Chromatography

Chromatography

1. All Methods have

   • Stationary Phase
   • Mobile Phase

2. Components can

   • Adsorb
     • The process of components in a solution being stopped from moving.
   • Desorb
     • The process of moving on after being adsorbed.

3. Thin Layer Chromatography (TLC)

   • Stationary phase
     • Thin powder spread on plate or glass
   • Mobile phase
     • Ethanol or Water
   • Techniques
     • A small spot of sample first placed onto end of the plate, higher that the solution depth - ORIGIN
       • The plate submerged in solution (solution BELOW the origin)
         • As solvent rises, the components in sample being tested separate
   • Identifying
     • Running standards (known samples) against the unknown sample
     • Calculating the Rf values
   • Compared to Paper
     • Faster
     • Detect smaller amounts
     • Better separation for less polar compounds
     • Corrosive materials can be used (eg. HCl)

4. Rf Values

   • Always be <1
   • Component most STRONGLY adsorbed with have LOWEST Rf value.
   • Rf = Distance moved from the origin by component/ Distance moved from origin by solvent

5. Column Chromatography

   • Phases
     • Stationary Phase
       • Solid or Solid thinly coated in viscous liquid, packed in a glass column
     • Mobile phase
       • Dripped in slowly from a reservoir above
       • Tap at the bottom allows the solvent (ELUENT) to leave at the SAME RATE it enters
   • High Performance Liquid Chromatography (HPLC)
     • Extremely Sensitive
       • Separate compounds RMM >1000
     • Differs from traditional chromatography
       • Solid particles - 10-20 x's smaller
       • small sample size better separation
       • Small particles = high resistance, so needs HIGH PRESSURE (14000 kPa)
     • Identification
       • In the ELUENT stream.
         • Passes through UV light (it absorbs UV light, and a reduced signal is picked up by detector)
       • Time taken to pass through is Retention Time (Rt)
   • Gas Chromatography (GC)
     • Features
       • Most sensitive
       • Detect tiny amounts ( 10^-12)
       • But compounds have to be
         • RMM < 300
         • Perfect for analysis of TRACE compounds
     • Two types
       • Gas - Liquid
         • Stationary - Liquid
         • Mobile - Gas (usually N)
       • Gas - Solid
         • Stationary - SOLID
         • Mobile - Gas (usually N)
     • Components repeatedly pass in and out of solution
       • Least soluble are swept out first
     • Most useful detector - flame ionisastion

6. Interpretation

   • Samples are injected at t=0
   • Each component forms one peak
   • Conc. of a compound can be determined by running standards
   • GC - Spiking with known can help compare to unknown sample

7. All methods can be combined together, along with Mass Spectroscopy