Chapter 28 - Chapter 28 High-Performance Liquid...

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Chapter 28 High-Performance Liquid Chromatography Mobile Phase: Liquid Stationary Phase Separation Mechanism - Solid Adsorption - Liquid Layer Partition - Ion exchange resin Ion exchange - Microporous beads Size Exclusion - Chemically modified resin Affinity
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HPLC Advantages vs GC Not limited by sample volatility or thermal stability Two interacting phases Room temperature analysis Ease of sample recovery
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Instrumentation Solvent Reservoirs Pump Sample Injector Column(s) Detector Data System
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Mobile Phase Reservoirs Inert container with inert lines leading to the pump are required. Reservoir filters (2-10 mm) at reservoir end of solvent delivery lines Degassed solvent - Vacuum filtration - Sparge with inert gas (N 2 or He) - Ultrasonic under vacuum Elevate above pumps
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Isocratic elution: A separation that employs a single solvent or solvent mixture of constant composition. Gradient elution: Here two or more solvent systems that differ significantly in polarity are employed. After elution is begun; the ratio of the solvents is varied in a programmed way, sometimes continuously and sometimes in a series of steps. Separation efficiency is greatly enhanced by gradient elution.
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HPLC Pump Criteria Constructed of materials inert toward solvents to be used Deliver high volumes (flow rates) of solvent (to 10 mL/min) Deliver precise and accurate flow (<0.5% variation) Deliver high pressure (to 6000 psi) Deliver pulse free flow Have low pump-head volume Be reliable
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HPLC Pumps: Types Reciprocating pumps Syringe pumps Constant pressure pumps
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Reciprocating Pumps One, two, or three pump heads - more heads, less pulse Small head volumes (50 to 250 mL) Short piston stroke Inert pistons (generally sapphire) Continuous use (no refill time) Pulse dampeners
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Syringe Pumps Constant flow rate pump Non-pulsating flow Low flow rates (1 to 100 mL/min) Isocratic flow only Refill required when reservoir (~50mL) expended
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Constant Pressure Pump Constant pressure pump, not constant flow Can deliver high pressures Stable flow during delivery stroke Stop flow on refill stroke Low cost
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Sample Introduction Valve-type injectors - Six port fixed volume Rheodyne reproducible injection volumes variable loop size easy to use, reliable - Six port variable volume Waters variable injection volumes without loop change increased maintenance, operator skill required more expensive
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Auto Injectors Continuous injections operator free Comparable precision and accuracy to manual Much more expensive initially Much more convenient Up 100 samples and standards with microprocessor control
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Liquid-Chromatographic Columns Liquid-chromatographic columns are ordinarily constructed from smooth-bore stainless steel tubing, although heavy- walled glass tubing is occasionally encountered. The latter is restricted to pressures that are lower than about 600 psi.
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This note was uploaded on 05/06/2010 for the course CHEM 4414 at Arkansas Tech.

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Chapter 28 - Chapter 28 High-Performance Liquid...

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