Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Concept Videos

High-Performance Liquid Chromatography: Introduction01:11

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: Instrumentation00:57

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.
High-Performance Liquid Chromatography: Elution Process01:05

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...
Chromatographic Resolution01:15

Chromatographic Resolution

In chromatography, a solute moves through a chromatographic column and tends to spread, forming a Gaussian-shaped band. The longer the solute spends in the column, the broader the band becomes. The broadening can lead to overlaps within the column, affecting separation effectiveness.
The effectiveness of separation can be evaluated by determining the level of separation between two neighboring peaks in a chromatogram, which represents the individual components of a sample.
In chromatography,...
Optimizing Chromatographic Separations01:15

Optimizing Chromatographic Separations

Optimizing chromatographic separations is crucial for obtaining clean separations in a minimum amount of time. Optimization is required for several factors, including kinetic effects related to band broadening, plate height, capacity factor, and separation factor.
Band broadening refers to spreading solute bands as they travel through the column. This broadening can impact resolution. Plate height (H) represents the length required for one theoretical plate. A lower plate height corresponds to...
High-Performance Liquid Chromatography: Types of Detectors01:15

High-Performance Liquid Chromatography: Types of Detectors

The role of the detectors in High-Performance Liquid Chromatography (HPLC) is to analyze the solutes as they exit from the chromatographic column. The detector recognizes the solute's property and generates corresponding electrical signals, which are converted into a readable graph of the detector's response versus elution time called a chromatogram at the computer. There are several types of HPLC detectors, each with its own advantages and limitations, depending on the analyte properties and...

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

Small molecule stabilization of diverse amyloidogenic immunoglobulin light chains revealed by hydrogen-deuterium exchange mass spectrometry.

Journal of molecular biology·2026
Same author

Magnesium as a conformational gatekeeper of KRAS: Structural dynamics and therapeutic implications.

Protein science : a publication of the Protein Society·2026
Same author

Structures of DnaA domain I reveal a dimer conserved across Actinomycetes.

Nucleic acids research·2026
Same author

Low-Temperature HILIC Provides Enhanced Separations and Stability for LC-MS-Based Metabolomics.

Journal of proteome research·2026
Same author

More than an attachment module: covalent inhibitor warheads influence BTK dynamics and function.

bioRxiv : the preprint server for biology·2026
Same author

Magnesium as a Conformational Gatekeeper of KRAS: Structural Dynamics and Therapeutic Implications.

bioRxiv : the preprint server for biology·2026

Related Experiment Video

Updated: Jul 3, 2026

Automated HPLC Separation Using LC-Mate: An Integrated Repetitive Autosampler and Fraction Collector for Microscale Purification
07:11

Automated HPLC Separation Using LC-Mate: An Integrated Repetitive Autosampler and Fraction Collector for Microscale Purification

Published on: February 27, 2026

High-speed and high-resolution UPLC separation at zero degrees Celsius.

Thomas E Wales1, Keith E Fadgen, Geoff C Gerhardt

  • 1The Barnett Institute, Northeastern University, Boston, Massachusetts 02115, USA.

Analytical Chemistry
|August 5, 2008
PubMed
Summary
This summary is machine-generated.

This study introduces a custom ultraperformance liquid chromatography (UPLC) system for hydrogen/deuterium exchange mass spectrometry (HXMS). The new UPLC system enables rapid, low-temperature separations for improved protein analysis.

More Related Videos

Improved Polymerase Chain Reaction-restriction Fragment Length Polymorphism Genotyping of Toxic Pufferfish by Liquid Chromatography/Mass Spectrometry
09:34

Improved Polymerase Chain Reaction-restriction Fragment Length Polymorphism Genotyping of Toxic Pufferfish by Liquid Chromatography/Mass Spectrometry

Published on: September 20, 2016

Curtain Flow Column: Optimization of Efficiency and Sensitivity
06:44

Curtain Flow Column: Optimization of Efficiency and Sensitivity

Published on: June 12, 2016

Related Experiment Videos

Last Updated: Jul 3, 2026

Automated HPLC Separation Using LC-Mate: An Integrated Repetitive Autosampler and Fraction Collector for Microscale Purification
07:11

Automated HPLC Separation Using LC-Mate: An Integrated Repetitive Autosampler and Fraction Collector for Microscale Purification

Published on: February 27, 2026

Improved Polymerase Chain Reaction-restriction Fragment Length Polymorphism Genotyping of Toxic Pufferfish by Liquid Chromatography/Mass Spectrometry
09:34

Improved Polymerase Chain Reaction-restriction Fragment Length Polymorphism Genotyping of Toxic Pufferfish by Liquid Chromatography/Mass Spectrometry

Published on: September 20, 2016

Curtain Flow Column: Optimization of Efficiency and Sensitivity
06:44

Curtain Flow Column: Optimization of Efficiency and Sensitivity

Published on: June 12, 2016

Area of Science:

  • Biochemistry
  • Analytical Chemistry
  • Proteomics

Background:

  • Hydrogen/deuterium exchange mass spectrometry (HXMS) probes protein conformational properties.
  • Rapid, low-temperature chromatography is essential for maintaining deuterium labels during HXMS.
  • Traditional HPLC methods exhibit limitations for analyzing large proteins and complexes using HXMS.

Purpose of the Study:

  • To construct and validate a custom ultraperformance liquid chromatography (UPLC) system specifically for HXMS applications.
  • To overcome the chromatographic limitations of traditional HPLC for HXMS analysis of large proteins.

Main Methods:

  • Development of a custom UPLC system based on the Waters nanoACQUITY platform.
  • Integration of a Peltier-cooled module for maintaining 0°C during separations.
  • Utilized rapid chromatographic separations (6-min) with sub-2-µm particles for HXMS.

Main Results:

  • Achieved near-baseline resolution in 6-minute separations at 0°C.
  • Demonstrated a median chromatographic peak width of approximately 2.7 seconds at half-height.
  • Validated the system with single proteins >95 kDa and a four-protein mixture >250 kDa, showing comparable deuterium recovery to conventional HPLC.

Conclusions:

  • The custom UPLC system significantly advances HXMS technology.
  • This system enhances accessibility and broadens the application of HXMS for large proteins and complexes.
  • The developed UPLC system is expected to make HXMS a more mainstream analytical technique.