Chromatography Forum

Hello everyone!

I am doing separation of water soluble products from polymer degradation with aqueous SEC. Eluent: methanol/water 30/70, column: PL Aquagel-OH Mixed 8 um, flow rate: 0.7 ml/min, ELSD temperature: 60 C.

I did the calibration with PEG standards with MW from 12000 to 300 g/mol.

The problem is that when I inject Lutrol F127 (poly(oxyethylene-b-oxypropylene-b-oxyethylene)) with a MW of approx. 11000 g/mol, the sample is eluted after 13 min (which is after my smallest PEG standard of 300 g/mol). Besides this, the peak is very broad and is split on top as if 2 polymers are coeluting.

Do you have any suggestions for why is this ahppening?

Your nonionic is a big mixture of molecules, not a single finite molecule. Your chromatographic behavior is typical of nonionic surfactants we use in consumer products, whether EO, PO, or mixed ethylene oxide-propylene oxide nonionics.

Yes, this would explain the broad peak, but what about elution time?

I would suspect that there is some interaction between your polymer and the packing and since your polymer is more hydrophobic than your standards it is probably hydrophobic interaction. You could try a higher concentration of MeOH. Looks like your column is rated up to 50% organic but double check with the manufacturer since this high an organic concentration sometimes permanantly modifies the packing.

I also found a reference that had your same problem and they switched to a TSKgel column with good results.

See below:

Development of a size exclusion chromatography method for the determination of molar mass for poloxamers
Bengt Erlandsson, , a, Bengt Wittgrenb and Gunnar Brinkmalmb

a AstraZeneca R&D Södertälje, Analytical Development, SE-151 85, Södertälje, Sweden

b AstraZeneca R&D Mölndal, Analytical Development, SE-431 83, Mölndal, Sweden


Received 28 June 2002; accepted 27 October 2002. ; Available online 2 April 2003.

Abstract
An aqueous size exclusion chromatography (SEC) method for determination of the molar mass of poloxamers 188 and 407 has been developed as an alternative to the pharmacopoeia methods. During the development work two different columns and several different eluent compositions were investigated. With a PL-aquagel-OH column, non-exclusion behaviour was obtained. A TSKgel column gave good separation of both poloxamers. The best separation was obtained with an eluent consisting of sodium chloride (0.01 M)–methanol (90:10, v/v) on the TSKgel column. The method was shown to be linear within the elution time of the two poloxamers and to have acceptable precision. The results from the SEC method was compared to results obtained using SEC with online multi angle light scattering detection (MALS) and to results obtained with matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS).

Author Keywords: Aqueous size exclusion chromatography; Poloxamers; Non-exclusion behaviour; Multi angle light scattering; Matrix-assisted laser desorption/ionisation mass spectrometry