Separation and Purification Methods

Separation and purification methods Separations and Purifications (OC, BC) • Extraction: distribution of solute between two immiscible solvents o Separates based on solubility o Procedure ▪ 1) Create an aqueous layer and a less dense organic layer (which contains the things you want to extract) ▪ 2) You can use the weak acid to protonate strong bases, causing them to become polar and mix with the aqueous layer ▪ 3) You can use a strong acid to protonate the remaining weak bases, causing them to become polar and mix ▪ 4) You can use a weak base to deprotonate strong organic acids ▪ 5) You can use a strong base to deprotonate weak acids which remain o Phenol-chloroform extraction ▪ Used to separate nucleic acid from cellular proteins, isolates DNA and RNA • Distillation o Separates based on boiling point o Procedure: ▪ 1) Get mixture of two volatile liquids with BPs at least 20 degC apart ▪ 2) Boil the mixture slowly so that the more volatile liquid boils first ▪ 3) First liquid to boil is condensed in a cool tube o Effect of positive deviations to Raoult's Law ▪ This can occur in non-ideal solutions. Predicted vapor pressure of the solution is higher than it should be at a given molar fraction ▪ Causes the solution to boil at a lower temperature than either compound, cannot be separated by distillation o Fractional distillation ▪ More precise method, separates liquids with closer BPs ▪ Vapor is run through glass beads, allowing compound with the higher BP to repeatedly condense and fall back into the solution • Crystallization o Separates based on pure substances forming crystals easier than impure substances o Usually an exothermic process ▪ Why? Same reason Hsoln can be exothermic • IMF's in salts must be broken (endothermic) and bonds with solution form (exothermic) • In this case, bonds that form when creating crystal lattice are more stable than those in the original salts • Chromatography: Basic principles involved in analytic process o Column chromatography ▪ Mobile Phase: solution containing mixture; Stationary phase: column containing polar beads, (usually SiO2) ▪ Procedure • Solid phase traps polar compounds as solution is passed through column ▪ Variants • Gas-liquid chromatography o Mobile phase: mixture dissolved into a heated carrier gas; Stationary phase: liquid phase bound to a column o Procedure ▪ 1) Gas passed over liquid phase ▪ 2) Compounds in mixture equilibrate with liquid phase at different rates ▪ 3) Outputs a graph that shows peaks corresponding with different gaseous components that eluted at different times. Area under each peak corresponds to amount of each molecule eluted o Used for more complex separations • High pressure liquid chromatography o Column and solution system is put under high pressure • Size exclusion o Use beads to separate out molecules based on size, molecular weight o Larger molecules elute first • Ion-exchange chromatography o Separates based on net surface charge • Affinity chromatography o Separates based on highly specific binding interactions o Paper chromatography ▪ Mobile phase: sample spotted on polar paper and nonpolar solvent; Stationary phase: polar paper ▪ Procedure • 1) A mixture is spotted on some polar paper, one end of which is then placed in a nonpolar solvent • 2) Capillary action causes nonpolar solvent to move up paper, dissolving the same as it passes over it • 3) More polar elements of sample are attracted to polar paper, move more slowly up the paper ▪ Rf factor: way of determining polarity of a component, identifying different components 𝐷𝐼𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑒𝑑 𝑏𝑦 𝑐𝑜𝑚𝑝𝑜𝑛𝑒𝑛𝑡 • Rf for a component = 𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑒𝑑 𝑏𝑦 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 • • Nonpolar compounds: Rf close to 1; Polar compounds: lower Rf o Thin-layer chromatography ▪ Like paper chromatography but use coated glass or a plastic plate for stationary phase and visualize results using an iodine vapor chamber Separation and purification of peptides and proteins (BC) o Electrophoresis ▪ Nucleic Acids • Gel electrophoresis o Separates based on charge and size o Electric field causes negatively charged nucleic acids to move. Porous mixture traps larger nucleic acids while smaller nucleic acids can move faster through the matrix ▪ Pores can't be used to separate proteins (they're too big) ▪ Proteins • SDS PAGE o Separates based on size o Denatures proteins and coats them with detergent to give them same charge to mass ratio. Then runs them through electrophoresis • Isoelectric focusing o Separates based on isoelectric points o Gel with a stable pH gradient is stationary phase. Proteins start off at low pH end and travel down this gradient via electrophoresis. Negative cathode end pulls positively charged proteins down gradient. Protein stops moving when it reaches its isoelectric point (no net charge, can't be pulled anymore) • o Qualitative Analysis o Chromatography ▪ Size-Exclusion ▪ Ion-Exchange ▪ Affinity Racemic mixtures, separation of enantiomers (OC) o Differences in crystallization ▪ Enantiomers form crystals that rotate light differently. Visualizing these crystals directly can be used to separate enantiomers o Stereospecific enzymes ▪ Only react with one enantiomer, creating a mixture that can be separated with other separation techniques o Conversion to diastereomers ▪ Enantiomers can be converted to diastereomers. Diastereomers have more different physical and chemical properties that are better for separation