HPLC Extra Column Volumes

December 1, 2014

It is of particular importance to reduce extra column volumes when using small volume columns or UHPLC. However, where do these extra column volumes come from, how can they be minimized, and what effect do they have on chromatography?

It is of particular importance to reduce extra column volumes when using small volume columns or UHPLC. However, where do these extra column volumes come from, how can they be minimized, and what effect do they have on chromatography?

Question:   If the HPLC system contains a large extra-column volume should it not be that the later eluting peaks are broader and have a higher plate count than the early eluting peaks?  

Answer: Firstly, I thought I would detail a couple of definitions in relation to the subject of efficiency and extra column volumes and their effects.

Definitions:

Extra column volume – the volume between the injection point and the detection point, excluding the part of the column containing the stationary phase.  It is composed of the injector, connecting lines, frits, and the detector; and it is this that determines the extra column effects.
Extra column effects – the total band broadening effects of all parts of the chromatographic system outside of the column itself.  Extra column effects must be minimized to maintain the efficiency of the column.  Areas of band broadening can include the injector, injection volume, connecting tubing, end fittings, frits, detector cell volume, and internal detector tubing.  The variances of all these contributions are additive.

W = bandwidth

The following are bandwidth contributions from:

Wc = column

Ws = injector/autosampler

Wlc = lines and connectors

Wfc = detector flow cell

As long as the bandwidth contributions Ws, Wlc, and Wfc are each less than 1/3W their effect on W can be neglected.

 

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