Chromatography Forum

I am running an impurity test for a polypeptide in a C18 column and using 226nm detection. My problem is that repetitive peaks appear at retention times that overlap the impurity I'm supposed to test. These peaks are a set of tree repetitions of 4 peaks each, always at the same retention times. These peaks appear even when running blanks.
I'd really appreciate any suggestion to solve this problem. Below I describe some details of the run and the solutions I've tried. thanks a lot!

Peaks grow with every injection until reaching a maximun size after 20 injections approx. Peaks appear between times 30 and 40 minutes.
I could not eliminate these peaks by:
-washing the column
-replacing every component of mobile phases
-running the blank in 3 different hplc (always the same peaks are there...)

Gradient is composed of mobil phase A: H2O 93%/ACN 7% with TFA 0.13%, and mobile phase B is: H2O 30%/ACN 70% with 0.1% TFA.
Gradien is as follows:
0-30%B
25-55%B
40-100%B
45-100%B
50-30%B
55-stop

Several additional steps you can take to diagnose the problem:

1. Look at the width of the interfering peaks. Are they about the same width as the neighboring (real) peaks? If they are, that suggests something coming in with that injection. On the other hand, if the interfering peaks are significantly wider than the neighboring peaks, that suggests that they are late eluters from a previous run.

2. Run the "three blank gradients" test described on our web site (http://www.lcresources.com/resources/TSWiz/hs400.htm). If the size of the peaks increases with increasing equilibration time, then you have *something* contaminating your "A" mobile phase. If the size does *not* increase with increasing equilibration time, then the interfering peaks are probably coming from carryover in the autosampler or contamination from the needle wash.

If the problem is mobile phase contamination, there are a couple of tricks you can used to get rid of them, depending on your pump configuration. There is a description near the end of the mini-tutorial on artifact peaks that I posted here:
viewtopic.php?f=31&t=19085

There is no precision regarding the C18 column you are using. Think also of trying different brands of the C18 column.

Thank you both for your answers.
I will try the three blank gradient test to figure out the problem. Additionally, I will try to post a chromatogram tomorrow since I think the pattern of the interfering peaks can be informative, they are three identical sets of 4 peaks. This repetitive pattern does not look like the usual garbage to me, but maybe someone has seen it before.

In terms of replacing the column, I’ve seen the interfering peaks with two C18 columns already. But I do not discard column problems altogether, since the pH of mobile phases is 1.8 and 1.6 and the column is not recommended for pH below 2. Should this be a concern? Unfortunately changing the column is not an option.

thanks!

I' ve tried the three blank gradient test and it seems to be a contamination of the mobile phase A. I could not find yet the cause, but I'd like to show you a chromatogram because this patter still bothers me.

I hope you can help me!

I would suspect the TFA as the source of your contamination. Try running the gradient test using just water and acetonitrile without the TFA. If the baseline is clean, you need to use the best quality TFA you can find. I always bought the 1-mL sealed vials of TFA to get the cleanest baseline.