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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...
¹H NMR Chemical Shift Equivalence: Homotopic and Heterotopic Protons01:03

¹H NMR Chemical Shift Equivalence: Homotopic and Heterotopic Protons

Protons in identical electronic environments within a molecule are chemically equivalent and have the same chemical shift. The replacement test is a useful tool to identify chemical equivalence and predict NMR spectra. A substituent replaces each of the protons being examined and the resulting molecules are compared. If the same molecule is obtained, the protons are equivalent or homotopic. Replacement of any hydrogens in ethane by chlorine yields chloroethane because all six protons are...
¹H NMR Chemical Shift Equivalence: Enantiotopic and Diastereotopic Protons00:58

¹H NMR Chemical Shift Equivalence: Enantiotopic and Diastereotopic Protons

Replacing each alpha-hydrogen in chloroethane by bromine (or a different functional group) yields a pair of enantiomers. Such protons are called prochiral or enantiotopic and are related by a mirror plane. Enantiotopic protons are chemically equivalent in an achiral environment. Because most proton NMR spectra are recorded using achiral solvents, enantiotopic hydrogens yield a single signal.
In chiral compounds such as 2-butanol, replacing the methylene hydrogens at C3 produces a pair of...
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.
NMR Spectroscopy Of Amines01:19

NMR Spectroscopy Of Amines

In proton NMR spectroscopy, primary amines and secondary amines showcase their N–H protons as a broad signal in the chemical shift range between δ 0.5 and 5 ppm. The exact position in this range depends on several factors, including sample concentration, hydrogen bonding, and the type of solvent used. Since amine protons undergo fast proton exchange in solution, the protons are labile and therefore do not participate in any splitting with adjacent protons. Thus, the observed peak is broad and...

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Related Experiment Video

Updated: Jun 11, 2026

Analyzing Protein Dynamics Using Hydrogen Exchange Mass Spectrometry
11:37

Analyzing Protein Dynamics Using Hydrogen Exchange Mass Spectrometry

Published on: November 29, 2013

D/H amide isotope effect in model alpha-helical peptides.

Zhengshuang Shi1, C Anders Olson, Neville R Kallenbach

  • 1Department of Chemistry, New York University, 100 Washington Place, New York, NY 10003, USA.

Journal of the American Chemical Society
|November 21, 2002
PubMed
Summary

The Cm experiment measuring D/H amide isotope effects reveals denaturant concentration impacts protein stability measurements. Adjusting for guanidinium chloride (GdmHCl) concentration is crucial for accurate comparisons of protein stability and hydrogen bond contributions.

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Last Updated: Jun 11, 2026

Analyzing Protein Dynamics Using Hydrogen Exchange Mass Spectrometry
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Published on: May 4, 2020

Area of Science:

  • Biochemistry
  • Structural Biology
  • Protein Dynamics

Background:

  • Protein stability is influenced by hydrogen bonds.
  • The Cm experiment measures D/H amide isotope effects to quantify hydrogen bond contributions.
  • Denaturant concentration can affect experimental outcomes.

Purpose of the Study:

  • To investigate the impact of denaturant concentration on D/H amide isotope effects in isolated alpha-helical peptides.
  • To determine if corrections for denaturant concentration are necessary for comparing protein stability.
  • To evaluate the energetic contribution of hydrogen bonds in isolated helices versus helical proteins.

Main Methods:

  • Utilized isolated alpha-helical peptides.
  • Employed the Cm experiment to measure D/H amide isotope effects.
  • Varied the concentration of guanidinium chloride (GdmHCl) as a denaturant.

Main Results:

  • A significant effect of denaturant concentration on the measured D/H isotope effect was observed.
  • Correction for GdmHCl concentration is essential for valid comparisons between different proteins.
  • Hydrogen bonds in isolated alpha-helices may be more stabilizing due to reduced strain compared to helical proteins.

Conclusions:

  • Denaturant concentration must be standardized or corrected for when using the Cm experiment to assess protein stability.
  • Buried hydrogen bonds in helical proteins are not inherently more favorable than solvent-exposed ones in isolated helices.
  • This study refines the understanding of hydrogen bond energetics in protein structure and stability.