Chromatography Forum

Hi,
I am working on nucleosides,they are weak basic highly polar compounds.I have tried many buffers and ion pair reagents on most of columns,but its eluting at 2 min in all cases.atlast it retained on Primesep100 column,but peak shape was not good.It is very broad.Can anypne plz suggest me if ur working or dealt with this situation.I want to have sharp peak.My mobile phase with Primesep column in 0.5 % formic acid and ACN(85:15).
Thank u

Go to the Waters website and look for the Chemistry eApplications Booklet. You can also look directly at the location below, but you may need to have signed in already.

http://www.waters.com/WatersDivision/pd ... ntis30.pdf

The bottom line is: you need to wrok with a C18 that is compatible with 100% water.

prathima_chem,

Primesep 100 should work and peak shapes suppose to be good. Please make sure that this are not compounds coming from previous injection.
Also I would try to change formic acid to TFA or sulfuric acid (both at 0.05-0.1%). Use 5-10% of ACN.
Please post list of your compounds here or email it to me and we will rerun this in the lab.

Here is application on Primesep 200 column. Your retention on Primesep 100 will be even longer:
http://www.sielc.com/application_013.html

If you reduce buffer strength (acid concentration) you will achieve longer retention. Going to 0% organic will increase retention even further. Primesep columns are compatible with 100% water and 100% CAN. You don’t need another column, just troubleshoot /optimize your separation

Hi ,
Thank u very much for ur reply.I am working on azacytidine,I have a plan to try TFA instead of formic acid.I observed an improved peak shape with increase in concentration of formic acid from 0.1 to 0.5 %.I will try with 1.0% formic acid ,then switch to TFA.
I have to sharpen the peak,then my goal is achieved.Once again I thank u for ur interest in sorting out the problem.

If you need LC/MS method with Primesep 100 column, I would suggest using ammonium formate instead of formic acid. Ammonium formate will create more ions in comparison to formic acid (as it dissociates completely). If your detection technique is UV then you can use TFA, sulfuric or phosphoric acids and corresponding Na or K buffers. TFA and sulfuric will give you sharp peaks.

Below are some nucleoside applications:

Unison UK-Silica:
http://www.imtakt.com/TecInfo/TI167E.pdf

Unison UK-C18:
http://www.imtakt.com/TecInfo/TI088E.pdf

Mononucleotides on Unison UK-Amino:
http://www.imtakt.com/TecInfo/TI363E.pdf

hi,
I thank u SiELC tech for ur guidance,Me method of nucleoside is finalised with Primesep100 column with 0.1%TFA and ACN(90:10) as mobile phase.
Peak shape has improved greatly with introduction of TFA rather with formic acid.
Thank u very much and we ordered 2 primesep100 columns for the analysis.