CHROMATOGRAPHY

 

• Chromatography is the separation of molecular mixtures depending on the differential affinity of the solute between 2 immiscible phases: one is a stationary phase & the other is a mobile phase. According to the nature of these 2 phases chromatography is classified as:

d    Adsorption chromatography: If the stationary phase is solid.

d    Partition chromatography: If the stationary phase is liquid.

• Theories of Chromatography:

 d    Plate theory                                         d    Rate theory

• Techniques of chromatography:

 d   Adsorption (non-ionic / non-polar)                  d   Partition (non-ionic / polar)

 d   Ion Exchange (ionized)                                    d   Size Exclusion (# mol wt)

• The choice of a particular technique depends upon:

 d    Complexity of Sample            d   Its physical & chemical properties

                d    The resolution required           d    The ease & speed of technique

                d   The availability & cost of equipment

• Accordingly:

d    If the material is volatile &stable in the gas phase    à        Gas chromatography is used

d    If the material is of # m wt (Protein, Triglyceride)   à        Liquid chromatography is used

d    If the material is ionized in solution (amino acid)    à        Ion exchange liquid chromatography

d  If it is # polar, hydrophilic of intermediate m wt (sugar) à Partition chromatography is used

d    If non-ionizable,  hydrophobic or non-polar             à        Liquid adsorption chromatography

• The stationary phase:

d    In GSC          à        Activated charcoal, silica gel, alumina, glass beads

d    In GLC         à        Diatomaceous earth

• Detectors are classified in 2 general gps:

d    Mass flow rate detectors                   à        E.g. flame ionization detectors

d    Concentration sensitive detectors     à        E.g. thermal conductivity detector

• For GLC we can use Mass Spectroscopy

Classification of Chromatography:

Chromatography can be classified according to the nature of the stationary & mobile phases:

If classified according to the mobile phase: chromatography can be either gas or liquid chromatography

• Gas Chromatography (GC): where the mobile phase is a gas (it is suitable for volatile substances). According to the nature of the stationary phase it can be further sub-classified as:
  •      Gas Liquid Chromatography (GLC): The stationary phase is liquid which may be supported by an inert support as silica, diatomaceous earth or alternatively no support, mainly relying on capillary columns (Volatility & solubility) for separation.
  •      Gas Solid Chromatography (GSC): The stationary phase is solid (silica, alumina or activated charcoal) & separation relies on volatility & adsorption at the temp. used.
• Liquid Chromatography: where the mobile phase is a Liquid. According to the nature of the stationary phase it can be further sub-classified as:
  • Gel Chromatography: the stationary phase is a cross-linked polymer; it is used in the separation of different molecular weights /sizes.
  • Ion-exchange Chromatography: the stationary phase is an ion-exchange resin; it is used in the separation of amino acids & alkaloids.
  • Partition Chromatography: the stationary phase is an liquid supported on inert support as silica or cellulose.
  • Adsorption Chromatography: the stationary phase is solid e.g. Al₂O₃.

If classified according to the technique used: liquid chromatography can be classified as:

• Column Chromatography:
  • Simple column chromatography (SCC)
  • High performance liquid chromatography (HPLC)
• Planner Chromatography:
  • Thin layer chromatography (TLC)
  • Paper chromatography (PC)

N. B.:

d  Separation by the use of silica gel relies on the m wt. (particle size) (gel chromatography).

d  In gel chromatography, adsorption of the solid solute occurs.

d  HPLC resolution means separation of one analyte from others in a mixture.

• The use of Silica gel in TLC relies on size exclusion.
• In Paper Chromatography consists of 2 phases: Stationary phase (solute in paper) & mobile phase (organic solvent).
• Paper Chromatography: in cellulose paper chromatography:

–        High affinity to water (water is mobile phase).

–        Low affinity to organic solvent (Chloroform).

–        Solute in paper phase.

Substance to be separated

Volatile                                                                             Non-volatile

Gas Chromatography                                                        Liquid Chromatography

Ionized                                                Non-ionized

        Ion Exchange      Polar                Non-polar        # M. wt

1. Partition    Adsorption          Gel
   1.   In TLC:
   1. a.      The organic phase is moving, silica gel is stationary.
   2. The organic phase is stationary, silica gel is moving.
   3. The aqueous phase is stationary.
   4. The aqueous phase is moving.
2. Chromatography is believed to be:
   1. a.      Solute in paper phase.
   2. Organic solvent phase.
   3. Paper phase.
3. What role does cellulose play in paper chromatography:
   1. Retains the polar solvent (water) (it has #er affinity for water than organic solvents)
   2. Retains the organic solvent
4. Gas chromatography is used for:
   1. Volatile substances.
   2. Polar substances.
5. The presence of alcohol can be detected by:
   1. Respirometer.                              c.   TLC
   2. GLC.
6. Which does not affect the HPLC:
   1. a.      The recorder.
7. Which is not in HPLC:
   1. a.      In line filter.
8. How to improve resolution in GLC:
   1. a.      For polar compounds à Increase polarity.
   2. b.      For non-polar compounds à Increase molecular weight.
9. Solid body chromatography is an example of:
   1. M. wt                                            c.   Ion exchange
   2. Adsorption                                    d.   Partition coefficient.

10.  Flame ionization detectors are used in:

1. Gas chromatography                                          b.   Liquid chromatography

 

11.  In gel chromatography, resolution depends upon:

a.   Particle size                                        b.   Extent of ionization

12.  Resolution in HPLC means:

1. a.      Separation of a compound from others in a mixture

13.  In gas chromatography:

1. Use the derivative of the substance that is volatile
2. Effluent gas can pass through a flame ionization detector

14.  TLC, depends on:

1. Capillarity

15.  Reversed phase HPLC means:

1. Reversed phases of mobile / stationary phases (mobile is water)
2. Mobile phase more polar with increased retention time
3. Mobile phase is more polar than the stationary