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• Chromatography


  An important biophysical technique that enables the separation, identification, and purification of the components of a mixture for qualitative and quantitative analysis

  
• Chromatography


  • Makes use of differences in size, binding affinities, charge, and other properties to separate materials
  • Is a powerful separation tool used in all branches of science and is often the only means of separating components from complex mixtures
  • Allows the separation of components of a mixture on the basis of their nature, structure, size, and other properties

  
• Stationary phase


  The phase that is either a solid or liquid particle attached to a glass or a metal surface on which the components of the mixture to be separated is absorbed selectively

  
• Mobile phase


  The phase that is either liquid or gas that is passed through a chromatographic system where the components of the mixture are separated at different raters by adsorbing them to the stationary phase

  
• Affinity chromatography


  A separation technique where the components of a mixture are separated based on their affinity towards the stationary phase of the system

  
• Affinity chromatography


  1. The column is prepared by loading it with solid support like agarose or cellulose, onto which the substrate/ ligand with the spacer arm, is attached

  2. The mobile phase containing the mixture is poured into the column at a constant rate

  3. The ligand-molecule complex is eluted from the stationary phase by changing the conditions that favor the separation of ligand and components of the mixture

  
• Affinity chromatography


  • Is based on the principle that components of a mixture are separated when the element having an affinity towards the stationary phase binds to the stationary phase, while other components are eluted with the mobile phase
  • The substrate/ ligand are bound to the stationary phase so that the reactive sites for the binding of components are exposed

  
• Affinity chromatography


  • Purification of coli β-galactosidase from a mixture of proteins using the p-aminophenyl-1-thio-β-D-galactopyranosyl agarose as the affinity matrix
  • Removal of excess albumin and α2-macroglobulin from the serum albumin
• Anion exchange chromatography


  The separation technique for negatively charged molecules by their interaction with the positively charged stationary phase in the form of ion-exchange resin

  
• Anion exchange chromatography


  1. A column packed with positively charged resin is taken as the stationary phase

  2. The mixture with the charged particles is then passed down the column where the negatively charged molecules bind to the positively charged resins

  3. The anion exchange resin is then passed through the column where the negatively charged molecules now bind to the anion exchange resin displacing the positively charged resin

  4. An appropriate buffer is applied to the column to separate the complex of anion exchange resins and the charged molecules

  
• Anion exchange chromatography


  • Is based on the principle of attraction of positively charged resin and the negatively charged analyte, where the exchange of positively charged ions takes place to remove the negatively charged molecules
  • The stationary phase is first coated with positive charges where the components of the mixture with negative charges will bind

  
• Anion exchange chromatography


  • Separation of nucleic acids from a mixture obtained after cell destruction
  • Separation of proteins from the crude mixture obtained from the blood serum
• Cation exchange chromatography


  The separation technique for positively charged molecules by their interaction with negatively charged stationary phase in the form of ion-exchange resin

  
• Cation exchange chromatography


  1. A column packed with negatively charged resin is taken as the stationary phase

  2. The mixture with the charged particles is then passed down the column where the positively charged molecules bind to the negatively charged resins

  3. The Cation exchange resin is then passed through the column where the positively charged molecules now bind to the Cation exchange resin displacing the negatively charged resin

  4. An appropriate buffer is applied to the column to separate the complex of Cation exchange resins and the charged molecules
• Cation exchange chromatography


  • Is based on the principle of attraction of negatively charged resin and the positively charged analyte, where the exchange of negatively charged ions takes place to remove the positively charged molecules
  • The stationary phase is first coated with negative charges where the components of the mixture with positive charges will bind

  
• Cation exchange chromatography


  • Separation of positively charged lanthanoid ions obtained from the earth's crust
  • Determination of total dissolved salts in natural waters by analyzing the presence of calcium ions
• Column chromatography


  The separation technique where the components in a mixture are separated on the basis of their differential adsorption with the stationary phase, resulting in them moving at different speeds when passed through a column

  
• Column chromatography


  1. The column is prepared by taking a glass tube that is dried and coated with a thin, uniform layer of stationary phase (cellulose, silica)

  2. The sample is prepared by adding the mixture to the mobile phase and introduced into the column from the top

  3. The molecules bound to the column are separated by elution technique where either solution of the same polarity is used (isocratic technique), or different samples with different polarities are used (gradient technique)

  
• Column chromatography


  • Is a solid-liquid chromatography technique in which the stationary phase is a solid & mobile phase is a liquid or gas
  • Is based on the principle of differential adsorption where different molecules in a mixture have different affinities with the absorbent present on the stationary phase

  
• Column chromatography


  • Extraction of pesticides from solid food samples of animal origin containing lipids, waxes, and pigments
  • Synthesis of Pramlintide which is an analog of Amylin, a peptide hormone, for treating type 1 and type 2 Diabetics
  • Purification of bioactive glycolipids, showing antiviral activity towards HSV-1 (Herpes Virus)
• Flash chromatography


