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J. Biol. Chem., Vol. 280, Issue 7, 4, February 18, 2005
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Classics
Identification of Small Amounts of Organic Compounds by Distribution Studies. Application to Atabrine (Craig, L. C. (1943) J. Biol. Chem. 150, 33-45)
Identification of Small Amounts of Organic Compounds by Distribution Studies. II. Separation By Counter-current Distribution (Craig, L. C. (1944) J. Biol. Chem. 155, 519-534)
Lyman Creighton Craig (1906-1974) was born on a farm in Iowa. Influenced by his older brother's enthusiasm for chemistry, Craig graduated from Iowa State College in 1928 with a B.S. degree. He remained at Iowa State for graduate school where he majored in chemistry and minored in entomology, partly due to his appreciation for the role of insecticides in agriculture. After earning his Ph.D. in 1931, Craig went to Johns Hopkins University to study with E. Emmet Reid. During his three years at Iowa State and his two years at Johns Hopkins, Craig published twelve papers, mostly in the Journal of the American Chemical Society, on the chemistry of nicotine alkaloids and their insecticidal action.1
These accomplishments earned Craig an appointment in 1933 as a research assistant in chemical pharmacology at the Rockefeller Institute for Medical Research in New York. At Rockefeller, Craig worked on the ergot alkaloids with Walter A. Jacobs, the subject of a previous Journal of Biological Chemistry (JBC) Classic (1). Craig was a gifted experimentalist with skill in equipment design, and his first of many contributions to chemical instrumentation was in 1936 when he designed and built a microdistillation apparatus (2).
Craig was forced to shift his research focus during the war and applied his efforts to aspects of the wartime program on antimalarials. It was during the course of examining whether the antimalarial drug atabrine, or a product of its metabolism, was the active parasiticide in humans that Craig invented a laboratory apparatus for the separation of organic compounds by the technique of counter-current distribution (CCD).
Faced with having to identify small amounts of atabrine and its transformation products in urine and blood, Craig developed a method that could be used to accurately detect microgram amounts of compounds. This method is the subject of the first JBC Classic reprinted here. Milton T. Bush of Vanderbilt University School of Medicine suggested to Craig that the distribution coefficient might be a useful physical constant for the identification and purity determination of an organic compound that could be used along with the melting point and the boiling point. Craig enlarged upon this idea by measuring a series of distribution constants ("the ratio of the weight per cc. in ethylene dichloride solution to the apparent weight so derived for the aqueous layer") for atabrine while progressively diluting the solution with methyl alcohol. He repeated the experiment with several compounds closely related to atabrine. The resulting plots of distribution coefficients versus volume percentage of water in the aqueous phase gave characteristic curves for each compound. Craig then used these standard curves to analyze the blood and urine from patients and dogs receiving atabrine.
A. J. P. Martin and R. L. M. Synge at the Wool Research Laboratory in
Leeds, England used CCD to separate different types of N-acetyl amino
acids, which were prepared by acteylation of the amino acids in acid
hydrolysates of wool. They did this manually in separatory funnels by
transferring the dissolved N-acetyl amino acids partitioned between
the upper and lower phases of two immiscible solvents from one funnel to
another. This then led them to use the principle of CCD to develop partition
chromatography in which one of the liquid phases was immobilized on a column
of gel, and the other phase, the mobile phase, was passed down the column.
Partition chromatography led then to the development of paper chromatography
that opened new worlds to biochemists. Craig, however, decided to build an
apparatus for the separation of mixtures of organic compounds by CCD. In the
second JBC Classic reprinted here, Craig describes an apparatus that could
accomplish, simultaneously, 20 quantitative extractions in a single step. The
device, which was made of stainless steel, consisted of a series of 20 small
separatory funnels (plates) with the capability, after each shaking and
settling, of sliding the top phase from one funnel into the lower phase of the
next. The distribution of the compounds being extracted was based on a
Gaussian curve that could be calculated from the number of plates, the
distribution constant, and the concentration. He tested his apparatus on
-naphthoic acid in an ethylene dichloride-water-methanol solvent system
and showed that the actual performance of the machine was very close to
theoretical predictions. The novelty and originality of this work is attested
by the fact that Craig is the sole author and there are no references cited at
the end of the paper.
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CCD has also been used by other scientists to isolate hormones such as
oxytocin, vasopressin, adrenocorticotrophic hormone (ACTH), growth hormone,
lactogenic hormone, melanophore-stimulating hormone, and parathyroid hormone.
It has also aided in studies on the structure of the tobacco mosaic virus
protein, human hemoglobin 
and chains, angiotensin, and other
biologically active peptides. Also, CCD was used by Robert Holley at Cornell
University to fractionate crude RNA from liver to give a pure tRNA, which was
then fully sequenced for the first time.
In addition to his CCD apparatus, Craig was involved in the design of several other instruments widely used in biochemistry. For example, his rotary evaporator for removal of solvents (5) increased the speed of the process and eliminated the problem of bumping of the solution often encountered in distillation. He also devised ways to use dialysis for the separation of compounds on the basis of size. He developed a dialysis cell in which the entering solution flows over a cellophane membrane, which is stretched or acetylated to vary the pore size (6). Because a molecule's ability to diffuse through a membrane depends on its conformation, Craig also used rotatory dispersion, nuclear magnetic resonance, tritium-hydrogen exchange, circular dichroism, and fluorescent probes to get additional information on the shapes of molecules in solution. This broad approach to studying polypeptide conformation was the principle theme of Craig's research at Rockefeller during the last decade of his life.
Lyman Craig received wide recognition and many honors for his scientific work. He was elected to the National Academy of Sciences in 1950 and received the Albert Lasker Award for Basic Medical Research in 1963. In 1966 he received the Fisher Award in Analytical Chemistry from the American Chemical Society and the Kolthoff Medal of the American Pharmaceutical Association in 1971.
FOOTNOTES
1 All biographical information on Lyman C. Craig was taken from Ref.
7. 
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