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HPLC-UV method for nucleotides

Posted: Tue Aug 01, 2017 2:33 pm
by ikik
Hi all,

I need to analyse nucleotides using HPLC-UV. I have a supelco c18-T column. Does anyone have a hplc method I could use?

Re: HPLC-UV method for nucleotides

Posted: Wed Aug 02, 2017 12:27 pm
by HPLC chemist

Re: HPLC-UV method for nucleotides

Posted: Wed Aug 02, 2017 1:14 pm
by ikik
Hi HPLC chemist,

Yes I did and only one of the methods worked but i had baseline problems where the baseline drifted quite significantly affecting my chromatogram.

The method I tried:

Mobile phase A: 0.03M KH2PO4, 10mM TBAHS(tetrabutylammonium hydrogen sulphate) adjusted to ph 6 with NaOH.

Mobile phase B: Mobile phase A:methanol (80:20 v/v)

injection vol: 10 microlitre, flow rate: 0.7 ml/min, column temp: 35oC

When I run it without TBAHS, I have no problems with the baseline. However I need TBAHS to separate the nucleotides.

Re: HPLC-UV method for nucleotides

Posted: Fri Aug 04, 2017 6:02 pm
by HPLC chemist
Sounds like your 'buffering/ion pairing' is not stable for your sample matrix. Increase the concentration of the TBAHS to 25 mM.

Re: HPLC-UV method for nucleotides

Posted: Fri Aug 04, 2017 6:04 pm
by HPLC chemist
Also increase the concentration of the Phosphate buffer to 50 mM (not 0.03 mM).