MEASUREMENT OF COLUMN EFFICIENCY IN WHOLE COLUMN DETECTION CHROMATOGRAPHY

The efficiency of a high-performance liquid chromatography (HPLC) column is measured independently of extra-column contributions to peak variance by means of whole column detection (WCD). The apparatus consists of a commercially available column instrumented with 14 individual UV absorbance detection zones. The variance of the peak at each detector is determined by fitting the peak to a gamma density function. A plot of total variance versus column position gives a straight line whose slope is equal to the height equivalent to a theoretical plate, H, and whose intercept is the sum of all contributions to extra-column variance. Variance measurements obtained on column for the three isomers of nitroaniline over the range 20-100% acetonitrile in water gave the dependences of H and extra-column variance on the capacity factor, k'. It was found that the extra-column variance is proportional to eta/(k' + 1)2, where eta is the viscosity of the mobile phase. This result is explained in terms of broadening within the connecting tubing by an amount proportional to the viscosity and focusing at the head of the column by 1/(k' + 1)2. Contributions to extra-column variance by the injector and electronic integration time are calculated from first principles and found to be small, while the average variance contribution from the detectors is deducted from the intercept of a plot of extra-column variance vs eta/(k' + 1)2.