Chromatography Forum

Hello !
I'm looking for a specific method for oligosides. I use a carbohydrate WATERS column with refractometric detector but I wonder if it is really specific. Does a method more specific exist ? with derivatization and PDA detector for example ? I'd rather LC method but GC and HPTLC method are welcome.

Thank you for your answers

Entire books have been dedicated to the analysis of carbohydrates...

For the measurement of oligosaccharides, other techniques are possible. For example, size-exclusion-type of techniques are possible, a reversed-phase method is possible. Derivatization is usually not recommended, but it is also not impossible. Detection can be with a refractometer or at low wavelength with the right mobile phase. In your case a detection at 210 nm should be suitable.

I can get you more specific information, if you tell me what problem you would like to solve by changing the method.

Here's an example of ABEE-Converted Oligosaccharide method:

http://www.imtakt.com/TecInfo/TI204E.pdf

Dionex has the CarboPac line of anion exchange columns designed for sugar/carbohydrate analysis. (Carbohydrates are ionized at pH 13.) The detection is usually pulsed amperometry, which is not all that specific, since it detects anything oxidizable. It is possible to interface with MS instead.

Check out http://www1.dionex.com/en-us/columns_ac ... p5498.html

Thanx to everybody for your numerous answers.

Uwe Neue,
My problem is the following : I have to quantitate fructose and glucose or sometime identify other oligosides with the WATERS method and a refractometric detector in complex vegetal extracts. I often meet other peaks and want to know if they are carbohydrate. I' m afraid that refractometer is as specific as ELSD and detect everything going through the cell

Bryan Evans,
The application coming from imtakt is very interesting and is a bit what I was looking for. However Uwe Neue does not recommend derivatization and I wonder why. Because of yield ?

If someone has other reference of this type and more detailed, I'm ok.

Hi Syntaxerror,

I realize you are dealing with crude vegetal extracts, but would a strong cationic exchange column (calcium or lead type) be suitable to your purpose I wonder. You may need to perform sample clean-up prior to analysis.

Cheers.

I think your right. cationic exchange column could be the solution. I have precisely a brand new still in its box. I should try it soon. However is this type of retention specific with sugars ? Is it possible that this kind of column retain other compound than oligoside ?

Finally to sum up, there is (at least) two way for a LC method to become specific : choose a specific detector or using a specific stationary phase.

Tell me if I'm wrong.

Hi syntaxerror,

Derivatization is often avoided because it can cause poor % recovery, is labor intensive, and is not always reproducible.

However, if done correctly, it can be a powerful tool for specific applications.

The derivatization used in that chromatogram was done for a specific reason - to monitor the positional isomers of alpha-Gal2 during a reaction. Here's the reference if you are interested:

Reverse Reaction of Aspergillus niger APC-9319 α-Galactosidase in a Supersaturated Substrate Solution: Production of α-Linked Galactooligosaccharide (α-GOS)

Akiko YAMASHITA1), Hiroyuki HASHIMOTO1), Koki FUJITA2), Masamichi OKADA3), Shigeharu MORI3) and Sumio KITAHATA1)

Bioscience, Biotechnology, and Biochemistry
Vol. 69 (2005) , No. 7 pp.1381-1388

Hmm... Due to the fact that you are working from vegetable extracts, what you need is a method that is insensitive to random interferences but gives you a reasonably selective detection of carbohydrates. There may be nothing wrong with the analytical method, but you would like a more specific detection scheme. First, MS comes to mind. Maybe Mark Tracy's method could be OK, but I know nothing about it; he will need to advise you. More specific detection can be achieved with derivatization, either off line, or post column, but in either case you will depend on the specificity of your reaction chemistry.

The method that you are using is not bad from the standpoint of retention specificity for very polar compounds such as your carbohydrates. You need an improvement in the detection sensitivity. MS, post-column derivatization come to mind, but maybe Mark can advise on PAD after post-column addition of base (I do not know if MeCN would be a problem for this).

Hi Syntaxerror,

Not sure as I have not worked with sugars from crude plant sources before. You will need to try it out, or perhaps someone more experience in this forum could help.

The strong cationic column I have used was from Benson. I have also used an amino column (Phenomenex) for glucose and sorbitol, but from memory, peak shapes were not too good.

Perhaps a quick flip through a manufacturer's catalog might help too.

Here, we have done glucose and fructose by GC and GCMS after derivatization to trimethylsilyl derivatives. We've used HPLC-RI for them in the past when assaying pancake syrups (sorry, it's a living !!!).

Dear Syntaxerror:

There are many columns available for sugar separation (based on affinity/ion exchange). But the "application note" from a vendor should be confirmed against your sample matrix.

Alltech and Supelco (now Sigma) have a lot of columns for carbohydrate separation. You should discuss your situation with an application specialist (because the columns for carbohydrates are expensive). I have tried a Benson column, and it didn't work for us (even though the chromatogram in vendor's catalog looked good).

Disclaimer: I am not associated with Alltech nor with Sigma.

Alfred.

Hi syntaxerror,

Derivatization is often avoided because it can cause poor % recovery, is labor intensive, and is not always reproducible.

(...)

Dear Bryan,
I agree with you but don't you think sample preparation (SPE for example), is as labor intensive, reproducible as derivatization ?

Since last week, I tried ion exchange (Pb2+) mode. I got another profile with other interferences...
But searching in my library, I found out the sugar analyse bible. The name of this precious book is : "Analytical Chemistry of Carbohydrates" of Heimo Scherz & Günther Bonn.

I hope find in my happiness

While I do not have this book, a book by Guenter Bonn is certainly a good choice. You can come back here, if you need additional help.

hi synthaxerror,

i don't have a great experience about carbohydrates analysis but i find that amino polymer based columns give good performances (for example astec amino columns that i used for glucosamine analysis with ELSD detection).
cheers