Chromatography Theory

How to get speed in HPLC


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.


Communication problem with Chromeleon 6.8

Dionex Logo 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.

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.


High temperature vs. Ultra high pressure liquid chromatography

The last CASSS Discussion group focused on the possible advantages and disadvantages of high temperature and/or high pressure in a liquid chromatography. The Discussion group was hold as a debate – two experts against each other. The high temperature approach was defended by Nebojsa M. Djordevic (SANO CRO) and Michael W. Dong (Genetech) advocated the use of ultra high pressure in HPLC.

High temperature or High pressure?
High temperature or High pressure?

High temperature in liquid chromatography

The first speaker was Nebojsa Djordevic. First of all, he started with short introduction of the influence of the temperature on the separation in HPLC. The most important equation in the liquid chromatography – the resolution equation – is temperature dependent. Change in the temperature causes change in all three parts of the equation: efficiency, selectivity and retention.

The higher temperature also decreases the mobile phase viscosity. With lower viscosity of the mobile phase, the pressure of the system decreases and then we can use higher flow rates (= faster analysis). At elevated analysis temperature the solubility of the samples increases and it is not necessary to use high concentration of the organic modifier in the mobile phase. Thus, high temperature liquid chromatography is another step to green chemistry.

Using a high temperature liquid chromatography one has to consider also some limitations. The secondary equilibrium (pH) changes, the kinetics varies (chiral separations) and the conformational changes of the sample can occur.

Using a high temperature is not only “heating” a column. The instrumental demands have to be also considered. The heater itself can form radial and axial temperature gradients, the solvent needs to be preheated; unheated detection cell can causes the precipitation of the sample, etc. Last but not least, the column and sample stability can change significantly using a high temperature.

The main advantage of high temperature HPLC is possible control of the elution selectivity. The high temperature can switch the elution order of (critical) peak pair and help to separate compounds which are not separated at ambient temperature. As Nebojsa Djordevic rightly mentioned ‘you don’t need a hundred thousands of plates if you have good selectivity’.

Ultra high pressure liquid chromatography

The next speaker, Michael W. Dong focused on the ultra high pressure liquid chromatography. His presentation was devoted mainly on the instrumental aspect of high pressure in HPLC. According M. Dong, high pressure instruments together with a low dispersion are new platform of HPLC. Currently, all main chromatography manufacturers offer the UPLC systems with pressure limit around 80 – 130 MPa (12 – 19 000 psi).

The UPLC allows fast and selective separation with high resolution for complex mixtures, enhanced peak capacity and fast method development. On the other hand, one has to take special care about injection precision, detector sensitivity (column bleeding) and method portability. Another issues rise from the high pressure safety, viscous heating of the mobile phase and system costs.

The main application of the UPLC system is connected with the high throughput, repeatability and speed, e.g. pharmaceutical industry. On the end of his presentation, M. Dong mentioned, that HPLC systems will be fully replaced by the UPLC instrumentation.


In the following discussion the pros and cons of both high temperature and high pressure systems were compared. While the high pressure allows only increase in the efficiency (and the increase in pressure is still more the penalty we have pay with using small particles), the elevated temperature changes also selectivity and retention of the separation. And if you are able to control the selectivity (the peak resolution) you don’t need (super) high efficiency.

The significant argument for the temperature is financial expenses. The implementation of the high temperature in HPLC instrumentation can be done easily and cheaply then in the case of the high pressure application.

To conclude, the elevated temperature in liquid chromatography was slightly forgotten during last couple of years. With proper implementation, however, the high temperature can bring significant improvement of current and future separations. Cheaply.

What do you think?


Costless improvement of your HPLC instrument

The recent progress in the column development brought to the market new type of highly efficient columns. These columns can be use either with standard HPLC instruments or with the instruments allowing separations at ultra high pressure (900 MPa). The conventional size of the column (150 x 4.6 mm) has been decreased significantly. The typical size of column for ultra high pressure liquid chromatography (UPLC) is 50 x 2.1 mm with sub 2 μm ID particles.

Reduction of the extra-column volume of instruments

The decrease in the column size increases the significance of the extra column volumes – the volume of the liners between the injector and column, as well as column and detector, injector itself and detection cell. The contribution of the extracolumn volumes can be almost neglected using the conventional size of the column (150 x 4.6 mm) and flow rate as high as 1 mL/min (ok, almost neglected;). On the other hand, if we are using the identical instrument with small columns (50 x 2.1 mm) the influence of the extracolumn contribution is much higher and can devastate our efficiency and separation.

In recent paper, F. Gritti et al. compared several currently commercially available HPLC instruments in terms of the influence of the extracolumn volumes on their separation power. They found, that the only optimization of the extracolumn volume in standard Agilent 1100 HPLC system from 15.2 μl to 3.8 μl, together with the change in the volume of detection cell (13 μl to 1.7 μl) dramatically improve the columns efficiency. The average increases in the average column efficiencies were 28, 41, and 278% for the 100 x 4.6 mm, the 50 x 4.6 mm, and the 50 x 2.1 mm I.D. Kinetex columns, respectively.

Using this very simple and almost costless modification of the current instruments, the full performance of large diameter column can be achieved. However, other approach has to be applied in case of the resolution power of small diameter columns.

Sample focusing with weak solvent

In the same article, authors showed the possibility of column efficiency improvement with the injection of the weak solvent plug after the sample. After the sample injection, the weak solvent (water in reversed-phase chromatography) is injected and if there is no retention of this weak solvent on the stationary phase, the width of the sample band adsorbed at the column inlet can be reduced by diluting the sample with the weak solvent. Following picture (adapted from the discussed article) describes the whole idea.

Sample focusing with weak solvent
Sample focusing with weak solvent

With this approach, the sample dispersion on the column inlet is almost eliminated and it is possible to achieve apparent column efficiency close to the maximum possible for most columns currently available, including the short 2.1 mm I.D. columns packed with 2.6 μm superficially porous particles.

These examples show that it is possible to improve the HPLC instrument performance using very simple and costless approaches. However, the further improvement in the instrumentation (especially decrease in the instrumental extracolumn volumes) is necessary to be able to reach the full possible performance of the new, highly efficient, columns.