law for dilute solutions of gases in liquids. It has been assumed throughout this development that the gas dissolves in the membrane material in its original molecular form and does not experience changes in its structure which would alter the equilibrium relationship defined by Henry’s law. Henry’s law is sufficiently accurate for gas partial pressures ranging from 0 to 1.5 atmospheres for most polymers. Thus, under normal circumstances of gas detection where the electroactive molecular species is the diluent gas, the pressure response characteristics will be completely defined by the flux and/or signal level equations presented earlier. The concentration of a gas dissolved in a polymer at relatively high pressures may be expressed by: C = Kpn
(14)
where
C = concentration of dissolved gas in the material, moles/cc K = solubility coefficient, moles/cc-mm Hgn n = exponent determined experimentally (n = 1 for Henry’s law) p = gas partial pressure, mm Hg The pressure response characteristics at these relatively high pressures can be defined by substitution of Equation 14 into Equation 13 : i = [nFA(P,)o/(Ax)m]pa”e-E’RT RECEIVED for review October 13, 1967. Accepted January 24. 1968.
Complete Identification of Chromatographic Effluents Using Interrupted Elution and Pyrolysis-Gas Chromatography John Q. Walker and Clarence J. Wolf McDonnell Co., St. Louis, Mo. 63166 One of the major problems remaining in gas chromatography is concerned with the identification of eluted compounds. An identification system which utilizes pyrolysis-gas chromatography is described. The instrument performs the same basic function as does a gas chromatograph-mass spectrometer combination. Organic compounds are separated in a temperature programed gas chromatograph (the separation GC). The effluent from this chromatograph passes into a high temperature pyrolysis unit in which the complex compounds are degraded into simpler ones. The products so formed are analyzed by means of a second gas chromatographic unit (the analysis GC). The technique of interrupted elution chromatography is adapted to the separation GC so that all the unknowns can be pyrolyzed and identified in a single GC analysis. The chromatograms resulting from the pyrolysis of several classes of compounds including hydrocarbons, methyl esters of fatty acids, and alcohols are presented and discussed.
ONEOF THE MAJOR problems remaining in gas chromatography today is concerned with the positive identification of eluted compounds. Identification of unknowns can be made by comparison with known calibration samples, by making characteristic derivatives, or by the determination of unique physical properties such as infrared or mass spectra. The combination of a gas chromatograph (GC) with a mass spectrometer or infrared spectrometer forms a versatile analytical instrument. The most desirable arrangement consists of a G C connected directly to the spectrometer through a molecular separator. However, spectrometers are not accessible to all who require their use, and even if available they require a specialist in order to interpret the data. The relatively simple and inexpensive technique of pyrolysis-gas chromatography (PGC) yields a unique signature characteristic of the sample pyrolyzed. In this method the material being analyzed is thermally fragmented in a controlled manner and the product distribution is recorded with
a GC. Recently, Levy has carefully and in great detail reviewed the entire field of PGC (I). Dhont (2,3) and Weurman ( 4 ) have successfully applied PGC to the analysis of chromatographic effluents. They trapped the eluted vapors at the outlet of the chromatograph and then pyrolyzed the compound. The resulting chromatogram was used t o identify the compound. Keulemans together with Perry ( 5 ) and Cramers (6) studied the techniques required for reproducible pyrolysis of organic vapors in order to obtain structural information about compounds eluted from a GC. Levy and Paul (7) described a two-unit G C system built into a single instrument. Their apparatus consisted of a conventional GC, a flow delay-line trap, pyrolyzer, and a second GC. A few selected compounds from the original mixture could be trapped and pyrolyzed. They showed that the pyrolysis of normal alkanes, olefins, alcohols, mercaptans, and saturated and unsaturated methyl esters of fatty acids was reproducible and analogous t o mass spectra. Sternberg et al. (8)also described a two-unit G C which contained a high pressure electrical discharge between the chromatographs. As a compound emerges from the first GC, it is fragmented in the discharge and the stable products identified and recorded in the second GC. This gives rise to fragmentation spectra which are analogous to mass spectra. (1) R. L. Levy, Chromatog. Reu., 8,48 (1966). (2) J. H. Dhont, Nature, 206, 882 (1963). (3) J. H. Dhont, Analyst, 89, 71 (1964). (4) C. Weurman, Chem. Weekblud, 59,489 (1963). ( 5 ) A. I. M. Keulemans and S. G. Perry, “Gas Chromatography,” M. Van Swaay, Ed., Butterworth, Washington, D. C., 1962, pp 356. (6) C. A. J. G. Cramers and A. I. M. Keulemans, J . Gus Chromarog., 5, 58 (1967). (7) E. J. Levy and D. G. Paul, Ibid., 5, 136 (1967). (8) J. C. Sternberg, I. H. Krull, and G. D. Friedel, ANAL.CHEM., 38, 1639 (1966). VOL 40, NO. 4, APRIL 1968
