Chromatography

Chromatography

All Methods have
1. Stationary Phase
2. Mobile Phase
Components can
1. Adsorb
  1. The process of components in a solution being stopped from moving.
2. Desorb
  1. The process of moving on after being adsorbed.
Thin Layer Chromatography (TLC)
1. Stationary phase
  1. Thin powder spread on plate or glass
2. Mobile phase
  1. Ethanol or Water
3. Techniques
  1. A small spot of sample first placed onto end of the plate, higher that the solution depth - ORIGIN
    1. The plate submerged in solution (solution BELOW the origin)
      1. As solvent rises, the components in sample being tested separate
4. Identifying
  1. Running standards (known samples) against the unknown sample
  2. Calculating the Rf values
5. Compared to Paper
  1. Faster
  2. Detect smaller amounts
  3. Better separation for less polar compounds
  4. Corrosive materials can be used (eg. HCl)
Rf Values
1. Always be <1
2. Component most STRONGLY adsorbed with have LOWEST Rf value.
3. Rf = Distance moved from the origin by component/ Distance moved from origin by solvent
Column Chromatography
1. Phases
  1. Stationary Phase
    1. Solid or Solid thinly coated in viscous liquid, packed in a glass column
  2. Mobile phase
    1. Dripped in slowly from a reservoir above
    2. Tap at the bottom allows the solvent (ELUENT) to leave at the SAME RATE it enters
2. High Performance Liquid Chromatography (HPLC)
  1. Extremely Sensitive
    1. Separate compounds RMM >1000
  2. Differs from traditional chromatography
    1. Solid particles - 10-20 x's smaller
    2. small sample size better separation
    3. Small particles = high resistance, so needs HIGH PRESSURE (14000 kPa)
  3. Identification
    1. In the ELUENT stream.
      1. Passes through UV light (it absorbs UV light, and a reduced signal is picked up by detector)
    2. Time taken to pass through is Retention Time (Rt)
3. Gas Chromatography (GC)
  1. Features
    1. Most sensitive
    2. Detect tiny amounts ( 10^-12)
    3. But compounds have to be
      1. RMM < 300
      2. Perfect for analysis of TRACE compounds
  2. Two types
    1. Gas - Liquid
      1. Stationary - Liquid
      2. Mobile - Gas (usually N)
    2. Gas - Solid
      1. Stationary - SOLID
      2. Mobile - Gas (usually N)
  3. Components repeatedly pass in and out of solution
    1. Least soluble are swept out first
  4. Most useful detector - flame ionisastion
Interpretation
1. Samples are injected at t=0
2. Each component forms one peak
3. Conc. of a compound can be determined by running standards
4. GC - Spiking with known can help compare to unknown sample
All methods can be combined together, along with Mass Spectroscopy

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