Separation of Large Proteins or Polymers by Non SEC Methods

Discussions about gel permeation chromatography / gel filtration chromatography / size exclusion chromatography

15 posts Page 1 of 1
Hey All

I was interested to pose a question. Generally when it comes to separating large proteins from one another or large polymers from one another, it is generally accepted that Size Exclusion is the only way to do this. And that Reversed Phase separations and Normal Phase separations will not work.

But now I wonder why this should be so. We are all familiar with the On/Off mechanism by which large molecules separate on a column. So if, for example we had to separate two polymers and one was 700,000 and the other was 900,000 (same polymer just different sizes), shouldn't this be possible. Maybe we run a fairly standard gradient and find that they both come out around 35% organic, and then we go back and run a very very shallow gradient around that area.

Shouldn't it be possible to separate them. It seems like it should be possible but I've never seen this done.

Anyone have any experience with this??
300A, C4 is a standard phase to separate proteins. But with RP we have denaturating conditions for the proteins. This is one reason why SEC is so popular.
For fast screening also with large proteins non porous phases are available on the market and work well.
Limitations are pore size and particle size, and denaturating conditions.
Gerhard Kratz,
Your view of the possibilities is too constrained. Large proteins can be separated nicely using either ion-exchange (via differences in charge) or hydrophobic interaction chromatography (HIC) via differences in polarity. It's possible to get a separation to baseline based on differences in a single residue, which is a lot better than with SEC. Also, these are nondenaturing modes, unlike reversed-phase (per Gerhard's comment). For proteins larger than, say, 30 KDa, I would recommend materials with pore diameter of 1000- or 1500-A, not 300 A. These afford sharper peaks and better selectivity.
PolyLC Inc.
(410) 992-5400
Hmm. Interesting.

Most of you guys comments has been on proteins. So let me ask: what about polymers. Is there any way to baseline resolve a 100,000 molecular weight polymer from a 150,000 polymer.

I've never seen anything that even comes close...

Any ideas are appreciated.

Thank You
Well, what's the composition of your polymers? Are they charged? What are the functional groups? We need more details in order to provide a coherent response.

Your past experience seems to have been based on SEC, at least in part. The general rule in SEC is that two analytes of the same geometric shape must differ by at least 2x in molecular weight in order to get a baseline separation. That accounts for the problem you described.
PolyLC Inc.

(410) 992-5400
The polymers we're currently dealing with are Povidone. You can easily google the structure.

I appreciate any feedback.

My original thought was that, due to the on/off mechanism, a slow gradient should work (with reversed phase or hilic). But I have never seen that done (nor have I been able to do it myself).
Some people would argue that "on-off mechanism" for big molecules in reversed-phase is something of an exaggeration. The retention mechanism is fundamentally the same for big and small molecules, it's just that the slope of the relationship between retention and solvent composition is molecular weight dependent (roughly proportional to the square root of the MW), so with big molecules, it's very steep.

More to the point, if you had monodisperse polymers at 100 kDa and 150 kDa, you might well be able to separate them with a shallow gradient. Polymer peaks in reversed-phase tend to be very broad because you're getting partial separation within the distribution (i.e., the 100k separates a bit from the 99k and the 101k which separate a bit from . . . ) so that what you see is essentially the "envelope" of those slightly separated peaks.
-- Tom Jupille
LC Resources / Separation Science Associates
+ 1 (925) 297-5374
Put your sample on a HILIC column with wide pores. For polymers this large I'd recommend 500-A or even 1000-A. Elute the column isocratically with whatever % ACN provides a retention time around 5-10' (probably around 60%). Then, increase the % ACN to, say, 65% and assess the retention. Continue to increase the % ACN in modest increments until you have a retention time around 40-50'. That's probably going to afford the best separation you're going to get for these polymers. If you want even better separation, then slow down the flow rate or use a longer column.
PolyLC Inc.

(410) 992-5400
Thanks Andy, we'll give that a try. Two last follow up questions:

Since the on/off mechanism should be in effect for these molecules. Why would you suggest isocratic. Wouldn't a slow gradient around the organic range where they elute be the best.

