Anal. Chem. 1995, 67,4565-4570
Imaging of Thin-Layer Chromatograms Using Matrix-Assisted Laser Desorption/ionization Mass Spectrometry Arkady 1. G u s ~ v ,Olivier ~ * ~ J. Vasseur,t Andrew Pmctor,t Andrew 0. Sharkey,* and David M. Hewules*~5 Departments of Chemistry and Geology and Planetary Science, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, and Department of Chemistry, VandettM University, Nashville, Tennessee 37235
A new approach for directly coupling thin-layer chromatography (TLC) with matrix-assisted laser desorption/ ionization mass spectrometry is introduced. To avoid excessive analyte spreading along a TLC plate, a matrix layer was first generated on a different substrate, from which it was transferred to the plate. The transfer was accomplished by pressing the matrix onto a previously wetted TLC plate. Successful direct imaging of silica gel TLC plates with high spatial resolution (250-500 pm) and high sensitivity has been demonstrated for several compounds, including rhodamine B, guinea green B, bradykinin, and angiotensin11, using nanogram quantities of analyte. The ultimate spatial resolution was estimated to be -50 pm due to matrix heterogeneity and the minimum laser beam diameter required to produce good signal intensity. The current protocol induced minimal analyte planar diffusion (-0.5 mm) and resulted in an absolute detection limit in the picogram range.
TLC/MS SIMS and FAB experiments usually involve the use of a liquid matrix deposited onto the TLC plate; mostly conventional FAl3 matrices, Le., glycerol, triethanolamine, and thioglycerol, are sed.^,^,^.^ These matrices induce soft ionization and promote analyte transfer (extraction) toward the surface of the TLC plate. (The term analyte extraction in this paper means diffusion/transfer of the compounds toward and onto a plate surface but not out of a plate.) However, use of a liquid matrix also induces lateral spreading of the analyte along the plate. Herein lies the main dilemma of imaging. A liquid matrix provides efficient analyte extraction and good sensitivity but exhibits planar diffusion; amorphous matrices show limited planar diffusion but poor analyte extraction. One of the successful approaches, developed by Busch and co-workers, was to use a “meltable” matrix.loJ1 This technique allowed image analysisll with high spatial resolution.6 TLC/LD, utilizing either ultraviolet 0or infrared (IR) laser radiation, does not require a liquid matrix/extraction solvent. TLC/UV-LD produces high sensitivity and spatial r e s o l u t i ~ n ~ ~ - ~ ~ but induces significant fragmentation and suffers from poor Coupling of thin-layer chromatography (EC) with mass reproducibility. To minimize fragmentation, IR laser desorption spectrometric (MS) detection combines the simplicity of TLC with from a TLC plate followed by multiphoton ionization (MUPI) has the specific detection capabilities of MS.1,2 Indirect coupling been u ~ e d . l ~ -Although ’~ a low detection limit has been demonusually involves analyte elution from a TLC plate followed by MS strated for several compounds using this method, there are several analysis of eluted fractions or thermal evaporation of volatile drawbacks associated with MUPI. compounds from a plate. Separated analytes can also be introThus, complicated imaging analysis and complex instmmentaduced directly into a mass spectrometer on a TLC stationary phase tion, fragmentation, and relatively poor sensitivity have been the (adsorber) . The advantages of such direct TLC/MS analysis are main drawbacks of TLC/MS coupling to date. These problems its suitability for TLC imaging (two-dimensional scanning), nonare primarily connected with the protocol of TLC/MS coupling destruction of the TLC plate, and minimal sample preparatior~.~,~ and the method of ionization. The coupling protocol should Several MS methods have already been used for direct TLCI produce efficient analyte transfer toward the TLC surface and MS coupling: secondary ion mass spectrometry (SIMS), fast atom preserve spatial resolution. The crucial point in the coupling bombardment ,4-11 and direct laser desorption (LD) .13-18 +
Department of Chemistry, University of Pittsburgh.
