Chromatography Forum

There are two vendors that I'm familiar with that make mixed mode columns: Dionex and SIELC.

Has anyone had any experience with these. I am wondering if they really perform as standard reversed phase columns as well as having ion exchange capabilities. I know, for example, that the maximum content of organic in the mobile phase can be limited with these types of columns.

If anyone can suggest which of these vendors columns performs more like standard reversed phase column I would be interested in hearing the feedback.

Thank You

Don't take this the wrong way, but if you need a standard reversed-phase column, get a standard reversed-phase column! The mixed-mode columns are tremendously powerful for mixtures of ionizable and neutral compounds (kind of like ion-pair, but without the equilibration time issues) but would not be my first choice for general-purpose use (if for no other reason than there is a limited choice of columns available).

There is no limitation on the amount of organic you can use with mixed-mode columns in terms of stability, reproducibility, etc. Our columns are compatibe with 100% water and 100% ACN. You need to realize that most of mixed-mode columns have polar embedded groups which are ionizable, So when you use high organic, for example, you might easy go from reversed-phase ion-exchange mode to HILIC/ion-exchange mode. Also one of the mistakes people usually do is running gradients without considering both mechanism: reversed-phase and ion-exchange. If you are running gradient of ACN and got from 0% ACN to 100% ACn you are increasing strength of the mobile phase in-terms of RP properties but you usually decrease strength of the mobile phase in-terms of ions, so you peaks might not be as sharp as if you go from 0% ACN to 60% ACN increasing amount of ions from 5 mmol to 50 mmol (for example). In most cases our columns will show at least same efficiency as corresponding RP columns (plate count and symmetry), you just need to take in consideration ionic properties of your molecule: do you have ion-exchange interaction or ion-exclusion? Once you realize mechanism of retention you have no problem being a highly efficient "Method Developer"

My BIAS Opinion: If you can use mixed-mode column in reversed-phase mode, cation- and anion-exchange mode, HILIC mode and mixed-mode, why not unify your tools and have one or two columns for any case which might come your way?

Contact me if you have any questions - I've been developing these phase for the last 7 years, so I might advice you on few things

Vlad Orlovsky
SIELC Technologies
www.sielc.com

That's a good point (about running gradients). I guess the best thing in many cases whould be to do the ion-exchange part 1st (when the mobile phase is predominantly aqueous) and then run the reversed phase gradient.

If we were to use this kind of approach would the equilibration time be especially long. In other words, if we run up to 100% organic (where the organic phase is 100% ACN for example) then we re-equilibrate with an aqeous phase which has some type of salt to modify the ion-exchange process, would all of this result is a longer than usual equilibration.

Thanks

You don't need to do 100% organic and do switching, your usual gradient from 10% ACN to 60% ACN should work. Gradient of ions can be from 5 to 100 mmol. Equilibration is the same as in RP column (4-5 column volumes) unless you are changing buffer, then you need to consider buffering capacity of the column, but all this is manageable if you know what are you doing:
http://www.sielc.com/MethodDevelopment_ ... ation.html

Most of the mixed-mode columns are less hydrophobic than RP, for example our column have C12 carbon chain. Polar ionizable groups on the surface reduce hydrophobicity of the column. You can do single, double and triple gradients with mixed-mode columns and obtain extremely sharp peaks, almost GC type:
http://www.sielc.com/application_117.html
http://www.sielc.com/application_122.html

This happens due to focusing effect which you can achieve on our mixed-mode columns.

RP columns, such as C18, are most commonly used for good reasons. They are easy to use, more familiar to most chromatographer, readily available, etc. Thus I agree with Tom that RP columns should be the first choice to consider when encountering a new separation challenge. On the other hand, C18 columns have limitations, such as not fully compatible with aqeuous mobile phase, not suited for separating highly hydrophilic analytes, and limited selectivity range. Mixed-mode columns provide remedies to these shortcomings.

In my opinion, the main benefit of mixed-mode column is adjustable selectivity, which provides greater flexibility in method development. Although mixed-mode columns have lots of potentials and can be considered (at least in theory) to be a repalcement to many other HPLC columns, considering the long history and much familarity of RP columns, I believe it is more useful for most chromatographers to use mixed-mode columns as the complementary or "niche" columns. You will find mixed0mode columns can do magics when the RP columns fail to produce desirable result.

At Dionex, we develop each mixed-mode column to address specific analytical challenges, such as Acclaim Trinity P1 (a RP/SCX/WAX trimode column) for pharmacutical counterion and API analysis, Acclaim Mixed-Mode WAX-1 for organic acids and a broad range of anoinic molecule analysis, Acclaim Surfactant for a a variety of surfactants (anionics, cationics, nonionics and amphoterics) analysis, and etc. All these applications can't be fully addressed by any other types of HPLC columns alone.

In terms of method development, good understanding of retention mechanism is essential. When using a mixed-mode column, the sparation process in the column may be a complex one, but the developed method can be very simple.

The following link and sources will provide more information on Acclaim Trinity P1:
1. http://www.dionex.com/en-us/webdocs/707 ... 239-02.pdf
2. X. Liu, C. Pohl, J. Sep. Sci. 33 (2010) 779–786.
3. K. Zhang, L. Dai, N. P. Chetwyn, J. Chromatogr. A 1217 (2010) in press.
4. B. N. Tran, R. Okoniewski, R. Storm, R. Jansing, K. M. Aldous, J. Agric. Food Chem. 58 (2010) 101 – 107.

For other mixed-mode columns from Dionex, please check: http://www.dionex.com/en-us/products/co ... 71735.html

We use mixed-mode columns more and more in our lab, mostly to be able to achieve retention of very hydrophilic molecules (and to get rid of the old ion-pair methods).

One thing to remember: you have to consider that you also may get retention of sample components that you usually don't worry about using RP. That can be excipients and components in the sample solvent. We have one method where the chloride ions in the sample caused a well retained negative peak (k' about 5) using Primesep D. It no problem move this peak to an area where it did no harm though.

XL: How much organic can the Dionex mixed mode columns tolerate.

Thanks
H

You can use any ratio of organic solvent you need with Dionex mixed-mode columns, or anywhere from 0 to 100%. Accordingly, you can use any ratio of aqueous portion from 100 to 0%.

The question is not how much mixed-mode columns can tolerate, but rather what mechanism of retention you are controlling at high organic. There is no RP/ion-exchange at very high organic.
Mixed-mode columns are stable at any organic and water concentrations, and it is not a function of the particular manufacturer, but rather of a polar groups on the surface.

When using a mixed-mode column at high organic solvent, reversed-phase retention is minimized but ion-exchange retention still exists. Conversely, using a mixed-mode column at high ionic strength and high aqueous mobile phase, ion-exchange interaction is "masked" and the main retention mechanism is reversed-phase retention. Anywhere in between, retention is governed by different combination of RP and on-exchange interactions. The goal of method development is to identify the optimal combination for the specific separation at hand.