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For PGC to be applicable t o identification of chromatographic effluents, it is desirable that compounds be introduced one at a time into the pyrolyzer. It is also desirable that a determination be made of the identity of all the unknowns in a single GC analysis. If a system of traps is used, it is impractical t o use more than two or three separate traps. If no traps are used and the carrier gas flow passes directly from one GC into the pyrolyzer and then into the second GC, the pyrolytic fragments from one compound will overrun and mix with those of another. These limitations can be circumvented by the use of interrupted elution chromatography (sometimes called stop-start) as described by Scott (9). In this technique, the chromatographic process within the first GC (called the separation GC) is stopped after a peak emerges. This is accomplished by depressurizing the helium carrier gas in the column while continuing the gas flow in the rest of the system. Thus, while the separation GC is deactivated, the pyrolytic fragments from the first compound are swept through the second GC (the analysis GC). After a prescribed time interval the separation GC is repressurized with carrier gas and the chromatographic process continues. The system employing interrupted elution in the separation GC, the flow-through pyrolyzer, and the analysis GC is called a tandem gas chromatograph. For many applications such a system can rival that of a gas chromatographmass spectrometer combination instrument.
Ns I l l o w c~nli~ller an6 rclamelei
Figure 1. Block diagram of tandem gas chromatograph
Apparatus. A modified F and M 5750 equipped with a flame ionization detector was used as the separation chromatograph. It is t o be emphasized that essentially any column can be used in the separation GC provided it is capable of resolving the individual components of the mixture being studied. A 4-meter by 0.65-inch i.d. stainlesssteel column containing 15 % stabilized diethylene glycol succinate on S0/90 mesh Anakrom ABS was used for the separations reported here. Helium flowing a t a rate of 53 ml/min was used as the carrier gas. The pyrolyzer consisted of a 36-inch long gold tube having a 0.040-inch i.d. and wound into a 1i2-inch diameter helix. This was placed inside a nickel cylinder having a large thermal mass. The cylinder was contained in a Hevi-Duty combustion furnace controlled with a Wheelco 407 capacitrol coupled with a 610 Pilot Amplifier and saturable core reactor so that the temperature could be maintained to within + 5 C". This system when operated at 700" C and with a helium flow of 48 ml/min, decomposes approximately 5 to 10% of the parent compound. Fanter, Grayson, and Wolf (10) investigated several reaction chambers and concluded that gold pyrolyzers gave more reproducible results with flowthrough systems than did stainless steel or quartz. A modified Perkin-Elmer Model 11 chromatograph was used as the analysis GC. The stainless steel column was 15 feet long with a 0.65-inch i d . , and contained 25% Dow Corning DC200 on 80/100 mesh Chromosorb P. This is a versatile nonpolar column and was chosen for its ability t o elute compounds according t o their boiling point. Therefore, considerable structural information can be derived from the elution time of the pyrolytic fragments in the analysis GC. The column was operated a t 165" C with a helium flow of 48 ml/min and used a flame ionization detector. All peak
areas were determined by means of an Infotronics Model CRS-100 digital integrator. Positive identification of the pyrolytic products was made by mass spectrometry. The output from the analysis 700' was connected to a Bendix Model 12-101 time-of-flight mass spectrometer by a fritted glass molecular separator (11). All reagents used were chromatographic or high purity reagent grade and were used without further purification. Procedure. The block diagram for the tandem gas chromatograph is shown in Figure 1. In normal operation, helium carrier gas flows through No. 1-flow controller into the four port valve along the path indicated by the solid line. The vent and valve to the No. 2-flow controller are closed. The sample is introduced in a conventional manner with a hypodermic syringe a t the injector. The temperature programed separation column resolves the mixture into individual components which are detected with separation detector Ds. Each compound is fragmented at the preselected temperature in the pyrolyzer. The separation of the pyrolytic fragments is performed rapidly in the analysis column and the products are sensed by the analysis detector Da. The analysis time can be minimized by either temperature or flow programing the analysis column. Flow programing is analogous t o temperature programing except that the pressure (therefore the flow) across the column is raised rather than the temperature (12, 13). A flow programer (Perco Supplies, San Gabriel, Calif.) was connected between the pyrolyzer and the analysis column. A check valve was used to ensure that the gas flowed through the analysis column only and did not back up into the pyrolyzer. When the first compound from the separation GC leaves the detector Ds, the four port valve switch is rotated so that the helium from No. 1-flow controller passes through the valve in the direction shown by the lower dashed line. The separation GC is now isolated from the pyrolyzer and the analysis column. The latter continues the analysis. The helium in the separation GC is released t o the atmosphere by the vent through the precolumn; the vent is then closed. Scott (9) has reported that band spreading in the column during the stop action is minimized when the pressure is relieved and the overall band resolution is essentially uneffected by the stop. A stainless-steel restrictor, 6 ft long and of a 0.010-inch i.d., is placed between the four port valve and the vent. This prevents any rapid gas surge from occurring and removing organic material from the edge of the separation column. When the analysis of .pyrolytic fragments is complete, the separation column is repressurized by opening the valve connecting the No. 2-flow controller to the separation GC.
