Because of lack of small pores it is difficult to separate small molecules with polymer monoliths in isocratic mode. We have prepared monolithic capillary columns and then hypercrosslinked them to afford a monolith containing an array of small pores [1].

This monolithic column affords good separation of uracil and alkylbenzenes in isocratic mobile phase mode (a column efficiency as high as 73 000 plates/m was determined for uracil) and also proved useful for separations in size exclusion mode.

Organic polymer monoliths and small molecules

Compare to silica based monoliths, porous polymer monoliths contain very small or even no concentration of small pores in their porous structure. Therefore, they exhibit much smaller surface areas (tens of square meter per gram) and usually are not suitable for separation of small molecules. Several approaches were explored to improve this drawback of organic polymer monoliths: copolymerization of dimethacrylates differing in the length and branching of the fragment connecting the polymerizable units[2]; the termination of the polymerization reaction at an early stage [3,4] to achieve large surface areas; and the use of high polymerization temperatures [5,6].

However, it has always proven difficult to prepare polymer monoliths possessing both large through pores and a multiplicity of small pores in a single step and alternative approaches needed to be developed.

Hypercrosslinking modification

Separation of uracil (1) and small alkylbenzenes (2-7) with organic polymer monolith. See Ref. 1 for more details.

Hypercrosslinking, pioneered by Davankov several decades ago [7-10] enables the preparation of large surface area materials from preformed polymer precursors. The original implementation used linear polystyrene, which was cross-linked via Friedel-Crafts alkylation to afford materials containing mostly small pores [11].

The typical porous monolithic structure consisting of interconnected microglobules results from phase separation during polymerization of a mixture of monomers and porogens. For poly(styrene-co-vinylbenzyl chloride-co-divinylbenzene) monoliths less than ideal reactivity ratios for monomers such as styrene, chloromethylstyrene, and divinylbenzene lead to polymer microglobules amenable to hypercrosslinking. The divinyl monomer polymerizes faster, and the remaining monomer mixture becomes significantly richer in the monovinyl monomers as the polymerization reaction nears completion. This mixture then affords only slightly cross-linked chains attached to the surface of highly crosslinked microglobular scaffolds. When the pores are filled with a thermodynamically good solvent such as 1,2-dichloroethane, this surface polymer layer is solvated.

Capillary liquid chromatography

The precursor column performs poorly as all alkylbenzenes are less retained and eluted in a single broad peak. In contrast, baseline separation of all alkylbenzenes is obtained with the column after hypercrosslinking (see Figure). On the other hand, gradient separation of the proteins is better on the non-modified column because of negative effect of the small pores on the gradient separation [12]. Finally, because of significant concentration of small pores, these columns can be used for separation of polymers in size-exclusion chromatography.

Our work clearly demonstrates the possibility of postpolymerization hypercrosslinking of the monolithic stationary phase to afford columns for efficient isocratic separation of small molecules in reversed phase and polymers in size exclusion modes.

References

1. Urban, J., Svec, F., Fréchet, J.M.J. Anal. Chem. 2010, 82.
2. Xu, Z., Yang, L. and Wang, Q. J. Chromatogr. A 2009, 1216, 3098 – 3106.
3. Wang, Q., Svec, F. and Fréchet, J. M. J. Anal. Chem. 1995, 67, 670 – 674.
4. Trojer, L., Bisjak, C. P., Wieder, W. and Bonn, G. K. J. Chromatogr. A 2009, 1216, 6303 – 6307.
5. Peters, E. C., Svec, F. and Fréchet, J. M. J. Adv. Mater. 1999, 11, 1169 – 1181
6. Meyer, U., Svec, F., Fréchet, J. M. J., Hawker, C. J. and Irgum, K. Macromolecules 2000, 33, 7769 – 7775.
7. Davankov, V. A., Rogozhin, S. V. and Tsyurupa, M. P. Macronet Polystyrene Structures for Ionites and Method of Producing Same. U.S. Patent 3,729,457, April 24, 1973.
8. Pastukhov, A. V., Tsyurupa, M. P. and Davankov, V. A. J. Polym. Sci., Polym. Phys. 1999, 37, 2324 – 33.
9. Davankov, V. A. and Tsyurupa, M. P. React. Polym. 1990, 13, 27 – 42.
10. Davankov, V. A., Tsyurupa, M., Ilyin, M. and Pavlova, L. J. Chromatogr. A 2002, 965, 65 – 73.
11. Tsyurupa, M. P. and Davankov, V. A. React. Funct. Polym. 2006, 66, 768 – 779.
12. Urban, J., Moravcova, D. and Jandera, P. J. Sep. Sci. 2006, 29, 1064– 73

Dr. J. Kirkland during the award talk

Yesterday, I had a great opportunity to participate in Discussion group organized by the An International Separation Science Society (CASSS). The main topic was the Scientific Achievements Award for one of the founders of modern HPLC – Dr. Jack Kirkland.

