Chromatography Forum

Dear colleagues:

Is there any way to convince Aspartic Acid and Arginine to spend few minutes in RP-HPLC column?
I tried C18, C8 and AQ columns but none of them worked!

I tried acetonitrile/water acidified with 0.1% phosphoric acid, ACN/0.1TFA, and Water 100% with no success!

In most of the cases the two compounds are not retained at all!

Any IDEAS?

Thanks in advance.

You will find a way, I am sure. But first you will have to convince a circle to transform itself into a square

But seriously: You will have to use some kind of derivatisation (see this method by Agilent, or another type of column. Primesep columns come to my mind at first.

Cheers,
Jörg

first you will have to convince a circle to transform itself into a square

Is it that difficult!

Thanks for the suggestions.
I am trying to avoid the derivatisation and also trying to work with the available equipment and columns because I need the method for only few runs!

Can anyone add more help in the negotiations with the two acids?

My first choice would be HILIC, I'd suppose that should be quite straight forward.
If you MUST use a C18, I'd think you might have a chance by using a low-pH mobile phase (in order to protonate the carboxylic and amine groups, so that both analytes are in their cationic form) in combination with an anionic ion-pairing reagent such as SDS.

If you MUST use a C18, I'd think you might have a chance by using a low-pH mobile phase

I used a C8 column with 0.1% TFA in both ACN and Water with a pH of 2 but I got no retention at all!

I hope I am not going to the wrong direction!

The catch ist to use an ion-pairing reagent. At low pH your analytes will be positively charged, therefore interaction with your hydrophobic stationary phase ist next to nothing. TFA actually has some ion-pairing properties, but not as strong as a "true" ion-pairing agent like SDS or some sort of long-chain alkyl sulfonate.
Mixed-mode columns like the Primesep mentioned by Jörg would be a much more convenient choice...

your answer is RP/mixed-mode or HILIC/mixed-mode:
http://www.sielc.com/Compound-Aspartic-Acid.html

for RP/mixed-mode you retain compounds by weak RP and stronger cation-exchange
For HILIC/mixed-mode you repaint compounds by HILIC and cation-exchange

You will need a gradinet of ions for your two compounds as one contains one basic/two acidic groups and second one contains one acidic and two basic groups.