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?

Couple of days ago, I mentioned on Chromatographer’s facebook page article by Peter Carr about speed in HPLC published in Analytical Chemistry. There are two parts in the article: (i) critical comparison of different approaches how to reach speed in HPLC and (ii) theoretical background of speed and efficiency optimization in high performance liquid chromatography.

Today, I would like to summarize first part of the article and compare various techniques how to reach speed (and efficiency)  in liquid chromatography separations.

UHPLC – ultra high pressure liquid chromatography

Small particles + very high pressure = fast separation.  Now, ever growing family of columns with sub 2 μm particles as well as high pressure instruments are available and there is no problem to reoptimize method applied for column with 5 μm particles and used it for small particles. With decrease in particle size, the working pressure in HPLC increases significantly which might be seen as a disadvantage, because (generally) new instrument is needed.

Another problem connected with the HPLC instrumentation are extra-column volumes which become more significant with columns packed with small particles. Significant improvement of current instruments is needed.

On the other hand, even small and very cheap adjustments of your HPLC instrument might improve its separation power the separation substantially. Last but not least, there are issues connected with temperature gradients at ultra high pressure chromatography.

Temperature

High temperature in HPLC is definitely not a new idea. However, today it might be seen from another perspective. Higher temperature changes viscosity of the mobile phase hence decrease the pressure of the system or allow us to use longer columns. High temperature HPLC can be carried out using conventional HPLC systems using superior columns with no additional costs.

Additionally, high temperature also affects selectivity of separation which is impossible with working at high pressure. From practical point of view, it is necessary to preheat the mobile phase before entering the HPLC column.

Core-shell particle

Or supperficially porous particles if you like. Again, not a new idea but because of their huge improvement during last couple of years core-shell particles now dominate over the other types of stationary phases and I would say that there are the best stationary phases currently available.

Their advatnage is in low diffusion rate and (associated) very high efficiency and speed. Moreover, sub 3 μm core-shell particles can show performance similar to sub 2 μm fully porous particles but at lower working pressure. Again, one have to be aware of extra-column volumes.

Monoliths

If anything can undermine the supremacy of core-shell particles then only monolithic stationary phases (yes, I am working with monoliths;). Couple of years ago, it looked like the monoliths are best columns ever in terms of speed. However, they do not show expected column efficiency. They are better suited for high efficient separations with long columns rather then for fast HPLC analysis.

At HPLC 2010, there were introduced second generations of silica-based monoliths with improved efficiency, as well as 2nd generation of organic polymer-based monoliths with hypercrosslinked surface.

I call the later ones as superficially porous (or core-shell) monoliths. With independent optimization of both flow-through pore diameter and size and distribution of small nanopores on the surface of monolithic skeleton we were able to improve significantly the efficiency (and analysis speed) of prepared columns. I believe that such columns might play significant role in further development of columns for fast HPLC.

Chemometrics

Less obvious, but still valid argument. The proper peak recognition by chemometrics software can help to decrease the time of the analysis especially in case of coeluting peaks or not satisfactory resolution. It migh also decrease time of deep method optimization.

The role of method optimization

À propos method optimization. I would like to follow this topic in one of the next posts (knowing my posting frequency I really don’t want to say when;). But let me now cite part of the Peter Carr’s article:

For separation that will be done many, many times, use of a thoroughly optimized method can save a tremendous amount of time in contrast to the brute force approach of throwing more plates at the problem. Conversely in an assay is not going to be repeated, UPLC techniques can save a lot of time in developing optimized assays. (Anal. Chem. DOI: 10.1021/ac102570t)

Nothing to add.

Uff, long one again. I hope I might be able to increase my posting frequency. I don’t have always time to update this blog, but I am updating Chromatographer’s facebook page always when I find interesting paper. So if you are interested you might “like it” and be part of the discussion.

What do you think about speed in HPLC? Can monoliths ever overcome core-shell particles?

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?

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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?

When two do the same thing, it’s not always the same. In the last issue of LCGC North America, Ronald Majors summarized his highlights of HPLC 2010. In my HPLC 2010 flashbacks I focused exclusively on oral presentations I have attended during the meeting. Ronald Majors summarized whole symposium in a detailed and comprehensive way, including the comparison of attendance this year vs. last year, percentage distribution of individual topics, as well as plenary lectures, major applications and usage of various detection techniques.

