Biomass hydrolysates - A challenging sugar separation

[NBC]

We routinely analyze the sugars listed below as part of the compositional analysis of biomass (e.g., wood, grasses, agricultural crops). We combine several HPLC techniques to do this, but this is slow and the peak resolution is often poor.

I'd like to hear if people know of a column that can give baseline resolution of all these monosaccharides in a single run. Ideally I'd like to have a run time of 10 minutes or less to increase throughput, but I won't hold my breath for that one.

These sugars are typically at the 1 to 20 g/L concentration:
Sucrose
Cellobiose
Glucose
Xylose
Galactose
Arabinose
Mannose
Fructose

We get satisfactory results HPAEC-PAD but the detector is so sensitive we have to do huge dilutions to get into the linear range of the detector, which introduces its own set of errors.

Ligand exchange on a Pb-column with RI detection (e.g., Biorad Aminex HPX-87P, water mobile phase @ 85C, 0.6 mL/min) is mediocre. Sucrose/cellobiose and arabinose/mannose co-elute. Xylose and galactose have significant peak overlap. All the peaks are broad. The poor resolution causes problems with integration and quantitation. Other ligand exchange columns are worse (Ca, H, Ag, etc).

For those who are interested Supelco has a guide with sugars and their RT's on various ligand exchange columns. See Bulletin 887 https://www.sigmaaldrich.com/Graphics/S ... 0/4525.pdf

Amino columns with ELSD are again OK for some sugars. They have their own problems which I believe have been discussed elsewhere in the forum.

If you know of a better choice out there please let me know. If I can’t find a reasonable solution by LC I may consider purchasing a CE. Have a look at the separation of sugars and acids by CE here: http://www.chem.agilent.com/scripts/Lit ... WHID=13832
Its Agilent application note 5968-7715E if the link doesn’t work.

-Thanks all for your input

[Kostas Petritis]

How about this one (see below)?

They seem that they can analyze all the sugars that you mention by using a Prevail Carbohydrate column and ELSD...

Title: Improved method of analysis of biomass sugars using high-performance liquid chromatography
Author(s): Agblevor FA, Murden A, Hames BR
Source: BIOTECHNOLOGY LETTERS 26 (15): 1207-1210 AUG 2004
Document Type: Article
Language: English
Cited References: 8 Times Cited: 1
Abstract: The precise quantitative analysis of biomass derived sugars is a very important step in the conversion of biomass feedstocks to fuels and chemicals. However, the most accurate method of biomass sugar analysis is based on the gas chromatography analysis of derivatized sugars either as alditol acetates or trimethylsilanes. The derivatization method is time-consuming but the alternative HPLC method cannot resolve most sugars found in biomass hydrolysates. We have demonstrated for the first time that by careful manipulation of the HPLC mobile phase, biomass monomeric sugars ( arabinose, xylose, fructose, glucose, mannose, and galactose) can be analyzed quantitatively and there is excellent baseline resolution of all the sugars. This was demonstrated for both standard sugars and corn stover hydrolysates. Our method can also be used to analyze dimmeric sugars (cellobiose and sucrose).

[NBC]

Yes, I'm very familiar with that paper. It has been (cough) difficult to reproduce their results. However, we have found the Prevail column to be useful for glucose/fructose/sucrose analysis.

Let me back up to say that we have dug deep in to the literature on this topic. There are many, many good references on sugar chromatography. For those interested, a great place to start is this monograph:

Carbohydrate Analysis by Modern Chromatography and Electrophoresis
Ziad El Rassi
2002-10 2nd ed.
English Book 1170 p.; ill; 09.450x06.500 Inches
San Diego :; Elsevier Science [Imprint]; Elsevier Science & Technology Books ; ISBN: 9780444500618 0444500618


The reason I posted my question on this forum is that none of the published methods I've found quite do what we need to do. Sugar chromatography is challenging. It may take some "outside of the box" thinking to get the best solution to this HPLC separation .

[Uwe Neue]

Here are a few data taken from the Waters catalogue on the retention (in minutes) of a range of sugars under standard conditions. The column is the Carbohydrate Analysis Column.

Xylose 4.3
Arabinose 4.8
Fructose 5.7
Mannose 6.4
Glucose 7.3
Galactose 7.7
Sucrose 11.9
Cellobiose 15.3

The closest pair is the glucose - galactose pair. I do not recall at the moment if is is cleanly resolved (it should be according to the retention data), and it is too long ago when I worked on this project, but you should get this information if you call up Waters.

