Preparative layer chromatography basics

As explained in Chapter 1, classical preparative layer chromatography (PLC) involves flow of the mobile phase by capillary action. The method uses relatively basic equipment and is not expensive. 

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... 

As a rule, chemical methods used in the examination of writing materials require initial preparation of a sample for study. Paper chromatography, thin-layer chromatography and capillary electrophoresis are experimental techniques often applied. These methods lead primarily to separation of the dyes contained in the ink under examination and to the discrimination of ink samples. The techniques are simple to use, require a small amount of sample for examination, are selective and give reproducible results. Their basic disadvantage, however, is the necessity to isolate the ink from the substrate (e.g. paper) on which the examined document has been prepared. Solvent extraction of the ink often leads to partial damage of the document. 

The parent substance, 8-azapurine, was first prepared in 1957, but its alkylation was not studied until 1969. Dimethyl sulfate in cold, aqueous sodium hydroxide (the pH held above 7) gave equal proportions of the 7-, 8-, and 9-methyl derivatives. These were separated by taking advantage of the lower basic strength of the 9 isomer (Table I), then proceeding by thin-layer chromatography. ... 

Seiler (76) has reported the separation of P-, P-, P-, and P-O-P-acids by thin layer chromatography. Pure silica gel, 28 g., is well ground with 2 g. of pure starch, mixed with 50 ml. of water, and then diluted with 10 ml. of water. TTiis amount of the mixture is just enough to prepare five plates of 20 X 20 cm. After the water on the thin layer is removed, the plate is dried at 120°C. for 2 hr. and then kept in a desiccator containing calcium chloride. A basic solvent is prepared by mixing 50 ml. of methyl alcohol, 15 ml. of concentrated aqueous ammonia, 5 ml. of... 

Many of the qualitative uses of open column chromatography have been replaced by thin layer chromatography and HPLC, and the availability of preparative HPLC systems has further reduced the use of the technique. It does, however, find continued application for the large scale separation(s) (>10g) of reaction mixtures encountered in synthetic organic chemistry, especially as with minor modifications to the basic apparatus, extremely inexpensive systems (c/. HPLC) with moderate resolution (/if >0.10) can be set up [1]. These techniques known as flash chromatography and short path chromatography are discussed in more detail herein. Gel and affinity chromatography are also still practised extensively in open column mode in the biosciences. 

The purpose of this chapter is to present an overview of all important aspects of thin-layer chromatography (TLC). It will briefly review information and references on topics covered in the following chapters in Part I and will refer readers to these specific chapters. It will treat in more detail topics not covered further in separate chapters, such as sampling and sample preparation and the more classical procedures of TLC. A suggested source of additional basic, practical information on the practice and applications of TLC is the primer written by Fried and Sherma (1). 

Isolation of the active constituents was done using a bioassay-guided procedure. Depending on the extract, various combinations of chromatographic methods and solvent systems were used. Basic procedures included thin layer chromatography (analytical and preparative), as well as standard column chromatography. Structures were elucidated by a combination of NMR and MS spectrometric techniques. Details of the analyses will be reported elsewhere. 

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