Role of Sample load and Sample volume in chromatography

Sample preparation for the chromatographic separation is an important step in the chromatographic purification process. There are certain parameters of the sample to be checked before loading to the chromatographic column. Sample load, sample volume, concentration and viscosity of the samples, these are the major parameters which interfere or influence the resolution.

Filtering a Protein sample - Image Source

Ion Exchange chromatographic(IEC) separation is based on the charge, for principle of Ion Exchange chromatography click here. In IEC, for binding, ionic strength of the sample should be less and in Hydrophobic Interaction Chromatography binding takes place at higher salt concentration. Both these chromatographic techniques are independent of sample volume.

Binding capacity of the matrix should be checked, and amount of matrix required is calculated according to the quantity of protein to be bound.

This is calculated using the formula,

chromatographic matrix / media volume (L) =

Quantity of protein to be bound (g) * (1 + safety margin/100) / dynamic binding capacity(g/L)

Sample applied to the column should be filtered to remove any particulates, and if the sample is viscous it need to be diluted or else it will cause problem while passing through the stationary phase.

HIC and IEX are independent of sample volume.

In case of gel filtration / size exclusion chromatography sample volume is an important factor, gel filtration chromatography is independent of sample concentration (but not more than 70mg/ml). Sample volumes are expressed as percentage of volume of the packed bed.