Secondly, again because of the on/off mechanism, would we be best off with a shorter HILIC column.
I believe that the on-off mechanism can be used to describe adsorption to a reversed-phase or ion-exchange column. However, HILIC is a partition mode. Think of it as partitioning your polymer between two phases in a separatory funnel. You want to add just enough ACN to bias the partitioning in favor of the aqueous phase to some extent. Such being the case, longer is better with regard to column length. Also, isocratic separation confers the best separation of all, since partitioning will be occurring along the full length of the column.

Whichever % ACN you have in the mobile phase, be sure to have it in the sample solvent as well. It would also be helpful to have at least 10 mM salt overall in the mobile phase and sample solvent.
PolyLC Inc.

(410) 992-5400
OK, I'm back. This time I have two questions in one. A Friday morning challenge for you all.

First question: Previously it was recommended to use HILIC as a good way to separate polar polymers. We have begun working on this (with an amide column). Only made a few injections so far but everything is coming out late (around 80-85% water). So my question is: might it be a good idea to use a less polar - hence less retentive - HILIC column such as cyano. I believe cyano is the least retentive of the HILIC options (doesn't bind as much water I guess). This is same logic as why C4 columns are often better for protein separations. Because big molecules can have too much retention and hence not give as good separation.

My second question is: can it be a problem to have too much water in a HILIC separation. Commonly HILIC methods go from 5% water to 50% water, and usually no more than 50% water is needed. But for large polymers I'm thinking more water may be necessary to elute them. So I'm wondering: is there any concern with going above 50% water. For example, does it cause the sorbed layer of water to be lost, and thereby completely kill the separation.

Thanks for any suggestions.
Hajdaei, I don't like repeating myself, so this is the last time I'm going to say this. For polymer molecules as large as yours, you need a column material with pore diameter of 500 or 1000 Å. Example: Our company's PolyHYDROXYETHYL A material. I expect that this amide column you tried was TSK Amide-80. It has a pore diameter of about 80 Å, totally unsuited for your application. The polymer molecules will encounter appreciable steric hindrance diffusing into and out of the pores, resulting in badly skewed peaks (by the way, what was the peak shape like in your runs?). An additional advantage of the wider-pore material is the lower surface area, which will permit your polymers to elute earlier.
PolyLC Inc.

(410) 992-5400
Yes I recall that. We don't have a large pore column handy and don't want to purchase one for this "one off" analysis. I've run large molecules before with standard pore columns. Not optimal but it does work well enough in many cases.

As much as I enjoyed your reprimand, what I would enjoy even more, would be answers to the questions I posed.
In regards to the question above "is there any concern with going above 50% water (in hilic).....does it cause the sorbed layer of water to be lost, and thereby kill the separation".

I don't know exactly what the critical point is, but I think it is true that if you go much above 50% water, you will start to lose the sorbed layer, and the hilic separation will fall apart.

The sorbed water layer forms in the first place because water has more affinity for polar surfaces than for a mobile phase made of something like 80/20 ACN/Water. But if the water content in the mobile phase gets too high, this is no longer the case. Similarly, hilic doesn't work very well with methanol as the weak solvent. I think this is because water has a high affinity for methanol and therefore there is less tendency to form a sorbed layer.
I completely agree with Tom that the “on-off mechanism" for big molecules in reversed-phase is an exaggeration. And that statement is based on my on experience as well as other chro0matographers. Over the time I’ve separated a number of proteins from other proteins as well as impurities of large molecular sizes by means of isocratic elution – some times mixed mode i.e. isocratic followed by gradient elution. And it wouldn’t be possible if on-off mechanism was “the mechanism”
Saying that I don’t underestimate the usefulness of the gradient mode, but it’s best advantages are narrower peaks and shorter runs when at its best.
Something else: I should it be so bad denaturing the molecules that are to be separated? Sometimes it represents some advantages and some times it very useful in SEC elutions as well.
Finally – as Andy too addressed the matter, the pore size can be (will be) a useful parameter in reversed phase separations as well in SEC.

Best Regards
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Dancho Dikov
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