Separations and Purifications

Extraction

• Transfer of dissolved compound from starting solvent into another solvent where product is more soluble
• Based on “like dissolves like”
• Need 2 immiscible solvents (can’t mix)
• Temporarily mixed by shaking so solute can pass between solvents

Example

• Solution of isobutyric acid and diethyl ether → can extract acid with water
  • Both acid and water are polar and soluble
  • Acid transfers to aqueous layer when mixed
• Ether layer = organic phase
• After mixing, layers separate
  • Use separatory funnel to isolate
  • Turn stopcock to drain one layer
• Organic layer usually on top, but can be opposite
  • Position of layers determined by density
• Often repeat draining of aqueous layer to transfer all of acid
• Evaporate solvent using rotary evaporator (rotovap)
• Can also use same principle to remove unwanted impurities
  • Small amount of solute used to extract/remove them
  • This is called a wash

Filtration

• Isolates a solid from a liquid
• Pour mixture onto paper that only allows solvent to pass through
  • Like a coffee filter
• Solid leftover = residue
• Flask full of liquid = filtrate
• Gravity filtration:
  • Uses its own weight
  • Common when the product of interest is in the filtrate
• Vacuum filtration:
  • Solvent forced through by vacuum
  • Solvent kept warm/hot to keep product soluble so it doesn’t precipitate

Recrystallization

• Method for purifying crystals in solution
• Dissolve product in minimum amount of hot solvent, then let it recrystallize
• Solvent chosen so product is soluble at high temp but not at low temp
• When solution cools, product recrystallizes and leaves impurities behind

• Takes advantage of differences in boiling points
• Vapor → condensate drips into vessel → end product = distillate
• How you make ethanol from mixtures with water, since ethanol boils at a lower temp

Simple distillation

• Used to separate liquids that:
  • Boil below 150 ºC
  • Have at least ~25 ºC difference in boiling points
• Prevents compound degradation and minimizes second compound going into distillate
• Setup:
  • Distilling flask with combined liquid solution
  • Distillation column with thermometer and condenser
  • Receiving flask collects the distillate
• Superheating:
  • Happens when liquid heated above its boiling point without vaporizing
  • Gas bubbles can’t overcome atmospheric pressure and surface tension

Vacuum distillation

• Used when you want to distill a liquid with boiling point over 150 ºC
• Lower ambient pressure → decreases temp needed to boil
• Helps avoid degrading product

Fractional distillation

• Separates liquids with similar boiling points
• Fractional column has increased surface area (glass beads, steel wool)
• As vapor rises:
  • Condenses on surfaces and refluxes back down
  • Rising heat causes it to evaporate again
  • Condenses again higher in the column
• Each evaporation–condensation cycle enriches the lower-boiling component
• When vapor reaches top of column, it’s mostly the lowest BP compound and goes into receiving flask

• Uses physical and chemical properties to separate and identify compounds in a mixture
• Place sample onto solid medium (adsorbent)
• Run mobile phase (liquid or gas) through stationary phase
• Compounds adhere to stationary phase with different strengths
  • Migrate at different speeds = partitioning
• Different compounds have different partitioning coefficients
• Often separate based on polarity, but can also use size, charge

Thin-layer and paper chromatography

• Very similar, differ only in medium:
  • Thin-layer: silica gel or alumina on an inert backing
  • Paper: cellulose paper
• Sample placed directly onto adsorbent (spotting)
• Plate is developed by placing it upright in a developing chamber
  • Bottom has a shallow pool of solvent (eluent)
  • Spots must sit above solvent level
• Solvent creeps up plate by capillary action, carrying compounds at different rates
• If using more polar solvent:
  • Polar compounds may not travel as fast
  • Rf values decrease
• When solvent front nears top, plate is removed and dried
• Mobile phase is often an organic solvent that doesn’t bind strongly to gel
  • Nonpolar compounds move more quickly while polar ones stick to gel
  • More nonpolar samples end up further up the plate
  • Less polar compound → moves fastest → highest Rf
  • Compound that travels fastest is also first to elute

  

Reverse-phase chromatography

• Exact opposite of normal TLC:
  • Stationary phase is nonpolar
  • Polar molecules move up plate quickly
  • Nonpolar molecules stick to stationary phase
• Plate is usually white, so:
  • Put under UV light to see spots
  • Or stain with iodine, acid, or vanillin
  • Staining usually destroys ability to recover the compound

Rf (retardation factor)

• $R_f = \dfrac{\text{distance spot moved}}{\text{distance solvent front moved}}$
• Used in preparative TLC:
  • Larger plate with larger spot of sample
  • Sample separates into bands
  • Bands scraped off and washed to isolate pure compounds

Column chromatography

• Column filled with silica or alumina beads as adsorbent
• Gravity pulls solvent and compounds down the column
• Can force solvent through using gas pressure (flash column chromatography)
• Can change polarity of eluent to help elute compounds
• Solvent drips out end of column, and fractions collected over time
• Solvent evaporated, leaving behind compound

Ion-exchange chromatography

• Beads coated with charged substances
  • Attract/bind molecules with opposite charge
• Example: positively charged beads bind negatively charged DNA backbone
• After other compounds pass through, a salt gradient elutes the charged molecules that stuck

Size-exclusion chromatography

• Beads contain tiny pores of varying size
• Small compounds enter pores and are slowed down
• Large compounds can’t enter and travel around beads → move faster
• Vary pore size to tune for different molecular weights

Affinity chromatography

• Protein of interest bound by column with high affinity for that protein
• Beads coated with receptor or specific antibody for protein
• Stationary phase can also be metals like nickel for engineered proteins
• To elute:
  • Wash column with free receptor/target/antibody
    • Competes with bead-bound receptor and frees protein
  • Or use eluents with varying pH or salinity to disrupt bonds
• Recovered substance can sometimes bind to eluent
  • If eluent is something like an inhibitor, it may be hard to remove

Gas chromatography (GC) / vapor-phase chromatography (VPC)

• Eluent is a gas instead of liquid
• Adsorbent is crushed metal or polymer in a long (e.g., ~30 ft) column
• Column is coiled inside an oven to control temp
• Mixture injected and vaporized
• Gaseous compounds travel at different rates based on how strongly they adhere to adsorbent
• Separate in space by the time they reach end of column (retention time)
• Injected compounds must be volatile:
  • Low melting point
  • Sublimable solids or vaporizable liquids
• Compounds registered by detector
• Often:
  • Separate by GC, then inject into mass spectrometer (GC–MS)
  • Mass spectrometer:
    • Ionizes and fragments compounds
    • Separates fragments by mass-to-charge ratio
    • Used to determine molecular weight

High-performance liquid chromatography (HPLC)

• Eluent is liquid, passing through a packed column
• Very similar to column chromatography, but more controlled
• Sample injected into column and separates as it flows through
• Compounds pass a detector and are collected as solvent exits
• Interface is computerized, similar to GC
• “High performance” comes from computer control of:
  • Solvent gradients
  • Temperature