Chromatography Forum

Hi, all -

As a newbie to sugar analysis by HPLC, I'm wondering a lot about monomers and oligomers.

We are running a pretty typical sugar/acid/alcohol analysis using a BioRad Aminex 87H column with H2SO4 mobile phase (0.01N) and RI detection.

We have some samples that we are sure (based on processing of the product) will have both oligomeric and monomeric forms of the sugars.

If we inject the sample, we see some monomeric peaks, and a few small lumps throughout the chromatogram. No huge peak at the void.

If we hydrolyze the sample, thus converting any oligomers to monomers, we see the monomeric peaks grow substantially (indicating oligomers WERE PRESENT in the un-hydrolyzed sample). These peaks are much larger than any of the little lumps in the unhydrolyzed sample.

Where were the oligomers in the first, unhydrolyzed sample chromatograms? We filter the samples, but the oligomers aren't big enough to be filtered out.

Does anyone know of any standard HPLC method for oligomeric sugars (that can be run using RI detector)? I was thinking of maybe SEC - to sort the oligomers by size - but they will be chains and branched chains, so I don't know that SEC will be able to tell the difference.

Thank you for reading!

H_H

The column you are using is USP L17:

Strong cation-exchange resin consisting of sulfonated cross-linked styrene-divinylbenzene copolymer in the hydrogen form, 7 to 11 µm in diameter

I don't know why, if oligos are present, they are not detected. Perhaps they are not eluted from the column.

Anyways, if you wish to separate monosaccharides from oligosaccharides, you will most likely require gradient elution.
Since a). RI is not ammendable with gradient and b). oligosaccharides have poor chromophores; you have 2 choices.
Normal phase and gradient elution (ELS or MS detection) or derivitize the oligos for (NP) LC-Fl.

Below is LC-ELSD method for laminarioligosaccharides on Unison UK-Amino:
http://www.imtaktusa.com/site_media/fil ... TI306E.pdf

If there is hydrophobicity to the USP L17 column, then perhaps you need organic to elute the oligomers from the column.

But, please consult the manufacturer before doing that.

Thanks, Bryan. The obvious potential answer (the stuff never eluted) didn't even occur to me!

It's too bad I can't gradient-elute them on one system without a detector then run them through an RI on a second system. I don't think we have the financial capability to buy new detectors. We do have a UV detector or two, but without a chromophore ...

Thanks again. Any other suggestions are welcome.

H_H

The calcium form of that cation exchange resin will separate glucose oligomers up to about dp12 or so. BioRad sells that as HPX87C.

Thanks, Tom. Bio-Rad also recommended the 42C and 42A Aminex columns. I found an old paper using the 42A to separate oligomers. I may give it a whirl.

H_H

In one of my former lifes I used to work for Transgenomic in San Jose. They have/had a pretty extensive line of carbohydrate columns. I am not trying to push columns but there is some useful generic information on their web site:

http://transgenomic.com/lib/ug/482214-00.pdf
http://transgenomic.com/pd/Chrom/Carboh ... tChart.asp
http://transgenomic.com/pd/Chrom/Carboh ... cChart.asp

thanks,

rh

Odd about the oligomers not showing up. They should elute between void and monomer for most as a result of the exclusion mechanism.

A fairly novel and little known trick would be to use one of the Thermo hypercarb columns. With mostly water you can get slight retention for monomers and good retention, thus separation of oligomers.

Odd about the oligomers not showing up. They should elute between void and monomer for most as a result of the exclusion mechanism.

It would be if the mechanism were exclusion -- which it's not on those columns. Closer to liquid-liquid partition, with elements of ligand exchange on the Ca++ or heavy-metal form columns.