  A separation technique where smaller sizes of gel particles are used as the stationary phase and a higher pressure is applied to increase the flow rate of the mobile phase

  
• Column chromatography


  • Separated molecules can further be analyzed for various purposes

  
• Column chromatography


  • Extraction of pesticides from solid food samples of animal origin containing lipids, waxes, and pigments
  • Synthesis of Pramlintide which is an analog of Amylin, a peptide hormone, for treating type 1 and type 2 Diabetics
  • Purification of bioactive glycolipids, showing antiviral activity towards HSV-1 (Herpes Virus)
• Flash chromatography


  Separation technique where smaller sizes of gel particles are used as stationary phase, and pressurized gas is used to drive the solvent through the column

  
• Flash chromatography


  1. Column is prepared by taking a glass tube that is dried and coated with a thin, uniform layer of stationary phase

  2. Sample is prepared by adding the mixture to the mobile phase and introduced into the column from the top

  3. Molecules bound to the column are separated by elution solution where either solution of the same polarity is used (isocratic technique), or different samples with different polarities are used (gradient technique)

  4. Elution solvent is applied with a constant minimum pressure required to move the solute down the column

  
• Gas chromatography


  Separation technique in which the molecules are separated on the basis of their retention time depending on the affinity of the molecules to the stationary phase

  
• Gas chromatography


  1. Sample is injected into the column where it is vaporized into a gaseous state

  2. Column is set with the stationary phase where the molecules are separated on the basis of their affinity to the stationary phase

  3. Components of the mixture reach the detector at different times due to differences in the time they are retained in the column

  
• Gas chromatography


  • Identification of performance-inducing drug in the athlete's urine
  • Separation and quantification of a solid drug in soil and water samples
• Gel filtration chromatography/ Gel permeation chromatography/ Size exclusion chromatography/ Molecular sieve chromatography


  Form of partition chromatography used to separate molecules of different molecular sizes

  
• Gel filtration chromatography


  1. Column is filled with semi-permeable, porous polymer gel beads with a well-defined range of pore sizes

  2. Sample, mixed with the mobile phase, is then injected into the column from the top of the column

  3. Molecules bound to the column are separated by elution solution where either solution of the same polarity is used (isocratic technique), or different samples with different polarities are used (gradient technique)

  4. Elution conditions (pH, essential ions, cofactors, protease inhibitors, etc.) can be selected, which will complement the requirements of the molecule of interest

  
• Gel filtration chromatography


  • Separation of recombinant human granulocyte colony-stimulating factor (rhG-CSF) from inclusion bodies in high yield by urea-gradient size-exclusion chromatography
  • Separation of hen egg lysozyme using both acryl amide- and dextrin-based gel columns
• High-performance liquid chromatography (HPLC)


  Modified form of column chromatography where the components of a mixture are separated on the basis of their affinity with the stationary phase

  
• HPLC


  1. Column is prepared by taking a glass tube that is dried and coated with a thin, uniform layer of stationary phase

  2. Sample is prepared by adding the mixture to the mobile phase and introduced into the column from the top, and a high-pressure pump is used to pass the sample at a constant rate

  3. Mobile phase then moves down to a detector that detects molecules at a certain absorbance wavelength

  
• Hydrophobic interaction chromatography


  Separation technique that separates molecules on the basis of their degree of hydrophobicity

  
• Hydrophobic interaction chromatography


  1. Column is prepared with a glass tube applied with solid support like silica gel, upon which hydrophobic groups like phenyl, octyl butyl, are attached

  2. Sample is prepared by adding the mixture to the mobile phase and injected into the column from the top

  3. Molecules with hydrophobic groups form an interaction with the hydrophobic groups of the stationary phase, while molecules without such groups move out of the column with the mobile phase

  4. Elution solution with decreasing salt gradient is then passed into the column that removes the bound molecules from the stationary phase

  
• Ion exchange chromatography


  Separation technique for charged molecules by their interaction with the oppositely charged stationary phase in the form of ion-exchange resin

  
• Hydrophobic interaction chromatography


  Extremely important for the separation of proteins with hydrophobic groups

  
• Hydrophobic interaction chromatography


  • More appropriate than other methods, as this technique results in minimum denaturation activities
  • Can also be applied to the separation of other organic compounds with hydrophobic groups
  • Allows the separation of hydrophilic and hydrophobic biological molecules from each other

  
• Hydrophobic interaction chromatography


  • The separation of plant proteins from the crude extracts

  
• Principle of Ion exchange chromatography

  1. Attraction of charged resin and the oppositely charged analyte

  2. Exchange of negatively/ positively charged ions takes place to remove the charged molecules

  3. Stationary phase is first coated with particular charges where the components of the mixture with opposite charges will bind

  4. Cation or anion exchange resin with a higher affinity to the charged components then binds the components, displacing the oppositely charged resin

  5. Cation or anion exchange resin-component complex then is removed by using different buffers