Experiment 5

Experiment 5 - Experiment 5: The Determination of the...

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Experiment 5: The Determination of the Molecular Weight of Hemoglobin by Gel Permeation Chromatography Pledge Results and Calculations: In order to determine the molecular weight of Hemoglobin by Gel Permeation Chromatography, a solution of proteins with varying molecular weights was allowed to flow through a GPC column. The column was packed with G-75 and G-25 and a buffer solution was used to keep the column from drying out. A solution of Sephadex containing Blue Dextran 2000,
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Cytochrome C, and Phenol Red was added and each of the components was separated based on size depending on their ability to move through the gel pores. Blue Dextran 2000 was viewed as a distinct narrow band and was the first eluted component. Next, a brown band was seen and determined to be Cytochrome C. Finally, Phenol Red was eluted as a redish-orange broad band. Phenol red was the most intense fraction. Total elution of 44mL of solution took 28 mins. Table 1 shows the retention volume, molecular weight, and the log of the molecular weight. Solution Retention Volume (mL) Molecular Weight (Da) Log(Molecular Weight) Blue Dextran 2000 11 8.0x10^4 4.90 Cytochrome C 17 1.2x10^4 4.08 Phenol Red 29 3.0x10^3 3.48 Hemoglobin 12 47676 4.68 Table 1: Retention Volume, Molecular Weight and Log(MW) of Sephadex Components and Calculated MW of Hemoglobin The retention volume describes the sum of the volumes required to reach the most intense fraction. One mL samples were observed in test tubes in order to notice the most intense fraction for each component. Figure 1 shows a plot of Log(MW) versus Retention Volume. This plot was constructed to obtain the equation for the line of best fit, which would then be used to determine the weight of Hemoglobin. Figure 1: Plot of Log (MW) vs. Retention Volume (mL) Using the equation for the line of best fit, y = -0.0751x + 5.5795, the molecular weight of Hemoglobin was calculated. The y-value is the molecular weight and the x-value is the retention volume. Over the course of 20 minutes, the retention volume of Hemoglobin was found to be 12 mL, giving a molecular weight of 47676 Da. Table 2 lists the calculated MW and the known
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Experiment 5 - Experiment 5: The Determination of the...

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