It would be like carrying coals to Newcastle* to say that in Anaheim, CA, USA is the most important chromatographic meeting of the year (HPLC 2012, June 16 – 21, click for scientific program). And it is my great pleasure to announce that there are four representatives of our university and each of them is going to present an oral presentation with his results:

• my colleague Michal Holčapek will talk about Complex Lipidomic Characterization of Patients with Cardiovascular Diseases (Monday, June 18, Biomarkers, 11:20 – 11:40)
• at the same time but on Tuesday, I am going to present my results about Preparation, Characterization and Application of Hypercrosslinked Monolithic Stationary Phases with Gradient of Porosity (Tuesday, June 19, Monoliths I, 11:20 – 11:40)
• then on Thursday, my boss Pavel Jandera will focus on Combinations of Columns with Different Polarities in RP-RP and HILIC-RP Two-dimensional LC with Programmed Elution (Thursday, June 21, Multidimensional Separations II, 11:00 – 11:20)
• and right after him, my colleague Petr Česla will describe Novel Approach to Highly Efficient Fast Multidimensional Separations Employing Micellar Electrokinetic Chromatography with Gradients of Pseudostationary Phases (Thursday, June 21, Multidimensional Separations II, 11:20 – 11:40). Petr is also Csaba Horváth Young Scientist Award Nominee (fingers crossed;)

You can see that our department has quite strong participation at this year’s HPLC meeting. I hope there will be four very nice talks presented by Pardubice guys.

If you are going to be there please come and see me, you might use password chromatographer, to let me know that you are reader of this ;) I am also going to be part of volunteering squad.

See you in Anaheim!

*I hope this is a correct translation of the proverb I am trying to say;)

PF 2012

Finally! A blog update. After almost six months I have something to tell. Even though only shortly. I am going to describe my past chromatographic months in one of the next posts. Until then, I would like to wish you nice year 2012 and especially good health. Everything else will come along.

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!

HPLC 2011

Last year, I summarized my HPLC 2010 notes after the meeting. This year I would like to share with you a list of talks I am going to see. If you like, you might share your favorite talks presented on HPLC 2011 (link to program at HPLC2011.com).

Sunday – Opening Ceremony

No discussion. You have to be there and not only because there is welcome reception after that. I am looking forward to seeing both presenters: G. Guiochon and A. Perczel.

Monday

Again – (almost) mandatory plenary lectures (Gy. Vigh, G. Bonn and M. Mobidelli). After that I am going to see section focused on Particle technology with keynote lecture by F. Gritti. J. Omamogho won last year Csaba Horvath Award and I still remember his talk and nice results. I am curious what kind of talk it will be this year. Than I will probably move to Proteomics where Bas Eeltink (my former colleague from Amsterdam) is going to talk about potential of polymer monoliths in analysis of proteins isoforms. On the end again Particle technology and T. Farkas with talk about core-shell particles.

After lunch it is In memory of Uwe Neue (talks of W. Lindner, T. Walter, and M. Gilar) with transfer to Electrodriven methods where I want to see Csaba Horvath Award nominee J. Chin.

Tuesday

Almost the same situation as during Monday’s afternoon – session dedicated to the memory of Csaba Horvath with only one exception which is my colleague drom Pardubice Michal Holčapek in Metabolomics session (and I am still thinking about keynote lecture in Metabolomics presented by R. Kennedy).

Column technology it is after that with two Award nominees (M. Abi Jaoudé and Ivo Nischang) where I am very curious about my former Berkeley’s colleague Ivo’s  presentation.

Chemometrics with Microfluidics. First I would like to either J. Kutter talk about carbon nanotube based columns or Y. Vander Heyden talk about experimental design, I don’t know which one yet. Then it will be S. Fanali with monolithic frits in Microfluidcs and J. Boccard and P. Boswell as Award nominees in Chemometrics.

Wednesday

On Wednesday morning I would like to start with purification and characterization of large biomolecules topic presented by A. Jungbauer in Biopharmaceuticals and then move to Retention mechanism for talks by B. Buszewski, G. Desmet, and (Award nominee) S. Bruns.

I am not sure what presentations I am going to see during the second morning session, but it would probably be P. Haddad with recent advances in ion chromatography, V. Kertész with laser ablation and/or G. Dicinoski with talk about chromatography in the prevent acts of terrorism. The talks by U. Tallarek, A. Cavazzini and M. Trudgett will follow.

