Gel Filtration Lab

SCHA 319

Gel Filtration

Basis of the Experiment

In this experiment the student is introduced to the use of gel filtration as a method of separation. The principles of this method are illustrated and molecules of different sizes are separated on macroporous materials.

Theory

Gel filtration is one of the two size exclusion chromatography methods commonly encountered in chemical labs and is similar is PAG Electrophoresis. The other method is gel permeation chromatography and the difference between the methods lies in the nature of the mobile phase used to carry out the separation. Gel filtration uses a polar mobile phase, often an aqueous solution, and is used in the separation of biologically important molecules whereas gel permeation a non polar mobile phase is used and is more commonly used in the separation of synthetic macromolecules (polymers). In both methods a porous bead is used in the separation mechanism. The size of these pores is the key to the separation process. Sephadex, along with other materials, is commonly used in gel filtration. Sephadex gels are synthesized by the cross-linking of soluble dextrans and the conditions of this process control the size of the pores in the bead. The gels are then sold with different molecular weight ranges. Sephadex has the disadvantage of being compressible and not suitable for work under pressure. This lab has been modified to use a silica based stationary phase. It is made up of porous silica microspheres with a surface stabilized by zirconia to make it more chemically rugged.

The solid material is packed into an HPLC column and eluted with a buffered aqueous solution. The total volume of the system is expressed as equation 1,

Vt = Vo + Vi + Vg + Vec (1)

where Vt is the total system volume, Vg is the volume occupied by the support itself, Vi is the volume accessible to the mobile phase that is internal to the beads, Vec is the volume between the injector and the column and from the column to the detector (you may assume it is small and may be ignored) and Vo is the volume outside of the beads. Very large, high molecular weight, compounds will not fit into the pores of the gel and therefore will be excluded from the inner volume of the resin bed. The molecules will pass through the column with the void volume (Vo or Vm). Very small molecules will be able to pass into the inner volume of the resin and will therefore elute from the column at the sum of the void and inner volumes. Molecules of intermediate molecular size will be able to pass into some pores but not others and will elute at the volumes between the two extremes. The fraction of pores that a given compound can gain access is represented by the letter K. Therefore an expression for a compound’s elution volume, Ve, will be

Ve = Vo + KVi (2)

K will fall between 0 and 1.

K = 1 for very small molecules.

K = 0 for very large molecules.

As mentioned above, gels come in a variety of pore sizes and are usually characterized by the following parameters.

1. Exclusion Limit: molecular weight (size) above which a molecule is totally excluded from the

gel and elutes at Vo. (K = 0)

2. Total permeation limit: molecular weight (size) below which a molecule can freely move

throughout the porous gel elutes at Vo + Vi. (K = 1)

3. Working range: molecular size range in which a molecule may penetrate some but not all of

the gel interior.

The value K can be considered similar to the partition coefficient in ordinary partition chromatography and this value will be constant for a given compound and is independent of physical column dimensions. If the value of K is found to be larger than 1 this would indicate that there is another mechanism (i.e. surface/solute interaction etc.) other than that size exclusion involved for that analyte.

Apparatus and Reagents

You will assist in the development of the method. Discuss what you will need to do with the instructor.

HPLC System: HP 1090 or Agilent 1100

Column: Zorbax GF 250 Ref Material

Mobile Phase: 0.200 M Phosphate pH 7.00 Use 100% Bottle C

Detection: 280 nm

Flow Rate: 2.00 mL

Standard Mix: Biorad 151-1901

Thyroglobulin MW 670,000

Gamma Globulin MW 158,000

Ovaalbumin MW 44000

MyoglobulinMW 13,500

Vitamin B12MW 1350

Unknown

Protein - Prepare a solution that is about 1 mg/mL

Data Work Up:

Prepare a plot of log MW vs retention volume.

Estimate the MW of your unknown protein from the plot above.

Prepare a Table with the protein name, retention time and associated K value.

Prepare a Table with the various volumes for your separation system. Estimate Vt from the dimensions of the column.

Questions:

1)What would be the retention of a very large molecule such as blue dextran (MW 2,000,000 Daltons)?

2)What would be the retention of a very small molecule such as aspartame?

3)What would be the retention time for a protein with a formula weight of 75,500 Daltons

Notes:

This work will be passed in with the data collected using electro-spray ionization Mass Spectrometry molecular weight. This will show you an alternate way of FW determination.

Use the web tutorial to assist you in the calculations needed for this lab.

To Shut Down the system.

Run 100% water (Bottle A) for 10 minutes.

Be sure to turn off the pump and detector.

Shut off the air tank (drives the auto sampler)