Lab Techniques- Chromatography

5.2 Chromatography

Basic features that are present in all types of chromatography:

  1. All are used to separate mixtures of compounds (whether its for identification or for purification)

  2. involves a mobile phase and a stationary phase

Analyte: the substance of interest that will be separated by chromatography

Eluent: a solvent that carries the analyte

Thin Layer Chromatography (TLC)

  • Compounds are separated based on differing polarities

  • Frequently used in OChem labs

    • Used for separating very small amounts of material

    • fast separation

  • TLC is a solid-liquid partitioning technique where the mobile liquid phase ascends a thin layer of absorbent (typically silica) that is coated on a glass plate

    • absorbent acts as a polar stationary phase

    • sample (~1 microliter of sample) is spotted near the base of plate (approx. 1cm from the bottom)

    • plate is then placed upright in a sealed container with a shallow layer of solvent

    • capillary action draw the solvent up the plate (in a process called developing or running)

    • Separation of compounds is due to polarity

      • more polar molecules travel at a slower rate

      • less polar molecules travel at a faster rate

    • Visualization of TLC can be through colors or in the process of iodine vapor or other chemical staining techniques

  • The distance molecules traveled can be measured through Rf (ratio to front) values

    • Calculated by dividing the distance traveled by an individual component by the distance traveled by the solvent front

    • Rf is always positive but is never greater than 1

      Rf compound 1: 26/53 =0.49

Column (Flash) Chromatography

  • Uses same principles as TLC to isolate bulk compounds

  • Column is filled with silica gel and is saturated with chosen organic solvents and mixture of compounds at the top of the column

    • excess solvent is periodically added to the column and the flow of solvent (along with separated compounds is collected at the bottom)

    • More polar molecules travel slower than less polar molecules

    • compounds can be expected to leave in order of least polar to most polar

Ion Exchange Chromatography

  • Used when materials need to be separated based on varying charge states

  • frequently used in the separation of mixtures of proteins

    • because at any given pH proteins within a mixture may exist protonated or deprotonated

  • Solid stationary phase uses a polymeric resin functionalized with either positive or negatively charged moieties on a polymer surface

    • positive charged moieties in resin capture negative charged groups (making them move slower), allowing positive charged and neutral charged groups to pass freely

    • negatively charged moieties in resin capture positive charged groups (making them move slower), allowing negatively charged and neutral charged groups to pass freely.

High Performance Liquid Chromatography (HPLC)

  • Takes advantage of differing affinities of various compounds for a mobile phase or stationary phase

  • Improvement over Flash Chromatography

    • because HPLC forces mobile phase under high pressures, which increases speed and efficiency of separation

  • Pumping unit is where pressurization of the mobile phase first occurs and the sample to be separated is solubilized and injected by a syringe

    • the mobile phase carries the sample to the column to be separated and detected when they exit the column

    • eluent is collected after detection and the components can be isolated after evaporation of solvent if desired

  • Most HPLC contains a nonpolar stationary phase

    • more polar compounds travel faster in HPLC than nonpolar

    • why HPLC is also called reverse phase HPLC

Size Exclusion Chromatography

  • Used to separate bulk materials based on molecular size

  • Uses a column where stationary phase is packed in

    • stationary phase employs chemically inert porous polymer beads and allow for the permeation of small molecules

  • frequently used for the separation of large polymers from small oligomeric fragments or the separation of full proteins from smaller peptide chains

  • Relatively speedy elution (compared to silica chromatography)

  • Not effective at separating different compounds of similar sizes

  • larger molecules elute first (since they travel interparticle instead of intraparticle)

Affinity Chromatography

  • commonly used to purify proteins or nucleic acids from complex biochemical mixtures (growth media, blood, cell lysates)

  • based on highly specific interactions between macromolecules

  • target molecules is trapped on stationary phase, which is then washed to remove the unwanted components of the mixture

    • target protein is then eluted off the solid phase in a highly purified state

  • Isolation of protein involves antibody interactions

    • antibodies are added so that the protein can bind to the antibodies

    • beads and microbe-derived proteins are then added to bind to the antibodies to create a bead-antibody-microbe protein-and target protein complex

    • target is then isolated after centrifuging and decanting the supernatant

  • magnetic beads can also be used in place of centrifuging

    • magnetic beads can bind to target protein

    • magnet is used to collect bead and target protein complex

  • Affinity tags can be used (created through recombinant technology) instead of antibodies

    • popular tag is His tag

Purifying protein using antibody and protein a-linked beads

Affinity Chromatography using beads

Gas Chromatography (GC)

  • form of column chromatography in which partitioning of components to be separated takes place between a mobile gas phase and a stationary liquid phase

    • separation based of various volatilities (boiling points)

  • Sample is loaded into a syringe and injected into a device through a rubber septum

    • sample is then vaporized by a heater in the injection port and carried along by a stream of inert gas (typically helium)

    • sample gas is moved into a column composed of particles that are coated with liquid absorbent

    • each component of sample interacts differently with the absorbent based of relative volatilities

      • less volatile components speed more time dissolved in liquid stationary phase

      • more volatile components spend less time in stationary phase

    • upon exiting the column, components are burned and results in ions that are detected and recorded to determine number of components and their relative amounts

  • smaller hydrocarbons have the lowest boiling point

  • intramolecular hydrogen bonding decreases melting and boiling points