CLASSICS
The Use of Chromatography in Biochemistry
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ARTICLE |
Carotene. VIII. Separation of Carotenes by Adsorption
(Strain, H. H. (1934) J. Biol. Chem. 105, 523-535)
Harold H. Strain received his Ph.D. from Stanford University and
worked most of his career at the Carnegie Institution of Washington,
Plant Biology Division, located on the Stanford campus. He was hired as
an organic chemist specifically to aid in the characterization of
pigments from photosynthetic organisms. In the course of this work, he
developed a procedure for the separation, isolation, and
characterization of carotenes by adsorption chromatography. Adsorption
chromatography was first described in 1906 by the Russian botanist
Michael Tswett (1) who successfully fractionated petroleum ether
extracts of chlorophyll and other plant pigments on narrow glass
columns packed with dry calcium carbonate. Many of the leading chemists
at the time did not value Tswett's chromatographic method and made
little use of it. However, several chemists gradually appreciated its
value, and by the 1930s adsorption chromatography was widely used.
Strain's Journal of Biological Chemistry (JBC) Classic is
an excellent example of how organic chemists applied the principles of
adsorption chromatography first enunciated by Tswett for the separation
of complex mixtures. The power of adsorption chromatography must have
been recognized by those who subsequently went on to develop different
kinds of chromatographic methods that depend on other principles for
separation including partition chromatography, ion exchange
chromatography, gas chromatography, and gel filtration. These methods
provided the tools needed by biochemists in the last half of the
twentieth century to separate and characterize the complex mixtures of
compounds in living things that were present in very small amounts, had
very similar properties, were often extremely labile, and were not
amenable to separation by the conventional methods of the organic chemist.
In this JBC Classic, Strain explores the use of various adsorbents for
the purification of individual carotenes from mixtures. There was
motivation for a new approach because many of the traditional chemical
methods for carotene purification altered them irreversibly, calling
into question the structures of the natural material. Strain tested
various adsorbents including metallic oxides, charcoal, and fuller's
earth. After preliminary testing, he concluded that magnesium oxide had
the most desirable properties for separation of 
- and 
-carotenes
and that the adsorbent did not chemically alter the carotenes being
separated. The experimental description is as useful today as it was in
1934. He reports that using a glass column to hold the adsorbent was
more effective than batch adsorption. He describes packing the columns
and the use of siliceous earth mixed with the adsorbent to increase the
flow rate. He also describes differential elution with solvents of
different polarity. Ultimately, he proves that the carotenes purified
by these methods were pure and unaltered by the purification
technique.1
Robert D.
Simoni,
Robert L.
Hill, and
Martha
Vaughan
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FOOTNOTES |
1
We thank Dr. Arthur Grossman, Carnegie Institute
for Plant Biology at Stanford University, where Strain did his work,
and Pat Craig for providing biographical information and correspondence that she has collected about Strain for a history of the Carnegie Institution that she has written.
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REFERENCES |
| 1.
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Tswett, M. S.
(1906)
Physikalisch-chemische studien uber das chlorophyll. Die adsorptionen.
Ber. Bot. Ges.
24,
316-332
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Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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