Chromatography Forum

Hello I am trying to separate some sterols in the product by using C18 column. During my experiment I have to use methanol as MP on the first four components and methanol:water 92:8 for the other components. I have got a low pressure gradient pump. Can anyone let me know how to set up the gradient programme please. I have tried some different programmes but they are not working. Thanks a lot.

92% methanol is a weaker mobile phase than 100% methanol. Your gradient should start with 92% methanol and go to 100% methanol.

92% methanol is a weaker mobile phase than 100% methanol. Your gradient should start with 92% methanol and go to 100% methanol.

Thanks for help, Uwe. If I use weak methanol (92%) as eluent by isocratic method I can sepearte all compounds, But the retention time will be very long. That' s why I want to use 100% methanol first as the first 4 components will be eluented out within 10mins. The other two components (only can be sepearted at methanol:water 92:8) then will be coming out later. (around 50 minutes at weak mobile phase). This is only my thoughs. I am a newbird for using gradient pump. The results I got were not like what I have thought. Can you give me some advice please. Thanks!

So you have reasonable retention of all your compounds still in 100% methanol, but the last two do not resolve. In this case, a gradient back to 92% methanol is not going to help, and you need to figure out other ways to find a solution.

How about an isocratic separation with an intermediate solvent composition, say maybe 95% methanol?

May I suggest checking with Klaus Bischoff at Bischoff Chromatography. He introduced a new approach to HPLC at Pittcon with his new POPLC which is as I understand it Phase Optimization LC. This uses a new approach which will allow you to basically build your own column in segments to create the optimal separation. (This is not an advertisement...I saw it at Pittcon and was impressed.)

I hope this helps you.
Jeff

Have you tried MeOH/THF?

http://www.imtakt.com/TecInfo/TI169E.pdf

I would try another column Type (if availible check C8 columns) or
blend your methanol with other solvents like acetonitrile or some THF.

Alex

Thanks for everyone's help. I have tried 95% methanol but the last two peaks can not resolve until 92% methano was used. I have another option: under isocratic condition the first four peaks can resolve very well in 12 minutes by using 100% methanol as mobile phase. The other components can be well seperated in the acetonitrile :water (97:3) mobile phase around 35mins. Can I use gradient pump to put this together? I have tried a bit. But it was not working to me. I haven't used THF as I have beening thinkging the gradient method will work for me. I am going to try THF if it is not working.(but I have tried acetic acid before). Thanks again.

Have you tried a gradient with starting composition of 92:8 and hold for 2 to 5 min then ramp the methanol conc to 99% over 8-10 min then hold the 99% as long as needed to elute the other components. I try to never run 100% organic in a c18 column. also sounds as though you need a weaker stationary phase, try c8 or phenyl. Whenever I need gradients starting at organic conc around 90% I start to think I have the wrong column.

KC:

try to never run 100% organic in a c18 column.

Hi KC,
I wonder what discomfort may it cause?

I do not know the disconfort of KC, but mine might be similar.

If you have to use 100% organic is because you are having excessive retention for your analytes, which means your chromatography has a major probability to become problemaic!

I would move to a less polar column, such as C8 or to a different specificity, such as Phenyl.