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241L final review sheet

241L final review sheet - Chem 241L Review Experiment 5 The...

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Chem. 241L Review Experiment 5: The Determination of MW of Hemoglobin by Gel Permeation Chromatography Important Concepts: The Stationary phase particles are highly hydrated, contains range of pore sizes, and they are assumed to be spherical Small molecules(Phenol Red MW 3,000 Da) are retained the longest, and K= 1 Large particles (Dextran Blue MW 80,000 Da) are eluted first, and K=0 The range for K it from 0 to 1. Molecules that fit in the range for the column will have a K in this range Volume is proportional to time The column is packed with Sephadex (cross-linked Dextran) Void volumes vary from column to column based on how they are made Elution time is dependent on log of MW Objective: to set up a gel permeation column and use it separate three molecules (Dextran Blue 2000, Cytochrome C and Phenol Red) to create a calibration plot, and then to test a sample of the Hemoglobin and determine its molecular weight from the calibration plot. Procedural Things: 30 test tubes marked to 1 mL mark, used to catch elution from column. Add up the test tubes to know the amount eluted for each sample. Collect on a basis of color Make sure that there is always enough buffer solution at the top of the column, if there is not the gel will crack and the column will not work. A layer of G-25 slurry is places on top of a long layer of G-75 slurry to create the column Total volume of the column is 30 mL Collect the first part of the solution, before the colors start to elute in a graduated cylinder Same procedure for Hemoglobin. The test tube with the most intense color is the test tube of interest The G-25 slurry can be poured down the drain, the G-75 gel phase should be poured back into the container.
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