CHROMATOGRAPHY

• Chromatography


  Technique for the separation, purification, and testing of compounds
• Chromatography


  • Ability to separate a mixture of compounds or analyte and determine their respective identity (chemical structure) and concentration
• The term "chromatography" is derived from Greek, chroma meaning, "colour," and graphein meaning "to write"
• Chromatography


  1. Analyte is combined within a liquid or gaseous mobile phase

  2. Separation of colored compounds on a suitable adsorbent
• Chromatography


  A process in which a solution of a mixture containing inert material, drug principles, and impurities is separated into its components while moving
• Principal objectives of chromatography

  • Resolution of mixture into constituent parts
  • Determination of homogeneity
  • Comparison of substance suspected of being identical
  • Purification
  • Concentration of substance from dilute solutions
  • Identification and control of technical products
  • Quantitative separation from the complex mixtures
  • Identification of molecular structure
• Types of chromatography


  • Adsorption chromatography
  • Thin layer chromatography
  • Column chromatography
  • Partition chromatography
• Adsorption chromatography


  • Different compounds are adsorbed on the adsorbent to different degrees based on the absorptivity of the component
  • A mobile phase is made to move over a stationary phase, thus carrying the components with higher absorptivity to a lower distance than that with lower absorptivity
• Thin layer chromatography


  Mixture of substances is separated into its components with the help of a glass plate coated with a very thin layer of adsorbent, such as silica gel and alumina
• Column chromatography


  • Technique used to separate the components of a mixture using a column of suitable adsorbent packed in a glass tube
  • Adsorbent: the material placed in the column to adsorb the drug (e.g. purified siliceous earth, activated alumina, silica gel and calcium carbonate)
• Partition chromatography


  • Continuous differential partitioning of components of a mixture into a stationary phase and mobile phase
  • Example: Paper chromatography where chromatography paper is used as a stationary phase suspended in a mixture of solvents that act as a mobile phase
• Eluant


  A solvent used in the separation of substances
• Eluate


  A solution obtained by elution
• Differential extraction


  Method of separation of any organic component present in an aqueous solution using an organic solvent with higher solubility for the desired compound compared to water, and which is immiscible with the aqueous solution
• Applications of chromatography


  • Separation, isolation and purification of proteins from complex sample matrices
  • Separation of proteins of interest from other proteins and further purification
• Chromatography is mainly used for the separation, isolation and purification of proteins from complex sample matrices in bioanalytical chemistry
• Proteins must be separated from all other cell components like lipids and nucleic acids in order to be analysed
• Rf value


  Measure of how far a compound moves up a thin layer chromatography plate relative to the solvent front
• Potentiometry


  Branch of electrochemistry which deals with the study and measurement of electrode potentials
• Redox titration


  Titrations based on the transfer of electrons between the titrant and the analyte, usually followed by potentiometry
• Oxidation-reduction (redox) reaction


  Chemical reaction that involves a transfer of electrons between two species, where the oxidation number of a molecule, atom, or ion changes by gaining or losing an electron
• Redox reaction components


  • Oxidizing agent - ion or molecule that accepts electrons
  • Reducing agent - ion or molecule that donates electrons
• In a redox reaction, what is oxidized is the reducing agent and what is reduced is the oxidizing agent
• Reduction potential


  • Measure of how thermodynamically favourable it is for a compound to gain electrons
  • A high positive value indicates a compound is readily reduced and is a strong oxidising agent
• A substance with a higher reduction potential will oxidise one with a lower reduction potential
• Chroma - colour
• Graphein - to write
• separated by chromatograph:

  1. Inert material
  2. drug principle
  3. impurities
• mobile phase - is made to move over a stationary phase, thus carrying the components with higher absorptivity to a lower distance than that with lower absorptivity. 
• Thin layer absorbent:

  1. silica gel
  2. alumina
• thin layer - glass plate
• column chromatography - glass tube
• Adsorbent : the material place in the column to adsorb the drug.
• Example of absorbents:

  1.  purified siliceous earth
  2. activated alumina
  3. silica gel
  4. calcium carbonate.
• Eluant : a solvent use in the separation of substances
• Eluate : a solution obtained by elution
• Separating funnel - use in separating the layers created by the organic solvent w/ aqueous solution in differential exctraction
• organic solvent - is chosen such that it is immiscible with the aqueous solution so that it can form layers and can be separated easily using a separating funnel.
• bio analytical chemistry - mainly used chromatography for the separation, isolation and purification of proteins from complex sample matrices
• Redox reactions are common and vital to some of the basic functions of life:

  1. photosynthesis
  2. respiratiom
  3. combustion
  4. corrosion
  5. rusting.