Chromatography Forum

Hello everyone,

I would like to ask your advise in achieving peak separation between two organic compounds: ribulose 1,5-biphosphate (RUBP) and 3-phosphoglyceric acid (3-PGA) in HPLC using ELSD as detector. In the literature, they achieved peak separation between the two compounds by using the following mobile phases:

Mobile A : 20 mM ammonium acetate and 20 mM ammonium hydroxide in water (pH 9.45)
Mobile B : acetonitrile (ACN):water (85:15, v/v)

Unfortunately, we only have the Shimadzu GIST NH2 column whose working pH range is limited within 2 - 7.5, hence I am very limited in adjusting the pH of my mobile phases. I have tried using just 50 mM ammonium acetate as mobile phase A without adjusting the pH. Unfortunately, I could not manage to achieve peak separation despite using gradient elution program.

I would like to still explore different ways to make it work with our current column and only buy one if it is really necessary (last option). However, I have limited experience with chromatographic separation, let alone for similar organic compounds.

Would anyone have some helpful advice for this?

What is your gradient profile? What is the column specified for the method you are trying to use?

Unless your time/effort are abundant its probably cheaper to buy the column that you know will work vs designing a whole new method.

I'd expect a -3 vs -4 charge difference at pH 9.5 would be easy to separate here by your HILIC + anion exchange method, however you are around pH 7 without adjusting the pH of the mobile phase at all and the compounds could have a similar -3 charge state.

Have you tried running the separation at pH 3?