Chromatography Forum

Hi,

I am trying to run 5' monophosphate nucleotide standards on HPLC. I am using ammonium acetate buffer at pH 5.8 with ACN at 5%. I have tried a few C18 columns but am always having the dCMP come off the column at the same retention time as the void volume. The chromatograph barely separate my other dGMP and dAMP standards. I was wondering what I might be doing wrong that is causing these fast retention times. Thanks.

These are very polar analytes, and to use 5% acetonitrile will elute them too early. You will need to go to 100% water to get good retention. If you have a standard C18, it won't work in water. You need a C18 that has been designed to work in 100% water, such as the Atlantis T3 from Waters. It gives the highest retention in water.

The reason you get no retention is that these analytes are very hydrophilic. Reversed phase chromatography is not suited for compounds that prefer the water eluent to the unpolar stationary phase.
These compounds are much better separated by HILIC (which in essence is the opposite of reversed phase), a hydrophilic stationary phase and an acetonitrile rich eluent.

http://www.sequant.com/files/documents/ ... nd_CTP.pdf

If you have no previous experiance with HILIC you can order the practical guide to HILIC free of charge on our webpage.

http://www.sequant.com/default.asp?ml=11810

Below are 3 ways to analyze nucleotides (RP + ion-pairing, RP + IEX, NP):

Unison UK-C18 (RP + ion-pairing): http://www.imtaktusa.com/site_media/fil ... TI133E.pdf

Scherzo SM-C18 (RP + IEX): http://www.imtaktusa.com/site_media/fil ... TI529E.pdf

Unison UK-Amino (NP): http://www.imtaktusa.com/site_media/fil ... TI363E.pdf