Chromatography Forum

Hi,

I would like to have an LC-method for sulfosuccinic acid, using eluents that are MS-compatible.

http://www.sigmaaldrich.com/catalog/Pro ... =ON&F=SPEC

I have tried anion-exchange, but the molecule sticks so hard to the column that I need sulfuric or phosphoric acid to get it out. In ordinary reversed-phase, it has no retention what so ever. Could this be a candidate for HILIC?

thanks,
Mattias

If you used a strong anion-exchange (SAX) column, then try a weak anion-exchange (WAX) column at a pH around 7.5. SAX columns maintain their charge density up to pH 9.5 or so. WAX columns lose their charge density in a continuum between pH 5-9. Our PolyWAX LP material is an example. Also, you could decrease the overall retention by the simple method of choosing a WAX material with a wide pore diameter, such as 1000 or 1500 Å. These materials have 25% the surface area of a 300-Å material and the disparity is even greater relative to a 100-Å material.

Did you try HILIC?

Thanks,

Andy Alpert> I have used a SAX column, and I don't think I have any WAX columns in-house. This molecule has three negative charges, which is probably too much for the SAX. I will see if I try that or HILIC.

Bintang> After more than 12 years of work with LC, I have still never tried HILIC . I assume that the molecule needs to be soluble in a high percentage of acetonitrile (needs to be tested)? Could you recommend column and conditions that could work as a starting point?

If you try HILIC, be prepared to include at least 40 mM salt in the mobile phases and sample solvent, and possibly more. Otherwise, you're going to get bad tailing or even multiple peaks for a solute like this with so many ionizable groups. Ref: A.J. Alpert, Anal. Chem. 80 (2008) 62 [specifically, Fig. 14]. It might be simpler to get a WAX column and then run an increasing pH gradient.

Specialty organic acid column ?

organic acid column ought to work by ion-exclusion mechanism. I think the Phenomenex H form
is probably the cheapest one and you could try it on a guard first to see if retention is appropriate.

You need a low capacity ion-exchange column to elute sulfosuccinic acid. In addition, try to control the pH low enough to make sulfosuccinic acid less charged (net charge less than 2 and preferrably close to 1). HILIC might work, too. As Andy suggested, the mobile phase should have some ion strength and pH control for better peak shape.

If you use HILIC/anion-exclusion (Primesep S or Primesep S2) you don't need to go to 40 mmol of buffer. Almost any anion-exchange column will require much high buffer concentration due to strong multiple interactions.

What is wrong with the method we developed on Primesep B4 column? High concentration of ammonium formate?

Mattias,

later today I will have method for you for this compound on Primesep S column (75% ACN with 15 mmol ammonium acetate buffer). Retention is about 10 minutes with good symmetry (0.9-1.1).

Vlad, can you post the chromatogram and do you have any more for S? There is an application tab
on your webesite but no examples.

I need to see where did all applications go.

Here is one of the applications:

http://www.sielc.com/upload/file/pdf/Su ... column.pdf

Primesep S is a mixed-mode HILIC/cation-exchange/anion-exclusion column. It allows to use less ACN to retain polar ionzable analytes
You can also look at Primesep S2:
http://www.sielc.com/Products_Primesep_S2.html

I never posted chromatograms here, so I need to see how to do this.

Vlad

Here is the method for analysis of sulfosuccininc acid on Primesep S column with LC/Ms compatible conditions:

http://s1139.photobucket.com/albums/n55 ... icacid.jpg