Even well-packed columns will lose some of their efficiency over long usage periods of many cycles, especially when used with samples of high impurity level. If your column is used for synthetic peptide or recombinant peptide purification, or, in a worst-case, for aggregating, fibrillating peptide purification, the column performance can decrease rather quickly.
The first warning sign is usually the rising backpressure of the column. Peak shape deterioration or the drop in the number of theoretical plates may be another sign that you need to clean off the fibrillated peptide building up on the inlet side of the silica bed in your column.
Useful tip: perform the washing step with reversed flow. You do not want the garbage to travel through the column!
Your stationary phase is an expensive investment, so how can you extend its lifetime, or regenerate your packed column that has started to degrade in performance?
As a first step, washing your column with 90 – 100 % organic solvent is never harmful, and this simple step often helps! Useful tip: perform the washing step with reversed flow. You do not want the garbage to travel through the column!
What solvent should be used for regenerations?
Peptides normally interact through multi-interactions to silica: hydrophobic, silanophilic electrostatic interactions, hydrogen bonds, etc. Therefore, it is fundamentally important to suppress ALL interactions for a successful column regeneration. It is also important to suppress intermolecular peptide-peptide interactions.
Urea, ammonium hydroxide and Tris are well known agents to suppress peptide interactions. Tris
(10 mM, pH = 9-10) is an excellent agent to prevent peptide aggregation and to dissolve them. And of course, the pH will also have a large impact to suppress and dissolve. The proper pH to deploy is difficult to predict and depends largely on the isoelectric point of the peptide.
Generally, you will need around 50-60% organic solvent to suppress the hydrophobic interactions, and you will need cations (e.g., ammonium, sodium, amines) to suppress silanophilic interactions.
This is the key to allow degradation of the latched impurities to occur; the disengagement of the aggregation combined with the desorption effect of the organic solvent, which carries away the disengaged peptide fibril fragments. This is the necessary “double action” for successful column cleaning and will help extend your column lifetime.
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to regenerate your DAISOGEL packed column.