Chromatography Forum

Hello everyone,

I am developing a method for sugar analysis (sucrose, glucose etc) using Agilent 1100 series refractive index detector using a TSKgel Amide-80 column. I can see my sugar peaks but they are very small even when I am injecting 2% wt/wt solution.

My conditions are as follows:
Equilibration time = 2 hours
RID heater temp = 35 C
purge time = 20 min
flow rate = 1mL/min
Injection volume = 5, 10 or 20 uL
Mobile phase = ACN/H20 80:20 in same bottle
Column temp = 40 C ( I started out with 50 C. Results comparable to 40 C)

Also I see a huge peak in the beginning (~3.4 minutes for a 30 min run), even in blanks. This peak intensity is around 400,000 nRIU whereas those of the compounds of interest are around 40,000 units.


I would appreciate any suggestions as to how to increase sensitivity.


Thank you.

Does the huge peak at 3.4 minutes correspond to the void time?

itspip's suggestion is right on. Assuming this is a 250 x 4.6mm column, the dead time should be a bit over 2.5 minutes (so 3.4 fits).

Check with Agilent and see if you can get a copy of their recommended PQ (Performance Qualification) procedure for that detector and then run it. That will tell you if the detector is functioning to spec. If it's not, you might have to check that the flow cell is clean and properly aligned.

I had a similar result when autosampler was not working properly and injected mostly air instead of the sample. If the line coming out of the detector is transparent,you can see the bubble coming out. It did indeed come at the void volume point.

This huge peak sounds like a solvent front...are you sure this isn't an issue of scaling the chromatography?