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Related Concept Videos

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...
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.
Peptide Identification Using Tandem Mass Spectrometry01:33

Peptide Identification Using Tandem Mass Spectrometry

Tandem mass spectrometry, also known as MS/MS or MS2, is an analytical technique that employs two mass analyzers. Essentially it is a series of mass spectrometers that helps isolate a particular biomolecule and then helps study its chemical properties.
This technique helps gather information regarding the protein from which the peptide was obtained and to study the peptides’ amino acid sequence. Identifying peptides from a complex mixture is an important component of the growing field of...
Gas Chromatography: Types of Columns and Stationary Phases01:17

Gas Chromatography: Types of Columns and Stationary Phases

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Principles Of Column Chromatography01:13

Principles Of Column Chromatography

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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Automated Sample Multiplexing by using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT)
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Peptide mapping with liquid chromatography using a basic mobile phase.

Hongji Liu1, Bi Xu, Manas K Ray

  • 1Shire HGT, 700 Main Street, Cambridge, MA 02139, USA. hliu@shire.com

Journal of Chromatography. A
|October 8, 2008
PubMed
Summary

A novel liquid chromatography method using a basic mobile phase improves peptide mapping sensitivity, sequence coverage, and separation. This advancement enables confident identification and quantification of deamidation products.

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Area of Science:

  • Analytical Chemistry
  • Biochemistry

Background:

  • Traditional peptide mapping often uses acidic mobile phases like trifluoroacetic acid (TFA) or formic acid (FA).
  • These acidic conditions can present limitations in sensitivity, sequence coverage, and separation capacity for liquid chromatography (LC).

Purpose of the Study:

  • To introduce and evaluate a new peptide mapping technique utilizing an ammonia-containing basic mobile phase.
  • To compare the performance of this basic method against traditional acidic methods (TFA and FA).

Main Methods:

  • Peptide mapping was performed using liquid chromatography (LC).
  • A novel basic mobile phase containing ammonia was employed.
  • The new method was compared to traditional methods using trifluoroacetic acid (TFA) and formic acid (FA).

Main Results:

  • The basic mobile phase method demonstrated superior ultraviolet (UV) and mass spectrometry (MS) sensitivity compared to TFA.
  • It offered enhanced sequence coverage for tryptic maps over TFA.
  • The new method surpassed the FA method in UV sensitivity, sequence coverage, and separation capacity.
  • Significantly different chromatographic selectivity was observed, enabling previously difficult applications.
  • Baseline separation of peptides from deamidated products was achieved, facilitating accurate LC/MS identification and quantification.
  • No on-column deamidation occurred under the tested conditions.

Conclusions:

  • The ammonia-containing basic mobile phase offers significant advantages for peptide mapping over traditional acidic methods.
  • This new approach enhances sensitivity, sequence coverage, and separation, particularly for analyzing deamidation products.
  • The method provides complementary information when used alongside acidic conditions for comprehensive protein analysis.