Chromatography Forum

I am currently working on some HPLC methods for artificial sweeteners. I was curious about the advantages of the RID detector over just using the DAD detector at UV 200-210nm for analytes with minimal UV absorbance such as Rebauside A and Seviocide in Stevia. I am aware RID is only an option for isocratic and that it can be a pain the equilibrate.

I have an Agilent 1260 with both DAD and RID.

The RID is a "bulk property detector", and as such responds to *everything*. This means you have to pay closer attention to things like:
- mobile phase degassing
- pump operation (minor flow rate fluctuations or pulsations)
- temperature control
- injection volume if diluent doesn't exactly match mobile phase

I was able to test the RID. Steviol glycosides have an unusually low molar absorbance at 210nm about 10% of my other analytes like aspartame and acesulfame. The RID signal of the sg's were approximately 1/10 of that.

Injecting 10ul on my 1260 I can see aspartame and acesulfame down to a bit below 1ppm, steviol glycosides to about 10ppm and on the RID 100ppm was about as low as I could go limit of detection was about 50ppm.

One on my collegues was able to do sucralose down to 6.25ppm but they injected 50ul.

But yea it is a very insensitive detector.

Also one of my analytes is close to the void volume of about 2min and there was a huge negative peak there from the sample injection solvent which was water 10% CH3CN much weaker than the mobile phase. Evaporating and redissolving in mobile phase helped.

"Also one of my analytes is close to the void volume of about 2min"

If that is the case, then your analysis method may be invalid. For most modes of chromatography (not SEC) you will need to demonstrate that the method is selective for the sample(s) being analyzed so each one needs to have a proper K prime value (>1.5,>2.0 is preferred). If any peak elutes near at or near Tzero, then you do not have enough retention.

I realize that but in order to get Mogroside V, Rebaudioside D, M, A and Stevioside in one run without a gradient the only composition that worked was 55deg, 5% methanol, 30% CH3CN, 65% water acidified H2SO4 pH 2.5. When I tried reducing the CH3CN to 25% my critical 2 analytes Reb A and Stevioside went from 8 to 25 minutes and from resolved to barely resolved at all (almost 1 peak).

All in all though I am probably going to stick with the gradient-DAD method.