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Title: Analysis of Hydrophobic and Hydrophilic Silica TLC Plates by LAESI-MS Authors: Trust Razunguzwa, Pamela Williams PAN00006 Introduction Thin Layer Chromatography (TLC) has been an indispensable tool to separate complex mixtures in analytical chemistry since the pioneer studies by Kirchner et al. in the 1, 2 1950s . TLC is convenient, simple, fast, and a cost efficient alternative to LC (liquid chromatography) methods, in which the latter uses a significantly larger volume of solvents. In addition, multiple samples can be spotted and separated simultaneously on a single TLC plate in comparison to conventional liquid chromatography. Currently, TLC remains an important separation technique and there are still TLC research applications in food, pharmaceutical, forensic 2, 3 sciences, clinical, and synthetic chemical industries . Traditionally, TLC plates are stained or labeled to provide information on the retardation factor (Rf) based upon the 3 position of the analyte . Traditional TLC staining techniques do not give a clear identification of sample spots; however, coupled with mass spectrometry (MS), analytes separated by TLC can be identified and confirmed with MS/MS analysis. TLC and MS have been used in combination for 2 the past 30 years . The majority of the experiments focused on Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) analysis of TLC plates. MALDIMS has a lot of instrumental challenges for the direct analysis of TLC plates including: 1) Application of MALDI matrix causing spreading and elution of the sample 2) MALDI matrix background noise in the low mass range that can interfere with detection of small analytes, 3) specialized TLC plates with a conductive aluminum back are required, 4) the MALDI UV laser only penetrates the surface of the TLC plate and cannot achieve multiple ablations per location without application of matrix between ablations, 5) MALDI 1, 2, 4 analysis is carried out in a vacuum chamber . To overcome some of the challenges, there have been recent advances in atmospheric pressure sample introduction technologies including DESI (desorption electrospray ionization). In DESI-MS, a pneumatically assisted electrospray emitter directly impacts the sample, which desorbs the analytes into the mass spectrometer. Van Burkel et al (2005) reported the following DESI-MS instrumental challenges with TLC separation: 1) Physical damage to TLC plate occurs due to mechanical forces of the pneumatic jet, 2) the TLC separation must be near plate edge or excised, 3) frequent maintenance of the mass spectrometer is required to remove TLC stationary phase, 4) the electrospray elutes part of the analyte out of desorption the range. by an electrospray jet prior to detection in the mass spectrometer. The LAESI-MS technology is a novel approach in which virtually no sample preparation is needed 5 prior to analysis . In this report, we utilize LAESI-MS to image molecules separated on a bare silica TLC plate using ® the Protea LAESI DP-1000 system and its proprietary ProteaPlot™ imaging software. After separation, the TLC plates were loaded and directly analyzed by LAESI-MS coupled to a Thermo LTQ XL™ mass spectrometer. Experimental The C18 reversed phase (RP) TLC plates were purchased from Whatman (Piscataway, NJ). The TLC plate size was 2.5 x 7.5 cm with a layer thickness of 200 µm, and 60 Å silica bead pore size. The fluorescent hydrophilic plates were purchased from EMD chemicals (Gibbstown, NJ). For separation of peptides on the RP silica plate, 1 µL of a sample mixture containing three peptides was spotted onto the TLC plate and separated using a solution of 0.1% TFA in 40% acetonitrile. The peptides ([des-Arg] Bradykinin, substance P and ACTH), were present in at a concentration of 1 µg/µL in solution. Two synthetic compounds, 4-[2Methoxy-5-nitro-4-(1-(2,2,2-trifluoroacetamido)ethyl) phenoxy]-N-[2-(2,2,2,2-trifluoroacetamido)ethyl]butanamide and 4-[2-Methoxy-5-nitro-4-(1-acrylamidoethyl)phenoxy]-N(2-acrylamidoethyl)butanamide were spotted and separated on the hydrophilic silica TLC plate at 1 µg level for each compound. These compounds were denoted 1 and 2 respectively. Three separation lanes were used