voltage dividing circuit. Mixtures of the trimethylsilyl derivatives of epiandrosterone and dehydroepiandrosterone were determined with as little as 2 0 nanograms of sample. A special application of the method, the determination of stable isotopic abundance in volatile organic compounds, was investigated with mixtures of the
penta-0-trimethylsilyl derivatives of glucose and glucose-d,. Samples as small as 0.1 microgram were analyzed conveniently; average deviation from true values was 5.5%.
combination of mass Api$k2netry and gas-liquid chromatography (GLC) has been achieved, using pressure reduction systems (6, 9) for the direct coupling of the two instruments and fast-scanning techniques for recording mass spectra. Combined instruments of this type offer several important advantages: e:g., mass spectral data can be obtained with extremely minute quantities of a volatile organic compound, and grossly impure samples may be used provided the desired compound is eluted as a single component from the GLC column. The effectiveness of this technique has been established for studies which involve structural elucidations, determinations of reaction mechanisms, and identifica-
tions of trace components of a mixture of organic compounds. When the retention times of two or more compounds are exactly or nearly identical, these compounds are eluted from typical short packed GLC columns in an unresolved or only partially resolved mixture. Another potentially important application of GLC-mass spectrometry is the determination of the composition of such mixtures. Several mass spectrometric methods may be employed for these determinations. The intensities of m/e values characteristic of each species in the mixture can be plotted us. time, using data from multiple mass spectra recorded every few seconds during elution of the mixture (6). Individual curves are obtained in this manner, and their areas Present address, Department of Biochemistry and Nutrition, Graduate School of Public Health, University of Pittsburgh. 'Present address, De artment of Biochemistry, St. Louis 8niversity School of Medicine. VOL 38, NO. 1 1 , OCTOBER 1966
1549
can be related to composition of the mixture. Data obtained from a single mass spectrum presumably suffice in those cases in which no separation of the individual components has occurred during GLC. We have evaluated another, more automated technique in which the combined instrument can be used for analyses of two-component mixtures. A specific application of this method, of potential value in biochemical studies utilizing stable isotopes as tracers, is the determination of stable isotopic abundance in volatile organic compounds at the submicrogram level. Preliminary reports of the results of this investigation were given at the Third International Symposium on Advances in Gas Chromatography, Houston, Texas, October 1965, and a t the Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, March 1966. EXPERIMENTAL
Materials. D-Glucose containing seven atoms of deuterium was purchased from Merck, Sharp and Dohme of Canada, Ltd., Montreal, Canada. This sugar, in the form of a n aqueous equilibrium mixture, was converted to penta - 0 - trimethylsilyl - D - glucopyranose-& (TMSi glucosed7) as described previously (7). Small quantities of the P anomer of TMSi glucosed, were then isolated by gas chromatography on 3% SE-30 at 150" C. Separate solutions of TMSi P-glucose and TMSi P-glucosed7 in isooctane were adjusted in concentration so that equal aliquots produced exactly the same areas on a gas chromatograph equipped with a flame ionization detector. Mixtures of the protium and deuterium forms were prepared from these stock solutions with volumetric pipets; the total concentration of TMSi glucose in each mixture was adjusted to about 0.5 /.kg.(pl. TMSi derivatives of epiandrosterone and dehydroepiandrosterone and a mixture of TMSi steroids prepared from human plasma were kindly furnished by J. Sjovall and R. Vihko, Karolinska Institutet. Instrumentation. Mass spectrometric analyses were made with a single focusing, rapid magnetic-scanning mass spectrometer (prototype of LKB 9000, LKB Produkter AB, Stockholm) coupled through molecule separators with a gas chromatographic system (6). The coiled glass GLC column was 2 meters in length by 3-mm. i.d., and was packed with 3% SE-30 on 100 to 120 mesh, acid-washed and silanized Gas Chrom S from Applied Science Laboratories, Inc., State College, Pa. Isothermal analyses were made at column temperatures which gave retention times of 15-20 minutes for TMSi ,%glucose (ca. 160' C.) and, in separate studies, for the TMSi dehydroepiandrosterone (216' C.), using a helium inlet pressure of 0.9 kg./cm.* The molecule separators and ion source
1550
ANALYTICAL CHEMISTRY
1
Figure 1.