Jack Kirkland is the inventor of superficially porous particle stationary phases. These particles have solid core covered with small layer of porous nanoparticles. Maybe you know them as Poroshell or Halo columns. Columns packed with these particles show extremely strong separation power for different kinds of applications. In the past, I had a opportunity to compare various types of chromatographic stationary phases (including totally porous, non-porous, superficially porous particles and monolithic stationary phases) and I have to confirm their strengths.

In his very inspiriting presentation Dr. Kirkland mentioned not only history of development but also current state of the art and possible future steps for superficially porous particles as stationary phases. Do you know, that the size of the Halo particles (2.7 μm) is chosen according the theory calculations (the highest achievable efficiency for certain conditions) as well as because of the end frits in the column (the holes in the frits are 2 μm wide so the particles with 2.7 μm i.d. can’t go through them therefore same frits as for 5 μm i.d. particles can be used)?

One of the most important message in the presentation was that in chromatography the separation is always a compromise. Either you are looking for high throughput (then your choice will be probably UPLC or monoliths) or you are looking for the best selectivity and peak capacity (and you are going to choose porous particles). I know, this paragraph is very schematic conclusion, however it is usually like this. You have to always choose what you want and what is your goal in separation and to separate.

I was also happy to meet another founder of the (high performance) liquid chromatography: Dr. Lloyd R. Snyder.

Superficially porous particles with thicker outer shells were used extensively for liquid-liquid chromatography [1] and as the support for early bonded-phase packings in reverse phase HPLC [2].

Structure of particles

Nowadays superficially porous particles typically have a 5-µm solid core and a ~ 0.25 – 1 µm thick outer shell with 30-nm pores. But, the thinner the shell, the smaller is the particle surface area and the less is the sample loadability of these particles. Therefore, there must be a compromise between the thickness of the sh

Scheme of superficially porous particle

ell to obtain good separation efficiency and good sample loadability. Thin porous shells lead to low surface areas, low retentivity, and low sample loadability; thicker shells of higher surface area produce higher retentivity and higher sample loadability.

Advantages

Superficial porous particles have unique characteristics that favour certain applications. Because of their size, macromolecules have very poor diffusional qualities. Therefore, the advantage is significant for the very short diffusional path lengths within the porous shell on the surface of superficial porous particles. Compared to conventional totally porous particles of the same size, superficial porous particles show improved mass-transfer kinetic properties that permit more rapid separations of macromolecules [3].

References

1. J. J. Kirkland, Anal. Chem., 41 (1969) 218.
2. J. J. Kirkland, J. J. DeStefano, J. Chromatogr. Sci., 8 (1970) 309.
3. J. J. Kirkland, Anal. Chem., 64 (1992) 1239.

Non-porous particles (crawfordscientific.com)

It is known that the kinetics of mass transfer in wide pore bonded silica can be slow, because of restricted intraparticle diffusion and, furthermore, remaining active surface sites can give rise to undesired interactions.

All together, these effects cause additional peak dispersion in high performance liquid chromatography and often considerable loss in recovery of biological activity [1].

Particle’s porosity elimination

A clear way around this dilemma is to eliminate the porosity, which minimizes the pore diffusion and mass transfer resistance (longitudinal diffusion) effects. The diffusion paths that analytes must traverse between stationary phase and mobile phase are very short with nonporous media and column efficiency essentially becomes independent of the flow rate [2]. Commercially available HPLC columns with nonporous stationary phases are stable at the low pH and high temperature [3].

Because of the reduction in surface area, nonporous supports exhibit much lower retention times for the same organic modifier content compared with porous columns [2]. The extremely low external surface area of nonporous supports can be regarded as a drawback since it leads to a considerably lower loadability compared with porous materials [1].

Drawbacks

Disadvantages of non-porous silica packings are that the column length is restricted because high pressure is required for an adequate mobile phase flow and it is difficult to pack a column densely and homogenously [4]. Guiochon and Martin [5] indicated that it would be almost impossible to use a high-efficiency HPLC column packed with less than 1-µm diameter particles. This suggests that the decrease in the particle diameter reaches a limit if particles are packed into columns directly.

References

1. M. Hanson, K. K. Unger, LC-GC Int., (1996) 741.
2. T. J. Barder, P. J. Wohlman, C. Thrall, P. D. Dubios, LC-GC Vol. 15, No.10 (1997) 918.
3. R. Ohmacht, I. Kiss, Chromatographia, Vol. 42, No. 9/10 (1996) 595.
4. H. Giesche, K. K. Unger, U. Esser, B. Eray, U. Truedinger, J. N. Kinkel, J.Chromatogr., 465 (1989) 39.
5. G. Guiochon, M. Martin, J. Chromatogr., 326 (1985) 3.