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Related Concept Videos

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...
¹H NMR Signal Integration: Overview00:58

¹H NMR Signal Integration: Overview

The intensity of a signal, which can be represented by the area under the peak, depends on the number of protons contributing to that signal. The area under each peak is shown as a vertical line called an integral, with the integral value listed under it, as seen in the proton NMR spectrum of benzyl acetate. Each integral value is divided by the smallest integral value to obtain the ratio of the number of protons producing each signal. The ratio reveals the relative number of protons and not...
NMR Spectroscopy: Chemical Shift Overview01:15

NMR Spectroscopy: Chemical Shift Overview

The position of the absorption signal of a sample is reported relative to the position of the signal of tetramethylsilane (TMS), which is added as an internal reference while recording spectra. The difference between the absorption frequencies of the sample and TMS (in Hz) is divided by the spectrometer operating frequency (in MHz) to obtain a dimensionless quantity called the chemical shift. It is reported on the δ (delta) scale and expressed in parts per million.
For instance, the proton...
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.
¹H NMR: Interpreting Distorted and Overlapping Signals01:02

¹H NMR: Interpreting Distorted and Overlapping Signals

Spin systems where the difference in chemical shifts of the coupled nuclei is greater than ten times J are called first-order spin systems. These nuclei are weakly coupled, and their chemical shifts and coupling constant can generally be estimated from the well-separated signals in the spectrum.
As Δν decreases and the signals move closer, the doublets appear increasingly distorted. The intensities of the inner lines increase at the cost of those of the outer lines as the signals are slanted or...
Interpreting ¹H NMR Signal Splitting: The (n + 1) Rule01:10

Interpreting ¹H NMR Signal Splitting: The (n + 1) Rule

In the AX proton spin system, proton A can sense the two spin states of a coupled proton X, resulting in a doublet NMR signal with two peaks of equal (1:1) intensity. When proton A is coupled to two equivalent protons (AX2 spin system), the spin states of each X can be aligned with or against the external field, creating three possible scenarios. This results in a 1:2:1  triplet signal, where the central peak corresponds to the chemical shift of A and is twice as large or intense as the others.

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Related Experiment Video

Updated: Jun 28, 2026

Using Solution NMR to Characterize Biomolecular Condensates Under Biphasic Conditions
09:01

Using Solution NMR to Characterize Biomolecular Condensates Under Biphasic Conditions

Published on: April 17, 2026

Solvent signal as an NMR concentration reference.

Huaping Mo1, Daniel Raftery

  • 1Purdue Interdepartmental NMR Facility, Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana 47907, USA. hmo@purdue.edu

Analytical Chemistry
|November 15, 2008
PubMed
Summary

This study introduces using NMR solvent signals, like water, as a universal concentration standard. This method accurately quantizes analyte concentrations without needing external references or probe tuning.

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Last Updated: Jun 28, 2026

Using Solution NMR to Characterize Biomolecular Condensates Under Biphasic Conditions
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Published on: January 30, 2019

Area of Science:

  • Analytical Chemistry
  • Nuclear Magnetic Resonance (NMR) Spectroscopy

Background:

  • Accurate concentration determination is crucial in chemical analysis.
  • Existing NMR quantification methods often require external standards or complex calibration.
  • Solvent signals in NMR are ubiquitous and their concentrations can be independently verified.

Purpose of the Study:

  • To propose and validate the use of NMR solvent signals as a universal concentration reference.
  • To establish a robust and self-contained method for quantitative NMR analysis.

Main Methods:

  • Utilizing the inherent NMR signal of a highly protonated solvent (e.g., water) as an internal standard.
  • Employing small pulse angle excitation to mitigate radiation damping effects from strong solvent signals.
  • Directly referencing analyte proton concentrations against solvent proton concentrations.

Main Results:

  • Demonstrated accurate analyte concentration determination across a wide dynamic range (4 microM to >100 M).
  • Validated the method using water as a reference solvent at concentrations exceeding 10 M.
  • Showcased the robustness of the method, independent of probe tuning.

Conclusions:

  • NMR solvent signals provide a universal, stable, and readily available concentration reference.
  • The proposed method eliminates the need for external concentration standards, simplifying NMR quantification.
  • This approach offers a robust and efficient alternative for precise analyte concentration measurements in NMR.