Anal. Chem. 1983, 55,2285-2289
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Thin-Layer Chromatographic Plate Scanner Interfaced with a Mass Spectrometer Louis Ramaley,* Margaret E. Nearing,' and Margaret-Anne Vaughan Department of Chemistry, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4J3
Robert G. Ackman Canadian Institute of Fisheries Technology, Technical University of Nova Scotia, Halifax, Nova Scotia, Canada B3H 2 x 4
W. David Jamieson Atlantic Research Laboratory, National Research Council of Canada, 1411 Oxford Street, Halifax, Nova Scotia, Canada B3H 321
A device which scans a 1 X 10 cm TLC plate past a source of desorption energy has been developed. This scanner is interfaced to a quadrupole mass spectrometer by means of an isoiatlon valve and a heated transfer line inserted into the direct sample inlet port. Chemical ionization reagent gas transports the desorbed compounds into the ion source. Two sources of desorption energy have been employed, a pulsed COP laser and a 150-W incandescent lamp. Sensitivity depends on the compound studied and the desorption energy available, with a minimum detectable amount of about 1 ng. Quantitation can be performed by using an internal standard with a precision of about 20%. Response with silica gel plates is not linear. The method Is nondestructive; plates can be rescanned or subjected to other detection methods.
The combination of mass spectrometry with various chromatographic techniques has been extremely fruitful. Both gas and liquid chromatographs have been successfully coupled to mass spectrometers, providing sophisticated debction capability. Thin-layer chromatography (TLC), although simple and inexpensive, suffers from lack of good detectors. To develop TLC methods and as a referee technique to solve complex problems, a mass spectrometer would be a most useful detector. However, it is unlikely that an expensive and complicated mass spectrometer will be used routinely as a detector. The most popular methods of sample introduction from thin-layer plates into mass spectrometers involve removing individual spots and introducing them one at a time using the direct probe inlet. Many authors (1-5) report scraping the spot from the plate, eluting the sample from the adsorbent and evaporating the eluent before direct probe introduction. Spots of about 0.1 to 1pg are needed for good results. Several authors (6-8) have introduced the samples and adsorbent without the elution step. Although faster, this method provides less satisfactory results and requires spots greater than 1 pg. Henion et al. (9) have recently compared these two methods. Kruegar (10) has eluted the sample directly from the TLC plate and electrosprayed the resulting solution onto an aluminum foil for californium fission fragment mass spectrometry. Issaq et al. (11)have used a Camag Eluchrom system to elute spots directly into a mass spectrometer. All of these methods require a knowledge of spot positions on the 'Present address: "Youth Science News", Suite 805, 151 Slater St., Ottawa, Ontario, Canada K1P 5H3.
plate, destroy the chromatogram, and are cumbersome and time-consuming. Kaiser (12) first directly coupled thin-layer chromatography and mass spectrometry by using a small H2-02flame to desorb samples directly from a silica plate, Helium was used to sweep the desorbed materials simultaneously into a flame ionization detector and a mass spectrometer. The amount of sample reaching the mass spectrometer was small and certain samples readily decomposed. Parkhurst and McReynolds (13) have patented a method for introducing TLC samples directly into a mass spectrometer which involves heating small segments of the TLC plate. To the best of our knowledge no results using this method have been published. More recently Unger et al. (14) have subjected silica gel and cellulose plates to secondary ion mass spectrometry directly in the ion source. Good spectra were obtained but spots of at least 10 pg were needed and the analysis required long periods of time. In any scanner, the sample must be desorbed from the plate and then transferred to the ion source while a correlation is maintained between the spectrum scan number and the spatial distribution of samples on the plate. To maintain this correlation, the plate can be moved linearly past the source of desorption energy or the energy source can be moved linearly along the plate. The former is by far the simpler method and has the fewest disadvantages. Two approaches can be taken to the transfer of the desorbed sample: the plate can be moved through the ion source with desorption and ionization taking place almost simultaneously or the plate can be scanned externally with respect to the mass spectrometer and the desorbed molecules transferred to the ion source through a heated line. The former is cumbersome, requiring major modifications to the spectrometer and involving pump-down and background problems, but would provide the best spectra for a wide range of compounds. The latter is simple but may involve sample loss or breakdown during transfer to the ion source. A scanner of the second type (15, 16) is described below which will couple to a wide variety of mass spectrometers through the direct inlet port, requires little or no modification of existing instrumentation, can be inserted or removed in 10 min without venting the mass spectrometer, and can completely scan a small TLC plate in less than 15 min. Desorption energy is provided in two forms: broad band infrared and visible radiation from a tungsten filament incandescent lamp (15)or pulses of 10.6-pm radiation from a small COZ laser (16).The incandescent source is simple and inexpensive. Laser desorption (LD) experiments (Hillenkamp (17) presents a recent overview of LD) have indicated that this type of desorption causes less decomposition than de-
0003-2700/83/0355-2285$01.50/0@ 1983 American Chemical Society
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sorption by slow heating. Although most LD work involves both desorption and ionization, there is good evidence (18-20) that LD produces many more neutrals than ions, even at high laser power. This is important when desorption occurs external to the ion source.
