2-D plot shows separation of benzyl alcohol, benzene and butylbenzene using remote NMR/MRI with a monolithic chromatography column.

During the time I spent in Berkeley I had the honor to work on the beginning of the project leading to the portable system for highly sensitive multi-dimensional chemical analysis. This work included hyphenation of NMR with liquid chromatography separation using organic polymer monoliths.

I have to admit, it was my first touch of NMR ever. I knew the theory, principle and technique, but I had never worked with it. Fortunately, we were four of us, Tom and Nick as NMR guys and Stuart and me responsible for chromatography. We have used hypercrosslinked monolithic columns which already proved to be suitable for fast separation of small molecules.

Read the press release

Since yesterday, I wanted to describe the whole project with all the background, theory, results and so on. But there are people who did it before me and in much better shape than I can ever do. So if you are interested in this very nice topic, you can read article about Application of NMR/MRI to microfluidic chromatography published at the Berkeley Lab website. It describes rationale and inspiration behind the work, as well as achieved results and future plans. Moreover, you might get more information in the paper published in Anal. Chem.

LC-NMR hyphenation

Although there is (almost) nothing to add, I would like to share my view and experience I got working on this topic. First of all, capillary liquid chromatography and NMR are quite contradictory techniques. To get better results you need low injection volume in LC, but then you have no signal (low sensitivity) in NMR. The same applies with speed – you get higher efficiency at lower flow-rates of mobile phase (LC) but you are loosing signals in NMR with their slow transfer. Last but not least, all metallic parts have to be in certain distance from the NMR magnet.

Monolithic capillary column has been placed inside the magnet and connect with injector via a long fused silica capillary. First, we have started with 100 μm I.D. monolithic column and splitter who divided the flow from the pump. Later, we increased internal diameter of our monolithic column up to 530 μm which allowed increase in signal and avoid using splitter. Thus, we could connect the column with injector via 250 cm long capillary (50 μm I.D.).

We had to inject mixture of pure compounds (benzyl alcohol, benzene and butylbenzene) to be able to get any signal. The separation showed in the figure is quite fast and if there is no tailing of butylbenzene peaks it would be possible to separate these test compounds in less then 60 s.

Thanks guys

Thanks to this project I had a nice opportunity to learn something new and work with techniques and their hyphenation which open door to a future portable system for very sensitive chemical analysis. No surprise that at the end I would like to thank my co-workers Tom Teisseyre, Nick Halpern-Manners and Stuart Chambers. It was my pleasure to work with you, guys!

Generally, methacrylic acid is used as a charge-bearing agent for generation of electroosmotic flow in capillary electrochromatography. However, methacrylic acid has a significant effect on the morphology of the monolithic stationary phases based on styrene – divinylbenzene system as showed recently by group in Prague.

Figure 1 A separation of small organic molecules using poly(styrene-co-divinylbenzene) columns (A) without methacrylic acid and (B) with methacrylic acid. Mobile phase 65% ACN, flow rate, 4 μl/min; column length, 170 mm. Peaks: thiourea (1), phenol (2), aniline (3), benzene (4), toluene (5), ethylbenzene (6), propylbenzene (7) and butylbenzene (8). Figure adopted from J. Chromatogr. A 1218 (2011) 1544.

The monolithic material prepared without methacrylic acid in the polymerization mixture showed a very low surface area of 0.1 m²/g, whereas the surface area of organic polymer monolith with methacrylic acid increased significantly up to 261 m²/g. The addition of methacrylic acid in to the polymerization mixture improves also separation power of prepared monolithic columns. Figure 1 shows the separation of the mixture of small molecules on the column without (A) and with (B) methacrylic acid in the polymerization mixture.

Surface area vs. Gel porosity

My question is, which property is responsible for the separation of small molecules on the organic polymer-based monoliths? Is it surface area, as showed in discussed article or in our work concerning the hypercrosslinked materials with very high surface area? Or is it the gel porosity as suggested by Ivo Nischang? He has showed that monolithic material with very low surface area is capable of the separation of small molecules and thus the gel porosity (and pore accessibility) is probably the reason of high efficiency and good separation of small molecules.

Or is the combination of both? The pore size and its distribution? The pore accessibility? The swelling of monolith? There are still questions in the air.

