Chromatography Forum

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

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 !

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.

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

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

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.

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!

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.