* Department of Geology and Planetary Science, University of Pittsburgh. Department of Chemistry, Vanderbilt University. (1) Poole C. F.; Poole, S. K. Anal. Chem. 1994,66, 27A (2) Busch, K L. Thin layer chromatography coupled with mass spectrometry. In Handbook of thin layerchromatography; Sherma, J., Fried, B., Eds.; Marcel Dekker: New York, 1991; p 183. (3) Busch, K. L. Trends Anal. Chem. 1992,11, 314. (4) Oka, H.; Ikai, Y.; Kondo, F.; Kawamura, N.; Hayakawa, J.; Masuda, K; Harada. IC; Suzuki, M. Rapid Commun. Mass Specfrom. 1992,6, 89. (5) Nakagawa. Y.; Iwatani, K. J Chromafogr. Biomed. Appl. 1991,562, 99. (6) Busch, K L.; Mullis, J. 0.;Chakel J. A. /. Planar Chromatogr. 1992,5 , 9. (7) Busch, K L.; Mullis, J. 0.;Carlson R E. J Liquid Chromafogr. 1993,16, 1695.
(8) Duffin K. L.; Busch, K. L. /. Planar Chromafogr.-Mod.TLC 1988,1, 249.
0003-2700/95/0367-4565$9.00/0 0 1995 American Chemical Society
(9) Monaghan, J. J.; Morden, W. E.; Johnson, T.;Wilson, I. D.; Martin, P. Rapid Commun.Mass Specfrom. 1992,6, 608. (10) DiDonato, G. C.; Busch, K L. Anal. Chem. 1986,58, 3231. (11) Duffin K L.; Flurer, R A; Busch, K. L.; Sexton L. A; Dorsett, J. W. Rev. Sci. Instrum. 1989,60, 1071. (12) Novak, F. P.; Hercules, D. M. Anal. Left. 1985,18, 503. (13) Novak. F. P.; Wilk, 2. A,; Hercules, D. M. J. Trace Microprobe Tech. 1985, 3, 149. (14) Kubis, A J.; Somayajula, K. V.; Sharkey, A G.; Hercules, D. M. Anal. Chem. 1989,61, 2516. (15) Roger, IC;Milnes, J.; Gormally, J. Inf.J Mass. Specfrom.Ion Processes 1993, 123, 125. (16)Fanibanda, T.; Milnes, J.; Gormally, J. Inf./. Mass. Specfrom. Ion Processes 1994,140, 127. (17) Li, L.; Lubman, D. M. Anal. Chem. 1989,61, 1911. (18) Krutchinsky, A N.; Dolgin, A I.; Utsal, 0. G.; Khodorkovski, A. M.1. Mass Spectrom. 1995,30, 373.
Analytical Chemistry, Vol. 67, No. 24, December 15, 1995 4565
protocol is matrix selection. The best matrix should provide soft ionization and efficient extraction toward the surface while preserving spatial resolution. In other words, the matrix should be a liquid for optimal extraction and a solid after extraction to prevent planar analyte spread. We have previously reported a combination of matrix-assisted laser desorption/ionization (MALDI) with TLC which was used to compensate for some of the above pr0b1ems.l~ Some MALDI featuredparameters make the method extremely attractive for TLC/MS coupling. First, the MALDI method has broad utility for qualitative and quantitative analysis.20-2i MALDI has been used for peptides and proteins, oligosaccharide^,^^^^^ enzymatic digests,29and underivatized DNA oligomer^.^^,^^ Second, ionization is efficient using a large excess of matrix compared to analyte, the optimum matrix/analyte ratio being -lOOOO/l (see, for example, ref 321. Third, a MALDI matrix can be applied initially as a solution to improve analyte extraction and thereafter can be crystallized to prevent analyte spreading. In addition, MALDI matrices have good vacuum stability. An absolute detection limit of 2-4 ng was demonstrated for the TLC/MALDI combination using bradykinin, angiotensin, and enkephalin derivative^.'^ However, this detection limit is still poor compared to the conventional MALDI detection limit, which is in the picogram range. Another problem concerns imaging. Even with a simplified'coupling protocol, analyte spreading was found to be -1.5 mm. To compensate for sample heterogeneity, the signal intensities were averaged for five successive laser spots, which limits the spatial resolution to -1 ".I9 Thus, the purpose of the present work is to develop an approach which will primarily compensate for analyte spreading and allow imaging with higher spatial resolution. This protocol is also optimized for maximum extraction efficiency (low detection limit) and minimal analyte spreading. EXPERIMENTAL SECTION
The TOF laser mass spectrometer employed in these investigations was a modiiied LAMMA 1000 (Leybold-Heraeus GmbH) laser microprobe using a nitrogen laser (VSL 337 ND, Laser Science Inc., Cambridge, MA) emitting at 337 nm. The laser beam was focused using a telescopic lens arrangement. Changing the distance between lenses allowed beam defocusing. The results presented here were obtained with the laser beam defocused to (19) Gusev, A I.; Rabinovich. Y. I.; Proctor, A,; Hercules, D. M.Anal. Chem.
