Separations and Purifications

Solubility-Based Methods

  • Extraction: the transfer of a dissolved compound (desired product) from a starting solvent into a solvent where the product is more soluble
    • Simplest ways of dissolving and is based on the concept that like dissolves like.

  • Two solvents must be immiscible – form two layers and do not mix.
    • Two layers are temporarily mixed by shaking so that the solute can pass from one solvent to another.
    • Aqueous phase: polar layer, usually water
    • Organic phase: nonpolar layer
  • Separatory Funnel: equipment used to isolate the two phases after they have been given time to spread out again.
    • Denser layer sink to the bottom due to gravity, and can be removed.
    • More common for organic layer to be on top, but ultimately depends on density.
    • Process is repeated several times to ensure all of the desired product is extracted
  • Once the desired product has been isolated, it can be obtained by evaporating the solvent by using a rotary evaporator.
  • Wash: reverse of the extraction process in order to remove unwanted impurities.
    • Small amount of solute is used to extract and remove impurities

  • Isolates a solid from a liquid
    • Solid is called the residue and the flask full of liquid is called the filtrate.
  • Gravity Filtration: solvents own weight pulls it through the filter and is most commonly used when the filtrate is the desired product.
  • Vacuum filtration: solvent is forced through the filter by a vacuum connected to the flask, used when the solid is the desired product.

  • Used to further purify crystals in solution
  • Product is dissolved in a minimum amount of hot solvent and is cooled down to let it recrystallize.
  • Solvent should be chosen so that the solute (product) is only soluble at high temperatures


  • Used when product is a liquid that is soluble in the solvent.
  • Takes advantage of differences in boiling point to separate two liquids by evaporation and condensation.
    • Liquid with the lower boiling point will vaporize first, and the vapors will rise up into the distillation column to condense in a water cooled condenser
      • Forms a condensate which drips down into the vessel and forms the distillate

Simple Distillation
  • Same technique as above and should only be used to separate liquids that boil below 150 degrees Celsius and have at least a 25 degree’ difference.
  • Apparatus consists of a: distilling flask (contains the combined liquid), distillation column (thermometer and a condenser), receiving flask (collects distillate).
  • Superheating: when a liquid is heated to a temperature above boiling point but does not vaporize. Gas molecules stay trapped within liquid due to surface tension and atmospheric pressure.

Vacuum Distillation
  • Used whenever liquid has a boiling point above 150°C
  • Vacuum lowers the ambient pressure, which subsequently lowers the boiling points
  • Higher temperature may degrade the product

Fractional Distillation
  • Used to separate liquids with similar boiling points
  • Fractionation column connects the distillation flask to the condenser.
    • Increases the net surface area by including inert objects on the column
    • Vapor condenses on these surfaces and refluxes back down
    • Each time the condensate evaporates, the vapor consists of a higher proportion of the compound with a lower boiling point
  • Only the desired product drips down to the receiving flask once it goes through the “rungs” of the column.


  • The more similar a compound is to its surroundings, the more it will stick to and move slowly through its surroundings.
  • Sample is placed onto a solid medium (stationary phase), a liquid or a gas (mobile phase) will then run through the stationary phase.
    • Displaces (elutes) the sample and carries it through the stationary phase.
  • Partitioning then occurs after the sample is eluted. The mobile phase will adhere to the stationary phase with differing strengths which causes the substances to move at different speeds.
    • Represents an equilibrium between two phases
    • Different compounds will have different partitioning coefficients and will elute at different rates.
      • Results in the separation within the stationary phase and allows for the isolation of each substance individually
  • Different media can be used for the stationary phase and the property most commonly exploited on the MCAT is polarity.

Thin-Layer and Paper Chromatography
  • Two above only vary in the medium used for the stationary phase. Thin-layer uses silica gel or an alumina adherent to an inert carrier sheet is used. Paper chromatography uses paper (cellulose)
  • Spotting: sample that is to be separated is placed directly onto the adsorbent itself
  • Developed: adsorbent is placed upright in a developing chamber (usually a jar or beaker)
    • Eluent: shallow pool of solvent at the bottom of the jar.
  • Solvent will then creep up the plate by capillary action and carry various compounds in the sample with varying rates. When the solvent reaches the top of the plate, the plate is removed for drying.
  • In TLC, silica gel is polar and hydrophilic while mobile phase is usually a moderately polar organic solvent.
    • Non-polar compounds dissolve in the organic solvent and move quickly as the solvent moves up the plate
    • The more nonpolar the sample is, the further up the plate it will move.
  • Reverse-Phase Chromatography: Stationary phase is instead nonpolar so that polar molecules move up the most.
  • Retardation factor: compounds are identified using this factor. Relatively constant for a particular compound in a given solvent
    • Rf = distance spot moved/distance solvent front moved
  • Most frequently performed on a small scale. Preparative TLC is a larger scale means to purify substances.

Column Chromatography
  • Uses an entire column filled with silica or aluminum beads as a adsorbent, which allows for greater separation.
  • Uses gravity as opposed to capillary action to move the solvent and compounds down the column.
  • Flash Column: solvent can be forced through the column using gas pressure.
  • Solvent eventually drips out of the end of the column and the different fractions that leave the column can be collected.
    • After collection, the solvent can be evaporated to leave behind the substance of interest.
  • Can be used to separate and collect macromolecules such as proteins and nucleic acids (biochemistry)

Ion-Exchange Chromatography
  • Beads in the column are coated with charged substances so that they attract or bind compounds.
    • A positively charged compound will attract and hold a negatively charged backbone of DNA or protein as it passes through the column.
  • Salt gradient can be used to elute the charged molecules that have stuck

Size-Exclusion Chromatography
  • Beads used in the column contain tiny pores of varying sizes.
    • Pores allow small compounds to enter the beads and thus slows them down.
  • Small compounds are slowed down and retained longer

Affinity Chromatography
  • Protein of interest is bound be creating a column with high affinity for that protein.
    • Accomplished by coating beads with a receptor that binds to the protein or a specific antibody to the protein.
  • Protein can be eluted by washing the column with a free receptor
    • Or Eluents can be created to alter the pH or salinity levels that disrupts the bonds between the ligand and the protein of interest
      • Recovered substance can be bound to the eluent, which can be difficult to remove.

Gas Chromatography
  • GC is also known as vapor-phase chromatography (VPC). Main difference between it and other methods is that the eluent is a gas. Instead of a liquid.
    • Adsorbent is crushed metal or polymer which is coiled into a column and placed in an oven to control the temperature.
  • Gaseous compounds travel through the column at different rates since they adhere to the adsorbent in the column at different degrees.
  • Injected compounds must be volatile: low melting-point, sublimable solids or vaporizable liquids.
  • Common to separate molecules using GC and then inject the pure molecules into a mass spectrometer for molecular weight determination.
    • Mass spectrometry: the ionization and fragmentation of compounds which then runs the fragments through a magnetic field which separates the masses based on charge.
      • Relative concentration of the different fragments can be calculated and compared against reference values to identify the compounds.

High-Performance Liquid Chromatography
  • Liquid eluent travels through a column of defined composition.
  • Needed high pressure before but not anymore.
  • Small sample is injected into the column and separation occurs as it flows through. The compound then passes through a detector and is collected as the solvent flows.
  • Difference is that the process is computerized by the detector
    • Allows for sophisticated solvent/temperature gradients can be applied to the column to help resolve the various compounds in the sample.