hplc - instrumental analysis · pdf fileperformance of hplc detectors table 28-1 mass lod*...

HPLC  CH  28  

CHEM  314  

Solvent  mixing    Sample  injec<on  

Separa<on  in  Packed  column  

detec<on  

Solvent  mixing    Sample  injec<on  

Separa<on  in  Packed  column  

detec<on  

ELUTION  METHODS  

Example:  caffeine  lab    50-­‐50  Methanol-­‐H2O  mixture  

SAMPLE  INJECTION  

PARTICLE  SIZE  SMALLER  PARTICLES  MEANS  SMALLER  H  

PARTICLE  SIZE  SMALLER  PARTICLES  MEANS  SMALLER  H  

VAN  DEEMTER  PLOT  

•  Par<cle  size  α  plate  height  •  For  small  par<cle  sizes,  H  is  invariant  with  flow  rate  

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  

DERIVATIZE  SILICA  TO  REDUCE  TAILING  

NORMAL  AND  REVERSE  PHASE  CHROMATOGRAPHY  

DETECTORS  

1.  UV-­‐VISIBLE  SPECTROPHOTOMETER  

2.  EVAPORATIVE  LIGHT  SCATTERING  DETECTOR  

COMPARING  UV-­‐VIS  AND  ELSD  

Soluble  components  of  a  drug  

3.  Mass  Spectrometry  Ioniza<on  and  

ion  accelera<on  

selec<on  of  ions   detec<on