Liquid Chromatography (Chapter 28) Four types of high performance liquid chromatography (HPLC): ·partition adsorption (liquid-solid) ·ion exchange ·size exclusion or gel Increasing polarity Water-insoluble Water-soluble Nonpolar Ionic Nonionic polar 人 、Partition Adsorption lon /(Reversed exchange (Normal phase partition) partition) 103 10 Exclusion 10 (Gel permeation) (Gel filtration) 0 Fig28-1: CEM 333 page 16.1
Liquid Chromatography (Chapter 28): Four types of high performance liquid chromatography (HPLC): • partition • adsorption (liquid-solid) • ion exchange • size exclusion or gel Fig 28-1: CEM 333 page 16.1
Instrumentation for HPLC: For reasonable analysis times,moderate flow rate required but small particles (1-10 um) Solvent forced through column 1000-5000 psi-more elaborate instrument than GC Solvents degassed-"sparging" ·High purity solvents Single mobile phase composition-isocratic elution Programmed mobile phase composition-gradient elution Fig 28-4 Pum nlet rain valye Priming syringe Solvent proportioning valve -To detector Column Backpre Filter ransduce regulator CEM 333 page 16.2
Instrumentation for HPLC: • For reasonable analysis times, moderate flow rate required but small particles (1-10 mm) • Solvent forced through column 1000-5000 psi - more elaborate instrument than GC • Solvents degassed - "sparging" • High purity solvents Single mobile phase composition - isocratic elution Programmed mobile phase composition - gradient elution Fig 28-4 CEM 333 page 16.2
Reciprocating Pump (Fig 28-6) Column 凵Pulse Motor Seal damper Ball check valves Reciprocating Solvent Up to 10,000 psi,small internal volumes ·Produces pulsation Sample injection(Fig 28-7): Load sample Inject sample 00p column From pump From pump ·Similar to FIA,GC Introduce small sample (0.1-100 uL)without depressurization Microsyringe/septum system (only <1500 psi) CEM 333 page 16.3
Reciprocating Pump (Fig 28-6): • Up to 10,000 psi, small internal volumes • Produces pulsation Sample injection (Fig 28-7): • Similar to FIA, GC • Introduce small sample (0.1-100 mL) without depressurization • Microsyringe/septum system (only <1500 psi) CEM 333 page 16.3
HPLC Columns: ·Stainless steel ·10-30 cm long 4-10 mm internal diameter .1-10 um particle size-40,000-60,000 plates/m High Speed Isocratic Separation (Fig 28-8): ·100,000 plates/m 10 Time. CEM 333 page 16.4
HPLC Columns: • Stainless steel • 10-30 cm long • 4-10 mm internal diameter • 1-10 mm particle size - 40,000-60,000 plates/m High Speed Isocratic Separation (Fig 28-8): • 100,000 plates/m CEM 333 page 16.4
Gradient Elution: Solvent polarity (composition)continuously varied or stepped Fig 28-5 (a)Gradient elution Peak identity -trichlorobenzen (b)Isocratic elution 二 10.Hexachlorobenzene 5 1015202530 Retention time.min CEM 333 page 16.5
Gradient Elution: • Solvent polarity (composition) continuously varied or stepped Fig 28-5 CEM 333 page 16.5