Separation and Purification

Distillation

the action of purifying a liquid by a process of heating and cooling

simple distillation requires compounds to have boiling points that differ by at least 25 C

Fractional Distillation

can be used to separate miscible solvents by using the difference in boiling points of the two liquids when the difference in boiling points is less than 25C

fractionation column improves separation by providing more surface area for condensation and evaporation (enables liquid to spread out, making it easier to boil)

fractionation column is packed with glass beads or steel wool which increases the surface area for condensation and evaporation

Volatile Compounds

can easily evaporate since they usually have lower boiling points

Liquid - Liquid Base Extraction

Polar compound on the bottom; non polar compound on the top

Acid Wash/Base Wash

-NH2 → -NH3+/COOH → COO-

Both modifications may allow for a compound to be more soluble in aqueous solutions, without really changing the compound itself

Keys of Chromatography

Stationary Phase: solid support

Mobile Phase: solvent moving past stationary phase

the more the compound of interest interacts with the mobile phase the faster it moves; the less it interacts with the mobile phase, the slower it moves

uses polarity and charge to separate compounds from a complex mixture

Paper Chromatography

-polar stationary phase (water absorbed on paper)

-nonpolar mobile phase

-poor separation

-fragile

Thin Layer Chromatography

-polar stationary phase (silica)

-nonpolar mobile phase

-better separation

-rigid support

Retention Factor R_^f

distance traveled by molecule/distance traveled by solvent

the higher the mobility (far distance traveled), the more similar the compound is to the mobile phase

Reverse-phase Chromatography

Stationary Phase: non-polar (C18)

Mobile Phase: polar

Column Chromatography

-stationary phase is most often a viscous liquid that’s coating solid beads

-properties of the liquid phase can vary, depending on the type of chromatography being done

-mobile phase is an inert gas, the sample, usually in some liquid form is also vaporized into gas

-ability for the sample to be vaporized, or the compound’s boiling point also dictates the order of elution

-allows for greater separation than TLC

Gel Filtration

separate by size

Affinity Chromatography using antibodies

Conjugating, or coupling, antibodies to beads, then adding in a mixture of proteins will allow for the isolation of the protein that’s the proper antigen to the antibody

most specific and efficient method for isolating proteins, it can be tough to create antibodies for a protein of interest

Affinity chromatography using engineered tag

-add 6 sequential histidine residues to the end of the protein sequence

-His6 has high affinity for NI2+

Ion Exchange Chromatography

net charge of molecule binding to column

-anion exchange → negative (anions) → COOH-

-cation exchange → positive (cations) → NH3+

pH of solution will affect the charge state and its ability to bind to the column

buffer used

-anion exchange → pH above pI of molecule

-cation exchange → pH below pI of molecule

Slowly increasing the concentration of a buffered salt solution will allow the ions to compete for binding sites

Less charge compounds will elute in low salt concentrations, and the more charged compounds will elute in high salt concentration