tailieunhanh - HPLC for Pharmaceutical Scientists 2007 (Part 6)

Size-exclusion chromatography (SEC) separates polymer molecules and biomolecules based on differences in their molecular size. The separation process in simplified form is based on the ability of sample molecules to penetrate inside the pores of packing material and is dependent on the relative size of analyte molecules and the respective pore size of the absorbent. The process also relies on the absence of any interactions with the packing material surface. Two types of SEC are usually distinguished: 1. Gel permeation chromatography (GPC)—separation of synthetic (organic-soluble) polymers. . | 6 SIZE-EXCLUSION CHROMATOGRAPHY Yuri Kazakevich and Rosario LoBrutto SEPARATION OF THE ANALYTE MOLECULES BY THEIR SIZE Size-exclusion chromatography SEC separates polymer molecules and biomolecules based on differences in their molecular size. The separation process in simplified form is based on the ability of sample molecules to penetrate inside the pores of packing material and is dependent on the relative size of analyte molecules and the respective pore size of the absorbent. The process also relies on the absence of any interactions with the packing material surface. Two types of SEC are usually distinguished 1. Gel permeation chromatography GPC separation of synthetic organic-soluble polymers. GPC is a powerful technique for polymer characterization using primarily organic solvents. 2. Gel filtration chromatography GFC separation of water-soluble biopolymers. GFC uses primarily aqueous solvents typically for aqueous soluble polymers proteins etc. . Physical and chemical properties of polymers are dependent on their molecular weight and molecular weight distribution. The separation principle in SEC is based on the forced transport of the polymer molecules through the porous stationary-phase media under the conditions of suppressed interactions of the HPLC for Pharmaceutical Scientists Edited by Yuri Kazakevich and Rosario LoBrutto Copyright 2007 by John Wiley Sons Inc. 263 264 SIZE-EXCLUSION CHROMATOGRAPHY polymer analyte with the surface. The mobile-phase eluent is selected in such way that it interacts with the surface of packing material stronger than the polymer. Under these conditions the smaller the size of the molecule the more it is able to penetrate inside the pore space and the movement through the column is retarded. On the other hand the bigger the molecular size the higher the probability the molecule will travel around the particles of the packing material and thus is eluted earlier. The molecules are separated in order of decreasing .