Chromatography multiple spotting

Beckett, A.H., Beavan, M.A., and Robinson, A.E., Paper chromatography multiple spot formation by sympathomimetic amines in the presence of acids, J. Pharm. Pharmacol., 12, 203T, 1960 Chem. Abs., 55, 9785c, 1961. 

The pigments in green leaves are to be extracted into an organic solvent, and the extract is to be analyzed by thin-layer chromatography (TLC). The presence of multiple spots on the developed TLC plate will indicate that more than a single pigment is contained in the leaves. 

In the field of steroid analysis, thin-layer chromatography (TLC) is still the method of choice, especially when many simultaneous analyses have to be carried out hundreds of analyses can be performed in a short time and with small demands on equipment and space. Samples can be analyzed with minimal cleanup, and analyzing a sample by the use of multiple separation steps and static postchromatographic detection procedure is also possible because all sample components are stored on the layer without the chance of loss. The time required in TLC analysis is about 10-60 min. As little as 0.01 /tg of steroids/spot can be detected by TLC. Using a TLC plate with thicker adsorbent layers (0.5 -2 mm), several grams of substance can be isolated (preparative TLC). 

An example of the harmful effects of the multiple use of the solvent system is shown in Fig. 51. Four plates, each with two 10-mm lanes, each containing 20 pg metoc-lopramide hydrochloride, were developed successively with the solvent system chloroform + methanol + concentrated ammonia solution (56 + 14 + 1 ml). The continuous downwards shift of the hRf value can be clearly seen, combined with an increasing broadening of the spots of the substance during the chromatography. These effects were caused by the reduction in the amount of base in the remaining solvent. 

The usual two-dimensional chromatography is a multiple development method in which the sample is spotted in the corner of a square of paper and ascending or descending development is made successively with two different solvents in two directions perpendicular to each other. Between developments, the paper is thoroughly dried to remove all traces of the first solvent. The best separations of complex mixtures are obtained if different types of solvents are employed. 

This compares well with an RSD of 50% in MALDI signal intensities reported for multiple peptide spots deposited from a single capillary using an optimized matrix seed layer technique. Further improvements in deposition volume uniformity were realized for an eight-channel chip containing a methacrylate monolith developed for reversed-phase chromatography in the second separation dimension, with negligible variations at flow rates down to 200 nL/min per channel. 

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