The one method which might help to shine more light on this question is the inverse size-exclusion chromatography. This technique is more than suitable for the analysis of porous properties of (monolithic) stationary phases in the range of interest – micro and mesopores with size lower than 50 nm. The pore size and distribution in swollen state (in the presence of mobile phase) will be probably the most important parameter.

Maybe in the future there will be such study with more information.

What is your oppinion?

One column fits all was title of my poster presented at HPLC 2010 in Boston that described the preparation and characterization of hypercrosslinked monolithic stationary phases and their application in several chromatographic modes. We summarized poster’s results and submitted them as a paper in Journal of Chromatography A.

Hypercrosslinking modification is not a new technique. Several decades ago, Davankov prepared large surface area polymers using this approach. However, this approach is a new in the field of monolithic stationary phases. It allows us to prepare materials with both large flow-through pores and small pores on the surface of monolithic scaffold.

On the beginning of this year, we published short letter describing the application of hypercrosslinking modification of monolithic stationary phases in Analytical Chemistry.

So what exactly is hypercrosslinking modification and what is new in the newest paper?

Hypercrosslinking modification

The difference in reactivity ratios for monomers lead to porous polymer which allow hypercrosslinking. The divinylbenzene polymerizes faster then monovinyl styrene and vinylbenzyl chloride. Thus, remaining monomer mixture becomes significantly richer in the monovinyl monomers as the polymerization reaction approaches completion and affords only slightly crosslinked chains attached to the surface of polymerized monolith. After solvation with a thermodynamically good solvent, this layer can be crosslinked via the Friedel-Crafts reaction. The polymer chains become fixed in their solvated state during the reaction thus forming pores that persist even after the solvent is removed.

Following scheme shows each step of hypercrosslinking modification.

Hypercrosslinking modification step by step (click for large image)

As I said, hypercrosslinking allows to prepare monolithic materials with both large and small pores. With this approach we can overcome the usual problem of organic polymer monolithic stationary phases – their weak separation power in terms of separation of small molecules (although, I have to say that other directions, such as short time of polymerization, provide also monoliths suitable for these kind of separations).

The presence of small pores in the monolithic material enhances significantly its surface area. Using this approach we were able to prepare monolithic materials with surface area higher than 600 m²/g.

Polymerization mixture composition

In the first paper about hypercrosslinked monoliths, we have shown that the final properties of hypercrosslinked monolithic stationary phases strongly depends on the composition of the polymerization mixture. In the new one, we studied the influence of the polymerization mixture composition more deeply using mixture design approach. We systematically varied the composition of the polymerization mixture and compared the resulting properties (efficiency and porosity) with concentration of individual compounds in the mixture.

As expected, we found out that extend of hypercrosslinking depends on the percentage of divinylbenzene in the monomer mixture, which controls the number and length of hypercrosslinkable loose chains, and the ratio of functional monomer vinylbenzyl chloride and inert styrene that affects the frequency of reactive sites along these loose chains.

Time and temperature of hypercrosslinking modification

We tested influence of time and temperature of hypercrosslinking modification. There is no change in column mesopore porosity or efficiency after 2 hours of modification, which agrees with previously published data.

In terms of temperature, the concentration of mesopores inside the hypercrosslinked monoliths as well as an efficiency of the column increases with increase in modification temperature.  However, there is no increase in these properties with temperatures higher than 90°C. Thus, the best columns are prepared with hypercrosslinking modification for 2 h at 90°C.

Mobile phase composition

Since the beginning of this project, we were facing the problem with peak tailing, especially for more retained compounds. Then I found in the literature (and here), that peak tailing is quite common problem for styrene-divinylbenzene type of the stationary phases.

This issue can be solved by adding thermodynamically good solvent in the mobile, such as tetrahydofuran (THF). With higher concentration of THF in the mobile phase the peak shape improves and retention decreases. The optimum composition of the mobile phase was found to be 20% water, 20% THF and 60% acetonitrile.

Effect of temperature

High temperature liquid chromatography is experiencing kind of come back during last couple of months/years. We tested influence of higher analysis temperature on the separation power of hypercrosslinked monoliths. With higher temperature the separation time of six alkylbenzenes (benzene through amylbenzene)  decreases from 8 minutes at 20 °C to less then 4 minutes at 80°C.

