Reversed phase partition development

In principle, h.p.Lc. arose from conventional liquid column chromatography, following the development of g.l.c. and realisation that it was a rapid and accurate analytical method. This led to a reappraisal of the liquid column chromatographic system, which in turn resulted in research developments in instrument design and in the manufacture of column-packing materials. These now have precise specifications to make them suitable for adsorption, normal and reversed phase partition, ion exchange, gel permeation, and more recently affinity chromatography. 

Most critical pairs may be separated through reversed phase partition chromatography by working at low temperature (see p. 94). Another way is to develop the unsaturated lipids through quantitative oxidation with a peroxidic solvent, which has no influence on the separation... 

Fig. 149. Separation of the triglycerides of sunflower oil (Helianthus annuus) by combination of argentatic i -TLC and reversed phase partition-TLC [89]. A pre-fractionation of the triglycerides through argentation-TLC adsorbent silica gel G-silver nitrate (25%) solvent benzene-diethyl ether (80 + 20) time of run 3.5 h spray reagent 2, 7 -dichlorofluorescein in ethanol (UV light) amount 20 mg B separation of fractions 1—9 (A) through reversed phase partition-TLC stationary phase paraffin on kieselguhr G solvent acetone-acetonitrile (80 + 20) (80% saturated with paraffin), developed twice time of run 40 min each time visualisation aqueous-alcoholic a-cyclodextrin solution-iodine vapour amounts ca 12 [xg of each of the fractions 1—9 and 20 jxg sunflower oil...

Figure 18.2 shows the sequence of pigments obtained in the reversed-phase partition system. The chromatogram of the saponified extract should indicate the four carotenoids in the same locations, without the chlorophylls. As expected, the order of migration is reversed compared to the adsorption system illustrated in Figure 18.1. The chromatogram in Figure 18.2 results from spotting 5 xl of leaf extract solution. The application of a lower initial zone volume would improve the separations of the upper and bottom two zones. Alternatively, the use of a different mobile phase can emphasize these resolutions at the expense of the overall separation. Neoxanthin and violaxanthin were separated from each other and all other pigments by development for 35 min with methanol-acetone-water (2 2 1) (respective Rt values 0.23 and 0.18). Carotene Rf 0.40) and chlorophyll a (R 0.53) were completely resolved in butanol-acetone (8 7).

Tswett s initial column liquid chromatography method was developed, tested, and applied in two parallel modes, liquid-solid adsorption and liquid-liquid partition. Adsorption ehromatography, based on a purely physical principle of adsorption, eonsiderably outperformed its partition counterpart with mechanically coated stationary phases to become the most important liquid chromatographic method. This remains true today in thin-layer chromatography (TLC), for which silica gel is by far the major stationary phase. In column chromatography, however, reversed-phase liquid ehromatography using chemically bonded stationary phases is the most popular method. 

Precoated plates are also available for reversed-phase liquid-liquid partition thin-layer chromatography. Here the silica gel has been treated with an octadecyl silylating reagent thus coating the particles with a non-polar chemically-bonded thin film. The solvent employed is more polar than the film and chromatographic development results from partition between these two phases. 

Two-dimensional planar chromatography (2D-TLC) is frequently used in combination with autoradiography or digital autoradiography (DAR) in studies on metabolism. Examples of 2D-TLC-DAR will be given in the analysis of pharmaceutical products. Other applications generally use either different types of development, or utilise different interactions for separation, or different stationary phases, such as elution-displacement absorption-partition normal phase-reversed phase ion exchange-normal phase. 

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