Desalting of Oligonucleotide, Peptide
and Protein Solutions
The desalting of peptide and protein solutions has been performed using various techniques, including dialysis, gel filtration or ultrafiltration. Desalting can also be rapidly and simply accomplished using reversed phase chromatography (RPC). Most biomolecules of commercial interest can be retained at high capacity on the reversed phase media, while salts flow through unretained. The target is then eluted using a step or linear gradient of an organic solvent. This means of desalting has the added benefit of being able to further purify the target molecule, as desired.
Two examples of this technique are illustrated below.
The first example illustrates the fact that RPC can be an excellent desalting method for synthetic oligonucleotides. Figure 1 demonstrates the desalting of a phosphodiester 12-mer oligonucleotide using a 1.0cm ID x 7cm L column of Amberchrom™ CG300M with a 10mM TEAA mobile phase.
Figure 1 - Desalting of a 12-mer Phosphodiester
on Amberchrom™ CG300M
The oligonucleotide is loaded in a 0.9% NaCl solution, and the salt is allowed to flow through. By increasing the acetonitrile concentration to 30%, the desalted oligonucleotide can be eluted from the Amberchrom™ column.
The second example demonstrates that RPC can also be used to desalt peptides, as demonstrated in Figure 2.
Figure 2 - Desalting of Vancomycin on Amberchrom™ CG161M
In this example, a 5mg/mL solution of vancomycin in 5% sodium chloride was loaded on a 1cm ID x 6.3cm L Amberchrom™ CG161M at a flow rate of 2mL/minute. A total of 970mg of vancomycin was loaded on the column, and the column was then rinsed with four column volumes (CV) of Milli-Q water. During the first cycle, the column was eluted using 34 CV of methanol. In order to improve the recovery of vancomycin and decrease the solvent usage, the elution conditions were changed to 20 CV of 50% methanol/50% Milli-Q water.
The results of both desalting cycles are shown in Table 1.
Table 1: Desalting of Vancomycin on Amberchrom™ CG161M |
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Cycle |
Step |
Load (mg) |
Recovered (mg) |
% Recovered |
1 |
Load |
970 |
314 |
|
Rinse |
102 |
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Elution |
443 |
80 |
||
2 |
Load |
970 |
249 |
|
Rinse |
199 |
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Elution |
481 |
92 |
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 |
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