New directions in separation science

CASSS Discussion GroupIn May, I announced that CASSS organizes a discussion group about consulting.

Today, I would like to mention next discussion group focused on New directions/developments in Separation Science.

The meeting is taking place on September 15th, 2010 in Woodfin Suites in Emeryville, CA at 6 pm.

Personally, I am looking forward to attending this meeting. The topic as well as a list of panelist (see below) are very interesting. It will be nice to hear opinions about new directions in separation science from people with different background and experience.

I have to admit that my knowledge, interest, and predictions focus mainly on liquid chromatography (and monoliths, of course;)  so I am very curious about other topics, such as sample preparation, miniaturization and/or new materials in separations, which are possibly going to be part of the discussion too (I don’t know, just guessing;)

The information from CASSS website

Today’s sophisticated separation and analytical instruments and techniques bear scant resemblance to the “absorption analysis” technique reported by M.S. Tswett in 1903.  But that same drive for innovation and improvement is alive and well in 2010.

Join us for a lively discussion on the latest trends and technical innovations presented at the Pittcon, ASMS and HPLC conferences this year.  Question our expert panelists on where the trends might be taking the industry – and what to watch out for.

One thing is constant in this field … change.  From mergers and acquisitions to plenty of new products – stay abreast of the trends that will affect you most.

Invited panlists

Registration starts at 5:30 p.m., dinner at 6:00 p.m., and Panel discussion at 7:00 p.m.

The prize for registration before Wednesday, September 8 is $35 for Discussion only and $49 for Discussion and Dinner. On-site registration are not eligible for dinner. More info on CASSS website.

Future post

I will try to remember (ie. make notes of;) discussed topics and possible conclusions and bring them to you here on soon after the meeting. Keep in touch.

PS: if you would like to be informed about new posts, you might consider subscribtion to RSS chanel or email newsletter.

Chromatography Theory

Chromatographic coffee cup – do it yourself

Recently, I was browsing products related to the chromatography keyword. To my big surprise, the number ten (at least in my results) was a “16 oz. Double Wall Insulated Tumbler with chromatography column alone – Paper Insert”.

This is what I call a present for a chromatographer!

I waited until our next order (Harry Potter Playstation 3 game for my wife) and included the coffee tumbler in the order. Partly because I didn’t (want) understand, partly because of wishful thinking but I thought that the (chromatographic) paper is inserted in between the tumbler’s walls. It wasn’t. My bad. Instead, there was inserted paper with the picture of chromatographic column (ehmm, old fashioned burette). As advertised.

Anyway, I have decided to modify the tumbler to way I see it – original tea/coffee cup with the chromatographic separation on it. My only next condition was to avoid using any laboratory equipment.

So – if you are interested – you can very easily repeat the experiment in your kitchen and prepare your own original coffee cup.

To summarize, the aim of this small experiment is to perform thin layer chromatography of black office marker on a paper as stationary phase and use this paper as an original sign of a tea/coffee tumbler. No one else will touch it and everyone will ask about the way how to do it.

Ok, that’s plan.

A little bit of theory

The black markers usually contain more then a black color with several basic colors. Therefore, the black line traced on the filtration paper immersed with one side in the mobile phase is drifted towards the other side via capillary forces. During this journey the marker’s pigments are separated into the individual colors. Pretty much as a principle of thin layer chromatography ;-) Let’s start.


For our experiment we need: black marker as a sample, filtration paper as a stationary phase and some mobile phase. Further you might need a glass, cup or pot, pencil, scotch-tape, and scissors. That’s pretty it.

Preparation of a stationary phase

As a stationary phase I have used filtration paper from our lab. This is only one violation against my no lab staff condition. The filtration paper from the coffee machine can be successfully use too. We just don’t have any. The filtration paper I have had was smaller than the paper inserted in the tumbler, thus I used two of them and cut a right size and shape according the original paper.

Home made thin layer chromatography
Original tumbler and preparation of filtration paper as a stationary phase

The reaction vessel

An empty glass. Or a cup, pot; whatever fits your paper and purpose. Better if you can have one with a lid. With the lid, vapor of your mobile phase fills vessel and speed up and improve the separation. In my case, I have used an ordinary kitchen glass (made by ikea).


As a sample I have used black Expo Vis-à-vis wet erase marker. I have tested two different markers: Expo and Sharpie. The reason why I used the Expo is that with Sharpie I didn’t get a nice separation.

Hint #1: you better try the separation before the one you want to use in your tumbler. In this case, you can select the marker you like the most.

From analytical point of view, this can be used to indentify unknown marker: just compare their traces.

Mobile phase

That’s my favorite part. As a mobile phase I have used vodka. I told you: no lab staff ;-) You can probably use any distilled brandy. I would prefer the transparent one since I am not sure about the color of (evaporated) whisky on the stationary phase paper. Since the vodka (or any kind of home made brandy) is in the range of 40 – 50% the further dilution is not necessary. In lab I would use acetonitrile : water mixture (70 : 30 or 80 : 20 ratio). The concentration composition of this mobile phase can vary depending on the desired speed and resolution of separation.

