High-Performance Liquid Chromatography (HPLC) System Configuration

Basic System Configuration of HPLC

An HPLC system is composed of several integrated modules that work together to deliver the mobile phase, introduce the sample, separate components, detect them, and process the resulting data.(Fig. 1). Understanding the flow path helps clarify how each component contributes to reliable and reproducible analysis. Below is a simplified schematic of a typical HPLC flow path:

Flow Path Overview

The mobile phase travels from the solvent reservoirs through the degasser and pump, where it is pressurized and delivered at a controlled flow rate. The sample is introduced via the injector or autosampler and carried into the column, where separation occurs. As compounds elute from the column, they pass through the detector, generating a signal that is processed and displayed by the data system as a chromatogram.

Key System Components

Solvent Reservoirs

Hold the mobile phase solvents. These may contain a single solvent (isocratic elution) or multiple solvents blended during gradient elution.

Degasser

Removes dissolved gases from the mobile phase before it reaches the pump.

• Prevents formation of air bubbles in the flow path
• Reduces baseline noise and signal spikes
• Minimizes baseline drift caused by outgassing in the detector flow cell
  

Degassing improves detector stability and overall system performance.

Pump

Delivers the mobile phase at high pressure through the system.

• Provides precise and consistent flow rate
• Enables gradient mixing when multiple solvents are used

Stable flow is critical for reproducible retention times and accurate quantification.

Injector/Autosampler

Introduces a precise volume of sample into the flowing mobile phase.

• Manual injectors use a fixed sample loop
• Autosamplers automate injections for multiple samples

 Accurate injection volume ensures reliable quantitative results.

Column Oven

Maintains the column at a controlled temperature.

• Improves retention time reproducibility
• Enhances seperation consistency
• Can improve peak shape and reduce viscosity-related pressure changes

Temperature stability is especially important for sensitive or highly reproducible methods.

Column

The heart of the HPLC system.

• Packed with stationary phase material
• Responsible for separating compounds based on chemical interactions

Different column chemistries enable different separation modes.

Detector

Monitors compounds as they elute from the column.
Common detector types include UV/Vis, diode array (DAD), fluorescence, and refractive index detectors. The detector converts chemical information into an electrical signal.

Data System

Controls the instrument and processes detector signals.

• Displays chromatograms
• Integrates peaks
• Calculates retention times and peak areas
• Generates quantitative reports

Manual Injection Versus Automatic Sampling

There are two types of sample injector modules: a manual injector and an autosampler. A manual injector requires the user to load and inject the sample via a syringe into the injection valve in the load position to fill the sample loop (Fig. 3). The user then switches the injection valve to the inject position, connecting the sample loop to the pump and column, allowing mobile phase to pass from the pump through the sample loop onto the column (Fig. 4). An autosampler uses an automated injection valve with a mechanism to inject multiple samples sequentially.