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Related Concept Videos

High-Performance Liquid Chromatography: Introduction01:11

High-Performance Liquid Chromatography: Introduction

3.8K
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:
3.8K
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
When the...
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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

1.7K
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...
1.7K
Size-Exclusion Chromatography01:08

Size-Exclusion Chromatography

2.2K
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,...
2.2K
Principles Of Column Chromatography01:13

Principles Of Column Chromatography

9.2K
The chromatography technique was first invented in 1901 by Michael S. Tswett, a Russian botanist, to separate plant pigments using organic solvents. Further, in 1941, Archer John Porter Martin and R. L. M. Synge modified the technique by packing silica gel into a column. A mixture of amino acids was then separated on the packed column using chloroform and water mixture as the mobile phase. This was the first report on column chromatography. At present, column chromatography is a widely used...
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Related Experiment Video

Updated: Mar 2, 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

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High-performance liquid chromatography column length designed for submicrogram scale protein isolation.

J D Pearson

    Analytical Biochemistry
    |January 1, 1986
    PubMed
    Summary

    Short high-performance liquid chromatography (HPLC) columns improve protein recovery for gas-phase sequencing. Columns under 1 cm enhance recovery of hydrophobic proteins without losing resolution, crucial for analyzing small protein samples.

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    Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification
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    Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification
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    Related Experiment Videos

    Last Updated: Mar 2, 2026

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    Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification
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    Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification
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    Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

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    Area of Science:

    • Biochemistry
    • Analytical Chemistry
    • Proteomics

    Background:

    • Current gas-phase sequencing requires small protein amounts (5-100 pmol).
    • High-performance liquid chromatography (HPLC) often yields low recoveries for these small protein quantities due to column inefficiencies.
    • Protein surface hydrophobicity and size impact HPLC recovery rates.

    Purpose of the Study:

    • To investigate the effect of reduced HPLC column length on protein recovery and resolution.
    • To optimize HPLC methods for analyzing minute protein samples for sequencing.

    Main Methods:

    • Fabrication and evaluation of six HPLC columns with lengths ranging from 0.2 to 25 cm.
    • Assessment of protein loading capacity and separation resolution for each column.
    • Testing recovery rates for surface-hydrophobic proteins using a standard protein profile.

    Main Results:

    • HPLC columns shorter than 1 cm significantly increased recovery of surface-hydrophobic proteins.
    • Reduced column length did not compromise resolution, maintaining analytical accuracy.
    • Optimal protein resolution was achieved with column loading under 10 micrograms, yielding >90% recovery.
    • All columns utilized standard 4.1 mm internal diameters compatible with typical HPLC pumps.

    Conclusions:

    • Short HPLC columns ( < 1 cm) are effective for analyzing small protein amounts (nanogram to microgram range).
    • This approach enhances recovery of challenging hydrophobic proteins without sacrificing resolution.
    • The optimized method supports efficient primary sequence information acquisition for proteomics applications.