Chromatography Forum

One can actually run quite comfortably isocratic ion-exchange of large molecules.

Hi Uwe,

Would be nice if you could provide some examples of that (i.e. isocratic ion exchange protein separations)

Best Regards

If you give me your e-mail, I can send you a bunch of chromatograms of immunoglobulins obtained isocratically. Alternatively, you can look up isocratic ion-exchange chromatograms of proteins in some old publications by Fred Regnier.

Do the chromatograms contain proprietary methodology, or otherwise something you do not wish to share publicly?

If not, please feel free to upload some of the most illustrative to a zero-cost picture host such as www.tinypic.com

Thanks

Hi Uwe,

I looked up Fred Regnier, but found no detailed methods nor data/chromatograms demonstrating isocratic separation of proteins by IEC. There were quite a bit of IEC publications associated with him but I couldn’t find this particular topic.
I also searched for/googled ion-exchange protein separation and got >1500000 hits. I probed some of the promising hits and found exclusively gradient separations of proteins.
I’ll drop you a mail afterwards, so that you’ll get my mail address. I’m quite interested in seeing some of the data you mention (hopefully some nice, actual separations). Because I’ve seen (not that seldom) people thanking God for seeing a single peak, which most probably consists of 5 – 6 or more not separated peaks, and call it separation.
By the way, while searching the net, I fond some kind of protein separation guide, written by Timothy Bradshaw and I took a short excerpt, just because it’s nicely condensed in a few sentences. See text bellow.

Ion-exchange chromatography occurs as a multi-step process, including the movement of the peptide or protein solute from the mobile phase into the stationary phase environment, ionic binding to the solid support, and finally, the selective displacement and elution of the solute. Ultimately, separation occurs because those charged solutes displaying relatively weak interactions with the ion-exchange stationary phase will be retained less on the column, eluting earlier than those charged solutes
that react more strongly with the column and elute later. It is important to note that the diffusional movement (mass transfer) of solutes through the stationary phase is relatively slow in IEC.

He also mentions the relatively slow mass transfer in ion-exchange, as a whole, which makes isocratic elution in this technique even less feasible.
It’s important to note; I’m not saying that some kind of protein separation in isocratic ion-exchange mode is out of the question. But I am convinced that gradient elution/separation of proteins is by far the most feasible and efficient choice in IEC.

Best Regards

Isocratic elution of proteins: M. E. Rounds, F. E. Regnier, J. Chrom 283 (1984) 37:
"Proteins analyzed were beta-lactoglogulin A, conalbumin, ovalbumin, and soybin trypsin inhibitor...
At each pH, samples were eluted isocratically, starting at 100% eluent B (high ionic strength) and repeating at decreasing concentrations of B until the protein was completely retained on the column. This generated a series of retention times for each protein."

I also went back to the data from my lecture at the last HPLC conference. The constant of the mass transfer term for the proteins for which I determined this coefficient under retained conditions in ion-exchange chromatography did not show anything unusual, i.e. no slow mass transfer. The MW of the proteins varied between 13000 and 37000, and the constant of the C-term was about 1/10, which is nothing unusual. I can't confirm slow mass transfer in ion-exchange of proteins, at least not for the MW range investigated here.

What would mass transfer have to do with favoring gradients over isocratic, anyway, unless one went from high viscosity to low viscosity?

At each pH, samples were eluted isocratically, starting at 100% eluent B (high ionic strength) and repeating at decreasing concentrations of B until the protein was completely retained on the column. This generated a series of retention times for each protein."

It seems to me that there was some kind of pH gradient – stepwise or whatever. Which is the alternative option (although not very often utilized) to a salt gradient.

What would mass transfer have to do with favoring gradients over isocratic

Slow (or no) mass transfer would usually mean that the analyte is stuck in the stationary phase, so it’s needles to point out that isocratic conditions will get you nowhere. A gradient (increasing ion strength) on the other hand will displace the captured analyte (selectively) and thus facilitate the last phase of the separation (i.e. elution)

Best Regards

I would call this a strong absorption, not a slow mass transfer.

When Fred Regnier says that the elution was carried out isocratically, we do not need other people making second guesses about what one of the best chromatographers in bioseparations really did.

Danko, go and read the papers first!

Hi Uwe,

I can’t find this particular paper. Would you link to it, please?
Or better yet, refer to more instances of isocratic proteins separations on IEC. There should be many more, since there are some benefits to it.
I wouldn’t miss the chance to optimize all my gradient methods to run isocratic and I believe I speak on behalf of thousands of other chromatographers.

