Chromatography Forum

Hi all:

would you please help recommend me how to separate like Glucose and Galactose?and I want to know more about how to use carbohydrate column.what kind of eluent?RP/NP?can I add ion pair?will the pH value affect the eluent order?

thanks and regards

Glucose and galactose determinations on sugar columns may be performed with alkaline eluents and pulsed amperometric detections.

Please find three Application Notes on the Metrohm Website:

AN-P-011 Six carbohydrates in a sugar solution
AN-P-013 Free sugar components in an instant coffee powder
AN-P-014 Eight sugar components in an explosive material

Below is an LC-ELSD application of glucose and galactose separated
by Unison UK-Amino under normal phase conditions:

http://www.silvertonesciences.com/files/TI322E.pdf

There are many approachs to separate these molecules by HPLC. This is general:
1. You can use HILIC mode on Sil NH2 (or on Sil with NH(Et)2 1 mM-0.1 mM and other organic amins additives in mobile phase), eluent: 75-80%v MeCN in water with RF detection (you also can use UV indirect detection if you add some additives in mobile phase, or using acetone), but this approach take much of organic solvent.
2. You can sepate this molecule on SAX coloumn using H2SO4aq. 0.01N-0.005N.
3. Ligande-exchange mode on polimeric sorbents modified (ion impriting)by the ions of Ca2+ and other. In this mode temp. of coloumn is about 75-80 oC. Eluent: water.

In some cases, if the concentration of sugars relatively is very low, used derivatization methods and RP HPLC UV (FL) or electrochemical detection.

Hey you can also look at these columns/applications aslo

http://www.dionex.com/en-us/columns_acc ... 60686.html

Check this Method:
Column: Aminex HPX-87P column, 300 x 7.8 mm (Bio-Rad)
Sample:Wood pulp hydrosylate, model solution, 20 μl
Eluant:H2O
Flow rate:0.6 ml/min
Temperature:85 °C
Detection:RI @ 16x
Peaks:
1. Cellobiose, 0.1%
2. Glucose, 10% (ca. 14 min)
3. Xylose, 0.1%
4. Galactose, 0.1% (ca. 17 min)
5. Arabinose, 0.1%
6. Mannose, 0.5%

I use these columns and the HPX 87H version for organic acids.... They simply rock! Check the mobile phase and the detector. Nothing complicated...

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You will find an alternative in this new Technical Note.

http://www.sequant.com/files/SeQuant_Te ... TN-006.pdf

What is the matrix that glucose and galactose are present in and at what concentrations - this may have a significant bearing on what method route you choose...