Question

1. Make a table to summarize the liquid chromatography techniques we have learned, including normal phase LC, reversed-phase LC, HILIC, ion-exchange chromatography, affinity chromatography, and SEC/GPC. It should cover general principle/rationale, advantages, disadvantages, etc. for each of them. 2. If you receive an unknown protein to be analyzed (newly made, just a name offered, no relevant information provided or found from website), what do you plan to do using chromatography? 3. Find a chromatography instrument in campus and do some investigation on it. What is it, mode, manufacturer, application, etc.

Answer 1

Chemical reactions usually result in number of impurities, side products. Chromatography is a technique used to separate various types of molecules in a mixture, so that one can get purified material. There are several types of chromatographic techniques based on the molecule to be purified. Molecules are polar, non polar, ionic, high molecular weight and ionic. Each chromatographic technique requies stationary and mobile phases

	General priniciple	Advantage	Disadvantage
Normal phase	Used to purify non polar molecules Stationary phase: Polar (ex: Silica) Mobile phase: Non polar (ex: Less polar organic solvents) Priniciple: Stationary phase absorbs high polar molecules and mobile phase elutes non polar molecues.	Easy to purify non polar molecules by using volatile organic solvents. Low cost technique, since stationary phase is normally silica which is abundant.	Since the stationary phase is polar, water is adsorbed on the suface of the stationaly phase leading to decrease in its activity
Reverse phase	Used to purify polar molecules Stationary phase: Non polar (ex: C-18-silica; ) Mobile phase: Polar (ex: water, buffers) Priniciple: Stationary phase absorbs low polar molecules and mobile phase elutes high polar molecues.	Used to purify high polar molecules like protiens.	Expensive technique as silica is modified to make it as non polar. Difficult to remove the solvent from the product (Lyophilization technique can be used which consumes lot of energy and time)
HILIC	Hydrophilic interaction chromatography (HILIC) is used to seperate water soluble molecules which are often poorly retained under reversed phase HPLC conditions. Mechanism can be considered as normal phase chromatography	Can purify polar molcules when it is difficult to purify with reverse phase chromatography	Shortage of solvents (ex: Acetonitrile); Incompatibility of uv detection system (ex: by using acetone as the solvent)
Ion-exchange	The separation is based on the formation of ionic bonds between the charged groups of biomolecules and an ion-exchange gel/support carrying the opposite charge. Mobil phase: Aqueous buffer system Stationary phase: Inert organic matrix chemically derivative with ionizable functional groups	only one interaction involved in the separation	columns typically are much more expensive than reversed-phase columns
Affinity	Substrate has been bound covalently on stationary phase in such a way that the reactive groups are exposed. When the mixture of proteins passed through the chromatography column, those proteins that have a binding site will bind to the stationary phase, while all other proteins will be eluted	Great technique to purify vaccines and enzymes	Only small volumes can be managed. Lot of skill is required
SEC/GPC	GPC separates molecules by their size. It is mainly used to separate the polymers.	Good separation of large molecules from the small molecules with a minimal volume of mobile phase	Filtration will be difficult if the molar masses of the polymers are too close