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  Home > JCE Print > Journal of Chemical Education > Issues > 2008  > September  >
In the Laboratory
A Static Method as an Alternative to Gel Chromatography
An Experiment for the Undergraduate Biochemistry Laboratory
Alex D. Burum and Allan G. Splittgerber
Department of Chemistry, Gustavus Adolphus College, St. Peter, MN 56082
Cover
September 2008
Vol. 85 No. 9
p. 1257

Abstract
This article describes a static method as an alternative to gel chromatography, which may be used as an undergraduate laboratory experiment. In this method, a constant mass of Sephadex gel is swollen in a series of protein solutions. UV–vis spectrophotometry is used to find a partition coefficient, KD, that indicates the fraction of the interior space of the gel bead available to the protein molecule. For a large protein that penetrates the bead pores only marginally, the partition coefficient will be small. For a small protein that penetrates substantially into the interior pore space, the partition coefficient will be large. In practice, a constant weighed quantity of dry gel is swollen in a series of protein solutions of different volumes. After equilibration, the supernatant is sampled and the absorbance at 280 nm is measured and compared to the absorbance of the original protein solution. A linear plot is made according to equations developed in this article, and the partition coefficient is found from the intercept. The method is reliable so long as sufficient care is taken in sample preparation.
Supplement
Student handouts; Instructor notes
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Citation
Burum, Alex D.; Splittgerber, Allan. G. J. Chem. Educ. 2008, 85, 1257.
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Keywords
Biochemistry; Chromatography; Hands-On Learning / Manipulatives; Instrumental Methods; Laboratory Equipment / Apparatus; Laboratory Instruction; Proteins / Peptides; Separation Science; Upper-Division Undergraduate; UV-Vis Spectroscopy
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History
Created:
Last Updated:
8/4/2008
8/4/2008
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Experiments, laboratory exercises, lecture demonstrations, and other descriptions of the use of chemicals, apparatus, instruments, computers, and computer interfaces are presented in the Journal of Chemical Education as illustrative of new or improved ideas or concepts in chemistry instruction and are directed at qualified teachers. Although every effort is made to assure and encourage safe practices and safe use of chemicals, the Journal of Chemical Education cannot assume responsibility for uses made of its published materials. Many chemicals are hazardous. Precautions for the safe use of hazardous chemicals and directions for their proper disposal are described in the Material Safety Data Sheets and on the labels. We strongly urge all those planning to use materials from our pages to make choices and to develop procedures for laboratory and classroom safety in accordance with local needs and situations.
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