need some help with SEC principles

[Username]

Hello all,

I use a GFC column in order to quantify a polymeric compound of MW about 3000 Da.
the column i use is a PSS suprema 5µm 30Å, and the eluent is 4% MeOH in water, with 100mM of ammonium acetate. (1ml/min)

When i inject a 100 ppm std, i see a peak at around 8 min (the polymer i guess) and an other one at 14 min, wich, i guess, has to be the sodium present in my sample.
but why, when i inject a sample of NaCl, do i see 2 peaks, one, for the sodium at 14 min, and an other at about 9 min, the chlorides??

I thought that, with a MW < 100 da, the chlorides and the sodium in the samples would have come together, at the permeation limit of the column (which has to be 14 min)?


I use a corona CAD detector. The UV detector at 220 nm shows me a peak at 8 min (the polymer) but also 2 negative peaks, at 9 and 11 min.

I'm not event sure i should use such a column, the goal is to separate the polymer from the matrix (sea water), and then, quantify this polymer..

[Jeffrey Bodycomb]

It is usually illustrative to inject a blank (pure eluent) to see what peaks appear. Then, move on to pure solvent (if different from the eluent). With an RI (and presumably a CAD) you will certainly observe "system" peak(s). And knowing where these appear is helpful for finding out which features of your chromatogram are from your polymer analyte.

I have seen cases where there were multiple system peaks at the end of the separation. Your Na and Cl should come out together. It seems that you may have an MeOH peak distinct from the NaCl peak.

Or, you may have another lower molecular weight component in your sample (maybe monomer?).

What do you wish to quantify about this polymer?

[Bryan Evans]

Have you considered trying RP for this application (quantitative analysis of 3kDa polymer)?

[kurian]

I suggest like Jeffrey to inject a blank with the same composition of sample but without polymer.
What's the appearence of your chromatogram with Water injection ?

You will see at the end of all of your chromatograms a negative peak. This peak is the salinity difference between sample solution and mobile phase, it's perfectly normal.
The start of your negative peak is the start of your total volume column.

You have in SEC a dynamic range between the initial (or dead) volume of the column (not the system) and total volume.

Your compounds will emerge later when smaller.
So your Na and Cl (in the same peak of total volume) will emerge later.