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Related Concept Videos

High-Performance Liquid Chromatography: Introduction01:11

High-Performance Liquid Chromatography: Introduction

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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:
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Mass Spectrometry: Complex Analysis01:21

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Mass spectrometry is an important technique for the identification of pure compounds. However, it has some limitations for the analysis of complex mixtures, often due to excessive fragmentation making the spectrum too complicated to decipher. Mass spectrometry can be combined with suitable separation methods in sequence, forming hyphenated methods, which are useful in the analysis of complex mixtures.
GC–MS is a powerful hyphenated method commonly used in forensics and environmental...
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High-Performance Liquid Chromatography: Elution Process01:05

High-Performance Liquid Chromatography: Elution Process

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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...
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High-Performance Liquid Chromatography: Instrumentation00:57

High-Performance Liquid Chromatography: Instrumentation

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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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Chromatographic Methods: Terminology01:18

Chromatographic Methods: Terminology

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Chromatography is an analytical technique widely used in fields such as chemistry, biology, environmental science, and pharmaceuticals to separate the components of a mixture and identify substances between them. The process of chromatography is based on the interactions between two distinct phases: the stationary phase and the mobile phase. The stationary phase is fixed in place by a supporting material, while the mobile phase moves over it, carrying the solutes. As the mobile phase travels,...
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Analyte Adsorption and Distribution01:09

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In certain chromatographic separations, solutes transfer between the mobile phase and the stationary phase via sorption, which typically refers to the process of adsorption. For many chromatographic systems, the sorption process often depends on the polarity of the compounds—an expression of the overall dipole moment within the molecule. During the separation process, there is competition between the solute and solvent for adsorption to the stationary phase. Highly polar compounds and...
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Related Experiment Video

Updated: Aug 23, 2025

Tuning a Parallel Segmented Flow Column and Enabling Multiplexed Detection
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Quantifying Coexistence Concentrations in Multi-Component Phase-Separating Systems Using Analytical HPLC.

Anne Bremer1, Ammon E Posey2, Madeleine B Borgia1

  • 1Department of Structural Biology, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA.

Biomolecules
|October 27, 2022
PubMed
Summary
This summary is machine-generated.

We developed a new analytical High-Performance Liquid Chromatography (HPLC) method to precisely measure biomolecular concentrations in phase-separated condensates. This technique quantifies dilute and dense phases in complex mixtures, advancing quantitative models of cellular compartmentalization.

Keywords:
biomolecular condensatescoexistence linephase separation

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Area of Science:

  • Biochemistry
  • Cell Biology
  • Biophysics

Background:

  • Cellular compartmentalization is often driven by biomolecular phase separation.
  • Existing in vitro methods lack precision for quantitative modeling of phase separation.
  • Accurate measurement of coexisting dilute and dense phases is crucial, especially in multicomponent systems.

Purpose of the Study:

  • To develop a precise analytical technique for quantifying phase separation in vitro.
  • To enable quantitative modeling of multicomponent biomolecular condensates.
  • To provide insights into the interactions governing phase separation.

Main Methods:

  • Analytical High-Performance Liquid Chromatography (HPLC) was employed for in vitro separation and quantification.
  • The method allows label-free detection of biomolecules.
  • Concentrations in both dilute and dense phases were measured.

Main Results:

  • The developed HPLC method accurately quantifies coexisting dilute and dense phase concentrations.
  • This technique is sensitive, applicable to various macromolecules, and allows for recovery of samples.
  • It provides quantitative data for reconstructing coexistence curves in multicomponent mixtures.

Conclusions:

  • Analytical HPLC offers a precise and versatile tool for studying biomolecular phase separation.
  • The method facilitates quantitative modeling of complex biological systems.
  • It advances our understanding of homotypic and heterotypic interactions in phase-separating biomolecules.