My peptide is 2000Da, hydrophobic and soluble only in DMF and DMSO. So, when I am doing RP-HPLC with Sunfire C-18 column (4.6x250mm, 5micron) with solvent system ACN+0.1%TFA and Water+0.1% TFA, whenever I inject the sample, peak elutes at 1-2 min irrespective of the gradient. Can anyone suggest what may be the possible reason of it or what should I do to avoid such problem?
Your column may be bad. Try another one.
An alternative explanation is that your peptide binds to the column and never elutes. The peak at 1-2 min is the injection front rather than the peptide.
Use some slow gradient method for peptide separation using RPLC. Try following gradient:
A= ACN+0.1%TFA , B= Water+0.1% TFA
From 10% to 45% of B in 20 min, switched to 100% buffer B for 50 min, followed by 10 min. Optimize your flow rate according to your column.
Try slower gradient system. It might help youThe peak at 1-2 mins. may not be your peak of interest, it may be because of dead volume of the column.
If your column does not retent the solute though it should, check your column performance with the method given in columns performance report. This performance report is always included in the delivery package of the column. If the column does not retent the test solutes, your column has lost the C18; if it retents, then modify your analysis method (i.e. slower the gradient).
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