High‐speed gel permeation chromatography. A study of operational variables

Systematic studies of operational variables affecting GPC separation using μ‐Styragel of nominal porosity 10 6 , 10 5 , 10 4 , and 10 3 Å are reported. The dependences of (1) flow rate, (2) injection volume, and (3) concentration on elution volume ( V e ), the theoretical plate number ( N ), and peak width at half‐height were examined. The values of V e changed with changing (1), (2), and (3). The values of N were inversely proportional to the root of flow rate. The relation between sample load and N showed that lower concentration and larger injection volume were desirable for N than the opposite. The concentration dependence for high‐speed GPC was significantly higher than classical low‐speed GPC. The methods of minimizing the effects produced by the individual variables and the optimum operational conditions are discussed. Recommended operational conditions are as follows: flow rate, 1 to 2 ml/min; injection volume, 0.1 ml; sample concentration, below 0.4% (or if injection volume 0.5 ml, sample concentration below 0.1%).