1995, 67,1805. (20) Hillenkamp, F.; Karas, M.; Beavis, R. C.; Chait, B. T. Anal. Chem. 1991, 63,1193A. (21) Vertes, A; Gijbels, R. In Laser Ionization Mass Analysis; Vertes, A,, Gijbels, R.,Adams, F.,Eds.; John Wiley & Sons: New York, 1993;p 127. (22) Duncan, M. W.;Matanovic, G.; Cerpa-Poljak, A. Rapid Commun. Mass Spectrom. 1993, 7,1090. (23) Nelson, R W.; McLean, M. A,; Hutchens. T. W. AnaLChem. 1994,66,1408. (24) Muddiman, D. C.;Gusev, A I.; Proctor, A; Hercules, D. M.; Venkataramanan, R.; Diven, W. Anal. Chem. 1994,66, 2362. (25) Jespersen, S.; Niessen, W. M. A; Tjaden, U. R; van der Greef, J. J. Mass Spectrom. 1995,30,357. (26) Lidgard, R; Duncan, M. W. Rapid Commun.Mass Spectrom. 1995,9, 128. (27) Harvey, D. J. Rapid Commun.Mass Spectrom. 1993, 7, 614. (28) Stahl, B.; Steup, M.; Karas, M.; Hillenkamp, F. Anal. Chem. 1991,63,1463. (29) Schar, M.: Bornsen, K 0.;Gassman, E. Rapid Commun. Mass Spectrom. 1991, 5,319. (30)Wu, K J.; Steding, A; Becker, C. H. Rapid Commun.Mass Spectrom. 1993,
7,142. (31) Wu, K J.: Shaler, T. A; Becker C . H. Anal. Chem. 1994,66, 1637. (32)Gusev, A I.; Wilkinson, W. R.; Proctor, A; Hercules, D. M. Anal. Chem. 1995, 67.1034.
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Analytical Chemistry, Vol. 67, No. 24, December 15, 1995
-120 x 80 pm unless otherwise stated. Positive ions were postaccelerated to 16 keV onto a discrete dynode type secondary electron multiplier (SEW (EM119/2, Thorn EMI, Rockaway, NJ). The spectral acquisition system consisted of a 200 MHz transient recorder (TR 8828 B, LeCroy Inc., NY) with a PC-based data processing system. Modifkations are described in detail elsewhere.32 The pressure was (1-2) x mbar in the sample chamber and 3 x mbar in the analyzing chamber. The instrument contains a precise motorized X I Z manipulator and a microscope which allow control of plate position, visual selection, and examination of the point of laser interaction with the sample. Spatially resolved mass-spectral data (images) were obtained by moving the plate using the XIZmanipulator. All data acquisition and most secondary processing software (except threedimensional) were written in-house (GOOGLY). Standard processing included data acquisition, spectral smoothing, mass calibration, measurement of the intensities, and plotting of contour and/or 3-D images. Sample Preparation. The matrix used in this study was acyano-4hydroxycinnamic acid (a-CHCA)33 mixed with a+ fucose, both purchased from Sigma Inc. (St. Louis, MO). The matrix solution concentration was 15 mg/mL (a-CHCA)/5 mg/ mL (fucose), prepared daily by dissolving in acetone. The following analytes were used: bradykinin (FW 1060.2), angiotensin I1 (FW 1046.2), rhodamine B (FW 479.0), and guinea green B (FW 690.8). The analytes were dissolved in methanol. All solvents were HPLC grade. TLC Separation. Analytes were separated on silica gel 60 aluminum-backed TLC plates having an organic binder (E.Merck, Germany) and silica gel 60 plastic-backed plates with a gypsum binder or binder free adsorbent (Polygram SIL G and SIL N-HR, Macherey Nagel, Germany). Both plates had a fluorescent indicator. The solvent systems used for development were as follows: for bradykinin, water/methanol/acetic acid (44:506) with either 3% KCl or 3% NaCl or without salt; for angiotensin 11, 1-butanol/pyridine/aceticacidhater (15:15:312); and for rhodamine B and guinea green B, ethyl acetate/methanol/water (80:20:20). The solvents for bradykinin and angiotensin separation on silica gel were used as described in refs 8 and 19 and produced similar Rfvalues. Samples were applied to the plates as small spots with diameters less than 2 mm. Separation was carried out at room temperature. To avoid a large abundance of sodium and potassium adducts, the plates were pretreated by developing and washing in a methanol/water solution as described e1~ewhere.l~ TLC plates were cut to 20 x 20 mm to 50 x 50 mm after separation. Ninhydrin (0.2% in ethanol, Sigma) was used for bradykinin and angiotensin sample visualization. RESULTS AND DISCUSSION
Coupling Protocol. The coupling protocol addresses the following: analyte extraction from the adsorber (transfer/ transportation toward the surface), matrix deposition, and crystallization. These processes are closely connected, and each step can induce analyte spread. Different MALDI matrices and methods of direct matrix deposition/crystalliiation Cgently spraying, vacuum evaporation, air-forced evaporation, etc.) on the TLC plate were tried. Some of the methoddmatrices produced better detection limits, but all induced additional analyte spreading of (33)Beavis, R. C.; Chaudhary, T.; Chait B. T. Org. Mass Spectrom. 1992, 27, 156.
