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Related Concept Videos

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.
In HPLC, two phases play a critical role in the separation process:
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High-Performance Liquid Chromatography: Instrumentation00:57

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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

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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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Optimizing Chromatographic Separations01:15

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Optimizing chromatographic separations is crucial for obtaining clean separations in a minimum amount of time. Optimization is required for several factors, including kinetic effects related to band broadening, plate height, capacity factor, and separation factor.
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High-Performance Liquid Chromatography: Types of Detectors01:15

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The role of the detectors in High-Performance Liquid Chromatography (HPLC) is to analyze the solutes as they exit from the chromatographic column. The detector recognizes the solute's property and generates corresponding electrical signals, which are converted into a readable graph of the detector's response versus elution time called a chromatogram at the computer. There are several types of HPLC detectors, each with its own advantages and limitations, depending on the analyte...
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Supercritical Fluid Chromatography01:18

Supercritical Fluid Chromatography

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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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Simple In-House Ultra-High Performance Capillary Column Manufacturing with the FlashPack Approach
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Performance in (Ultra-)high-performance liquid chromatography-How to qualify and optimize instruments in practice.

Alexander Jaekel1, Michaela Wirtz1, Stefan Lamotte2

  • 1Department of Natural Sciences, University of Applied Sciences Bonn-Rhein-Sieg, Rheinbach, Germany.

Journal of Separation Science
|February 9, 2023
PubMed
Summary

Hybrid chromatography systems offer limited suitability for ultra-high-performance liquid chromatography (UHPLC) applications. Optimizing extra-column volume improves performance, but gradient precision and dwell volume remain challenges for UHPLC.

Keywords:
extra column band broadeningliquid chromatographysuperficially porous particlessystem optimizationvan Deemter curve

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Area of Science:

  • Analytical Chemistry
  • Chromatography

Background:

  • Ultra-high-performance liquid chromatography (UHPLC) offers significant advantages in speed and resolution over conventional high-performance liquid chromatography (HPLC).
  • Hybrid systems combining UHPLC and HPLC capabilities are explored to bridge performance gaps.

Purpose of the Study:

  • To evaluate the suitability of a UHPLC/HPLC hybrid system for UHPLC applications.
  • To investigate and optimize the impact of extra-column band broadening, gradient system, and injection system on hybrid system performance.
  • To compare the performance of superficially porous particles with fully porous particles in approximating UHPLC performance.

Main Methods:

  • Optimization of extra-column volume to UHPLC specifications (<10 μl).
  • Testing and optimization of gradient and injection systems.
  • Recording of Height/Velocity (H/u) curves for 5 μm superficially porous and 3 μm fully porous particles.

Main Results:

  • Optimization of extra-column volume increased theoretical plate number up to twofold, within UHPLC range.
  • Injections for qualitative UHPLC purposes demonstrated precision, but the gradient system lacked precision and accuracy.
  • Superficially porous particles showed comparable performance to fully porous particles, approximating UHPLC capabilities.

Conclusions:

  • Hybrid systems present a compromise between HPLC and UHPLC, with limited suitability for true UHPLC due to gradient system limitations.
  • Extra-column volume optimization is crucial for enhancing hybrid system performance towards UHPLC standards.
  • Superficially porous particle technology offers a viable alternative for achieving UHPLC-like performance on HPLC systems.