9-10. Chromatography, Electrophoresis and Quality Control

• Instrumental analysis

  • a field of analytical chemistry that investigates analytes using scientific instruments
  • Block diagram of an analytical instrument showing the stimulus and measurement of response.
• Chromatography

  • is the separation of the components of a mixture based on the different degrees to which they interact with two separate material phases
• CHROMATOGRAPHY

  1. Mobile phase – moving phase
  2. Stationary phase – does not move
• CHROMATOGRAPHY

  • The mixture (sample) is usually introduced into the mobile phase, which is then made to move through the stationary phase.
  • The components are attracted to and slowed by the stationary phase to varying degrees, and as a result, they move along with the mobile phase at varying rates, and are thus separated
• CHROMATOGRAPHY

  • The mobile phase can be either a gas or a liquid,
  • while the stationary phase can be either a liquid or solid
• CHROMATOGRAPHY

  • All techniques which utilize a gas for the mobile phase come under the heading of gas chromatography.
  • All techniques that utilize a liquid mobile phase come under the heading of liquid chromatography
• Partition Chromatography

  • The mobile phase is a liquid that moves through a liquidstationary phase as the mixture components partition or distribute themselves between the two phases and become separated.
• PARTITION CHROMOTOGRAPHY

  • The separation mechanism is thus one of the dissolving of themixture components to different degrees in the two phasesaccording to their individual solubilities in each.
• Partition Chromotography

  • This form of chromatography is based on a thin fim formed on the surface of a solid support by a liquid stationary phase.
  • Solute equilibriates between the mobile phase and the stationary liquid.
• Partition Chromatography

  • The stationary phase actually consists of a thin liquid film chemically bonded to the surface of finely divided solid particles
  • Such a stationary phase cannot be removed from the solid substrate by heat, reaction, or dissolving in the mobile phase.
• Partition Chromatography

  • The mobile phase can also be a gas
• Partition chromatography

  • a type of chromatography in which the stationary phase is a liquid chemically bonded to the surface of a solid substrate, while the mobile phase is either a liquid or gas.
• Partition Chromatography

  • The mixture components dissolve in and out of the mobile and stationary phases as the mobile phase moves through the stationary phase, and separation occurs as a result
• Adsorption Chromatography

  • The separation mechanism is adsorption
• Adsorption Chromatography

  • is probably one of the oldest types of chromatography around.
  • It utilizes a mobile liquid or gaseous phase that is adsorbed onto the surface of a stationary solid phase.
  • The equilibriation between the mobile and stationary phase accounts for the separation of different solutes.
• Adsorption Chromatography

  • The stationary phase consists of finely divided solid particles packed inside a tube, and the mixture components adsorb or stick to the surface of the solid
• Adsorption Chromatography

  • The nature of the adsorption involves the interaction of polar molecules, or molecules with polar groups, with a very polar solid stationary phase.
  • The mobile phase can be either a liquid or a gas
• Ion Exchange Chromatography

  • It is a method for separating mixtures of ions, both inorganic and organic.
• Ion Exchange Chromatography

  • The stationary phase consists of very small polymer resin beads that have many ionic bonding sites on their surfaces. These sites selectively exchange ions with certain mobile phase compositions as the mobile phase moves.
  • Ions that bond to the charged site on the resin beads are thus separated from ions that do not bond
• Ion Exchange Chromatography

  • In this type of chromatography, the use of a resin (the stationary solid phase) is used to covalently attach anions or cations onto it.
  • Solute ions of the opposite charge in the mobile liquid phase are attracted to the resin by electrostatic forces.
• Ion Exchange Chromatography

  • The stationary phase material can be either:
  • an anion exchange resin, which possesses positively charged sites to exchange negative ions,
  • or a cation exchange resin, which possesses negatively charged sites to exchange positive ions.
• Ion Exchange Chromatography

  • The mobile phase can only be a liquid
• Ion Exchange Chromatography

  • Positively charged protein binds to negatively charged bead
  • Negatively charged protein flows through
• Ion Exchange for Deionization of Water

  Wide columns packed with a mixture of:

  1. an anion exchange resin that exchanges dissolved anions for hydroxide ions,
  2. a cation exchange resin that exchanges dissolved cations for hydrogen ions

  ... are used because water that is passed through such a column becomes free of ions (deionized) since the hydrogen and hydroxide ions combine to form more water.
• Partition Chromatography (Paper & TLC)

  • Thin film of liquid formed of a solid inert support
  • Liquid or Gas
  • Solutes equilibrate between the 2 phases according to their partition coefficients
• Adsorption Chromatography

  • Solid that attracts solutes
  • Liquid or Gas
  • Solutes move at different rates according to the forces of attraction to the stationary phase
• Ion Exchange Chromatography

  • Solid resin that carries fixed ions & mobile counterions of opposite charge attached by covalent bonds
  • Liquid containing electrolytes
  • Solute ions of chage opposite to the fixed ions are attracted to the resin by electrostatic forces & replace the mobile counterions
• Size Exclusion Chromatography

  • It is a technique for separating dissolved species on the basis of their size
• Size Exclusion Chromatography

  • The stationary phase consists of porous polymer resin particles. The components to be separated can enter the pores of these particles and be slowed from progressing through this stationary phase as a result.
  • The mobile phase for this type can only be a liquid
• Chromatography Configurations

  Configurations can be broadly classified into 2 categories:

  1. Planar methods
  2. Column methods
• PLANAR METHOD

  • utilize a thin sheet of stationary phase material
  • the mobile phase moves across this sheet, either upward (ascending chromatography), downward (descending chromatography), or horizontally (radial chromatography).
• PLANAR METHODS:

  • Paper Chromatography
  • Thin Layer Chromatography
• COLUMN METHODS:

  • Open Column Chromatography
  • Instrumental Chromatography
• Paper chromatography

  • makes use of a sheet of paper having the consistency of filter paper (cellulose) for the stationary phase
• Paper Chromatography

  • Since filter paper is hydrophilic, the stationary phase is actually a thin film of water unintentionally adsorbed on the surface of the paper. Thus, paper chromatography represents a form of partition chromatography only.
  • The mobile phase is always a liquid
• Paper Chromatography

  • a technique used for the separation & identification of relatively small chemical substances by a moving solvent on sheets or strips of filter paper
• Paper Chromatography

  • Substances to be identified are 'spotted' near one end of the filter paper
  • As the solvent moves up the paper, different molecules move at different rates with the smallest molecules moving the fastest.
• Paper Chromatography

  • The technique is used for small molecules such as amino acids, small peptides and sugars
• Thin Layer Chromatography

  • The stationary phase is a thin layer of material spread across a plastic sheet or glass or metal plate.
• Thin Layer Chromatography can be any of the four types: including adsorption, partition, ion exchange, and size exclusion

  • The mobile phase for TLC is always a liquid.