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.

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 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.

Discussion

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?

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

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.

I had to solve connection problem in between the Bruker MS and Agilent LC (Agilent shutdown). On the very end, I found out there was no problem in their mutual communication. However, it shows me the future of the instrumental services. WebEx communication.

WebEx is software delivered as a service which you can use it from any computer with an Internet connection. WebEx combines real-time desktop sharing with phone conferencing, so everyone sees the same thing as you talk. And that is exactly what happened.

I asked people from Bruker for advice with my (instrument;) communication problem and we scheduled the WebEx seminar (let’s call it seminar). At exact time I connected to the website they sent me and our online communication started.

For me, it was brand new experience. Ok, I have to say I have no problem with online communication (chat, blogs, social media, …) but it was for first time for me to join the online communiation because of the problem with chromatographic instrument. And I found it very useful.

The online comminication

• saves cost expenses – we all know that it is not always necessary to set up service visit,
• saves time – very important, the seminar can be scheduled on every possible time, which meets requirements of both side
• enhances productivity – majority of the problems can be solve with some kind of advice

I know, all this can be done also using the phone. And for sure, phone is very useful. But using the online communication, you can allow the servicing company the full control over your instrument computer and you don’t have to worry about (almost) anything.

I had this experience with the Bruker company. I am sure others companies offer the same service (or will offer very soon). I belive, that online service is the future of the instrumental service and that majority of the troubleshooting will be solve in this way.

Agilent 1100

From time to time, we all face problems with HPLC instruments. Then, we all search the internet for the solution for our problem. In this troubleshooting section I would like to point out my problems with instruments and their solutions. Hopefully, it can help also you.

Recently, I was asked to run the HPLC-MS instrument which was off for several months. The LC part was Agilent 1100 and MS was from the Bruker (HCT+). The instruments themselves worked fine, but there was a problem with their connection. At least I thought so. The Agilent HPLC went always to power off mode after couple of minutes. No matter what I have done, no matter what I have tried. It always went to shutdown mode.

As I said, I thought the problem is in their internal (software) connection. The Bruker HyStar software was started by the ChemStation. Because of this I have contacted Bruker company and asked them about the problem. I will speak about the webex communication sometimes in the future, but I have to say it was a nice and new experience. The guy from the Bruker told me, that there is no problem with the connection at all. Therefore I have continued to ask – Agilent this time.

They told me, that the problem I am describing can be caused by the instrument degasser. After the long time, when the instrument is not used at all, the degasser can be broken. It is still working, still degassing the mobile phase, but it can send the wrong input to the instrument and switch it off.

So, if you have a problem with the Agilent instrument (1100) which is going to the power off mode without any reason, check the connection on the back of the instrument. If there is a line between the degasser and the main unit of the instrument (usually pump, but can be autosampler too) than disconnect this line. It is probably different type of connection than the LAN type.

It is probable, that the (autosampler) control light will go red after while. But as soon it can not comunicate with the instrument, it cant tell that something is wrong. And you have still a lot of time to contact the company service to solve the problem with the degasser. Alternatively, you can ultrasonicate the mobile phase or degass it with the stream of helium.

Calibration curve in inverse size-exclusion chromatography (Ve/Vc - elution volume of the polymer divided by the volume of the column, log Mr - logarithm of (polystyrene) molar mass)

The inverse application of the size-exclusion chromatography (SEC) concept, inverse size-exclusion chromatography (ISEC) [1], utilizes a set of molecular probes with defined sizes to determine pore dimensions, and is also referred as chromatographic porosimetry [2].

ISEC provides an alternative to mercury porosimetry or nitrogen adsorption for the determination of the pore size dimensions and the surface area of chromatographic stationary phases. This technique was developed by Halász [3], and has subsequently been extended and refined [4,5,6].

ISEC methodology has been applied to the characterization of a variety of chromatographic stationary phases, including silica [4,5,7,8], silica modified with bonded-phases [9,10] or coated with formamide [11], alumina [8], series of carbohydrate-based size-exlusion gels [6] and synthetic polymer-based adsorbent [12]. The non‑destructive nature of ISEC is and advantage also in structural characterization of monolithic columns [13].

