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?

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?

Recently, I was browsing amazon.com 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.

Materials

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.

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).

Sample

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.

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.

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.

One of the most important characteristics describing the column properties is column permeability. Term permeability refers to the column packed with a stationary phase (particles or monolith) and describes how easy flows the liquid (mobile phase) through the column.

At given pressure, the higher is flow through the column the higher is the permeability of the column. More exact definition of the permeability is described as  the volume flow of fluid per unit time per unit area per unit pressure gradient.

In case of liquid chromatography, there is limiting pressure that has practical uses, so if particle s with small diameter are used (to have as low plate height as possible), then the column with limited length and mobile phase with a low viscosity have to be used.

Column permeability calculation

The column permeability can be easily calculated from the flow and column characteristics using the embeded equation. Here the F_m is the mobile-phase flowrate, η is the mobile-phase viscosity, Δp is the pressure drop across the column, L is the column length, and r is the column inner radius.

One can see that the flow of the mobile phase through the column is directly proportional to the pressure across the column and the fourth power of the particle diameter and inversely proportional to the product of the mobile phase viscosity and the length of the column.

Example of liquids with different viscosity

UPDATED – One of the general property of the liquids is their resistance to change a form. This resistance is called viscosity and can be expressed as a resistance to flow. In case of liquids, the viscosity can be simply expressed as “thickness”. For example, water is “thin” with low viscosity, while honey is “thick” having a higher viscosity (see example on right). More technically speaking, viscosity is classically defined as the tangential force per unit area necessary to maintain unit relative velocity between two parallel plates in a liquid unit distance apart.

Viscosity unit conversion

The unit of viscosity is called the poise (P). The SI unit for the viscosity is Pascal-second (Pa.s).

1 cP = 0.001 Pa.s

Viscosity of the mobile phase

In liquid chromatography, the viscosity of the mobile phase plays crucial role. It influences the maximum pressure used. Clearly, the mobile phase with lower viscosity shows lower instrumental pressure. Thus, higher flow rates of the mobile phase can be used and lead to the shorter analysis time.

Following tables and plots show dependence of the viscosity for a acetonitrile-water and a methanol-water mixtures at different composition of the binary solvent and temperature. Generaly, the viscosity decreases with increase in concentration of the organic modifier, acetonitrile or methanol, respectively. Further, as you can see from the following figures, the viscosity decrease with higher temperature, which forces further research in the field of high temperature liquid chromatography.

Acetonitrile-water mixture

Methanol-water mixture

Source

These data are based on the table presented in Introduction to Modern Liquid Chromatography (L.R. Snyder, J.J. Kirkland, J.W. Dolan, Willey, 2010, 3rd ed.).

For different solvents look in the Mixtures of Water and Organic Compounds file in Landolt-Börnstein Database.

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.

Thin layer chromatography is probably the easiest way how to perform chromatographic separation. At least you do not need any instrument. In thin layer chromatography (TLC) the solvent flows through the stationary phase which covers the thin plate. One part of plate is submerged into the mobile phase which travel across the plate using capillary forces.

The sample spots drift towards the second end of the plate according their interaction with the stationary phases. Some of them travel faster then other, hence resulting separation occurs.

One part of thin layer chromatography uses paper as a stationary phase and is accordingly called paper chromatography.

Paper chromatography

Paper chromatography is probably the simplest form of any kind of chromatography and (probably therefore) is widely used. Most people meet chromatography in its paper version at school.

The fibres of paper (cellulose) may be used either directly as a stationary phase or can provide support for liquid stationary phase (for example water).

Sample preparation and separation development

First, you need to draw a thin pencil line at a distance of a 1 – 2 cm from the bottom of the paper. This line servers as a starting reference for the calculation of R_f values. Why pencil? Because ink contains soluble pigments that will separate when the mobile phase is running over the paper.

The solution of sample is then introduced on the line at the paper. It is important to make sample spots both as concentrated as possible and as small as possible. You can add several very small spot additions on the top of previously dried sample. Very useful for sample introduction are glass capillaries. The smaller the better.

The paper with samples should be hung from a support to allow the bottom end of the paper to be immersed into a chromatography tank – in this case, the mobile phase then travel up the paper forced by capillary action.

Alternatively, the paper can be curl into the form of tube and secured by paper clip. This tube can then stay vertically inside the tank.

In both cases, be careful and immerse paper cautiously. The mobile phase should be about 1 cm below the line with the samples. Otherwise, samples can be washed out by the mobile phase. And you don’t want it, don’t you?

Finally, lid should be placed on the tank, so the atmosphere surrounding the paper is saturated with the mobile phase’s vapor.

Before the solvent reaches the end of the paper, second pencil line is to mark the distance traveled by the mobile phase. Paper can be then removed and dried.

Identification and detection

The retention factor, R_f , which characterizes the retention of each compound is than calculated as the ratio between the distance of the spot from the beginning and the distance of the solvent front:

R_f = distance traveled by the spot / distance traveled by solvent

The R_f of compound should be the same both in case of separation of complex mixture and in case of individual compound traveling through the paper. Thus, it is possible to identify spots by their R_f values if the individual compounds traveled together with the analyzed mixture.

Retention factor values depend highly on experimental conditions and, if we want to identify spots in the mixture, we should always run mixture together with individual compounds.

In case of colored compounds it is very easy to see where the spots have traveled. A number of dyes and inks can be separated into their individual components. In this way, we might find out how many different pigments are in blue, black inks, or food dyes.

You might check the composition of ink even at home with very easy paper chromatography experiment.

Non-colored components can be detected by post-analysis derivatization. For example, amino acids can be analyzed with separated in the mobile phase formed from a 4 : 1 : 5 mixture of 1-butanol, glacial acetic acid, and water. The paper can be then sprayed with ninhydrin, which change the color of amino acids to purple and allows the identification of spots on the paper.

Thin layer chromatography

Thin-layer chromatography (TLC) is very similar to paper chromatography. The advantage of TLC is that offers better separation with higher reproducibility.

TLC uses as stationary phase solids such as alumina or silica immobilized on a glass or polymer plate. Alumina is is very polar and separation between the stationary and mobile phase may involve adsorption, partition, and/or ion exchange process. The mobile phase may be water, aqueous ammonia solution, mixtures such as an alcohol/water/acetic acid solution or other organic solvents.

Thin layer chromatography plates are developed in the same way as a paper in paper chromatography. Pencil lines are drawn above the mobile phase level and plates are placed upright in a chromatographic tank. Special care should be taken so the plates are not scratched. Otherwise it might impair the separation.

Alumina as a stationary phase is very often coated with fluorescent material which helps visualize compounds as they are separated on the plate. The compounds quenches the fluorescence, so they are visible as a black spots under UV light.

Detection in thin layer chromatography

Again, the retention factors can be calculated and applied for the component identification. TLC plates are often treated with reagents such as iodine or derivatizing agents to visualize compounds that cannot be seen by naked eye.

Thin layer chromatography is normally used for a qualitative analysis of non-volatile compounds such as pharmaceuticals or dyes. In organic chemistry, TLC is very often used to determine if synthetic samples contain impurities (single spot – pure compound, several spots – impurities).

Quantification in thin layer chromatography is difficult, since it is hard to quantitatively deposit known quantities of mixture on the plate.

to be continued …

The gas chromatography is special type of chromatography, where the mobile phase is gas, such as helium or nitrogen. The stationary phase is usually solid support covered with liquid layer.

After the sample injection the mobile phase carries the sample compounds through the column. Usually, the temperature gradient is applied and compounds are then separated according theirs boiling points.

Gas chromatography instrumentation

The figure shows typical scheme of gas chromatograph. The gas (mobile phase) flows through the column placed in the oven with controlled temperature. After the separation is finished the individual compounds elute from the column and specific detector registers signal.

Applications

Gas chromatography is very useful for the analysis of small volatile compounds with boiling points lower than 300 °C. Gas chromatography is applied in chemistry industry (especially petrochemistry) to control the quality chemical products. GC can be also used for analysis of toxic compounds, environmental analysis and so on.