Chromatography Forum

Hi.
I am struggling with some Glucose in my Organic acid experiment.
I am using Synergi Hydro RP column for seperating organic acids from bacterial cultures.
(Flow rate 0.7 ml/min, 20mM KH2PO4 pH 2.9 detection at 220 nm, isoratic)

I was able to obtain very clean peak for all my standards ( Oxalic acid, D-gluconic acid, Citric, Acetic, Lactic, Malic, etc..)
But whenever I examine my bacterial culture samples D-gluconic acid peak rises too high.
I think it has something to do with D-glucose in the media. (retention time of D-glucose was exactly same as D-gluconic acid.. as expected....)

So, here is my question..
Because I can't use sucrose or lactose for my bacterial culture media, I need to use D-glucose for my experiment.
Is there any ways to distinguish D-glucose from D-gluconic acid by HPLC? something like, extra sample preparation steps perhaps? or do I need to try out a new column?


Any suggestions will be greatful.
Thanks in advance

Yours
Young

Hellow
1) Big peak.
D-gluconic acid has a "conjugate relationship" from carboxylic to aldol groups , pair of electrons multiplies the absorption of light at 220 nm. Its normal . Solve problem - dissolve (dilute) the sample.
2) Glucose and gluconic acids not selected on hydro-RP.
This substances were polar and good dissolve in water . Glucose slighty sorbed in hydrophobes (RP-C8 RP--C18) and semi-hydrophilic (Hydro-RP) sorbents and jump out in a first minutes.
Ideal - use the aminophase column . Aminophase good sorbed aldehyde (pairing electrone mechanism) and acids (ion pair mechanism).
Variant => use SPE which aminophase .
Anohter (bad) variant- use dinamic modification column (such hexylamine buffer).

The use of an HILIC column maybe also a good alternative.

You should not have any problem separation these compounds if you are using correct tools. Sugars are neutral and polar, acids are polar and acidic. Just use HILIC/Ion exchange:
http://www.sielc.com/Application-Effect ... Acids.html
http://www.sielc.com/Application-HILIC- ... Acids.html