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Types of HPLC – Explained with Examples

Types of HPLC – Explained with Examples 🔬

High-Performance Liquid Chromatography (HPLC) has different types depending on how the separation is done. Each type is designed to handle specific compounds and sample properties. In this post, you’ll learn the main types of HPLC used in the pharmaceutical industry and how they work.

1. Reverse-Phase HPLC (RP-HPLC) 🔄

Reverse-phase HPLC is the most commonly used type in pharma labs. In this method, the stationary phase is non-polar (hydrophobic), and the mobile phase is polar (water-based solvents).

  • Used for: Non-polar or moderately polar compounds
  • Example: Testing drug substances like paracetamol, ibuprofen, or vitamins

2. Normal-Phase HPLC (NP-HPLC) ⚖️

This method uses a polar stationary phase (like silica) and a non-polar mobile phase (like hexane). It separates polar compounds better than RP-HPLC.

  • Used for: Separation of isomers, sugars, amino acids
  • Example: Identification of small polar impurities in raw materials

3. Ion-Exchange HPLC ⚡

Ion-exchange chromatography separates compounds based on their charge. The column contains charged groups that attract oppositely charged molecules.

  • Used for: Ions, charged drugs, amino acids, peptides
  • Example: Analysis of insulin or water-soluble vitamins

4. Size-Exclusion HPLC (Gel Filtration) 🔍

This method separates molecules based on their size. Larger molecules come out first because they cannot enter the pores of the packing material, while smaller ones take longer.

  • Used for: Proteins, polymers, and enzymes
  • Example: Molecular weight analysis in biotech products

Comparison Table 📊

Type Stationary Phase Mobile Phase Best For
Reverse-Phase Non-polar Polar (water, methanol) Non-polar drug compounds
Normal-Phase Polar (silica) Non-polar (hexane) Polar isomers and small molecules
Ion-Exchange Charged resins Buffer solutions Ionic compounds, peptides
Size-Exclusion Porous beads Buffer or water Proteins, large biomolecules

Summary ✅

  • Reverse-Phase HPLC – Most used, for non-polar drugs
  • Normal-Phase HPLC – For polar compounds
  • Ion-Exchange HPLC – For charged molecules
  • Size-Exclusion HPLC – For large molecules based on size

Keywords: Types of HPLC, Reverse-Phase HPLC, Normal-Phase HPLC, Ion Exchange HPLC, Size Exclusion Chromatography, pharma testing, HPLC method selection

Written by Nirmal Patel | o2h.space

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