I am not going to repeat what was written. You can read Ronald Majors’s paper yourself (and maybe compare it with my HPLC 2010 flashbacks). Majority of the meeting focused on HPLC (33% of all papers). In the group of HPLC presentations, 25% described news in the field of monolithic stationary phases (organic polymers vs. silica-based monoliths 2:1), 17% covered superficially porous particles (or shell particles, poroshell, fused-core particles), and 16% papers presented HILIC as a separation technique for polar compounds.

So, if you want to know what is going on in liquid chromatography separations, read HPLC 2010 highlights.

The Urbans on HPLC 2010

A little bit of shameless self-promotion. I am sorry about that ;-)

Ronald Majors mentioned recent progress in the field of monolithic stationary phases.  He describes both organic polymer monoliths and silica-based inorganic materials. In group of Frantisek Svec, we are preparing polymer monoliths suitable for separation of small molecules. To do that, we use postpolymerization hypercrosslinking modification to synthesize monolithic phase with small pores on the surface of monolithic skeleton. The paper describing our attempts is submitted. Once published, I will let you know for sure ;-)

Iva with winning poster and received diploma

Last but not least.

I already said it in my HPLC 2010 flashbacks, but now when it is published in LCGC it’s official even more: my wife Iva won a first prize poster for a work focusing on “Monolithic Polymer Layers for Separation of Peptides and Oligonucleotides Using Pressurized Planar Electrophoresis and Electrochromatography“. The work has been done in cooperation with University of Indiana, Purdue and authors recently published Letter in Analytical Chemsitry.

I am proud of her.

End of self-promotion, thanks for your patience ;-)

Couple of days ago I wrote about September Discussion group organized by CASSS International separation science society. As I promised I made notes and I would like to share them with you now.

Although I expected slightly different format of meeting (selection of few topics and their detailed discussion with the attendants) I was able to find some new ideas and directions in current and future analytical chemistry.

The speakers (Robert Stevenson, Tom Jupille, and David Sparkman) presented their views about new directions in analytical chemistry, liquid chromatography, and mass spectrometry.

Robert Stevenson – New technologies in analytical chemistry

Robert Stevenson started with the importance of semantic technologies and data processing and control. With emerging techniques more and more data is acquired and analyzed. In comparison to “regular” database with x and y coordinates, semantic analysis allows to add another flexible and dynamic dimension(s) using RDF framework.

Next, he focused on high content analysis – relatively young technique (6 – 7 years) connected mainly with fluorescence microscopy and further data analysis. Using this technique human stem cells can be tested and thus testing on animals avoided. The drawback is large data output and higher prize ($0.25/well and in usual experiment 10⁵ to 10⁷ wells is analyzed).

Software enabled microscopy was a last part of Robert Stevenson’s talk. With this approach correction of optical imperfection is done using software. Using this approach, fluorescent photo-activated localization microscopy enables imaging of DNA strains during the cell division. Unbelievable.

Tom Jupille – High performance liquid chromatography

Tom Jupille summarized history of liquid chromatography separation, which started in 1905 by Michael Tswett. There was not any giant step in the instrumental development during first fifty years. Columns in 1955 looked almost the same as those used by M. Tswett and used gravity as driving force for the mobile phase (although they were much bigger).

Oppositely, next 50 years changed the instrumentation completely. New techniques such as HPLC were introduced thanks to rapid development of a high quality engineering and computers technology. Then, instrumentation in 2005 looked like self-standing, robust, machines controlled with a PC producing hundreds and thousands results every day.

And 2055? Miniaturization and Black-boxes. Tom Jupille compared the evolution of chromatographic system to personal computers. Nowadays, only small group of people knows how computers work. Majority of people uses computers in their daily life but has no clue about bits, RAM or binary system.

HPLC systems

The same applies for chromatographic instruments. In the future, people are going to use LC system and they may not even notice using one. For them, it will be the only way how to reach a result– composition of the sample, level of compound of interest, sample preparation for further analysis and so on.

My (footnote) heretical question: is liquid chromatography sample preparation technique for mass spectrometry?

Future of HPLC instrument began with introduction of Dionex ICS 5000, which is a system with minimal extracolumn volumes and flexible and modular approach. In this I agree with Tom Jupille and his “As instruments evolve, they became appliances” statement. We as method development crowd should provide end-customer with required technology/instrument/column. And end-customer does not need to know value of efficiency at minimum of van Deemter curve.

