High Performance Liquid Chromatography

High Performance Liquid Chromatography (HPLC) is an analytical separation technique that pumps a liquid mobile phase at high pressure through a column packed with stationary-phase particles, separating a sample’s components based on their differing affinity for each phase.

HPLC has played a key role in the development of separation science, with major contributions in pharmaceuticals, the food industry, life sciences, and basic research. The application base is widening day by day due to advances in instrumentation and technological progress in basic chemistry. Employment prospects are strong for anyone with a good understanding of HPLC, and the growing number of quality control, method development, and drug development laboratories has further increased demand for skilled HPLC professionals.

Our HPLC programme provides insight into both the theoretical aspects and practical hands-on experience of analyzing a diverse range of samples, along with the added benefit of interaction with our technical experts.

What Is HPLC?

HPLC is an analytical technique used for the separation of components of an organic mixture of compounds when such compounds are nonvolatile, thermally unstable, and have relatively high molecular weights. Its earlier name was High Pressure Liquid Chromatography, because it involved the use of a liquid mobile phase requiring higher pressures than the gases used in Gas Chromatography.

A liquid carrier stream, termed the mobile phase, carries the injected sample through the separation column and to the detector. In the column, the individual components are separated based on physicochemical interactions, and the elution order depends on those interactions. The separated components are then detected based on the absorption of light, changes in refractive index, electrochemical/conductivity changes, or the size distribution of eluting molecules.

The detector response takes the shape of peaks with an area directly proportional to the amount present. Operating software handles the required calculations and controls parameters like injection volume, injection sequence, detection wavelength, and wash cycles — most of a user’s time is spent on mobile phase preparation, buffers and standards, and record-keeping rather than the run itself.

Key Components of an HPLC System

Mobile Phase

The mobile phase transports the sample through the system. It must be inert to the sample components — pure solvents or buffer combinations are commonly used — and free of particulate impurities, and it should be degassed before use.

Mobile Phase Reservoirs

Inert containers for mobile phase storage and transport. Generally transparent glass bottles are used for visual inspection of the mobile phase level, with stainless steel particulate filters inside to remove impurities.

Pumps

Variations in mobile-phase flow rate affect the elution time of sample components and introduce errors. Pumps provide a constant flow of mobile phase to the column under constant pressure.

Injectors

Injectors provide constant-volume injection of the sample into the mobile-phase stream. Inertness and reproducibility of injection are necessary for a high level of accuracy.

Column

A stainless-steel tube packed with a stationary phase. It’s a vital component and must be maintained properly per supplier instructions to get reproducible separation efficiency run after run.

Column Oven

Temperature variation during a run can change the retention time of separated components. A column oven maintains constant column temperature via air circulation, ensuring a constant mobile-phase flow rate through the column.

Detector

Gives a specific response for the components separated by the column, independent of changes in mobile-phase composition. Most applications use UV-VIS detection, though detectors based on other techniques are also popular.

Data Acquisition & Control

Modern HPLC systems are computer-based; software controls operational parameters (mobile-phase composition, temperature, flow rate, injection volume and sequence) and handles acquisition and treatment of output.

Specialized Versions

Specialized systems may include solvent selection valves, vacuum degassers, autosamplers, column switches, pre- or post-column derivatization, and fraction collectors — all covered in our certificate programme.

Recent Advances in HPLC

Scale of Operation

HPLC can analyze samples across a vast concentration and molecular-weight range — from picogram to gram-level quantities — through different column dimensions and operational modes, enabling work on both small molecules and large biomolecules weighing several million daltons.

Stationary Phase Technologies

Silica-based columns remain the backbone of stationary phases, but monolithic phases, zirconia-based phases, and core-shell technologies have contributed to greater analysis speed, wider temperature and pressure ranges, and studies over extended pH ranges.

Choice of Detectors

Common options include UV, RI, photodiode array, fluorescence, conductivity, and light-scattering detectors, plus mass-selective detectors. LC-MS-MS is widely used for highly sensitive analysis of complex mixtures, including bioanalytical studies and pesticide-residue analysis.

Choice of Mobile Phases

Reverse-phase chromatography — using polar solvents as the mobile phase — is the most popular mode. Combinations of solvents with different polarities, buffers, and isocratic/gradient operation can help resolve complex mixtures with closely spaced peaks.

Learn More

See our full Glossary of HPLC Terms if you’re unfamiliar with any terminology.

Want to master the fundamentals and operation of HPLC? Join our certificate course on HPLC and become a certified expert online.