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High-Performance Liquid Chromatography: Introduction01:11

High-Performance Liquid Chromatography: Introduction

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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, 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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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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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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Supercritical Fluid Chromatography01:18

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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.
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Chromatography is a technique used to separate compounds based on differences of partitioning between two phases, the stationary phase and the mobile phase.
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Comprehensive two dimensional liquid chromatography as analytical strategy for pharmaceutical analysis.

Marion Iguiniz1, Florent Rouvière2, Estelle Corbel3

  • 1Université de Lyon, Institut des Sciences Analytiques, UMR 5280, CNRS, Université Lyon 1, ENS Lyon, 5 rue de la Doua, 69100 Villeurbanne, France; Oril Industrie, 13 rue Auguste Desgenetais, 76210 Bolbec, France.

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|September 14, 2017
PubMed
Summary

Comprehensive two-dimensional liquid chromatography (LCxLC) offers high peak capacity for pharmaceutical analysis. Three generic RPLCxRPLC configurations achieved nearly 1000 effective peak capacity, separating known compounds and unknown impurities.

Keywords:
Impurity profilingOn-line LCxLC–MSOrthogonalityPeak capacityPharmaceutical analysis

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Area of Science:

  • Analytical Chemistry
  • Chromatography

Background:

  • Comprehensive two-dimensional liquid chromatography (LCxLC) provides high peak capacity for complex sample analysis.
  • Pharmaceutical analysis demands methods capable of resolving intricate mixtures.

Purpose of the Study:

  • To identify optimal generic on-line LCxLC conditions for pharmaceutical sample analysis.
  • To evaluate chromatographic systems based on orthogonality and peak capacity.

Main Methods:

  • Comparative study of 190 stationary phase, pH additive, and organic modifier combinations.
  • Implementation of selected RPLCxRPLC configurations for UV/MS analysis.
  • Evaluation of linear gradient conditions and practical sample peak capacity.

Main Results:

  • Three RPLCxRPLC configurations demonstrated high performance for two pharmaceutical samples.
  • Effective peak capacity near 1000 was achieved in under 50 minutes.
  • Significant improvement in detected compounds, including unknown impurities, compared to 1D RPLC.

Conclusions:

  • Generic LCxLC conditions are suitable for pharmaceutical analysis.
  • Selected 2D-systems provide excellent separation power for complex pharmaceutical samples.
  • On-line RPLCxRPLC significantly enhances compound detection and impurity profiling.