Size Exclusion Chromatography Course

Lesson 1 • Principles of Chromatographic Separation

Covers stationary/mobile phase interactions and band broadening fundamentals. Establishes vocabulary used throughout the course.

Lesson 2 • Physical Basis of Size Exclusion

Explains steric exclusion and pore diffusion as the sole separation mechanism. Connects pore geometry to molecular size discrimination.

Lesson 3 • Key SEC Terminology and Parameters

Defines void volume, elution volume, and distribution coefficient Kd. Provides the quantitative language needed for all subsequent chapters.

Lesson 1 • Column Care and Lifetime

Covers storage, regeneration, and performance monitoring to maximize column lifespan. Directly impacts data reproducibility and cost efficiency.

Lesson 2 • Pore Size and Fractionation Range

Explains how nominal pore size determines the molecular weight fractionation window. Guides column selection for proteins, polymers, and small molecules.

Lesson 3 • Porous Particle Chemistries

Surveys silica, cross-linked dextran, agarose, and synthetic polymer supports. Links chemistry to chemical compatibility and operating range.

Lesson 1 • Sample Preparation for SEC

Covers filtration, buffer exchange, and concentration steps before injection. Prevents column contamination and ensures accurate molecular weight data.

Lesson 2 • Flow Rate and Pressure Optimization

Relates flow rate to resolution via the van Deemter equation adapted for SEC. Balances analysis time against peak efficiency.

Lesson 3 • Temperature Effects on SEC

Describes how temperature alters viscosity, diffusion, and secondary interactions. Guides temperature selection for reproducible separations.

Lesson 4 • Mobile Phase Composition Principles

Explains how ionic strength, pH, and organic modifiers suppress non-specific interactions. Establishes criteria for choosing aqueous vs. organic SEC solvents.

Lesson 1 • UV-Vis Absorbance Detection

Explains wavelength selection, detector linearity, and baseline stability for UV detection. Covers protein detection at 280 nm and nucleic acid detection at 260 nm.

Lesson 2 • HPLC and FPLC System Architectures

Compares high-pressure and low-pressure liquid chromatography platforms used for SEC. Guides instrument selection based on pressure, scale, and sample type.

Lesson 3 • Pumps, Injectors, and Tubing

Details pump types, injection valve operation, and tubing dead volume. Minimizes extra-column band broadening for accurate results.

Lesson 4 • Viscometry and Mass Spectrometry Coupling

Covers online viscometry for intrinsic viscosity and SEC-MS coupling for identity confirmation. Expands analytical information from a single SEC run.

Lesson 1 • Molecular Weight Averages and Dispersity

Calculates Mn, Mw, Mz, and dispersity Ð from chromatographic data. Connects distribution statistics to material properties and product quality.

Lesson 2 • Relative Calibration with Standards

Uses narrow-distribution standards to build a log MW vs. elution volume curve. Establishes the most common calibration approach in polymer and protein analysis.

Lesson 3 • Absolute Molecular Weight with MALS

Derives absolute MW directly from light scattering without calibration standards. Eliminates column-chemistry bias for complex macromolecules.

Lesson 4 • Validation of Calibration Methods

Applies accuracy, precision, linearity, and range criteria to calibration validation. Ensures data integrity for regulated and research environments.

Lesson 1 • Injection Volume and Sample Load Optimization

Determines maximum injection volume and mass load before resolution degrades. Prevents overloading artifacts in both analytical and preparative SEC.

Lesson 2 • Defining Method Goals and Requirements

Translates analytical objectives into measurable method performance criteria. Aligns column, mobile phase, and detector choices with the intended use.

Lesson 3 • Resolution Enhancement Strategies

Applies column coupling, flow rate adjustment, and temperature tuning to improve resolution. Provides practical tools for difficult separations.

Lesson 4 • Method Robustness and Ruggedness Testing

Evaluates method sensitivity to small deliberate changes in key parameters. Identifies critical parameters and sets acceptable operating ranges.

Lesson 1 • Protein Aggregation Analysis

Uses SEC to quantify monomer, dimer, and higher-order aggregate content in protein samples. Directly supports drug substance quality assessment.

Lesson 2 • Formulation and Stability Studies

Monitors aggregation and fragmentation during accelerated and real-time stability studies. Links SEC data to formulation decisions and shelf-life predictions.

Lesson 3 • Nucleic Acid and Oligonucleotide Analysis

Adapts SEC conditions for DNA, RNA, and oligonucleotide size profiling. Addresses unique mobile phase and column requirements for nucleic acids.

Lesson 4 • Monoclonal Antibody Characterization

Applies SEC to assess mAb size variants, fragments, and conjugate integrity. Covers method conditions specific to IgG and antibody-drug conjugates.

Lesson 1 • Technical Report Writing for SEC

Structures SEC reports with methods, results, discussion, and conclusions sections. Communicates findings clearly to scientific and non-scientific stakeholders.

Lesson 2 • Data Integrity and Electronic Records

Applies audit trail, access control, and data review principles to SEC software systems. Meets expectations of data integrity frameworks in regulated industries.

Lesson 3 • Chromatogram Interpretation and Peak Integration

Applies baseline correction, peak detection, and integration algorithms to SEC chromatograms. Ensures accurate area percent and molecular weight calculations.

Lesson 4 • Method Validation for Regulated Environments

Executes validation parameters including specificity, linearity, accuracy, and precision. Prepares documentation for regulatory submission and audit readiness.