Chromatography Forum

Hello everyone,

Any ideas how to separate the two isomers sucrose and turanose?
Till now both of them appear as one peak in the chromatogram. I ‘ve tried different temperatures, flow rate, mobile phase ratio but nothing seems to work. They still appear as one peak. I ‘m thinking also about using ethyl acetate with ACN/water but I don’t know if it will work.

Column: Shodex Asahipak NH2P-40 3E
Mobile phase: ACN/ Water (75:25)
Flow: 0,35 ml/min
Column Temperature: 30 ºC

Any ideas are welcome.
Thanks.

Aren't carbohydrate isomers separated well by ion exchange columns in the Pb or Ca forms ??

Given that Shodex's own literature shows those two as coeluting on that (polymer-based) column, I'm not surprised it failed. They *do* show a separation on a silica-based column: http://www.shodex.com/en/dc/03/02/64.html. My guess is that secondary interactions are critical here. You can certainly try tweaking the mobile phase, but I wouldn't be optimistic.

As JMB suggested, a "ligand exchange" column (cation exchanger in Ca++ or, less likely, Pb++ or Ag+ form) is another possibility, although I couldn't find any references to its use.

Why not try a column with immobilized boronic acid ligands? They should retain turanose via transient ester formation with the cis-diols. Sucrose hasn't any such and so should elute earlier.

unfortunately at the moment there is no other column available in the lab as we acquired the hplc system about a month ago.
i was looking for a solution with the existing column but it seems a bit hard though.
there is no problem with other other carbohydrates such as fructose, glucose but only with sucrose and turanose.
thanks everybody anyway
any other suggestion would be aprreciated.

If you haven't got the budget to buy a new column(!), and you've only got hplc, you're probably stuffed. You could potentially assay sucrose in other ways, though, for example an enzymic assay coupled to change in absorbance if you have access to a spectrophotometer. If you have access to GC, you could go that way.

-Andy, wouldnt boronic acid in the mobile phase also interact with the cis-diols making them less hydrohilic?

Petrus -

Sure, if you wanted to conduct a basic research project to ascertain how much boronic acid to use in the solvent.

About 20 years ago, a graduate student from Germany presented a poster at the Prep HPLC conference in Washington, DC, about including boric acid in solutions for this purpose. Unlike a boronic acid, when boric acid forms a complex with cis-diols, the third -OH group of the boric acid can ionize and thereby add an additional (-) charge to the analyte with which it's complexed. The poster exploited this to separate things with cis-diols from their analogues without them by ion-exchange chromatography. I haven't seen any publications on this approach since then.

I come back to the topic as I tried to modify the mobile phase after manufacturer’s suggestion with acetic acid or formic acid. The result was the retention time to increase about 3 minutes for all compounds and the undesired broadening of peaks.

I returned to initial mobile phase but the problem with retention time and peaks remains.
I assume that it's probably because of the acidic pH in the pores of the column but will it be permanent?
Any way to "regenerate" the column?How can I return to the initial state of the column?

Column: Shodex Asahipak NH2P-40 3E
Mobile phase: ACN/ Water (75:25)
Flow: 0,35 ml/min
Column Temperature: 30 ºC

Thanks in advance.

unfortunately at the moment there is no other column available in the lab as we acquired the hplc system about a month ago.
i was looking for a solution with the existing column but it seems a bit hard though.

Since you have now killed your existing column, it's time to try something that has a chance of working, per earlier suggestions.

Any way to "regenerate" the column?How can I return to the initial state of the column?

Amino columns, in my experience, are fragile, so there is a good chance that you have irreversibly damaged to column. Check with Shodex and follow their recommendations for this specific column, but don't get your hopes too high. Sorry!

We have had luck with similar molecules (lactobionic acid and lactobionolactone) using betacyclodextrin functionalized silica. See the article below (if you have access)

http://www.sciencedirect.com/science/ar ... 7394890528

Hypercarb (porous graphitic carbon) HPLC columns are reputedly very good for separation of isomers, including oligosaccharides.