Question

What does "Salt boundary and Void volume" represent in Size Exclusion Chromatography?

Answer 1

Solution:

Salt boundary in Size Exclusion Chromatography (SEC)

Size-exclusion chromatography (SEC), also known as molecular sieve chromatography. It is a chromatographic method in which molecules in solution are separated by their size, and in some cases molecular weight. It is usually applied to large molecules or macromolecular complexes such as proteins and industrial polymers. Basically, when an aqueous solution is used to transport the sample through the column, the technique is known as gel-filtration chromatography, versus the name gel permeation chromatography, which is used when an organic solvent is used as a mobile phase. The chromatography column is packed with fine, porous beads which are composed of dextran polymers, agarose, or polyacrylamide (BioGel P). The pore sizes of these beads are used to estimate the dimensions of macromolecules. SEC is a preparative, non-destructive analytical technique that permits the separation of molecules by their size. This is especially useful in protein purification because while there may be many proteins in a sample, their molecular weights can vary widely. This allows one to separate a mixture of proteins based on this wide size distribution. It also provides a simple procedure for removing salt (desalting) from a protein sample. Salt boundary is the limitations to the size of the salts present in the protein which can be separated.

Void volume in Size Exclusion Chromatography (SEC)

This is the space that is outside of the gel particles. The volume of liquid collected from the minute that the sample is applied to the column until an unretained molecule is eluted from the column. This represents the volume that non-retained molecule experienced on its travels through the column. Each molecule will experience this volume, whether retained or not. This value must be measured each time a SEC column is prepared and used because it is unique to each column (based on packing density, gel composition, length etc.).