Pharamcogonocy

• Ether


  • CaCO3
  • Chlorophyll
  • Chromatography
  • Colors
• Comparing Chromatography to the Flow of a River

  • Base
  • Water flow
  • Light leaf
  • Heavy stone

  
• Mobile Phase / Stationary Phase

  • A site in which a moving phase (mobile phase) and a non-moving phase (stationary phase) make contact via an interface that is set up
  • The affinity with the mobile phase and stationary phase varies with the solute
  • Separation occurs due to differences in the speed of motion
• Mobile phase


  Stationary phase
• Chromatography


  Analytical technique
• Chromatogram


  Obtained "picture"
• Three States of Matter and Chromatography Types

  • Mobile phase: Gas
  • Liquid
  • Solid
  • Stationary phase: Gas
  • Liquid
  • Solid
  • Gas chromatography
  • Liquid chromatography

  
• Liquid Chromatography


  • Chromatography in which the mobile phase is a liquid
  • The liquid used as the mobile phase is called the "eluent"
  • The stationary phase is usually a solid or a liquid
  • It is possible to analyze any substance that can be stably dissolved in the mobile phase

  
• Interaction Between Solutes, Stationary Phase, and Mobile Phase

  Differences in the interactions between the solutes and stationary and mobile phases enable separation
• Chromatography Types


  • Column Chromatography
  • Paper Chromatography
  • Thin Layer Chromatography (TLC)

  
• Separation Process and Chromatogram for Column Chromatography

  1. Output concentration

  2. Time

  3. Chromatogram

  
• Chromatogram


  • tR: Retention time
  • t0: Non-retention time
  • A: Peak area
  • h: Peak height
• High Performance Liquid Chromatography (HPLC)

  • Higher degree of separation
  • Refinement of packing material (3 to 10 µm)
  • Reduction of analysis time
  • Delivery of eluent by pump
  • Demand for special equipment that can withstand high pressures
• Flow Channel Diagram for High Performance Liquid Chromatograph

  1. Pump

  2. Sample injection unit (injector)

  3. Column

  4. Column oven (thermostatic column chamber)

  5. Detector

  6. Eluent (mobile phase)

  7. Drain

  8. Data processor

  9. Degasser

  
• Advantages of High Performance Liquid Chromatography

  • High separation capacity, enabling the batch analysis of multiple components
  • Superior quantitative capability and reproducibility
  • Moderate analytical conditions
  • Unlike GC, the sample does not need to be vaporized
  • Generally high sensitivity
  • Low sample consumption
  • Easy preparative separation and purification of samples

  
• Fields in Which High Performance Liquid Chromatography Is Used

  • Biogenic substances
  • Medical products
  • Food products
  • Environmental samples
  • Organic industrial products
• High-performance liquid chromatography (HPLC) columns

  • Guard column: Protective column or cartridge installed between the injector and the analytical column
  • Derivatization column: Post-column derivatization, also known as post-column reaction, renders visible certain compounds that are normally invisible
  • Fast column: Quickly and simply separate common inorganic anions from a variety of sample matrices
  • Preparatory column: Used to purify sufficient quantities of a substance for further use, rather than analysis

  
• Stationary Phases


  • Normal-phase Partition Chromatography
  • Reverse-phase Partition Chromatography
  • Ordinary Stationary phase as silica or alumina
  • Chiral stationary phases in separating isomers

  
• Reverse-phase Partition Chromatography


  Stationary phase: chemically bonded or non polar substance

  Mobile phase: a polar solvent

  Used for separation of polar compounds

  
• Normal-phase Partition Chromatography


  Stationary phase: polar substance

  Mobile phase: a non polar organic solvent

  Used for separation of non polar compounds

  
• Adsorption Chromatography


  Stationary Phase: Silica, Alumina

  Mobile Phase: Isocratic elution, Gradient elution, Polytypic Mobile Phase (Mixed Mode Chromatography), Chiral stationary phase

  
• Polytypic Mobile Phase (Mixed Mode Chromatography)

  Versatile technique

  Several chromatographic modes can be employed using the same column

  The column contains hydrophobic resin covalently bonded to a hydrophilic organic layer

  By changing the mobile phase, the mode of separation is changed to achieve desired selectivity

  
• Detector


  Component of HPLC instrument that emits response due to the eluting sample compound and signals a peak on the chromatogram

  Positioned immediately after the Stationary Phase to detect the eluted compounds

  
• MS detector


  Based on the fact that the sample molecule is ionized (by several methods)

  As it passed through a mass analyzer the ions current is detected and recorded

  Methods of Ionization: Electron impact method, Chemical ionization method, Fast Atom Bombardment method
• Importance of HPLC to US
• Advantages of HPLC


  • Run samples, print out chromatograms and spectra automatically

  The versatility of stationary phases made it popular method for bioassay-guide isolation

  Different mode of mobile phase run (gradient, isocratic)

  Can be range-up to preparative scale

  It can be used for the majority of natural compounds that are soluble in organic solvents and can be adapted to ion exchange for isolation of highly polar compounds

  Excellent separating power

  Speed and reproducibility

  
• Disadvantages of HPLC


  • It is expensive (analytical instrument may cost upwards of 20000$, and preparative HPLC may be 30000$)

  Preparative column (2000$) have short life

  Needs high purity solvents (HPLC grade)
• Example of HPLC application for separation of natural products
• Chromatography


  A separation method that relies on differences in behavior between a flowing mobile phase and a stationary phase to separate the components in a mixture

  
• Chromatography


  • The components to be separated are distributed between 2 phases one of which is stationary phase (fixed) while the other moves in a definite direction
  • Used to Separate
  • Analyze
  • Identify
  • Purify
  • Quantify components in a mixture

  
• Chromatography


  1. Mixture loaded onto stationary phase

  2. Mobile phase flows through stationary phase

  3. Components separate based on affinity to stationary vs mobile phase

  
• Adsorption Chromatography


  Stationary phase is solid (silica, alumina), mobile phase is liquid, polar adsorbents have polar OH groups which interact with solute molecules

  
• Partition Chromatography


  Stationary phase is liquid supported on solid matrix, mobile phase is gas or liquid, solute distributes between phases based on partition coefficient

  
• Mobile phase


  Solvent moving through the column
• Stationary phase

  Substance that stays fixed inside the column
• Eluent


  Fluid entering the column
• Eluate


  Fluid exiting the column (that is collected in flasks)
• Elution


  The process of washing out a compound through a column using a suitable solvent
• Analyte


  Mixture whose individual components have to be separated and analyzed
• Chromatography steps

  1. Analyte loaded onto stationary phase (silica bed)

  2. Mobile phase flows through stationary phase

  3. Components separate based on affinity to stationary phase