RI question

[pinkpanther]

Hi!

Can someone please explain this to a beginner in HPLC.

I have a standard solution that contains one polar analyte, and that is only diluted with water. I run it in a mobile phase that consists of 80% acetonitrile and 20% water, the detector is a RI detector.

At low concentrations (0.1 to 20 mg/mL) of the standard I get 2 resolved peaks, one for the analyte and one for the solvent (?) =water.

At higher concentrations (20 mg/mL and higher) of the standard, the analyte peak migrates into the solvent peak, which results in one very distorted positive peak, with a negative valley on its right side. Sometimes the result is one large distorted peak and one smaller peak with a valley between them.

My questions:
Why is the solvent peak positive at low concentrations? Shouldn´t it be negative since water has a lower RI value than acetonitrile?

And why do I get a negative peak and distorted positive peaks at higher concentrations? Is it due to column overload or what?

I am very thankful for any suggestions.

[Rolandas Plausinaitis]

Hi,

I'm not so experienced to answer your questions. Some of possible problems can be caused by dissolved air, pressure swing during the injection...

You should modify your sample preparation procedure in such way that final sample solution would be in the same solvent as mobile phase (80/20). That should solve at least some problems.

Good luck!

[sksrinivas]

Hi

I think you have a column overload problem. Column overload results in weird peaks when using RI detectors.

I'd suggest you do a blank run, without the sample. That would give you an idea of what's going on.

I'd agree with Rolandas - dissolve your sample in the mobile phase, to mimimise peak shape distortion.

Also, keep an eye on injection volume. It should not exceed 20 microliters. Large injection volumes could cause peak distortions in RI detectors.

Hope this helps.

S.K. Srinivas
K-Prime Chromatography Training
Bangalore, INDIA

[pinkpanther]

Hi!

I´ve made some test runs with sample blanks and I got 1 negative peak = valley. But when I inject my standard solution, I get 2 positive peaks.

The solubility of my standard compound is 100 mg/mL water, and I´ve tested to dissolve it in the mobile phase but with bad results.

The mobile phase is degassed with an inline vacuum degasser, so I don´t think there is any dissolved air in the system.

My injection volume is 10 uL, is that really too high?

[HW Mueller]

One can easily inject air with the sample. My guess for most of your problems is also column overload.

[tom jupille]

As I recall, with ACN/water mixtures, RI is a non-linear function of concentration (I can't find specific values, though), so should be quite possible to see either positive or negative (or just plain "strange") peaks for water in an ACN/water mobile phase

[sksrinivas]

Hi

I still think it's a column overload issue, coupled with the fact that your sample is not soluble in the mobile phase.

Your injection volume is OK, no problems there. I think the problem is the concentration. So perhaps you shouldn't exceed 20 mg/ml, since you get acceptable peaks at that concentration.

Try a different mobile phase composition and see if your sample is soluble in that. Say, ACN/water 50:50, or so.

Seems to me your sample is a carbohydrate, if I'm not mistaken. In which case, it won't be completely soluble in acetonitrile. So perhaps you have no other choice but to reduce ACN and compromise on the run time a bit.

Hope this helps.

S.K. Srinivas