HPLC

2. A technique where in components of a mixture gets
separated when passed in a solution through a
medium.

4. Very small particle size
High pressure is applied to solvent flow

5. When the particle size is decreased the total surface area
increases, which thereby improves the resolution but also
increases the resistance faced by the mobile phase, which
leads to the decrease in the flow rate (time increases). So a
pump is installed to apply high pressure across the column
thereby increasing the flow rate and decreasing the time
required.

6. Porous layer beads (porous outer shell, 20-45μm)
Microporous particles
• Longer pores: 20-40μm
• Shorter pores:5-10μm
Chemically bonded phase (siloxanes)

7. Desired characteristics of mobile phase:
• Purity
• Reactivity
• Detector compatibility
• Degassed
Reservoir should be at least 500ml

8. Pressure: 500-5000 psi
Flow delivery:
a. Analytical: 1ml/min
b. Preparative: 100ml/min
Pulse free solvent flow

9. Syringe injector: ml syringe; Teflon septum; 3000 psi
Loop injector: metal

10. Conventional (4-6mm, 3-25cm, 1-3 cubic cm/min)
Microbore (1-2mm, 25-50cm, 5-20 cubic mm/min)

11. 1. U.V. detector
2. Refractive index detector
3. Fluorescence detector
4. Scanning wavelength detector
5. Electrochemical detector
6. Mass spectrometer detector
7. NMR spectrometer detector
8. Evaporative light-scattering detector

12. Resolution and speed
Reusability
Reproducibility
Automated
Adaptable

13. Separation & identification of amino acids,
carbohydrates, lipids, nucleic acids, proteins,
pigments, steroids, and many other biologically
active molecules.

14. Biochemistry Laboratory by Rodney Boyer
Principles & Techniques of Biochemistry and Molecular
Biology by Keith Wilson & John Walker
Introduction to Chromatography by V. K. Srivastava & K.
K. Srivastava