How to seperate close peaks in C18 HPLC

[Hana97]

Hello everyone.

I am currently trying to seperate compound from an active fraction of a sea star. Based on the nmr i ran earlier, there are sugar and nucleoside compounds in it. I had tried various methods, and this is the method with the best seperation:

This is the link to the chromatogram:
https://drive.google.com/file/d/1jaWg6J ... p=drivesdk

Solvent A: 0.1% TFA in H20
Solvent B: pure Acetonitrile
Flow rate: 1.0 ml/min
Temperature: Ambient
Tyoe of column: Purospher Hibar C18 encapped( 5 micrometer)
Method: 0 to 10th minute: Solvent B from 0% to 25%
10th to 15th minute: Solvent B from 25% to
50%

Can anyone please suggest methods to improve this chromatogram?
I have tried reducing the flow rate to 0.5ml/min and tweaking the gradient, the peaks became wide and separation is not good.

Thabk you in advance.

[Consumer Products Guy]

For me, the easiest thing to try is to make the temperature 10C cooler, try that. Then make 10C warmer than ambient, try that. See if better/worse/same.

[Hollow]

for me, some infos are missing:
- dimension of column (diameter x length)
- type of detection (wavelength; do you have PDA or MS at hand?)
- full gradient table (including re-equilibration)
- solvent in which your sample is dissolved
- how does a chromatogram of a solvent blank look like

[JackSilver]

Those peaks that I see look to be eluting early. The reply above mine has a lot of good questions that would allow us to help solve the problem. Also, please indicate which peaks are of interest to you.

[Vlad Orlovsky]

Slowing flow rate is not going to to too much. You compounds elute close to the void and you might run into a problem of interference coming from sample matrix, large injection, column contamination, etc. If you have any ionizable groups in your compound your best choice is mixed-mode chromatography - either HILIC/ion-exchange or RP/ion-exchange. Mixed-mode is all about selectivity and if you can employ two mechanisms you should be able to have retention and separation. Contact me if you have questions.

Here are couple of examples (including nucleotides):
https://helixchrom.com/compounds/guanosine-diphosphate/
https://helixchrom.com/compounds/raffinose/