Thin layer chromatography interface

J. W. Hofstraat, S. Griffioen, R. J. van de Nesse and U. A. Th Brinkman, Coupling of narrow-bore column liquid chromatography and thin-layer chromatography. Interface optimization and characteristics for normal-phase liquid chromatography , J. Planar Chromatogr. 1 220-226 (1988). 

The question as to the potential availability of the requisite amphiphilic precursors in the prebiotic environment has been addressed experimentally by Deamer and coworkers, [143,145] who looked into the uncontaminated Murchison chondrite for the presence of such amphiphilic constituents. Samples of the meteorite were extracted with chloroform-methanol and the extracts were fractionated by thin-layer chromatography, with the finding that some of the fractions afforded components that formed monomolecular films at air-water interfaces, and that were also able to self-assemble into membranous vesicles able to encapsulate polar solutes. These observations dearly demonstrated that amphiphiles plausibly available on the primitive Earth by meteoritic infall have the ability to self-assemble into the membranous vesides of minimum protocells. ... 

Figure 9.29 Membrane formation by meteoritic amphiphilic compounds (courtesy of David Deamer). A sample of the Murchison meteorite was extracted with the chloroform-methanol-water solvent described by Deamer and Pashley, 1989. Amphiphilic compounds were isolated chromatographically on thin-layer chromatography plates (fraction 1), and a small aliquot ( 1 p,g) was dried on a glass microscope slide. Alkaline carbonate buffer (15 p,l, 10 mM, pH 9.0) was added to the dried sample, followed by a cover slip, and the interaction of the aqueous phase with the sample was followed by phase-contrast and fluorescence microscopy, (a) The sample-buffer interface was 1 min. The aqueous phase penetrated the viscous sample, causing spherical structures to appear at the interface and fall away into the medium, (b) After 30 min, large numbers of vesicular structures are produced as the buffer further penetrates the sample, (c) The vesicular nature of the structures in (b) is clearly demonstrated by fluorescence microscopy. Original magnification in (a) is x 160 in (b) and (c) x 400.

The most recent modification of the NBD-Cl method involves a further improvement in its qualitative support (616). It involves the infusion of the extract employed for thin-layer chromatography via an electrospray interface into a mass spectrometer operating in the multiple-stage mass spectrometry mode, thus allowing confirmation of suspect results. The cleanup of the thyroid gland samples was also performed with a selective extraction procedure, based on the specific complex formation of the thiouracil, methylthiouracil, propylthiouracil and phenylthiouracil, tapazole, and mercaptobenzimidazole residues with mercury ions bound in a Dowex 1-X2 affinity column. 

The lower, chloroform-rich phase is separated carefully from the protein-containing interface, and then it is washed twice with methanol-water (10 9, v/v) and the washes are discarded. The chloroform layer contains the phosphatidic acid (as a sodium salt) and can be isolated by acetone precipitation. The yields can be of the order of 90-95%. One alternative route to identification of the chloroform-soluble material is to analyze it for total phosphorus and total fatty acid ester (see procedures described earlier). In the case of diacylphosphatidylcholine as the substrate, the fatty acid ester/P molar ratio should be 2.0. Another approach is to subject the chloroform-soluble fraction to preparative thin-layer chromatography on silica gel H (calcium ion free) in a two-dimensional system with a solvent system of chloroform-methanol-28% ammonium hydroxide (65 35 6, v/v) in the first direction and a solvent system of chloroform-acetone-methanol-glacial acetic acid-water (4.5 2 1 1.3 0.5, v/v) in the second direction. The phosphatidic acid will not migrate far in the basic solvent Rf 0.10) and will show an Rf value one-half of that of any remaining starting substrate (fyO.40) in the second solvent. Of course with a simple substrate system, one can use the basic solvent in one dimension only... 

In an interesting experimental protocol, Silvestro et al. (1993) utilized HPLC-mass spectrometry with an ion spray (electrospray) interface for determination of PAF and lysoPAF in human PMN (neutrophils). Both unstimulated and stimulated (with complement-activated zymosan) cells were used as starting material. The total lipids were isolated in the usual way, and the PAF was isolated and purified by a combination of thin-layer chromatography, HPLC, and silica chromatography. This final PAF preparation was subjected to a bioassay with the inclusion of 3H 16 0 PAF to monitor recoveries. 

The problems to be solved in interfacing thin-layer chromatography to mass spectrometry (TLC-MS) are largely different to those experienced in LC-MS and other column separation techniques [95-99]. At the completion of the separation the chromatogram is fixed in time and space with the major portion of the mobile phase eliminated by evaporation. The thin-layer plate can be considered as a storage device effectively de-... 

Many FT-IR spectrometers have external ports for optical coupling to dedicated accessories. The IR radiation is conveniently directed to/from the external ports by computer-controlled flip mirrors. A large variety of accessories, like an IR microscope, interfaces for gas chromatography (GC/FT-IR), liquid chromatography (HPLC/FT-IR), thin layer chromatography FT-IR (TLC/FT-IR), etc., is commercially available. This type of method combination is usually called a hyphenated technique. FTIR spectrometers can even be supplemented by a FT-Raman accessory. The versatile combination of FT-IR spectrometers with other instruments has substantially contributed to their abundance in most analytical laboratories. 

Because the vast majority of samples are complex mixtures, they generally require the separation of their components, by GC or LC, prior to their introduction into the ion source. GC is usually carried out on fused silica capillary columns. LC is available in two formats in conventional LC the flow rates are O.l-l.O ml/min, while nano-LC operates at sub pl/min flow rates. Capillary electrophoresis (CE) can be interfaced to mass spectrometers (similarly to LC). Thin-layer chromatography (TLC) is compatible with the newer surface ionization methods. 

The use of MS in addition to GC/MS includes methods such as capillary zone electrophoresis (CZE/MS), thin layer chromatography (TLC/MS) and others. The major problem in employing these separation-MSD methods is the interface. The very high vacuum required for MS operation requires complete removal of the carrier gas or liquid . Since the present review treats specifically the use of the MSD for the analysis of alkanes and cycloalkanes, we concentrate on the GC/MSD/C (gas chromatography/ mass spectrometry/computerized) method. The MS used can be quadrupole or magnetic and its configuration will, of course, control the power of resolution (PR), the mass range, etc. The ionization mode is also important, e.g. whether El or Cl (see Sections, III.A.l and III.A.2). 

In the following section, three approaches are focused on and exemplarily described, for which instrumentation and support are commercially available and for which own research experience are given. One HPTLC-MS application is based on an interface using an elution head (high-performance thin-layer chromatography-electrospray ionization mass spectrometry, HPTLC-ESI-MS) the other two applications are using either laser light (HPTLC-MALDI-MS) or a gas beam for desorption (HPTLC-DART-MS). They all are soft ionization techniques however, when combined with analyzers suited for structure elucidation, like ion trap or tandem mass spectrometry (MS/MS), structural information is also available. 

Anderson, R.M., Busch, K.L. (1998) Thin-layer chromatography coupled with mass spectrometry interfaces to electrospray ionization. Journal of Planar Chromatography, 11, 336-341. 

Hsu, F.L., Chen, C.H., Yuan, C.H., Shiea, J. (2003) Interfaces to connect thin-layer chromatography with electrospray ionization mass spectrometry. Analytical Chemistry, 75, 2493-2498. 

Qrinak, A., Ariinghaus, H.F., Vering, G., Orinakova, R. (2004) Modified chromatographic thin layer surface as an interface to couple thin layer chromatography with ToF-SIMS. Surface and Interface Analysis, 36, 1122-1125. 

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