Estimation of the band broadening contribution of HPLC equipment to column elution profiles

Summary Methods to determine the contribution of the chromatographic equipment to the total band broadening which involve replacing the column by a union or a capillary tube are not suitable as they involve a fundamental change in the chromatographic system. The linear extrapolation method, based on the estimation of the relative influence of the instrument variance on solutes with different capacity factors, is a more attractive alternative method since the column remains in the chromatographic system. This method is only valid when a number of conditions are satisfied. By meeting these conditions the error in the instrument variance by using the linear extrapolation method was determined. At the same time, ways to minimise these errors were studied. Use of the linear extrapolation method in combination with conventional columns of 4.6 mm i.d. appears to yield inaccurate results. In combination with microbore columns the method can be used, provided the columns have a maximum length of 5cm and contain a packing material with a particle size of 2 or 3µm. The error in the determined instrument variance is then of the order of 2µl2.