Refractive Index Detection question

[tristanewalters]

Hey guys, I have a question about RID on the HPLC. I'm a newb when it comes to this kind of detection

We have a method analyzing a disaccharide in which there are two forms. One has two closed rings and the other has one closed ring and one straight chain.

We are attempting to monitor the change between the two forms over time, but we see a 2x decrease in one of the peak's area and a 5x increase in the others.

Is RID similar to UV in a way that different molecules will have different "response factors", presumably based on its refractive index, and is that why we are not seeing equal total peak areas over our time points?

Thanks for any help you can provide

[tom jupille]

Is RID similar to UV in a way that different molecules will have different "response factors", presumably based on its refractive index, and is that why we are not seeing equal total peak areas over our time points?

The short answer is "Yes".

[t_morris]

Here are some tips for a better baseline on RID:

1. RID is very temperature sensitive (so have your hplc tubing encased with a tygon tubing as insulation & keep the compartment as closed as much as possible)
2.Always run water through system at end of run to prevent any buffer salts depositing in the detector.
3.To clean and store detector run 50/50 IPA/Water.
4.Gradients won't work on a RID.
5.These systems take a long time to equiibrate. (Try equilibrating overnight at 0.3 ml/min.)

Hope this helps...

[balamus]

Just to add,
Always flush the reference cell with the mobile phase before starting the measurements.
If the flow is stopped at the end of the day, flush the reference cell again before starting the measurements next day.
A simple solution would be to leave the system overnight with low flow (e.g. 0.1 ml/min).

[Consumer Products Guy]

I'm going to add as a tip to improve RID baseline (needed most when maximum sensitivity is needed):

If a quaternary type pump, by pass the mixing valve, to eliminate any chance of slight changes there to the mobile phase. Agilent makes an adapter for this #0100-1847.

[Meerkat]

The RID is the preferred detector for analysis of sugars, polysaccs, monosacs and analysis of degradation of such parent compounds. Don't be dismayed if your chrom looks "grassy" and not like the pristine pictures of chrom shown in advertisements.
Run a 0.1% sodium azide solution thru your system when it's idle to keep microbial growth from damaging your tubing and flow cell. Also a good idea to equilibrate overnight on low flow to improve readiness.

[remesquaddie]

Some great tips, very real. Also remember that RI detector cells are very pressure sensitive, and easily crack at low back pressures. Therefore, NEVER connect another detector on its WASTE side, and keep the WASTE tubing reasonably short, and under .009 ID.