Question

why must the M-ligand sample solution be free of salt when it is loaded onto the column?

Answer 1

The M-ligand sample solution contains charge particle (Metal will be in positive charge and ligand will be in negatively charged or neutral containing lone pair). If it contains salt while loading to the column then it will interact with that salt and it will be stuck into the column. Then it will be very difficult to elute it. To avoid, this difficulty M-ligand sample solution must be free of salt when it is loaded onto the column.

Ion exchange chromatography Ion exchange chromatography is based on the charge of the protein you are trying to isolate. If your protein has a high positive charge, you'll want to pass it through a column with a negative charge. The negative charge on the column will bind the positively charged protein, and other proteins will pass through the column. You then use a procedure called "salting out" to release your positively charged protein from the negatively charged column. The column that does this is called a cation exchange column and often uses sulfonated residues. Likewise, you can bind a negatively charged protein to a positively charged column. The column that does this is called an anion exchange column and often uses quaternary ammonium residues.

Salting out will release, or elute, your protein from the column. This technique uses a high salt concentration solution. The salt solution will out-compete the protein in binding to the column. In other words, the column has a higher attraction for the charge of salts than for the charged protein, and it will release the protein in favor of binding the salts instead. Proteins with weaker ionic interactions will elute at a lower salt, so you will often want to elute with a salt gradient. Different proteins elute at different salt concentrations, so you will want to be sure you know well the properties of your protein best results. Also be aware that changes in pH alter the charges in proteins. Be sure you know the isoelectric point of your protein (the isoelectric point is the pH at which the charge of a protein is zero) and make sure the pH of your system is adjusted and buffered accordingly.

The basic steps in using an ion exchange column are:

1. Prepare the column Pour your buffer over the column to make sure it has equilibrated to the required pH.

2. Load your protein solution Some proteins in the solution don't bind and will elute during this loading phase.

3. Salt out Increase the salt concentration to elute the bound proteins. It is best to use a salt gradient to gradually elute proteins with different ionic strengths. At the end bump the system with a very high salt concentration (2-3M) to make sure all proteins are of the column.

4. Remove salts Use dialysis to remove the salts from your protein solution.