Methods Based on Spot Size

As with densiometric analysis, methods based on spot size avoid all the difficulties connected with the recovery of the sample and with the extraction of impurities from the adsorbent [31]. An additional advantage is that they may be applied with equal facility to adsorption or partition chromatograms. To illustrate the method, the determination of the cholesterol content of wool wax alcohols by adsorption chromatography will be described. 

A series of standard solutions of cholesterol were prepared by serial dilution. Equal volumes (2 pi) of a solution of known concentration of the sample and of each of the standard solutions were spotted on to the adsorbent alternatively. For high accuracy a micrometer syringe should be used, but also, a capillary pipette (Drummond Microcap), could be used. 

Purdy and Truter [33] discovered that the square root of the area is a linear function of the logarithm of the weight of material in the spot. Statistical tests by these workers showed that the goodness of fit with their relationship is more satisfactory than for any of the earlier proposals. 

The advantages of using the linear relationship between VA and log W are that the experimental work can be substantially reduced, and statistical evaluation of the accuracy is possible. Only two standard solutions, one of which is prepared from the other by dilution, are required to determine the equation of a straight line. Equal volumes of the two standard solutions and the test solution are chromatographed simultaneously, and the weight of the compound in the sample is calculated from the equation  

Where the subscipts s and d refer to the standard and the diluted standard, respectively, and D is the dilution factor. 

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