I
Schematic diagram of accelerating voltage alternator
were held at 240' C. and 250' C., respectively. All mass spectra and alternator measurements were recorded at 20 e.v. electron energy with 3500 volts accelerating voltage; the filament emission current was 60 pa. A schematic diagram of the accelerating voltage alternator, used in the studies of the composition of unresolved mixtures, is shown in Figure 1. The high voltage relay switch (K1) was single pole, single throw, and normally closed, so that the voltage divider was by-passed and full voltage of 3500 volts was delivered from the accelerating voltage power supply to the ion source. Manual focusing of the lower of the two selected m/e values was made by adjustment of magnetic field strength with controls on the mass spectrometer, with switch S1 off and gang switch (SZ) open (Position 1). T o ease the problem of obtaining perfect focus on the top of the peak for a given m/e, and to assure maximum stability of the system for prolonged periods of time, a 20 C.P.S. filter was used in the galvanometer amplifiers for two pens of an oscillographic recorder, and a second filter (4 c.P.s.) was inserted between the electron multiplier preamplifier and the galvanometer amplifiers. The power switch (Sl) was then closed, with gang switch still open, and the higher m/e was focused by decreasing accelerating voltage with the coarse and fine controls of the high voltage divider. Although the appropriate difference in accelerating voltage for focusing two ions a t a given field strength can be calculated, i t was convenient to count masses visually on the oscillographic recorder during this adjustment and to ascertain perfect focus by observing the amplitudes of the galvanometer pens. The system was designed so that any two ions of as much as 10% difference in mass could be studied. The entire high voltage and relay section of the alternator unit was carefully insulated. Focusing two, m/e values by this technique requires some time and
patience. It is normally carried out before injection of the sample, making use of the mass spectral ions from the background of organic "bleed" from the column. An expanded spectrum of 20 or 30 mass units in the range of interest, accurately showing relative intensities in this range, enables the operator to locate the desired m/e values without difficulty. Of course, prior recording of the mass spectrum of each component is prerequisite, and some understanding or interpretation of these spectra is desirable, since the presence of partially labeled species may contribute to an unknown extent to a particular m/e value, leading ultimately to incorrect results. If possible, a check of the method with a synthetic mixture of the compounds should always be made prior to determinations of unknown mixtures. An additional benefit of this preliminary standardization with known mixtures is the recognition of an isotope effect in mass spectral fragmentation, if this phenomenon should occur in a particular case. Actual measurement of the changing intensities of two m/e values during elution of a two-component mixture was begun by setting the gang switch for a selected frequency of high voltage switching (Positions 2, 3, or 4), thus activating the multivibrator. Recordings were made with two galvanometer pens of an oscillographic recorder operating a t a chart speed of one inch per minute. Areas of the curves produced in this way (see Figures 4 and 8) were determined from peak height and width a t half height. When one of the two components represented less than 10% of the mixture, its area was determined from the curve at higher sensitivity (X10) and the result was corrected to lower sensitivity ( X 1). Cross contributions by one component to the intensity of the other's characteristic ion (background) were corrected by obtaining recordings of the intensities of both ions during GLC of each pure component.
