Georges Guiochon pointed out in his excelent reivew about monolithic stationary phases four directions from which we can expect a serious improvement in (monolithic) columns performance.

High temperature chromatography

High temperature chromatography, which causes a reduction in the viscosity of the mobile phase. So far, monolithic stationary phases have not yet been used at high temperatures but this is only a matter of time. High temperature liquid chromatography currently pioneered by Peter Carr and his group is going to be one of the major research areas in analytical chemistry for the next ten years. A significant reduction of analyses times by a factor between 3 and 4 is quite likely.

Increase in the pressure

An increase in the maximum pressure available to the analyst. Most commercial instruments can operate at inlet pressures of up to 40 – 50 MPa. A few of them can reach inlet pressures of 100 – 120 MPa and pumps able to reach 900 MPa are available. The use of high pressures requires far more caution than chromatographers are used to apply. This may create new, some times unexpected, safety hazards against which analysts should be forewarned. One advantage of monolithic columns is that extremely efficient columns, able to generate one or even several millions of theoretical plates could be operated with conventional HPLC instruments if long enough columns could be prepared.

Optimize the structure

A decrease in the minimum value of the height equivalent to theoretical plate (HETP) of the columns used. This will come from a reduction of the heterogeneity of the radial distribution of the flow-through pore sizes, also from a reduction of the average size of the domains of the monolithic column used and from a reduction in the variance of the domain sizes.

We have to be able to control (and suppress) monolith heterogeneity. My small prediction: one who is able to prepare the (monolithic) stationary phase with no or limited heterogeneity will be able to achieve unimaginable efficiency and column performance. Like for example homogeneous pillars.

Higher column permeability

Internal heterogeneity of organic polymer monolith

An increase in the column permeability. This requires an increase in the average flow-through pore size. Since this size is included in the domain size, this requirement is in conflict with the previous one. Both can be achieved only by decreasing the average size of the porons, which would increase the external and total column porosity at the expense of the internal column porosity and the total surface area of adsorbent in the column. There is no clear limit here but it does not seem that much can be gained. Most probably, a reduction in the variance of the domain size accompanied by an increase in the degree of radial homogeneity of the monoliths constitute the most promising avenues for the monolith designers and makers.

Solutions?

One of the possible ways how to connect these last two conflicting requirements can be preparation and optimization of hypercrosslinked monolithic stationary phases. The porous structure (flow through pores) can be optimized independently on the structure of the thin hypercrosslinked layer prepared on the surface of the monolith (micro- and mesopores). Firstly, the generic monolith is prepared (flow through pores) and then the surface of the stationary phase is modified with the hypercrosslinking reaction and thin layer of small pores is formed.  Then, only the general models connecting the preparation and modification of the hypercrossllinked monoliths with their chromatographic properties have to be developed and understand.

What do you think about these suggestions?

PS: if you haven’t done yet – look at the review written by Georges Guiochon. There is all you need to know about monoliths but were afraid to ask.

Analyst 1957, 77, 964 - 969.

Although the monolithic stationary phases suitable for separations were introduced in the 1990s [1,2,3], the idea of using a “continuous block of the porous gel structure” as stationary phase was discussed in Analyst by R. L. M. Synge, A. J. P. Martin, and A. Tiselius as longs ago as in 1952.

Both equilibrium and kinetic aspects of the molecular-sieve properties of zeolites have been studied in detail by Barrer, and it is clear that these equilibria could be used for the separation of small molecules on chromatographic columns of zeolites. Zeolites could not be used with larger molecules, as the spaces in them are too small. However, from dialysis and ultrafiltration studies enough is known of the properties of membranes and gel structures to suggest that these, though their pores could not be expected to possess the regularity of those of zeolites, could nevertheless be used for more refined separations than have hitherto proved possible. If used as powders in ordinary chromatograms, however, these substances would exhibit the disadvantages already discussed, namely that adsorption, increasing with molecular weight, would work in the opposite sense to molecular-sieve effects. An alternative possibility, suggested in discussions between Dr. A. J. P. Martin, Prof. A. Tiselius and one of us (R.L.M.S.), is to use electro-endosmosis to move a solution through a continuous block of porous gel structure. In this way the equivalent of movement of liquid through a very thick ultrafiltration membrane is attained without the necessity of great hydrostatic pressures, which would destroy the membrane structure. Here adsorption and molecular-sieve or frictional effects would all act in the same sense, tending to retard more the larger molecules.

And conclusions?

1. Smart people have smart ideas.
2. Each idea we are reading today in scientific journals may have a huge impact in comming years. At least in same way, as monoliths have changed the chromatography.

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

Many applications of porous materials in areas such as catalysis, adsorption, ion exchange, chromatography, and solid phase synthesis rely on the intimate contact with a surface that supports the active sites.

In order to obtain large surface area, a significant number of smaller pores should be incorporated into the polymer.

Types of pores

The most substantial contribution to the overall surface area comes from micropores, with sizes smaller than 2 nm, followed by the mesopores ranging from 2 to 50 nm [1,2]. Larger pores (macropores) contribute very little to the surface area. However, these pores are essential to allow liquid to flow through the material at a reasonably low pressure. This pressure, in turn, depends on the overall porous properties of the material [3].

