Chromatography Forum

Hi, all -

Does anyone know if there are any columns (mixed mode, similar to BioRad Aminex columns) for UPLC for sugars, acids and ethanol?

I haven't even begun to search, but I usually stop here first to ask questions.

H_H

Polymer columns do not have the strength to work under UPLC conditions. However, Waters has introduced last year a packing that will separate such analytes in HILIC mode. The packing is called ACQUITY UPLC BEH Amide. You should find a lot of applications at the Waters website, or I can send you some (I presented on this material last year at the HPLC conference).

You should look at Unison UK-Amino (3um). If can tell us which sugars, I can post data on here.

Also, which detector will you be using?

Like Uwe, I am unaware of similar Aminex columns that can operate under UHPLC pressure. For operation under HPLC pressure, you can consider

Sepax Carbomix http://www.sepax-tech.com/Carbomix.php

Macherey-Nagel Nucleogel Sugar http://www.mn-net.com/HPLCStart/Special ... fault.aspx

HI, all -

Thanks for the tips.

We're looking at C5 and C6 monomeric sugars, mostly xylose, arabinose and glucose. Our detectors are RI.

We currently have 2 HPLC methods - one that's very long (25 min) with great sugars resolution, and one that's rather short (10 min) with so-so sugars resolution.

I was thinking with UPLC I might be able to run a very short method with great resolution.

Will the HILIC separation work on organic acids and ethanol, too? I'm unfamiliar with HILIC.

H_H

Below are applications for arabinose, xylose, and glucose on Unison UK-Amino:

http://www.imtaktusa.com/site_media/fil ... TI455E.pdf
http://www.imtaktusa.com/site_media/fil ... TI404E.pdf

The UHPLC will help for this application - not because of its pressure capabilities,
but because the system dead volume (dispersion) is so low.

HILIC is a very versatile technique. It requires a polar stationary phase designed for HILIC, such as the ACQUITY UPLC BEH Amide column, and a mobile phase with a larger proportion of acetonitrile (or acetone) and a smaller proportion of water. A classical method for sugars is the separation with acetonitrile/water on an amino column. This is a HILIC technique.

I have shown the analysis of ethanol and sugars in fermentation broth (beer) with UPLC at the HPLC conference, so it does work.

Here is a brief brochure that gives you two examples on how this column works: http://www.waters.com/webassets/cms/lib ... 3122en.pdf

RI detectors are fine. We have used both RI and ELSD for such applications. The advantage of ELSD is that you can use gradients, as you can see in the application example.

Uwe -

As per IUPAC, the definition of normal-phase chromatography:
An elution procedure in which the stationary phase is more polar than the mobile phase. This term is used in liquid chromatography
to emphasize the contrast to reversed-phase chromatography.

So, can we agree that separation of sugars on NH2 phase is normal phase?

Uwe -

Any data for arabinose / xylose separation?

Xylose and arabinose are not a problem at all with the UPLC amide column...

I'd rather check that the RI detector is compatible with the small peak volumes coming off an UHPLC. I would expect some issues.

Alex

Hi,

This is an interesting thread to me as I am constantly looking for a way of resolving the common monomeric sugars quickly. The new waters UPLC column will do this. However, I do not think it will also allow the quantification of Ethanol as well. The Dionex IC method has the same drawback - by the way, they have just brought out a new fast carbohydrate column that I Beta tested and it provides great resolution of all major sugars with a 17 minute run time, including regeneration/wash.

Uwe - do the columns that you speak of allow the quantification of ethanol on the same run as the sugars?

Elliot.

No, not yet. I asked a collegue to check this out.

Instrument : Shimadzu SCL-10A system
Column : Supelcosil LC-NH2 250 X 4.6 mm\
Flow rate : 1.2ml/min
Detector : RID 10A

I'm using this system to detect d-sorbitol. After the solvent peak, a negative peak appear just after d-sorbitol peak. This negative peak is absent when I ran the system in Jan.

This negative peak is absent when I inject the mobile phase.

The mobile phase for this system is 75 : 25 MeCN : ultrapure water