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Related Concept Videos

Ion-Exchange Chromatography01:09

Ion-Exchange Chromatography

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Ion-exchange chromatography, or IEC, is a technique for separating ions based on their affinity for the stationary phase. The stationary phase is a cross-linked polymer resin with covalently attached ionic functional groups. The functional groups can be either positively charged (cation exchangers) or negatively charged (anion exchangers). A cation exchanger consists of a polymeric anion and active cations, while an anion exchanger is a polymeric cation with active anions. The choice of...
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High-Performance Liquid Chromatography: Introduction01:11

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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.
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High-Performance Liquid Chromatography: Elution Process01:05

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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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High-Performance Liquid Chromatography: Types of Detectors01:15

High-Performance Liquid Chromatography: Types of Detectors

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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...
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Ion Exchange01:17

Ion Exchange

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Ion exchange chromatography separates charged molecules from a solution by reversibly exchanging them with mobile, or 'active', ions associated with the oppositely charged stationary phase. This method can be used to separate ions, soften and deionize water, and purify solutions. The polymers comprising the ion-exchange column are high-molecular-weight and chemically stable polymers, crosslinked to be porous and essentially insoluble. They are also functionalized with either acidic or...
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Related Experiment Video

Updated: May 3, 2026

Ion Exchange Chromatography IEX Coupled to Multi-angle Light Scattering MALS for Protein Separation and Characterization
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Ion exchange-high-performance liquid chromatography (IEX-HPLC).

Marie Corbier1, Delphine Schrag, Sylvain Raimondi

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

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

Ion exchange-high-performance liquid chromatography (IE-HPLC) is a fast method to analyze antibody charge variants. This study details sample preparation, chromatographic conditions, and data analysis for accurate isoform proportion calculation.

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Area of Science:

  • Biochemistry
  • Analytical Chemistry
  • Protein Science

Background:

  • Purified antibodies exhibit charge heterogeneity due to post-translational modifications and degradation.
  • Accurate characterization of antibody charge profiles is crucial for quality control and therapeutic efficacy.
  • Existing methods may lack the throughput or resolution required for comprehensive analysis.

Purpose of the Study:

  • To describe a detailed protocol for ion exchange-high-performance liquid chromatography (IE-HPLC) for antibody charge profiling.
  • To provide guidance on sample preparation, chromatographic conditions, and data analysis for reproducible results.
  • To enable accurate quantification of different antibody isoforms.

Main Methods:

  • High-throughput analytical method utilizing ion exchange-high-performance liquid chromatography (IE-HPLC).
  • Detailed procedures for sample preparation, including buffer formulation and salt gradient optimization.
  • Specific chromatographic parameters: injection volume, flow rate, run time, and column selection.
  • Method for chromatogram integration to calculate isoform proportions.

Main Results:

  • Established a robust IE-HPLC method for determining the charge profile of purified antibodies.
  • Demonstrated the ability to resolve and quantify various antibody isoforms.
  • Provided a framework for validating the analytical method for routine use.

Conclusions:

  • IE-HPLC is a powerful and high-throughput technique for antibody charge variant analysis.
  • The described methodology facilitates accurate assessment of antibody isoform distribution.
  • This approach supports comprehensive characterization of antibody products in biopharmaceutical development.