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
Source: Skoog, Holler, and Nieman, Principles of Instrumental Analysis, 5th edition, Saunders College Publishing.
Comparison of Reversed-Phase Mediaof Different Chain Length
Peak ID1 – Uracil2 – Phenol3 – Acetophenone4 – Nitrobenzene5 – Methyl Benzoate6 – Toluene
Source: Rubinson and Rubinson, Contemporary Instrumental Analysis, Prentice Hall Publishing.
General Schematic of LC
Source: Skoog, Holler, and Nieman, Principles of Instrumental Analysis, 5th edition, Saunders College Publishing.
Effect of Gradient Elution
Source: Skoog, Holler, and Nieman, Principles of Instrumental Analysis, 5th edition, Saunders College Publishing.
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
Source: Skoog, Holler, and Nieman, Principles of Instrumental Analysis, 5th edition, Saunders College Publishing.
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.
Source: Skoog, Holler, and Nieman, Principles of Instrumental Analysis, 5th edition, Saunders College Publishing.
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.
Source: Skoog, Holler, and Nieman, Principles of Instrumental Analysis, 5th edition, Saunders College Publishing.
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
Source: Rubinson and Rubinson, Contemporary Instrumental Analysis, Prentice Hall Publishing.
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.
Source: Skoog, Holler, and Nieman, Principles of Instrumental Analysis, 5th edition, Saunders College Publishing.
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