HPLC

HPLC

1. Intro

   • MP under high pressure
   • SP PS ↓ so ↑ SA and better separation
     • PS 5 μm and 400 bar → HPLC PS 2 μm and 1500 bar → UPLC
   • VS LC
     • detect small amounts by detectors
     • High pressure and controlled flow of MP
     • Rapid and high resolution
     • Column Packing

2. Pumps (MP from solvent Reservoir), may reach 500 atm

   • Reciprocating
     • Cylindrical, piston back and forth
     • Unlimited Solvent Vol
     • Gradient and pressure applied
     • Dis: Pulsed flow ∴ Damper/ dual piston
   • Syringe
   • Pneumatic/ constant pressure
   • Binary (high pressure , flow rate control ratio), Quater. ( Low pressure, proportioning vavles)

3. Columns

   • precolumn (Guard Column )
     • Packing =analytical
     • remove impurites X contamination
     • Saturation ∴ no stripping
   • Analytical Column
     • SS or glass
   • Preparative ( 300 L)

4. Phases

   • SP technology (RP)
     • zobrax SB
       • Bulky , Protect siloxane , pH (1-8 )
     • z eclipse XDB
       • Double endacpped
     • Z bonus RP
       • selectivity , trple endcapped
     • z Extend C18
       • pH (2-11.5) , bidentate
     • Hypersil BDS( Base deactivated Silica )
     • Kintex (nano ) less band broadening
     • Gemini (cross linking ) pH (1-12)
   • Mobile Phase
     • pure, low visc, compatible wiz detector
     • filtered and degassed (X impurities / o2) disturb detector
       • Degasser
       • Passing He
       • Sonicator
     • Elution
       • Elution power = adsorpability on SP
       • Elutropic series
       • eluent strength , Nomograph
       • UV Cut off
       • isocratic Vs gradient ( better)

5. Detectors

   • Selective (Solute)
     • Absorbance
     • Fluorescence
     • electrochemical
     • Mass/ Radio/ Chiral
   • Universal (Bulk) (poor sensitivity and limited)x gradient
     • RI
     • Evaporting LS
     • Conductivity / corona

6. injectors

   • Single / automated
   • 0.1-100 μl under 4000 psi
   • overflow when loop out of solvent stream
   • Loading position: fill loop/ Injecting: sample pass by solvent

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