HPLC-Principle, Instrumentation, Parameters and Applications

Learn the basics of hplc, high pressure liquid chromatography, including principle, types of hplc techniques, and how a mobile phase and stationary phase enable component separation.

Learn how high performance liquid chromatography uses small particles and high pressure to separate components as the mobile phase flows from injector to detector, producing peaks proportional to amounts.

Explore HPLC techniques by modes and principles—normal and reverse phase, adsorption and size-exclusion, affinity chromatography—plus isocratic and gradient separations for analytical and preparative, qualitative and quantitative analysis.

Describe core HPLC components and two pump types, including manual and auto injectors, guard and analytical columns, detectors, recorders, and integrators, and explain how constant high-pressure delivery reduces detector noise.

The mixing unit blends solvents via static or dynamic mixers, with low-pressure helium degassing and high-pressure variants, while the gradient controller enables separation with same solvent or with changing polarity.

Explore how HPLC injectors introduce samples with constant flow and pressure, using septum injectors, flow injectors, and rearranging injectors to deliver precise microliter volumes free of bubbles and particulates.

Explore guard and analytical columns, their roles in protecting the analytical column and enabling sample filtration, while comparing normal phase and reverse phase column types.

Explore ion-exchange and size-exclusion HPLC columns, where charged stationary phases and mobile phases drive separation. Learn how column material, particle size, and functional groups affect ion exchange and size-based separation.

Explore the hplc detectors including UV-visible, array (photodiode array), refractive index, fluorescence, electrochemical, light scattering, mass, and optical rotation detectors.

Examine refractive index detectors—deflection and Fresnel types—whose low sensitivity limits analytical use; compare calorimeter detectors emitting fluorescent radiation, conductivity detectors for ions, and electrochemical amperometric (parametric) detectors with three electrodes.

Explore hplc detectors, including operating light scattering detectors (universal) and lc-ms systems. Examine optical dispersion detectors based on refractive index differences and circularly polarized light.

Recorders capture detector responses, baseline, and peaks over time in HPLC, enabling retention time detection; integrators, as enhanced recorders, measure peak retention time, height, width, and areas.

Explore key HPLC parameters—retention time, retention volume, resolution, separation factor, theoretical plates, and asymmetric factor—and show how stronger interaction with the stationary phase increases retention time.

Explore how resolution and separation factor quantify how two components separate in HPLC, using retention times, peak widths, and baseline separation criteria.

Explore theoretical plates and their role in HPLC column efficiency, including plate height, HETP, diffusion, and the impact of flow rate and packing on peak sharpness.

Understand the asymmetric factor in HPLC, including tailing and fronting from adsorption sites and stationary phase saturation, and how pretreatment and polar mobile phases influence it; compute from peak heights.

HPLC is a versatile, sensitive technique used in pharmaceutical, chemical, environmental, forensic, and biotechnological applications. Identify compounds by retention time against standards and assess purity and impurities from peak areas.

This lecture covers quantitative analysis methods to determine component quantity, including direct comparison method, calibration curve method, and internal standard method, using peak areas and interpolation.

Apply multi component analysis with HPLC to quantify drug mixtures in formulations, isolate and identify drugs and metabolites in urine, plasma, and serum, and perform preparative-scale purification and stability studies.