High-Performance Liquid Chromatography (HPLC) is a widely used analytical technique

in the field of chemistry and biochemistry for separating, identifying, and quantifying
compounds in a mixture. It is based on the principles of liquid chromatography, where a
liquid mobile phase is used to carry the sample through a stationary phase.

Principle of HPLC: HPLC works on the principle of chromatography, which involves the
separation of components in a mixture based on their differential affinity for a stationary
phase and a mobile phase. In HPLC, the stationary phase is typically a solid adsorbent
(e.g., silica) packed into a column, and the mobile phase is a liquid (usually a solvent or a
mixture of solvents). The separation is achieved by varying the composition of the
mobile phase and the interaction between the sample compounds and the stationary
phase. Key principles include:

1. Partitioning: The compounds in the sample partition between the mobile phase
and the stationary phase based on their chemical properties, leading to
differential migration rates.
2. Adsorption: Compounds interact with the stationary phase through various
forces such as Van der Waals, hydrogen bonding, and ionic interactions, causing
different retention times.
Working
1. Sample Preparation: The sample is dissolved in an appropriate solvent and
filtered to remove particulate matter.
2. Column Selection: A column with the appropriate stationary phase (e.g., C18 for
nonpolar compounds) and dimensions is selected based on the sample's
properties and the desired separation.
3. Mobile Phase: The choice of mobile phase (composition and flow rate) is critical
for achieving separation. Gradient elution, where the mobile phase composition
changes over time, is often used for complex mixtures.
4. Injection: The prepared sample is injected into the HPLC system.
5. Separation: The sample components pass through the column and are separated
based on their interactions with the stationary phase. Each compound elutes at a
specific retention time.
6. Detection: The eluted compounds are detected using various detectors like UV-
Vis, fluorescence, or mass spectrometry.
7. Fraction Collection: In preparative HPLC, the fractions containing the target
compound(s) are collected for further processing.

Flow Chart of HPLC Working: Here's a simplified flowchart of the HPLC process:

1. Sample Preparation
  Dissolve sample in appropriate solvent.
 Filter to remove particulates.
2. Column Selection
 Choose column type and dimensions.
3. Mobile Phase Preparation
 Prepare the mobile phase with the desired solvent composition.
4. Injection
 Inject the sample into the HPLC system.
5. Separation
 Sample components pass through the column.
 Separation based on interaction with stationary phase.
6. Detection
 Compounds are detected as they elute from the column.
7. Data Analysis
 Analyze chromatograms to identify and quantify compounds.
8. Fraction Collection (for preparative HPLC)

Components of an HPLC System: An HPLC system comprises several essential

components:

1. Pump: Delivers the mobile phase at a constant flow rate.

2. Injector: Allows the introduction of the sample into the system.
3. Column: Contains the stationary phase for separation.
4. Detector: Detects and quantifies the compounds eluting from the column.
5. Data System: Collects and analyzes data from the detector.
6. Mobile Phase Reservoirs: Store the solvents used as the mobile phase.
7. Gradient Controller: Manages the composition of the mobile phase over time
(for gradient elution).
8. Auto-sampler (optional): Automates sample injection.
9. Column Oven (optional): Controls and maintains the column temperature.
10. Fraction Collector (for preparative HPLC): Collects and stores separated
fractions.