PENTA-0-TRIMETHYLSILK8-GLUCOSE MW 540
80 v)
z W
- 60 z G W
40
2
;20 I-
Lz
. 80-
206
PENT*-0-TRIMETHILSIL~L8-GLUCOSE-d,
YW 547
*
Figure 3. Changes in fragment ion intensitites during elution of glucose and glucose-d, mixture from 3% SE-30 column
192
of the changes in intensities of two With this technique the results are not affected by differences in volatility nor by the degree of resolution of the components. Previous evidence suggested that TMSi glucose and its heptadeutero form are slightly separated on short packed columns of SE-30 ( I ) , even though mixtures of the two gave a single gas chromatographic peak which was perfectly symmetrical to the eye. Partial mass spectra of the two TMSi fl anomeric forms are shown in Figure 2. Fragment ions of m/e above 250 were of very low intensity. Three appropriate pairs of ions for investigation were those a t mle 191 and 192, 204 and 206, and 217 and 220. Multiple mass spectra were therefore taken of this region of the mass spectrum at 15-second intervals during elution of a mixture containing about 40% of TMSi pglucose-d7. Plots of total uncorrected ion intensities us. time are shown in Figure 3. These curves provide conclusive evidence that partial isotopic fractionation resulted on GLC with a short packed column (2 m. X 3 mm. i d . ) containing 3% SE-30; they also corroborate previous investigations with long packed columns of high efficiency, which demonstrated that the perdeutero form of TMSi @-glucosehas the shorter retention time, compared with that of the protium form. It is also clear from inspection of the curves shown in Figure 3 that the ratio of deutero to protium glucose changes continuously across the GLC peak, as would be expected when slight separation has occurred. Equally important, the ratios of the areas of the paired curves were approximately equal to that of the starting mixture, The m/e pair 217 and 220 was particularly suitable for investigation with the accelerating voltage alternator since the background abundance of m/e 220 in pure protium T l E S 0-glucose
m/e values.
Figure 2. Partial mass spectra of trimethylsilyl derivatives of glucose and heptadeuteroglucose
RESULTS AND DISCUSSION
Recent studies have demonstrated that short, packed GLC columns have sufficient resolving power for partial fractionation of some isotopic-labeled compounds from their nonlabeled species (1-3). There are now known to be many instances in which this phenomenon occurs during chromatographic procedures of all kinds; Klein recently summarized the effects observed in a number of authenticated cases of isotope fractionation (4). I t is apparent that this phenomenon is not limited to a particular type of isotopic atom, nor to compounds of very low molecular weight. Although the combined technique of gas chromatography-mass spectrometry is of considerable potential value in the determination of isotopic abundance in submicrogram quantities of organic compounds, the frequent occurrence of partial isotope fractionation poses an analytical problem. This is considered to be a special case of the general problem of determining, from mass spectral data, the composition of a mixture of unresolved components in the gas chromatographic effluent stream. If the nature of the mixture is known and certain m/e values are unique for each substance in the mixture, mass spectrometry can be used to determine the proportions of each component. If no fractionation has resulted in GLC and all of the components have exactly the same retention times, a single mass spectrum provides sufficient data for accurate calculations of composition.
If, however,
the components are partially resolved, a single mass spectrum is inadequate since the composition of the GLC effluent stream is changiug continuously. Lindeman and Annis demonstrated this in studies of incompletely resolved mixtures of low molecular weight hydrocarbons (5). They determined compositions from plots of the intensities of particular m/e values us. time, obtained from mass spectra recorded every few seconds during elution of the peak. This technique is a useful one for partially resolved mixtures, although it is time consuming and may not be accurate if a mass spectrum has not been taken at the exact peaks of intensity change for each of the components. Either unresolved or partially resolved mixtures of two components could be analyzed with a double collector on the mass spectrometer, providing some flexibility existed for selecting appropriate m/e values. Unfortunately, such devices are not easily incorporated into commercially available combined instruments. Separate collection of the entire mixture from an analytical gas chromatograph and subsequent analysis by mass spectrometry with a direct probe inlet will also serve for the determination of composition. Even slight differences in relative volatility of the components can be expected to affect the accuracy of data collected in this manner, however. Our results suggest that a simple solution is afforded by the use of a device which, by switching accelerating voltage a t frequent intervals, allows more or less continuous recording
