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HPLC CH 28
CHEM 314
Solvent mixing Sample injec<on
Separa<on in Packed column
detec<on
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Solvent mixing Sample injec<on
Separa<on in Packed column
detec<on
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ELUTION METHODS
Example: caffeine lab 50-‐50 Methanol-‐H2O mixture
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SAMPLE INJECTION
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PARTICLE SIZE SMALLER PARTICLES MEANS SMALLER H
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PARTICLE SIZE SMALLER PARTICLES MEANS SMALLER H
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VAN DEEMTER PLOT
• Par<cle size α plate height • For small par<cle sizes, H is invariant with flow rate
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SMALLER PARTICLE SIZE REQUIRES INCREASED PRESSURE
Ultra Performance Liquid Chromatography (UPLC) Up to 1,000 bar
HPLC 70-‐400 bar
P= pressure f=par<cle shape and packing ux= linear flow rate n=solvent viscosity L= column length r= column radius dp= par<cle diameter
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DERIVATIZE SILICA TO REDUCE TAILING
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NORMAL AND REVERSE PHASE CHROMATOGRAPHY
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DETECTORS
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1. UV-‐VISIBLE SPECTROPHOTOMETER
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2. EVAPORATIVE LIGHT SCATTERING DETECTOR
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COMPARING UV-‐VIS AND ELSD
Soluble components of a drug
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3. Mass Spectrometry Ioniza<on and
ion accelera<on
selec<on of ions detec<on