Download - Chapter 25 lc harris
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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 Chromatography2. High Performance Liquid Chromatography
http://www.youtube.com/watch?v=kz_egMtdnL4
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High Performance Liquid chromatography
Normal Phase LCpolar spless polar mp
Reverse Phase LCnonpolar spmore polar mp
Classification
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A. Classical Liquid Chromatography
Column: glass tube (1-5 cm dia, 50-100 cm length)sp: solid particles (150-200 m dia)mp: liquid, gravity fed
Limitations:Slow flow ratesLong separation timesResolution not great
Use: preparative chemistry/biochemistry
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Smaller particle size narrower peaks
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Smaller particle size small H
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High Performance Liquid Chromatography
Instrument
1. Solvent delivery system
2. Pump
3. precolumn4. Injection valve
5. Column6. Detector
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High Performance Liquid Chromatography
1. Solvent ReservoirsIsocratic: single solventGradient elution: vary ratio of solvents
2. PumpSteady, reproducible, constant
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3. Precolumn (Guard Column)Protect more expensive analytical columnIdentical to analytical columnHelps avoid stripping stationary phase from
analytical column
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4. InjectionValvesSyringe/septum
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5. Analytical Column
A. Stationary phase• Liquid on solid surface
High Performance Liquid Chromatography
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Bonded phase packings
R: C8
C18
R: (CH2)n NH2
(CH2)nCN
Reverse Phase vs. Normal Phase
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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
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General Elution Problem in Chromatography
Much shorter time
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High Performance Liquid Chromatography
DetectorsSensitiveLinear responseSmall volume cellsInsensitive to changes in T, solvent
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A. UV-Vis
Must absorb radiation
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B. Refractive Index
Universal
Less sensitive
T, P solvent sensitive
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C. ElectrochemicalMust be able to be
reduced/oxidizedSelective
D. FluorescenceVery sensitiveSelectiveDerivatization
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High Performance Liquid Chromatography
5. Detectors - Comparison
Detector Detection limits, ng
UV 0.1-1
RI 100-1000
Echem 0.01-1
Fluorescence 0.001-0.01
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Qualitative and Quantitative Analysis
QuantitativePeak heightPeak areaCalibration with
standardsInternal
standard method
QualitativeLimited # of componentsAbsence of componentUse hyphenated
techniques
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From Skoog, West, Holler,