four basic types of column chromatography where mobile phase is a liquid partition chromatography...

Four basic types of column chromatographywhere mobile phase is a liquid

Partition ChromatographyBonded-PhaseLiquid-Liquid

Adsorption ChromatographyLiquid-Solid

Ion-Exchange Chromatography

Exclusion (or Gel) Chromatography

General Advantages of LC

Sensitivity

Quantitative

Separation of nonvolatile and/or thermally fragile compounds

Wide applicability

Applications of LC

Source: Skoog, Holler, and Nieman, Principles of Instrumental Analysis, 5th edition, Saunders College Publishing.

LC Separation Mechanisms

Source: Rubinson and Rubinson, Contemporary Instrumental Analysis, Prentice Hall Publishing.

Effect of Particle Size of Packing Materialand Flow Rate on Plate Height in LC


Comparison of Reversed-Phase Mediaof Different Chain Length

Peak ID1 – Uracil2 – Phenol3 – Acetophenone4 – Nitrobenzene5 – Methyl Benzoate6 – Toluene


General Schematic of LC


Effect of Gradient Elution


LC Pumping Systems

General Requirements:Generate pressures up to 6000 psiPulse-free outputFlow rates from 0.1-10 mL/min0.5% or better flow control reproducibilityCorrosion resistant

LC Pumping Systems

Reciprocating Pumps• Pulsed flow must be damped• Small internal volume• High output pressures• Adaptable for gradient elution• Constant flow rates independent of column back-pressure or solvent viscosity

Displacement Pumps• Flow independent of viscosity and back-pressure• Limited solvent capacity• Inconvenient to change solvents

Pneumatic Pumps• Inexpensive• Pulse free• Limited capacity and pressure• Dependent on solvent viscosity and backpressure• Not good for gradient elution

LC Columns

10-30 cm long x 4-10 mm internal diameter

Packing usually 5 or 10 m diameter

Microcolumns: 1-4.6 mm internal diameter with 3-5 m packings

LC Packing Materials

Pellicular•Spherical, nonporous, glass or polymer beads•30-40-m diameter•Thin porous layer of silica, alumina, or ion-exchange resin deposited on surface

Porous•Most common•3-10-m diameter•Silica (most common), alumina, or ion-exchange resin•Thin organic film bonded to surface

LC Detectors

General•Similar characteristics to GC detectors, except temperature range•Minimal internal volume to avoid peak broadening

Types:•Respond to bulk property of mobile phase, modulated by presence of solute•Respond to specific property of solute•General response to solute following volatilization (removal) of mobile phase

Common LC Detectors

UV – one of most common

Fluorescence – much greater sensitivity than UV

Refractive Index – widely used general detector

Electrochemical – based on amperometry, polarography, coulometry,or conductometry. High sensitivity, wide applicability range

Mass Spectrometry – becoming increasingly used since interfacingproblems figured out. Expensive.

LC Mobile Phase Qualities

•High purity•Reasonable cost (and disposal)•Boiling point 20-50 °C above column temperature•Low viscosity•Low reactivity•Immiscibile with stationary phase•Compatible with detector•Safety – limited flammability and toxicity

LC Mobile Phase Selection

k’ of 2-5 for two or three component mixturek’ of 0.5-20 for multicomponent mixture

Match analyte polarity to stationary phase polarityMobile phase of different polarity

Normal Phase:•nonpolar solvent, polar stationary phase•least polar component elutes first•increasing mobile phase polarity decreases elution time

Reversed Phase:•polar solvent (water, MeOH, ACN), nonpolar stationary phase•most polar component elutes first•increasing mobile phase polarity increases elution time•most widely used

Relationship between polarity and elution times fornormal-phase and reversed-phase LC.


Reversed-Phase Ion-Pair ChromatographyMobile phase: aqueous buffer containing organic solvent andcounter-ion of opposite charge of analyte.

Ion-pair forms neutral species soluble in nonaqueous solvent.


Ion-Exchange Processes

Based on exchange equilibria between ions in solution andions of like charge on surface of essentially insoluble, high-molecular weight solid.

Most common cation exchangers:•The strong acid sulfonic acids, –SO3

-H+

•The weak acid carboxylic acids, –COOH

Most common anion exchangers:•The strong base ternary amines, -N(CH3)3

+OH-

•The weak base primary amines, -NH3OH

Mechanism of Ion Chromatography


IC Detection

Typically done with conductivity detection•Sensitive•Universal for charged species

Key to column regeneration and avoid high eluent conductanceare suppressor columns. Suppressor column packed withsecondary ion-exchange resin to convert solvent ions toa molecular species.

Size-Exclusion Chromatography

Packing contains network of uniform pores into which soluteand solvent can diffuse.

Solute is “trapped” in pore until carried away by solvent.

Residence time in pore related to effective molecular size of solute.•Molecules larger than average pore size are excluded from pore, not retained.•Molecular diameter significantly smaller than pore can penetrate throughout pore, so

elute last.•Fractionation of intermediate-sized molecules. Some shape dependence.

Calibration Curve for SEC

Exclusion limit definesMW beyond which noretention occurs.

Beyond permeation limitall molecules elute in oneband since they can allfreely (completely)penetrate the pores.


Types of SEC

Gel Filtration Chromatography•Aqueous solvent•Hydrophilic Packings

Gel Permeation Chromatography•Nonpolar Organic Solvents•Hydrophobic Packings

Advantages of SEC

•Short, well-defined separation times•Narrow bands, good sensitivity•No sample loss since solutes do not interact with stationary phase•Absence of column deactivation

Disadvantages of SEC•Limited number of bands accommodated since short time scale•Not applicable to similar-sized molecules, like isomers

Comparison of LC and GC

BothEfficient, highly selective, widely applicableOnly requires small sampleMay be nondestructive of sampleMay have quantitative analysis

Advantages Favorable to LCCan separate nonvolatile or thermal unstable samplesGenerally applicable to inorganic ions

Advantages Favorable to GCSimple, less expensive equipmentRapidMore efficient, higher resolutionEasily interfaced with mass spectrometry

four basic types of column chromatography where mobile phase is a liquid partition chromatography...

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