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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This document compares gas chromatography (GC) and high performance liquid chromatography (HPLC). GC uses a gas mobile phase and separates compounds based on volatility, while HPLC uses a liquid mobile phase under high pressure and separates compounds based on interactions with the stationary phase. Some key differences discussed are that GC uses simpler, less expensive equipment than HPLC but can only analyze more volatile compounds, whereas HPLC can analyze a wider range of compounds including thermolabile ones. Both techniques have different instrumentation requirements and a variety of detectors can be used for analysis.
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Liquid chromatography separates analytes based on their differential migration between a stationary and mobile phase. The stationary phase is fixed within the column, such as C18 or silica, while the mobile phase carries the sample through the column. Samples are injected into the column and separated components are then detected to create a chromatogram displaying the separation over time. High performance liquid chromatography has applications in chemical, environmental, pharmaceutical, clinical, and bioscience analyses.
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Overview
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- 1. 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
- 2. High Performance Liquid chromatography Normal Phase LC polar sp less polar mp Reverse Phase LC nonpolar sp more polar mp Classification
- 3. A. Classical Liquid Chromatography Column: glass tube (1-5 cm dia, 50-100 cm length) sp: solid particles (150-200 µm dia) mp: liquid, gravity fed Limitations: Slow flow rates Long separation times Resolution not great Use: preparative chemistry/biochemistry
- 4. Smaller particle size narrower peaks
- 5. Smaller particle size small H
- 7. High Performance Liquid Chromatography Instrument 1. Solvent delivery system 2. Pump 3. precolumn 4. Injection valve 5. Column 6. Detector
- 8. High Performance Liquid Chromatography 1. Solvent Reservoirs Isocratic: single solvent Gradient elution: vary ratio of solvents 2. Pump Steady, reproducible, constant
- 9. 3. Precolumn (Guard Column) Protect more expensive analytical column Identical to analytical column Helps avoid stripping stationary phase from analytical column
- 11. 5. Analytical Column A. Stationary phase • Liquid on solid surface High Performance Liquid Chromatography
- 12. Bonded phase packings R: C8 C18 R: (CH2)n NH2 (CH2)nCN Reverse Phase vs. Normal Phase
- 14. 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
- 16. General Elution Problem in Chromatography Much shorter time
- 17. High Performance Liquid Chromatography Detectors Sensitive Linear response Small volume cells Insensitive to changes in T, solvent
- 19. A. UV-Vis Must absorb radiation
- 20. B. Refractive Index Universal Less sensitive T, P solvent sensitive
- 21. C. Electrochemical Must be able to be reduced/oxidized Selective D. Fluorescence Very sensitive Selective Derivatization
- 22. 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
- 24. 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
- 25. From Skoog, West, Holler,