Best Regards

HW Mueller wrote:
I would call this a strong absorption, not a slow mass transfer

I’d love to hear your definition of slow mass transfer.

Best Regards

JA

In an earlier post you stated:

I'll have a go at the mental experiment:

For a column of given capacity I would expect the same retention time for both the 2 mmol and 4 mmol sample load assuming that the latter does not cause 'mass overload' (charge site overload or whatever the equivalent nomenclature is in IC.)

However, I feel this example is distinctly different from the case where load is kept constant and capacity is changed. As capacity refers to the density of retentive sites on the sorbent I visualise this simply as the distance the ion or analyte travels before being re- ad/absorbed. Analogously, reversed-phase retention will increase on a C18 with increased bonding density.

While it's understandable that you might think the capacity is reflected in the charge density of the ion exchange phase, the reality is that unless the ion exchange phase is homogeneously distributed throughout the stationary phase (which is rarely the case with modern ion exchange materials), there is generally no direct connection between the charge density and ion exchange capacity. A case in point is the DNAPac column mentioned at the beginning of this thread. It consists of a thin film of ion exchange particles (< 100 nm) on the surface of inert EVB-DVB substrate particles. The charge density of the surface film is the same as that of a particle of high capacity ion exchange material of the same composition as the surface film. The capacity is just low because of the low percentage of stationary phase in the column not because it has low charge density. If I want to double the column capacity, I just use an ion exchange coating particle with double the diameter. In this case the capacity would be doubled but the charge density would be unchanged.

Chris

Oops

Welcome back, Chris.

Thank you for elaborating on the nature of the DNAPac column, which is quite intriguing. If I picture it correctly, it is characteristically distinct from a pellicular support where the 'retention giving film' (words are failing me) is a porous extension to the underying particle?

Is the core particle truley inert, because I don't picture the underlying polymer to be completely nonporous (is it just achieved through high crosslinking?) Are ions/molecules able to diffuse into this structure and experience hydrophobic attraction? While asking this I also wonder about the absolute lack of porosity in, for example, the traditional silica-based pellicular reversed phase supports or the more modern "fused core" packings.

In incorrectly making the association between capacity and charge density I must of assumed a homogeneous, monomolecular layer of absorptive sites. I guess my example of the C18 bonding density is equally flawed if one considers a less dense but thicker stationary phase coating.

With regard to your example of doubling the microbead diameter to double the capacity, it could be closer to 4x the capacity due to the r^2 relationship on surface area.

edit: Now I'm a bit confused... particles on particles every time for pellicular packings?..

JA,

Regarding your question about whether or not the polymeric substrate particle is porous, this is certainly a potential consideration with polymeric substrates. In the case of the DNAPac PA100, the core consists of 55% cross-link "microporous" ethylvinylbenzene-divinylbenzene. The old terminology "microporous" certainly indicates that the substrate has defined porosity but it's a bit like describing a billiard ball as being porous. There are essentially no pores is larger than 10 Ã…. On top of this, the exterior surface of the particle is lightly sulfonated in order to allow for electrostatic attachment of the nanoparticles. At least in the case of DNA, the sulfonation layer excludes DNA from access to the core since the sulfonation layer has the same net charge as DNA.

Regarding the relationship between latex diameter and capacity, it's understandable that you might think that there is a square function relationship between coating particle diameter and capacity (I had the same thought before I determined experimentally that it was otherwise many years ago) but I assure you that if you create a geometric model you'll see that it's actually only proportional to the diameter of the coating particle. From a geometry point of view, the rationale stems from the fact that if you double the diameter of the coating particle there are significantly fewer particles on the surface which partially compensates for the geometric effect that particle volume has on capacity. But, if you think of it is a film the thickness of which is equal to the diameter of the coating particle it's easier to visualize why capacity is actually directly proportional to coating particle diameter.

Chris

It is too tedious to ascertain which diameter you guys are talking about. That of the supporting particle or the fused stat phase spheres?? Could you clarify? Also it seems that any correlation between any diameter and capacity is going to be starkly nonlinear somewhere. If the particle gets so big that it doesn´t fit into a column one will not have any capacity.
Also, I have considerable problems with the apparent dichotomy between the number of ionic sites in a colunmn and capacity in IC. For instance: It seems to me that if the equilibrium of an analyte ion lies completely on the side of the stat phase the capacity equals the number of ion sites.