Hi all!

I'll just like to seek some opinions with regards to the subject matter. One of my columns (Phenomenex Luna 150x20 I.D, 5uM) is experiencing this problem. Prior to this problem, we have tried flushing it with 95%ACN and 5%DMSO because it was blocked. After the which, the problem of peak tailing and peak splitting occur whenever we inject an DPH standard.

Please advice. Thank you!

Peak splitting (assuming analyte peak did not split under similar conditions previously), is generally caused by at least two fluid pathways through the column with different flow velocities. In general, the problem is symptomatic of either a plugged inlet column frit, plugging of the surface of the column inlet or an irregular void at the top of the column. The first of these problems can be readily checked by replacing the inlet frit. The latter problem can be readily checked by visual inspection. Partial plugging of the chromatographic media at the top of the column bed may or may not be apparent upon inspection. However, since you observed a blockage problem already, there's a good chance that this is the cause of the problem. I would suggest that you increase the duration of your 95% acetonitrile and 5% DMSO flush to see if this alleviates the problem. Otherwise, you may wish to backflush the column to remove the residual particulate. As a last resort, you can sometimes get away with scraping away the topmost layer of column packing material and replacing it with fresh material from another spent column using a small spatula.