The Beginning

Open-column LC was invented by Michael (Mikhail) Semenovich Tswett (1872-1919) at the beginning of the 20th century and he was responsible for naming the method. Paper, thin-layer, closed-column liquid and gas chromatography were later invented by other scientists who varied the method set-up described by Tswett.

The father of chromatography was not a chemist but a botanist. Although born in Asti, Italy, and educated in Geneva, Switzerland, he was a Russain national. In 1896 he obtained a PhD from the University of Geneva. Later, when he was an assistant professor at the University of Warsaw, Poland, he needed a method to isolate plant pigments in pure form. At that time the standard technique for this purpose was the partition of plant extracts between immiscible organic solvents. However, Tswett was also interested in the problem of how the pigments are fixed, i.e. adsorbed, within the plant cell. He carried out numerous experiments with adsorbents and tested

Table 1 Important historical papers on liquid chromatography

Tswett MS (1906) Adsorptionsanalyse und chromatographische Methode. Anwendung auf die Chemie des Chlorophylls. Berichte der Deutschen Botanischen Gesellschaft 24: 384 Tswett MS (1910) Khromofilly v Rastitel'nom i Zhivotnom Mire.

Warsaw: Karlassiakov Palmer LS and Eckles CH (1914) Carotin - the principal natural yellow pigment of milk fat: its relations to plant carotin and the carotin of the body fat, corpus luteum and blood serum. Journal ofBiological Chemistry 17: 191 Kuhn R, Winterstein A and Lederer E (1931) Zur Kenntnis der Xanthophylle. Hoppe Seyer's Zeitschrift für Physiologische Chemie 197: 141 Martin AJP and Synge RLM (1941) A new form of chromatogram employing two liquid phases. Biochemical Journal 35: 1358 Glueckauf E (1955) Theory of chromatography. IX. Theoretical plate concept in column separations. Transactions of the Faraday Society 51: 34 Van Deemter JJ, Zuiderweg FJ and Klinkenberg A (1956) Longitudinal diffusion and resistance to mass transfer as causes of nonideality in chromatography. ChemicalEngineeringScience 5: 271

Moore S, Sparkman DH and Stein WH (1958) Chromatography of amino acids on sulfonated polystyrene resins. An improved system. Analytical Chemistry 30: 1185 Porath J and Flodin P (1959) Gel filtration: a method for desalting and group separation. Nature (London) 183: 1657

more than 100 different powdered materials. Finally he found that inulin, sucrose and calcium carbonate were best suited for the separation of the pigments of green leaves - chlorophylls (two compounds) and xanthophylls.

Unfortunately the new method was only described in lectures, in a paper in a German botanical journal (1906) and in a book in the Russian language (1910). It is obvious that most chemists did not read either botanical journals or a Russian book published in Warsaw. Moreover, when reading these texts today, many details of the experimental set-up are missing. These facts, together with the outbreak of World War I in 1914 and Tswett's untimely death at the age of 47 in 1919, hindered the acceptance of the new method.

Tswett mainly used small glass columns of 2-3 mm diameter packed with adsorbent to a height of c. 30 mm. The plant extract was applied on top of the column and transported into the packing by a solvent - the mobile phase - which was added on top and sucked through the adsorbent by a slight vacuum or forced through with a slight pressure. The best elu-ents were found to be benzene and carbon disulfide; solvent mixtures and even gradients could also be used. Several columns could be used in parallel (Figure 1).

Tswett did not publish more than three 'chromato-grams'; one is shown in Figure 2. Experimental details of all these drawings are missing. The pigments were not eluted but the whole packing was pushed out of the glass tubing and the different zones were then cut apart with a knife. The pigments were extracted from the adsorbent by an appropriate solvent and identified by UV spectroscopy (which was a tedious and time-consuming method). Figure 3 makes clear how similar the structures of chlorophylls a and b are and, from our current perspective, we get the impression that Tswett was a very skilled experimenter. It was difficult to reproduce his separations using only his descriptions.

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