Chromatography Course

Lesson 1 • The Chromatographic System Components

Identifies stationary phase, mobile phase, injector, column, and detector as the five core components. Explains how each component influences separation quality.

Lesson 2 • History and Scope of Chromatography

Traces chromatography from Tswett's plant pigment work to modern hyphenated techniques. Contextualises why separation science is central to analytical chemistry.

Lesson 3 • Band Broadening and Plate Theory

Explains theoretical plates, HETP, and the van Deemter equation as efficiency metrics. Connects each broadening term to practical operating variables.

Lesson 4 • Retention and Selectivity Concepts

Introduces retention time, retention factor, and selectivity factor as quantitative descriptors. Demonstrates how adjusting phase chemistry shifts selectivity.

Lesson 1 • TLC Plate Preparation and Spotting

Covers commercial plate selection, sample preparation, and correct spotting technique. Proper spotting directly determines spot quality and Rf reproducibility.

Lesson 2 • Principles of Planar Chromatography

Defines capillary-driven mobile phase migration on flat stationary phases. Distinguishes TLC from paper chromatography by sorbent and mechanism.

Lesson 3 • Mobile Phase Selection for TLC

Applies eluotropic series and polarity matching to choose effective solvent systems. Systematic solvent optimisation reduces trial-and-error development time.

Lesson 4 • High-Performance TLC Techniques

Introduces HPTLC plates, automated spotting, and densitometric scanning for quantitative work. HPTLC bridges qualitative TLC and instrumental quantitative methods.

Lesson 1 • GC Detectors and Their Selectivity

Compares FID, TCD, ECD, NPD, and FPD detectors by sensitivity, selectivity, and linearity. Detector choice is matched to analyte class and required detection limits.

Lesson 2 • GC Columns and Stationary Phases

Classifies packed and capillary columns by polarity, film thickness, and inner diameter. Column selection determines selectivity and is the primary method development variable.

Lesson 3 • GC Injection Techniques

Compares split, splitless, on-column, and PTV injection modes for different sample types. Injection mode selection controls discrimination, discrimination artefacts, and detection limits.

Lesson 4 • Temperature Programming in GC

Explains isothermal vs. programmed temperature runs and their effect on resolution and run time. Optimised temperature programmes separate wide-boiling-range mixtures efficiently.

Lesson 1 • Ion-Exchange and Ion Chromatography

Explains cation and anion exchange retention and suppressed conductivity detection for ions. Ion chromatography enables simultaneous quantification of inorganic and organic ions.

Lesson 2 • Reversed-Phase HPLC Fundamentals

Establishes reversed-phase retention on alkyl-bonded silica using aqueous-organic mobile phases. RP-HPLC is the dominant mode and the reference point for all other HPLC modes.

Lesson 3 • HPLC System Components and Flow Path

Describes pump, degasser, autosampler, column compartment, and detector as the HPLC flow path. Each component's performance specification directly limits overall system accuracy.

Lesson 4 • Normal-Phase and HILIC Modes

Contrasts normal-phase retention on polar sorbents with HILIC retention of hydrophilic analytes. HILIC fills the gap for polar compounds poorly retained in reversed-phase systems.

Lesson 1 • Defining Method Requirements

Translates analytical objectives into measurable performance targets before experimentation begins. Clear requirements prevent over-engineering and guide efficient development.

Lesson 2 • Sample Preparation for Chromatography

Covers extraction, cleanup, and concentration techniques that precede chromatographic injection. Adequate sample preparation determines method sensitivity and matrix compatibility.

Lesson 3 • Systematic Method Development Strategy

Applies design-of-experiments and one-variable-at-a-time approaches to optimise chromatographic conditions. Structured strategies reduce development time and improve robustness.

Lesson 4 • Robustness Testing and Transfer

Uses Plackett-Burman designs to identify critical method parameters and set operational limits. Method transfer protocols ensure equivalent performance across laboratories and instruments.

Lesson 1 • Tandem Mass Spectrometry in Chromatography

Explains product ion, precursor ion, and MRM scan modes for structural confirmation and quantification. MRM is the gold standard for trace quantification in complex biological and environmental matrices.

Lesson 2 • Quantitative MS Method Design

Applies internal standard selection, matrix-matched calibration, and isotope dilution to achieve accurate quantification. Proper quantitative design compensates for matrix effects and instrument variability.

Lesson 3 • Mass Spectrometry Fundamentals for Chromatographers

Introduces ionisation, mass analysis, and detection as the three MS stages relevant to hyphenated methods. Understanding MS fundamentals is prerequisite to interpreting chromatographic MS data.

Lesson 4 • GC-MS Ionisation and Spectral Interpretation

Covers electron ionisation and chemical ionisation modes and their resulting fragmentation patterns. Spectral interpretation skills enable compound identification from library matching and manual analysis.

Lesson 1 • Protein A and Antibody Purification

Explains Protein A ligand selectivity for IgG Fc regions and its role in monoclonal antibody purification. Protein A chromatography is the platform capture step in biopharmaceutical manufacturing.

Lesson 2 • SEC Column Selection and Operation

Compares dextran, agarose, and silica-based SEC media for analytical and preparative scales. Column selection balances resolution, pressure tolerance, and compatibility with target molecules.

Lesson 3 • Affinity Chromatography Fundamentals

Defines biospecific ligand-target interaction as the basis of affinity retention and elution. Affinity chromatography achieves purification factors unattainable by other single-step methods.

Lesson 4 • Size Exclusion Chromatography Principles

Explains pore size distribution, exclusion limit, and permeation as the basis of SEC separation. SEC separates without chemical interaction, making it ideal for native protein analysis.

Lesson 1 • Supercritical Fluid Chromatography

Describes CO2-based mobile phases, modifier addition, and backpressure regulation in SFC systems. SFC excels at chiral separations and offers orthogonal selectivity to reversed-phase HPLC.

Lesson 2 • Ultra-High-Performance Liquid Chromatography

Explains sub-2-micron particles, elevated pressure operation, and reduced dispersion in UHPLC systems. UHPLC delivers faster runs and higher resolution compared to conventional HPLC.

Lesson 3 • Capillary Electrochromatography and Microfluidics

Covers electroosmotic flow-driven separations in packed capillaries and chip-based formats. Miniaturised formats reduce solvent consumption and enable high-throughput screening applications.

Lesson 4 • Chiral Chromatography Principles

Explains three-point interaction model and chiral stationary phase types for enantiomer resolution. Chiral separations are critical for pharmaceutical purity and stereochemical characterisation.