Column development

The general trend in column development is reduction of particle size. From tens of micrometer on the beginning of HPLC history to current highly efficient sub 2 μm particles. The advent of ultra high pressure liquid chromatography (UPLC) is closely related to pressure issue of HPLC columns packed with small particles (high pressure is a prize for speed and efficiency).

The question is is there always need for very high efficient columns. No. We need resolution and selectivity (as Peter Schoenmakers mentioned during HPLC 2010 and Tom Jupille during his talk).

Another issue related to HPLC columns packed with very small particles is contribution of extracolumn volumes to a separation (I already mentioned it a bit). By reducing extracolumn volume you can very easily improve separation power of your HPLC system with very low or no additional cost. However, the sub 2 μm superficially porous particles are probably going to be future packing material.

Monoliths, on the other hand, have efficiency as 3 μm particles with back-pressure of particles with 10 – 15 μm. According Tom Jupille, they are not going to dominate over other separation materials mainly because of patent issues.

From my point of view, I see room for monoliths in special application using stationary phases tailored for certain separation problem with desired selectivity (as well as efficiency and back pressure). Another advantage of monoliths is easy preparation in special format of separation devices (lab on chip).

I believe that 2nd generation of organic polymer monoliths (hypercrosslinked materials) may contribute significantly as a new family of stationary phases. I call them superficially porous monoliths.

David Sparkman – Mass spectrometry as a separation technique

I had an opportunity to share a table with a last speaker – David Sparkman. He likes the Czech Republic, which he visited during an international conference a few years ago.
David Sparkman said that mass spectrometry can be considered as a separation tool since the beginning of this technique. Main improvement of MS detection is attributed to development of MS/MS detection in 1978 . Surprisingly, in 1990s the GC-MS technique almost disappeared.

Nowadays, the main driving forces in development of mass spectrometry instrumentation focus on tandem quadrupole arrangement and/or ion mobility mass spectrometry. In later case, the ions are separated not only using MS, but also according their different mobilities based on size, charge and so on.

During his talk David Sparkman described later development in the field of mass spectrometry instrumentation. He started with Waters Xero TQ with quadrupole – collision cell – quadrupole arrangement, which improve signal to noise ration and allows analysis of femtograms of sample.

AB/Sciex developed Triple TOF, which is NOT instrument with three time-of-flight analyzers, but it looks like the analysis is done using three TOF systems. Triple TOF uses 40 GHz time to digital detector which allows very high sensitivity and detection.

QIT (Thermo, I believe) is quadrupole – ion trap mass spectrometer with dual cell arrangement (low and high pressure) and shows very high resolution.

Because of time, David Sparkman only quickly mentioned other producers, such as Bruker and his maXis, Shimadzu with LCMS 8030 system or Agilent 6490 using jet-stream technology.

From chromatographic point of view, the most important characteristics for mass spectrometry are robustness, repeatability and reproducibility of chromatographic separation for subsequent analysis with mass spectrometry.

“If you do LC and don’t know MS you may be out in the cold” David Sparkman

Conclusion

During the discussion I have asked about the sample preparation. With decrease in time of anlysis (seconds, minutes) the importance of fast and robust sample preparation increases. If your analysis is 5 minutes, you don’t want to prepare your sample for one hour. According the panelist this topic needs special attention, however because of time they did not elaborate on this topic further.

I am glad to be here in Bay Area and attend such meetings. I believe that in the future CASSS Discussion groups can be webcasted on the internet (live or for download). Since then you need to trust me ;-)

I would be more than happy to discuss your opinion about future of analytical chemistry (and separation techniques in particular). Please feel free to comment on this article. If you prefer more open communication, you can use chromatographer.com facebook page.

As I mentioned several weeks ago, there was an international symposium on the separation science – HPLC 2010 – held in Boston last week. It was my second North American conference (together with San Francisco 2006) and third in total (plus Stockholm 2005).

Allow me to summarize my remarks I made during the lectures I have attended. Fortunately, I have the opportunity to see majority of my pre-selected talks as a volunteer with the microphone in presentation halls.

This post is quite long. However, I didn’t want to chop it in several different posts rather to place all the information together. One more note: your selection (and conclusions) of talks can be completely different, I was mainly visiting sessions focusing on the new columns material, columns characterization and multidimensional techniques as well as monolithic stationary phases.

