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Related Concept Videos

2D NMR: Heteronuclear Single-Quantum Correlation Spectroscopy (HSQC)01:19

2D NMR: Heteronuclear Single-Quantum Correlation Spectroscopy (HSQC)

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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...
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NMR Spectroscopy Of Amines01:19

NMR Spectroscopy Of Amines

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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...
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NMR Spectroscopy of Aromatic Compounds01:14

NMR Spectroscopy of Aromatic Compounds

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Aromatic compounds can be identified or analyzed using proton NMR and carbon‐13 NMR. Typically, aromatic hydrogens or hydrogens directly bonded to the aromatic rings are strongly deshielded by the aromatic ring current. Therefore, they absorb in the range of 6.5–8.0 ppm in proton NMR spectra. For instance, aromatic hydrogens directly bonded to the benzene ring absorb at 7.3 ppm. However, aromatic hydrogens of larger rings absorb farther upfield or downfield than the ideal range.
6.2K
NMR Spectroscopy of Benzene Derivatives01:34

NMR Spectroscopy of Benzene Derivatives

11.1K
Simple unsubstituted benzene has six aromatic protons, all chemically equivalent. Therefore, benzene exhibits only a singlet peak at δ 7.3 ppm in the 1H NMR spectrum. The observed shift is far downfield because the aromatic ring current strongly deshields the protons. Any substitution on the benzene ring makes the aromatic protons nonequivalent, and the protons split each other. The peak is, therefore, no longer a singlet and the splitting pattern and their associated coupling...
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NMR Spectroscopy: Chemical Shift Overview01:15

NMR Spectroscopy: Chemical Shift Overview

3.1K
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...
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NMR Spectroscopy: Spin–Spin Coupling01:08

NMR Spectroscopy: Spin–Spin Coupling

3.0K
The spin state of an NMR-active nucleus can have a slight effect on its immediate electronic environment. This effect propagates through the intervening bonds and affects the electronic environments of NMR-active nuclei up to three bonds away; occasionally, even farther. This phenomenon is called spin–spin coupling or J-coupling. Coupling interactions are mutual and result in small changes in the absorption frequencies of both nuclei involved. While nuclei of the same element are involved...
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Extraction and Quantification of Soluble, Radiolabeled Inositol Polyphosphates from Different Plant Species using SAX-HPLC
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SAX-HPLC and HSQC NMR Spectroscopy: Orthogonal Methods for Characterizing Heparin Batches Composition.

Franco Spelta1, Lino Liverani1, Alessandra Peluso1

  • 1R&D Department, Opocrin S.p.A., Formigine, Italy.

Frontiers in Medicine
|May 7, 2019
PubMed
Summary

Comparing two key heparin analysis methods, Strong-Anion-Exchange HPLC and NMR-HSQC, reveals their strengths and weaknesses. This study validates these techniques for accurate heparin characterization.

Keywords:
HSQCSAX-HPLCbuilding blockscharacterizationcompositionheparinquantitative NMR

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Area of Science:

  • Biochemistry
  • Analytical Chemistry
  • Pharmacology

Background:

  • Heparin's complex structure necessitates robust analytical methods for characterization.
  • Existing methods for determining heparin's building blocks, such as Strong-Anion-Exchange HPLC and NMR-HSQC, have not been fully compared.
  • Understanding the complementary nature of these techniques is crucial for accurate heparin quality control.

Purpose of the Study:

  • To critically compare Strong-Anion-Exchange HPLC and quantitative bidimensional 1H-13C NMR (HSQC) for heparin structural analysis.
  • To evaluate the capabilities and drawbacks of each method.
  • To establish a reliable approach for the regular characterization of commercial heparin preparations.

Main Methods:

  • Analysis of over 30 heparin batches from 8 manufacturers using both SAX-HPLC and NMR-HSQC.
  • SAX-HPLC involved enzymatic digestion and separation of oligosaccharides.
  • NMR-HSQC analysis was performed on intact heparin to quantify monosaccharides and disaccharides.

Main Results:

  • Direct comparison of results from SAX-HPLC and NMR-HSQC highlighted similarities and differences.
  • The study identified specific structural features best evaluated by each method.
  • Concordance between the two methods served to verify their accuracy.

Conclusions:

  • The combined use of SAX-HPLC and NMR-HSQC provides a powerful tool for comprehensive heparin characterization.
  • This comparative approach enhances the accuracy and reliability of heparin structural analysis.
  • The findings support the routine application of these complementary methods for quality control of commercial heparin.