Analysis temperature has also positive effect on the efficiency of the (hypercrosslinked) columns. The minimum of van Deemter curve for benzene at optimized ternary mobile phase decreases from 28 μm at 20 °C to 16 μm at 80 °C.

Sample loading

Couple of years ago, I have studied effect of injected volume and mass on the efficiency of the monolithic columns (silica-based Merck). At that time, I did not find any significant decrease in column performance with higher injected volume or mass. It was part of another study and we never published those data but since then I always thought something in the sense that it is very difficult to overload monolithic column.

I have to say I would never believe that the injected concentration might play significant role in capillary format organic polymer monoliths. It did.

With decrease in injected amount of benzene the column efficiency increased significantly. Keeping all conditions constant and changing only the amount of benzene, the efficiency increases twice with decrease in the mass from 17.5 to 0.14 pg.

Over 80 000 theoretical plates/m

The van Deemter curve for benzene (0.14 pg) measured at 80°C using optimized ternary mobile phase (20% water, 20% THF and 60% acetonitrile) shows the minimum at 12 μm. This value corresponds to the column efficiency 83 200 theoretical plates/m and represents the highest column efficiency found for a organic polymer monolithic column used in isocratic mode.

Optimized separations

Taking into account all previously mentioned steps of optimization (polymerization mixture composition, hypercrosslinking time & temperature, mobile phase composition, and analysis temperature) we are able to show very fast and efficient separation of small molecules.

Our testing mixture contains small alkylbenzenes (benzene, toluene, ethylbenzene, propylbenzene, butyl benzene and amylbenzene) and we are able to separate them in less then 2 min at elevated temperature. Moreover, this type of the columns proved to be successful also in separation of peptides in gradient mode.

Separation of small molecules at a temperature of 80°C using the column hypercrosslinked at 90 °C for 2 h and the ternary mobile phase. Conditions: Column 100 μm x 130 mm; mobile phase 20% water, 20% tetrahydrofuran, 60% acetonitrile; flow rate 0.5 μL/min; UV detection at 254 nm; back pressure 26 MPa. Analytes: uracil (1), benzene (2), toluene (3), ethylbenzene (4), propylbenzene (5), butylbenzene (6), pentylbenzene (7).

Size-exclusion chromatography

Last but not least, the advantage of hypercrosslinked monolithic stationary phases is significant amount of small pores in their pore size distribution. Thus, they can be used for size-exclusion type of separations. Indeed, we were able to separate four polystyrene standards and toluene in less then 10 minutes using two hypercrosslinked columns connected with zero-volume union (total length 670 mm).

It is the first demonstration of the use of monolithic column in size-exclusion chromatography of polymers using an organic solvent as the mobile phase.

And I have to say – finally! – because I already tried to prepare such a column couple of years ago.

Uff, that is it. Long post. If you are interested in hypercrosslinked monolithic stationary phases you might either read the paper published in Journal of Chromatography A or download the poster I presented at HPLC 2010 in Boston (pdf, 5.7 MB).

What do you think about hypercrosslinked monolithic stationary phases? Do you think that this 2^nd generation of organic polymer monoliths can catch up current highly efficient columns with superficially porous particles?

If you like this post or maybe even whole website you might consider to subscribe to RSS channel or my newsletter.

Today, I would like to describe my favorite chromatographic books: from one I bought even before I (really) knew what chromatography is to one which has chapter with my name on it.

Úvod do vysokoúčinné kapalinové kolonové chromatografie

My very first HPLC book

I am sorry to all of you who does not understand Czech language. This is my very first chromatographic book and I bought it even before I knew the term “chromatography” itself. The book was written by prof. Jandera and prof. Churacek and the name of the book in English means “Introduction to high performance liquid column chromatography“. The book was published in 1984.

It was on the end of my first year at university. I walked aimlessly through the library shop and in the corner I found shelf full of these books. They were already a little bit damaged and each one of them costs less than a big beer (which is actually the cheapest drink you can get in almost any restaurant in Czech Republic). I had no idea what chromatography means, who are the authors and what is going to be my main direction during years at university. So I bought it.

In couple of months I met chromatography again – during our analytical chemistry II lessons. In that time, I started slowly eplore the beauty of (liquid) chromatography separations and moved my attention from analysis of biological materials (which was my main direction) towards analytical chemistry itself and particularly liquid chromatography.

