Anal. Chem. 1986, 58,3231-3232
3231
CORRESPONDENCE Phase-Transition Matrix for Chromatography/Secondary Ion Mass Spectrometry Sir: Several investigators have analyzed static ( I ) chromatograms by particle-induced desorption mass spectrometry (2-7). Most experiments involve an excision of the chromatographic spot of interest, which is located on the chromatogram by an independent means of detection. A solvent is then used to extract the sample material from the chromatographic matrix, and the solution is analyzed as a discrete sample. For example, both glycerol and diethanolamine have been used to extract samples from silica thin-layer chromatographic plates. Such analyses can compromise the unique analytical advantages of the static chromatography/mass spectrometry experiment, namely, the retention of chromatographic integrity, the use of a dimensional probe to gain an independence of access order, and a spatial resolution which can be varied as necessary to define sample coordinates. The most obvious disadvantage of solvent extraction of sample from the chromatographic matrix is the loss of spatial resolution. The liquid solvent not only extracts material from within the bulk of the matrix but, if it is absorbed into the chromatogram, can redistribute sample across a much larger spot than that inherent to the chromatographic separation. Yet direct analysis of chromatograms without the extraction solvent is not generally feasible. Each of the particle-induced desorption techniques that has been used analyzes material at the surface of the sample. Secondary ion mass spectrometry (SIMS), for example, samples from perhaps the top 100 A of the surface. The bulk of the sample residing in the chromatogram is inaccessible, unless a depth profiling experiment is used. Unfortunately, a t the higher primary ion current densities necessary to sputter through the chromatogram, significant damage to the relatively fragile organic molecules also occurs. Lower primary ion current densities have been used to sputter organic samples from chromatograms,but the absolute flux of secondary ions was low (2, 3). Such a restriction is not necessary if a reservoir of sample, extending into the bulk of the chromatogram, can be established. However, the surface resolution of the chromatogram should not be compromised. Extraction in the z plane without diffusion in x and y is accomplished in our experiments with the use of a matrix that can be controllably cycled through the solid/liquid phase transition. In the solid phase, no diffusion of samples through this matrix occurs. With liquefication, samples in the chromatogram are extracted into a solvent reservoir, which provides a stable, persistent signal of organic secondary ions. An appropriate balance of properties between the solvent and the chromatographic matrix limits the xy diffusion to acceptable values. We here demonstrate the use of such a phase-transition matrix for the direct analysis of several classes of samples from silica gel thin-layer chromatograms and from a paper matrix is used used for electrophoresis. Threitol(1,2,3,4-butanetetrol) as the phase-transition matrix, although other low melting point matrices can be used as well. Threitol melts at 70-72 "C,a temperature easily reached with mild heating. The ability of threitol to dissolve a variety of organic samples appears to be similar to that of glycerol. The secondary ion mass spectrometer used for these experiments has been de-
scribed elsewhere (8,9). Cesium ions (0.2-mm-diameterspot at the surface, 10-keV energy, A/cm2) were used as the primary ions. Secondary ions are extracted through a Bessel Box energy filter into a quadrupole mass analyzer. Several methods have been developed for the incorporation of threitol into the chromatographic system. A small amount of threitol(5-10%) can be dissolved in the solvent system used for chromatographic development. This approach has been used in the direct analysis of phenothiazine-based drugs from thin-layer chromatograms, but some tailing of the sample spots has been observed. For chromatograms that have been developed independently, the threitol is overlayered onto the surface of the-chromatogram by means of spray deposition. The intact chromatogram is then placed in the SIMS instrument for analysis. In some experiments, the threitol can be maintained in the liquid state simply by heat transfer from the heated metal sample support. A charge-compensation filament inside the vacuum system also provides sufficient heat to liquefy the threitol. At higher primary ion current densities, heating by the primary ion beam itself is sufficient to melt the threitol precisely at the point of analysis, providing a spatially discrete extraction. The extraction behavior of threitol is compared to that of glycerol, which has been used in other studies (5, 6). With thin silica gel plates, the glycerol efficiently extracts the sample from the chromatographic matrix, but, after only a few minutes, is absorbed into the silica gel. Threitol, by contrast, is not readily adsorbed by the silica and, as a liquid, remains at the surface. Since the chromatogram is thin (100 pm), in-depth extraction occurs, but no xy diffusion is observed. Furthermore, no charging of the sample is observed, since as the threitol is liquefied, a conductive path between the bombarded surface and the aluminum backing plate is created at the site of analysis. Methionine enkephalin was used as a model compound to demonstrate the efficiency of the chromatographicextraction. Absolute sensitivity for discrete samples of this pentapeptide is %fold better with use of threitol as compared to glycerol, presumably because of differing surfactant properties. When these solvents are compared in use as extractants of the sample from the chromatogram, the long-term stability of the signal for the protonated molecular ion is much better for threitol, which is not absorbed into the chromatogram. In fact, 10 pg of peptide provides a stable ion current for 20 min. The spectrum of methionine enkephalin extracted from a chromatogram is the same as that of discrete sample and from a threitol matrix, similar to that reported from a glycerol matrix (IO, 11). Threitol itself does contribute to a background spectrum below m / z 300, including abundant ions at m / z 123 (M + H)' and m / z 245 (2M + H)+. The relative contribution of matrix signals from glycerol and threitol is currently being quantitatively assessed. The chromatography/SIMS instrument is being evaluated for the direct analysis of mixtures of phenothiazine-based drugs separated by thin-layer chromatography with a silica gel substrate. Threitol is used to extract the drug from the chromatographic matrix and provide a persistent current of
0003-2700/86/0358-3231$01.50/0 0 1966 American Chemical Society
3232
ANALYTICAL CHEMISTRY, VOL. 58, NO. 14, DECEMBER 1986 245
Cortioortsrone/Oirrrd'r P Silica TLC
Acepromrzine 5iliar TLC Threitol
I.*
,
I
a+@+ 321 *
1
3.0
154
~
I
nlz I F@ue 1. Positive ion SIMS spectrum of acepromarineseparated by
silica gel thin-layer chromatography and extracted from the chromatogram by iiquefled threitol. 245
Cortioo8toronel Glrard'i P Whatman 3YY Threltol
I
C+ 480
I I i-J* I
300
400 MlZ
Flgure 2. Positive ion SIMS spectrum of corticosterone(derivatized with Girard's P reagent) extracted from an electrophoretic matrix with liquefied threitoi.
