Separation and Purification Techniques

Extractions

important that the two solvents are immiscible; organic layer and aqueous layer - position of layers determined by relative densities (usually organic layer on top)

Rotary Evaporator

evaporating the solvent

Filtration

isolates a solid from a liquid

Gravity filtration

solvent's own weight pulls it through the filter, is more commonly used when the product of interest is in the filtrate

vacuum filtration

Solvent is forced through the filter by a vacuum connected to the flask, more often used when the solid is the desired product

Recrystallization

method for further purifying crystals in solution, dissolve product in minimum amount of hot solvent and let it recrystallize as it cools

Distillation

takes advantage of different boiling points; liquid with lower boiling point will evaporate first and condense in a water-cooled condenser and drip into a vessel - called the distillate

Simple Distillation

should only be used for liquids that boil below 150 degrees Celsius and have a boiling point difference of at least 25 degrees Celsius

What is superheating?

when a liquid is heated to a temperature above its boiling point without vaporization, gas bubbles within a liquid are unable to overcome the combination of atmospheric pressure and surface tension

Vacuum distillation

for liquids with bp over 150, lower the ambient pressure using a vacuum to decrease the temperature at which liquid must reach in order to have sufficient vapor pressure to boil

Fractional Distillation

to separate liquids with bp less than 25 degrees Celsius apart; uses fractionation column where the surface area is increased by the inclusion of inert objects (glass beads, steel wool); vapor pressure rises and condenses back down on the column continually with the liquid that has the lower bp reaching top of column first

Chromatography

separates compounds based on how strongly they adhere to the solid, or stationary, phase, or how easily they come off with the mobile phase

Thin-layer Chromatography

separation technique that uses silica gel (polar) and a nonpolar, organic eluent which leads to nonpolar substances traveling further up the silica gel

Paper Chromatography

separation technique that uses cellulose (polar) as stationary phase and an organic solvent

Rf Calculation

(distance spot moved) / (distance solvent front moved)

Reverse-phase Chromatography

Stationary phase is nonpolar while the mobile phase is polar, so polar molecules move up the plate more quickly

Column Chromatography

entire column filled with silica or aluminum beads as an absorbent; uses gravity to move the solvent and compounds down the column

ion Exchange chromatography

Beads in the column are coated with charged substances so that they attract or bind compounds with the opposite charge; after all other compounds move through the column, a salt gradient is used to elute the charged molecules that are stuck to the column

Size-exclusion Chromatography

Beads in the column contain tiny pores of varying sizes; allow small compounds to enter the beads, which slows them down.

Common approach in protein purification

Using ion exchange chromatography followed by size-exclusion chromatography

Affinity Chromatography

protein of interest is bound by creating a column with high affinity for that protein; coating beads with a receptor that binds the protein or a specific antibody to the protein - common stationary phase is nickel, eluted by washing the column with a free receptor that competes with bead-bound receptor

Gas Chromatography (Vapor-phase Chromatography)

eluent is a gas (usually helium or nitrogen), absorbent is a crushed metal polymer inside a 30-foot column with the column coiled and kept inside an oven to control its temperature; travel through column at different rates because they adhere to the absorbent in the column to different degrees and separate in space and time

High Performance Liquid Chromatography (HPLC)

Eluent is liquid and travels through a column of defined composition

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Stationary Phase varies depending on the compound of interest

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Sample is injected into column and liquid passes through the adsorbent, can do so under low pressures

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Compounds pass through a detector and are collected as solvent flows through the end of the apparatus

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Entire process is computerized -

\*temperatures and solvent gradients can be applied to a compound to help resolve various compounds in the sample\*

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\*higher performance as entire process is computerized\*