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Related Concept Videos

¹H NMR of Labile Protons: Deuterium (²H) Substitution00:48

¹H NMR of Labile Protons: Deuterium (²H) Substitution

This lesson illustrates the role of deuterium substitution in simplifying the NMR spectrum of compounds comprising labile protons. One method employed is the use of deuterium. Amongst the three isotopes of hydrogen, deuterium (2H) has a nucleus composed of one proton and one neutron. When the D2O solvent is added to a pure dry ethanol solution, its labile proton is substituted with deuterium.
¹³C NMR: Distortionless Enhancement by Polarization Transfer (DEPT)01:20

¹³C 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...
Two-Dimensional (2D) NMR: Overview01:12

Two-Dimensional (2D) NMR: Overview

The 1D NMR spectrum of large and complex molecules like natural products has complicated splitting patterns and overlapping signals, which can be easily interpreted using 2-dimensional (2D) NMR. Unlike 1D NMR, 2D NMR has two frequency axes that provide the coupling information between the nucleus A and nucleus B in a molecule. The process from which 2D spectra are obtained has four steps.
The first step is the preparation period, during which nucleus A is excited with a radiofrequency pulse.
¹H NMR of Conformationally Flexible Molecules: Variable-Temperature NMR01:15

¹H NMR of Conformationally Flexible Molecules: Variable-Temperature NMR

The axial and equatorial protons in cyclohexane can be distinguished by performing a variable-temperature NMR experiment. In this process, except for one proton, the remaining eleven protons are replaced by deuterium. The deuterium substitution avoids the possible peak splitting caused by the spin-spin coupling between the adjacent protons. The remaining proton flips between the axial and equatorial positions.
Applications Of NMR In Biology01:25

Applications Of NMR In Biology

Nuclear magnetic resonance (NMR) spectroscopy is a very valuable analytical technique for researchers. It has been used for more than 50 years as an analytical tool. F. Bloch and E. Purcell formulated NMR in 1946 and won the 1952 Nobel Prize in Physics  for their work. Biological macromolecules such as proteins, nucleic acids, lipids, and organic molecules including pharmaceutical compounds, can be studied using this versatile tool that exploits the magnetic properties of certain nuclei.
The...
Chemical Shift: Internal References and Solvent Effects01:17

Chemical Shift: Internal References and Solvent Effects

In an NMR sample, precise measurement of the absolute absorption frequencies of nuclei is difficult. A standard internal reference compound is added, and the frequency difference between the reference signal and sample signals is measured.
The internal reference compound generally used in NMR spectroscopy is tetramethylsilane (TMS). TMS is preferred because it is chemically inert, soluble in NMR solvents, and easily removable. Also, the highly shielded methyl protons in TMS yield an intense...

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Related Experiment Video

Updated: Jul 16, 2026

Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy
14:55

Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy

Published on: September 17, 2017

Solution NMR techniques for large molecular and supramolecular structures.

Roland Riek1, Jocelyne Fiaux, Eric B Bertelsen

  • 1Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Zürich, CH-8093 Zürich, Switzerland.

Journal of the American Chemical Society
|October 10, 2002
PubMed
Summary

This study combines Transverse Relaxation-Optimized Spectroscopy (TROSY) with polarization transfer techniques to analyze large biomolecules using 2D NMR. Optimized experimental parameters enable high-resolution spectra for macromolecules up to 800 kDa.

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Using Solution NMR to Characterize Biomolecular Condensates Under Biphasic Conditions
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Using Solution NMR to Characterize Biomolecular Condensates Under Biphasic Conditions

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Area of Science:

  • Biophysical Chemistry
  • Structural Biology
  • Nuclear Magnetic Resonance (NMR) Spectroscopy

Background:

  • Analyzing large homo-oligomeric macromolecules (110-800 kDa) using solution NMR presents challenges due to signal broadening.
  • Standard NMR techniques often struggle to provide high-resolution spectra for such large biological structures.

Purpose of the Study:

  • To develop and optimize 2D NMR methods for obtaining high-quality correlation spectra of large (15)N,(2)H-labeled homo-oligomeric macromolecules.
  • To investigate the performance of combined TROSY with CRIPT/CRINEPT techniques for large biomolecular structures.

Main Methods:

  • Combined Transverse Relaxation-Optimized Spectroscopy (TROSY) with Cross-Correlated Relaxation-Induced Polarization Transfer (CRIPT) or Cross-Correlated Relaxation-Enhanced Polarization Transfer (CRINEPT).
  • Acquired 2D solution NMR correlation spectra of (15)N,(2)H-labeled homo-oligomeric macromolecules ranging from 110 to 800 kDa.
  • Systematically optimized polarization transfer times, relaxation delays, and water-handling routines.

Main Results:

  • Achieved TROSY-based spectra with manageable line widths (e.g., ~75 Hz for 15N at 800 kDa).
  • Determined optimal polarization transfer times that are inversely proportional to molecular size (e.g., 1.4 ms for 800 kDa).
  • Established short recycle times (< 1 s) due to rapid proton longitudinal relaxation in H2O.

Conclusions:

  • The combined CRIPT/CRINEPT-TROSY approach effectively yields high-resolution 2D NMR spectra for large homo-oligomeric macromolecules.
  • Optimization of experimental parameters, particularly transfer times and water suppression, is crucial for successful NMR analysis of large structures.
  • The developed methods significantly advance the NMR study of large biomolecular assemblies in solution.