secondary ions. Figure 1 is the positive ion SIMS spectrum of acepromazine separated by silica gel thin-layer chromatography and extracted with the liquid threitol matrix. The total sample size was 5 pg, and the spectrum shown persisted for 15 min. Similarly, steroids can be analyzed directly from chromatographic matrices by spatially resolved secondary ion mass spectrometry. The use of electrophoresis requires the molecules to be separated be in an ionic form, accomplished in this case by derivatization of the steroid molecule with Girard's P reagent (12). The positive ion SIMS spectrum of corticosterone so derivatized, and extracted directly from an electrophoretic paper medium, is given in Figure 2. The ion at mlz 480 is the intact cation C+ of the derivatized molecule. Since the SIMS spectrum is generated from the liquid solvent, the nature of the underlying chromatographic matrix does not affect the mass spectrum. Thus, the SIMS spectrum of derivatized corticosterone extracted from an electrophoretic medium is the same as that of derivatized corticosterone extracted from a silica gel. Of practical importance is the extent of liquid absorption into the matrix and the preservation of spatial resolution. Threitol is slow to absorb into either the silica gel or the electrophoretic paper. The ultimate experiment for which the chromatography/ SIMS instrument was constructed is the acquisition of spatially resolved mass spectra for mixture components separated by chromatography. Figure 3 illustrates this capability. The intact cation of the derivatized corticosterone is monitored by the mass spectrometer. The chromatographic spot is ex-
x
u
10
x (mm) Figure 3. Spatially resolved data for the intact cation of derivatized corticosterone ( m l z 480) monitored over x and y . On the left is the threedimensional plot for the relative abundance of the m l z 480 ion. The contour plot for the same data is given on the right. tracted in situ with liquefied threitol, and the intensity of the signal is recorded at constant x and varying y . The signal is recorded every 0.1 mm along each axis, and the data are assembled into a three-dimensional plot of ion abundance vs. x and y . The variation in ion abundance is that expected for the spot size and the sample size. A finely focused liquid metal ion gun is presently being added to the system that should allow a spatial focus as fine as 1 pm, variable between this value and 0.5 mm, and rasterable across up to 15 mm ( x and y ) of the chromatographic surface. Electronic variation of the spot size of the primary ion beam is used in the "step-back, stop-down" experiment, an algorithm that automatically varies the spot size as necessary to resolve sample boundaries (13). Registry No. Threitol, 7493-90-5.
LITERATURE CITED Fenirnore, D. C.; Davis, C. M. Anal. Chem. 1981,53, 252A. Ramaley, L.; Vaughan, M.; Jarnieson. W. D. Anal. Chem. 1985,5 7 , 353-358. Unger, S. E.; Vincze, A.; Cooks, R. G.; Chrisman, R.; Rothrnan, L. D. Anal. Chem. 1983,5 3 , 976-981. Unger, S. E.; Ryan, T. M.; Cooks, R. G. S I A , Surf. Interface Anal. 1981,3 , 12-15. Chang, T. T.; Lay, J. O., Jr.; Francel, R. J. Anal. Chem. 1984, 5 6 , 109-1 11. Tantsyrev, G. D.; Povoiotskaya, M. I.; Saraev, V. A. Bioorg. Khim. 7984, IO, 648-852. Kushi, Y.; Handa, S. J . Biochem. 1985,9 8 , 265-267. Fiola, J. W. M.S. Thesls, Indiana University, Bloornington, IN, 1986. Fiola, J. W.; DiDonato, G. C.; Busch, K. L. Rev. Sci. Instrum., in press. Barber, M.; Bordoli, R. S.; Garner, G. V.; Gordon, D. 5.; Sedgwick, R. D.; tetler, L. W.; Tyler, A. N. Blochem. J . 1981, 197, 401-404. Katakuse, I.; Desiderio, D. M. In!. J . Mass. Spectrom. Ion Processes lM3, 5 4 , 1-15. DiDonato, G. C.; Busch, K. L. Blomed. Mass Spectrom. 1955, 12, 364-366. DiDonato, G. C.; Busch, K. L. Abstracts of the Pittsburgh Conference and Exposition of Analytical Chemistry and Applied Spectroscopy, 1988; p 991.
Gerald C. DiDonato Kenneth L. Busch* Department of Chemistry Indiana University Bloomington, Indiana 47405 RECEIVED for review April 1,1986. Resubmitted June 2,1986. Accepted July 25, 1986. The Whitaker Foundation and the Society of Analytical Chemists of Pittsburgh have supported the development of the chromatography/SIMS instrument. The Research Corporation has provided partial funding for the liquid metal ion gun.
