Chromatography Forum

Hi all, I am having trouble determining sucrose (1000ppm) which is prepared from >95% purity sucrose– the area observed is 1/3 of what it used to be observed previously, same system but newer column. The other 2 sugar came out fine with concentration closed to the expected concentration. A different sugar standard is used (the instrument was shifted from another site, hence new sugar standards were used), I tried preparing using the previous sugar standards, the surcose area is slightly higher, abt 50-60% of what it used to observe when tested in the other site (no label on the standard, higher area observed could due to hgher purity of Sucrose used). This also lead to an extremely small sucrose peak at 25 ppm. Could someone please advice me what I should look at to troubleshoot it.

So far I have done the following;

- Prepared standards using new and old sugar standards
- Back flashed the column with 100% water for 3 hours

Column in used: Phenomenex Rezex RNM – Carbonhydrate Na+ (8%) 33 X 7.8mm
Mobile Phase: 100% water
Temp: 75 degrees
Injection volume: 20ul

Alternatively, could I standardise the Surcose standard value to calculate total sugar in my samples?

Thank you all in advance.

Try reducing your temperature. Sucrose decomoses at high temps.

Sucrose can hydrolize in aqueous media under certain conditions. When you inject sucrose you should see some peaks which are related to decomposition products. Reducing temperature to room will increase your recovery drastically.

Thank you both for the advise but I am just trying to analyse with the same method that was used in the other lab. It was working fine hence I am very puzzle with the loss of area for sucrose only! Hence lowering the temperature will not be a visible option for me.

Please any other advise?

Thanks again.....

Then try to see if you can run this analysis on different lots of the column. If it is hydrolysis, then something changed in your method.

Temperature should be not the problem. But when I read "newer column" and different sugar standards I would do the following:
1. wash the column and do the conditioning
2. use fresh, new purchased sugar standards from a reliable manufacturer/supplier.
Good luck

How are you detecting the sugars? I assum that the lower response is lower detector signal? Is this ratioed to an internal standard or otherwise processed with a calulation?