Chromatography Forum

Hey guys,

New to the forum, I appreciate any advice you might have for someone fairly new to HPLC. Unfortunately I don't have any data on this, my home computer, so I'll do my best to describe it. Otherwise I can attach chromatograms on Monday.

Here's the breakdown:
Analytes: Capreomycin sulfate, antibiotic #2 (AB2)
Column: brand new Zorbax Eclipse Plus C18, 3.5 um, 4.6x150 mm (NO GUARD COLUMN)
Mobile Phase A: 0.1% TFA in H2O (pH 1.95-2.05)
Mobile Phase B: 0.1% TFA in ACN
Problem: The first few runs were great. We had three peaks, one for capreomycin IIA/IIB, one small for capreomycin IA/IB, and one for AB2, all around expected elution times. Eventually I had problems with clogging somewhere in the sample delivery system (no peaks, the auotinjector leaked, and the injection port overflowed). I gave up for the day and washed the column with about 5 mL mobile phase B at the advice of my PI. The next day my PI helped me backwash the 6-way valve and we got peaks again. However, my capreomycin peaks were shifted about 15 minutes back with the AB2 peaks unaffected. I then did the following things:
1) tightened some loose junctions where mobile phase was leaking slightly
2) changed my mobile phase A since there was visible evaporation at the top of the bottle
3) changed my sample
From then on, I got the consistent result of four strange peaks, two of them broad, early in the run for capreomycin.

Do y'all have any idea what could be going on? Might the low pH of my mobile phase A have destroyed the column? What about backpressure from the earlier clog? Why would this affect ONLY the capreomycin and not the second antibiotic? I appreciate any insight or troubleshooting advice you could offer. We're probably ordering a new column and guard column on Monday just in case.

The pH is not too low for a C18 solumn.

I'm using 0,1% TFA all day long on C18 columns.

How is your backpressure?

You should have 100-130bar with ACN/Water on this column at a flow of 1ml/min.

It depends a little from system to system.

With a high backpressure you could have ruined your column.

When the backpressure is too high, bypass the column and look if the column is glogged or something else.

If the column causes this you should replace it.

Furthermore I'll always recommend using a guard column. (Not only an inline-filter.)

My pumps haven't reported a pressure higher than 1500 psi (so ~100 bar), but since I had had fluid leakage I didn't know if that accurately reflected the pressure on the column. The more I think about it, the less that makes sense though. Do you have any idea what else could cause such a stark change in the way my system is interpreting capreomycin? Could we have done something funky when backwashing?

When the pressure is ok and stable and there are no leaks, then I think the problem is something else.

If possible post a screenshot of the run with gradient and pressure over time.

How do you prepare the samples?