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Related Concept Videos

Mass Spectrometry: Complex Analysis01:21

Mass Spectrometry: Complex Analysis

Mass spectrometry is an important technique for the identification of pure compounds. However, it has some limitations for the analysis of complex mixtures, often due to excessive fragmentation making the spectrum too complicated to decipher. Mass spectrometry can be combined with suitable separation methods in sequence, forming hyphenated methods, which are useful in the analysis of complex mixtures.
GC–MS is a powerful hyphenated method commonly used in forensics and environmental...
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-Resolution Mass Spectrometry (HRMS)01:15

High-Resolution Mass Spectrometry (HRMS)

The resolution of a mass spectrometer depends on the efficiency of separating ions with different ion masses. The mass of an atom is approximated to the sum of the masses of protons and neutrons inside, considering the masses of protons and neutrons as equal. However, the masses of the proton (1.6726 × 10−24 g) and neutron (1.6749 × 10−24 g) are not truly equal. There is a minor error in the expression of atomic masses relative to the simplest atom of hydrogen. For example, the mass of helium...
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...
Supercritical Fluid Chromatography01:18

Supercritical Fluid Chromatography

Supercritical fluid chromatography (SFC) provides a beneficial substitute for gas chromatography (GC) and liquid chromatography (LC) for certain samples because it merges the top attributes of both techniques. SFC allows the separation and analysis of compounds that GC or LC does not easily manage. These compounds are traditionally nonvolatile or thermally unstable, making GC unsuitable and lacking functional groups required for HPLC analysis.
SFC utilizes a supercritical fluid mobile phase,...

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Related Experiment Video

Updated: May 29, 2026

Multi-step Preparation Technique to Recover Multiple Metabolite Compound Classes for In-depth and Informative Metabolomic Analysis
11:25

Multi-step Preparation Technique to Recover Multiple Metabolite Compound Classes for In-depth and Informative Metabolomic Analysis

Published on: July 11, 2014

Low-Temperature HILIC Provides Enhanced Separations and Stability for LC-MS-Based Metabolomics.

Yifan Liu1, Madison L Jastrab1, Michael Xiao1

  • 1Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts 02115, United States.

Journal of Proteome Research
|May 28, 2026
PubMed
Summary
This summary is machine-generated.

We developed a low-temperature zwitterionic hydrophilic interaction liquid chromatography (Z-HILIC) method for liquid chromatography-mass spectrometry (LC-MS) metabolomics. This enhanced method improves metabolite separation and stability in human plasma and cell extracts.

Keywords:
HILICmetabolomicsnucleotidesseparation

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Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)
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Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Published on: May 20, 2013

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Last Updated: May 29, 2026

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Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)
11:00

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Published on: May 20, 2013

Area of Science:

  • Metabolomics
  • Analytical Chemistry
  • Biochemistry

Background:

  • Liquid chromatography-mass spectrometry (LC-MS) is crucial for metabolomics but faces challenges with peak shape, metabolite coelution, and retention time stability.
  • Conventional hydrophilic interaction liquid chromatography (HILIC) methods often struggle with polar metabolites and reproducibility in complex biological samples like human plasma and cell extracts.

Purpose of the Study:

  • To develop and optimize a stable and high-resolution HILIC method for untargeted LC-MS metabolomics.
  • To improve the separation and detection of polar metabolites in human plasma and cell extracts.
  • To enhance the robustness and reproducibility of HILIC-based metabolomics workflows.

Main Methods:

  • Optimization of a zwitterionic HILIC (Z-HILIC) column using a high-pH ammonium bicarbonate/acetonitrile mobile phase.
  • Investigated the effect of column temperature, specifically cooling to 5 °C (LT-ZHILIC), on peak shape and retention time stability.
  • Validated the optimized method using a 471 metabolite library and applied it to analyze cellular extracts and human plasma.

Main Results:

  • Cooling the Z-HILIC column to 5 °C significantly improved peak shape and retention time stability compared to ambient temperatures, especially with an acetonitrile-based mobile phase.
  • The low-temperature Z-HILIC (LT-ZHILIC) method achieved high-resolution separation of 471 metabolite standards and demonstrated robust performance over 100 injections.
  • Application to cellular extracts under nutrient deficiency revealed significant changes in nucleotide phosphates, a perturbation missed by a standard ZIC-pHILIC method.

Conclusions:

  • The LT-ZHILIC workflow provides a robust and stable platform for untargeted metabolomics, enhancing metabolite coverage and resolution.
  • This optimized method offers improved insights into cellular metabolic rewiring and the human plasma metabolome.
  • LT-ZHILIC represents a promising advancement for LC-MS-based metabolomics research.