High Performance Size Exclusion Chromatography

High Performance Size Exclusion Chromatography:  This technique is for separating 

dissolved species on the basis of their size and particularly applicable to high-molecular-  

weight species like oligomers and polymers to determine their  relative sizes and molecular 

weight distributions. Here, the stationary phase is polymer resin, which contains small pores. 

If the components to be separated are passed through the column the small sized particles can 

easily enter into these pores and their mobility is retarded. Whereas the large sized particles, 

which can’t enter into these pores can come out of the column fast and elude first. Thus the 

separation of various sized particles is possible  through variations in the elution time. It is 

classified into two categories based on the nature of the columns and their packing as:

Gel Filtration Chromatography - which uses hydrophilic packing  to separate polar species 

and uses mostly aqueous mobile phases. This  technique is mostly used to identify the 

molecular weights of large sized proteins & bio-molecules.  

Gel Permeation Chromatography -  which uses hydrophobic packing to separate nonpolar 

species and uses nonpolar organic solvents. This technique is used to identify the molecular 

weights of polymers.

Instrumentation :  The basic HPLC system consists of  a solvent (mobile phase) reservoir, 

pump, degasser, injection device, column and  detector. The pump draws the mobile phase 

from the reservoir and pumps it to the column through the injector. At the end of the column 

(effluent end), a detector is positioned. Mostly UV absorption detector is used. In the case of 

analytical studies, after the detection the eluents are collected in waste bottles. In the case of 

preparative studies the eluents are fractionally collected for further studies. Most of the 

HPLC design will be the same as described for all the four main groups previously described. 

However, there can be differences in selecting the specific detectors for particular type of 

analysis, say for example, with ion-exchange chromatography, detectors commonly used are 

conductivity detectors for obvious reasons. Other important detectors for HPLC separations 

include refractive index detector, fluorescence  detector and mass selective detector. The 

following is the most generalised outlay of the HPLC system:   

Disadvantages :  Column performance is very sensitive, which depends on the method of 

packing. Further, no universal and sensitive detection system is available.