Chromatography Forum

Hi everyone

I never really understood the complex interactions on primesep columns. Let's take Primesep SB and the retention of sulfamic acid (http://www.sielc.com/Compound-Sulfamic-Acid.html). What influence do the concentration of acid or buffer, and acetonitrile have? How will the retention time change when adding more acetonitrile or more acid?

Thanks for enlightenment
Jörg

It is Easy .
In mixed-mode you always have multiple interactions. In bi-modal columns it is reversed -phase and anion-exchange or RP/cation-exchange. Amount of ACN controls reversed-phase interaction and to some degree ionization of analyte and stationary phase. Buffer concentration affects ion-exchange interaction. Buffer pH affects ionization of your compound and ionization of the stationary phase and thus affects ion-exchange too. You need to have both, stationary phase and compound to be in ionized state to benefit from ion-exchange in addition to the reversed-phase interaction. It is very hard to find two compounds with exactly the same RP and ionic properties and mixed-mode explores a tiny difference in these properties to achieve separation. Here is a graph representing this statement:
http://www.sielc.com/Technology_2D_Properties.html

Depending on the properties of the molecules you have a different degree of interactions for RP and ion-exchange. In case you are referring it is mostly ion-exchange since sulfamic acid is hydrophilic and basic. In two methods you have different retention due to pH and amount of ions generated by ammonium formate and formic acid.
You your compound is hydrophobic, then more ACN will shorten retention of the compound. If you compound is ionic and there is ion-exchange and not ion-exclusion, then more buffer will shorten you retention.
Mixed-mode is not much complicated then RP chromatography, if you know mechanisms of retention and how to control them. I use comparison between car with automatic transmission and manual transmission. In automatic you have two pedals to get you car moving stopping, in manual you have 3 and a stick shift. Once you learn how to drive manual you will never go back to automatic - it is more fun, it is faster and more economical...plus ladies like cool cars . One of the reason we are offering free method development is that we are very effective in screening, we barely spend more than 4 h on any method, unless people are trying to separate multiple compounds with multiple impurities...then we need 8 h

Contact me if you have questions and I will be more than happy to help you. If you are interested in a seminar or presentation also let me know.

Hello Vlad

I try to do the math:
0.3% HCOOH is ca. 65 mM at pH 2.5
while the buffer is only 5 mM at pH 3.5
So in this example, is the pH of Ammonium formate or the ion thrength more relevant for the shorter RT in Example 1?

Jörg

PS: Thanks for the link to the 2D matrix. I clicked through your website, but somehow I have skipped this interesting information!

Two factors are playing role here, lower pH of the formic acid suppresses ionization of the analyte and also you need to consider dissociation constants of additives to the mobile phase.