1. Principle of HPLC

High-Performance Liquid Chromatography (HPLC) works on the principle of separation by differential partitioning of sample components between a mobile phase (liquid) and a stationary phase (solid adsorbent material inside a column).

• Components with less affinity towards the stationary phase elute faster.

• Components with more affinity elute slower.

• Retention time is unique for each compound under fixed conditions, allowing both qualitative and quantitative analysis.

The separation can be based on:

• Polarity (Normal Phase & Reverse Phase HPLC)

• Ion exchange

• Size exclusion

• Affinity interactions

2. HPLC System Working

Step-by-step process:

1. Solvent Reservoirs

   • Hold mobile phase(s) — could be a single solvent or a mixture.

2. Pump System

   • Delivers mobile phase at high pressure (up to 6000 psi) to push it through the column at a constant flow rate.

3. Injector

   • Introduces a fixed volume of the sample into the mobile phase stream.

4. HPLC Column

   • Packed with stationary phase (e.g., silica particles in RP-HPLC coated with C18 chains).

   • Separation occurs here due to different affinities of analytes for the stationary phase.

5. Detector

   • Detects separated analytes as they elute. Common: UV-Vis, PDA, fluorescence detectors.

6. Data System / Chromatogram

   • Converts detector signals into peaks on a chromatogram.

   • Retention time → identification, Peak area → quantification.

Schematic Flow:
Mobile Phase → Pump → Injector → Column → Detector → Data System

3. Applications of HPLC

• Drug assay & impurity profiling in pharmaceuticals

• Stability testing

• Bioanalytical studies

• Food & beverage analysis

• Environmental contaminant detection