VOL 38, NO. 1 1 , OCTOBER 1966
1551
‘I
m/e 22
-
u u 0 IO 5
MINUTES
15
20
Figure 5. Gas chromatogram of mixture of TMSi dehydroepiandrosterone ( 1 ) and TMSi epiandrosterone (2) on
2. ng
3% SE-30 I
epiandrosterone, with the accelerating voltage alternator was carried out as described with the isotopic-substituted glucose; a typical recording is shown in Figure 8. Simple calculations of the observed areas from six determinations gave a reasonably accurate estimate of the composition of this mixture (average 29.3% dehydroepiandrosterone found). Each of the analyses required less than a microgram of sample; one determination was made with 36 nanograms (found, 37.6% dehydroepiandrosterone). A crude mixture of ThlSi steroids, prepared from human plasma, was known to contain dehydroepiandrosterone, epiandrosterone and another steroid (androsterone) whose TMSi derivative also had a fragment ion a t m/e 272 (M-90) (8). Analysis of this sample (Figure 9) gave clear evidence for the presence of these compounds or other steroids with identical retention times and with mass spectral fragment ions a t m/e 270 and 272 (an unlikely combination). Comparisons of the GLC record with the mass spectral record show that, of a great many peaks in the mixture, there were three TMSi steroids in the mixture in appreciable
Figure 4. Continuous recording of m/e 217 and 220 during elution of TMSi glucose and glucose-d7 mixture from 3% SE-30 column Calcd. 2.67% Found 2.71%
was considerably lower than were the backgrounds at in/e 206 and 192. The recording shown in Figure 4 was obtained from the gas chromatographic effluent stream containing approximately one microgram of mixed ThlSi sugars. The areas of the upper curve ( x10) for m/e 220 and the lower curve ( x 1 ) for m/e 217 were compared after corrections for background and the difference in sensitivity of the two traces. The observed proportion of glucosed, in this mixture (2.71%) compared favorably witjh the calculated value of 2.67%. The small peak observed earlier in the chromatogram was calculated to be from 2.1 nanograms of an impurity. A summary is given in Table I of the results obtained with six synthetic mixtures of TlLlSi p-glucose and TMSi p-glucose-d7. The general problem of analysis of an unresolved mixture of two dissimilar organic compounds is illustrated by a GLC study of the TMSi derivatives of two steroids, epiandrosterone and de-
Table l. Determination of Isotopic Abundance in Mixtures of Glucose and Heptadeuteroglucose
EX:
penment 1 2 3 4 5 6
Sample Glucose-d7, % size ( p g . ) Calculated Found 0.55 0.77 1.37 1.30 0.57 1.12
45.04 21.45 12.02 6.41 2.67 1.35
44.48 20.86 11.92 7.60 2.72 1.25
hydroepiandrosterone. These compounds are partially resolved on short packed columns of 3% SE-30, as shown in Figure 5 ; TMSi dehydroepiandrosterone was eluted slightly before the other steroid. The mass spectra of these compounds, shown in Figures 6 and 7, were examined to select an a p propriate ion pair for the determination of composition of mixtures. Three ions which appeared to be suitably intense +, and yet selective were &I (M-15)+ and (M-90)f. The fragment ions a t m/e 270 and 272 (M-SO) were chosen for study since they afforded greater ease in focusing than the higher m/e pairs and because the intensities of these two ions represented approximately the same proportion of total ionization of the sample. Analysis of a mixture of the two TMSi steroids, 32.8% dehydro-
F e3
5z
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-
@
W O O TT W
60-
ANALYTICAL CHEMISTRY
li
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1
z
I
W-80
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I
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60
80
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Figure 6.
1552
MW 3 6 2 MW 3 6 2
4
120 110
160
im
200
220
240
260
I
-
2B9
300
* 320 340 360 mlc
Mass spectrum of trimethylsilyl epiandrosterone
i
great promise for the biochemist or chemist who is faced with a paucity of analytical sample. Although the present studies were carried out with two-component mixtures, the method should be applicable to multiple components by inclusion of a relay with multiple positions in the accelerating voltage alternator.
