R&D and GLP/cGMP NMR Services

Nuclear Magnetic Resonance (NMR) