Time-Averaged Proton Magnetic Resonance Analysis of Micro Samples from Open-Tube Gas Chromatographs R. E. LUNDIN, R. H. ELSKEN, R. A. FLATH, N. HENDERSONl T. R. MON, and R. TERANISHI Western Regional Research laboratory, Albany, Calif. EXPERIMENTAL
has always confronted the chemist who utilizes gas chromatography to separate closely related compounds. The 5O-pl. spherical glass microcell made by KMR Specialties permits a fourfold sample reduction and gives adequate resolution. Spectrum averaging increases PMR sensitivity far more markedly but a t the expense of longer experimentation times (1, 4). h reduction in sample concentration by a factor of 30 is practical. To determine the practical limits on sample size afforded by these developments a study was undertaken using a mixture of terpene alcohols that was of a difficult GC separafelt to be typical .~ tion.
b Proton magnetic resonance spectra can now b e obtained of fractions isolated by a single pass through an open-tube gas chromatograph column. With a microcell and time averaging the output o f an analytical 60-Mc. per second spectrometer over a period o f approximately 1 day, a sample containing 0.3 pmole of hydrogen provides a satisfactory spectrum. An all-electronic field sweep and other necessary interface circuitry for use in averaging are described.
T
HE PROBLEM of obtaining adequate amounts of pure material for proton magnetic resonance (PMR) analyses
Commercial isopulegol was used in this study. The trapping techniques used with the open-tube column gas chromatographs that were made in our laboratory (6, 7“) have been described (6)’ A standard microcell with a 1-mm. capillary was used rather than the 2-mm. type that has recently been described (.2) ’
Special sweep and trigger-control circuits as shown in Figures 1 and 2 were required to average digitally the ogtput of a Varian A-60 spectrometer with a Technical Measurements Corp. 1024-channel analyzer eauimed - - _ with a 5-kc. CAT plug-in.
RAMP ADJUST Ramp Information AMPLIFIER
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M E A S . CORP., NORTH HAVEN, CONN. HEWLETT-PACKARD CO.. P A L 0 A L T O , CALIF.
Figure 1.
Interconnection diagram
of CAT-A-60 combination VOL. 38, NO. 2, FEBRUARY 1966
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Figure 2.
The A-60 sweep circuit was modified to accept an alternate all-electronic ramp. This sweep mode conserves the limited life of the sweep potentiometer and adapts more readily to unattended repetitive operation. The ramp is provided by electronic integration using a high-gain, chopper-stabilized, operational amplifier and a computer-quality polystyrene feed-back capacitor in a conventional Miller circuit, After special care was taken to eliminate system ground loops (Figure 2), the sweep showed no deviation from linearity nor any effect of severe linevoltage transients. Discharge of the integrating capacitor with a relay, either energized manually or by the address overflow or ramp level circuitry, resets the sweep. Because the CAT and 1-1-60 are scanning independently, this technique would appear to be intrinsically less reliable than one directly relating CAT address to magnetic field. However, no loss of registration has been observed. Line widths remain those determined by the resolution or the size of the memory even after many hundreds of scans. 292
ANALYTICAL CHEMISTRY
Detailed interface-circuit diagrams
It is often convenient to use a modulation sideband as the CAT trigger. In the variable temperature probe, however, modulation causes a calibration shift of several cycles per second because i t is applied primarily t o the control sample. T o correct this situation, the transformer and associated circuitry shown in Figure 2 a t the modulation input were added. The modulation field a t the control sample is completely canceled, and a more satisfactory sideband level a t the analytical sample is provided. Trigger-signal search is simple because the ramp automatically and independently resumes after reset and its starting point can be set a few cycles per second ahead of the triggering peak with the sweep-offset control of the spectrometer. If the CAT fails to trigger because of degraded resolution, the avalanche current through the IN3020B diodes across the sweep output will reset the ramp when it approaches its maximum value. Thus, the ramp generator will continue to cycle so that the CAT can resume operation if good resolution should return.
