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If you have ever seen ink or plant pigment spread into separate coloured bands on a strip of paper, you have watched chromatography. It is now used everywhere from pharmaceutical labs to forensic science. It began with a botanist who wanted to know what the green colour of leaves was made of. Mikhail Tsvet, who published in German as M. Tswett, packed a glass tube with chalk powder, poured leaf extract through it, and watched the pigments separate. This post covers what he actually did, the idea behind it, a worked Rf calculation, common mistakes, and a lesson from how he worked.
Who was Mikhail Tsvet?
In 1906 the German Botanical Society's member list recorded him as "Dr. Michael Tswett, Privatdozent der Botanik", a lecturer in botany at the University of Warsaw. His reference lists show that he had already reported a new kind of adsorption phenomenon to the Warsaw Society of Naturalists in 1903.
That year, 1906, he published two short papers in the society's journal. The first, Physikalisch-chemische Studien über das Chlorophyll. Die Adsorptionen, was received on 21 June 1906. The second, Adsorptionsanalyse und chromatographische Methode, was received on 21 July 1906. An English translation of the second was published in the Journal of Chemical Education in 1967.
What problem was Tsvet trying to solve?
His first paper opens with a puzzle. Alcohol or ether pulls a deep green extract out of leaves, but petroleum ether gives only a yellow extract coloured by carotene. If chlorophyll dissolves well in petroleum ether, why does it not come out of the leaf?
His answer was adsorption. The green pigments cling to solid surfaces when dissolved in petroleum ether. He tested more than a hundred powdered substances and found the same basic effect. As adsorbents he recommended precipitated calcium carbonate, inulin, or powdered sugar. Shaken with a petroleum ether solution of leaf pigments, these powders pulled the green pigments out and left carotene in solution, because carotene was not adsorbed.
How did the first chromatography column work?
In the second paper he turned this into a column method. Here are the details he gives:
A narrow glass tube was packed firmly with dry, fine powder. He dried his calcium carbonate for two hours at 150 °C and used columns typically 20 to 30 mm high in his small tubes.
The pigment solution was pushed through under a slight extra pressure, then pure solvent was run through after it.
Each pigment stuck to the powder to a different extent, so the pigments spread out into separate coloured zones down the column.
He then pushed the packed column out of the tube and cut the zones apart with a knife to study each pigment separately.
He called the banded column a chromatogram and the procedure the chromatographic method. He compared it to other ways of analysing pigments as being like spectral analysis compared with looking through coloured glasses. Fine powder mattered: coarse powder, he wrote, gave blurred chromatograms.
Why did it take so long to catch on?
The 1952 Nobel ceremony speech for Archer Martin and Richard Synge says chromatography "was discovered in 1906 by the Russian-Polish Michael Tsvett", who separated the pigments of green leaves, and that Martin and Synge's advance was a new principle, partition between two liquids. In between, his method was largely set aside, and a 2009 paper in Studies in History and Philosophy of Science asks exactly why his adsorption analysis was rejected.
Worked example: calculating an Rf value
NCERT's Class 11 Chemistry Unit 8 covers adsorption chromatography, column chromatography and thin layer chromatography, and defines the retardation factor:
Rf = distance moved by the substance from the base line ÷ distance moved by the solvent from the base line
Suppose you run leaf pigments on a thin layer plate. These are sample numbers for illustration: the solvent front travels 8.0 cm, a yellow-orange spot travels 7.2 cm and a green spot travels 3.2 cm.
Yellow-orange spot: Rf = 7.2 ÷ 8.0 = 0.90
Green spot: Rf = 3.2 ÷ 8.0 = 0.40
The yellow-orange spot is held least strongly and moves furthest, the same behaviour Tsvet saw when carotene escaped adsorption on his chalk. For more on the lab techniques, see our post on purification and chromatography.
What mistakes do students make with chromatography?
Measuring from the paper edge. Both distances are measured from the base line where the sample was spotted.
Getting Rf above 1. A substance cannot move further than the solvent, so Rf always lies between 0 and 1.
Thinking colour is required. Tsvet himself noted that colourless compounds are adsorbed too; they just need another way to be seen.
Comparing Rf values across different solvents. Rf depends on the solvent and the adsorbent, so compare only under the same conditions.
If the ideas of adsorption and polarity behind these bands feel fuzzy, the Fermi Concept Library covers every concept in physics, chemistry, biology, mathematics and computing, one idea at a time.
What can a student learn from how Tsvet worked?
Tsvet started from a small, odd observation (petroleum ether giving a yellow extract) and refused to let it go. He then tested over a hundred materials before choosing an adsorbent, and wrote down practical details such as drying time and powder fineness so that others could repeat the work. When something in a practical does not behave as the textbook says, write down exactly what you saw. It may be the most useful result of the session.
Take three felt-tip pens, run their ink on a strip of filter paper with water, and calculate the Rf value of every band you see.