Thursday

E. Hilder with monoliths, J. Griffith with porous graphitic carbon and E. Uliyanchenko (Award nominee) with fast 2D UHPLC of polymers. All in section Advances in separation technology.

Then, there is no room for anything else than Stationary phases section. I would like to see talk of my boss Pavel Jandera in Multidimensional separations, but I have to be ready for my talk in my section (I would really appreciate if you come and see talk about hypercrosslinked monoliths).

After that I am sure I can really enjoy lunch and plenary lectures presented by P. Schoenmakers (it will be very nice and funny talk, as always), by Frantisek Svec (hyphenation of hypercrosslinked monoliths and NMR to get image of flow inside the column and to detect very fast separations of small molecules) and G. Xu about metabolomics.

And then? Closing ceremony and evening’s train back home.

As I said, I would love to read your pre-selected talks and, additionally, if you are going to be in Budapest and want to talk to me, look for a badge with Jiri Urban on it ;)

International Science Olympiad for high school students from 20 European countries. One particular example in an analytical chemistry part focuses on the iodometric titration with 3 maximum available points. The organizers decided to use “all or nothing” approach. Either you have a correct result with maximum points or you have made (even very small) mistake and there are no points for you. Nothing. Zero. For all teams.

As a member of organizers, I have attended the final discussion between the organizers and mentors from other teams. We have compared and discussed the results each team achieved and tried to find an agreement in number of points.

There was only one big problem – the above example.

The sentence I have used as a title for this post arose from a discussion with the most dissatisfied mentors’ team. How do you want to distinguish between a student with correct way of thinking vs. the guy who knows nothing?

I tried to explain our point of view. If you are going to work in an analytical chemistry lab no one will ask you about the way how you have reached the result. Only the correct answer counts. Incorrect result = no money/no promotion/… .

On the other hand, these guys are (high school) students. Best out of the best, but still students. There should be (some) incentive reward for the guy who knows what he is doing.

So, as you see there are valid arguments on both sides. I am still thinking of it and really can’t decide which approach is better.

Now, as time goes, I am slightly more convinced that there should be partial points for this example.

And what do you think?

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?

Last year, I tried to use “chromatographic” New Year’s greeting for first time. It was kind of easy.

This year, I would like to continue with this tradition and – additionally to strip presented below – I would like to wish you good health and welfare in the upcoming year. May the bad stuff from 2010 end and good persit.

Pick a good resolution for 2011

]]> http://www.chromatographer.com/pf-2011/feed/ 0 http://www.chromatographer.com/communication-problem-with-chromeleon/ http://www.chromatographer.com/communication-problem-with-chromeleon/#comments Wed, 15 Dec 2010 17:19:16 +0000 Jiri Urban http://www.chromatographer.com/?p=1001

Usually, I leave instrument’s computer up and running for long periods of time. No surprise that after several weeks there is significant amount of Windows Updates ready for installation. This week I had to reboot my system (Dionex Ultimate 3000) and during this occasion several new updated were installed.

After that I started to have problems with connection to the Chromeleon server.

And I did not know why.

The Dionex server and Chromeleon  software were running without any problems, I could open  Chromeleon browser, open and close any file and so on. But when I wanted to open panel of tabs to really control the HPLC system I couldn’t do that. The system kept screaming at me “Unable to connect to a server”. Ok, let’s check server again. Nothing. Same result.

I continued with regular try and error route:

A) Ok, let’s reboot computer once more. Twice more. Several times. Nothing.

B) I tried to manually connect the server selecting the various communication protocols. In dialogs that are used to make connections I found that computer with running instrument server does not display Chromeleon Server; instead, a red X and the phrase “The array bounds are invalid” were displayed. At least I found a “name” for an error. So next step is search through the software help. It did not find any answer.

C) I am going to ask big uncle Google. Copy and Paste “The array bounds are invalid” add “Chromeleon” and “Dionex” and “communication problem” and hit the search button. FINALLY! The first page in the results page linked to the Dionex website with a description of the issue followed by the solutions.

This website describes the background of the problem and answers with hotfixs for fast solution. Page concludes on general guidance on Windows Updates (usually there are safe, but if you might have problem with not running systems don’t do that or plan a good timing for it).

I downloaded hotfix, copied it over the suggested file and it worked. Thanks!

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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