Relays are provided to cut off the trigger and modulation voltages 10 seconds after the start of the sweep to eliminate spurious modulation signals and improper triggering on a strong
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Figure 3. Chromatogram of commercial isopulegol, 1000-foot, 0.03inch i.d., open-tube stainless-steel column coated with SF 96 (50) silicone oil, 150°, helium carrier gas at 20 cm. per second
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peak when the trigger signal falls below the discriminator level. The digital timing circuits used in the accumulate mode of the CAT make possible a reliable calibrated chart readout in this mode which cannot be obtained in the standard readout mode. However, the addition of a four-binary board t o the time-division circuitry is highly desirable t o have a series of readout times compatible with the various chart ranges and with the 250-second recorder sweep time, For readout in thij mode Dymec (I) amplifies the lowlevel analog display voltage to the level required by the recorder. It is also necessary to disable the voltage-tofrequency converter during this readout. RESULTS AND DISCUSSION
Figure 3 shows the separation of commercial isopulegol with a 1000-foot, 0.03-inch i d , open-tube column coated with SF-96 (50) silicone oil. The peaks were identified as: 1, isopulegol; 2, neoisopulegol; 3, epiisopulegol. Figure 4 shows the spectra of 0.8 mg. of isopulegol (peak 1) in carbon tetrachloride run in a spherical microcell under various instrumental conditions. The doublet-split triplet centered a t 6 = 3.32 in curve B arises from the carbinol hydrogen and provides a convenient measure of the sensitivity of the various methods. In A the triplet is undetectable, whereas in C it is readily observable, although the magnitude of the doublet splitting would be open to some question. To establish the minimum practical
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Figure 4. PMR spectra of 0.8 mg. of isopulegol in 50 pl. of carbon tetrachloride in spherical glass microceII A.
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Figure 5. 60 Mc. per second, time-averaged spectra of 50 pg. of neoisopulegol in 50 MI. of carbon tetrachloride in microcell A. B.
sample size for the microcell-A-60-CAT combination several samples containing from 10 to 100 pg. of neoisopulegol (peak 2) were averaged to obtain a 25-fold enhancement. This enhancement required more than two days at a sweep rate of 2 cycles per sq. second and was selected because spectrometers normally are idle over weekends. It appears that 0.3 pmole of hydrogen represents about the lower limit on sample size for a fairly usable spectrum under these conditions. If broad multiplets are absent or can be sacrificed, this amount can be appreciably reduced. For a sharp, uwplit resonance having a line width of 1 cycle per second or less, a sevenfold reduction is possible. It has been shown recently that an appreciable enhancement can be obtained by operating with a somewhat higher radio-frequency field and sweep rate than normally employed at the cost, however, of some shifting of peak positions, line broadening, and "abnormal" intensities (3). On the basis of this work, a sweep rate of 5 cycles per sq. second and an effective sideband radiofrequency field of about 200 pgauss were selected to provide the maximum signal for the minimum acceptable resolution and position accuracy (about 1 cycle per second). Figure 5 compares 853 scans of a 50-pg. sample of neoisopulegol run under these conditions over a period of 24 hours ( A ) with 667 scans of the sample taken over a period of 2 days ( B )under the same conditions used for spectrum B of Figure 3. It is
8 5 3 scans over 1 - d a y period 667 scans taken over 4 8 hours
obvious that 0.3 pmole of hydrogen provides a usable spectrum in 1 day of scanning. Thus, a weekend of scanning would probably provide a reasonable spectrum from 0.2 pmole of hydrogen. Because the sensitivity of the 100-Me. per second research instrument is approximately sis times that of the A-60, one collection from the open-tube column will often provide sufficient material for a single scan analysis. With multiple scanning it should be possible to obtain useful spectra from 0.03- to 0.05-pniole of hydrogen. LITERATURE CITED
(1) Allen, L. C., Johnson, L. F., J . Am. Chem. SOC.85, 2668 (1963). (2) Brame, E. G., Jr., ANAL.CHEM.37, 1183 (1965).
(3) Ernst, R., Anderson, W. A,, Rev. Sci. Inst?. (to be published). (4) Klein, 31. P., Barton, G. W., Ibid., 34, 754 (1963). (5) Teranishi, R., Buttery, R. G., Mon, T. R., Ann. S.Y . Acacl. Sci. 116. 583 (1964). (6) Teranishi, R., Flath, R. A., Mon, T. R., Stevens, K. L., J. Gas Chromatog. 3, 206 (1965). (7) Teranishi, R., Mon, T. R., ANAL. CHEW36, 1490 (1964). RECEIVEDfor review June 29, 1965. Accepted November 22, 1965. Presented at the International Symposium on .4dvances in Gas Chromatography, University of Houston, Houston, Texas, October 18-21, 1965. Western Regional Research Laboratory is a laboratory of the Western Utilization Research and Development Division, Agricultural Research Services, U. S. Department of Agriculture. Reference to a company or product name does not imply approval or recommendation of the product by the U. S. Department of Agriculture to the exclusion of others that may be suitable. VOL. 38, NO. 2, FEBRUARY 1966
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