[mobfesk2]

Hi,

I've been trying to separate a glycopeptide product from its starting material. I'm using a C8 column and detecting by LC-MS. My mobile phase is 0.1% TFA in water and 0.1% TFA in AcN. Temperature is 25C. My product has a tR of 12.29 mins and my starting material has a tR of 12.42 mins, which leads to 2 peaks that are not completely resolved. i.e. co-elution. Does any one know how I can get better separation. I'm using the following gradient:

Time (mins)    %B
0......5
3......5
10....15
15....18
23....30
27....95
29....95
30....5
32....5

I have even tried running from 15% - 18% B over 15 mins (10 - 25 mins) but still no fully resolved peaks

Thanks in advance for any suggestions

[enahs]

Can you not find different product ions? If so, it does not matter if they co-elute. Or are you trying to collect the products?


I am assuming you are following general practice and the organic is B.
If you did not gradient up to more B, as in, stay at 95% A and 5%B for the whole time (except for the wash at the end) I would expect you to get better separation (but it would increase your assay time). This will lead to not as sharp peaks, and so I would increase the column temperature some more, say up to ~30-35 C. Maybe even 99:1 (A:B). Increasing the proportion of water should seperate them more.


Also, most people tend to not use TFA with mass spec, some have found that it suppresses ionization. If you are having trouble getting low detection limits, I would try maybe acetic acid.

[JGK]

I would definitely replace the TFA preferably with formic acid