UPLC and UHPLC

Instrumental Technique

UPLC and UHPLC

Objective

To develop fast, accurate, sensitive, and highly efficient chromatographic methods using sub-2 µm particle columns and high-pressure systems for pharmaceutical analysis, quality control, bioanalysis, and research applications.

Instrument flow diagram of Ultra-Performance Liquid Chromatography (UPLC): solvent, pump, injector, sample, UPLC column, detector, PC for data analysis, and waste
Instrument Flow of Ultra-Performance Liquid Chromatography (UPLC)

Introduction

Ultra Performance Liquid Chromatography (UPLC) and Ultra High Performance Liquid Chromatography (UHPLC) are advanced forms of High-Performance Liquid Chromatography (HPLC). They employ columns packed with sub-2 µm stationary phase particles and operate under high system pressures (up to 1000–1500 bar), resulting in superior chromatographic efficiency, faster analysis, higher sensitivity, and lower solvent consumption. UPLC/UHPLC has become the preferred analytical technique in pharmaceutical industries, research laboratories, and bioanalytical studies due to its excellent resolution and high-throughput capabilities.

Purpose

To achieve efficient chromatographic separation by reducing particle size and increasing system pressure.

Principle

UPLC/UHPLC follows the same separation mechanism as HPLC but utilizes columns packed with particles smaller than 2 µm. The reduced particle size shortens the diffusion path of analytes, increasing separation efficiency and peak resolution while reducing analysis time.

Van Deemter Equation

The efficiency of chromatographic separation is described by the Van Deemter equation:

H = A + (B/u) + (C × u)

Where:

  • H = Height Equivalent to a Theoretical Plate (HETP)
  • A = Eddy Diffusion
  • B = Longitudinal Diffusion
  • C = Mass Transfer Resistance
  • u = Linear Velocity of the Mobile Phase

Smaller particle sizes reduce both eddy diffusion and mass transfer resistance, resulting in higher efficiency.

Importance

  • Higher theoretical plates.
  • Better chromatographic resolution.
  • Faster separations.
  • Improved analytical efficiency.

Purpose

To enhance chromatographic performance compared to conventional HPLC.

Performance Comparison

Parameter Conventional HPLC UPLC/UHPLC
Particle Size 3–5 µm <2 µm (typically 1.7 µm)
Operating Pressure 200–400 bar Up to 1000–1500 bar
Analysis Time Longer 2–10 times faster
Resolution Good Higher
Solvent Consumption Higher Reduced by 50–80%
Sensitivity Good Improved

Benefits

  • Faster analysis.
  • Higher chromatographic resolution.
  • Better peak shape.
  • Reduced solvent usage.
  • Increased laboratory productivity.

Purpose

To configure the UPLC/UHPLC system for high-pressure chromatographic analysis.

Major Components

Solvent Manager

Delivers mobile phase accurately at high pressure.

Autosampler

Introduces samples with high precision and reproducibility.

UPLC/UHPLC Column

Performs chromatographic separation using sub-2 µm particles.

Typical Specifications:

  • Particle Size: 1.7–1.9 µm
  • Length: 30–100 mm
  • Internal Diameter: 2.1 mm
  • Pressure Capacity: Up to 1500 bar

Column Oven

Maintains constant column temperature for reproducible retention times.

Detector

Common detectors include:

  • UV Detector
  • PDA (Photodiode Array)
  • Fluorescence Detector
  • Mass Spectrometer (LC-MS)

Data System

Acquires chromatographic data, processes results, and generates reports.

Analytical Advantages

  • Higher chromatographic resolution.
  • Much faster analysis (2–10 times faster than HPLC).
  • Lower solvent consumption.
  • Better sensitivity.
  • Improved peak symmetry.
  • Higher efficiency.
  • Excellent reproducibility.
  • Suitable for high-throughput laboratories.
  • Ideal for complex pharmaceutical samples.
  • Easily coupled with LC–MS/MS systems.
  • Environmentally friendly due to reduced solvent use.

Purpose

To utilize UPLC/UHPLC for advanced pharmaceutical and bioanalytical applications.

Pharmaceutical Applications

Pharmaceutical Quality Control

  • Assay of active pharmaceutical ingredients (APIs)
  • Finished product testing
  • Batch release analysis

Stability Studies

  • Stability-indicating methods
  • Forced degradation studies
  • Shelf-life evaluation

Impurity Profiling

  • Identification and quantification of impurities
  • Related substance analysis

Dissolution Testing

  • Quantitative analysis of dissolution samples
  • Drug release profiling

Bioanalysis

  • Plasma drug quantification
  • Pharmacokinetic studies
  • Bioequivalence studies

Drug Discovery

  • High-throughput screening
  • Lead optimization
  • Metabolite analysis

LC-MS/MS Applications

  • Highly sensitive quantitative analysis
  • Biomarker studies
  • Therapeutic drug monitoring