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Major topics of this course are: General Instrumentation, Spectroscopy Theory, Molecular Spectroscopy, Chromatography, Electrochemistry, Coulometric Methods, Voltammetric Methods. This lecture covers following points: High-Performance Liquid Chromatography, Liquid Chromatography, Partition Chromatography, Adsorption Chromatography, Ion Chromatography, Size Exclusion Chromatography, Environmental, Pharmaceuticals, Mobile Phase, Ultrasonicate
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Three basic ways to degas solventscolumn or detectorbubble forming gases that get trapped inparticles that can clog components & free ofpumping system – must be free ofin special reservoirs connected to theSolvents (mobile phase) – are stored
Can purchase HPLC solvents & water - still
piston pumpsemploy two reciprocating orHPLC pumping systems typically
achievedreallyflow is neverPulse-freereplacedneed to be& pump sealsCheck valves
Gradient elution separationefficiency ofimproves thedramatically
HPLC sample injectors are volumes are 10 to 50 μL but can be largerextreme accuracy & precision – typicalare overfilled by syringe givingexclusively 6 port valves that
6 port rotary valveInjector for HPLC
Ideal characteristics same as GC
Exception is temp range
Low dead volume 1 to 10 μL
Most common detector is UV-vis
absorbance detector
Variable wavelength – monochromator
Diode array detector- provide spectra
For universal & selective detectionMany HPLC detectors available
Cell for for HPLCdetectorUV-vis
Diode Array Detector
Refractive index detector (RI) - TCD in GCdifferent RI than solvent – analogous topasses through as long as solute haschanges in refractive index as samplebut has poor sensitivity – detectsresponds to nearly all solutes
Can use 2 or 3 electrode design with Pt or• Potentiometric – measure potential• Voltammetric – vary potential & measure i• Coulometric – fix potential & integrate i• Conductometric – measure conductivity carbon electrodes (glassy C or C paste)
Electrochem. detector nearly universal