What is HPLC ? Structure of HPLC.

 

High-Performance Liquid Chromatography (HPLC) is an advanced form of liquid chromatography used to separate, identify, and quantify components in a mixture. The structure of an HPLC system typically consists of the following main components:

 

1. Solvent Reservoirs

These are containers that hold the mobile phase (solvents). They can contain one or more solvents that are used to carry the sample through the system. Common solvents include water, methanol, and acetonitrile.

 

2. Degasser

The degasser removes dissolved gases from the mobile phase. Gases can form bubbles that interfere with the detector and affect the performance of the column.

 

3. Pump

The pump delivers the mobile phase at a high pressure (up to several hundred bar) and at a controlled flow rate. This high pressure is necessary to push the mobile phase and the sample through the densely packed stationary phase in the column.

 

4. Injector

The injector introduces the sample into the mobile phase stream. This can be done manually using a syringe or automatically using an autosampler.

 

5. Column

The column is the heart of the HPLC system. It contains the stationary phase, which is typically made of small particles of silica or polymer. The column is where the separation of the sample components occurs. Columns can vary in size, shape, and the nature of the stationary phase depending on the application.

 

6. Detector

The detector identifies and quantifies the separated components as they elute from the column. Common types of detectors include:

i. UV-Vis Detector: Measures absorbance of UV or visible light.

ii. Fluorescence Detector: Measures fluorescence emission.

iii. Refractive Index Detector: Measures changes in the refractive index.

iv. Mass Spectrometer (MS): Identifies compounds based on their mass-to-charge ratio.

7. Data System

The data system collects and analyzes the signals from the detector. It typically consists of a computer with software that controls the HPLC system and processes the data to generate chromatograms.

 

8. Waste Container

After passing through the detector, the mobile phase and any remaining sample are collected in a waste container.

 

Typical HPLC Flow Diagram

Solvent Reservoirs -> 2. Degasser -> 3. Pump -> 4. Injector -> 5. Column -> 6. Detector -> 7. Data System -> 8. Waste Container

Summary of the Process

Preparation: Solvents are degassed and pumped at high pressure.

Injection: The sample is injected into the mobile phase stream.

Separation: The sample travels through the column, where components are separated based on their interactions with the stationary phase.

Detection: Separated components are detected and quantified by the detector.

Analysis: Data from the detector are processed to produce a chromatogram, showing peaks corresponding to different components.

HPLC is widely used in various fields, including pharmaceuticals, environmental monitoring, and food analysis, due to its high resolution, speed, and ability to handle complex mixtures.