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Ancestry of Chromatography
Runge experiment Tswett experiment
FEB ‘2012
at a glance
The century old separation technique called “chromatography” has its origin in the middle of 19
century was spreaded into all branches of science. The chromatographic technique has branched out to
different types (like TLC, HPLC, GC, SEC, etc.,) and joined in hand with the technological
development to recognize the chromatographic instruments as a common appliances in almost all
scientific industries.
Archeology of chromatography:
The archeology of chromatography begins from 19th century scientist
F.F Runge(1) whose investigations could be considered as a
“precursor of chromatography”. The name “Chromatography” was
coined by M.S.Tswett almost 40 years after Runge’s book “The
chemistry of color” and “Preparation of dyes”.
Runge was interested on the production of textile dyes. He
demonstrated that a piece of filter paper is very useful to test the
completion of dye formation in solution. In his quantitative
experiments he illustrated precipitation of natural plant dye by the
addition of lead acetate solution. After adding the lead acetate to the
plant dye solution, a drop of the supernatant solution was spotted on a
filter paper strip and also spotted one drop of potassium chromate
solution in the other end of the filter paper strip. The spots started to
diffuse away from the spotting points and met in one spot. If
precipitation is completed, the excess lead acetate remaining in the
supernatant solution reacts with potassium chromate to form lead
chromate and appears as a dark yellow colour on the filter paper. Thus
the completeness of the dye formation was easily identified.
In 1861, Groppelsroeder(2) used a form of paper chromatography
which is called “capillary analysis” to separate the colored pigments.
He was considered as a “Grandfather of chromatography”. He used
the strip of paper with one end dipped on to an aqueous solution, to
separate the colored pigments. The components are moved to the end
of paper by capillary action.
In 1903, Mikhail Tswett(3) was influenced by the work of
Groppelsroeder capillary analysis experiment. The first step of
investigation was the extraction of pigments in the leaves. Tswett used
a column packed with a stationary phase of calcium carbonate. The
plant extract was placed at the top of the column and carried through
the stationary phase using petroleum ether as a mobile phase. Due to
the gravity force, samples are moved through the column, the
pigments in the plant extract separated into individual colored bands.
After adding more solvents to the column, the bands became separated
and moved down the column.
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Mikhail Tswett
This process was named as chromatography. The word ‘chromatography ‘is derived from Greek words ‘Chroma
‘and ‘graphein’. Chroma means “color” and graphein means “to write” which refers to ‘color writing’. After
the Tswett discovery the concept of chromatography did not evolve more than two decades.
Fig-2 Tswett experiment
In the beginning of 20th century, chromatographic technique has been used only for purifying very small
amounts of natural pigments in solution for spectroscopic studies. For nearly thirty years, chemists remained
reluctant to the use of chromatography. The outcome of the product yield was very less, when they use
chromatographic technique. In 1930, Kuhn and Lederer(4) demonstrated the power of chromatography as a well
preparative method for the separation of α and β carotene from carrots. They have reported the expected yield of
the carotene. Further they also demonstrated and proved by separation and isolation of egg yolk pigments using
calcium carbonate as a stationary phase.
In mid of 1940’s the development of large scale purification schemes was introduced for the separation of rare
earth element. speeding et al(4) has purified the rare earth elements by ion exchange chromatography. They have
separated three rare earth ions like samarium, Neodymium and praseodymium.
In 1941, Martin and Synge(5) has began their work on proteins,
which are made up of amino acids. They were trying to
characterize a particular protein by determining the different
types of amino acids present in it. They were developing a
separation procedure by using a packed column which is an
impregnated silica gel with water packed in a glass tube. They
added acetyl amino acid mixture to the top of glass tube and
poured the chloroform from the top. In this experiment they
could separate acetylproline and acetylleucine. This method
was successful to separate the amino acids as per their partition
coefficients. This process was named as “Partition
chromatography”. In this work they were awarded the nobel
prize in 1952.
In 1944, Martin and James(6) developed the “gas chromatography”(GC). In this technique, mixtures or
substances that can be vaporized or gasified by heat were separated. Helium gas is used to force the mixture
through a column to separate the gaseous components. The growth of liquid chromatography was eclipsed by the
introduction of gas chromatography .
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Between 1950 and 1960, size exclusion chromatography (SEC) became a popular technique in two branches:
The fractionation of synthetic polymers, described as “gel permeation chromatography” (GPC) and in the
resolution of biopolymers termed as “gel filtration chromatography” (GFC).
The real advancement of liquid chromatography began during the late 1960’s .First liquid chromatography
instrument was constructed by Casaba Harwath at Yale university(7), which was termed as “High Performance
Liquid Chromatography”(HPLC). Its principle remained the same as used by Tswett in the first decade of the
20th century, but the results were much improved.
In 1962, Klesper et al(8) introduced the supercritical fluid (small changes in pressure and temperature, fluid
density changed) as a mobile phase in chromatographic separation, it’s growth was relatively slow until the
early 1980’s. This method was named as “supercritical fluid chromatography” (SFC). It is considered as an
intermediate technique between gas chromatography and liquid chromatography. It is used for the analysis and
purification of thermally labile molecule and chiral compounds.
Recently developed chromatography technique is “capillary electro chromatography (CEC)”. The flow of
mobile phase is driven through the column by electric field. This phenomenon known as electroosmosis. The
electro osmotic flow is generated by applying a large voltage across the column. Strain(9), first reported the
use of electro osmotic flow in chromatography.
Fig -3 Development of chromatography over one century.
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Researchers found that the main principles of Tswett Chromatography could be applied in many different
ways, resulting the different varieties of Chromatography such as Thin Layer Chromatography (TLC), Liquid
Chromatography, Gas Chromatography (GC), etc.
1. Leslie S. Ettre , LCGC north America, Milestones in chromatography, volume 22
number 3, March 2004 page 262-270
2. A. Braithwaite, F.J.Smith (1995), Chromatographic methods, 5th edition, Chapter 1
page 2-4
3. Leslie S. Ettre, LCGC Europe, M.S Tswett and the invention of chromatography,
September 2003, page 2-7.
4. Georges Guiochon, Dean G. Shirazi (2006), Fundamentals of preparative and non linear
chromatography, second edition, Elsevier inc, chapter 1, page 3-5.
5. Archer J.P. martin (1952) , The development of partition chromatography, nobel lecture,
December 12,1952. Elsevier publishing company, Amsterdam 1964.
6. Harlod M. MC Nair, James m. Miller, Basics of gas chromatography, second edition, wiley
publication, chapter 1.
7. Lloyd R. Snyder, Joseph J. Kirkland and John W.Dolan (2010) Introduction to Modern
Liquid Chromatography, Third Edition A John Wiley & Sons Inc., chapter 1 page 7-8.
8. 8 Yasuhiko Arai, Takeshi Sako, supercritical fluids: molecular interactions, physical
properties and new applications, springer publication, chapter 4, page 260
9. Keith D. Bartle, Peter Myers (2001), capillary electro chromatography, chapter 1, page 1-3
U. Rampriya
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