m \
I
A. Matrix deposition ~
B.Drying
Rhodamine-B
,I
Stainless steel
f
.
D. Pressing
-\
7
1 1
F. MALDI analysis
matrix layer
matrix laver
C. Spraying of extraction solution on TLC plate
1
k
-
350
400
450
500
E. Stainless steel removed
C Figure 1. TLC/MALDI coupling procedure. 0
200
400
600
800
1000
MASS (Da)
1.5-5 mm. Siice analyte spreading exists even without extraction, the ultimate cause of analyte spreading is matrix crystallization, which causes extensive convection (mixing) and, hence, analyte spreading. To overcome this problem, we decided to separate the process of analyte extraction and matrix deposition/crystallization. Consequently we proposed to first crystallize the matrix onto a substrate other than the TLC plate, followed by transfer of the matrix layer onto the TLC plate. We called such an approach indirect matrix defiosition compared to the direct deposition of a matrix on a TLC plate.Ig There are at least three major problems that must be addressed: (i) matrix crystallization over a large substrate area, (ii) matrix transfer, and (iii) analyte incorporation into the matrix structure. To create a large area (50 x 50 mm) of a matrix layer, we used a modified approach of the fast matrix evaporation meth~d.A ~ ~mixture , ~ ~ of a-CHCtVfucose in acetone was deposited onto a stainless steel substrate (0.003 in. thick up to 50 x 50 mm) and dried using gentle air flow. The fucose comatrix did not completely dissolve in acetone but created a suspension which served as crystal nucleation points. This gave superior results over deposition of neat a-CHCA The sample preparation protocol is illustrated in Figure 1. After matrix crystallization on stainless steel (Figure lA,B),the matrix layer was transferred to the TLC plate. Transfer was accomplished by pressing the matrix substrate (matrix layer facing toward the plate) onto a TLC plate previously wetted by the extraction solvent, (Figure lC,D). The pressing step also provides analyte extraction by forcing analyte diffusion when squeezing the adsorber layer under pressure. The pressure should be high enough to maximize matrix transfer and to force (induce) analyte extraction toward the surface. After the pressure was released, the stainless steel substrate was removed (Figure 1E). The critical point of the protocol is use of an extraction solvent. The extraction solvent serves for (i) matrix layer transfer, (ii) analyte extraction, and (iii) analyte incorporation into the matrix structure. It should only partially dissolve the matrix to prevent complete matrix (34) V o m , 0.;Roepstorff, P.; Mann, M. Anal. Chem. 1994, 66, 3281. (35) Nicola, A J.; Gusev, A I.; Proctor, A; Hercules, D. M. Rapid Commun. Mass Spectrom., in press.
Figure 2. Mass spectra of rhodamine B separated on plastic-backed
TLC plates (binder-free adsorbent) with different quantities of analyte: (A) 10 ng, (6) l ng, and (C) blank sample (no rhodamine B).
dissolution or recrystallization. It should also promote analyte diffusion. A mixture of 1:l (v/v) methanol/water was found to be a good solvent for the a-CHCtVfucose matrix. It produced complete transfer of the matrix layer onto the TLC plate for a pressure range of 4-15 kg/mm2. (Higher concentrations of methanol or addition of acetonitrile led to complete dissolution of the matrix crystals.) After the matrix transfer and analyte extraction procedure is completed, the TLC plate with a matrix can be attached to the XYZ manipulator and introduced into the mass-spectrometerfor MALDI analysis, Figure 1F. The homogeneity of the matrix crystal structure on a silica gel plate was examined using a light microscope (Heerbmgg, Switzerland) and controlled during analysis using the microscope incorporated into the MS. The surface coverage was found to be 100% over the 50 x 50 mm plates for a matrix deposition of 0.20.4 pL/mm2 (4-8 pg/mm2). On a small scale, the matrix crystal layer on silica gel showed a little heterogeneity, 15-10pm. Thus, the optimum parameters of TLC/MALDI coupling were (i) 0.20.4 ,uL/mm2 (4-8 pg/mm2) of matrix solvent deposited on stainless steel, (ii) 4-8 kg/mm2 pressure for matrix transfer, and (i) 0.15-0.25 pL/mmz of extraction solvent (1:l methanol/water) deposited on a TLC plate. Several technical details should also be noted. Spraying of the extraction solvent should be gentle and homogeneous. The solvent amount should be a minimum,just enough to wet the gel. Excess solvent led to analyte spreading along the top of the plate. To produce a good quality (homogeneous) matrix layer on a TLC plate, the plate should be precisely parallel to the matrix stainless steel substrate during pressing. Even a small misalignment can result in migration of the extraction solvent. Furthermore, silica gel is very fragile, so pressure deviations can easily crack the layer. For the same reason, removal of the matrix substrate from a TLC plate (after pressing) should be very gentle. Detection Limit. Figure 2 shows mass spectra of rhodamine Analytical Chemistry, Vol. 67, No. 24, December 15, 1995
4567
i
A Rhodamine B
300 400
III
.-
200
>
400
c
;'
-6,
11
1
A
'i
I!
-
6 -
(d
a:
300 -
-
C 600
800
1000
1200
1400
MASS (Da) 0
500
1000
1500
MASS (Da)
Figure 3. Mass spectra of rhodamine B separated on aluminumbacked TLC plates (organic binder) with different laser irradiances: (A) 40% above the threshold, ( 5 ) 100% above the threshold, and (C) 100% above the threshold, blank sample (no rhodamine 5).
obtained on plastic-backed plates with different quantities of analyte. The main peak at 444 Da corresponds to the cation, formally (M - Cl)+. Matrix peaks usually occur up to 250-400 Da, which may possibly limit TLC/MALDI analysis of low-mass compounds. However, applications of MALDI in the low-mass range have been A detection limit was estimated on the basis of the spectral S/N ratio. The relatively large analyte quantities used in this experiment were necessary for analyte visibility on the TLC plate, which is essential for the imaging experiments. Using the S/N criterion, the detection limit for rhodamine B was found to be -40 pg. The pressure used for matrix transfer was 6 kg/mm2. The optimal transfer time (time of extraction) was found to be 2-3 min; longer extraction times did not produce an increase in signal intensity. This indicates that equilibrium is reached in less than 2-3 min. Since diffusion of similar compounds was found to take longer times,Igthis confirms that the analyte is extracted due to adsorber squeezing rather than diffusion. It is interesting to note that an increase in a polymer signal in the range of 800-1500 Da was seen for aluminum-backed TLC plates. We believe that this effect resulted from a polymer used as an organic binder. This polymer has a repeat unit of 44 Da, indicating that it is a poly(ethylene glycol). No high-mass signal (> 350-400 Da) was found for the plastic-backed TLC plates which utilize gypsum or have a binder-free adsorbent (see Figure 2). The optimum laser energy was found to be -40-60% above the threshold for all analytes. This is a very critical parameter, particularly for a Tu:plate with a polymer binder. Excessive laser energy led to suppression of the analyte signal and an increase in background. Figure 3 shows mass spectra obtained with normal laser power (40-60% above the threshold) and high power (-100% above the threshold). The ultimate detection limit was found to be almost the same for both plastic-backed TLC plates 4568
Analytical Chemistry, Vol. 67, No. 24, December 75,7995
Figure 4. Mass spectra of angiotensin II (20 ng) on stainless steel (A) and silica gel plastic-backed TLC plate (B).
