Writing colors, the role of chromatography in natural products research

Writing colors, the role of chromatography in natural products research

In the field of pharmacognosy we study natural products containing compounds with potential pharmacological activity. In order to analyze such products, we start from an extract obtained from our natural source (plant, fungi, bacteria, etc)  which shall contain a mixture of different compounds with different chemical, physical, and bioactive properties. 

Chromatography comprises a group of separation techniques employed for the separation of compounds in complex mixtures. First devised by Mijaíl Tsvet in 1900, chromatography was first used to separate plant pigments such as chlorophyl, carotenes, and xanthophylls. Nowadays, there are many chromatographic techniques, having all in common the use of a stationary phase and a mobile phase. Based on this principle, preparative column chromatography employs a solid stationary phase such as silica or Sephadex®; and a solvent of convenience is used as mobile phase. The compounds pass through the stationary phase carried by the mobile phase and the different interactions of the compounds contained in the mixture with the stationary phase will make their transport faster or slower, depending on the physical-chemical properties of the compounds. As a result of this preparative column chromatography, we can get fractions enriched with certain group of compounds with similar properties. In some cases, pure compounds are obtained in one single chromatographic step, depending on the complexity of the fractionated mixture, but in most cases, the fractions may need further fractionations step. This is why combination of different stationary phases and/or mobile phases will help us to get pure compounds out of natural products. Also the use of other preparative chromatographic techniques, which we can describe in another time.

How to know when do we have a pure compound? Before I talked about preparative chromatography, now it is time to talk about analytical chromatography. There are several techniques employed to analyze the composition and purity of natural products. The most employed one in analytical laboratories may be analytical thin layer chromatography (TLC), which belongs to planar chromatography. TLC employs as stationary phase a thin layer of adsorbent such as silica gel, alumina or cellulose deposed on an inert flat substrate. The sample is placed in solution on the surface of the plate, near the bottom, and the plate is immersed in a camera containing the mobile phase. The mobile phase should cover just the bottom of the plate and by capillary it will reach the top of the plate, dragging the different compounds with it. Depending on the chemical affinity of the compounds for the mobile phase and stationary phase, they will move more or less along the plate. Once the separation is done, we have to reveal the position of our compounds, for this, the plate is exposed to UV light at different wavelengths, were we can detect their presence by fluorescence or quenching. Finally, a revealing solution is spread on the plate to make the compounds visible under normal light and in some cases to reveal other compounds under UV light. When one single compound is detected in TLC there are big chances that our fraction contains a pure compound. However, there are more advanced techniques which enable us to analyze more accurately our fractions such as, high performance liquid chromatography (HPLC), gas chromatography (GC), supercritical fluid chromatography (SFC), capillary electrophoresis (CE) just to mention some examples that we count with at the Pharmacognosy Department of the University of Innsbruck.

size exclusion chromatography column TLC analysis TLC 3

A size exclusion chromatography column. After, the TLC analysis of the fractions collected from the column under UV light 254nm, UV light 366nm, and white light after derivatizing with iodine vapors and starch solution.


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