Science Blog for 09-01/09-02
I was considering continuing on with what my students in Adv Genetics will be doing but, to me, it’s a bit too simple. The phenol/chloroform was relatively simple but their next task will be to run a gel and I think I can skip that one.
Instead I thought I would touch on some of the topics my Biochemistry course is inflicting upon us. I thought Chromatography would be perfect. There are a LOT of type of Chromatography so I can make a relatively simple summation of the lot of them while essentially counting it as Biochem study as well.
Chromatography can be described as a type of laboratory techniques (of various flavors) in which a solution is fractionated or separated out based on some property.
- Mobile Phase: The mixture of substances dissolved in a liquid that will be fractionated
- Stationary Phase: a porous solid matrix in which the liquid will be passed through, often coupled with binding components
Partition Coefficient: the ratio of concentrations of a compound in a mixture; measure of the difference in solubility of a compound in two immiscible phases at equilibrium (represents stationary and mobile phases)
Analyte: target substance to be separated
Chromatogram/Chromatograph: a graph in which the x-axis represents retention time and the y-axis is a signal (from a spectrophotometer or mass spec) in which the signal is proportional to the concentration of the specific analyte separated
Eluate: the mobile phase exiting the column
Eulent: the solvent that carries the analyte
Retention Time: the time it takes for a particular analyte to pass through the system
In Column Chromatography (the only one I know anything about), the stationary phase exists in a tube (column) filling it entirely [packed] or concentrated on or along the inside of the tube well [open tubular column]. Solutes flow through the column as the mobile phase and are slowed due to interactions with the stationary phase. With a long column, portions of the mobile phase that have higher rates of migration will be separated first, followed by those with slower rates of migration.
Gel Filtration/Size Exclusion/Molecular Sieve Chromatography
This is a common form of chromatography in which the analyte is separated according to size and shape. In this case the analyte does not have interact with the stationary phase chemically/biochemically but only physically. This method is based on the ability of the analyte to penetrate and a pass through the gel bead stationary phase. Large molecules will elute from the column quicker than small particles that pass through pores of the gel beads. The smaller molecules will require a larger amount of eluent to elute.
In this form of chromatography charged molecules bind to oppositely charged groups that have been attached to the stationary phase matrix. Anions will bind to cationic groups (anion exchangers) and cations will bind to anionic grounds (cation exchangers). Polyelectrolytes, which are polyionic polymers with both positive and negative charges, will bind both cation or anion exchangers.
The binding affinity of the protein passing through can be complicated by the presence of other ions that may compete for the binding along with pH which will influence the protein’s net charge depending on the protein’s pI (Isoelectric point – point at which it has a net zero charge profile). Proteins that successfully bind can be eluted by an eluent with a higher salt concentration (salt = ions) or a pH that reduces the affinity with which it is binding the cation/anion exchanger.
Hydrophobic Interaction Chromatography
In this case the stationary phase matrix possesses octyl, butyl, or phenyl groups. These hydrophobic groups will interact with the hydrophobic groups of the proteins passing through. A buffer with high ionic strength, usually ammonium sulfate, is first passed through which reduces the solvation of the solutes and permit hydrophobic regions to be adsorbed. The high salt conditions promote hydrophobic interactions and aggregations. The greater the hydrophobicity of the molecule the less salt is required for promoting binding and the salt concentration can be gradually decreased to elute from the column in order of increasing hydrophobicity. Concentrations of detergent, which will disrupt the hydrophobic interactions, or changes in pH can also be used to elute.
My ‘favorite’ due to the fact that it is so specific, in this case a ligand attached to the matrix will specifically bind to a protein while all other proteins are washed out of the column. Elution conditions will have to be adjusted to release the binding. One thing to consider is that the binding has to be weak enough to be disabled (so biotin-streptavidin is probably not a good idea). Immunoaffinity uses Abs for this purpose. Metal chelate affinity involves diavalent metal cation (Zn2+) or (Ni2+) is attached so proteins with metal chelating groups (His tags) can be retained.
I am now distracted so I think that is good enough J