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Carryover peak RP-HPLC
Posted: Sat Jul 13, 2019 10:51 am
by Aniaam00120
I work on RP-HPLC method for analysing a small peptide. My diluent is water. Mobile phase A is Water +0.065% TFA & Mobile phase B Acetonitrile +0.05% TFA. I use Inertsil OTS-3 column together with guard column. When the peptide is mixed with CMC (Carboxymethyl cellulose that is natural glue) and mannitol I have massive carry over which I believe is due to the presence of CMC. It takes about 5 water injections to remove the carryover peak. Do you have any ideas for removing the carryover? I have eliminated other obvious contamination factors and I came to conclusion that this must be column retaining the sample that did not elute in the first run. Thank you in advance for your help.
Re: Carryover peak RP-HPLC
Posted: Sun Jul 14, 2019 6:40 pm
by Andy Alpert
The carboxyl- groups of the polymer may be chelating heavy metals in the HPLC column. Try passivating the column by pumping a solution of 40 mM EDTA.2Na through it at a low flow rate for at least 16 hours. Then, flush out the EDTA, equilibrate with your mobile phase, and assess the degree of carryover that you get. If this solves the problem, then repeat this treatment every 3 weeks. If it improves the situation but doesn't solve it, then try treating with EDTA for 24-30 hours instead of 16 hours.
Re: Carryover peak RP-HPLC
Posted: Thu Jul 18, 2019 9:21 am
by tkubowicz
Hello
Few ideas:
1. Extend final holdup time (high ACN %) in your method and check few blanks after to confirm if all "rubish" is eluted from column
2. You can create method with 95:5 ACN/Buffer and run it after your sample to clean column. Then you can run blank/no injection to confirm there is no carryover
3. Don't forget to setup proper needle/needle seat washing for sampler - it is critical place for carryover issue.
Regards
Tomasz Kubowicz
Re: Carryover peak RP-HPLC
Posted: Thu Jul 18, 2019 9:21 am
by tkubowicz
Hello
Few ideas:
1. Extend final holdup time (high ACN %) in your method and check few blanks after to confirm if all "rubish" is eluted from column
2. You can create method with 95:5 ACN/Buffer and run it after your sample to clean column. Then you can run blank/no injection to confirm there is no carryover
3. Don't forget to setup proper needle/needle seat washing for sampler - it is critical place for carryover issue.
Regards
Tomasz Kubowicz