Chromatographic separation, elution

Figure 8-12. Examples of las eluted enantiomers In a chromatographic separation on chiral HPLC with teicoplanin stationary-phase.

The chromatographic separation should whenever possible be completed in one operation. If, however, shortage of time necessitates an interruption, this can most conveniently be made immediately after the first band has been completely eluted, whereupon the lower end of the tube is closed by a short piece of rubber tubing carrying a screw-clip. Great care should be taken however not to allow even the top of the column to run dry. 

Development of the Chromatogram. The term development describes the process of performing a chromatographic separation. There are several ways in which separation may be made to occur, eg, frontal, displacement, and elution chromatography. Frontal chromatography uses a large quantity of sample and is usually unsuited to analytical procedures. In displacement and elution chromatography, much smaller amounts of material are used. 

A chromatographic separation can be developed in three ways, by displacement development, by frontal analysis, and by elution development, the last being almost universally used in all analytical chromatography. Nevertheless, for the sake of completeness, and because in preparative chromatography (under certain conditions of mass overload) displacement effects occur to varying extents, all three development processes will be described. 

Elution development is by far the most common method of processing a chromatographic separation and is used in all types of chromatography. Elution development is best described as a series of absorption-extraction processes which are continuous from the time the sample is injected into the distribution system until the time the solutes exit from it. The elution process is depicted in Figure 1. 

In a chromatographic separation, the individual components of a mixture are moved apart in the column due to their different affinities for the stationary phase and, as their dispersion is contained by appropriate system design, the individual solutes can be eluted discretely and resolution is achieved. Chromatography theory has been developed over the last half century, but the two critical theories, the Plate Theory and the Rate Theory, were both well established by 1960. There have been many contributors to chromatography theory over the intervening years but, with the... 

Considerable care must be taken when accessing closely eluting peaks. If the resolution is inadequate, measurements must be taken on the individual solutes, chromatographed separately on the column. 

Reiterating the conditions for a chromatographic separation once again, for two solutes to be resolved their peaks must be moved apart in the column and maintained sufficiently narrow for them to be eluted as discrete peaks. However, the criterion for two peaks to be resolved (usually defined as the resolution) is somewhat arbitrary and is usually defined as the ratio of the distance between the peak maxima to half the peak width (a) at the points of inflection. To illustrate the various degrees of resolution that can be obtained, the separation of a pair of solutes 2o, 3o, 4o, 5o and 6o apart are shown in Figure 12. Although, for baseline resolution, it is clear that the peak maxima should be separated by at least 6o for most quantitative analyses. 

The submitters mixed active anhydrous silica gel with water (12% v>/w) and stored it in a sealed container for at least 24 hours prior to use. A ratio of 60-80 g. of silica gel per gram of crude product was used for column chromatographic separations, and a column was chosen that would give a 10 1 height diameter ratio of adsorbent. Columns were wet-packed with distilled petroleum ether (b.p. 60-68c), and after the crude product had been applied a step-gradient was run rapidly through 2% vjv ether in petroleum ether, 5% ether, and 10% ether. The column was then eluted with 20% vjv ether in petroleum ether until the bromohydrin acetate was obtained. 

Ito30 determined Tedion through the difference between absorption at 250 and 277 nm after thin-layer chromatographic separation and elution with methanol. 

It is important for the analyst to be able to select the best stationary phase to use for a particular chromatographic analysis. Silica gel can be used in two modes of chromatographic separations as a stationary phase in normal elution development or as a stationary phase in exclusion chromatography. 

Figure 5. Effect of column dimensions on the chromatographic separation (ordinate absorbance, 340 nm abscissa elution time, min). Column A 1.5 yi 25 cm 442 mL 397 theoretical plates/ft 325 theoretical plates 0.4 mL/min flow rate Column B three 0.9 X 25 cm 47.7 mL 112 theoretical plates/ft 275 theoretical plates 1.05 mL/min flow rate.

It is not recommended to use carbon tetrachloride as an elution solvent in borane chromatographic separation since serious accidents have been reported during such operations. 

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