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High-Performance Liquid Chromatography - Analytical Chemistry II - Lecture Slides, Slides of Analytical Chemistry

Sri Ramachandra Medical College and Research InstituteAnalytical Chemistry

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

Typology: Slides

2012/2013

Uploaded on 08/30/2013

kamin
kamin 🇮🇳

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Chapter 28: High-Performance
Liquid Chromatography (HPLC)
• Scope
• Instrumentation – eluants, injectors, columns
• Modes of HPLC
– Partition chromatography
– Adsorption chromatography
– Ion chromatography
– Size exclusion chromatography
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Download High-Performance Liquid Chromatography - Analytical Chemistry II - Lecture Slides and more Slides Analytical Chemistry in PDF only on Docsity!

• Modes of HPLC• Instrumentation – eluants, injectors, columns• ScopeLiquid Chromatography (HPLC)Chapter 28: High-Performance

  • Size exclusion chromatography– Ion chromatography– Adsorption chromatography– Partition chromatography
  • Suitable for separation of nonvolatile• Can be very sensitive, accurate & precise• Broad applicability – organic & inorganic• Most widely used separation techniqueHPLC species
  • Has found numerous uses in industry, pharmaceuticals, etc.clinical settings, environmental areas,

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

  1. He purge (sparge units often built in)2) ultrasonicate (with vacuum)1) vacuum or suction filter (0.4 or 0.2 μm)

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

  • Normally packed under 6000 psi pressure- N = 40,000 to 60,000- Most common 25 cm, 5 μ, 4.6 mm ID- Packings are 3, 5, or 10 μm particle size- Length 10-30 cm, ID 4-10 mm- may cost $200-$1000 packed- can be PEEK (poly ether ether ketone)- usually stainless steelColumns at factory as a slurry
  • Detectors 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

    1. Filter instrument – optical filters, Hg lampThree types

Variable wavelength – monochromator

Diode array detector- provide spectra

For universal & selective detectionMany HPLC detectors available

Cell for for HPLCdetectorUV-vis

  • Low vol

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

  • Amperometric – fix potential &Electrochemical Detection measure current (i)

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