Chromatography Forum

hello friends...

I am working on an analysis of a very complex mixture.

I'd like to know...

a) Does the retention time depends on the column length (for both GC and LC)?

b) While using a HPLC attached to a PDA (190-800nm) I noticed the compounds have their best results at many wavelenths, most of them have the best result at 254nm. But I have got 200, 204, 263, 280 and so on. The problem is, I am using ChromQuest 4.2 (Thermo) and this PDA only allows me to use 3 discrete channels per run.
Question:
Does that mean I have to run the same sample as many times as needed to get my samples analysed on all wavelenths that gave best results?

A simpler question would be: Why do I need to use discrete channels? Why not use the scan only?

Thanks

To answer your first question with a question: if the velocity of the mobile phase stays the same and you e.g. double the lenght of the column, what will happen with the retention time of a component?

I use a Waters PDA and you can go in two directions:
you let the detector scan between 190 - 300 nm, then extract the various chromatograms at the different wavelenghts, you could you MaxPlot (if it is available) which means that the chromatogram is plotted at the max absorbance at any given retention time. with a PDA you do not have to worry about the scan time, since the spectrum if recorded using an array of photodiodes.
you just pick one wavelenght wich is most suitable.
In any case, you would have to run calibration samples the quantity the analytes, and this takes the different extintion coefficients into account.
I hope this helps.