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EXPERIMENTAL SECTION Apparatus. The scanner was interfaced to a Finnigan-MAT Model 4000 quadrupole mass spectrometer coupled to an INCOS data system. A diagram of the entire scanner system with the plate chamber open is shown in Figure 1. More detailed diagrams of the plate chamber and its cover are shown in Figure 2. A 1 X 10 cm TLC plate rides on a carriage in the plate chamber base. Motion is provided by a stepping motor (Superior Electric No. M061-FD301) through a rotary motion feedthrough (Varian Associates, No. 954-5151) and a rack and pinion gear assembly. Optical limit sensors (Clarex No. CLIZOO) are used to detect end of scan and prevent excess forward or reverse plate motion. Desorption energy passes through a window in the chamber cover which atmospheric pressure seals to the base with a large “0” ring, since, in operation, the chamber pressure is about 1torr. A bellows valve (Nupro, No. SS-4H-TSW) serves to isolate the chamber from the mass spectrometer when the chamber is not evacuated. The valve is heated by a length of nichrome wire,
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insulated from the valve with asbestos tape and powered by a 24-V filament transformer. Valve temperature can be varied with an autotransformer connected to the primary of the filament transformer. Chemical ionization (CI) reagent gas, usually methane, is used to sweep desorbed samples through the isolation valve and down a glass-lined, stainless steel transfer line to the ion source. This line is heated by nichrome wire powered directly from the mass spectrometer’s “ballistic” circuit for the control of sample probe temperature. The temperature of the line is sensed by an ironconstantan thermocouple and indicated by the mass spectrometer.
ANALYTICAL CHEMISTRY, VOL. 55, NO. 14, DECEMBER 1983 SEMI- REFLECTIVE
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Figure 4. Laser desorption arrangement. The transfer line, heater, and thermocouple are housed in a 0.500 in. 0.d. stainless steel tube. As shown in Figure 3, energy from a 24-V, 150-W quartzhalogen projection lamp (Sylvania No. FCS) is focused through an Infrasil-2 window onto the plate by an ellipsoidal mirror (Melles Griot, No. 02REM013). This arrangement gathers about 70% of the available lamp energy but does not provide a sharp focus since the lamp is not a point source and the mirror exhibits a large off-axis aberration. The laser, shown in Figure 4, is modeled after one described by Loy and Roland (21) and was constructed at the Physics Division of the National Research Council of Canada. The final version consists of a 50-cm borosilicate glass inner tube of 8 mm i.d. centered in a 60-cm Teflon tube, 3.75 cm 0.d. and 2.5 cm i.d. These tubes are separated by two brass electrodes located near the ends of the inner glass tube and sealed to both tubes with Vyton “0”rings. Large nylon end caps containing NaCl Brewster angle windows are sealed to the outer tube, also with Vyton “0”rings. The laser cavity is completed by a 100% reflective gold mirror at the rear and a partially transmissive germanium mirror at the front. A 45O gold mirror reflects the 8-mm laser beam through a 15-cm focal length cylindrical NaCl lens onto the plate through a NaCl window in the chamber cover. The image of the beam on the plate is an 8 mm wide line with highest energy at its center. Lasing is induced by a 15-kV discharge passed through a mixture of 80% He, 10% COz,and 10% Nzat 30 torr in the inner tube. The laser power supply used is not that of Loy and Roland (21) but a superior thyratron gated
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Flgui’e 5. Scan and pulse rate control electronics.
