Chromatography Forum

I will be working on a new project to look at the oligosaccharide profile of enzymes produced by a specific bacteria in degrading cellulose and hemi-celluloses. The ulitmate goal of the research is to optimize the enzyme prduction by this organism for use in ethanol production (it degrades cellulose/hemi-celluloses and produces ethanol at the same time).

I have been looking at verious HILIC methods for separation as well as separation on beta-cyclodextrin-modified columns (Talanta 47(4):1001 (1998)).

Does anyone have suggestions for this type of work from glucose to oligo (16) and same with the hemi-cellulose breakdown products?

Thanks in advance...

There are many different ways in which this can be done. My suggestion is to look at a vendor catalogue that sells columns for this purpose (the Waters catalogue has a nice listing of options).

You can do a size-exclusion separation, commonly done on a sulfonated styrene-divinylbenzene packing. This is typically done for monitoring the amount of sugar in corn syroup. You can get better separations of the oligomeric species for example with a water-compatible RP phase (I have used Resolve C18 for this purpose) and a mobile phase of 100% water. You can do the same with a HILIC phase in roughly 70% acetonitrile/30%water. Special amino phases for sugar separations are available. Do not use standard amino phases, since the retention will change with time due to stability problems with such phases. An alternative solution is HILIC with silica (less retentive) or an amide phase with roughly the same mobile phase.

Hi David -

Imtakt has some nice data for this type of separation using
Unison UK-Amino. This column was introduced this year at
HPLC 2008. This column is durable against water elution -
a problem seen with conventional silica-based NH2 phases:

Maltooligosaccharides: http://www.silvertonesciences.com/files/TI347E.pdf
Isomaltooligosaccharides: http://www.silvertonesciences.com/files/TI349E.pdf
Xylooligosaccharides: http://www.silvertonesciences.com/files/TI353E.pdf
Sialic and uronic acids: http://www.silvertonesciences.com/files/TI333E.pdf
Pentoses: http://www.silvertonesciences.com/files/TI328E.pdf
Cyclodextrins: http://www.silvertonesciences.com/files/TI310E.pdf

What is your detection going to be, LC-MS? LC-Fl?
LC-MS would use similar conditions. If LC-Fl, let me know - we have lots of data for that as well.

Thanks!

I am currently just starting to look into to this as this is a long term goal for my group at this company. Currently, all the LC equipment was purchased before I joined the company so we are currently limited to IR and UV detection methods.

Any more information would be appreciated.

Dave

Is that RI?

Botyris,

There are also some very good methods for saccharide separations using ion-loaded ion exchange columns. The simplicity is htat these use water as a mobile phase. YOu choose the adsorbed ion to determine the selectivity. Ca is the most common, but Pb, and sodium are also used.

The ion-loaded ion exchange columns are probably the most robust and easiest to regenerate.

Waters has some published literature than can help with the selection as well as Hamilton.

You can do monosaccharides on LC-RI under isocratic condtions.

For LC-UV of oligosaccharides, you can try ABEE-derivitization.
This is an example done on Cadenza CD-C18:
http://www.silvertonesciences.com/files/TI204E.pdf

Yes, they purchased RI detectors, but I will be able to buy other detectors in the future as needed.

I already have the monosaccharide separation setup and working. It is our routine analysis - we will have 2 to 3 HPLCs running almost continuously on this analysis.

But, in the near future, I will be looking to identify products from enzyme assays and enzyme purification work. This is where the oligosaccharide separation will be used for. This company has a license on the use of a bacteria that grows on biomass (cellulose, etc) and converts it directly to ethanol. So, the analysis will be geared towards cellulo-oligomers and hemicellulo-oligomers.

I have used columns from Transgenomic which may be useful for you. They have a pdf catalog at www.transgenomic.com which gives examples. I hope this helps you.

The Shodex website is pretty comprehensive detailing separations for the applications you are looking at. Good luck with it!

PS. Keep the board updated as to how you get on - we could all have something to learn here...

Hi David,

The H form ion exclusion column is most often used with water or 5 mM sulfuric as the mobile phase. Most of these are 7.8 x 300 mm columns from Biorad, Toshoh, Phenomenex, Sepax etc. You can easily separate glucose, cellobiose and cellotriose. Higher oligomers will begin to coelute with less retention as the sugar gets larger. If you have significant hemicellulose you will be unable to separate xylose from galactose or even resolve them from the glucose in which case you may need different chemistry.

I had some luck with Hilic amino and amide phases but you will want ELSD detection rather than RI. If ELSD I suggest polymeric based amide (from Tosoh) or the Alltech polymeric amino as your LOD will be much lower. I have not gone to this analysis because I always have interferants that coelute with the sugars (salts!) but I'm still working on this.

Abee derivatization does work well and you can try this.

One last option is the hypercarb from Thermo. It will just retain glucose in pure water and you can elute the rest of the oligosaccharides with a quick gradient and ELSD detection.

Best of luck,
Marc

Thank you for all your replies. We are currently using the Bio-Rad HPX-87H columns since the fermentation broth contains organic acids produced during fermentation (acetic acid, lactic acid, and formic acid) and I know these are not ideal for the HPX-87P columns.

The future work, not so distant, is to separate the oligomers from reactions of pure cellulose/xylan/arabinan with the crude enzyme cocktail from the fermentation broth to determine the extent of degradation. I saw the paper by Berthod et al (1998) Talanta 47: 1001-1012 where they used a cyclodextrin column using HILIC conditions to get separation of the cellulo-, xylo-, and arabino-oiligomers from each other. I am still researching this but will have to make a decision soon on this.

Sepax Oligo IEX separation:

http://www.sepax-tech.com/Proteomix.php

If you ever get a chance, do have a look at capillary electrophoresis. It's very good at this sort of application, it's very automated nowadays (see Beckman's equipment), and it has a resolution that frankly makes UPLC look rather silly. But I appreciate that if you're in an LC-based environment, it's a high-risk, big change to buy into a different technology.

I agree with lmh. CE is definitely the future for oligosaccharide profiling. It is very easy to do with Beckman's CE instrument and their Carbohydrate Profiling kit. I
Have a few colleagues that have had great success with this. It might be something to try with a used CE instrument just in case this is not the way your company wishes to go but the kit works great. I have also had great experience in the cyclodextrin area with the CE instrumentation