saccharose in HPLC

[annethebault]

i want to separate saccharose in H+ column. i tryed with H2O + 50 µL / L H2So4 and 55°C 0,6mL/min: 2 pics are coeluted! what is the ideal concentration of H2SO4? Thank you to answer me !

[tom jupille]

The cation exchange resins used for that type of separation are available in a variety of different ionic forms (H+, Ag++, Ca++, Pb++), each of which has a somewhat different selectivity. Decide which sugars you wish to separate and then contact the vendors (Bio Rad, Shodex, Dionex, etc.) for a recommendation.

Note that only the H+ form column is used with H2SO4 eluant; the others used only DI water (think about the solubility of Calcium sulfate!). That column genrally provides the least selectivity for sugars (it was originally designed to separate organic acids). The acid concentration has essentially no effect on sugar retention or selectivity.

[h.piatkowski@comalc.com]

Hi Annet,

What detector are you using? And what column? We do fermentation sugars here and use a Biorad Aminex 87-H column, works pretty good with an RI detector.

- Hubert

[JMB]

Anne,

Saccharose (sucrose) is EXTREMELY acid-labile; your chromatography is guaranteed to give you 100% conversion to a 1:1 mixture of glucose and fructose !!!

Suggest you do a lit. search; you need a non-acidic column and mobile phase, maybe a HILIC column with ammonium acetate or formate.

JMB

[JMB]

I should have said aq. ammonium acetate (formate) with MeCN for HILIC.

JMB

[Bart]

JMB is correct about sucrose inverting under acidic conditions. If you are only analyzing for sucrose, you don't need the acidic eluent, and can use water to elute sucrose from the column. I've also seen a fairly sharp peak on ion-exclusion columns even with an acidic eluent if you test the column closer to room temperature, although you may still see some "shouldering". Most chemists that want to use the sulfonated gel type columns for analysis of sugars opt for the ligand exchange columns, usually in the calcium form. It is only if you need to separate weak organic acids in the presence of sugars that you would choose an ion-exclusion column.

[annethebault]

Hi Annet,

What detector are you using? And what column? We do fermentation sugars here and use a Biorad Aminex 87-H column, works pretty good with an RI detector.

- Hubert

we use an RI detector and a biorad aminex 87-H column

[tom jupille]

As I indicated earlier, BioRad sells the HPX-87 columns in almost a half-dozen ionic forms. Each has somewhat different selectivity. The fact that the hydrogen form column separates the sugars that h.piatkowski is interested does not guarantee that it will separate the sugars you are interested in.

Check with BioRad to confirm that the H column is able to separate your sugars. If it is, then you have a dead or defective column. If it is not, then you have the wrong column for your application.