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

High Performance Liquid chromatography

Normal Phase LCpolar spless polar mp

Reverse Phase LCnonpolar spmore polar mp

Classification

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

Smaller particle size narrower peaks

Smaller particle size small H

High Performance Liquid Chromatography

Instrument

1. Solvent delivery system

2. Pump

3. precolumn4. Injection valve

5. Column6. Detector

High Performance Liquid Chromatography

1. Solvent ReservoirsIsocratic: single solventGradient elution: vary ratio of solvents

2. PumpSteady, reproducible, constant

3. Precolumn (Guard Column)Protect more expensive analytical columnIdentical to analytical columnHelps avoid stripping stationary phase from

analytical column

4. InjectionValvesSyringe/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

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

General Elution Problem in Chromatography

Much shorter time

High Performance Liquid Chromatography

DetectorsSensitiveLinear responseSmall volume cellsInsensitive to changes in T, solvent

A. UV-Vis

Must absorb radiation

B. Refractive Index

Universal

Less sensitive

T, P solvent sensitive

C. ElectrochemicalMust be able to be

reduced/oxidizedSelective

D. FluorescenceVery sensitiveSelectiveDerivatization

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

Qualitative and Quantitative Analysis

QuantitativePeak heightPeak areaCalibration with

standardsInternal

standard method

QualitativeLimited # of componentsAbsence of componentUse hyphenated

techniques

From Skoog, West, Holler,