Anal. Chem. 1991, 63, 2188-2193
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collected during an experiment similar to the one shown in Figure 7b. The profiles in Figure 8a are those of the displacer impurities, while the profile in Figure 8b represents the band of the sample and the displacer. For these two figures, a considerably expanded time scale has been used, for the sake of clarity. As the impurities have not been identified, their concentrations are given in units of peak area in the analytical chromatograms. The concentration profiles of two early eluting components (symbols A and 0 in Figure 8a) are qualitatively similar to those in Figure 3a. The third profile (symbol +) appears to be qualitatively similar to Figure 3b, and the last eluting impurity (symbol 0)is qualitatively similar to Figure 3c. Therefore, the profiles of the impurities agree well with theoretical predictions. These results demonstrate the potentially dramatic influence of the displacer impurities on the band profiles of the sample components, the strong need to use a pure displacer or to purify it carefully before starting a displacement experiment, and the definite requirement to make sure that any remaining impurities are harmless to the performance of the products.
CONCLUSION As long as the displacer contains impurities that are less retained than the displacer, one band at least, the last one in the isotachic train, will be contaminated by this impurity. The smaller the retention of a displacer impurity, the larger the number of bands which it will contaminate. The higher the impurity concentration, the higher is its concentration in the solute band. In principle, the effect of an impurity could be reduced to some extent by increasing the sample loading. Accordingly,the displacer should be a high-purity chemical and strict specifications regarding its content in weakly retained components must be fulfilled. Alternately, the displacer should be chosen so that it and its impurities can be removed readily from the purified product and that their concentrations can be assayed easily. Only those impurities whose presence in the purified products is inconsequential can be tolerated. In some cases, these restrictions may render more difficult and costly the development of a separation method by displacement chromatography.
ACKNOWLEDGMENT We deeply thank VYDAC for the C18column used for the displacement chromatography and the adsorbent used in the column for the off-line fraction analysis. We acknowledge the help provided by Yong-Shen Zhang in drawing the figures. Registry No. Phenol, 108-95-2.
LITERATURE CITED Horvath, Cs. I n The Scknce of Chromatography; Bruner, F., Ed.; Journal of Chromatography Library; Elsevler: Amsterdam, The Netherlands, 1985,p 179;Vol. 32. Mlemerlch, F.; Klein, 0. MultJcomponent Chromato6xephy. A Theory of Interference; Marcel Dekker: New York, 1970. Rhee, H.-K.; Amundson, N. R. AIChE J. 1982. 28, 423. Kattl, A. M.;Guiochon, 0. J. Chromatogr. 1988, 449, 25. Gu, T.; Tsai, G.J.; Tsao, G. T. Biotechnol. Bioeng. 1991, 37, 65. MorbMelli, M.; Stortl, G.; Carra, S.; Niederjaufner, G.; Pontoglio, A. Chem. Eng. Sci. 1985,40. 1155. Subramanian, G.; Phillips, M. W.; Cramer, S. M. J. Chromatogr. 1988, 439, 341. Golshan-Shirazi, S.;Guiochon, G. Anal. Chem. 1989, 61, 1960. Gulochon, G.; Ghcdbane, S.; Golshan-Shirazi, S.; Huang, J. X.; Katti, A.; Lin, B. C.; Ma, 2. Talanta 1989, 36. 19. Frenz, J.; Horvath, Cs. AIChE J. 1985, 31, 400. DeCarll, J. P., 11; Carta, G.; Byers, C. H. AIChE J. 1990, 36, 1220. Cardinal, F.; Ziggiotti, A.; Viscomi, G. C. J. Chromatogr. 1990, 499,
37. Katti, A. M.;Dose, E. V.; Guiochon, G. J. Chromatogr. 1991, 540, 1. Antia, F.; Horvath, Cs. Ber. Bunsen-Ges. Phys. Chem. 1989, 93,
961. Frenz, J.; Bourell, J.; Hancock, W. S. J. Chromatogr. 1990, 512, 299. Ramsey, R. S.;Katti, A. M.; Guiochon, G. Anal. Chem. 1990, 62,
2557. Katti, A. M.;Guiochon, G. C. R . Acad. Sci., Ser. 2 1989, 309 (11),
1557. Jacobson, J.; Frenz, J.; Horvath, Cs. Ind. Eng. Chem. Res. 1987, 26, 43. Katti, A. M.; Guiochon, G. Am. Lab. 1989, 21 (lo),17. Golshan-Shirazi, S.;Guiochon, G. Anal. Chem. 1988, 60, 2364. Zhu, J.; Katti, A. M., Guiochon, G. J. Chromatogr.,in press.
RECEIVEDfor review April 2, 1991. Accepted July 1, 1991. This work was supported in part by Grant CHE-8901382 from the National Science Foundation and by the cooperative agreement between the University of Tennessee and the Oak Ridge National Laboratory. We acknowledge support of our computational effort by the University of Tennessee Computing Center.
