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Chapter 25 lc harris

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This document discusses liquid chromatography techniques. It describes liquid chromatography as using a liquid mobile phase and liquid or solid stationary phase. It then summarizes classical liquid chromatography and high performance liquid chromatography. High performance liquid chromatography uses smaller particle sizes in the stationary phase, stronger pumps, and detectors to allow for faster separations and better resolution compared to classical liquid chromatography. The document outlines the basic components of an HPLC system including the solvent delivery system, pump, injection port, analytical column, and various detector types. It also discusses different modes of liquid chromatography like normal phase and reverse phase.

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LC Liquid Chromatography Mobile phase: liquid Stationary phase: liquid or solid Solute: liquid Mechanism: stronger ads. longer tr 1. Classical Liquid Chromatography 2. High Performance Liquid Chromatography http://www.youtube.com/watch?v=kz_egMtdnL4
High Performance Liquid chromatography Normal Phase LC polar sp less polar mp Reverse Phase LC nonpolar sp more polar mp Classification
A. Classical Liquid Chromatography Column: glass tube (1-5 cm dia, 50-100 cm length) sp: solid particles (150-200 袖m dia) mp: liquid, gravity fed Limitations: Slow flow rates Long separation times Resolution not great Use: preparative chemistry/biochemistry
Smaller particle size narrower peaks
Smaller particle size small H
Chapter 25 lc harris
High Performance Liquid Chromatography Instrument 1. Solvent delivery system 2. Pump 3. precolumn 4. Injection valve 5. Column 6. Detector
High Performance Liquid Chromatography 1. Solvent Reservoirs Isocratic: single solvent Gradient elution: vary ratio of solvents 2. Pump Steady, reproducible, constant
3. Precolumn (Guard Column) Protect more expensive analytical column Identical to analytical column Helps avoid stripping stationary phase from analytical column
4. Injection Valves Syringe/septum
5. Analytical Column A. Stationary phase Liquid on solid surface High Performance Liquid Chromatography
Bonded phase packings R: C8 C18 R: (CH2)n NH2 (CH2)nCN Reverse Phase vs. Normal Phase
Chapter 25 lc harris
Normal Phase Chromatography sp: polar mp: less polar Increase solvent strength: add more polar solvent Reverse Phase Chromatography sp: nonpolar mp: more polar Increase solvent strength: add more nonpolar solvent High Performance Liquid Chromatography
Chapter 25 lc harris
General Elution Problem in Chromatography Much shorter time
High Performance Liquid Chromatography Detectors Sensitive Linear response Small volume cells Insensitive to changes in T, solvent
Chapter 25 lc harris
A. UV-Vis Must absorb radiation
B. Refractive Index Universal Less sensitive T, P solvent sensitive
C. Electrochemical Must be able to be reduced/oxidized Selective D. Fluorescence Very sensitive Selective Derivatization
High Performance Liquid Chromatography 5. Detectors - Comparison Detector Detection limits, ng UV 0.1-1 RI 100-1000 Echem 0.01-1 Fluorescence0.001-0.01
Chapter 25 lc harris
Qualitative and Quantitative Analysis Quantitative Peak height Peak area Calibration with standards Internal standard method Qualitative Limited # of components Absence of component Use hyphenated techniques
From Skoog, West, Holler,

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Chapter 25 lc harris