Topic 4 Flashcards
Normal Phase HPLC
Non-polar mobile phase
Polar stationary phase
Least polar elutes first
Reverse phase HPLC
Polar mobile phase
Non-polar stationary phase
Most polar elutes first
Ion-exchange column
Separation of ions and polar molecules
Size-exclusion column
Separation of molecular size
UV-Vis
Compound must be UV-sensitive
Advantages of UV-Vis
1) Inexpensive
2) Applicable to many organic compounds
3) Robust
Advantages of Evaporating Light Scattering detector
1) Applicable to all analyte species
2) Suitable for non UV sensitive compounds
3) Suitable for heat-sensitive compounds
Advantages of Mass Spectrometer
1) Extremely sensitive
2) Able to obtain MS profile
Limitation - only for ionisable compound
Abnormal pump pressure (High)
Caused by:
1) Column clogging
2) Precipitation of solvent lines
Solutions:
1) Flush with strong solvent
2) Flush with solvent the precipitate is soluble in
Abnormal pump pressure (Low)
Caused by:
1) Leaks
2) Air bubbles between pump and mobile phase reservoir
Solution:
1) Check for leaks
2) Purge solvent lines
Shifts in retention time
Caused by:
1) Column aging
2) Contamination of mobile phase
3) Insufficient equilibrium time
Solution:
1) Replace column
2) Flush column
3) Longer column equilibrium times
Detection issues
Noisy peaks
Drifting baseline
Noisy Peaks
Caused by:
1) Uv lamp reaching end of life
2) Air bubbles
3) Intermittent pulses from pump
Solution:
1) Change uv lamp
2) Flush lines / check degasser functioning
3) Check pump fuctioning
Drifting baseline
Caused by:
1) Column bleeding
2) Mobile phase contamination
Solutions:
1) Replace column
2) Reprepare mobile phase
