Analysis of biomas digestion compunds? Looking for column re

[h.piatkowski@comalc.com]

Hi folks..

I'm doing some work on biomass disgestion with specific enzymes. basically digestion of cellulose and hemicellulose into C5 and C6 sugars. I need to find a good column that will be able to seperate as much of these sugars in a single run as possible, and if that's not all, if I couold get toxins seperated as well such as acetic acid and furfural seperated at the same time I would be very happy.. Any recomendations? Does anyone have any experinace with C5 sugar analysis?

Thanks for taking the time to read this post..

- Hubert

[mmadson53]

Hello Hubert;

I can suggest a column, however, if I were you I would first find the identities of the monosaccharides I were looking for. This is usually species dependent and isolation procedure dependent. In addition one needs reasonable assurance that only monosaccharides are present. If we know these two variables then we may find the best column. The best general column for monosaccharides and small oligosaccharides that you may be interested in is Dionex's PA 1 column. But Dionex has other columns that can address more specific separation problems better. For one to find this column a better understanding of the component sugars is necessary. It may be possible to find the component sugars in the literature.

Michael

[Rande]

I would recomend you check out the Bio-Rad web site and look at their Aminex Carbohydrate Analysis Columns (good for carbohydrates and organic acids). Keep in mind that you will need to use a RI detector.

You said something about " Toxins" - you will need alternative methods for proteins.

[h.piatkowski@comalc.com]

I was looking at the Bio-Rad Pb column for this work, but I just don't know how good the results are. I currently use the 87H and 42A for carbohydrate and organic acid analysis with an RI detector. Very nice columns, stable and last forever with the use of a guard..

Is anyone using the Pb column for pentose sugars.

Thanks for your help..

- Hubert

[mmadson53]

If one looks at the Bio Rad website they give a test chromatogram of wood pulp hydrolysate. You'll notice that they are doing mgs /ml which is representative of the RI detection system. Also they are not able to get baseline separation of mannose and arabinose. In addition you'll need to run at 85C. I would consider a Dionex system. Dionex can do carbohydrates in general and specifically at quite high sensitivity with excellent resolution. Dionex can do organic acids as well and fermentation broths are aslo doable with high comparitive sensitivity.

[efirth]

Hi Hubert.

I am currently doing the same analysis. I use a Rezex RHM (H+) column. It works as well as the Aminex columns, but is less expensive. This column (using 50mM Sulfuric acid eluent @ ~42 degrees and flow rate of 0.6ml/min) will resolve Glucose, xylose arabinose and cellobiose. Unfortunately, fructose, mannose, glactose will all co-elute around 10 minutes, which is on or around the xylose peak. Also, Sucrose comes out as a mess due to the acid eluent. This column also resolves many organic acids (including acetate) and a couple of the furfuryls and Ethanol (you will be doing fermentations, right?)

As for the sugars that all co-elute around 10 minutes, I am about to start looking at other Rezex/aminex columns (Pb+, Na+ etc) to see which resolve these. Let me know if you already have this info please...would be very grateful.

I hope this helps.

Elliot.

[h.piatkowski@comalc.com]

Hi Efirth,

Can you send me an e-mail or let me know what your e-mail is. I'd love to discuss something with you in regards to these kinds of analysis.

h.piatkowski@comalc.com

Thanks and have a great day..

- Hubert

[mmadson53]

As a carbohydrate chemist one must remember the Lobry-deBrun Alberta van Eckstein transformations which produce not only isomerisations, say for example, from glucose to mannose to fructose in acid but also degradation to furfural and HMF and other molecules. Hence the difficult time with sucrose as has been stated. This nearly precludes the use of sulfuric acid as an eluent for these particular carbohydrates if one wants accurate measurements of quantities of monosaccharides.

[h.piatkowski@comalc.com]

Hi mmadson53,

Thanks for the e-mail, I am not to familliar with the transformation you are talking about, but here is a question..

We currently employ the BioRad 87-H column for fermentation analysis (sugars, organic acids, ethanol..), we run a weak H2SO4 eluant 0.05 N I believe. Would this have an effect on my sugars? Are the results that I'm seeing possibly skewed due to the isomerisations?

The biomass samples that I will be working with are not a result of acid pre-treatment as with the NREL process, we are investigating a new process which is not as harsh.

I'm not sure what compounds I will need to ID yet, but I think most of the ones on the following list will be in the group :

- glucose
- xylose
- arabinose
- mannose
- galactose
- cellobiose
- fructose ?
- sucrose ?
- xylitol
- succinic acid
- lactic acid
- glycerol
- formic acid
- acetic acid
- ethanol
- HMF
- furfural

I realize that it will be impossible to ID all of them on a single column, and most likelly I will not need to see all of them, but I would love to hear from you folks any possible solutions you might have..

Thanks again for taking the time to read this post..

- Hubert

[Wen Jiang]

Hi Hubert,
Since your interested compounds (sugar, organic acid and etc.) are mostly hydrophilic, another alternative can be hydrophilic interaction liquid chromatography (HILIC).
Please check the followed URL to find more information of separating those compounds in HILIC.
http://www.sequant.com/sn/ufiles/SeQuan ... 00-16A.pdf
http://www.lcgcmag.com/lcgc/data/articl ... rticle.pdf

[mmadson53]

Hi Hubert;

Yes it could possibly skew your results. The way to test this would be to run glucose alone and check for any fructose or mannose produced. You'd have to get fairly high purity glucose however.

Thanks for your acknowledgement
.