Size Exclusion Chromatography: Apparatus

APPARATUS

The apparatus for ‘size-exclusion chromatography’ essentially comprises of a chromatographic column generally made up of glass having a diameter to height ratio of between 1 : 10 and 1 : 20, packed with an appropriate separation material (e.g., different grades of Sephadex) which is capable of fractionation in the suitable range of molecular size and may be adequately temperature controlled. It is an usual practice to allow the mobile phase to pass through the column at a constant rate either by the aid of a suitably pump or simply by gravity.

1. APPLICATION OF SAMPLE

The sample is normally applied to the column by adopting one of the five following methods, namely :

(i) Directly to the drained-bed-surface with permitting the bed to dry,

(ii) Layered beneath the mobile-phase, provided the sample is denser than the mobile-phase,

(iii) Using a flow adaptor,

(iv) Using a syringe through a septum, and

(v) Using an injection valve.

2. DETECTION AND RECORDING

The outlet from the column is connected to a ‘detector’ usually fitted with an ‘automatic recorder’ that permits exclusively the monitoring of the relative concentrations of the various constituents present in the sample. However, one may also make use of an automatic fraction collector duly attached to the outlet from the column, if required. The various experimental parameters stated below are normally given in the official monograph, namely :

(a) Temperature of the column, if other than ambient,

(b) Nature of the packing material*,

(c) Composition of the mobile-phase,

(d) Flow rate of the mobile phase,

(e) Means of detection** of the sample components.