The goal if this lab is to simply determine the molecular weight of hemoglobin using size
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
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.
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-
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.
molecules are spherical, which allow them to travel in the stationary phase more fluidly.
Dextran 2000, with a molecular weight of 2 E 6 Daltons is a soluble polysaccharide that is very
Therefore, it cannot enter the Sephadex pores, and it will elute from the column the
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.