BIOMAT NOTES

• Chromatography


  Techniques for separating a mixture into individual components, their detection and quantification

  
• Chromatography


  • Based on differential distribution of components between a stationary phase and mobile phase

  
• Chromatography techniques are always destructive
• Chromatography samples


  • In solution or gas phase

  
• Chromatography always takes some part of the material

  
• Chromatography usually uses 1 mg of sample in 1 mL or less
• Chromatography separation


  1. Column

  2. Packed column is the stationary phase

  3. Sample is added on top

  4. Mobile phase is added

  5. Sample moves through stationary phase and is separated into individual components

  
• Retention time


  Time a component of a mixture takes to go out of the column and be detected
• Capacity factor (k')


  Difference between retention time and dead time, quantifies interaction of a solute with stationary phase
• Theoretical plates


  • Chromatographic process explained with theoretical plates (derived from distillation theory)
  • Each plate has distribution equilibrium of solute between stationary and mobile phase
  • Solute moves along column due to dynamic action of mobile phase

  
• HETP


  Height equivalent to a theoretical plate, defines efficiency of chromatographic column

  
• Number of theoretical plates (N)

  Defines efficiency of chromatographic column, N = 16(tr/W)^2 where tr is retention time and W is peak width

  
• Separation mechanisms


  • Interactions between analyte and stationary/mobile phases must be present for separation to occur
  • If interactions are weak, analyte is bound to either stationary or mobile phase and never separates

  
• Chromatography techniques based on mobile phase

  • Liquid chromatography (LC)
  • Gas chromatography (GC)
  • Supercritical fluid chromatography (SFC)

  
• Chromatography techniques based on stationary phase

  • Column chromatography
  • Planar chromatography

  
• Liquid chromatography (LC)


  • Used to separate non-volatile, neutral or ionic, and thermolabile (heat sensitive) substances
  • Can separate amino acids, proteins, hydrocarbons, carbohydrates, terpenes/terpenoids, inorganic ions

  
• Planar chromatography


  • Easy to apply techniques
  • Used for preliminary information
  • Most used stationary phases are silica gel, alumina for adsorption, cellulose for liquid-liquid chromatography

  
• Early liquid column chromatography used long glass cylinders requiring very long separation times
• Using small stationary phase particles (1-5 μm) allows shorter columns (10-30 cm) with high pressure (100 atm) - this is HPLC

  
• HPLC detectors


  • Refractive index
  • Conductimeter
  • UV-Vis spectrophotometer
  • UV-Vis diode array
  • Spectrofluorimeter
  • Electrochemical
  • Mass spectrometer

  
• UV-Vis spectrophotometric detector


  Most used HPLC detector, based on UV absorption so suitable for many organic and inorganic substances, good sensitivity (ppb), non-destructive
• In gas chromatography (GC), the sample is vaporized and injected into the column, with a gas as the mobile phase which acts as a carrier
• GC applications


  • Characterization of terpenes, esters, ketones, short chain hydrocarbons, carboxylic acids, biochemical compounds, volatile organic compounds
• Gas-liquid chromatography


  • Stationary phase is a non-volatile liquid chemically bound to an inert solid support, mechanism is partition

  
• Gas-solid chromatography


  • Stationary phase is an inert solid, mechanism is adsorption, used for low molecular weight permanent gases

  
• GC column types


  • Packed columns
  • Tubular columns (wall coated open tubular, support coated open tubular, porous layer open tubular)
• Packed GC columns


  • Inert support finely ground, able to absorb liquid stationary phase or used as stationary phase, inner diameter 2-4 mm
• Tubular GC columns


  • Stationary phase is a thin layer on the inner column wall, diameter around 1 mm, can be very long for high number of theoretical plates
• GC advantages


  • Quick
  • Best resolution for volatile substances
  • Many detectors available
  • Broad range of components detectable
  • Can be coupled with mass spectrometry
• GC disadvantages


  • Only for volatile or made volatile substances
  • Not for thermolabile substances
• LC advantages


  • Can separate non-volatile, neutral, ionic, and thermolabile substances
  • Quantitative analysis possible
• LC disadvantages


  • Relatively slow
  • Not as good resolution as GC
  • Fewer detectors available
• Tubular columns


  • Stationary phase is layer in inner column wall
  • Diameter is around 1mm
  • Wall coated open tubular (WCOT) - thin layer of liquid or polymer (stat. phase) covers inner wall surface of column
  • Supported wall coated open tubular (SWCOT) - porous solid layer, easily wetted absorbing liquid stat. phase, lined in tubular column
  • Porous layer open tubular (PLOT) - porous solid layer inorganic or polymeric lined in tubular column, can be very long with huge number of theoretical plates
• Open tubular columns


  • Most efficient chromatographic technique
• Advantages of Gas Chromatography


  • Quick
  • Best resolution for components <400 g/mol
  • Many detectors available
  • Not thermolabile substances
  • Broad range of components detectable
  • Coupling with Mass Spectrometry
  • Quantitative
• Disadvantages of Gas Chromatography

  • Relatively volatile, or made volatile, substances
  • Not suitable for many thermolabile or non-volatile species
• Advantages of Liquid Chromatography

  • Relatively quick
  • Good resolution
  • Thermolabile/non-volatile species
  • Coupling with Mass Spectrometry
  • Quantitative

  
• Disadvantages of Liquid Chromatography

  • Solubility problems
  • Detectors specific for compounds (limited range)
• Supercritical Fluid Chromatography (SFC)


  For volatile (Tb < 300°C) and not thermolabile (no decomposition & T > 200°C) substances

  
• Ion Exchange Chromatography (IEC)


  Specifically designed for ionic compounds, based on exchange equilibria between ions present in mobile phase and ions present on stationary phase