Chromatography Forum

Hello Folks!

My First post on the forum!

I have been struggling for a while, with this new peptide that I am working on. I have relatively less experience with RP-HPLC. So, I have to develop a method that is able to detect as well as quantify 4 species in the sample. Let us call the peptide, Cyc-1, its reduced form, another peptide - T2 and T2's oxidized form, which is a homodimer of T2 itself, these four things need to de detected as well as quantified.

While my method is able to detect all four, a baseline improvement would make it possible to confidently quantify the species as well. However, the problem is that the reduced form of the peptide is pretty difficult to resolve as T2 and the reduced form in other methods elute very close. Suggestions would be greatly appreciated! I a, attaching my chromagtogram as well as the accompanying gradient change in the figure. It would be best if I am able to keep the duration of run in the same time range.

Mobile phase: ACN+0.1% TFA and Water+ 0.1% TFA @ 1 ml/min. MWD-215nm Ref lambda (nm): Off

PS: I have looked through the forum for suggestions and applied some of those (Eg: maintaining temp constant and changing % TFA, turning ref wavelength on)




the baseline marginally impoves when I try this other method:



However, I am not able to detect the reduced form of cyc-1

thanks!
Ansh

Hey,

what is the problem with the baseline?

When you use acetonitrile you'll ever have a baseline with a gradient comparable to the gradient of your solvent. You'll just detect it later because of the way between the pump and the detector.

First thing I would change is the gradient. Try to use a linear gradient, so the baseline is rising linear too.

Additionally I think a gradient from 12% to 35% in 2 minutes is a little bit too much. (Depends on your used column.)

As a shot in the dark:

10% organic -> 15 mins -> 90% organic -> 5 mins -> 90% organic

But I have no idea, what you've tested and if your column is able to work with this settings.

But the baseline would look much better with this gradient.

One issue with MeCN/TFA/H2O gradients is the greater absorbance of 0.1 TFA/MeCN vs. 0.1 TFA/H2O.

Running at 0.085% TFA/MeCN with 0.1% TFA/H2O will help to flatten the baseline.

JMB

Hey,

what is the problem with the baseline?

When you use acetonitrile you'll ever have a baseline with a gradient comparable to the gradient of your solvent. You'll just detect it later because of the way between the pump and the detector.

First thing I would change is the gradient. Try to use a linear gradient, so the baseline is rising linear too.

Additionally I think a gradient from 12% to 35% in 2 minutes is a little bit too much. (Depends on your used column.)

As a shot in the dark:

10% organic -> 15 mins -> 90% organic -> 5 mins -> 90% organic

But I have no idea, what you've tested and if your column is able to work with this settings.

But the baseline would look much better with this gradient.

Thanks for your reply! The Problem with the baseline is that the drifting of signal prevents quatification of the homodimer in a confident manner. I can't use a linear gradient as I lose the capacity detect other hydrophobicity sensitive peaks. I will try to use the suggested gradient and see if it works!

One issue with MeCN/TFA/H2O gradients is the greater absorbance of 0.1 TFA/MeCN vs. 0.1 TFA/H2O.

Running at 0.085% TFA/MeCN with 0.1% TFA/H2O will help to flatten the baseline.

JMB

That is a great suggestion! Thanks! I was thinking about trying that too, as I figured that the drifting baseline could be a UV absorbance issue. However, I am a bit skeptical about the difference 0.085% vs 0.1% is going to make as there is very little TFA/ml, anyway. But I will definitely try this as well!

The baseline is not the worst problem here.
Look at the T-2 peak shape. It's awful.
Either there are more than 1 substance eluting there or there are some unspecific interactions on the loose.
My guess is a ruined column.

Best Regards