Home-made thin layer chromatography

Ok, all stuff is ready. Let’s go. First, I labeled the paper with the marker. The length of your line is up to you. You would prefer either the long line through the width of the paper or short line covering only small part of the paper. I made a 5 cm (2”) long line roughly 2.5 cm (1”) from the edge of paper. This side (under the line) is then going to be immersed in the mobile phase.

Hit #2: To help paper fits the glass I curled the paper and used hairpins to hold it. Use new ones. Otherwise you can very easily get dirty paper.

Finally, to hold the paper in a vertical position I have used a pencil. You should have avoided any touch of the paper and glass wall. The mobile phase then flows equally without any restrictions, dispersions or speed ups.

Home made thin layer chromatograpy
Development of the separation (togerther with a test trace comparing two different markers – top left)

After immersing the paper in your favorite mobile phase, the liquid starts to rise via capillary forces and takes a sample with it. The low retained colors are faster than the more retained ones and “run” towards the opposite end of the paper faster. In our case you can see almost immediately after a start quick separation of four colors: black, yellow, red, and blue. As separation continues, the colors are separated more and more and later on you can notice total separation of least retained blue color from other colors. When the mobile phase reaches the other end of the paper, the separation is done. To cover only specific space of the paper, you might wish to stop the run sooner. All you need to do is then remove paper from the glass.

Happry chromatographer with final product ;-)
Happy chromatographer with a final product ;-)

When the separation is finished, move the paper to other glass and let it dry. Your original sign of chromatographic society membership is ready for use.

Have fun and enjoy your coffee, tea or any kind of tasteful mobile phase.


About me

Every thing you always wanted to know about monoliths

… but were afraid to ask.

You don’t have to be afraid any more. Really, if you have any kind of question related to (mainly organic polymer based) monolithic stationary phases, please do not hesitate and send me the puzzle.

The quickest way is to use contact form. Alternativelly, you might sing up for my newsletter and you will never miss exclusive email-only information.

So, what are you waiting for? :)

More than decade with monoliths

I started to work with monolithic stationary phases in 2000 as a new member of the group of Prof. Pavel Jandera at University of Pardubice, Czech Republic. The topic of my master thesis was Preparation and characterization of capillary based monolithic columnsfor HPLC and at that time I was able to prepare and chracterize whole three columns in the time range of six month. Sweet beginnings ….

Half of Ph.D. study abroad

After my master study I continued as a Ph.D. student (again in the group of Prof. Jandera) and I was lucky enough to spend half of my Ph.D. abroud in the labs of well known and respected scientists (Prof. Mike Cooke at Royal Holloway University of London, Dr. Henk Claessens and Prof. Cor Koning at Technical University Eindhoven, Prof. Peter Schoenmakers at University of Amsterdam, and Dr. Didier Thiébaut at ESPCI ParisTech). You can find more information together with topics I have done abroad in my curriculum vitae.

The dreams came true

The best working view I ever had. And will have. Bay Area together with Golden Gate Bridge from 6th floor of Molecular Foundry.

Since the beginning of my work with monolithic phases I found a lot of interesting and groundbreaking articles written by Frantisek Svec at University of California Berkeley. It would be nice to spend few months in his lab were my (very deep) thoughts and wishes. After few years of dreaming and few years of finishing my Ph.D. the dreams came true and from 2009 to 2011 I could spend two very nice and unforgettable years in his group in Berkeley. Never give up your dreams!

Back to beginnings

Now I am back in Czech Republic and I try to continue in what I have learned in Berkeley and whole Europe. I hope I will be able to prepare columns with completely new properties suitable for any kind of tailored separation.

Again, I hope that dreams come true. Eventually.


Who is Mr. Monolith

I used this question as one of my opening slide when I have presented my results to a broader audience than only chromatographic people.

So if you ask who is Mr. Monolith you have to also know when you ask. If before 1990 or after that year. Because …

In past monolith was considered to be a large single upright block of stone, especially one that was shaped into a column by people living in ancient times, and that may have had some religious meaning.


Currently and in the future is monolith understand as porous rod inside the capillary, which arise during the in-situ polymerization. There are silica-based and organic polymer based monoliths, which are very easy to prepare and used like a stationary phase for HPLC and CEC.

And I have no information about its religious meaning. Yet.


The difference in internal structure for packed (a) and monolithic (b) column

The main difference (when compared to conventional particulate packing materials) is in the internal structure of monolith. Monolith is one piece of porous material which fills whole volume of chromatographic column (usually capillary). The material contains significant amount of interconnected pores which allow flow of the mobile phase through the column at very low pressure. Thus, (when simplify) we can say that monoliths might be used at higher flow-rates of mobile phase and therefore significantly decrease the separation times.

The picture on the right shows you the main difference in their internal structure for packed (a) and monolithic (b) chromatographic column (H. Oberacher, C.G. Huber, Trends in Analytical Chemistry 21 (2002) 166 – 174.).

And this is what I do

I prepare monoliths by in situ polymerization of liquid organic monomers inside the capillary and use them as a stationary phases in capillary liquid chromatography. By changing the composition of polymerization mixture I can tune the properties of resulting columns and prepare the columns tailored for each and one application – from fast gradient elution of proteins to quick separation of small polar molecules at constant composition of mobile phase.

In case you are interested you might find more information on my Research page.


HPLC 2010 flashbacks

HPLC 2010 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.


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.


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.


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.


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.


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 ;-)


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.