Chromatography Forum

Dear all,

I have been facing some problem with my current assay.
My RT of my peaks are shifting by at least 0.4 mins after only 40 injections. The RT is slowly shifting further after every few injections.
I have not faced this problem when i was doing the same assay previously.
During this assay, column pressure also starts increasing slowly after every few injections. In previous assay, pressure still remains the same after about 70 plus injections (I run at least 70 plus injections for each batch/day). Increase of pressure during each run might b just around 1-2 bar but it never affects the RT. I have had certain batches where pressure does increase significantly but RT remains constant because the pressure increase was caused by a blocked frit or guard column.

I always flush my column after bout 150 injections. I initially thought that the slight increase of pressure and the shifting of RT might be caused by a faulty frit or guard column but both are new.

I have suspected that maybe my column (up to now, the column has been injected at least bout 1000 injections) is dying but not too sure about it. Peak shapes are all still good though.
It would be nice to have other views or opinions regarding this situation. Thanks!

1000 injections is not the longest column lifetime on record, but it's within what I would consider the "normal" range. Of couse, the lifetime can also be affected by the operating conditions (pH, temperature, organic solvent), column chemistry, and storage conditions. The first thing I would do is to flush the column one more time and then try again with a fresh batch of mobile phase. If the retention times do not return to their expected values, then put in a new column.

e3lyn - (1) is your mobile phase premixed, or does the HPLC mix them for you?

(2) is your column compartment temeperature-controlled, to a temperature at least 5C above your lab/overnight temperature?

Tell your boss that columns are consumable items, and like Tom hinted, always good practice to have at least one brand-new column in the drawer so you could readily swap that in and evaluate. And by evaluate, whether chromatography or hardware issues, I mean: is the issue better, worse, or the same. Only change one "thing" at a time.

Before I performed last rites on the column, I'd review what has changed since the last good series, especially with regard to potential particulates/insolubles from...

- inadequate/different sample preparation ( including pH, filtering, storage temp etc ). One indication that sample prrparation may be an issue may be that multiple injections of a mobile phase blank between a sample replicate doesn't increase the pressure, and doesn't increment the the retentuon time change as much.
- different mobile phase components ( including any filtering, temp changes - if buffered, ensure buffer compents are soluble )
- recent mobile phases through the instrument, especially if it has a membrane degasser, and any subsequent flushing.
- storage solvents used in the column, ensure they were fully flushed.
- condition of instrument, eg mixing valves ( if mobile phase is not premixed ) to ensure that mobile phase composition is consistent, and pump flowrates.

Bruce Hamilton

I'm running a gradient run as my mobile phase with a premix on one channel and another channel with methanol only. Temperature is 38C.

I would say that 1000 injections are not too bad but it is known that the sample tends to clogged up the frit or spoil the guard column after 2-3 batches. But never had any problem with the column if i flushes it.

I will try with another column to see if the retention shifts again. I'm still not sure if the column really is dying because peak shapes are still good.

Thanks for all the suggestions!