Sunday

Peter Carr in his plenary lecture awarded with a Martin Gold Medal of The Chromatographic Society described the advantages of the fast second dimension in two-dimensional comprehensive liquid chromatography (2D-LC). He compared time of 2D-LC analysis in 1990 (6 hours) with the current analysis time of twenty-thirty minutes with very fast second dimension (20 seconds!). The combination of perfluorated column together with zirconia type of the stationary phase seems to be satisfactory for several different applications of real samples from corn extract to Starbucks coffee or Minnesota’s red wine.

Short time travel: Peter’s lecture was next day followed by a 2D-LC tutorial led by Dwight R. Stoll. He focused on the necessity of the 2D-LC (is it really necessary and/or better?), column selection for multidimensional techniques and the biggest problem in the field of 2D-LC: lack of 2D instruments with very low gradient delay volume. He also focused on the fraction transfer and second dimension analysis time (why the 20 s looks like good compromise).

In the second plenary lecture, George M. Whitesides describes his efforts in preparation of no or low cost diagnostic tools. Nice talk. Why are we developing separation methods with the highest selectivity, capacity, retention, efficiency … when we are not able to provide their results for majority of people? I am sure symposium such as HPLC (2010) can significantly attribute to discussion like this.

Monday

Nobuo Tanaka introduced next generation of silica-based monolithic columns. Connecting several columns together (1 – 2 m) it is possible to achieve efficiency of several hundreds to million of plates. Moreover, the separation can be done at linear velocity as high as 11mm/s.

My colleague Stuart Chambers talked about the modification of the methacrylate-based monolithic columns with carbon nanotubes or methacrylate modified fullerenes. Surface modification significantly enhanced the column efficiency and values such as 80 000 tp/m for small molecule (benzene) can be achieved using this type of modification.

Ulrich Tallarek described mathematical approach towards the characterization of 3D structure of stationary phases. From my point of view, I am really looking forward to seeing such a model for organic polymer-based monoliths (taking into account their heterogeneity).

Fabrice Gritti discussed why the shell particles are so good and if there is still space for improvement? He described the mass transfer in these particles and compared several different types of the core-shell particles.

Tuesday

Wolfgang Lindner introduced new zwitterionic type of chiral stationary phase which can be used for both weak anion and strong cation exchange chromatography only by tuning the composition of the mobile phase.

Paola Dugo mentioned recent progress in comprehensive LC in the separation of small molecules (flavonoids) as well as larger ones (peptides).

My former boss from Pardubice, Czech Republic Pavel Jandera showed how to optimize gradients in 2D-LC. The gradients in the second dimension can be described as full in fraction (0 – 100%), segment in fraction (x – y%) and continuous shifting with separate run of gradient in second dimension. Optimization of the gradient in second dimension can be described as optimization of three separated steps – isocratic (dwell volume) – gradient – and isocratic again.

Matthew Lindford presented nanodiamond modified stationary phases. The diamond (nanoparticles) was attached to the impervious core using layer by layer addition using polyaminoallyl. These column show enhanced stability, as well as decent efficiency (55 000 tp/m).

Mary Wirth described submicron colloidal crystals nanoparticles packed in capillary format with submicron plate heights. This can be one of the new/next steps in the future of liquid chromatography – packing with ultra small particles and achieving ultrahigh column efficiency. However, the whole process has to be studied more deeply.

Gert Desmed asked where is ultrahigh pressure needed and if. The first part of his talk focused on the application of several connected column to provide the desired efficiency, whereas second one discussed the possibility of the gradients at constant pressure, which significantly speed up the separations (roughly about 10 – 20%).

Susan Olesik presented probably only one talk about the thin layer separations. She described the application of electrospinned polymer as stationary phase in TLC.

Peter Schoenmakers in his first talk (substituing his student Elena) introduced application of regular HPLC column for fast size-exclusion separation of polymers in UPLC mode in less than 1 min. In this case, the extracolumn volumes play very significant role and has to be minimize as much as possible.

Wednesday

Georges Guiochon has begun his talk with historical summary of core-shell type of the particles. Their superior performance is due to the reduced heat effect, short diffusion path and subsequently low contribution of A and C terms of van Deemter equation. The columns are getting smaller and smaller and therefore the role of the instrument is more and more important (why are you using highly efficient column if you are loosing all its performance in the extracolumn connections?). On the end of his talk Georges Guiochon pointed out the importance of column packing method and its quality.

Magdalena Titrici showed the possibility of the surface modification with either NIPAM or PEG-methacrylate based monomer to achieve a thermoresponsive stationary phase. With this kind of polymers the surface can be changed from highly hydrophilic to hydrophobic one.

