Chromatography Forum

Hi, folks -

I'm running RP, on peptide, AcN/H2O/TFA gradient, C18 column.

Should my AUC be changing significantly on my standards?

Check this out: (all std's at the same concentration, in the same matrix, prepared fresh).

Day 1: Avg. AUC = 262. %RSD (5 inj's) = 6.6
Day 2: (after column cleaning) Avg. AUC = 787. %RSD = 1.8.
Day 3: (after column cleaning) Avg. AUC = 1125. %RSD = 0.8
Day 3: (after ~12 samples were run; same std prep as Day 3 above) Avg. AUC = 975. %RSD = 1.2 (11 inj's)
Day 4: (after column cleaning) Avg. AUC = 403. %RSD = 10.2.

Initially, cleaning the column seemed to be a sure path toward better chromatography. When my AUC dropped so significantly on Day 3, its seemed like a good choice. But I think now the column is just beyond repair. The RT isn't shifting, peak shape is still adequate, but it's getting so that I can't even get ballpark results.

Sample matrix is an issue too: samples at high pH are now recovering ~600%!!!

Any suggestions/technical info/papers/empathy is much appreciated.

EM

AUC = Absorbance Units?

I would wonder if you are actually reading the wavelength of the top of the peak with a suitable spectral bandwidth or, alternatively, whether you are reading at a low wavelength where the solvent is absorbing.

If not, then I would suspect your sample solution is giving you some of the problems, and you could investigate whether the AUC stays constant if you clean the column and only inject standards.

The high results for samples could be co-elution of junk solubilised by the pH, or low results from your standards.
Treat standards same as samples ( eg high pH ) and see what happens.

Please keep having fun,

Bruce Hamilton

Sorry, Bruce. AUC = area under the curve. Peak area. (My group always says "AUC." I never used the term til I started here!)

I am now pretty certain that what I'm seeing is the result of surfactant in my samples building up on the column over time and affecting the retention of my analyte. Depending on the composition of the samples I run, the subsequent injections will (in effect) see a different stationary phase [that is, the built-up surfactant.] I think some of the analyte in each sample is hanging out among the built-up surfactant, then when the high pH samples hit it, it pulls the sample out. Thus the 600% recoveries.

I think the areas change because the stationary phase is changing after each run of samples, based on how many/composition of samples I ran. However, the standards are relatively reproducible because when I inject the standards, they're all "seeing" the same stationary phase 'cause no samples have yet been injected.

I don't know. It's a theory. My hat's off to anybody who actually read this entire post!

EM

If the surfactant was modifying the stationary phase, wouldn't the peak retention time also be changing?.

I would guess the surfactant and/or other material in your sample are solubilising more of your standard, or coeluting.

In both cases, you have to address the sample preparation, perhaps by SPE.

Please keep having fun.

Bruce Hamilton

Bruce -

I neglected to mention the flaw in my theory, which is that I would expect my RT to change, but it's not! That's what's so confusing. (I thought I could slide that by, but the folks here are too bright!)

I ordered some SPE cartridges today. I also have a plan in place to do more up-front investigation of my analyte (effects of pH, matrix, concentration, etc) before jumping into samples. [Although that's hard to do - they keep on sending me samples to run!]

EM

What's the surfactant (or approx. mwt of the surfactant if said
surfactant is proprietary).

One question, Are you filtering your samples? if so, what kind of membrane are you using? I am aksing this because at some point I was working with a surfactant in a formulation (small molecules, not peptides) and came to find out that I was loosing the surfactant to the PVDF membrane. I don't know much about peptide/proteing separations, but just wanted to mention it just in case.

What's the surfactant (or approx. mwt of the surfactant if said
surfactant is proprietary).

It's Polyvinyl Alcohol. I don't know the approx. MW they're using (but I could check the bottle.)

Are you filtering your samples?

Not yet, but I'm going to evaluate filters along with SPE cartridges next week.

Thanks, all.

EM