(9) R. P. W. Scott, I. A. Fowlis, D. Welti, and T. Wilkins, 6th International Symposium on Gas Chromatography and AssoI ciated Techniques, Rome, Italy, 1966. (10) D. L. Fanter, M. A. Grayson, and C. J. Wolf, 154th National American Chemical Society Meeting, Chicago, Sept. 1967, No. B60.
(11) M. A. Grayson and C. J. Wolf, ANAL. CHEM.,39, 1438 (1967). (12) A. Zlatkis, D. C. Fenimore, L. S. Ettre and J. E. Purcell, J . Gas Chromatog., 3, 75 (1965). (13) I. Halasz and F. A. Holdinghausen, "Advances in Gas Chromatography," ed., A. Zlatkis, Ed., Preston Technical Abstracts Co., Evanston, Ill., 1967, p 23.
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Tlme rm~wler
Figure 2. Pyrograms of n-decane and n-nonane recorded with the analysis GC The analysis column was heated to 165" C. The number above each peak represents an area normalized with respect to the largest pyrolytic product
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8
6
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a
2
Figure 3. Pyrograms of methyl esters of nonanoic, octanoic, and heptanoic acids recorded with the analysis of GC The analysis column was heated to 165' C. The number above each peak represents an area normalized with respect to the largest pyrolytic product When the pressure reaches its original value, the valve near the No. 2 controller is closed and the four port valve is rotated to its initial position. This process is repeated for every peak emerging from the separation GC. The precolumn serves as a trap for any material which may be removed from the separation column when the helium is vented; the material returns to the separation column when the system is repressurized. RESULTS AND DISCUSSION
In order t o avoid confusion the term pyrogram will be used throughout the discussion to refer to the chromatogram obtained with the analysis chromatograph. The analysis G C pyrograms of two typical hydrocarbons, n-nonane and ndecane, are shown in Figure 2. In this figure and all subsequent chromatograms, the current from the detector (ordinate) is shown as a logarithmic response. For the hydrocarbons, the total number of well resolved peaks is the same as the number of carbon atoms in the molecule. The first peak represents methane and, with the exception of the parent compound, the other peaks represent C2to C,-1 olefins (where n is the carbon number of the parent). The area of each peak can be normalized conveniently in one of three ways: with respect to the large parent peak, with respect to the sum of the nonparent peaks, and with respect t o the largest single peak other than the parent. Both of the last two methods are used to report mass spectra but we feel that normalization with respect to the largest peak other than the parent is desirable. In all figures, the numbers above each peak represent an area normalized in this manner. The normalization method serves to minimize small difference in the spectra resulting from such variables as flow rate or sample size. As long as the total per cent of parent decomposed in the pyrolyzer is small (less than 25z),the relative areas recorded for each peak in the spectrum are essentially independent of the amount decomposed. In the event that a
Figure 4. Pyrograms of nheptyl and n-hexyl alcohols recorded with the analysis GC The analysis column was operated at 165" C. The number above each represents an area normalized with respect to the largest pyrolytic product
digital integrator is not available, it may be desirable t o normalize with respect to the sum of the products rather than the largest single product. In addition to peak area the spectra requires an elution time (volume) for each peak in order to compare an unknown t o a standard. A convenient system of retention time (volume) is one based on the emergence of the first peak in the spectrum as zero time, all other retention times are therefore referenced t o the first peak. The analysis G C pyrograms of the methyl esters of heptanoic (C,), octanoic (C,), and nonanoic (C,) acids are shown in Figure 3. While the pyrograms are similar, they do show distinct differences, particularly with respect to the elution of higher boiling compounds. The first 6 peaks in all three spectra are the same; they are, from right to left, (1) C2H4 with a small amount of CHI, CO, and CO2, (2) C3H6, (3) C4H8, (4) C J h , ( 5 ) C2H3COOCH3, and ( 6 ) C3HsCOOCH3. The other peaks represent unsaturated methyl esters with carbon numbers up to one less than the parent ester. The pyrograms of n-hexyl and n-heptyl alcohols are shown in Figure 4. One of the largest peaks in both spectra corresponds t o an alpha olefin formed by the loss of H 2 0 from the parent. In hexyl alcohol the large peak which occurs 62 seconds after the methane-ethylene peak is 1-hexene. It has a relative area of 39. In heptyl alcohol the peak observed 140 seconds after the Cz's is 1-heptene and it has a relative area of 30. One of the most important parameters required for qualitative identification is the elution time in the separation GC. It is not immediately obvious that elution time remains a useful parameter when the interrupted elution technique is used. The effect of stop time in the separation G C on the total elution time of a CICmethyl ester is shown in Table I. The separations were interrupted for periods as long as 60 minutes with a negligible change in the true retention time. Thus, it appears that retention time in the separation G C is preserved and remains a useful chromatographic index in our system, VOL 40, NO. 4, APRIL 1968
