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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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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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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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High-Performance Liquid Chromatography: Elution Process01:05

High-Performance Liquid Chromatography: Elution Process

2.0K
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...
2.0K
High-Performance Liquid Chromatography: Types of Detectors01:15

High-Performance Liquid Chromatography: Types of Detectors

2.3K
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...
2.3K
Capillary Electrophoresis: Applications01:30

Capillary Electrophoresis: Applications

1.9K
Capillary electrophoretic separations offer various modes, each with unique applications. These modes include capillary zone electrophoresis, capillary gel electrophoresis, capillary array electrophoresis, capillary isoelectric focusing, capillary isotachophoresis, micellar electrokinetic chromatography, and capillary electrochromatography.
Capillary zone electrophoresis (CZE) separates ionic components based on their electrophoretic mobility. It has been used to separate proteins, amino acids,...
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Related Experiment Video

Updated: May 3, 2026

Rapid Assessment of Membrane Protein Quality by Fluorescent Size Exclusion Chromatography
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Rapid Assessment of Membrane Protein Quality by Fluorescent Size Exclusion Chromatography

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Size exclusion-high-performance liquid chromatography (SEC-HPLC).

Delphine Schrag1, Marie Corbier, Sylvain Raimondi

  • 1NovImmune S.A., Plan-Les-Ouates, Switzerland.

Methods in Molecular Biology (Clifton, N.J.)
|February 12, 2014
PubMed
Summary

Size exclusion-high-performance liquid chromatography (SE-HPLC) is a fast method to quantify antibody aggregates and fragments. This study details sample preparation, chromatographic conditions, and data analysis for accurate antibody characterization.

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Area of Science:

  • Biochemistry
  • Analytical Chemistry
  • Pharmaceutical Analysis

Background:

  • Antibody therapeutics require rigorous quality control.
  • Characterization of antibody aggregates and fragments is crucial for efficacy and safety.
  • High-throughput analytical methods are needed for efficient antibody assessment.

Purpose of the Study:

  • To describe a size exclusion-high-performance liquid chromatography (SE-HPLC) method for antibody analysis.
  • To provide detailed guidance on sample preparation and chromatographic conditions for SE-HPLC.
  • To outline the process for chromatogram integration and component proportion calculation.

Main Methods:

  • Utilized size exclusion-high-performance liquid chromatography (SE-HPLC) under isocratic conditions.
  • Detailed sample preparation protocols for purified antibodies.
  • Specified chromatographic parameters including buffer preparation, injection volume, flow rate, run time, and column selection.

Main Results:

  • The SE-HPLC method enables high-throughput determination and quantification of antibody aggregates and fragments.
  • Established conditions for valid analysis and accurate chromatogram integration.
  • Demonstrated the calculation of proportions for different antibody components.

Conclusions:

  • SE-HPLC is a robust and efficient method for assessing the purity and integrity of antibodies.
  • The described protocol facilitates reliable quantification of antibody variants.
  • This analytical approach supports quality control in antibody development and manufacturing.