on the TLC plate, with two of the lanes containing one of the compounds and the third lane containing a mixture of the two compounds. LAESI-MS was performed on the LAESI DP1000 system, using an electrospray voltage 4kV, syringe flow-rate of 2 µL/min, laser energy of 700 µJ at 10Hz repetition rate with 10 pulses at each location. The TLC plates were wetted with deionized water after TLC separation prior to LAESI-MS to loading onto the LAESI DP1000 system. Laser Ablation Electrospray Ionization Mass Spectrometry (LAESI-MS) is one of the most recent developments in atmospheric pressure mass spectrometry imaging. In LAESIMS, analytes are ablated at ambient pressure by a midinfrared laser. The ablation plume is intercepted and ionized © 2011 Protea Biosciences, Inc • 877.776.8321 • www.proteabio.com 1 Results To illustrate the capability of LAESI for imaging compounds separated on TLC plates, two plate types, a hydrophobic and hydrophilic TLC plate were used for LAESI-MS analysis. Compounds 1 and 2 were separated on the hydrophilic TLC plate as shown in the schematic and fluorescence optical image in Figure 1A. [des-‐Arg]-‐Bradykinin m/z = 452.7 Substance P m/z = 674.9 A) ACTH (7-‐38) 1687.96 2 200 600 1000 m/z 1400 1800 200 Figure 2: Ion maps of peptides (substance P, [des-Arg]-Bradykinin and ACTH (7-38)) separated on a C18 reversed phase silica bead plate. 1085.97 245.06 0 0 m/z = 1220 555.09 100 1 Rel ative Abundance B) 245.12 471.16 Rel ative Abundance 100 600 1000 m/z 1400 1800 Conclusion The Protea LAESI DP-1000 system can be used to analyze compounds separated on both hydrophilic and hydrophobic TLC silica plates, by simply wetting the TLC plates with water. This analytical method can be used to determine both the location and confirm the identity of analytes separated on TLC plates. C) Figure 1: A) Schematic of the separation scheme of the two compounds, and a fluorescence optical image of separated compounds 1 and 2. B) Resulting mass spectra from LAESI-MS analysis. C) Ion maps of the compounds from ProteaPlot software overlaid on TLC images of the analyzed TLC plate. The approximate position of the compounds optically visualized on the plate matched the position of the compounds indicated by the ion maps from LAESI-MS analysis. After performing LAESI-MS analysis on these plates, the data files were analyzed using ProteaPlot imaging software to determine the location of the separated compounds shown in Figure 1B (sodium adducts at m/z 471.2 and m/z 555.1). By overlaying an optical image of the TLC plate and the ion map of the compounds from ProteaPlot software (Figure 1C), very good correlation was achieved between the location of the compounds from ProteaPlot ion maps, and the locations indicated by optical fluorescence visualization (pencil circles). Similarly, an ion map from the separation of peptides on a hydrophobic TLC plate was achieved (Figure 2), showing the locations of three separated peptides, Bradykinin (m/z 452.7), Substance P (m/z 674.9) and ACTH (7-11) (m/z 1220). References (1) Han, Y, P. Levkin, I. Abarientos, H. Liu, F. Svec, and J.M.J. Fréchet. Monolithic superhydrophobic polymer layer with photopatterened virtual channel for the separation of peptides using two-dimensional thin layer chromatographydesorption electrospray ionization mass spectrometry. Analytical Chemistry. 2010, 82, 2520-2528. (2) Fuchs, B. R. Süß, A. Nimptsch, J. Schiller. MALDI-TOFMS directly combined with TLC: a review of the current state. Chromatographia Supplement. 2009, 69(1), 95-105. (3) Robards, K., P.R. Haddad, P.E. Jackson. Principles and practice of modern chromatographic methods (pp. 44, 182224). 1994. San Diego, California: Academic Press. (4) Van Berkel, G.J., M.J. Ford, and M.A. Deibel. Thin-layer chromatography and mass spectrometry coupled using desorption electrospray ionization. Analytical Chemistry. 2005, 77 (5), 1207-1215. (5) Nemes, P. and A. Vertes. Laser ablation electrospray ionization for atmospheric pressure, in vivo, and imaging mass spectrometry. Analytical Chemistry. 2007, 79, 80988106. Acknowledgements We acknowledge Dr. Miaosheng Li for preparing the hydrophilic TLC plates for analysis. © 2011 Protea Biosciences, Inc • 877.776.8321 • www.proteabio.com 2