The Use of Chromatography in Biochemistry
Year: 2002
Type: article
Abstract: 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 (1Tswett M.S. Physikalisch-chemische studien uber das chlorophyll. Die adsorptionen..Ber. Bot. Ges. 1906; 24: 316-332Google Scholar) 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 α- and β-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. 1We 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. 1We 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.
Source: Journal of Biological Chemistry
Language: English
Subfield: Analytical Chemistry
Field: Chemistry
Domain: Physical Sciences
Open Access status: hybrid
Estimated APC paid: $3,430
OpenAlex ID: W1534527252