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capacitive discharge design capable of firing the laser up to 50 pulses/s with no decrease in the pulse energy of 15 mJ. This power supply can be triggered manually or from an external source. Careful shielding of the laser and its power supply was needed to prevent interference with the computer system. The motor drive electronics are outlined in Figure 5. Originally the system was designed to operate in the full-step mode using a commercial drive assembly (Superior Electric No. STMlOl printed circuit board). To obtain better resolution and smoother operation, the circuit was redesigned to operate in the half-step mode with 400 steps per revolution or 0.05 mm of plate motion per step. This required modification of the STMlOl module to use only the driver amplifiers. The 555 timer, operating at 192 Hz, acts as the master clock for the circuit. This frequency is divided in steps of two from 96 Hz to 0.75 Hz by two 7493 4-bit binary counters. These frequencies can be individually selected to provide laser pulsing (1.5-96 Hz)and forward and reverse plate motion (2-256 s cm-’). A control is also provided for continuous speed variation if desired. Motion is initiated by setting the RUN flip-flop and can be halted at any time by resetting this flip-flop. Direction is switch selectable with the limit detectors overriding any inappropriate direction signal. Monostable multivibrator M1 clocks the two 4-bit 74194 left-right shift registers which provide signals of correct phase and period for the half-stepping mode. Motor direction is determined by the shift direction of this register. Monostable M2 triggers the laser through an optoisolator. The laser operates only during forward motion. The inverters at the outputs of the 7493 counters buffer these counters from noise induced in the lines to the speed and pulse rate selection switches by the laser. The counters act erratically in the absence of these inverters. A motor drive amplifier, four of which are used, is shown in Figure 6. These are part of the modified STMlOl module. A plate scanning speed of 64 s cm-’ was standard, requiring slightly more than 10 min for a complete scan. For laser desorption, 12 pulses per second was most commonly employed, providing four laser pulses per motor step. Reverse plate motion was always 2 s cm-l. The mass spectrometer was operated in the positive-ion mode using methane as CI reagent gas with an ionizing energy of 70 eV and a source temperature of 200 “C. All unnumbered logic shown in Figure 5 is standard TTL, series 7400. The forward and reverse limit detectors are identical.
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ANALYTICAL CHEMISTRY, VOL. 55, NO. 14, DECEMBER 1983
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Figure 8. Mass chromatogram at m l z 179 of five 1-pg spots of phenanthrene using laser desorptlon.
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Figure 7. Mass chromatograms at m l z 179 of five 0.5-pg spots of phenanthrene using lamp desorption: (curve A) freshly spotted plate; (cum B) rescan of the same plate. Regulated 5-V power is supplied by a Burr Brown No. 562 module, unregulated 24-V power is derived from a simple, filtered, full wave bridge rectifier. The mass spectrometer ballistic heater circuit was modified to provide feed-back control using the thermocouple. The wiring for this modification, the laser power supply wiring, and other construction details can be obtained from the authors. Reagents. All reagent gases were supplied by Canadian Liquid Air except that for the C02 laser which was provided by Liquid Carbonic Canada, Ltd. Other reagents and solvents were of reagent grade when available, otherwise they were of the best quality obtainable and were used as received. TLC plates were E. Merck No. 5673 Silica Gel 60 (without indicator) cut to 1 X 10 cm. Procedure. For plate scanning, the chamber is evacuated with a mechanical pump after having placed a plate on the carriage. The isolation valve is opened and the flow of CI reagent gas is adjusted to provide the desired pressure in the ion source (usually 0.2 torr). The source of desorption is turned on, scanning motion in initiated, and the data system signaled to acquire the chromatogram. The software normally used to acquire gas chromatographic data can be used for TLC data without modification. When the scan is complete, plate motion is reversed, the isolation valve closed, the chamber vented to the atmosphere, and the plate removed. Another plate can be run immediately. Most tests were carried out on plates spotted with various amounts of materials at fixed distances along the plate. This had the advantages of speed, since no development was performed, and precise knowledge of spot position, size, and identity. We refer to plates spotted in this manner as pseudochromatograms. We observed no significant differences between the results of scanning pseudochromatograms and chromatograms developed in the usual manner.
