Chromatography Forum

I'm setting up an lcms/ms method fot the determination of a zwitterion with a relative low mass (200 amu).
I'm now trying a Hilic silica column, but because it is an zwitterion i can't get the best retention.

About the questions what happens with salts, i can answer that for my situation. I have tested suppression today and it sucks
It has practically no retention but it's quite wide and my compound has got about the same retention as as the suppression.

The sample preparation is SPE.
Mobile phase is acetic acid 0,1% : MeOH 70:30
I have tried ACN and that has better retention as expected, but the sensitivity is much lower.

Does anybody have experience getting retention with an zwitterion on a ZIC pHilic for example? Or with adding HFBA as an ion pair to a zwitterion?

As alternative to HILIC you can try our new approach-BLIS (bufferless Ion Separation) for the separation/retention of zwitter ions. It involves ACN/water with no buffer in the mobile phase.

http://allsep.com/makeChr.php?chr=Chr_080

In the next two days we are sending out our next newsletter which is devoted to HPLC of zwitter ions (amino acids, small peptides, etc) and if you provide us with your email address we can send it to you

You also can check the following link (retention of underivatized amino acids on mixed mode columns)

http://allsep.com/makeCmp.php?cmp=Cmp_101

http://allsep.com/makeCmp.php?cmp=Cmp_025 (you can use ACN/water/ ammonium formate for LC/MS detection and sulfuric acid, TFA or phosphoric acid for low UV).

More retention is usually associated with less sensitivity. I do not think that this is the fault of the acetonitrile.

To get into a better spot, I would run a gradient from 95% acetonitrile to 50% acetonitrile with 0.1% acetic acid. I expect a better separation from the stuff that elutes early, and a sharper peak.

It is very hard to judge from the information given. However, we do have several customers that are successful in zwitterion separation and detection using the zwitterionic ZIC®-HILIC columns.

Here is the newsletter " LC Analysis of Zwitterions with Ion-Free Mobile Phase"

http://allsep.com/brochures/SielcNewsletter_0105.pdf

Contact us if you have questions

With those carboxyl phases and a H2O mobile phase: Can´t you get a huge change in retention time if for some reason your samples have lots of salts in them, or a pH far from7? For instance, ionic substances on silica based columns do this, example: M+ in H20 with a H2O mobile phase gets hung up on a silica based SEC column. If M+ is dissolved in PBS (0.15M phosphate + 0.15 NaCl) it comes out (very broad, though, 2µL injected on a 4.6x250mm column).

I have retention now.

Most of the problem was caused by an inconsistent column.

I still have problems with reproducibility tough.
And my peakshapes still aren't what i want, tailing.

How long does it takes for a hilic column to stabilize?
Is a stable backpressure enough? Especially if the gradient is with adding more aqeous acid, and at the end back in one step to original conditions? And then stabilize for a few minutes?

In our experience, a HILIC column stabilizes as quickly as a reversed-phase column. If there is trouble, check out the concetration of the ingredients.