In comparative studies between porosimetry techniques and ISEC, the ISEC method was perceived to require fewer assumptions than mercury porosimetry (e.g., contact angle and surface tension) [5], and to be superior to nitrogen adsorption for following the changes in the surface area, pore volume and pore dimensions that resulted from the grafting of polymeric coatings onto silica [7].

Advantages of inverse size-exclusion chromatography

ISEC has a number of advantages over alternative methods. Column experiments with intact samples packed in a bed can conserve sample integrity and are easy to carry out, as proposed to the special sample preparation procedures in electron microscopy. No other additional equipment other than a chromatography system is necessary for ISEC, so it is relatively inexpensive and convenient.

Operating conditions such as high pressure, low temperature and drying conditions, which are involved in gas sorption or mercury intrusion, are not imposed in ISEC. Experimental conditions similar to those in normal operations result in less significant morphological changes, thus assuring structural information that is relevant to properties of functional interest.

The working pore dimension range of 1 – 400 nm attainable by ISEC [3], which includes resolution not achievable by mercury porosimetry or gas sorption [3,14,15], is of major interest in studies of microporous materials for liquid chromatography.

Limitations

There are number of precautions necessary for realizing effective ISEC procedures. Retention differences are considered to result purely form steric interaction, so solute standards with low polydispersity, i.e., that are well-defined in size and shape, should be used for pore size distribution determination. Dilute standard solutions are typically used to reduce solute-solute interactions, especially aggregation. Appropriate ISEC probes and solvent conditions should be chosen to minimize solute-adsorbent binding and to avoid aggregation.

If these prerequisites for standard ISEC are not satisfied, alternative treatments of non‑standard ISEC must be used to extract the pore size distribution. Potential anomalies include solute adsorption that cannot be eliminated by manipulating solvent conditions and the polydisperse standards when monodisperse solutes are not available. Some adsorbents also contain large pores that are accessible even to the largest polymer standards typically used.

Consequently, the macropore volume cannot be quantitatively differentiated by ISEC, and it is difficult to determine accurately the interstitial volume in a column containing such macroporous media. Micrometer-size latex particles can be used as large probes for quantifying the composition of macropores [16], in this case extra care is needed in choosing the size of the filters and frits in the chromatography system.

References

1. L.G. Aggebrandt, O. Samuelson, J. Appl. Polym. Sci., 8 (1964) 2801.
2. A.A. Gorbunov, L.Y. Solovyova, V.A. Pasechnik, J. Chromatogr., 448 (1988) 307.
3. I. Halász, K. Martin, Angew. Chem. Inter. Ed. (Engl.)., 17 (1978) 901.
4. J.H. Knox, H.P. Scott, J. Chromatogr., 316 (1984) 311.
5. J.H. Knox, H.J. Ritchie, J. Chromatogr., 387 (1987) 65.
6. L. Hagel, M. Ostberg, T. Anderson, J. Chromatogr., 743 (1996) 33.
7. K. Jerabek, A. Revillon, E. Puccillli, Chromatografia, 36 (1993) 259.
8. L.Z. Vilenchik, J.A. Asrar, R.C. Ayotte, L. Ternorutsky, C.J. Hardiman, J. Chromatogr., 648 (1993) 9.
9. I. Mazsaroff, F.E. Regnier, J. Chromatogr., 442 (1988) 15.
10. W.Werner, I. Halász, J. Chromatogr. Sci., 18 (1980) 277.
11. R. Nikolov. W. Werner, I. Halász, J. Chromatogr. Sci., 18 (1980) 207.
12. P. DePhilllips, A.M. Lenhoff, J. Chromatogr. A, 883 (2000) 39.
13. H. Guan, G.Guiochon, J. Chromatogr. A., 731 (1996) 27.
14. A.J. de Vries, M. Lepage, R. Beau, C.I. Guillemi, Anal. Chem., 39 (1967) 935.
15. N.V. Saritha, G. Madras, Chem. Eng. Sci., 56 (2001) 6511.
16. Y. Yao, A.M. Lenhoff, J. Chromatogr. A, 1126 (2006) 107.

Superficially porous particles with thicker outer shells were used extensively for liquid-liquid chromatography [1] and as the support for early bonded-phase packings in reverse phase HPLC [2].

