carryover with HPLC

[tuksmum]

I am having carryover issues. I am new to chromatography and are not sure how to fix the following problem.

I typically run 3 blank injections before I run my sequence. Last week, my data started looking strange. The first blank injection ran clean, the second had major carryover and the the subsequent injections have carryover that elutes at the same retention time as my PDL and has an area greater than my PDL. Hence, my system fails suitability. I had flushed 50/50 ACN/H2O thru the system before I ran this sequence. But I did not do an injectionswhen flushing the 50/50 ACN/H2O solution. I suspect I have a serious carry over problem but not sure how to fix it.
Please help!

[mbulentcakar]

Hi,
Bear with me if i misunderstood, but the problem you described seems like the usual contaminated water/buffer in a gradient elution. It's very easy to confirm but somewhat difficult to pinpoint the exact cause, though forum has many discussions about the issue.
If this is not the case, you could provide more details about your mobile phase, injections, concentrations etc.
Best regards,
Bulent

[grzesiek]

like mbulentcakar said, seems like problem with contamination, please give us details of the method

[tuksmum]

mobile phase A: 95:5 (2%HClO4: ACN) Mobile phase B: 60% ACN:40% 2% HClO4 soln.
50 C column temp.
I inject 200 mcg of sample.

I suspect that I have to clean the injector. the contamination from previous runs is so strong and keeps increasing with each diluent injection. (diluent is 95:5 2%HClO4: ACN)

[mbulentcakar]

Hi Tuksmum,
I do not thing that the issue is related with the injector. The problem's most likely the contamination in your mobile phase A. You may check if your water or HClO4 supply has changed and you may track any contamination from pH electrodes, detergents or for some cases cosmetic hand creams.
This problem is common and discussed many times in this forum, one is below:
viewtopic.php?t=10475&highlight=gradient+contamination
Mr. Jupille posted a link to a very good mini seminar at that topic:
http://www.lcresources.com/more_resourc ... hp?f=3&t=5
Best regards,
Bulent

[grzesiek]

have yoy tried preaparing new mobile phase? I once had old TFA and this peak from injector that I couldn't get rid of

[HW Mueller]

Probably only a wizzard could get an increase in carryover after blanck injections.