RESULTS AND DISCUSSION Preliminary testing of the scanner was carried out by use of polycyclic aromatic hydrocarbons (PAH), in particular phenanthrene. Figure 7 is a typical example of a pseudochromatogram of five 0.5-pg spots of phenanthrene scanned with lamp desorption. Curve A is a mass chromatogram a t
m / z 179, the (M H)+peak of phenanthrene, of a freshly prepared plate. Curve B was obtained by rescanning the same plate immediately after curve A. Several features are obvious from these curves. The sensitivity a t the 1-pg level is good and the reproducibility is of the order of f20%. The technique is nondestructive since rescanning produces essentially the same curve but with lower sensitivity. The rescanned peak shapes, however, are slightly broader than the original and the return to base line is not as complete. This is probably due to migration of some of the samples to adjacent cooler areas on the plate right after desorption. Finally the peaks as recorded on the mass chromatogram are two to three times wider than expected since the spot diameters on the plate were 2 to 3 mm and the spots were 2 cm apart. The loss of chromatographic resolution was at first attributed to the lack of a sharp image of the lamp filament on the plate and to continued desorption of material from the heated plate after it had passed the actual image zone. Laser desorption was expected to improve chromatographic resolution since the image was less than 0.1 mm wide and very little bulk heating of the TLC plate occurred. Preliminary experiments with the laser produced very broad peaks with pronounced tails apparent in the mass chromatograms significantly after the time when the spot had passed the image zone. This was caused by desorbed material condensing on the cold chamber walls and only slowly redesorbing into the ion source. After three small 10-W cartridge heaters (Hotwatt, No. SC121) were installed in the chamber base near the desorption zone, the results shown in Figure 8 were obtained. Chromatographic resolution with both laser and lamp desorption is the same, but the sensitivity with lamp desorption is higher, since the lamp delivers far more total energy to the plate. Resolution seems limited by redistribution of desorbed materials within the plate chamber and by adsorption on cooler surfaces swept by the transport gas before it reaches the ion source, Image size does not appear to be critical. Laser energy can be varied by changing the pulse rate with the results shown in Figure 9. Sensitivity is almost directly proportional to pulse rate with a slight positive deviation from linearity. A rate of 12 s-' was chosen for routine work because the ion source remained clean much longer and less stress was placed on laser components at this rate. Day to day reproducibility was such that it was obvious an internal standard would be needed to attempt quantitative work. A series of samples with various amounts of phenanthrene and 2 pg of biphenyl as internal standard were spotted, developed with hexane in the normal manner, and then scanned. A plot of the logarithm of the ratio of peak areas vs. the logarithm of the amount of phenanthrene present was a straight line with a slope of 1.7. Essentially identical results
ANALYTICAL CHEMISTRY, VOL. 55, NO. 14, DECEMBER 1983
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sentially the same mass spectra by positive-ion chemical ionization with methane. Lamp desorption, simple, inexpensive, and providing as good or better sensitivity, might appear to be superior to laser desorption. Preliminary studies on materials less thermally stable than aromatic hydrocarbons indicated this may not always be so. Work on a wider variety of compounds and on various TLC surfaces is in progress.
