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Length of the size exclusion column will be proportional to the number of theoretical plates, so increasing length will enhance the separation resolution but upto a limit. The column width determines the limit for the sample volume that can be injected without affecting the resolution limits.

Column efficiency, the rate of zone spreading measured by taking a ratio of retention time to peak width and squaring it, is usually expressed as N. When peak width is measured at half height, the constant is 5.54. If the peak width is measured at base, a constant of 16 must be used to arrive at about the same N value.

Scientists consider a resolution of 1.0 or higher to represent an adequate separation. Measure the widths of two adjacent peaks in the chromatogram by noting where the x-axis values are at the base of each peak. The x-axis represents retention time, usually measured in seconds.

A longer column generally improves the separation. The trade-off is that the retention time increases proportionally to the column length and a significant peak broadening will be observed as well because of increased longitudinal diffusion inside the column.

K) The resolution (Rs) of a column for two analytes (A eluting before B) is given by Rs = tR(B) - tR(A)/(WA + WB)/2, where W is the baseline width of a chromatographic peak in the same units as the analyte retention time.

The resolution of a column is measured by its efficiency and the more efficient a column is the better the resolution. Column efficiency is: proportional to column length; a longer column is more efficient. inversely proportional to the stationary phase particle size.

The advantage of switching to longer columns is that the theoretical plates will increase and so will the resolution. The number of theoretical plates (N) is directly proportional to the length of the column and inversely proportional to the particle diameter2.

Decreasing particle size thus is a useful method for improving column efficiency and providing better separations. changing to particles that are half as big, while keeping the column length the same, will double the performance, but increase the required pressure by a factor of four.

Column efficiency, indicated as the number of theoretical plates per column, is calculated as N = 5.54 (tR / w0.5)2 where tR is the retention time of the analyte of interest and w0.5 the width of the peak at half height.

The resolution of a column is measured by its efficiency and the more efficient a column is the better the resolution. Column efficiency is: proportional to column length; a longer column is more efficient. inversely proportional to the stationary phase particle size.