Four basic types of column chromatography
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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

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Four basic types of column chromatography where mobile phase is a liquid

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


Four basic types of column chromatography where mobile phase is a liquid

General Advantages of LC

Sensitivity

Quantitative

Separation of nonvolatile and/or thermally fragile compounds

Wide applicability


Four basic types of column chromatography where mobile phase is a liquid

Applications of LC

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


Four basic types of column chromatography where mobile phase is a liquid

LC Separation Mechanisms

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


Four basic types of column chromatography where mobile phase is a liquid

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.


Four basic types of column chromatography where mobile phase is a liquid

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.


Four basic types of column chromatography where mobile phase is a liquid

General Schematic of LC

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


Four basic types of column chromatography where mobile phase is a liquid

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


Four basic types of column chromatography where mobile phase is a liquid

Effect of Gradient Elution

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


Four basic types of column chromatography where mobile phase is a liquid

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


Four basic types of column chromatography where mobile phase is a liquid

  • 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


Four basic types of column chromatography where mobile phase is a liquid

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


Four basic types of column chromatography where mobile phase is a liquid

  • LC Packing Materials

  • Pellicular

  • 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


Four basic types of column chromatography where mobile phase is a liquid

  • 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


Four basic types of column chromatography where mobile phase is a liquid

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


Four basic types of column chromatography where mobile phase is a liquid

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


Four basic types of column chromatography where mobile phase is a liquid

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.


Four basic types of column chromatography where mobile phase is a liquid

  • 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


Four basic types of column chromatography where mobile phase is a liquid

  • 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


Four basic types of column chromatography where mobile phase is a liquid

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.


Four basic types of column chromatography where mobile phase is a liquid

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.


Four basic types of column chromatography where mobile phase is a liquid

  • 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


Four basic types of column chromatography where mobile phase is a liquid

Mechanism of Ion Chromatography

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


Four basic types of column chromatography where mobile phase is a liquid

  • 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.


Four basic types of column chromatography where mobile phase is a liquid

  • 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.


Four basic types of column chromatography where mobile phase is a liquid

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.


Four basic types of column chromatography where mobile phase is a liquid

  • Types of SEC

  • Gel Filtration Chromatography

    • Aqueous solvent

    • Hydrophilic Packings

  • Gel Permeation Chromatography

    • Nonpolar Organic Solvents

    • Hydrophobic Packings


Four basic types of column chromatography where mobile phase is a liquid

  • 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


Four basic types of column chromatography where mobile phase is a liquid

Comparison of LC and GC

Both

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


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