Introduction to HPLC
Definition: An advanced liquid chromatography method for separating, identifying, and quantifying mixture components.
Pharmaceutical Use: Widely adopted for its accuracy and reliability.
Advancement: Unlike traditional liquid chromatography that uses gravity, HPLC employs high pressures to enhance separation and analysis precision.
Theory of HPLC
HPLC operates on the principle of partitioning analytes between a mobile phase and a stationary phase.
Key Concepts
Mobile Phase
Role: Solvent(s) that transport analytes through the column under high pressure.
Stationary Phase
Role: Packed column with coated particles that interact differently with each analyte, causing varied retention times.
Partitioning
Mechanism: Analytes distribute between mobile and stationary phases based on their affinities.
Separation
Outcome: Different retention times lead to the separation of compounds.
Detection
Process: Eluted compounds pass through a detector, generating a chromatogram (signal vs. time/volume).
Types of Interactions in HPLC
1) Normal Phase HPLC (NP-HPLC)
Stationary Phase: Polar (e.g., silica)
Mobile Phase: Less polar (e.g., hexane)
Separation Basis: Polarity
2) Reverse Phase HPLC (RP-HPLC)
Stationary Phase: Non-polar or weakly polar
Mobile Phase: Polar
Usage: Most common in pharmaceuticals
3) Ion-Exchange HPLC
Stationary Phase: Charged groups
Separation Basis: Charge properties
4) Size Exclusion HPLC (SEC/GPC)
Separation Basis: Molecular size
Mechanism: Larger molecules elute first by exclusion from pores
Modes of Mobile Phase Delivery
1) Isocratic Elution
Definition: Constant mobile phase composition throughout the run.
Advantages: Simple setup, high reproducibility.
Limitations: Less effective for complex mixtures, potential peak broadening.
2) Gradient Elution
Definition: Mobile phase composition changes during the run.
Advantages: Better separation for diverse compounds, sharper peaks, faster analyses.
Limitations: More complex equipment requires precise control for reproducibility.