HPLC baseline is not stable

[Zia]

Hello to all. I am Zia, I am a PhD student. I have a problem regarding my HPLC system, (LC-20AT, Shimadzu Corporation) equipped with a RID-10A refractive index detector and a HCT-360 column heater. The analytical column used is a Transgenomic ICSep ICE-ION-300 column (7.8 mm×300 mm). The mobile phase is 0.00085 N sulfuric acid at a flow rate of 0.4 mL/min, the injection volume is 20 microliter and the column temperature is maintained at 58 ℃. Despite of making the mobile phase flow for one hour, two hours and sometime more than that but the baseline is not getting stable on zero. Sometime even if it gets stable but when I inject the sample, it is sinusoidal and moves up and down the zero line. I am using fermentation samples to measure the concentration of glucose and ethanol produced during fermentation. I hope somebody can guide me how to solve this problem. I hope your cooperation and expert opinion in this regard. Thanks in advance to all of you.

[M Farooq]

Refractive index detectors require lot of patience. Unlike UV-Vis detectors, temperature effect on refractive index is more pronounced. Little fluctuation in temperature will cause a drift. Also the solvents need to be properly degassed for RI. Usually sonication under vacuum removes a large quantity of air from the solvents.

Follow the suggestions in this link to solve your problems:
http://www.shimadzu.com/an/hplc/support ... 63lab.html

By the way, there was a very interesting paper on determining sugars by absorbance, if you can do post-column arrangement just by mixing NaOH with the column effluent. The sugar is converted into a chromophore and it can be easily detected. It was published in J. Chrom. A very recently.

[kubowicz.tomasz]

The problem you have can be linked with temperature.
Your column is kept in 58 deg and RI detector cell is probably less than 40 so quite "warm" mobile phase is entering "cooler" detector cell. Refractive Index is very dependent from temperature (even small air fluctuation around detector can have impact on baseline)

Regards

Tomasz Kubowicz

[sivaraman_t]

This may be basic but still check if it is useful:
1) Ensure to fill Mobile Phase in the Reference Side of your RID-10A by selecting R-Flow. You have to press shift and some numerical key to activate R-Flow (Check with user manual). This should hopefully solve the problem.
2) If you do have an LC-20AD pump connect it instead of LC-20AT as it generates low pulsation.

Best of luck

Sivaraman