gel filtration question

[woow]

OK

If Gel Filtration (or Size Exclusion Chromatography) column has a specification mass range of 4000 to 400,000 Da; does this mean that the porous packaging limited of less than 4000 Da (smaller molecular weight) it loses accuracy, repeatability, etc.? (Is the GF column pore size or size range more important towards the limitation of a separation of a protein and small molecule?) And if there is a minimum is true, does anybody have any recommended GF columns going from mass sizes of say 100 to 100,000 Da?

Thanks
Winnie

[Uwe Neue]

These descriptors describe the range for which a particular material works well, often with a linear (or close to linear) calibration curve. Outside this range the separation deteriorates quickly: on the lower end, there is no pore space available for doing a separation, on the high end, the analyte molecules are excluded from the pores.

All manufacturers of SEC materials have several different columns for different ranges of molecular weight. Get yourself a catalogue or go on a website (for example the Waters catalogue or www.waters.com), and you will see the calibration curves for different materials. For example, the Biosuite 125 could be useful for your purpose.

[Matt Savage]

Try looking at the columns from Polymer Labs (recently bought by Varian) I use their organic SEC columns for GPC analysis and get good results with the 100A columns down to 100Da. In other words I can seperate monomers from dimers.

http://www.polymerlabs.com/gpc/aqueous.htm

Also in the UK they will let you try columns on your own kit if you ask them nicely.

Matt

[PJ8]

I tried some size exclusion chromatography to seperate boronic acid monomer and trimer (we de-hydrated some boronic acids to generate more obvious levels of the trimer.) However the trimer only had a molecular weight of around 500, around half of the minimum weight the column said it could seperate.

We saw some seperation, but the elution order disagreed with theory (i.e. the larger trimer eluted last rather than first. We had previously measured trimer levels of 75% by NMR.)

We surmised that since both compounds were way below the lowest molecular weight reccomended for the column both species passed through all the pores, the extra retention of the larger analyte due perhaps to extra pi-pi interactions... all a bit sketchy.

In hindsight I wish I had done some LC-MS to confirm peak identities, although you tended to form dimers and trimers in the source so maybe that wouldn't have helped.

If you have a column give it a go!

[Alfred88]

Dear all:

SEC will separate molecules based on dynamic sizes, not on absolute MW. And dynamic sizes depend on the solvent and mobile phase, so one molecule may be smaller (collapsed) in one solvent, but larger (expanded) in another solvent.

To use a SEC column, one must observe the MFG's recommended range. Too high MW will lead to total exclusion (i.e. molecules will elute with mobile phase with no retention!). Too low MW will lead to no separation (i.e. total permeation, molecules will travel along with all other small molecules, and solvent).

100 - 100kDA is a rather wide range. But you can try with the linear range column (which is a blend of several MW ranges, and will cost more) first, then "zoom" into the range of interest.

Alfred.

[woow]

so due to the fact that GPC (aka GF or SEC) separates by the analyte size large to small.

How does the following apply giving the hypothetical specifications of a column of 30cm x 7.5mm, size range of 4000 to 400,000 Da, pore size 125Å, and particle size of 5 to 10µm?

• 1. polymer size about 500 Da to 1000 Da:
  2. denatured polymer:
  3. aggregated polymer:
  4. unfolded or folded polymer:
  5. multicomponent compound and of different hydrophobicity:
  6. mobile phase pH slightly above the isoelectric point:
  7. an unclassified/unidentified silica-based stationary phase
  8. column was of smaller/longer length or particle or pore size
  9. PEEK or Stainless Steel cased column
  10. And finally, do GPC treated diffrently than Normal and Reversed Phase?

Need some opinion, theory, or facts toward understanding the methodology.
Thanks,
Winnie

[tom jupille]

If you are below the nominal size range of the column, you are not doing SEC.

At the risk of sounding self-serving, maybe your best bet would be to sign up for a training course. We do one that covers biopharmaceutical applications, although it is not limited to SEC. Polymer Labs (I think) does an SEC course. Probably so does Waters (if I'm leaving anyone out, speak up!). Even if your questions are not covered explicitly in the course, it would give you the opportunity to "brain-pick" the instructor. Another possibility would be to get a consultant in for a day or so.

[woow]

I would not mind applying for a course, my job has minimal focus or interest on theory and chemical background of chromatography.
I asked the column manufacturer and Waters tech if they have info to supply on methodology, but waiting for their reply.