High-Performance Liquid Chromatography (HPLC)

Introduction

• HPLC stands for High-Performance Liquid Chromatography or High-Pressure Liquid Chromatography.
• It is a modified form of column chromatography.

Column Chromatography vs. HPLC

• In column chromatography, a column is packed with an absorbent (e.g., silica) and the mobile phase moves by gravity.
• In HPLC, a high-pressure pump is used, generating pressures up to 40 MPa.
• HPLC uses an absorbent material with very small particles for increased surface area, leading to better separation efficiency and high resolution.

Components of HPLC

Column

• Made of stainless steel, can withstand pressures up to 50 MPa.
• Length varies from 5 to 25 cm; internal diameter of 4.5 mm.
• Flow rate of mobile phase: 1-3 ml/min.

Stationary Phase

• Made of absorbent material with uniform small particle size for better performance.
• Common materials: chemically modified silica, divinylbenzene.

Mobile Phase

• Mixture of different solvents, can be polar or non-polar.
• Stored in solvent reservoir, pumped into the column under high pressure.
• Introduced to the column via an injector.

Detector

• Detects the sample (analyte) coming out of the column.
• Types include UV, IR, fluorescence, refractive index, mass spectrometer, electrochemical detectors.
• Connected to a computer for data collection.

Working of HPLC

• Separated sample molecules are detected, resulting in peaks on a chromatogram (plot of retention time).
• Identifying components requires standards (known retention times).

Example

• Glucose peak at 5 minutes, sucrose peak at 8 minutes.
• Unknown sample analyzed; peaks at these times indicate the presence of glucose or sucrose.
• Standards are needed for identification.

Determining Concentration

• A standard curve is created by running known concentrations of a sample (e.g., glucose).
• Peak areas are plotted against concentration.
• Unknown sample concentration estimated from peak area using the standard curve.

Types of HPLC

Normal Phase HPLC

• Stationary phase: polar (e.g., silica with Si-OH groups).
• Mobile phase: non-polar.
• Polar molecules retained, non-polar molecules move quickly.

Reverse Phase HPLC

• Stationary phase: non-polar.
• Mobile phase: polar.
• Non-polar molecules retained, polar molecules move quickly.

Size Exclusion HPLC

• Based on size exclusion/gel permeation chromatography.
• Stationary phase particles are porous.
• Small molecules enter pores, move slowly; large molecules pass quickly.
• Separation based on size.

Ion Exchange HPLC

• Stationary phase with ionic charge.
• Positive stationary phase for negative analytes and vice versa.
• Separation based on molecular charge.