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High-Performance Liquid Chromatography: Introduction

High-Performance Liquid Chromatography: Introduction

High-performance liquid chromatography(HPLC), formerly referred to as High-pressure liquid chromatography, is a powerful technique used to separate, identify, and quantify components in complex mixtures. The term "high pressure" refers to using high pressure to push the liquid mobile phase through the tightly packed columns.
In HPLC, two phases play a critical role in the separation process:

High-Performance Liquid Chromatography: Elution Process

High-Performance Liquid Chromatography: Elution Process

In High-Performance Liquid Chromatography (HPLC), the elution process is critical to the separation of analytes and the quality of chromatographic results. Elution describes how compounds move through the column and separate based on their interactions with the mobile and stationary phases. This process determines the resolution, peak shape, and retention times in the chromatogram, which are essential for identifying and quantifying components in complex mixtures. Understanding the elution...

Peptide Identification Using Tandem Mass Spectrometry

Peptide Identification Using Tandem Mass Spectrometry

Tandem mass spectrometry, also known as MS/MS or MS2, is an analytical technique that employs two mass analyzers. Essentially it is a series of mass spectrometers that helps isolate a particular biomolecule and then helps study its chemical properties.
This technique helps gather information regarding the protein from which the peptide was obtained and to study the peptides’ amino acid sequence. Identifying peptides from a complex mixture is an important component of the growing field of...

High-Performance Liquid Chromatography: Instrumentation

High-Performance Liquid Chromatography: Instrumentation

High-performance liquid chromatography, or HPLC, is an analytical technique that separates liquid samples under high pressures. An HPLC instrument consists of glass bottles for storing solvents called mobile phase reservoirs. HPLC-grade solvents are used to maintain high purity, and the dissolved gases are removed using a degasser, such as a vacuum pumping system or sparging with helium. The solvents are then pumped into the analytical column using a screw-driven syringe or reciprocating pumps.

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Related Experiment Video

Updated: Jun 12, 2026

The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

Published on: April 11, 2014

Peptide mapping by reverse-phase high pressure liquid chromatography.

¹Department of Protein Chemistry, Imperial Cancer Research Fund, Lincoln's Inn Fields, London, England.

Methods in Molecular Biology (Clifton, N.J.)

|June 1, 2010

Summary

Reverse-phase high-performance liquid chromatography (HPLC) offers a versatile and rapid method for peptide separation and mapping. This technique provides excellent resolution, reproducibility, and quantitation, making it a valuable tool for biochemical analysis.

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Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

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Published on: August 17, 2015

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Last Updated: Jun 12, 2026

The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

Published on: April 11, 2014

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Published on: August 17, 2015

Area of Science:

Biochemistry
Analytical Chemistry

Background:

Reverse-phase high-performance liquid chromatography (HPLC) is a widely used technique for separating molecules.
Peptide mapping is crucial for protein identification and characterization.

Purpose of the Study:

To highlight the utility of reverse-phase HPLC for peptide mapping.
To present HPLC as an alternative to traditional two-dimensional separation methods.

Main Methods:

Utilized reverse-phase high-performance liquid chromatography (HPLC) for peptide separation.
Employed HPLC for one-dimensional peptide mapping.

Main Results:

Reverse-phase HPLC demonstrated excellent resolving power for separating complex peptide mixtures.
The technique enabled efficient peptide mapping, offering an alternative to conventional methods.

Conclusions:

Reverse-phase HPLC is a highly effective and versatile technique for rapid peptide separation and mapping.
HPLC offers advantages in reproducibility, quantitation, and speed, suitable for automation.