Sugars and Salts

[chromcat]

I am analyzing carbohydrates in fermentation media but am getting a lot of interference from salts. Any suggestions on removing the salts prior to analysis without removing any of the saccharides?
I'm using a hydrogen form, 9 µm particle size, 8% cross linkage, column with H2O as the mobile phase.
Thanks

[HPLC chemist]

Try filltering and ultrafiltering (0.22 um) your sample. Then pass it through Sephadex G-15 (if they still make it) or equivalent to desalt it. Sample is ready for injection.

[Karen01]

I am analyzing carbohydrates in fermentation media but am getting a lot of interference from salts. Any suggestions on removing the salts prior to analysis without removing any of the saccharides?
I'm using a hydrogen form, 9 µm particle size, 8% cross linkage, column with H2O as the mobile phase.
Thanks

If that column is what I think it is, it typically works by ion exclusion using a dilute sulfuric acid mobile phase.. great for organic acids ... not so great separation for saccharides.

For monosaccharides you will get much better results with a lead form column. And with that use a guard column that combines cation and anion exchange resins to remove the inorganic salts.

- Karen

[HPLC chemist]

Try this column;

https://www.phenomenex.com/Products/HPLCDetail/rezex

As I recall Thermofisher has a column specifically for fermentation mixtures. Good luck!

[chromcat]

Thank you all for your responses.
I'm having no difficulty separating the saccharides using the H+ column. The Pb column does a slightly better job than the H+ but the salt interference is much more noticeable and co-elutes with the analyte saccharides rendering quantification damn near impossible with the Pb column. Similar results when I tried a Ca column.
The current solution I have is what Karen01 mentioned: the cation and anion exchange "guard cartridges" installed prior to the analytical H+ column. This works great up until when the guard cartridges get overloaded and have to be replaced - ~$400/month.
The guard cartidges also don't allow a slightly acidic mobile phase as it would strip the cation exchange cartridge, though yes this would be better for the H+ column.
I wonder if Sephadex comes in syringe filter form.
The ideal situation, while not prohibitively expensive, would be a desalting syringe filter.

Cheers

[Karen01]

In my hands for monosaccharides P is better than Ca better than H (with sulfuric Acid mobile phase).

Can you get baseline separation of glucose and Xylose on the H?

The Sephadex solution probably won't work as it's size exclusion for desalting compounds with molecular weight > 1500 so it's a no go for mono and saccharides.

Any sort or syringe based desalting would have to use an ion exchange resin and likely more expensive per sample than the deashing pre-column

If you are dealing with high concentrations (so you can either inject very low volumes or dilute with ACN) and a sensitive enough detector, you could try HILIC with a HILIC Amide or amine column.

AFIK there are no really cheap solutions for this type of sugar analysis.

- Karen

[chromcat]

@Karen01

Yes, I can get baseline separation of Glucose and Xylose with the H+ column.

Did some tests with a 5mM H2SO4 mobile phase. It did improve the separation but since I'm using a RID matching the sample matrix to the mobile phase was too time consuming (~1000 injections/month).

I suppose it comes down to where I want to spend the money. Either on anion/cation exchange guard cartridges or labor/time to matrix match the sample with acidic mobile phase.

- Scott

[HPLC chemist]

Matrix match.