Chromatography Forum

Hi everybody;
is there any relationship between pore diameter and molecule size?
Here we analyze small molecules (usually MW less than 600) and we use columns packed with 100A pore diameter. Lately, somebody started working with MW ranging between 1200-1800, which gives us a big headache because nobody has experience outside small molecules.
When we run this compounds (any column we have, different gradients, different temp.) we usually get a blob. MS either gives the same spectrum across the blob or shows some impurities at the beginning and the end (in the later cases, looking at the UV you can see more than one compound in the blob but we couldn't get base line separation using normal or RP, in fact getting something like a peak is a miracle)
The NMR guy (doing his best, like all of us) thinks that there may be isomers (conformationals, diastomers, rotamers). This would explain the blobs.
Then came this particular sample. No difference for us but the NMR guy says that is quite pure (no isomers). So, I started to take a second look at our methods and I really don't know the part that the pore diameter is playing in the separation. I'm thinking on getting a column with bigger pore, but how big?

I'd appreciate any help on this.
Thanks,
Sergio

What are the large MW compounds that you are trying to analyze. If they are ionizable, then you might need a buffer of some sort. Please give more details of your chromatographic conditions.

is a natural product derivative, very non polar (eluting at about 90% ACN).
I'm using a grad 40-95% ACN in H2O (0.1% formic acid); there is no difference using NH4Ac (PH 6.5). In normal phase: 5-50% MeOH in HFE-7200 (is a fluorinated solvent we use instead of hexane, so we can use MS detector)

Yes, pore size can make a difference. A lot of protein & peptide work, for example, is carried out with packing whose pore sizes are in the 300Ã… range.

Note that this doesn't exclude the possibility that you can have multiple conformational states mutually equilibrating in solution. If the equilibration time is comparable to the residence time on the column, you can see just the kind of "blob" you are describing. One partial diagnostic is to increase the temperature (speeds up interconversion); if the peak sharpens up, that suggests that you do have multiple forms. If that's the case, the remedy is run at the higher temperature, or to stabilize one of the forms (temperature? pH? solvent?) so that it predominates.

It is not that large. My examples are peptides and oligonucleotides, and they still run fine on a 100A packing. However, if you want to make sure that the pore size is not the issue, you can use a 300A packing.

thanks everyody for your advice.

Sergio

I agree with Uwe that MW 1200-1800 is not too high and an 100A column should handle them just fine.