What is chromatography? 

Chromatography is a technique that can be used to separate various substances within a complex mixture. On the one hand, this enables the identification of individual substances (qualitative analysis). On the other hand, chromatographic separation can be used to determine the concentration of individual substances (quantitative analysis). [1-4] 

Why is it important for me? 

Chromatography is one of the most common chemical analysis techniques and is used for a wide variety of applications. For example, chromatography is used to analyze food and water for toxic substances to ensure our health. Furthermore, environmental pollution in air, water and soil can be detected. In addition to the analysis of environmental and food samples, chromatography is also used in (chemical) production for the quality control of raw materials and products. 

Chromatography can be used to solve many analytical challenges - maybe also some of yours? 

How does the separation happen? 

The different substances of a complex mixture (= sample) are separated in the column of the chromatography system. The sample is transported through the column, which contains the stationary phase, with the mobile phase. As the mobile phase flows through the column, interactions occur between the analytes and the stationary phase. This causes the analytes to be held back (= retention) so that they leave the column (= elution) with a time delay (= retention time). How strong and how long the analytes interact with the stationary phase depends on the chemical and physical properties of the analytes and the stationary phase. Different analytes are retained to different degrees, resulting in a separation of the different substances in a complex mixture. 

The most widely known and frequently used chromatographic analysis methods are high-performance liquid chromatography (HPLC) and gas chromatography (GC). Both differ, among other things, in the used mobile and stationary phase. In GC, (inert) carrier gases such as helium or hydrogen are used as the mobile phase. The stationary phase is located as a film on the inside of the column, which is usually between 15 and 60 m long and has an internal diameter of between 0.1 and 0.7 mm.  

In HPLC, liquid organic solvents are used as mobile phase. The stationary phase are surface-modified spherical particles, which are in a cylindrical column. Typically, the column is 50 - 150 mm long and the inner diameter is 2 - 5 mm. [1-6] 

What else do I need to know about chromatography? 

GC can be used to analyze substances that can be transferred undecomposed into the gas phase. Generally, the oven temperature is increased continuously and in a controlled manner during the analysis run (gradient elution) so that, for example, samples with analytes that have very different boiling points or vapor pressures can be analysed. The reason for the separation of substances in GC is, on the one hand, the interaction between the analyte and the stationary phase and, on the other hand, the vapor pressure (the boiling point is often used instead) of the analytes.  

HPLC is traditionally used to analyze substances or extracts that are dissolved in organic solvents. To ensure the best possible separation of a large number of analytes, the composition of the mobile phase is typically changed during the chromatographic separation (gradient elution). Normally, two or four solvents or mixtures are available, which are pumped using binary or quaternary pumps. In contrast to GC, in LC the interaction between analyte and stationary phase as well as the solubility of the analytes in the mobile phase are the main reasons for chromatographic separation. [2-6] 

How are the separated substances detected? 

There are various detectors for detecting the eluting analytes. For example, a diode array detector (DAD) is often used for HPLC and a flame ionization detector (FID) for GC. Nowadays, the mass spectrometer (MS) is being used more and more in comparison to the other detectors. You can use the MS, for example, if you do not know exactly which substances are present in your sample or if you need to achieve very low detection and quantification limits. Mass spectrometers are available in combination with both a GC and a LC. The operating principle of the mass spectrometer is very similar for both combinations. First, the substances eluting from the chromatography system are ionized. The resulting molecular ions are transferred to the mass analyzer by electric fields. In the mass analyzer, the molecular ions are separated according to the mass-to-charge ratio (m/z) and afterwards converted into an electrical signal so that they can be detected and plotted. 

The detailed design of mass spectrometers varies depending on the coupling technique. In the case of an MS coupled to a GC, the eluting molecules are usually bombarded with an electron beam in a high vacuum. This not only removes an electron from the molecule, but the excess energy leads to fragmentation of the molecular ion into so-called fragment ions with a lower mass. The analytes are usually not identified by means of the m/z ratio of the molecular ion, as this is sometimes no longer recognizable in the mass spectrum. However, the fragmentation is very reproducible, so that databases are used for identification. The match between the measured mass spectrum and the mass spectrum of the database is often used as one identification parameter. For LC mass spectrometers, ionization does not take place in a vacuum but under atmospheric pressure. In addition to ionization, evaporation of the mobile phase (solvent) also takes place in these kind of ion sources. [2-4,7] 

Who described chromatography first? 