From those kinds of pores, there are two main types in porous structure of monolith:

• the through pores enabling an easy flow of the mobile phase and
• the mesopores filled with the “stagnant” mobile phase in which the solute molecules should migrate to access the active adsorption sites.

The volume of the mesopores and the morphology of the mesopore space significantly affect the mass transfer resistance and hence the chromatographic band broadening. It also controls the phase ratio in the column and may influence the hydrophobicity of the monolithic material and the retention properties [4].

Therefore, the pore size distribution of the monolith must be adjusted properly to fit each type application. Important parameters such as temperature, composition of the pore-forming solvent mixture, and content of crosslinking monomer allow the tuning of the average pore size within a broad range, from tens to thousands of nanometres [3,5,6].

Temperature

The effect of temperature on the kinetics of polymerization allows the preparation of macroporous polymers with different pore size distributions from a single composition of the polymerization mixture. The effect of temperature can be readily explained in terms of the nucleation rates, and the shift in pore size distribution induced by changes in the polymerization temperature can be explained by the difference in the number of nuclei that result from these changes [3,6]. With higher temperature, monoliths with smaller pores are prepared.

Pore-forming solvents

Influence of the 60% (top) and 64% (bottom) of 1-propanol in the porogenic solvents on the porous properties of monolith

The choice of pore-forming solvent (porogen) is the mostly used tool for the control of porous properties without changing the chemical composition of the final monolith. In general, larger pores are obtained in a poorer solvent due to an earlier onset of phase separation. The porogenic solvent controls the porous properties of the monolith through the solvation of the polymer chains in the reaction medium during the early stages of the polymerization [3,5].

Properties of pore-forming solvents

Specific attention was paid to the design of the porogen mixtures for preparation of methacrylate monolithic columns. Ideally, this system should provide:

• preparation of a homogenous, single phase polymerization mixture from a charged, water soluble monomer and hydrophobic monomers;
• direct uniform incorporation of these monomers with widely differing polarities into a macroporous polymer monolith;
• exact control of the porosities of the monolithic material over a broad range; and finally
• facile initial washing and equilibration of the capillary column.

Extensive studies led to the development of a ternary porogen solvent system consisting of water, 1-propanol, and 1,4-butanediol in various proportions [7]. Besides the traditional ternary mixture binary porogenic solvents with only alcohols have also been adopted. Compared with ternary porogenic solvents, the design with binary ones allows for fine control of the pore size and tailoring of the specific surface area of the monolithic polymers. Monoliths prepared with binary porogenic solvents have a different pore distribution from those prepared with ternary porogenic solvents – with larger surface area and containing more of the small pores [8].

Cross-linker

In contrast, increasing the proportion of the cross-linking agent present in the monomer mixture affects the chemical composition of the final monoliths. At the same time, it also decreases their average pore size as results of early formation of highly cross-linked globules with a reduced tendency of coalesce. The experimental results imply that, in this case, the pore size distribution is controlled by limitations in swelling of cross-linked nuclei [3].

The control of porous properties of the organic polymer monolithic materials is a complex process influenced by various parameters.

References

1. K.K. Unger, Porous silica, J. Chromatogr. Library, 16, 1979, Elsevier, p. 15.
2. IUPAC, Manual of Symbols and Terminology, Apendix 2, Part I, Colloid and Surface Chemistry, Pure Appl. Chem., 31 (1972) 578.
3. C. Viklund, F. Svec, J.M.J. Frechet, U. Irgum, Chem. Mater, 8 (1996) 744.
4. D. Moravcová, P. Jandera, J. Urban, J. Planeta, J. Sep. Sci., 23 (2003) 1005.
5. B.P. Santora, M.R. Gagne, K.G. Moloy, N.S. Radu, Macromolecules, 34 (2001) 658.
6. F. Svec, J.M.J. Frechet, Macromolecules, 28 (1995) 7580.
7. E.C. Peters, M. Petro, F. Svec, J.M.J. Frechet, Anal. Chem., 69 (1997) 3646.
8. L. Zhang, G. Ping, L. Zhang, W. Zhang, Y. Zhang, J. Sep. Sci., 26 (2003) 331.

Typical structure of (polymethacrylate) organic polymer monolith

The generally accepted mechanism of pore formation in organic polymer monolihts during a typical polymerization in the presence of a precipitant is following [1,2]:

The organic phase contains both monovinyl and divinyl monomers, initiator and porogenic solvent. The free-radical initiator decomposes at a particular temperature and the initiating radicals start the polymerization process in solution.

Nuclei formation

The polymers that are formed by solution polymerization precipitate after they became insoluble in the reaction medium as a result of both their cross-linking and the choice of porogen. In this process, the monomers are thermodynamically better solvating agents for the polymer than the porogen. Therefore, the precipitated insoluble gels like species (nuclei) are swollen with the monomers that are still present in the surrounding liquid. The polymerization then continues both in solution and within the swollen nuclei.

