High Performance Liquid Chromatography (HPLC)

HPLC techniques enable determination and quantification of molecules across pharma, cosmetics, food, polymer, and environmental labs, serving as an integral tool throughout drug discovery, development, and commercialization.

Explore the history and principles of chromatography, highlighting mobile phase, stationary phase, and analyte, demonstrated by pigment separation with calcium carbonate and petroleum ether.

Master chromatography principles by examining how solubility and polarity drive affinity between analytes and stationary and mobile phases. Learn how hydrophilic and lipophilic interactions enable differential separation.

Explore types of chromatography, including column and planar methods, with a focus on affinity, ion exchange, and gel filtration techniques, and distinguish gas and liquid chromatography in HPLC.

Explore a simple design of high performance liquid chromatography, detailing solvent reservoir, pump, autosampler, and column, and explain how mobile phases, buffers, and detectors yield chromatographic separation.

Explore the technical design of HPLC systems, focusing on solvent reservoirs, binary and quaternary pump configurations, autosampler integration, and detector-driven chromatograms.

Learn how to prepare a clean, inert solvent reservoir for HPLC and manage freshly filtered and degassed mobile phases to prevent bubbles.

Explore the solvent delivery system in HPLC, detailing how pumps generate flow, mix solvents for gradient and isocratic conditions, and compare binary and quaternary pumps.

Explore how the sampler unit, injector, and autosampler introduce samples into HPLC system with 1–200 microliter volumes that determine peak areas, up to 100 samples in amber or clear vials.

Explore column properties, including stainless steel tubing and the stationary phase, to understand how packing materials, particle size, and modifiers influence selectivity and separation in HPLC.

Discover how detectors convert column effluent into chromatograms with quantitative and qualitative outputs. Learn about detector types and key characteristics—sensitivity, noise, and dynamic range—for optimal HPLC performance.

Operate the HPLC system through the PC and monitor to control modules, observe chromatograms, and derive elution times for sample components, enabling qualitative and quantitative analysis and accurate sample calculations.

HPLC in analytical chemistry enables qualitative and quantitative analysis by separating analytes on a column with a solid stationary phase and a liquid mobile phase, followed by detection.

Compare chromatogram retention times between standard and unknown samples to identify molecules, assess presence or absence, and evaluate mixture complexity, purity, and identity in qualitative analysis.

Explore quantitative analysis with HPLC by building calibration curves from standard samples and linking peak area to concentration to determine unknown concentrations.

Master quantitative analysis with HPLC by preparing standard and test concentrations, correlating peak areas to calibration curves, and determining exact tablet content and percent assay.

Normal phase HPLC, a subclass of affinity chromatography, separates analytes by polar interactions with a polar stationary phase using nonpolar mobile phases with small polar modifiers.

Explore reverse phase HPLC, preferred for low molecular weight analytes, using polar mobile phases and apolar C18-modified stationary phases. Consider solvents and alternative columns such as cyano or phenyl.

Understand retention time as a key descriptor in HPLC, using chromatograms and peak maxima to identify analytes and quantify concentrations, while noting how mobile phase flow rate affects retention.

Explore void volume, or dead volume, in HPLC by examining its impact on chromatograms, retention time, and the transfer lines from injector to column and to detector.

Learn how retention factor, or capacity factor, uses reduced retention time and void volume to compare HPLC conditions, guiding method development and column choice independent of flow rate.

Explore size exclusion, or gel filtration, chromatography that separates molecules by size using a porous gel stationary phase, steric hindrance, and retention time to distinguish analytes.

Explore selectivity as the chromatographic system's discrimination ability, enabling separation of multiple analytes and accurate peak area calculation through retention factors and stationary phase choices.

Explore how efficacy and theoretical plates determine chromatography performance, showing how reduced peak broadening yields narrower, sharper peaks for better separation and higher plate numbers.

Evaluate the combined efficacy and selectivity to understand the overall separation power of a chromatography system, considering analyte properties and stationary-phase chemistry.

Explore resolution in high performance liquid chromatography by examining how the difference between peak retention times and peak width yields efficacy and selectivity for a chromatographic system.

Celebrate completing the high performance liquid chromatography course while reinforcing core pharmaceutical quality concepts, including GMP, good laboratory practices, hygiene practices, and process validation guided by FDA and EMA approaches.