High-Performance Liquid Chromatography (HPLC): Principle, Instrumentation, Types, and Applications

Introduction to HPLC

High-Performance Liquid Chromatography (HPLC) is an advanced form of column chromatography widely used in pharmaceutical analysis, food science, environmental monitoring, and biological research.

It is a powerful separation technique that enables the identification, quantification, and purification of compounds from complex mixtures.

Unlike traditional column chromatography, HPLC applies high pressure (up to 400 atm) to push the mobile phase through a tightly packed column, resulting in faster, more efficient, and high-resolution separation.

Key Features of HPLC

• High resolution and accuracy.

• Suitable for thermally unstable and non-volatile compounds.

• Produces reproducible and reliable results.

• Can separate polar, non-polar, ionic, and biological molecules.

Principle of HPLC

The principle of HPLC is based on the differential distribution of analytes between two phases:

• Stationary Phase → column packed with adsorbent particles (e.g., silica, C18).

• Mobile Phase → liquid (single solvent or mixture) that flows under high pressure.

Mechanism of Separation

1. A sample mixture is injected into the HPLC column.

2. Each compound interacts differently with the stationary phase (adsorption, partition, ion-exchange) and mobile phase (polarity, solubility).

3. Because of these varying interactions, compounds migrate at different speeds.

4. Compounds elute at distinct times, known as Retention Time (tR).

5. A chromatogram is obtained, where each peak represents a compound.

Modes of Separation in HPLC

1. Normal Phase HPLC (NP-HPLC)

   • Stationary phase: Polar (silica).

   • Mobile phase: Non-polar (hexane, chloroform).

   • Application: Separation of polar compounds.

2. Reverse Phase HPLC (RP-HPLC)

   • Stationary phase: Non-polar (C18).

   • Mobile phase: Polar (water, methanol, acetonitrile).

   • Most widely used method.

3. Ion Exchange HPLC – separation based on ionic charge.

4. Size Exclusion HPLC – separation based on molecular size.

5. Affinity HPLC – separation based on specific biological binding (e.g., antigen–antibody).

Instrumentation of HPLC

The HPLC system is made up of several key components, each performing a specific role to ensure accurate and efficient separation.

1. Solvent Reservoir (Mobile Phase Container)

• Stores the mobile phase (solvent or solvent mixture).
• Must be degassed to remove air bubbles (by vacuum filtration, sonication, or helium sparging).
• Mobile phases should be filtered to remove impurities that can clog the column.

2. Pump

• Delivers the mobile phase through the system at high pressure (up to 6000 psi).
• Maintains a constant flow rate (0.1–10 mL/min).
• Types of pumps:
  • Reciprocating Pump: common, provides pulseless flow.
  • Syringe Pump: used for small flow rates.
  • Pneumatic Pump: less common.

3. Injector

• Introduces the sample into the mobile phase stream.
• Must be precise and reproducible.
• Types:
  • Manual injector (via a syringe through an injection valve).
  • Auto-sampler (used in advanced HPLC for automated multiple injections).

4. Column (Heart of HPLC)

• Most important part where separation occurs.
• Made of stainless steel tubes packed with stationary phase (e.g., silica particles).
• Length: 10–30 cm; Internal diameter: 2–5 mm.
• Common types:
  • Normal phase column (polar stationary phase).
  • Reverse phase column (RP-HPLC) (non-polar stationary phase like C18).
• Column efficiency depends on particle size, surface area, and porosity.

5. Detector

• Detects separated compounds as they elute from the column.
• Converts the signal into an electronic response (peak) on a chromatogram.
• Common detectors:
  • UV-Visible detector (most common) – measures absorbance.
  • Fluorescence detector – more sensitive.
  • Refractive Index (RI) detector – for compounds without chromophores.
  • Electrochemical detector – for redox-active compounds.
  • Mass spectrometer (LC-MS) – for highly sensitive detection.

6. Data Processing System (Computer/Integrator)

• Collects data from the detector.
• Converts it into a chromatogram (graph of detector response vs time).
• Used to calculate retention time, peak area, and concentration.

7. Waste Reservoir

• Collects the used mobile phase and sample after detection.
• Important for safe disposal.

Types of HPLC

(a) Based on Polarity

1. Normal Phase HPLC (NP-HPLC):

   • Stationary phase: Polar (silica).

   • Mobile phase: Non-polar (hexane, chloroform).

   • Used for separation of polar compounds.

2. Reverse Phase HPLC (RP-HPLC):

   • Stationary phase: Non-polar (C18, C8).

   • Mobile phase: Polar (water, methanol, acetonitrile).

   • Most common and versatile technique.

(b) Based on Elution Technique

• Isocratic Elution: Mobile phase composition remains constant.

• Gradient Elution: Mobile phase composition changes gradually during separation.

(c) Based on Separation Principle

• Ion-Exchange HPLC – separation of ionic/polar compounds.

• Size-Exclusion HPLC (Gel permeation) – separation based on molecular size.

• Affinity HPLC – separation based on biological interactions (antigen-antibody, enzyme-substrate).

• Chiral HPLC – separation of enantiomers (important in drug analysis).

Applications of HPLC

• Pharmaceuticals:

  • Quantitative assay of drugs.

  • Impurity and degradation product analysis.

  • Stability and bioequivalence studies.

• Clinical/Medical:

  • Measurement of antibiotics, hormones, vitamins, metabolites.

  • Therapeutic drug monitoring.

• Food and Beverages:

  • Analysis of preservatives, colors, sweeteners, flavors.

  • Vitamin and amino acid determination.

• Environmental Monitoring:

  • Detection of pesticides, pollutants, toxic chemicals in water, soil, air.

• Biotechnology:

  • Separation and purification of proteins, peptides, nucleic acids.

• UV Spectrophotometry: Principle, Instrumentation and Applications