Experiment 5 - Gel Permeation - Analytical Chemistry 241

Experiment 5 - Gel Permeation - Analytical Chemistry 241 -...

Info iconThis preview shows pages 1–4. Sign up to view the full content.

View Full Document Right Arrow Icon
Experiment 5: The Determination of the Molecular Weight of Hemoglobin by Gel Permeation Chromatography Flora Vo Lab Partner: Taylor Scott 26 March 2007 TA: BJ Privett Chem241L Section 410: Room 401 Thursday 1 PM – 4 PM Pledge: I pledge that no unauthorized assistance has been given or received in the completion of the work presented in this report. ________________________________
Background image of page 1

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Introduction The goal if this lab is to simply determine the molecular weight of hemoglobin using size exclusion chromatography. The student must first learn to build a column using Sephadex, and she then be able to calibrate the column using molecules of known molecular weights. Gel permeation chromatography separates molecules according to size, which explains this technique’s multiple names: size exclusion, molecular exclusion, and molecular sieve chromatography. This analytical technique uses an exclusion column, and it is filled with a spherical stationary phase. This stationary phase is hydrated, which means it possesses many hydrogen containing bonds, and it is also porous. This lab employs Sephadex particles, which is a highly crossed-link polysaccharide. This cross-linking is the basis for the porous quality of Sephadex, and it also turns the polysaccharide chains into three-dimensional spheres. The cross- linking is also regular, since this polysaccharide is just a long, repeated sugar molecule, and this regularity creates pores that are relatively uniform. The pore size range of Sephadex is 3000- 80000 Daltons. The above characteristics of the stationary phase allow the mobile phase to be separated according to size. The mobile phase consists of several spherical molecules of known weights. The molecules are spherical, which allow them to travel in the stationary phase more fluidly. Blue Dextran 2000, with a molecular weight of 2 E 6 Daltons is a soluble polysaccharide that is very large. Therefore, it cannot enter the Sephadex pores, and it will elute from the column the fastest. Cytochrome c, with a molecular weight of 12,327 Daltons, is too large for some pores and too small for other pores. As a result, it will elute from the column at an intermediate time. Last, Phenol Red, with a molecular weight of 354.38 Dalton. It is small enough to fit into almost every pore, so it will stay in the column much longer than the other two molecules. When the
Background image of page 2
compounds separate in the exclusion column, there will be a brown band (Phenol Red) on top, an orange band (Cytochrome c) in the middle, and a blue band (Blue Dxtran 2000) on the bottom. These three compounds and their respective retention times will be used to construction a
Background image of page 3

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Image of page 4
This is the end of the preview. Sign up to access the rest of the document.

This lab report was uploaded on 03/31/2008 for the course CHEM 241 taught by Professor Tiani during the Spring '08 term at UNC.

Page1 / 9

Experiment 5 - Gel Permeation - Analytical Chemistry 241 -...

This preview shows document pages 1 - 4. Sign up to view the full document.

View Full Document Right Arrow Icon
Ask a homework question - tutors are online