HPLC Techniques and Applications Quiz

HPLC utilizes different separation mechanisms based on the interaction of the analyte with the stationary phase and mobile phase. These include partition chromatography, adsorption chromatography, ion-exchange chromatography, size exclusion chromatography, affinity chromatography, and chiral chromatography.

A type of HPLC where the analyte distributes between two immiscible liquid phases: the stationary phase and the mobile phase. The stationary phase can be a liquid held on a silica support.

A specific form of partition chromatography where the stationary phase is chemically bonded to the solid support. This offers improved stability and reproducibility compared to physically adsorbed liquid phases.

HPLC offers several advantages over other analytical techniques, including high speed, excellent resolution, high accuracy, sensitivity capable of detecting very small amounts of analyte, and potential for automation.

Smaller particle size in the stationary phase leads to a greater number of theoretical plates (NTP), a lower plate height (HETP), higher pressure, shorter analysis time, and an improved limit of detection (LOD).

HPLC instrumentation consists of a mobile phase reservoir, a pump, a sample injection valve, a column containing the stationary phase, a detector, and data acquisition and analysis systems.

The stationary phase in HPLC is typically composed of highly pure, spherical, microporous particles like silica or polystyrene. It acts as the foundation for the separation process.

Bare silica can be used as the stationary phase for adsorption chromatography in HPLC. It's typically available in particle sizes ranging from 1.0 to 5.0 µm.

A type of HPLC where the solvent composition remains constant throughout the separation process.

A type of HPLC where the solvent composition changes continuously during the separation process, typically by increasing the polarity of the mobile phase.

Increasing the polarity of the mobile phase in reversed-phase HPLC (RP-HPLC) decreases the retention time (tR) of the analyte. Analytes spend less time interacting with the non-polar stationary phase and are eluted faster.

Gradient elution offers two main advantages: faster separation times and better resolution (Rs) compared to isocratic elution.

A common detector in HPLC that measures the absorbance of analytes in the ultraviolet (UV) range. It detects compounds with moderate UV absorption.

A highly sensitive HPLC detector that measures the fluorescence of analytes, which is the emission of light after absorbing UV radiation.

An HPLC detector that measures changes in the refractive index of the mobile phase caused by the presence of analytes. Not suitable for gradient analysis due to changes in mobile phase composition.

An HPLC detector that measures the electrical properties of analytes, such as conductance or potential. Useful for determining the presence of substances with unique electrical properties.

A type of HPLC where the stationary phase is polar and the mobile phase is non-polar. The least polar compound elutes first, and increasing the polarity of the mobile phase leads to shorter elution times.

A type of HPLC where the stationary phase is non-polar and the mobile phase is polar. The most polar compound elutes first, and increasing the polarity of the mobile phase leads to longer elution times.

The stationary phase in normal-phase chromatography is polar. It is typically made of silica or alumina, which have polar functional groups.

The stationary phase in reverse-phase chromatography is non-polar. It is typically a hydrocarbon chain, such as C8 or C18, covalently attached to a silica surface.

In normal phase HPLC, the least polar compound elutes first, followed by increasingly polar compounds.

In reverse phase HPLC, the most polar compound elutes first, followed by increasingly less polar compounds.

Increasing the polarity of the mobile phase in normal phase HPLC leads to shorter elution times. This is because the mobile phase becomes better at competing with the stationary phase for the analytes.

Increasing the polarity of the mobile phase in reverse phase HPLC leads to longer elution times. This is because the polar mobile phase becomes better at holding onto the polar analytes.