Chromatography Forum

I'm using naphthalene sulfonic acid as my UV-active additive, 0.0008M. running this at 100%, 1 ml/min.

the baseline is very noisy when I approach ug/ml levels. I'm using a column heater at 40C and filtering my mobile phase.

can anyone suggest anything please?

also I've run choline and DMAE samples at 1; 1:10; 1:100 and 1:1000 - I'm getting peaks (negative) but the areas don't make sense, i.e. I'm not seeing the expected changes in magnitude.

if anyone has any experience of this LC mode I'd really appreciate your suggestions, thanks

also I've run choline and DMAE samples at 1; 1:10; 1:100 and 1:1000 - I'm getting peaks (negative) but the areas don't make sense, i.e. I'm not seeing the expected changes in magnitude.

The background concentration of the UV-active additive must be much higher than the highest analyte concentration to be detected, at least 10 times higher, preferably 100 times. Indirect detection usually has a narrow linear range, in many cases the linear range is just one order of magnitude. I hope this helps.

why?

If memory serves me indirect UV is performed using a UV active additive with the same charge as the analyte. The measurement principle is based on maintaining solution electro-neutrality so the analyte displaces the UV active species having the same charge. This displacement is what gives the decrease (negative peak) in detector response.

This is from memory so hopefully someone will comment on the accuracy (or lack thereof) of this.

carls, read my post again. I'm expecting negative peaks - the problem is magnitude. I was hoping to see linear relationship, i.e., the peak area is 10 times higher when the conc. is 10 times higher...

is there no-one at all on this forum with significant ion-pair experience???

perhaps the following paper will help:

http://www.mdpi.com/1420-3049/10/9/1179/pdf

We tried an indirect UV method recently. We used 0.3 g/L benzoic acid in the mobile phase.

It did not work for us. Never really did figure out why.

Are there any inside tricks to getting this type of approach to work.

Thanks

Although I am not familiar with indirect UV, some general rules about UV detectors should still apply.

I guess the absorbance of your mobile phase is so high that almost no light passes through the cell. If you autozero your detector with water and then run your mobile phase through, you can then see what the absorbance of your mobile phase is. If it is above (approx) 1.5 AU, you will run into trouble. A lot of noise and non-linear behaviour.

is there no-one at all on this forum with significant ion-pair experience???

chromeleon - are you still following this?

The system you describe is more than simple ion pair chromatography or simple indirect UV detection.

Choline carries a permanent positive charge and your ion pairing agent (naphthalene sulfonic acid) is also your indirect UV visualization agent?

The article I posted above mentions such a case (competitive adsorption?) but this, in my experience, is not a common approach.