Insulin peak movement within a run

[Nikki]

Hi,

I'm having a problem with our RP method for insulin and I was wondering if anyone can suggest anything to help.

When we run our very shallow insulin gradient (30-32%) B the main peak elution time moves around by up to 5 minutes (it's only a 15 minute run including a 5min wash step and 2 min re-equilibration). This is causing me to miss some of the smaller peaks on either side we are interested in and sometimes moves the main peak out of range entirely, either into the wash step or into the phenol and cresol early eluting peaks. We have had this problem with both longer and shorter RP columns but it is a much bigger problem during the short runs.

Increasing the wash and re-equilibration times has helped a little but I still have this problem over the course of a run (~30 samples) and cannot increase the run time much further without having a serious impact on resources (the reason we switched to the small column). Initially the retention time got lower with every sample injected, however now I see the main peak moving without any apparent trends from one end of the chromatogram to the other.

I think that the presence of the aromatic preservatives in our samples is likely to be causing this problem but cannot pre-treat the sample to remove them.

Many thanks for any ideas

Nikki

Column: ACE 3 C18 100 x4 mm
Buffer A: Water/0.1% TFA
Buffer B: ACN/0.1% TFA
(a similar problem was encountered using buffers containing sodium sulphate)

[Peter Apps]

My guess would be that a gradient this shallow is beyond the flow precision of your pumps and mixer. You could try premixed 30% as A and premixed 32% as B, and run from 0 to 100 %.

Why are you running such a shallow gradient ?

Peter

[paulw]

I had a lot of trouble with the shallow gradient that a lot of published methods seemed to use. It was probably as Peter has already suggested an issue with the mixer capabilities. Insulin was very fickle about retention, I struggled for a long while trying to nail down an isocratic run, and I could never get a stable retention time. I also saw the problems you mentioned with the preservatives phenol and meta-cresol.

While I don't know how much flexibility you have, I found that insulin would not come off the column at ~20% ACN, however both of the preservatives would, so I ran a short period isocratic at 20%ACN to elute the preservatives then changed to a gradient to push the insulin off.

Paul

[Nikki]

Many thanks for your help, I am using a shallow gradient as that is what most of the published methods use. I will both suggestions and see how it all goes.

Cheers!

Nikki