Gel Permeation Chromatography

Gel Permeation Chromatography - Add the solution of...

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Andrew Castleman 2/28/11 Gel Permeation Chromatography Gel permeation chromatography is a type of size exclusion chromatography that uses porous gel beads in a column. The premise of separating by size is that smaller molecules will spend more time in the gel beads as they can enter the pores more freely. However, the larger molecules enter the pores less frequently and therefore are eluted earlier than the smaller molecules. The size of the molecule is proportional to the volume of eluent required to remove the molecule, meaning that the fraction number that indicates the volume is directly related to the molecular weight of the molecule. This type of chromatography can be used to estimate the molecular weight of a molecule. By first establishing the volumes required to elute molecules of known molecular weight, an equation can be obtained that can predict the molecular weight of a molecule based on the number of fraction required to elute it. The column should be set up using the porous gel beads.
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Unformatted text preview: Add the solution of proteins of known weight. Then add the eluent and collect fractions. To each fraction, add the blue protein dye. Analyze their absorbance using UV. Then add the hemoglobin to be characterized, and continue eluting until the hemoglobin has been eluted. Then test the absorbance of the hemoglobin and find the peak in its concentration. Compare the peaks of the first two proteins of known weight to the peak of hemoglobin. Figure 1. Based on the peaks of the two known molecules, BSA (66766 Daltons) and Lysozyme (17300 Daltons), it is estimated that the hemoglobin has a molecular weight of 61200 Daltons. The hemoglobin peaked at fraction 33 just one fraction later than BSA. So the estimation of 61200 is near the known weight of BSA. The literature weight for hemoglobin is 68000 Daltons. The experimental estimate is close to the literature weight indicating that is an efficient method for approximating molecular weight....
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