Four basic types of column chromatography where mobile phase is a liquid Partition Chromatography Bonded-Phase Liquid-Liquid Adsorption Chromatography Liquid-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 Material and 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 Media of Different Chain Length Peak ID 1 – Uracil 2 – Phenol 3 – Acetophenone 4 – Nitrobenzene 5 – Methyl Benzoate 6 – 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.

Source: Rubinson and Rubinson, Contemporary Instrumental Analysis, Prentice Hall 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 psi Pulse-free output Flow rates from 0.1-10 mL/min 0.5% or better flow control reproducibility Corrosion resistant

LC Pumping Systems Reciprocating Pumps Displacement 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 mm diameter Microcolumns: 1-4.6 mm internal diameter with 3-5 mm packings

LC Packing Materials Pellicular Porous Spherical, nonporous, glass or polymer beads 30-40-mm diameter Thin porous layer of silica, alumina, or ion-exchange resin deposited on surface Porous Most common 3-10-mm diameter Silica (most common), alumina, or ion-exchange resin Thin organic film bonded to surface

LC Detectors General Types: 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.

Source: Rubinson and Rubinson, Contemporary Instrumental Analysis, Prentice Hall 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 interfacing problems 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 mixture k’ of 0.5-20 for multicomponent mixture Match analyte polarity to stationary phase polarity Mobile 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 for normal-phase and reversed-phase LC. Source: Skoog, Holler, and Nieman, Principles of Instrumental Analysis, 5th edition, Saunders College Publishing.

Reversed-Phase Ion-Pair Chromatography Mobile phase: aqueous buffer containing organic solvent and counter-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 and ions 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 conductance are suppressor columns. Suppressor column packed with secondary ion-exchange resin to convert solvent ions to a molecular species.

Size-Exclusion Chromatography Packing contains network of uniform pores into which solute and 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 defines MW beyond which no retention occurs. Beyond permeation limit all molecules elute in one band since they can all freely (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 Disadvantages 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 Both Advantages Favorable to LC Efficient, highly selective, widely applicable Only requires small sample May be nondestructive of sample May have quantitative analysis Advantages Favorable to LC Can separate nonvolatile or thermal unstable samples Generally applicable to inorganic ions Advantages Favorable to GC Simple, less expensive equipment Rapid More efficient, higher resolution Easily interfaced with mass spectrometry