AIM; to separate milk proteins based on their size, allowing identification and characterization

individual proteins present in the milk

INTRODUCTION

A gel filtration column separates molecules and complexes by size. The separation is
not strictly by molecular mass, since the shape of the molecule or complex can affect
migration through the column. Unlike other types of column chromatography, gel
filtration chromatography does not involve any interaction of the sample or the solvent
with the matrix in a column; separation is achieved by physical means. This method can
be applied to purify milk proteins, which are a mixture of various sizes and molecular
weights.
A gel filtration column matrix has a defined porosity, allowing molecules of different
sizes to either pass through or be retained. The porosity can consist of either channels
within the matrix beads or pores on the surface of the beads. If a molecule or complex
can enter the pores, the retention time in the column will be longer because the
molecule follows a longer path through the column than any molecule that is too large to
enter the pores. The smaller the molecule, the more readily it will enter pores, the longer
its path through the column, and the longer its retention time. Molecules too large to
enter the pores move rapidly through the column with the mobile (solvent) phase.
The pore size of the beads in a gel filtration column determines the range of sizes that
can be efficiently fractionated. In a mixture of compounds that are above the column
size range, molecules cannot be fractionated because they are not able to enter the
pores and will thus elute together. Conversely, all molecules and compounds that have
molecular masses below the column size range, known as the permeation limit, will
have full access to the pores and thus will also elute together.
Elution time is inversely proportional to size. The molecules a of the largest size will
elute first, since they will be retarded only slightly by the pores. All molecules within the
size range will elute in tight, narrow bands, giving good sensitivity. Thus the principle
behind gel filtration is that smaller molecules are excluded from the pores of the gel
matrix and take longer to elute from the column, while larger molecules pass more
freely and elute earlier.