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.

Liquid chromatography offers numerous possibilities how to separate samples of interest. By changing the composition of mobile phase and/or character of stationary phase the separation efficiency and selectivity can be completely change. Read more about main liquid chromatography modes.

Size-exclusion chromatography (SEC)

Size-exclusion chromatography (also known as gel permeation chromatography, GPC) is separation technique, where no interaction of the analyzed sample and stationary phase takes place. The sample is separated only according size (related to the molar mass) and pore characteristics of the stationary phase.

Because of their size, large molecules are not able to enter small pores and are excluded from the column even before the front of the mobile phase. On the other hand, small compounds can penetrate in all small pores and elute later. Elution volume of very small compound (such as toluene) is than equal to the column hold-up volume.

In size-exclusion chromatography, usually all compounds are eluted in the elution window created by the very large sample with molar mass about 2 – 3 000 000 and very small compound such as toluene.

Applications

Size-exclusion chromatography (SEC) is usually used for separation and characterization of synthetic and natural polymer. In polymer chemistry is useful for determination of molar mass of synthesized polymers.

SEC can be used also for characterization of the porous properties of chromatographic column. Then, we are speaking about inverse size-exclusion chromatography (ISEC).

Normal phase chromatography (NP)

In one of the oldest chromatographic technique normal phase liquid chromatography (NP) the stationary phase is polar and the mobile phase non-polar. Silicagel, aluminuim oxide or polyamide are usual materials used as the stationary phases in normal phase liquid chromatography. Non-polar hydrocarbons such as hexane, heptane serves as mobile phase.

Retention is controlled by the surface area of the sorbent and polarity of the sample. More polar samples elute later (because of higher interaction with the polar stationary phase).

Reversed-phase chromatography (RP)

As the name suggests, the stationary phase – mobile phase arrangement is reversed to the normal phase chromatography. From my point of view, the normal phase chromatography names normal only because of the earlier development. Otherwise, the reversed-phase chromatography (RP) dominates in the liquid chromatography separations.

The compounds in RP are separated according the polarity – more polar elute first.

The stationary phase is non-polar. The majority of RP stationary phases is polar silica gel modified with the non-polar groups – usually alkyl chains. These alkyl chains have different length (4 – 18 carbon atoms) and functional end groups (amino, cyano, aromatic, …).

Usually, only 4 various solvents and their combinations are used in RP liquid-chromatography: acetonitrile, methanol, tetrahydrofuran and water.

Reversed-phase liquid chromatography can be used for endless different applications in pharmaceutical industry, biochemistry, polymer analysis to name a few.

Normal phase vs. reversed-phase chromatography

Are you always confused which one is which one? Usually, I had a problem to remember that in normal phase chromatography the stationary phase is polar and mobile phase non-polar, while the stationary phase is non-polar and mobile phase polar in reversed-phase chromatography.

You don’t have to remember all these differences. Only you have to remember, that -as mentioned earlier – there are only 4 solvents in reversed-phase chromatography (ACN, MeOH, THF and H2O). If you remember this you won. These solvents are polar, therefore the stationary phase has to be non-polar and therefore the chromatography uses reversed-phase. It always worked for me.

Ion exchange chromatography (IEX)

The retention in ion exchange chromatography is based on the ionic interaction between the ion on the surface of the stationary phase and oppositely charged sample ions. It can be used for both, cations (cation exchange chromatography) and anions (anion exchange chromatography).

Applications

Very common application of ion exchange chromatography is separation of proteins. These compounds have functional groups which can be positively or negatively charged. Each protein has a different charge in certain mobile phase and therefore can be separated from others. Very important characteristic of the mobile phase is its pH which controls the charges of proteins.

Affinity chromatography

Another technique very useful in separation of biological samples. In affinity chromatography the stationary phase contains functional groups with very specific reaction to the analytes. The general example can be antigen – antibody couple.

The specific reaction with the sample allows very selective separation of target compound.

Chiral separations

In chiral chromatography the compounds are separated according their chiral activity – chirality. Human hands are nice example of the chirality – same structure, mirror image.

The stationary phases in chiral chromatography have chiral selector on the surface, such as cyclodextrines, vancomycin or crown ethers.

Applications

Chiral chromatography dominates in pharmaceutical industry. Generally, only one chiral compound has any biological activity, whereas the other not. Therefore, it is very important to separate these two compound.

In liquid chromatography liquid mobile phase flows through the column with stationary phase. The main principle of separation remains the same.

Compounds have different affinity to the stationary phase and are separated while flowing through the column. The compounds separated with liquid chromatography are disolved in the mobile phase. They have lower difussion coefficients than gaseous compound separeted with gas chromatography.

Except thin layer chromatography, majority of liquid chromatography is performed in high-pressure arrangement. The liquid is pushed through the column using high pressure pumps. In this case we are speaking about high pressure liquid chromatography (HPLC).

According the polarity of the mobile and/or stationary phase, the liquid chromatography separations can be divided in numerous methods for different kind of samples.