Work in Progress (No, really)

Wednesday, October 13, 2010

Ion-Exchange Chromotagraphy

+ No comment yet
Ion-Exchange Chromatragraphy uses differences in the sign and magnitude of the net electric charge of proteins at a known pH. The column matrix is a synthetic polymer (resin says my book) that contains charged groups. Cation Exchangers are the bound groups with anionic groups and those with bound cations are called anionic exchangers.

The affinity of each protein for the charged groups on the column is affected by the pH (which as we know determines the ionization state of the molecule). Furthermore, the concentration of the competing free salt ions in the surrounding solution also affect the affinity.

How to optimize seperation?
  • Gradually changing pH or salt gradient

Cation-Exchange Chromatagraphy
The matrix is contains negatively charged groups and during the mobile phase, proteins with an overall positive charge move through the matrix slower than those with a net negative charge. (Makes sense I guess.)

In our favorite chromotagraphy (Ion-exchange), the expansion of the protein band in the mobile phase is a result of the seperation of protines with different properties and as a result of diffusional spreading.

As the length of the column increases, the resolution of two types of proteins with different net charges usually improves.

As the length of the column increases, the rate of the protein solution flow through the column decreases.

As the amount of time spend on the column increases, the resolution will tend to decline (due to diffusional spreading within each protein band).

So what do I do to finish this weird mess you've made me make?
Proteins of the effluent are collected in test tubes. The ones testing positive for the protein of interest are combined.

This has been a boder-line copy-paste/plagarism of Lehingers Principles of Biochemistry (The cinco edition). Page 86.

Know....errr. Now that I've typed it, I'm hoping it sticks for my test haha

I think the second ones sound better. It's a bit closer to the text and some other obvious reasons. Both are pretty good though.

Post a Comment