Different forms of Chromatography - Biagini

Question 1

What is the difference between the stationary phase and the mobile phase?

Answer 1

Mobile phase is a gas or liquid that passes through a ‘column’ containing the ‘stationary phase’

Stationary phase is a liquid or a solid that does not move, usually held inside a ‘column’

Question 2

What is adsorption chromatography?

Answer 2

• Consists of a solid stationary phase and liquid or gas mobile phase.
• The solute is adsorbed on the surface of the solid particles.
• The more strongly it is adsorbed, the more slowly it travels through the column.
• The solid phase is usually made of silica.

Question 3

What is partition chromatography?

Answer 3

• Uses a high-boiling stationary phase bonded to a solid surface (usually inside a GC column) and gas mobile phase
• The solute equilibrates between the stationary liquid and gas phases.

Question 4

What is size-exclusion chromatography?

Answer 4

• AKA Gel permeation chromatography, separates molecules according to their size (hydrodynamic value)
• Smaller molecules are able to enter the pores of the media and therefore, take longer to elute.
• Larger molecules are excluded from the pores and elute faster.

Question 5

What is Affinity Chromatography?

Answer 5

• A method of separating biochemical mixtures, based on a highly specific biological interaction such as that between antigen and antibody, or enzyme and substrate.

Question 6

What are the advantages of HPLC?

Answer 6

• High resolving power
• Speed of separation
• Continuous monitoring of the effluent
• Quantitatively accurate
• Reproducible using the same column
• Can be done repetitively
• Automation and data handling are possible.

Question 7

Which parameters govern retention time in Reverse Phase HPLC?

Answer 7

1. Chemical nature of the stationary phase
2. The type of solvents that compose the mobile phase and their ratio
3. The pH and ionic strength and additives of the mobile phase

Question 8

What eluents are typically used in HPLC?

Answer 8

• Common solvent used include any miscible combinations of water with various organic solvents such as methanol or acetonitrile.
• Water may contain buffers or salts or compounds such as trifluoroacetic acid which acts as an ion pairing agent.

Question 9

What are columns typically composed of in HPLC?

Answer 9

• Precision-bore stainless steel
• Typicall 10-30cm long, with frits at each end.

Packing columns:

• Polystyrene beads
• Silica shell on glass beads
• Totally porous silica particles.

Question 10

What is typical of the Detector?

Answer 10

• Must be sensitive - what is the minimum detectable concentration?
• Have a linear range - allows for easier quantitative analysis
• Types of detection:
  
  • Bulk properties e.g. refractive index
  • Solute properties - ideally independent of mobile phase, e.g. UV.
• Detectors inc UV/Vis, Fluorescence, chemiluminescence, FTIR, RI, MS etc.

Question 11

What is typical of UV detectors?

Answer 11

• Most commonly used system.
• Highly sensitive
• Temperature independent (relatively)
• Flow-rate independent
• Wavelengths used: 254 nm and/or 280 nm.
• Looks at the 210-800 nm rang.e

Question 12

What is isocratic elution?

Answer 12

• In isocratic elution compounds are eluted using a constant mobile phase composition.

Question 13

What is Gradient Elution?

Answer 13

In gradient elution, different compounds are eluted by increasing the strength of the organic solvent.