High Performance Liquid Chromatography (Lecture 24)

Question 1

What are the fundamental concepts of HPLC?

Answer 1

• Inject liquid sample into liquid mobile phase
• HPLC uses high pressure to force the solvent through closed columns containing very fine particles that give high resolution separations.

Question 2

Why is GC preferred over LC when there is a choice?

Answer 2

GC is normally less expensive and generates much less waste.

Question 3

What are the key facts regarding HPLC columns?

Answer 3

• Samples are filtered or centrifuged.
• Titanium frits distribute the liquid evenly
  over the diameter of the column.
• 5-30 cm in length.
• Inner diameters of 1-5 mm.
• Steel or plastic.
• Guard column protects the main
  chromatography column.

Question 4

What is the stationary phase?

Answer 4

• Liquid filament coated on packing material (silica)
• Pharmaceutical companies often separate the two enantiomers of a drug.
• Use optically active bonded phases

Question 5

What are the two types of HPLC?

Answer 5

• Normal phase chromatography with a polar stationary phase and a non-polar mobile phase.
• Reverse phase chromatography with a non-polar stationary phase and a polar mobile phase.

Question 6

Isocratic elution

Answer 6

mobile phase with fixed composition

Question 7

What is gradient elution?

Answer 7

• Gradient elution uses a continuous change of solvent
  composition to increase the mobile phase strength.
• Gradient elution is analogous to temperature programming in GC.

Question 8

What are the different types of sample detection?

Answer 8

• Spectroscopic (UV Vis)
• Electrochemical (voltammetry, coulometry, conductivity etc.)
• Spectrometric (Mass spec)