The technique of chromatography was first published in 1903 by the Russian botanist Mikhail Semyonovich Tswet. [8] He used this technique to separate plant pigments, e.g. chlorophyll. For this purpose, he used a glass tube filled with powdered chalk and/or aluminium oxide. By adding solvent and under the effect of gravity, the pigments moved downwards along the column and were separated during this process. As a result, Tswet observed different color edges along the column. Because of the resulting color bands, Tswet called this technique chromatography. The term chromatography is made up of the Greek word "chroma" for color and "graphein" for writing. [1,8] 

If I want to analyze a chocolate bar, how do I get it into the analysis system? 

As you can imagine, a bar of chocolate cannot simply be placed in the analysis system. Like other samples, such as coffee, tea or spices, sample preparation must be carried out in advance. The preparation until the sample is in an analyzable form can be very complex. In the simplest case, a liquid solvent is used for sample preparation, which extracts the analytes from the homogenized sample. In order to avoid contamination of the analyzer and to achieve low detection limits, further purification steps are often required. These include, for example, filtration, solid phase extraction (SPE) or centrifugation. Such sample preparation methods usually require a large amount of toxic solvents. Alternatively, so-called microextraction techniques, such as SBSE [9], TF-SPME [10,11] or SPME [12,13], can be used instead. Like the sample preparation methods for volatile analytes, such as the headspace technique [14], thermodesorption or dynamic headspace, microextraction techniques require little to no organic solvent. These methods are therefore more environmentally friendly and less costly, while improving occupational safety in the laboratory. 

References & literature recommendations 

K. Kaltenböck, Chromatographie für Dummies, 1. Auflage, Wiley-VCH, Weinheim, 2010. [Note: German only] 

G. Schwedt, T. C. Schmidt, O. J. Schmitz, Analytische Chemie: Grundlagen, Methoden und Praxis, 3. Auflage, WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim, 2017. [Note: German only] 

M. Otto, Analytische Chemie, 4. Auflage, Wiley-VCH, Weinheim, 2014. [Note: German only] 

K. Cammann, Instrumentelle analytische Chemie: Verfahren, Anwendungen, Qualitätssicherung, 1. Auflage, Spektrum Akad. Verl., Heidelberg, 2010. [Note: German only] 

S. Kromidas, Der HPLC-Experte: Möglichkeiten und Grenzen der modernen HPLC, Wiley-VCH, Weinheim, 2014. [Note: German only] 

S. Kromidas, Der HPLC-Experte II: So nutze ich meine HPLC / UHPLC optimal!, Wiley-VCH, Weinheim, 2015. [Note: German only] 

J. H. Gross, Mass Spectrometry: A Textbook, Springer Verlag, Berlin, Heidelberg, 2011. 

M. Tswett, Adsorptionsanalyse und chromatographische Methode. Anwendung auf die Chemie des Chlorophylls, Berichte der deutschen botanischen Gesellschaft 24 (1906) 384. 

E. Baltussen, P. Sandra, F. David, C. Cramers, Stir bar sorptive extraction (SBSE), a novel extraction technique for aqueous samples: theory and principles, J. Micro. Sep.,11.10 (1999): 737-747. https://doi.org/10.1002/(SICI)1520-667X(1999)11:10<737::AID-MCS7>3.0.CO;2-4 

I. Bruheim, X. Liu, J. Pawliszyn, Thin-film microextraction, Anal. Chem. 75 (2003) 1002–1010. https://doi.org/10.1021/ac026162q. 

R. Jiang, J. Pawliszyn, Thin-film microextraction offers another geometry for solid-phase microextraction, TrAC Trends in Analytical Chemistry 39 (2012) 245–253. https://doi.org/10.1016/j.trac.2012.07.005. 

J. Pawliszyn, Handbook of Solid Phase Microextraction, Chemical Industry Press, Peking, 2009. 

C. L. Arthur, J. Pawliszyn, Solid phase microextraction with thermal desorption using fused silica optical fibers. Anal. Chem. 1990, 62, 19, 2145–2148. https://doi.org/10.1021/ac00218a019 

B. Kolb, L. S. Ettre, Static Headspace-Gas Chromatography: Theory and Practice, Wiley-VCH, New York, 1997.