Chromatography, in its various forms, has become one of the most widely used analytical separation techniques in chemistry and biochemistry earning early developers of this technique A.J.P Martin and R.L.M Synge the 1952 Nobel Prize in Chemistry1. Chromatography is an incredibly important tool in the carbon scientist’s toolkit. Especially for someone like me that is trying to understand how specific fractions of the carbon pool cycle through ecosystems and how carbon diversity fits into a unfiied ecological theory. This is an interesting story about where this method came from.
For those unfamiliar with the jargon, a quick catch-up: Chromatography is a flow system-based technique that is used to separate complex mixtures. The sample is adsorbed onto a column (stationary phase) where it is then pushed along by a mobile phase moving at a constant flow rate. Components of the mixture may interact with the stationary phase differently, some slowed by the resistive forces of the column, and therefore pass through at different rates (differential migration).
It was a young botanist, M.S. Tswett who first presented a chromatography prototype in his investigation of isolation techniques for plant pigments from leaves (chlorophylls and carotenoids). His paper: On a New Category of Adsorption Phenomena and Their Application to Biochemical Analysis (1906)2 would set the foundation for the chromatographic method.
Early Work 3,4,5
Trained in botany and plant physiology in Geneva, Tswett was aware of the presence of different chloroplast pigments and recognized a need for methods to separate and isolate them. The fundamental observation that led to his development of chromatography began with an investigation of pigments in leaves. Tswett noted that carotenes could be extracted from leaves with relative ease using nonpolar hydrocarbon solvents (ether or naphtha). However, for chlorophyllic pigments, the addition of polar solvent such as ethanol was required (recall the general rule in chemistry that ‘like dissolves like’). Strangely, once extracted, the isolated chlorophyllic pigments were soluble in nonpolar solvent.
After extensive investigation he concluded that this inability to extract chlorophyll with the non-polar solvent and the easy accomplishment of this goal post-ethanol addition could not be ascribed to a chemical alteration of chlorophyll that rendered it soluble. Instead, he postulated that the chlorophyllic pigment complexes were adsorbed to the plant tissue. Therefore, one needed a solvent with dissolving power higher than the adsorption forces of the plant tissues to extract the chlorophyll. In addition, once extracted, chlorophyll should dissolve in a nonpolar solvent since there are no longer adsorptive forces acting upon it.
To test this assumption, Tswett created a model leaf using tissue paper. Both are composed of plant (cellulose) fibres. To make this “leaf” he extracted pigments from plant leaves using ethanol, evaporated the ethanol and then re-dissolved the residue in a nonpolar solvent. Next, he impregnated the filter paper with the solution. The now pigmented paper was not only leaf-green, but also behaved towards the extraction solvents in the same way: nonpolar hydrocarbons extracted carotene but not chlorophyll from the ‘leaf’ while after the addition of ethanol, the chlorophyll extraction was successful.
Having shown that filter paper had an adsorption affinity for chlorophyll present in hydrocarbon solution he wondered if other solvents and adsorbents could be used to further elucidate the chemical mysteries contained with a plant leaf. And so, he set out to continue his work feeling that the systematic investigation of this method:
“would elucidate the nature of adsorption, and furnish a new physical method based on adsorption for the separation of substances.”
Developing the Adsorptive Separation Method 6,7,8,9
Tswett reported the investigation of over 100 inorganic and organic solid substances to study the adsorption affinity for chlorophyll. He investigated these by packing powdered adsorbent into a small narrow tube. The nonpolar solvent with the pigment extract was then added to the adsorber column until the pigments formed a narrow initial zone at the uppermost regions of the adsorbent. He then washed the adsorbed pigment mixture through the column with fresh solvent, noting how green and yellow rings started to form. As more solvent was added, the rings separated, widening as they moved down the column. If a proper solvent was selected, it was possible to elute the pigments individually from the column, resulting in complete pigment separation.
Tswett also devised a second method for separating plant pigments in a stepwise selective adsorption and extraction. To do this he added the adsorbent to the pigment. He then selectively added different solvents that resulted in the separation of pure pigments, which could be identified by not only their colour but by their UV-absorption spectra.
On the basis of this work, Tswett coined the term “chromatography” and, in a widely quoted statement:
“Like light rays in the spectrum, the different components of a pigment mixture, obeying a law, are resolved on the calcium carbonate column and then can be qualitatively and quantitatively determined. I call such a preparation a chromatogram and the corresponding method the chromatographic method.”
The word chromatography is composed of two Greek roots, χρῶμα chroma (colour) and γράφειν graphein (to write), and its verbatim translation means “colour writing,” which refers to visualizing the separated multicoloured rings on the column. Another interpretation of this term links it to Tswett’s surname, the meaning of which is “colour” in Russian. According to this interpretation, “chromatography” actually could mean “Tswett’s writing”6.
In playing around with leaves, Tswett demonstrated the property of selective adsorption of carbon compounds from complex mixtures (mobile phase) onto other substances (tissue paper and other adsorbents) in columnar form (i.e, a solid phase). He explored the proper selection of solvent and adsorbents and also emphasized that although his work was focused on plant pigments, other substances could also be separated by chromatography (even non-coloured compounds). Thus, Tswett, with his visionary and creative experiments, planted an imaginative seed, from which the roots of modern chromatographic methods grew.
- “The Nobel Prize in Chemistry 1952”. nobelprize.org. Retrieved 04 September 2020.
- Tswett M. On a New Category of Adsorption Phenomena and their Application to Biochemical Analysis (translation from the Russian). The original paper is hardly found and the English translation has been published by G. Hesse and H. Weil in 1954 (Woelm Mitteilungen AL8, M. Woelm, Eschwege). The Proceedings of the Warsaw Society of Natural Sciences, Sect XIV (6). (1906).
- L.S. Ettre and K.I. Sakodynskii, Chromatographia, 35, 223–231 (1993).
- L.S. Ettre and K.I. Sakodynskii, Chromatographia, 35, 329–338 (1993).
- L.S. Ettre, Chromatographia, 42, 343–351 (1996).
- Ettre LS. MS Tswett and the invention of chromatography. LC GC North America. 21, 458-67 (2003).
- M. Tswett, Ber. Dtsch. Botan. Ges., 24, 316–323 (1906). Translation from German in: V.G. Berezkin, Ed., Chromatographic Adsorption Analysis: Selected Works of M.S. Tswett (Ellis Horwood, New York, USA 1990).
- M. Tswett, Ber. Dtsch. Botan. Ges., 24, 384–393 (1906). Translation from original German can be found in: M.S. Tswett, Khromofilly v Rastitel’nom i Zhivotnom Mire (Chromophylls in the Plant and Animal Kingdom) (Karbasnikov Publishers, Warsaw, 1910).
- H.H. Strain and J. Sherma, J. Chem. Ed., 44, 235–242 (1967).