CORRESPONDENCE Pulsed Rapid Heating Method for Volatilization of Biological Molecules in Multiphoton Ionization Mass Spectrometry Sir: Multiphoton ionization (MPI) mass spectrometry has been shown to be a very powerful technique for chemical analysis (1). In MPI, molecules are ionized by absorbing two or more photons from a pulsed laser beam and the resulting ions are detected by, usually, a time-of-flight mass spectrometer (TOFMS) (1-4)or a Fourier transform mass spectrometer (FTMS) (5-9). Advantages of using MPI as an ionization source for mass spectrometry include high sensitivity, high selectivity, and the ability of controlling the mass fragmentation patterns. Moreover, supersonic jet spectroscopy (SJS) has been combined with MPI mass spectrometry in a TOFMS to further enhance the sensitivity and selectivity of the MPI 0003-2700/91/0363-2188$02.50/0
technique (1,10,11). By incorporating SJS with MPI mass spectrometry, a two-dimensional detection scheme based on mass spectrum and jet-cooled wavelength spectrum can now be uniquely employed for identification of molecules. However, in the past, both MPI mass spectrometry and SJS were limited to the studies of volatile molecules. In order to extend the techniques for the study of thermally labile biochemicals, a method for the vaporization of these molecules without thermal decomposition must be developed. Thus, pulsed laser desorption (LD) (1, 12, 13) and, more recently, fast atom bombardment (FAB) (14)have been used for the generation of neutrals from nonvolatile and thermally labile 0 1991 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 63, NO. 19, OCTOBER 1, 1991
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Flgure 1. Schematic of the pulsed rapid heating probe and part of the experimental setup for muttiphoton ionization mass spectrometry and supersonic jet spectroscopy. Drawing is not to scale. The dimensions of the major components are given in the text.
molecules. In the LD or FAB experiments, a pulsed laser (e.g. C02laser) or a FAB gun is used to desorb molecules from a sample substrate placed close to the nozzle orifice. The resulting neutral molecules are then entrained into a pulsed supersonic jet that prevents them from thermal decomposition through collisional cooling and carries them into a TOFMS where MPI takes place. Both LD and FAB/supersonic jet MPI mass spectrometry methods have been applied for the detection of a variety of biochemicals (1, 12-14). Although the mechanisms for the generation of ions and neutrals by LD and FAB have not been completely understood, there is some evidence which suggests that neutral formation during the processes of LD and FAB can be described by a thermal heating model (15-20). Thus, in the pulsed LD and FAB/MPI experiments, we are using highenergy particles to deliver the energy to the sample and/or sample substrate; this energy is transferred into heat and vaporizes the molecules coated on the substrate. Clearly, a more straightforward and simple method to volatilize the sample is direct heating. But for the generation of gas-phase molecules of thermally labile molecules, the heating rate or the time taken for energy transfer to the sample molecules for vaporization of intact molecules is very important. I t is believed (21-29) that vaporization, i.e., dissociation of intermolecular bonds, is favored when a high heating rate is used and decomposition, i.e., dissociation of intramolecular bonds, is a dominant process if the heating rate is low. For a given compound, the optimal heating rate in favor of vaporization is difficult to measure currently and is dependent upon several factors including the sample property and its preparation. However, in general, the thermal decomposition is minimized with rapid heating. Friedman and co-workers (21-23) introduced a rapid sample-heating method to vaporize samples a t a heating rate of 12 K / s for electron impact mass spectrometry. Cotter and Fenselau (24,25) have also used heating rates of >10 K/s by inserting a Vespel sample probe directly into a hot ion source block, according to the method used by Hansen and Munson (26),to vaporize biochemicals. Daves and co-workers (27-29) used flash desorption to produce gas-phase molecules a t a heating rate of K/s. These techniques have been used for vaporization of biological molecules with some success. However, although these direct heating methods have advantages including low cost, simplicity, ease, safe operation, and adaptation compared with the pulsed LD and FAB desorption/vaporization techniques, one of the major disadvantages is that they cannot be used to introduce the sample into the system continuously without reapplying a new sample to the probe. Thus, the efficiency and sensitivity of this system suffers. Moreover, it is impossible to perform experiments such as supersonic jet spectroscopy, which require the sample to be introduced into the system in a continuous or pulsed form for a period of time. Therefore, it is desirable
to develop a rapid heating method with the capability of introducing samples into the system repetitively. It should be noted that even if the sample is thermally stable upon heating, repetitive pulsed heating of samples, compared with continuous heating, can enhance the sample utilization efficiency for pulsed laser ionization in a TOFMS. For example, Fassett et al. (30) have reported that a 30-fold improvement in sample utilization efficiency can be achieved for a pulsed thermal atom source using a miniaturizing Re filament relative to a continuous thermal source in a resonance ionization mass spectrometer system for the determination of atomic species such as iron. Here we wish to report a pulsed rapid heating method for volatilization of thermally labile and nonvolatile molecules for SJS and MPI mass spectrometry. This technique uses a heating probe that consists of an electrically heated plunger driven by a solenoid to desorb the sample in less than 210 ps a t a repetition rate of 10 Hz. In this report, the design of the heating device is first described. Preliminary studies on the performance of this technique are then presented. It is demonstrated that this method can be used to vaporize amino acids and dipeptides for SJS and MPI mass spectrometry. Finally, the potential applications of this technique for both biological molecule detection and the mechanistic studies of the heating nature of the LD and FAB processes are discussed.
EXPERIMENTAL SECTION The time-of-flight mass spectrometer setup has been reported previously (14,31,32). In brief, the system consists of an angular reflectron time-of-flight mass spectrometer (R. M. Jordan, Co., Grass Valley, CA) mounted vertically in a six-port cross pumped by a 6-in. diffusion pump (Varian Associates, Inc., Lexington, MA). A pulsed nozzle (R. M. Jordan Co., Grass Valley, CA) with a 50-ps pulse width is used to form a supersonic jet. C02 is used as the expansion gas throughout this work. The jet expands into the acceleration region of the TOF and a laser beam perpendicular to both the jet and flight tube ionizes the sample. No skimmer was used in this study. The 1 m long flight tube is differentially pumped by a 4-in. diffusion pump (Varian Associates, Inc., Lexington, MA). The pressure in the flight tube is usually below Torr, and the pressure in the ionization region is
