Chromatography Forum

Hello Everyone,

I am doing a bioanalytical method validation. The biological matrix is liver microsomes and I normally prepare my calibration standards by spiking the analyte into a blank microsomes containing both Mgcl2, sodium pyrophosphate and potassium phosphate buffer. My sample prep is mostly protein precipitation using equal volume of cold acetonitrile.

I have discovered that whenever I run my batch of standards, the peaks are splitting. This is usually resolved after back flushing the column but I cannot continue doing this since it is taking up the analysis time.

I would appreciate any suggestion towards solving this problem.

Thank you.

If you have multiple peaks and they are all splitting *the same way*, the chances are good that cause is a inlet flow anomaly (either a partially plugged inlet frit, or a headspace on the column, or an air bubble stuck in the inlet frit.

Air bubbles tend to come and go, so I don't think that's your problem. Backflushing the column would fix a partially-plugged frit, and might take care of a headspace temporarily. Unfortunately, the only way to determine for sure is to take the inlet fitting off the column and look. If the packing doesn't come all the way up to the top of the column (i.e., a headspace), then replacing the column is the best solution. If the packing *does* come up to the top of the column, then replace the frit and do a better job filtering your samples to prevent recurrence.

My guess at this point is that you have a headspace; it might be cheaper to just replace the column (and do a better job with sample filtration!) than to fuss with more diagnostics.

Thank you for the response