Need to seperate sample with maltose, maltulose and melibios

[h.piatkowski@comalc.com]

Hi folks..

I have a sample which has maltose, maltulose and melibiose in it. With my current setup I only get 1 peak, and need to find a column or make method changes to enable me to see the individual compounds.

My setup is:
BioRad Aminex HPX-87H column, MP : 0.01 M H2SO4, 0.6 ml/min, column @ 65°C, RI detector at 55°C.

I also tried another carbohydrate column I have BioRad Aminex HPX-42A with no success..

Does anyone have a method or a recomendation, or even an offer to run the sample for me...

Thanks for all your help..

- Hubert

[Bruce Hamilton]

If you go to the Bio-rad web site and download the Aminex HPLC column guide, a table in there will tell you what you already know, that maltose and melabiose co-elute on your HP-87H, however it does list other Aminex columns that separate them, and a Google search found a patent reference that implied that Aminex columns could also separate maltose and maltulose. Maltulose isn't mentioned in the Bio-Rad documents.

Your best option may be to ask Bio-Rad, or one of the companies that make similar columns to the Aminex columns, to recommend a suitable column, and they may even run your sample.

Please keep having fun,

Bruce Hamilton

[h.piatkowski@comalc.com]

Thanks Bruce, I got a hold of BioRad and they recomended using a diferent mobile phase. I will give that a try and see what I can see..

Thanks again for your help..

- Hubert

[Bart]

Hello Hubert,

I sent you a separate email with a retention chart for common carbohydrates separated on these types of columns. We (Transgenomic) have an equivalent line of columns that uses the same technology as Bio-Rad. Bruce is right that you need another type of column with a stronger ligand such as calcium or lead to separate these sugars. Even with the stronger ligand, from our charts it is a difficult separation. If you are interested, we would be willing to try your components on a couple of our ligand exchange columns. If you have the Bio-Rad 87P or 87C columns, I would recommend you try those first. We do have another option with a lower cross-linked material if those don't work, which could help separate the sugars. I'm surprised Bio-Rad would recommend trying a different eluent (the only option being weaker or stronger acidic eluent with an ion-exclusion column), because the mode of separation for carbohydrates on ion-exclusion columns such as the 87H has little to do with the eluent strength. The best option for a difficult sugar separation on these types of columns is a carbohydrate analysis column which uses a simple water eluent. Please let me know if you need our help.

Bart Poulsen
Director, Chromatography Product Management
Transgenomic, Inc.

[HPLCchemist]

Hubert,

In the past I have used a Biorad HPX-87C for carbohydrate and alcohol analysis in complex fermentation mixtures. The column you are using is for the analysis of more simple sugars.

Try using the same conditions as the Brazilians

Detector; RI
Injection Volume; 20 uL
Column; Shodex KS-801
Eluant; Water at 80 degrees Celcius
Flow; 0.6 mL/minute
Run Time; 30 minutes

Check out the Brazilian Archives of Biology and Technology,
"Production of Alcohol by Simultaneous Saccharification and Fermentation of Low Grade Wheat Flour"
Vol. 49, n. 3: pp. 481-490, May 2006

HPLC Chemist