Structure of particles

Nowadays superficially porous particles typically have a 5-µm solid core and a ~ 0.25 – 1 µm thick outer shell with 30-nm pores. But, the thinner the shell, the smaller is the particle surface area and the less is the sample loadability of these particles. Therefore, there must be a compromise between the thickness of the sh

Scheme of superficially porous particle

ell to obtain good separation efficiency and good sample loadability. Thin porous shells lead to low surface areas, low retentivity, and low sample loadability; thicker shells of higher surface area produce higher retentivity and higher sample loadability.

Advantages

Superficial porous particles have unique characteristics that favour certain applications. Because of their size, macromolecules have very poor diffusional qualities. Therefore, the advantage is significant for the very short diffusional path lengths within the porous shell on the surface of superficial porous particles. Compared to conventional totally porous particles of the same size, superficial porous particles show improved mass-transfer kinetic properties that permit more rapid separations of macromolecules [3].

References

1. J. J. Kirkland, Anal. Chem., 41 (1969) 218.
2. J. J. Kirkland, J. J. DeStefano, J. Chromatogr. Sci., 8 (1970) 309.
3. J. J. Kirkland, Anal. Chem., 64 (1992) 1239.

Non-porous particles (crawfordscientific.com)

It is known that the kinetics of mass transfer in wide pore bonded silica can be slow, because of restricted intraparticle diffusion and, furthermore, remaining active surface sites can give rise to undesired interactions.

All together, these effects cause additional peak dispersion in high performance liquid chromatography and often considerable loss in recovery of biological activity [1].

Particle’s porosity elimination

A clear way around this dilemma is to eliminate the porosity, which minimizes the pore diffusion and mass transfer resistance (longitudinal diffusion) effects. The diffusion paths that analytes must traverse between stationary phase and mobile phase are very short with nonporous media and column efficiency essentially becomes independent of the flow rate [2]. Commercially available HPLC columns with nonporous stationary phases are stable at the low pH and high temperature [3].

Because of the reduction in surface area, nonporous supports exhibit much lower retention times for the same organic modifier content compared with porous columns [2]. The extremely low external surface area of nonporous supports can be regarded as a drawback since it leads to a considerably lower loadability compared with porous materials [1].

Drawbacks

Disadvantages of non-porous silica packings are that the column length is restricted because high pressure is required for an adequate mobile phase flow and it is difficult to pack a column densely and homogenously [4]. Guiochon and Martin [5] indicated that it would be almost impossible to use a high-efficiency HPLC column packed with less than 1-µm diameter particles. This suggests that the decrease in the particle diameter reaches a limit if particles are packed into columns directly.

References

1. M. Hanson, K. K. Unger, LC-GC Int., (1996) 741.
2. T. J. Barder, P. J. Wohlman, C. Thrall, P. D. Dubios, LC-GC Vol. 15, No.10 (1997) 918.
3. R. Ohmacht, I. Kiss, Chromatographia, Vol. 42, No. 9/10 (1996) 595.
4. H. Giesche, K. K. Unger, U. Esser, B. Eray, U. Truedinger, J. N. Kinkel, J.Chromatogr., 465 (1989) 39.
5. G. Guiochon, M. Martin, J. Chromatogr., 326 (1985) 3.

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.

Chromatography is analytical chemistry method which is used (and useful) for the separation of complex mixtures of chemical compounds. The main mechanism of the separation is repeatable distribution of the tested compound in between two different phases.

Usually, one phase is solid, fixed in the separation device and the other is moving and flows through the unit. If gas is a second phase, we are referring to the gas chromatography, in case of liquid as a second phase the name is liquid chromatography.

The device where separation takes place is called chromatographic column. This cylindrical shape column is filled with the different kinds of materials – stationary phases. These materials are usually spherical silica particles with different, but well defined, surface chemistry.

The mobile phase flows through the column together with sample (mixture of compounds). Each compound has various affinity to the surface of stationary phase and therefore is separated form each other. In case of ideal state all compounds are eluted from the column in separated bands.

Various techniques are used to recognize these bands and transform them into the signal. In most of the cases the signal draws chromatographic peak – the “hill like” curve describing Gauss distribution.