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Analysis G C chr@malag!am
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Figure 6 . Tandem gas chromatograms of a mixture of n-nonane and methyl hexanoate. The upper curve shows the separation GC chromatogram including a 5minute stop. The lower curve shows the analysis GC (operated at 185 C) pyrograms of the two compounds Figure 5. Comparison of the pyrograms obtained from methyl hexanoate with and without interrupted elution The analysis column was heated to 185" C. The number above each peak represents an area normalized with respect to the largest pyrolytic product It is also important that the stopstart procedure not interfere with the repeatability of the analysis G C pyrogram. This point is illustrated in Figure 5 where the analysis G C pyrograms from the methyl ester of hexanoic acid are shown with and without interrupted elution. The lower tracing was obtained without stopping the separation G C while the upper curve shows the pyrogram observed after the C g ester was delayed 18 minutes on the separation G C column. Note that the relative area of each peak as well as the signature is preserved. The chromatograms from both the separation GC and analysis G C from a mixture of n-nonane and methyl hexanoate
Table I. Effect of Interrupted Elution on the Retention Time of Methyl Myristate Total True Stop time retention time0 retention timeb Run min min min 1 0.25 17.89 17.6 2 25.0 32.52 17.5 3 30.0 47.00 17.0 4 60.0 77.04 17.0 0 Clock time measured from time of sample injection to maximum peak height in the analysis GC detector. * Retention time corrected for stop time in the separation GC.
Table 11. Repeatability of Methyl Hexanoate Pyrolysis Standard Peak Compound Area0 deviationb 1 CHI C2H4 100 16 31 12 2 CaHa 21 il 3 C4Hs 6 il 4 CsHio 5 CzHa COOCHa 18 1 1 6 CsHT COOCHI 7 il Average area of 5 separate determinations. b The standard deviation of 5 determinations.
+
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ANALYTICAL CHEMISTRY
are shown in Figure 6. The upper curve shows the recorder tracing from the separation G C on a scale 100 times less sensitive than that of the analysis G C tracing shown in the lower portion of the figure. The nonane eluted approximately 1 minute after the mixture was injected. It passed directly into the pyrolyzer and analysis GC. Thirty seconds after the nonane eluted, the separation G C was stopped. When all the products from pyrolyzed nonane passed through the analysis detector (approximately 6 minutes), the separation G C was repressurized and the chromatographic process started. The methyl ester eluted and its pyrogram was recorded. It is important to note that reproducible signatures were obtained in the tandem G C from a mixture of two compounds which elute less than a minute apart on the separation column. The resolution of the analysis G C system is dependent on several parameters. It is necessary that the compound entering the pyrolyzer enter as a sharp peak and not as a broad flat peak. The volume of a single compound should not exceed 5 pl. The rate and limits of the flow programming, just as in temperature programming, must be adjusted to yield the desirable resolution for the particular analysis. Most of the analyses described above were performed with 1 to 3 p l samples, although we have routinely used samples as small as 0.01 pl. The actual minimum detectable amount of organic compound required for a complete analysis in the tandem G C will depend on the particular detectors, columns, and carrier gas flow rate used, Under optimum conditions, with high sensitivity low noise detectors and low bleed columns, we believe 10-9 grams would be sufficient for analysis with the tandem GC. The reproducibility of the normalized area for the six peaks observed from the pyrolysis of methyl hexanoate is summarized in Table 11. The standard deviation was calculated from 5 separate determinations and the areas of the larger peaks show slight variation. ACKNOWLEDGMENT
We thank L. Hammon for his help and care in obtaining some of the experimental data. RECEIVED for review October 9, 1967. Accepted January 5, 1968.
