Carbohydrate Analysis - Help Needed!

[jdyer51287]

Hello,

I am analysing samples from cellulose and hemi-cellulose hydrolysis of biomass, therefore I have a lot of monosaccharides to separate!

I am currently using:

Phenomenex Luna NH2 5µm 100A (250x4.6 mm) at 40°C, with a flow rate of 1mL/min

LC Gradient Profile: MeCN:H2O
Initial - 82.5:17.5
8 min - 82.5:17.5
15 min - 77.5:22.5
17 min - 82.5:17.5
24 min - 82.5:17.5

With ELSD detector at 45°C and 1.3 L/min

This gives adequate separation of all the cellulose sugars I am interested in (levoglucosan, fructose, glucose, sucrose + cellobiose) and mannose and rhamnose from the hemi-cellulose. However, I have been unable to resolve xylose from arabinose, am finding that galactose is co-eluting at the same retention as glucose, and that d-allose is unresolved from fructose.

I have the possibility of purchasing a new column to try resolve all of these compounds in a single injection. I have so far been pointed towards lead based columns such as the Agilent Hi-Plex Pb, Rezex RPM, CarboSep CHO782 or CHO682 and the Aminex HPX-87P. Does anyone have any experience with any of these columns? Or any other recommendations?

I am also interested in oligosaccharide separation, however I am aware that it would be very unlikely to find a single column that could resolve all my monomers of interest and oligosaccharides!

Thanks in advance for any help

[JMB]

My first advice would be to contact the applications group of Phenomenex, and also of the Aminex-type columns

Also, I believe that, within the last month or so, I have seen promotional material for the use of columns bearing bonded boronic acid groups for carbohydrate separations. The
-B(OH)2 groups react with -OH groups of carbohydrates, depending on sterochemical properties. Try a Google on this. Hope I'm not dreaming !

[Gerhard Kratz]

I would also check out the Shodex website. An NH2 column is good for many applications but for sugar analysis with complex samples I would recommend to use a specialized column.

[tristanewalters]

We have had luck using beta-cyclodextrin functionalized columns in separating carbohydrates. It may be a useful tool for you.

[Karen01]

The Aminex P is great for monosaccharides ... not so great for the Di's and up. It uses a water mobile phase (so isocratic) with an RI detector.

For Di's and polysaccharides we use Water's UPLC amide column 2.1 X150 on an HClass with an ELSD.

- Karen

[mattmullaney]

No one has mentioned these...if you've an Ion Chromatograph, Thermo Dionex and Metrohm, in addition to Shodex, also make excellent columns for monosaccharide and disaccharide separations.

[You may not have an Ion Chromatograph, though.]

Of the others, I would vote myself for the Aminex series of columns.

[jdyer51287]

Thanks for all the replies, I'll definitely look into the different suggestions.

I have now also run my standards on a Rezex Pb column (borrowed from a colleague) and have less success separating my particular mix of sugars!

[carlo.annaratone]

I would suggest you looking into HILIC as well. It has some desirable properties:

-solvents typically are MS and ELSD friendly
-do not require ion chromatographers
-modern columns with small particles or core shell particles are available (high plate count and better resolution)

just to name a few that seem to fit into your goal: Nucleodur HILIC (small particles), Nucleodur HILIC (2.7 µm core shell), Waters XBridge amide (small particles, extreme pH tolerant), ZIC-pHILIC (conventional particles but pH resistant)

avoid bare silica HILIC columns as those are known to give poor results with sugars.