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Related Concept Videos

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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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: Introduction01:11

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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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Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification
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Demonstration of continuous gradient elution functionality with automated liquid handling systems for high-throughput

Kamiyar Rezvani1, Andrew Smith2, Jannat Javed2

  • 1Purification Process Sciences, BioPharmaceuticals Development, R&D, AstraZeneca, Gaithersburg, US.

Journal of Chromatography. A
|November 30, 2022
PubMed
Summary

High-throughput purification using automated liquid handling systems improves bioprocess development. This study introduces a new system enabling continuous gradient elution for RoboColumns, yielding scalable and consistent chromatography performance.

Keywords:
ChromatographyGradient elutionHigh-throughputPurificationScale-down

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Area of Science:

  • Biopharmaceutical Manufacturing
  • Process Development
  • Chromatography

Background:

  • High-throughput systems are crucial for biopharmaceutical process development, enhancing experiment productivity and reducing material use.
  • Existing high-throughput purification tools, like RoboColumns on Tecan systems, offer scalability but lack optimized liquid delivery for preparative chromatography.
  • Limitations include inability to provide continuous liquid flow and ideal linear gradient conditions, impacting protein chromatography and gradient elution experiments.

Purpose of the Study:

  • To describe a modified Tecan Freedom EVO system for high-throughput purification.
  • To enable continuous gradient elution capability for RoboColumn experiments.
  • To demonstrate the system's ability to generate linear conductivity gradients and provide scalable data.

Main Methods:

  • Modification of a Tecan Freedom EVO liquid handling system to provide continuous liquid delivery.
  • Application of the modified system to RoboColumn experiments for high-throughput purification.
  • Generation of linear conductivity gradients and evaluation of RoboColumn performance against larger formats.

Main Results:

  • The modified Tecan system successfully enabled continuous gradient elution for RoboColumn experiments, generating highly linear conductivity gradients.
  • RoboColumn performance and product quality data were in agreement with larger, bench-scale chromatography formats for two model purification methods.
  • The gradient purification method showed more consistent performance between RoboColumns and larger columns compared to step elution methods.

Conclusions:

  • The developed Tecan Freedom EVO system enhances high-throughput purification by enabling continuous gradient elution for RoboColumns.
  • This advancement provides scalable and consistent chromatography data, bridging the gap between high-throughput screening and manufacturing scale.
  • New insights into discontinuous flow effects on RoboColumn performance were gained, aiding bioprocess development.