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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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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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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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Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification
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Blind column selection protocol for two-dimensional high performance liquid chromatography.

Niki K Burns1, Luke M Andrighetto1, Xavier A Conlan1

  • 1Centre for Chemistry and Biotechnology, School of Life and Environmental Sciences, Deakin University, Waurn Ponds, Vic 3216, Australia.

Talanta
|May 8, 2016
PubMed
Summary
This summary is machine-generated.

Selecting orthogonal columns for two-dimensional high-performance liquid chromatography (2D-HPLC) is challenging. This study presents a blind optimization method using OpenMS® to efficiently predict optimal column combinations for natural product separation.

Keywords:
Electrospray mass spectrometryMultidimensional high performance liquid chromatographyOpenMS®Plant extracts

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Area of Science:

  • Analytical Chemistry
  • Chromatography
  • Natural Product Analysis

Background:

  • Two-dimensional high-performance liquid chromatography (2D-HPLC) is crucial for complex mixture separation.
  • Column selection for 2D-HPLC is often empirical and time-consuming.
  • Optimizing column orthogonality enhances separation efficiency.

Purpose of the Study:

  • To introduce a blind optimization method for selecting orthogonal columns in 2D-HPLC.
  • To develop a data processing pipeline for predicting column performance.
  • To accelerate the column selection process for natural product extracts.

Main Methods:

  • Utilized OpenMS® software for data processing.
  • Developed a pipeline to map mixture components across various HPLC columns.
  • Employed fractional surface coverage (fcoverage) to assess separation space utilization.

Main Results:

  • A blind optimization method was developed and validated.
  • The combination of diphenyl and C18 stationary phases predicted high fcoverage (0.48) for opium poppy extract.
  • Predicted fcoverage closely matched experimentally observed values (0.43).

Conclusions:

  • OpenMS® and in-house algorithms significantly expedite orthogonal column selection for 2D-HPLC.
  • The blind optimization approach reduces trial-and-error in column selection.
  • This method is effective for optimizing the separation of crude plant material extracts.