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Mass chromatogram at m l z 179 (A) and RIC (e) of five 10-ng spots of phenanthrene using laser desorption at 48 pulses/s. were obtained by using peak heights. Since a slope of unity would be expected for linearity, these results show a strong positive deviation. This could be due to several factors, e.g., nonlinearity of adsorption energy with sample size or loss of sample due to adsorption on cooler surfaces. Precision varies somewhat from run to run, but was normally between 10 and 20%. The detection limit for substances such as phenanthrene was about 10 ng as shown in Figure 10. Identical results were obtained for both lamp and laser desorption. However, to achieve this limit with the laser, the pulse rate was set at 48 s-l and the mass scan range was reduced from 120-250 to 140-225 daltons in a 3-s scan. It is obvious that these small peaks are not detectable in the reconstructed ion chromatogram (RIC) and thus one must suspect their presence and search for them or use such special data processing techniques as the Biller-Biemann algorithm or reverse library searches. The technique of selected ion monitoring usually provides an improvement in detection limit of an order of magnitude or more. Although not verified with our scanner, this same improvement should be expected here. The background, exemplified by the RIC in Figure 10, is due to materials that desorb from the plates and chamber walls during a scan. The background is almost constant and much lower whenever a plate is rescanned. Figure 11 shows the results of a chromatogram of a PAH mixture spotted and developed with hexane in the normal manner. The chromatographic performance is not spectacular, as might have been expected when using silica gel for this particular mixture. However, the mass spectrometer and its data system allow easy analysis of overlapping peaks. Phenanthrene and anthracene cannot be distinguished because they do not separate chromatographically and also yield esFigure 10.
Figure 11. Mass chromatograms and RIC of a PAH mixture containing seven components of 1 pg each using lamp desorption. The m / z values for the mass chromatograms correspond to the (M + H)' peak for each component.
ACKNOWLEDGMENT The authors wish to thank Neil H. Burnett of the Physics Division, National Research Council of Canada, for the gift of the laser and associated optical components and his advice with this aspect of the project. Thanks are also due to D. J. Embree, Emyr Lewis, and F. G. Mason of the Atlantic Research Laboratory for help with other aspects of the research.
LITERATURE CITED Rix, M. J.; Webster, B. R.; Wright, I. C. Chem. Ind. (London). 1969, 452-454. Brewster, D.; Jones, R. S.; Parke, D. V. Biochem. J . 1977, 764, 595-600. Just, W. W.; Filipovic, N.; Werner, G. J . Chromatogr. 1974, 96, 189- 194. Weber, J. M.; Ma, T. S.Mikrochlm. Acta 1976, 227-242. Cox, P. J.; Farmer, P. B.; Jarman, M. Blochem. fharmacol. 1975, 24, 599-606. Heyns, K.; Grutzmacher, H. F. Angew. Chem., Int. Ed. Engl. 1962, 1 , 400. Down, G. J.; Gwyn, S. A. J . Chromatogr. 1975, 103, 208-210. Kraft, R.; Otto, A,; Makower, A.; Etzold, G. Anal. Biochem. 1081, 173, 193-196. Henion, J.; Maylin, G. A.; Thomson, B. A. J . Chromatogr. 1983, 277, 107-124. Krueger, F. R. Chromatographla. 1977, 10, 151-153. Issaq, H. J.; Schroer, J. A.; Barr, E. W. Chem. Instrum. ( N Y ) 1977, 8, 51-53. Kalser, R. Chem. Br. 1969, 5 , 54-61. Parkhurst, R. M.; McReynolds, J. H. U S . Patent 3896661, 1975. Unger, S. E.; Vincze, A.; Cooks, R. G.; Chrlsman, R.; Rothman, L. D. Anal. Chem. 1981, 53, 976-981. Ramaley, L.; Jamieson, W. D.; Ackman, R. G. "Abstracts"; 28th Annual Conference on Mass Spectrometry and Allied Topics; New York, May 1980; p 324. Ramaley, L.; Nearing, M. E.; Jamieson, W. D.; Ackman, R. G. "Abstracts"; 29th Annual Conference on Mass Spectrometry and AIlied Topics; Minneapolls, MN; May 1981; p 135. Hillenkamp, F. Int. J . Mass Spectrom. Ion fhys. 1982, 45, 305-313. Cotter, R. J. Anal. Chem. 1981, 53, 719-720. VanBreemen, R. B.; Snow, M.; Cotter, R. J. Int. J . Mass Spectrom. Ion Phys. 1983, 49, 35-50. Schueler, B.; Kruegar, F. R.; Felgl, P. Int. J . Mass Spectrom. Ion fhys. 1983, 47, 3-6. Loy, M. M. T.; Roland, P. A. Rev. Sci. Instrum. 1077, 48, 554-556.
RECEIVED for review June 14, 1983. Accepted September 6, 1983. This work was partially funded by contract support from the Marine Analytical Chemistry Standards Program of the National Research Council of Canada.
