High performance liquid chromatography (HPLC) is an essential technique used in chemistry and other sciences to separate, identify, and quantify compounds in a mixture. Using an HPLC machine, scientists can analyze samples like natural products, drugs, pollutants, and more. This guide will explain how HPLC works and how to operate an HPLC system for your own analyses.
Some recent news related to HPLC:
- New method analyzes trace toxins 10 times faster using HPLC
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- Improved method uses HPLC to analyze brain chemicals linked to anxiety
Key Points About HPLC
- HPLC works by pumping a pressurized mobile phase through a column containing a stationary phase.
- Components in a sample separate as they interact differently with the phases.
- A detector analyzes the separated components as they elute from the column.
- HPLC provides quantitative and qualitative analysis of mixtures.
Basic Components and Setup
An HPLC system consists of a pump, injector, column, detector, and data analysis software. The pump pushes the mobile phase like various solvent mixtures through the system at high pressure. An autosampler or manual injector introduces the sample into the mobile phase stream. The column contains tightly packed stationary phase particles that interact with sample components. As components elute from the column, the detector such as UV/vis measures them and sends data to the software for analysis.
Types of HPLC Separations
There are two main types of HPLC - normal phase and reverse phase. In normal phase HPLC, the stationary phase is more polar than the mobile phase. For reverse phase, the stationary phase is less polar than the mobile phase, making it the most common type. The type of separating columns and solvents used depends on the polarity of the sample components to be analyzed.
Performing an HPLC Analysis
To run a sample, you program the HPLC with parameters like solvent composition, flow rate, pressure limits. Load the stationary phase column, inject the sample, and let components separate and elute from the column into the detector over time. Peaks in the chromatogram correspond to each separated component. Retention time identifies components which can then be quantified. Proper method development and controls ensure quality results.
Applications of HPLC in Research and Industry
HPLC finds wide use in pharmaceutical development and quality control to analyze drug compounds and impurities. It's also used in fields like food science to test for adulterants, the environmental industry to detect pollutants, and biological research for analyzing proteins, sugars and other large molecules. With some basic training, HPLC enables many chemical analyses that were not possible before.
Conclusion
In summary, high performance liquid chromatography is a highly versatile and powerful analytical technique that has revolutionized chemical analysis since its development in the late 20th century. Using an HPLC system, researchers and industry can separate, identify and quantify many different compound types from various sample matrices. With appropriate methods, HPLC opens up new possibilities for discovery and quality assurance across many scientific disciplines.
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