Related Experiment Video
Updated: May 13, 2026
09:38
Single-throughput Complementary High-resolution Analytical Techniques for Characterizing Complex Natural Organic Matter Mixtures
Published on: January 7, 2019
HPLC-NMR revisited: using time-slice high-performance liquid chromatography-solid-phase extraction-nuclear magnetic
Kenneth T Johansen1, Sileshi G Wubshet, Nils T Nyberg
1Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
Analytical Chemistry
|February 26, 2013
Summary
This study introduces a novel HPLC-SPE-NMR method for natural product dereplication. The technique efficiently identifies compounds in complex mixtures using advanced spectral matching, minimizing false positives.
Area of Science:
- Analytical Chemistry
- Natural Product Chemistry
- Spectroscopy
Background:
- Natural product dereplication is crucial for drug discovery and chemical ecology.
- High-performance liquid chromatography coupled with nuclear magnetic resonance (HPLC-NMR) is a powerful tool for compound identification.
- Existing HPLC-NMR methods can be limited by sensitivity and throughput.
Purpose of the Study:
- To develop and validate a time-based trapping HPLC-SPE-NMR method for efficient natural product dereplication.
- To achieve sufficient mass sensitivity for analyzing complex natural product mixtures.
- To create and test an in-house algorithm for matching NMR spectra against a database.
Main Methods:
- Utilized time-based trapping of separated compounds onto solid-phase extraction (SPE) cartridges.
- Employed a state-of-the-art HPLC-SPE-NMR system with a cryogenically cooled probe head for enhanced sensitivity.
- Developed and applied a novel spectral matching algorithm allowing for chemical shift variations.
Main Results:
- Successfully obtained high-quality 1H NMR spectra (600 MHz) from complex natural product mixtures.
- The developed HPLC-SPE-NMR method coupled with the matching algorithm accurately identified known compounds in test extracts.
- Demonstrated a low number of false positives, which were easily recognizable.
Conclusions:
- The developed HPLC-SPE-NMR approach provides a sensitive and effective method for natural product dereplication.
- This technique facilitates the identification of compounds in complex mixtures, aiding in the discovery of novel bioactive molecules.
- The in-house spectral matching algorithm enhances the reliability and accuracy of compound identification in dereplication studies.
Related Concept Videos
01:11High-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.
In HPLC, two phases play a critical role in the separation process:
In HPLC, two phases play a critical role in the separation process:
01:192D NMR: Heteronuclear Single-Quantum Correlation Spectroscopy (HSQC)
Heteronuclear single-quantum correlation spectroscopy (HSQC) is a 2D NMR technique that reveals one-bond correlations between hydrogen and a heteronucleus. The HSQC experiment is similar to the heteronuclear correlation experiment (HETCOR) but is more sensitive. In the HSQC spectrum, the proton chemical shift is plotted on the horizontal F2 axis, while the 13C chemical shift is plotted on the vertical F1 axis. The corresponding proton and 13C spectra are also shown. The HSQC contour plot does...
01:182D NMR: Overview of Heteronuclear Correlation Techniques
Heteronuclear correlation spectroscopy is an analytical technique that investigates the coupling between different types of nuclei, often a proton and an X-nucleus, such as carbon-13 or nitrogen-15. This method is commonly used in nuclear magnetic resonance (NMR) spectroscopy to gain insights into complex chemical compounds' structural and compositional aspects. A typical heteronuclear correlation spectrum displays X-nucleus chemical shifts on one axis and a proton spectrum on the other axis.
00:57High-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.
01:13¹H NMR: Complex Splitting
A proton M that is coupled to a proton X results in doublet signals for M. However, NMR-active nuclei can be simultaneously coupled to more than one nonequivalent nucleus. When M is coupled to a second proton A, such as in styrene oxide, each peak in the doublet is split into another doublet.
Splitting diagrams or splitting tree diagrams are routinely used to depict such complex couplings. While drawing splitting diagrams, the splitting with the larger coupling constant is usually applied first.
Splitting diagrams or splitting tree diagrams are routinely used to depict such complex couplings. While drawing splitting diagrams, the splitting with the larger coupling constant is usually applied first.
01:20¹³C NMR: Distortionless Enhancement by Polarization Transfer (DEPT)
When proton-coupled carbon-13 spectra are simplified by a broadband proton decoupling technique, structural information about the coupled protons is lost. Distortionless enhancement by polarization transfer (DEPT) is a technique that provides information on the number of hydrogens attached to each carbon in a molecule. While the DEPT experiment utilizes complex pulse sequences, the pulse delay and flip angle are specifically manipulated. The resulting signals have different phases depending on...