High Performance Liquid Chromatography (HPLC)

High Performance Liquid Chromatography (HPLC) is a widely used analytical technique for separating, identifying, and quantifying components in liquid samples. It is essential in pharmaceutical, environmental, food, petrochemical, and ISO 17025 accredited laboratories.

Working Principle

HPLC separates compounds based on their interaction between a liquid mobile phase and a solid stationary phase inside a high-pressure column. Components with stronger affinity to the stationary phase elute later, producing distinct retention times in the chromatogram.

Main Components

• Solvent Reservoirs – Mobile phase storage
• Degasser – Removes dissolved gases
• High Pressure Pump – Delivers mobile phase at controlled flow
• Injector / Autosampler – Introduces sample
• Column – Packed with stationary phase
• Detector – UV, PDA, Fluorescence, RI etc.
• Data System – Chromatogram acquisition and integration

Common HPLC Detectors

• UV Detector – Most common in pharma analysis
• PDA (Photodiode Array) – Multi-wavelength detection
• Fluorescence Detector – High sensitivity applications
• Refractive Index Detector – Sugars and non-UV compounds

Modes of HPLC

• Reverse Phase (RP-HPLC)
• Normal Phase
• Ion Exchange
• Size Exclusion
• Gradient Elution
• Isocratic Elution

ISO 17025 Laboratory Relevance

In accredited laboratories, HPLC methods require validation, system suitability testing, calibration traceability, uncertainty estimation, and preventive maintenance documentation. Proper pump maintenance and column conditioning are critical for reliable performance.

Common Troubleshooting Areas

• High back pressure
• Baseline drift or noise
• Ghost peaks
• Peak tailing or splitting
• Pump leakage or unstable flow
• Air bubbles in detector cell

Part of Laboratory Engineering Hub

HPLC is one of the core analytical techniques covered under our complete laboratory analyzer engineering framework.

Explore the full Laboratory Engineering reference including GC, HPLC, UV-Vis, ICP, Karl Fischer and Titration: Laboratory Analyzers – Engineering Fundamentals

Chromatographic Calculations

Capacity Factor (k')

k' = (tR − t0) / t0

Theoretical Plates (N)

N = 16 (tR / W)^2

Resolution (Rs)

Rs = 2 (tR2 − tR1) / (W1 + W2)

These values are used during system suitability testing under ISO 17025.

Worked Example

Retention time = 4.5 min Dead time = 1.0 min Peak width = 0.20 min

k' = (4.5 − 1.0) / 1.0 = 3.5

N = 16 (4.5 / 0.20)^2 = 16 × (22.5)^2 = 16 × 506.25 = 8100 theoretical plates

Back Pressure Equation

Column back pressure is influenced by:

ΔP ∝ (Viscosity × Flow Rate × Column Length) / Particle Size²

Higher viscosity or smaller particle size increases system pressure.

Troubleshooting Logic Flow

• High pressure → Blocked inline filter
• Baseline noise → Air bubbles
• Peak tailing → Column degradation
• Drift → Lamp aging