[NBC]

I'm glad you pointed this column out. The Waters Carbohydrate Analysis Column has a propyl amine stationary phase. From the retention times listed in their catalog we'd think that the chromatography would be OK (they use 80:20 acetonitrile:water, 2 mL/min).

The issue is that the peak widths are broad, so there can be significant overlap. Fructose elutes relatively early on this column but is 0.5 minutes wide at the baseline. The later eluting sucrose peak is almost 0.9 min wide. (we'll save discussion of the effects of overlap on quantitation for a different thread)

Broad peaks tend to be the norm for sugar analysis. That’s the main problem with the Pb-type ligand exchange mentioned above or the SP-0810 sold by Waters.

There are trade-offs. The Carbohydrate Analysis Column could potentially give me better chromatography for some sugars. If that's my best alternative, I'd rather stick with a Pb-type column. They use water as the mobile phase which eliminates the acetonitrile disposal problem when using amino columns.

This is a great discussion. Keep those good ideas coming!

[Uwe Neue]

If you want, I can try to get a chromatogram next week from tech support. It has been too long ago, and I do not recall the resolution of the different peaks. Please note, that the carbohydrate analysis column is a 25 cm column. I am aware that the sugar peaks are typically broad on most columns, but it also can be manipulated with flow rate or temperature. The reason for broad peaks is the secondary equilibrium of the sugar anomers. If you can speed it up, the peaks become narrower.

[jamott]

You might want to look into the Shodex Asahipak NH2P-50 columns. They seem to be more rugged and give better resolution than the aminopropyl silica-based columns I've seen.

[NBC]

Uwe - Thanks for the offer to get the chromatogram. I'd like to see it.

jamott - The Asahipak is interesting. Here's what I have for RT's from their website:

Fructose 5.9
Arabinose 6.2
Xylose 6.6
Mannose 7.8
Galactose 8.1
Glucose 8.6
Sucrose 11.9
Cellobiose ???

I'd like to see where cellobiose elutes relative to sucrose (the other dimer). It looks like peak widths are relatively narrow, particularly at higher temperature (avoids the anomer issue). This is a step in the right direction.

Shodex has some applications where they modify the acetonitrile/water mobile phase with either H3PO4 or Tetrapropylammonium buffer (pH=10)

Has anyone else tried this?

[Fabiano]

NBC,


Good resolution with short analysis time? maybe a GC method will be better for you.

I know that derivatize hundread of samples sounds strange but you can lyophilize them simutaneously and use microwave assisted reactions.

You can prepare a sugar derivative in 6 minutes (TMS or Acetylated), separation (Ara, Xyl, Rham, Glu, Gal, Man and Fru -acetylated) took just a few minutes using a 10m column.

Good luck,
Fabiano

[Uwe Neue]

I have not studied, if the anomer equilibrium speeds up more, if you go to a higher pH. I do know that it slows down when you go an acidic pH, to the point that you will get two peaks. This might be worth exploring.
Temperature of course does speed up things and makes the peaks narrower.

[NBC]

Fabiano -

I've looked into the GC methods but had always thought that derivitization took a long time. Have you published your 6 minute protocol? If so, would you mind posting the reference? We are particularly interested in automated, high-throughput sample prep methods.


Uwe -

Raising the temperature takes care of the anomer problem. For example, people run Pb-coulumns at >80C w/neutral pH. However, at this high temp on this coulmn the peaks are still broad.

In contrast, the chromatogram I saw on the Shodex website for the previously mentioned Asahipak had relatively narrow peak widths. I'm wondering if that holds up for real samples.

[NBC]

bump

[Uwe Neue]

I assume that "bump" means to remind me that I promised you a chromatogram or other better information than I gave you before. Unfortunately, there is no new information. The peaks on the standard chromatogram do not include the critical pair glucose and galactose. So I have no information, if these two will be resolved well. Sorry!

[DR]

search Aminex Carbohydrate Analysis Columns @ www.biorad.com. The Aminex HPX-87P seems to cover most of these.

Basically the same columns but with additional sample chromatograms here: http://www.metachem.com/Applications/Sarcarb4.htm

[yangz00g]

I used to employ two different columns in tandem (not 2D) to seperate compunds that cannot be resolved by either signle one but can be resolved in part by each one.

You may be able to find one column to seperate some, say, use the Aminex Carbohydrate Analysis Columns DR recommended for most of the sugars , a second one for the others, and put them in tandem.

Hope this idea helps.