2-D plot shows separation of benzyl alcohol, benzene and butylbenzene using remote NMR/MRI with a monolithic chromatography column.

During the time I spent in Berkeley I had the honor to work on the beginning of the project leading to the portable system for highly sensitive multi-dimensional chemical analysis. This work included hyphenation of NMR with liquid chromatography separation using organic polymer monoliths.

I have to admit, it was my first touch of NMR ever. I knew the theory, principle and technique, but I had never worked with it. Fortunately, we were four of us, Tom and Nick as NMR guys and Stuart and me responsible for chromatography. We have used hypercrosslinked monolithic columns which already proved to be suitable for fast separation of small molecules.

Read the press release

Since yesterday, I wanted to describe the whole project with all the background, theory, results and so on. But there are people who did it before me and in much better shape than I can ever do. So if you are interested in this very nice topic, you can read article about Application of NMR/MRI to microfluidic chromatography published at the Berkeley Lab website. It describes rationale and inspiration behind the work, as well as achieved results and future plans. Moreover, you might get more information in the paper published in Anal. Chem.

LC-NMR hyphenation

Although there is (almost) nothing to add, I would like to share my view and experience I got working on this topic. First of all, capillary liquid chromatography and NMR are quite contradictory techniques. To get better results you need low injection volume in LC, but then you have no signal (low sensitivity) in NMR. The same applies with speed – you get higher efficiency at lower flow-rates of mobile phase (LC) but you are loosing signals in NMR with their slow transfer. Last but not least, all metallic parts have to be in certain distance from the NMR magnet.

Monolithic capillary column has been placed inside the magnet and connect with injector via a long fused silica capillary. First, we have started with 100 μm I.D. monolithic column and splitter who divided the flow from the pump. Later, we increased internal diameter of our monolithic column up to 530 μm which allowed increase in signal and avoid using splitter. Thus, we could connect the column with injector via 250 cm long capillary (50 μm I.D.).

We had to inject mixture of pure compounds (benzyl alcohol, benzene and butylbenzene) to be able to get any signal. The separation showed in the figure is quite fast and if there is no tailing of butylbenzene peaks it would be possible to separate these test compounds in less then 60 s.

Thanks guys

Thanks to this project I had a nice opportunity to learn something new and work with techniques and their hyphenation which open door to a future portable system for very sensitive chemical analysis. No surprise that at the end I would like to thank my co-workers Tom Teisseyre, Nick Halpern-Manners and Stuart Chambers. It was my pleasure to work with you, guys!

Before the last CASSS Discussion group debate on difference between high temperature and high pressure liquid chromatography started, there was a welcome slide projected on the wall. There was only one sentece (paraphrase):

Meet other people who like and understand what you do

I highlighted the most important part (for me), because I have always problems to explain what I am doing. I would like to ask you all for your thoughts.

• How do you define chromatography?
• Do you have problems to interpret chromatography to other people who don’t understand the chemistry at all?

How do you define chromatography?

In my case, I am always trying to use words as analysing what is inside a sample, separation of complex mixtures, etc. On the very end (when I see that the listener has no clue at all), I am always using examples such as “when you are visiting doctors, they can determine the level of your cholesterol in a blood with chromatography” or “it can be used for a quality control of gasoline in your car”.

Usually, people just answer “ahaa”. And I know, that they still don’t know what I am talking about.

Once I have read the definition of the chromatography as a running race. On the beginning there is a group of a runners and as time flows (mobile phase?;) the group is separated to a groups of the runners with a same speed (retention). On the end of the run, the winner is a non retained compound and the others are individual parts of the mixture. I am not using this expression often, though. But maybe I will.

On the end of the day – as the saying goes – if I am not able to explain what I am doing to my grandparents, then I dont know what I do.

What are your experience and expressions how to define chromatographic separations?

Your comments and suggestions are more than welcome.

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.

Happy New Chromatographic Year

Not only from the chromatographic point of view, I wish you in the year 2010:

• low pressure
• high efficiency
• sharp resolution
• large capacity
• and satisfactory results

… or (in case you dont want to use chromatographic expressions) you can use any of the translations.

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.

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.

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.