Dear Colleagues,
In brief, I have a method for protein resolution using a C18 column but it was developed in a 250 * 4.6 mm column, at the moment I have a C18 but short in length 150 * 4.6 mm. Would you recommend shortening the time of 40% (as it is 100 mm shorter) and keep the same gradients between solvent A (polar phase) and B (non-polar phase) or playing with the gradients too?
Thanks for your help.
Best,
Giovanni
For an identical, but shorter in length column running isocratic analysis by HPLC, the flow rate and composition would be the same as before (THE column ID is most important). A shorter column will have less load capacity (lower back-pressure too) so at the same concentration, the inj volume should be reduced.
For a gradient analysis conversion, you need to specify the exact gradient composition program (in detail) for anyone to suggest an answer. Since only the length of the column is changing, try to initially keep the steepness of the gradient the same, then re-optimize the composition as needed (always checking for proper retention and resolution).
Note: Be sure to re-develop and re-optimize the method after changing the length of the column. Even so called "identical" columns (same support, particle size, dimensions and so on) may show differences in elution. Also, as the column volume decreases, the HPLC's system volume increases and may have a greater impact on the results (e.g. dwell volume, flow cell volume, injection volume...).
Hi Giovanni,
Have a look here:
http://www.ace-hplc.com/technical-area/knowledge.aspx
This contains a tool in excel to make method transfers in LC.
Regards, Hendrik
Hi Giovanni,
maybe this whitepaper that I once downloaded from c&en will help you?
-Joseph
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