(gypsum and binder-free adsorbent) and aluminum (polymer binder)-backed plates, although the latter require more precise tuning of the laser power. Thus, the optimum parameters for matrix transfer/analyte extraction were the same for both silica gel TLC plates, i.e., 6 kg/mm2 of pressure and 2-3 min of extraction. It should also be noted that use of a silica gel substrate decreased resolution -20-50% compared to stainless steel. The same effect was reported for MALDI analysis of proteins on blotting nylon membranes.36 The authors suggested that this was a result of surface charging. Figure 4 shows the mass spectra of angiotensin I1 obtained on stainless steel (A) using the standard sample preparation and on a silica gel aluminum-backed TLC plate (B) (no separation) under optimum conditions. The same amount of matrix was used in both cases to keep the same matrix/analyte molar (M/A) ratio. Since the exact quantities of matrix and analyte on the plate surface after matrix transfer and analyte deposition are unknown, the initial amounts of analyte and matrix were used for the M/A calculations. Comparison of the intensities shows no significant difference, which demonstrates the efficiency of the proposed indirect method of matrix deposition and analyte extraction. It also confirms that sensitivity is determined by the M/A ratio. The detection limit was found to be -100 pg without separation and -200 pg after separation. Essentially the same detection limit was found for bradykinin. This is at least 10 times better than that reported for direct matrix depo~ition.'~ However, it should be noted that conventional MALDI analysis of angiotensin on stainless steel under optimum conditions produced detection limits of -5 pg.35 The M/A molar ratios obtained for 2 ng of angiotensin I1 and for the detection limits of angiotensin I1 using indirect and direct matrix deposition on Tu: plate and fast evaporation method on a stainless stee134a35 are presented in Table 1. One can see that the M/A ratios at the detection limits are essentially the same. The small difference between conventional MALDI and TLC/ (36) Zaluzec, E. J.; Gage, D. A: Allison,J.; Watson, J. T. J. Am. SOC.Mass Spectrom.
1994.5. 230.
Table 1. Matrix to Analyte Ratios Obtained for Different Ouantlties of Angiotensin II and Methods of Matrix Deposition
quantity of analyte method of analysis
2 ng
indirect matrix deposition on TLC plate 2 direct matrix deposition on TLC plate 4 conventional MALDI analysis
For 5
x
4 mm analyte spot. For 2
1 x
x x
lo5
lo6
104 b
detection limit 2 x lo6 a (0.2 ng) 4 x lo6 (2 ng) 4 106 b (5 pg)
2 mm analyte spot.
MALDI can be explained by the analyte recovery factor, which increases the "real" M/A ratio. Conversely, the M/A ratios obtained for 2 ng deposition differed by 2 orders of magnitude, which is related to the different matrix quantitiesused for sample preparation. These results suggest that the major factor which limits absolute detection limit is the M/A ratio, although extraction and separation efficiency cannot be totally neglected. Hence, improvement of the detection limit is primarily connected with a decrease in the M/A,ratio. However, attempts to use less matrix led to poor surface coverage and matrix heterogeneity on the TLC plates. This problem is under investigation. Direct TLC Plate Imaging. Two dyes were used to investigate analyte spreading: rhodamine B and guinea green B. The amount of dye deposited ranged from 1 to 100 ng. These compounds were used because they are visible at a concentration of 1-5 ng/spot. The two dyes were separated by TLC unless otherwise stated. Comparison of the size of the initial spots with the same spots after matrix transportation/crystation showed that indirect matrix deposition produced minimum analyte spreading, which did not exceed -0.5 mm for all plates used. The spread possibly results from excess extraction solvent on the top of a plate which occurred after the adsorber was squeezed and can lead to spreading onto the top. Three-dimensional (3-D) images were obtained on silica gel plates by plotting the spatial dimensions x and y versus absolute ion intensity, which should be proportional to the analyte concentration. The following conditions of matrix transfer and analyte extraction were used: 1:1 methanol/water extraction solvent: 5-7 kg/mm2 of pressure, and 3 min of extraction. The 3-D image of guinea green B obtained from a silica gel TLC plate (organicbinder) is shown in Figure 5. The peak at 669.8 Da was used for intensity measurements. The spectra were collected with 0.50 mm steps. (The smaller step size of the grids results from image interpolation and smoothing.) Similar results were obtained with rhodamine, bradykinin, and angiotensin 11. The 3-D image of angiotensin I1 (100 ng) and a contour plot obtained from a silica gel TLC plate (organic binder) are shown in Figure 6. The analyte was visualized with ninhydrin. Both protonated and ninhydrin adduct peakslgwere used for intensity measurements. The image was acquired with a step size of 0.50 mm. The 3-D image of rhodamine (5 ng) and its contour plot obtained from a silica gel TLC plate (binder-free adsorber) are shown in Figure 7. The peak at 444 Da was used for imaging. The image was acquired with a step size of 0.25 mm, which resulted in more than 15 x 25 pixels per analyte spot, which is more than enough to accurately describe the analyte distribution. The contour plot confirms that the analyte spread does not exceed
Figure 5. Three-dimensional image of guinea green B (20 ng), separated on silica gel plastic-backed TLC plate. The spectra were collected using a 0.5 mm step.
