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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...
Gas Chromatography: Types of Columns and Stationary Phases01:17

Gas Chromatography: Types of Columns and Stationary Phases

Gas chromatography (GC) relies on stationary phases to separate and analyze components in a sample. There are two main types of stationary phases: liquid and solid. Liquid stationary phases are non-volatile, thermally stable, and chemically inert liquids coated onto the column. Solid stationary phases are particles of adsorbent material, such as silica gel or molecular sieves.
For an analyte to remain on the column for a sufficient amount of time, it must exhibit some level of compatibility (or...
Ion-Exchange Chromatography01:09

Ion-Exchange Chromatography

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...
Types Of Column Chromatography01:29

Types Of Column Chromatography

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
When the...

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Related Experiment Video

Updated: May 23, 2026

Simple In-House Ultra-High Performance Capillary Column Manufacturing with the FlashPack Approach
13:36

Simple In-House Ultra-High Performance Capillary Column Manufacturing with the FlashPack Approach

Published on: December 4, 2021

Packing Capillary Columns for RP-HPLC.

Robert Moritz

    CSH Protocols
    |April 10, 2012
    PubMed
    Summary
    This summary is machine-generated.

    This study details adapting conventional high-performance liquid chromatography (HPLC) systems for capillary columns. The method enables accurate low-flow rates and gradients for protein and peptide separation using custom-packed fused-silica columns.

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    Post Column Derivatization Using Reaction Flow High Performance Liquid Chromatography Columns
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    Post Column Derivatization Using Reaction Flow High Performance Liquid Chromatography Columns

    Published on: April 26, 2016

    Related Experiment Videos

    Last Updated: May 23, 2026

    Simple In-House Ultra-High Performance Capillary Column Manufacturing with the FlashPack Approach
    13:36

    Simple In-House Ultra-High Performance Capillary Column Manufacturing with the FlashPack Approach

    Published on: December 4, 2021

    Post Column Derivatization Using Reaction Flow High Performance Liquid Chromatography Columns
    06:25

    Post Column Derivatization Using Reaction Flow High Performance Liquid Chromatography Columns

    Published on: April 26, 2016

    Area of Science:

    • Analytical Chemistry
    • Biochemistry

    Background:

    • Capillary or microcolumn high-performance liquid chromatography (HPLC) is crucial for separating small amounts of proteins and peptides.
    • Conventional HPLC systems often require modifications to achieve the low-flow rates necessary for capillary columns.

    Purpose of the Study:

    • To describe a protocol for adapting conventional HPLC systems to operate slurry-packed capillary columns.
    • To enable accurate low-flow rates (0.4–4 μl/min) and gradients for capillary HPLC.

    Main Methods:

    • Adapting conventional HPLC systems for low-flow rate operation.
    • Fabricating 0.32-mm internal diameter (I.D.) polyimide-coated fused-silica columns.
    • Slurry-packing columns with reversed-phase chromatographic supports.
    • Utilizing a commercial axial-beam longitudinal flow cell with UV detectors.

    Main Results:

    • Successful adaptation of conventional HPLC systems for capillary column operation.
    • Achieved accurate low-flow rates (0.4–4 μl/min) and gradients.
    • Demonstrated fabrication of functional fused-silica capillary columns.

    Conclusions:

    • The described protocol effectively enables the use of capillary HPLC for sensitive protein and peptide analysis.
    • This method provides a practical approach for researchers to perform high-resolution separations with limited sample quantities.