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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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Size-Exclusion Chromatography01:08

Size-Exclusion Chromatography

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In size-exclusion chromatography (SEC), also known as molecular-exclusion or gel-permeation chromatography, molecules are separated based on their sizes. This technique is important for separating large molecules such as polymers and biomolecules. The two classes of micron-sized stationary phases encountered in SEC are silica particles and cross-linked polymer resin beads. Both materials are porous, but their pore sizes vary significantly.
Silica particles offer advantages such as rigidity,...
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Supercritical Fluid Chromatography01:18

Supercritical Fluid Chromatography

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Supercritical fluid chromatography (SFC) provides a beneficial substitute for gas chromatography (GC) and liquid chromatography (LC) for certain samples because it merges the top attributes of both techniques. SFC allows the separation and analysis of compounds that GC or LC does not easily manage. These compounds are traditionally nonvolatile or thermally unstable, making GC unsuitable and lacking functional groups required for HPLC analysis.
SFC utilizes a supercritical fluid mobile phase,...
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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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Types Of Column Chromatography01:29

Types Of Column Chromatography

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The stability and compatibility of column material with samples are crucial for efficient purification in chromatographic techniques. Various operating parameters such as pH, temperature, or solvent affect the packing of the column material, thereby determining the purification efficiency. The choice of column material also plays an essential role in deciding the operating parameters and can be modified based on the proteins that need to be purified.
Gel Filtration Chromatography
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Online Size-exclusion and Ion-exchange Chromatography on a SAXS Beamline
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Inline small-angle X-ray scattering-coupled chromatography under extreme hydrostatic pressure.

Robert C Miller1, Cody Cummings2, Qingqiu Huang2

  • 1Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York, USA.

Protein Science : a Publication of the Protein Society
|November 2, 2022
PubMed
Summary

Researchers developed a high-pressure system for size-exclusion chromatography coupled with small-angle X-ray scattering (SEC-SAXS). This method enables structural analysis of biomolecules under extreme deep-sea conditions, revealing pressure-induced changes.

Keywords:
SEC-SAXSextreme biophysicshigh-pressure biologysize-exclusion chromatographysmall-angle X-ray solution scattering

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Area of Science:

  • Biophysics
  • Structural Biology
  • Deep-Sea Microbiology

Background:

  • Extreme hydrostatic pressure impacts deep ocean and subsurface microbial life.
  • Biological small-angle X-ray scattering (BioSAXS) provides structural insights into biomolecules.
  • Size-exclusion chromatography-coupled SAXS (SEC-SAXS) is a dominant BioSAXS technique, but its application under high pressure is challenging.

Purpose of the Study:

  • To investigate the feasibility of performing size-exclusion chromatography coupled with small-angle X-ray scattering (SEC-SAXS) under extreme hydrostatic pressure.
  • To develop and validate an apparatus for high-pressure SEC-SAXS.
  • To observe pressure-induced structural and functional changes in biomolecules relevant to deep-sea environments.

Main Methods:

  • Development of a novel apparatus for high-pressure SEC-SAXS.
  • Experimental validation at pressures up to 100 MPa.
  • Analysis of biomolecular structure and oligomeric state under varying pressure and temperature (0°C-50°C).

Main Results:

  • Demonstrated reproducible chromatographic separations coupled directly to high-pressure BioSAXS for the first time.
  • Achieved successful operation at pressures up to at least 100 MPa.
  • Observed pressure-induced changes in biomolecular folding and oligomeric state.

Conclusions:

  • High-pressure SEC-SAXS is a viable technique for studying biomolecules in extreme environments.
  • The developed apparatus expands opportunities for understanding life in deep ocean and subsurface habitats.
  • This method allows for the observation of critical pressure-induced biomolecular adaptations.