0.5 mm. Furthermore, the relative increase of the spot area did not exceed 10%. For some TLC points, because of matrix heterogeneity, we had to move the laser spot position *30 pm to maintain a signal. Attempts to use a laser beam spot size less than -30 x 20 pm led to signal degradation and poor signal reproducibility. Results recently published by Hillenkamp et al.37 showed that decreasing of the laser spot size to less than 25 x 15 pm caused a rapid decrease in signal intensity. Thus, assuming a matrix heterogeneity of 10-20 pm and minimum laser spot size of 30 pm, the ultimate spatial resolution can be estimated to be -50 pm. It should be noted that even though the detection limits were estimated to be 200 pg for angiotensin I1 and 50 pg for rhodamine B, imaging requires considerably more analyte. However, modification and further optimization of the matrix deposition protocol and analyte extra&on/transfer to the surface should improve signal reproducibility and decrease the detection limits and hence the amount of sample required for imaging. We consider imaging of nanogram or even hundreds of picogram quantities of an analyte to be feasible. CONCLUSIONS Successful imaging of several analytes directly from a thinlayer chromatographyplate with high spatial resolution and high sensitivity using matrix-assisted laser desorption/ionization mass spectrometry has been demonstrated. Indirect MALDI matrix deposition with transfer of the matrix layer to the TLC plate minimized analyte spreading along the plate and gave low detection limits. The proposed method of indirect matrix deposition/crystallization minimized analyte spreading to 0.5 mm and improved detection to 50-200 pg, which is at least 10 times better than that obtained with direct matrix deposition. Imaging was demonstrated for several compounds, including rhodamine B, guinea green B, bradykinin, and angiotensin I1 using nanogram quantities of analyte. Spatial resolution of the TLC plate image was (37) Dreisewerd, IC; Schiirenberg, M.; h a s , M.; Hillenkamp, F. Int. 1.Mass
Spectrom. Ion Processes 1995, 141, 127. Analytical Chemistry, Vol. 67,No. 24, December 15, 1995
4569
.
11 00-
I
10 00-
9 00-
a 007 00-
6 00-
3 00-
1
2 00
Figure 6. Three-dimensional image (A) and contour plot (8)of angiotensin II (100 ng) separated on silica gel plastic-backed TLC plate. The image was collected using a 0.5 mm step.
OOOJ
000
' I
'
200
400
6.00
1' 800 , I ?1000 1 distance, m m
Figure 7. Three-dimensional image (A) and contour plot (B) of rhodamine B (5 ng) separated on silica gel aluminum-backed TLC plate. The image was collected using a 0.25 mm step. The dotted line gives an outline of the initial spot.
established at 250 pm. The imaging spatial resolution is limited by matrix heterogeneity and the size of the laser spot required to produce good quality spectra. Thus, ultimate spatial resolution was estimated to be -50 pm. Three types of TLC plates with organic and inorganic (gypsum) binders and binder-free adsorber were used in this study. The plates with the organic binder produced a polymer signal [prob ably poly(ethy1ene glycol)], which was particularly strong for high laser irradiance. However, the ultimate detection limits were found to be almost the same for the three types of plates, although the plate with an organic binder required more precise tuning of the laser power.
4570 Analytical Chemistry, Vol. 67, No. 24, December 15, 1995
ACKNOWLEDGMENT
This work was supported by the U.S. Environmental Protection Agency under Grant R819809-01.
Received for review May 16, 1995. Accepted September
5, 1995.@ AC950472+ Abstract published in Advance ACS Abstracts, October 15, 1995.