1MSiO@so
I
n.80
M-56
I
I
1
1
N
ACKNOWLEDGMENT
The advice and guidance of Sirio Gattinara and Sune Melkersson in the design of the accelerating voltage alternator, and the technical assistance of Sten Wikstrom and Osten h’ilsson are gratefully acknowledged.
M-15
2olillLd 80
60
100
Figure 7.
120
Mass spectrum
of trimethylsilyl dehydroepiandrosterone
abundance, and that several minor components of much lower retention times showed evidence of containing mass spectral ions a t m/e 270 or 272. Even though the GLC effluent stream contziined a variety of additional substances with retention times similar to that of TMSi androsterone (peak 1 in Figure 9)) none of them contributed t o the peak for mje 272, which was due entirely to TMSi androsterone. h preliminary investigation was made of the ultimate sensitivity of this technique, since its value for determinations of isotopic abundance is partially dependent on the ability to analyze very small samples isolated from biological material. With present instrumentation and highest sensitivity, a sample containing 20 mg. of a mixture of TMSi ethers of epiandrosterone and dehydroepiandrosterone was sufficient for analysis. With lesser quantities than this, the steriods were apparently adsorbed to the SE-30 column because no peaks were observed a t m/e 270 and 272. Although further studies of useful ranges of sensitivity will be required it is apparent that determinations can be made with as little as 20 nanograms of misture injected into the system and possibly with much less, provided the GLC colunin has no adsorptive properties ait,h the c,ompounds investigated. The accuracy achieved in these measurements of isotopic abundance of a mixture of glucose and glucose-d, was not’ particularly impressive, especially
I
I
7
’ \ m/o
4/ 1’2
272
1
14
16 MINUTES
18
20
Figure 8. Continuous recording of m/e 270 (TMSi dehydroepiandrosterone) and m/e 2 7 2 (TMSi epiandrosterone) during elution of a mixture
when compared with results obtained by classical mass spectrometric methods which utilize much larger gaseous samples and a double collector. I t appears likely that this accuracy will be improved considerably with better circuitry in the accelerating voltage alternator and with electronic integrating equipment to provide greater accuracy in the measurement of peak areas. Preliminary studies in these areas support this contention. However, of the known methods for the determination of isotopic abundance, this technique holds
LITERATURE CITED
(1) Bentley, R., Saha, N. C., Sweeley, C. C., ANAL.CHEM.37, 1118 (1965). (2) Bieman, K., “Mass Spectrometry, Organic Chemical Applications,” p. 217, McGraw-Hill, New York, 1962. ( 3 ) Kirshner, M. A,, Lipsett, hI. B., J. Lapid Research 6 , 7 (1965). (4) Klein, P. D., “The Occurrence and
Significance of Isotope Fractionation During Analytical Separations of Large Molecules,” in “Advances in Chromatography,” J. C. Giddings, R. A. Keller, ed., Vol. 111, p. 3, Marcel Dekker, Inc., New York, N. Y., 1966. ( 5 ) Lindeman, L. P., Annis, J. L., ANAL. CHEM.32, 1742 (1960). (6) Rvhage, R., ANAL. CHEM.36, 759
(1964).( 7 ) Sweeley, C. C., Bentley, R., Makitn, M., Wells, W. W., J. Am. Chem. SOC. 85, 2497 (1963). (8) Siovall, J., Vihko, R., Steroids 6 , 597 (1965). (9) Watson, J. T., Biemann, K., ANAL. CHEM.36, 1135 (1964).
t-
0
I
1
1
a
b
0
1
-----.I1 0
Io
8
n
W
D
L
O
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Figure 9. GLC record (upper left corner) and continuous recording of m/e 270 and 272 during GLC of mixed TMSi steroids from human plasma
RECEIVEDfor review July 11, 1966. Accepted August 3, 1966. This investigation was supported partially by research grants (AM-04307 and HE-07878) from the U. S. Public Health Service, and from Therese and Johan Anderssons Minne. Ian Fries acknowledges a summer fellowship from Columbia College of Physicians and Surgeons. VOL 38, NO. 1 1 , OCTOBER 1966
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