Measurement of effluent volume

The retention volume of the sample is one of the most important data obtained by analytical gel chromatography. 

In gel chromatographs of the first generation, the volume of each fraction collected is measured separately. In the instruments of the second generation, the retention volume is measured continuously, most frequently by means of a siphon. This device can easily be automated and utilized for controlling the injection system, and, eventually also the pumping system and fraction collector. The drawback of the siphon is its sensitivity to the variations of interfacial tension of the effluent and to the formation of the film of grease on its walls. Both effects are pronounced especially in the aqueous mobile phases. Smaller retention volumes can be measured by means of the drop counters or capillary volumeters. From the point of view of aqueous eluents, the drop counters exhibit the same limitations as siphons. The capillary volumeters are based upon counting how many times the effluent fills up the fixed volume of a capillary. The results of both, drop counters and capillary volumeters, depend on the flow rate and on the physical characteristics of the effluent. 

According to the commercial literature, many modem pumping systems provide strictly constant flow rate so that it is sufficient to measure the retention time. It seems that this information must be taken into account with some precaution, especially in the cases of very low flow rates, long-term experiments and aqueous eluents. For measurements of very high precision, the retention volumes can be determined by automatic continuous weighing of the effluent (21). 

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The measurement of effluent volume is not very reliable because of the effect of the geometry and packing characteristics of any column. It is often more useful to use a reduced parameter, such as V/V0, which is not so dependent upon column characteristics and is comparable with the calculation for RF values in thin-layer chromatography. 

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