Injection volume, maximum, HPLC

The enzyme assay mixture contained in a final volume of 200 //L 20 //mol glycine-KOH buffer (pH 9.5), 0.30 //mol serotonin-HCl, 0.12 //mol acetyl-CoA, and 50 //L of enzyme solution with a maximum activity of 100 mU/mL. After 5 minutes of incubation at 35°C, the reaction was stopped by diluting 10-fold with 0.1 M perchloric acid. After filtering, a 25 //L aliquot of the filtrate was injected onto the HPLC column. 

A diluted solution contains component A, which is required to be isolated by preparative HPLC amongst accompanying impurities. The separation factor of A to the nearest adjacent peak is 1.5 and its retention factor is 4. The analytical column used has a theoretical plate number of 6400 and a void volume of 2 ml. Calculate the maximum allowed injection volume at which peak A actually touches its nearest neighbour. 

The user of preparative HPLC in general wants to obtain as much of a pure compound per unit time as possible. Therefore, it is necessary to work under conditions of overload. If sample solutions are diluted, volume overload will preferentially occur whereas mass overload is common with concentrated samples. Often both effects are present and the peaks become truncated, as can be seen at the bottom of Fig. 20.3 (with increasing retention the plateau is lost and the peaks become triangular). The maximum possible injected amount of a concentrated solution is determined empirically the injection volume is increased until the peaks touch each other. Non-diluted samples are not suitable. 

Additionally, the combination of trace enrichment and microbore columns can effectively increase the maximum sample volume injectable without seriously degrading efficiency. Slais et al. (29) evaluated this combination for the determination of polynuclear hydrocarbons and chlorinated phenols in water. By using reversed-phase HPLC and am-perometric detection, Slais et al. (29) reported lower limits of detection from 20 to 280 ng/L of water (parts per trillion) when 1-mL sample enrichments were carried out directly on the analytical microbore column. 

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