Pulse chromatography

1
Pulse chromatography
Mobile phase
Stationary phase
Effluent
ABC
Mobile phase
Mobile phase
C
A
B
Mobile phase
B
A
Mobile phase
A
2
Applications

• Used for the separation of a range of substances
  
• Proteins
• Nucleic acids
• Lipids
• Antibiotics
• Hormones
• Sugars
• Industry
• Pharmaceutical manufacture
• Pharmaceutical and biomedical analysis
• Environmental analysis
• Foods and nutraceuticals production
• Diagnostics
• Process monitoring

3
Chromatographic equipment
4
Columns
5
Separation mechanisms
6
Pumping requirements in chromatography

• High pressure chromatography
• Pressure typically greater than 1MPa
• Frequently referred to as High Performance Liquid
  Chromatography or HPLC
• Special pumps which deliver constant mobile phase
  flow rates at high pressures are required
• Plunger pumps commonly used
• Medium pressure chromatography
• Pressure typically in the range of 0.1 to 1 MPa
• Pumping requirements less demanding
• Plunger, diaphragm and peristaltic pumps
  frequently used
• Peristaltic pumps are particularly suitable for
  handling sterile material
• Low pressure chromatography
• Typically less than 0.1 MPa
• Peristaltic pumps
• Gravity flow

7
Sample injection
Sample
To waste
From pump
From pump
From pump
To column
To column
To column
Normal position
Load position
Inject position
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Detection

• Concentration of the individual components in the
  effluent stream is monitored
• Plot of concentrations versus time or cumulative
  effluent volume is called a chromatogram
• Two ways of monitoring
• Off-line Chemical analysis, visible/UV
  spectrophotometry, refractive index measurement,
  fluorescence spectroscopy, conductivity
  measurement, turbidity measurement, immunoassays
• On-line UV/visible absorbance, fluorescence,
  refractive index, conductivity and light
  scattering
• Other parameters
• pH
• Column pressure drop

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Chromatogram
Partitioning Inlet distribution Radial
dispersion Axial dispersion
Pulse Peak
10
Theory of chromatography
Free Bound
C
C
C
Diffusion
Diffusion
z
z
t