I missed the beginning of talk of Marja-Liisa Riekkola, however on the very end of her talk she spoke about the capillary packed with very low density lipoproteins (VLDL) “particles”. I have to check this idea again because it looks very originally.

Jesse Omamogho described the new types of core-shell particles prepared using the seeded growth method. Using this technique they are able to control both the diameter of inner core as well as the thickness of the porous layer independently. The pore size of the particles is about 90 A with surface area from 80 to 200 m2/g. On the very end of the HPLC symposium, this talk won a first prize of the Csaba Horvath Award.

Charles Lucy discussed the influence of the stationary phase hydrophilicity on the retention and selectivity of the inorganic ions.

Uwe Neue described the selectivity of the new type of the stationary phases with controlled surface charge. He introduced plots/ways how to characterize column selectivity and how to compare it with other types of the columns.

James Jorgenson, father of ultra high pressure chromatography, spoke about the columns packed with 1 – 1.5 porous and non porous particles used at very high pressure. He studied the influence of the packing slurry solvent on the quality of the column. In the following discussion, Georges Guiochon compared chromatographic particle to the city: there are cities with a lot of gates and streets which make them very easy to enter. On the other hand, there are towns with only one gate and main street. Those are difficult to enter. The particles inside the column have a same “behavior” – either it is easy to enter them (core-shell) or it is difficult (e.g. fully porous).

Anthony Edge described the usage of graphitic column at high temperature with water as mobile phase.

David McCalley focused on the relationship between the applied pressure and compound retention together with molecular structure. Main influence of the analysis pressure on the retention can be observed for ionizable compounds.

Tivadar Farkas compared the influence of the extracolumn volume on the chromatographic behavior of core-shell highly efficient columns. He claimed that with current instrumentation we are not able to fully exploit potential of such columns.

Thursday

Kazuki Nakanishi described the preparation of silica-based monoliths with improved homogeneity of stationary phases enabling high efficient separations at low back pressure. Columns prepared according a new protocol contain only 5% of solid material. However, the disadvantage is their mechanical stability. Such column can be applied either in solid phase extraction or bioreactors.

Emily Hilder presented monoliths in planar format for dried blood spot sampling.

Brett Paul introduced the organic polymer monoliths prepared inside a 1 mm ID titan tubing. He described optimization of preparation together with first results. The column provide efficiency of 50 000 tp/m. Such column can be used at high analysis temperature, e.g. 180 °C.

Peter Schoenmakers continued with the ultra pressure size-exclusion chromatography topic. He focused on the degradation of very large polystyrene standards (> 7 MDa) at ultra high pressure. Further, he described separation of branched polymers using molecular topology fraction inside the monolithic stationary phases with very narrow flow-through pores. Peter correctly pointed out selectivity is one of the main property we should focus on. With very high and specific selectivity it is not necessary to require high efficiency, especially in case of very specific and tailored separations.

Robert Kennedy described in his plenary lecture segmented flow methods for hyphenation of LC and detection techniques, as well as their application in sample handling and tailored injection.

My current boss Frantisek Svec summarized the work of our group. He mentioned hypercrosslinking modification of organic polymer-based monoliths suitable for separation of small molecules (my work), as well as results of my colleagues with modification of the monolithic surface with carbon nanotubes (Stuart Chambers), gold (Yan Xu) and hydroxyapatite (Jana Krenkova) nanoparticles.

Atilla Felinger focused on the description of the thernodynamics and kinetics of solute transfer in HPLC. His very last talk at HPLC 2010 was “spiced” with the sudden technical problem with microphone. Fortunately, the organization team worked quickly and efficiently. As a very good chromatographic column ;-)

Conclusions

So, there are my HPLC 2010 flashbacks. In summary, my feelings are that the future of HPLC separation lies between superficially porous core-shell particles for fast and highly efficient separations and multidimensional techniques for complex samples. The monolithic stationary phases can still play significant role, especially 2nd generation of monoliths with tailored surface modification providing high selectivity for compound(s) of interest. Due to their thermal stability they can be also used at very high temperatures.

I would be more than happy to read your opinion either about HPLC 2010 or my notes.

Thanks for reading.

PS: and as a very last note: the poster of my wife about pressurized electrochromatography and electrophoresis on the thin layer monolithic plates for separation of peptides and oligonucleotides was awarded with the first prize in poster competition.

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.

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