Chromatography Forum

Hi everyone,

I am beginning an assay of a large (50-300 kda) polymer. UVmax is ~254 nm.

The chain resembles a polysacharride. I have a few ideas of how to approach this. I was curious about one approach.

Does it make sense to try using a Cyano column in normal phase mode?

Thanks,

Unless you have one discrete "hook" somewhere on the molecule, what typically happens with polymers is that you get a "blob" representing the distribution of different sizes overlapped together. If that blob is separated from any interferences, they it might work, but I wouldn't be too hopeful.

If you need a size distribution, or if that's the only big molecule in your sample, then SEC would be my first choice.

By hook do you mean like a tethered API molecule? This sample will have that.

I am definitely expecting a blob. The functionalization of the polymer chain does not only occur at the terminals of the chain, but along the chain as well. Things contributing to the size of the blob will be varying chain length, varying functionalization, and related substances (and other things that I haven't thought of).

The goal is to have a stability indicating method. If we use SEC then my concern is that we won't be able to separate related substances.

Wouldn't you share this concern? Or how do you see the development of a stability indicating method for a large polymer molecule?

I agree with Tom and would recommend SEC for this polymer. Try to test an SEC column, packed with >5µm particles, ID 4,6mm with high number of theoretical plates. Or go with a standard SEC column, but I would recommend instead of a 30cm long column, go with a 60 cm long column. Using 2 x 30cm columns in series will not give the same resolution as you will get on the 60cm column, even if you use dead volume free direct column connector. Good luck.

A lot depends on what the related substances *are*. If they are subunits of your polymeric API, then either SEC or HILIC might work if the differences in size (presumably equivalent to a differnce in the number of polar functional group "hooks") are enough to move them out from the blob (don't you just love these highly technical terms?). I'd give the edge to SEC as a more strightforward mechanism in that case.

If they have different functionality, then HILIC would probably be the better bet (again, if you can get enough selectivity to pull them from the blob).

All of this is just speculation. You won't know until you try.

Thank you guys. I appreciate the help. I have never developed a HILLIC type of method.

I haven't found much info on HILLIC. Is HILLIC referred to as something else in textbooks? Where can I find some good info on this strategy?

Thanks.

Hello.
You can try. As we say: "If a long fret - something happens ( will be made acidents)".In the normal phase polysaccharide (if it does not dissolve in water) can sit on the column.
With a respected Tom Jupille I agree-Hilic on macroporous silica gel is more promising.

Most current information on HILIC is probably in the various column manufacturers' web sites. It's an acronym for "hydrophilic interaction chromatography" (the term was coined by Andy Alpert a couple of decades ago). Many people (including me) people consider it a subset of normal-phase chromatography.

To oversimplify greatly, HILIC is basically driven by partition of analyte molecules between a water-rich hydrated layer on the stationary phase and a relatively water-poor mobile phase (usually something like ACN with 5 to 50% or so of water or aqueous buffer). In this kind of system, water is effectively the strong solvent (i.e., more water = faster elution), which is the opposite of what we see in the more common reversed-phase.

Can cyano columns be used for HILIC? I read some articles that insinuated this. Or maybe a better question is what kind of columns are good for HILIC?

Your best bet is a column that the manufacturer specifies as a "HILIC" column. Most common types would be bare silica, diol, amino, or various zwitterionic phases. Remember that for HILIC to work, the stationary phase surface effectively has to "pull" some water out from the mobile phase.

Would you expect an alumina column to do well for this kind of separation? I believe they adsorb more water than silica. I would think it would require more water than silica to elute the sample.

Would you expect an alumina column to do well for this kind of separation?

I'm out of my depth with that question! You can always try.

Check elution sequences of alumina versus silica in old TLC injstructions. I don´t have anything on this with me here, but seem to remember that alumina was less polar than silica.

Can I ask is your polymer water soluble or not?

For HILIC, it stands for Hydrophilic interaction liquid chromatography. It has a selection of weak acidic, neutral, and basic stationary phases for separating basic, neutral and acidic compounds of high polarity, from Diol, Silica to Pyridine and Imidazole.

I just found that you mentioned your polymer is polysaccharide. Then SEC would be quite suitable.

Please find one related data (figure 11 in page 6) in our SRT SEC column catalog for your review.
http://www.sepax-tech.com/catalog/Catalog_SRT.pdf