Later on, I was lucky enough to be part of the class when founder of chromatography techniques in Czech Republic – Prof. Churacek – gave  lessons during his last year before retirement. So there is no surprise, that I asked Prof. Jandera if there is a space for me in his group – there was and since then I am part of his group at University of Pardubice, Czech Republic.

And the book I am talking about now was always with me, whenever I was working with chromatography abroad.

Actually, I have it on my desk even now.

HPLC Columns: Theory, Technology, and Practice

HPLC Columns

My second HPLC book in the list is HPLC Columns: Theory, Technology, and Practice written by Uwe Neue. This book describes thoroughly a theory of chromatography, columns packing, characterization, chemistry, selection, and maintenance. Large part of the book is devoted to individual modes of liquid chromatography, such as normal and reversed-phase, size-exclusion, hydrophilic interaction, and ion-exchange chromatography.

I still remember reading the parts about methacrylate-based packing, few paragraphs about monolithic stationary phases (page 72;) and trying to dip more and more in a liquid chromatography techniques and separations. Sweet first year of my PhD.

What I especially like on Uwe Neue’s book is its easy to read style and the way how he explains the problem. Reading the book I have feeling that I am on his lecture or (even better) listening to him.

Monolithic Materials: Preparation, Properties, and Applications

Bible of monoliths. Ok, let’s at least call it a fundamental book in area monolithic stationary phases edited by Frantisek Svec, Tatiana Tennikova and Zdenek Deyl. It took me while before I was able to look inside this very first book describing preparation, characterization and application of continuous porous stationary phases. Finally, I was able to borrow it from library of Eindhoven’s Technical University during my stay there. I immediately made a copy of first chapters focusing on organic polymer monoliths and read them the same evening.

There are two big advantages of this book: First, it was first. I don’t think I have to write more about it. Secondly, it describes the monolithic stationary phase from A to B. There is a description of all main types of monoliths, their preparation techniques, properties, and characterization: organic polymer-based monoliths, silica-based monoliths, ring-opening metathesis polymerization, water-soluble monomers-based monoliths,  polysaccharide materials, and high internal phase  emulsion materials, just to name a few.

Moreover, application description spans from separation of small molecules, through peptides and proteins to DNA and large polymer standards.

If you are new in a field of monolithic stationary phases, this book gives you nice overview of possible materials and their application in the separation you need.

Introduction to Modern Liquid Chromatography

Introduction to modern liquid chromatography

3rd edition of this introduction with almost 900 hundred pages covers probably all possible questions about theory of liquid chromatography and its application. Authors Lloyd R. Snyder, Joseph J. Kirkland and John W. Dolan focus on theory, instrumentation (detection, column, troubleshooting), method development and validation, and sample preparation. Of course, there is a deep description of individual modes of liquid chromatography, as in the case of Uwe Neue’s book: normal phase, reversed-phase, ion-exchange, size-exclusion, chiral separations, and preparative chromatography.

Individual chapters are divided according the type of sample and/or technique used. So, for example, you can find information about hydrophilic interaction chromatography (HILIC) as a part of chapter Normal phase chromatography as well as Separation of peptides and proteins.

Monolithic Chromatography and its Modern Applications

Monolithic chromatography

Reading all these books I always thought Maybe once I can have a chapter in such a book. And it happened. Roughly three years ago editor Perry Wang contacted Pavel Jandera with question about his contribution to book about monolithic stationary phases and its modern applications. We extended our review about polymethacrylate monoliths dedicated to Frantisek Svec on the occasion of his birthday and prepared chapter for forthcoming book.

In comparison to the first book I mentioned couple of paragraphs ago, this one does not cover such a broad range of different monolithic materials. It describes organic polymers, as well as silica-based monoliths, further ring-opening metathesis polymerization and monolithic cryogel beds.

The description of analysis of pharmaceutical-, ionic-, and phytochemicals, amino acids, and DNA and viruses separations is in an application part of the book.

I am especially looking forward to reading chapter about hyphenation of monolithic columns with chemiluminescence detection, because it reminds me time I spent in Paris working with supercritical fluid chromatography and chemiluminescence detection in analysis of crude oil residuals.

The book is now available for pre-order on amazon, it should be published very soon published.

What are your favorite books about chromatography?

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.
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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.