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Column Resolution

The resolution RS of a column tells us how far apart two bands are relative to their widths. The resolution provides a quantitative measure of the ability of the column to separate two analytes, Rs= 2(t R2 t R1) W A+ W B

It is evident that a resolution of 1.5 gives an essentially complete separation of A and B, whereas a resolution of 0.75 does not. At a resolution of 1.0, zone A contains 4%B and zone B contains 4% of zone A. The resolution of a given stationary phase can be improved by increasing the length of the column and thus increasing the number of theoretical plates. An adverse consequence of the added plates, however, is an increase in the time required for separating the components. Optimization techniques Variation in plate height The resolution of a column improves as the square root of the number of plates it contains increases. The increasing of the number of plates is expensive in terms of time unless the increase is achieved by reducing the plate height and not by increasing column length. Methods of reducing plate height, including the particle size of the packing material, the diameter of the column, and the thickness of the liquid film. Variation in the retention factor Often, a separation can be improved significantly by manipulation of the retention factor k B . Increases in k B generally enhance resolution (but at the expense of elution time). Usually, the easiest way to improve resolution is by optimizing k. For gaseous mobile phases, k can often be improved by temperature changes. For liquid mobile phases, changes in the solvent composition often permit manipulation of k to yield better separations. Variations in the Selectivity Factor Optimizing k and increasing N are not sufficient to give a satisfactory separation of two solutes in a reasonable time when approaches unity. A means must be sought to increase while maintaining k in the range of 1 to 10. Several options are available; in decreasing order of desirability, as determined by promise and convenience, the options are 1. changing the composition of the mobile phase 2. changing the column temperature 3. changing the composition of the stationary phase Skoog, West et al, Fundamentals of analytical chemistry, 8th ed, ISBN-13: 978-0-03-035523-3

4. using special chemical effects Increases in temperature usually cause increases in k but have little effect on values in liquidliquid and liquid-solid chromatography. In contrast, with ion-exchange chromatography, temperature effects can be large enough to make exploration of this option worthwhile before resorting to a change in column packing material. A final method of enhancing resolution is to incorporate into the stationary phase a species that complexes or otherwise interacts with one or more components of the sample.

Skoog, West et al, Fundamentals of analytical chemistry, 8th ed, ISBN-13: 978-0-03-035523-3