Branched or even cross-linked polymer molecules that can still be formed in the solution, are captured by the growing nuclei and further increase their size. The nuclei enlarged by the continuing polymerization, associate in clusters being held together by polymer chains that cross-link the neighbouring nuclei.

Later stage

In the later stages of the polymerization, the size of the clusters is large enough to allow contact with some of their neighbours thereby forming a scaffolding-like interconnected matrix within the polymerizing system [3].

Control of the kinetics of the overall process through the changes in reaction time, temperature, and overall composition allows the fine tuning of the macroporous structure and provides an understanding of the mechanism of large pore formation [3,4].

References

1. J. Seidl, J. Malinsky, K. Dusek, W. Heitz, Adv. Polym. Sci., 5 (1967) 11.
2. K.A. Kun, R. Kunin, J. Polym. Sci. A1, 6 (1968) 2689.
3. F. Svec, J.M.J. Frechet, Chem. Mater, 7 (1995) 707.
4. C. Viklund, F. Svec, J.M.J. Frechet, U. Irgum, Chem. Mater, 8 (1996) 744.

As a new type of chromatographic stationary phase, monoliths have been subjected to intensive study in the last years. They differ from other supports mainly in their characteristic structure, which results in the improved chromatographic properties.

Packed (a) and monolithic (b) chromatographic column. TrAC 21 (2002) 166.

While most of the chromatographic supports are particle shaped, monoliths consist of a single piece of highly porous material. In contrast to porous particle, the pores inside the monolith are open, forming a highly interconnected network of channels. Monoliths can be prepared in various ways and can have an inorganic or an organic based skeleton [1,2,3,4,5,6].

Silica-based monoliths

The first being silica-based monolithic columns, generally prepared using sol‑gel technology. This technology can be applied to create a continuous sol‑gel network throughout the column former by gelation of a sol solution within the capillary [7,8]. Alternatively, it can be used to glue LC silica-based particles, once the capillary has been packed conventionally, producing a continuously bonded bed [9].

Organic polymer-based monoliths

The second category is rigid organic polymer-based monolithic columns, and these include acrylamide-based [10,11], methacrylate-based [12], and styrene‑based polymers [13]. The polymer network is generally formed inside the capillary by a step-wise chain polymerization reaction.

Polymerization reaction mixtures usually consist of a combination of monomers and cross-linker, initiator and a porogenic mixture of solvents. A variety of monomers can be employed to fabricate the final monolith, being both charged and hydrophilic, to generate electroosmotic flow for capillary electrochromatography, or uncharged and hydrophobic, to allow reversed-phase interactions used in HPLC. The cross-linker concentration can be adjusted to change the degree of cross-linking which influences the overall porosity. An initiator is needed to begin the step-wise chain reaction, and it is often 2,2’‑azo‑bis‑isobutyronitrile (AIBN). The polymerization can be initiated using UV light or thermal treatment.

Precipitation of the polymer occurs after it becomes insoluble in the reaction medium. Solubility is influence both the cross-linking and choice of porogen (a poor solvent for the polymer), which is commonly a mixture of alcohols.

The formation of the monolith can be achieved in-situ within either untreated or pre-treated capillaries. The pre-treatment of the capillary often involves surface preparation for the introduction of double-bond functionality, allowing covalent bonding of the monolith to the capillary wall, which is of particular importance for HPLC application where the monoliths needs to withstand high pressures.

Preparation of organic polymer monoliths

The polymerization mixture is forced into the capillary and generally initiated thermally. The reaction then continues by free radical polymerization to form a macroporous rigid monolithic polymer. The unreacted components, such as porogenic solvents, are then washed away.

References

1. S. Hjerten, J.-L. Liao, R. Zhang, J. Chromatogr., 473 (1989) 273.
2. T.B. Tennikova, B.G. Belenkii, F. Svec, J. Liq. Chromatogr., 13 (1990) 63.
3. M. Merhar, A. Podgornik, M. Barut, M. Zigon, A. Strancar, J. Sep. Sci., 26 (2003) 322.
4. H. Zou, X. Huang, M. Ye, Q. Luo, J. Chromatogr A, 954 (2002) 5.
5. A.-M. Siouffi, J. Chromatogr A, 1000 (2003) 801.
6. E. F. Hilder, F. Svec, J. M. J. Fréchet, J. Chromatogr A, 1044 (2004) 3.
7. K. Nakanishi, N. Soga, J. Am. Ceram. Soc., 74 (1991) 2518.
8. K. Nakanishi, N. Soga, J. Non-Cryst. Solids., 139 (1992) 1.
9. R. Asiae, X. Huang. D. Farman, Cs. Horváth, J. Chromatogr. A., 806 (1998) 251.
10. S . Hjerten, J.-L. Liao, J. Chromatogr. 457 (1988) 333.
11. F. M. Plieva J. Andersson, I. Y. Galaev, B. Mattiasson, J. Sep. Sci., 27 (2004) 828.
12. F. Svec, J. Sep. Sci., 27 (2004) 747.
13. H . Oberacher, C.G. Huber, TrAC 21 (2002) 166.