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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: 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: 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.
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Gas Chromatography: Sample Injection Systems01:08

Gas Chromatography: Sample Injection Systems

In gas chromatography, the sample is introduced as a vapor plug into the carrier gas stream for high efficiency and resolution. A microsyringe injects the sample solution into a heated sample port, vaporizing it and mixing it with the carrier gas. This process is important to ensure the sample is properly prepared for analysis. Thermally sensitive samples can be injected directly into the column and volatilized by slowly increasing the column temperature.
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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...
Chromatography: Introduction01:10

Chromatography: Introduction

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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Using strong injection solvents with 100% aqueous mobile phase in RP-LC.

Eric Loeser1, Patrick Drumm

  • 1Chemical and Analytical Development, Novartis Pharmaceuticals, East Hanover, NJ 07936, USA. eric.loeser@novartis.com

Journal of Separation Science
|February 20, 2007
PubMed
Summary

Using a strong organic solvent in the diluent for High-Performance Liquid Chromatography (HPLC) can improve peak shape for weakly retained analytes, even under 100% aqueous mobile phase conditions. This counterintuitive approach offers analytical utility when the solvent elutes after the analyte.

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Area of Science:

  • Analytical Chemistry
  • Chromatography
  • Separation Science

Background:

  • High-Performance Liquid Chromatography (HPLC) analysis often requires dissolving samples in organic solvents to improve solubility.
  • Using a strong organic solvent in the sample diluent can cause peak shape distortions, especially for weakly retained analytes under 100% aqueous mobile phase conditions.
  • This distortion is problematic as it can lead to inaccurate results and reduced analytical sensitivity.

Purpose of the Study:

  • To investigate the effect of using a strong organic solvent in the diluent on peak shape for weakly retained analytes in HPLC.
  • To explore the potential analytical utility and limitations of this counterintuitive approach.
  • To provide guidance for optimizing sample preparation in specific HPLC scenarios.

Main Methods:

  • Investigated the impact of varying organic solvent concentrations in the diluent on analyte peak shape.
  • Focused on High-Performance Liquid Chromatography (HPLC) under 100% aqueous mobile phase conditions.
  • Examined analytes with weak retention and the elution behavior of organic solvents like Tetrahydrofuran (THF).

Main Results:

  • Contrary to expectations, using a strong organic solvent (e.g., THF) in the diluent did not always cause peak distortion.
  • Peak shape remained unaffected when the organic solvent in the diluent eluted significantly after the analyte.
  • Even small amounts of organic solvent in the diluent could cause significant peak broadening under certain conditions.

Conclusions:

  • The elution order of the organic solvent relative to the analyte is critical for peak shape in HPLC.
  • Employing a strong organic solvent in the diluent, provided it elutes late, can be a viable strategy for analyzing weakly retained analytes.
  • This approach offers potential analytical utility but requires careful consideration of its limitations and specific experimental conditions.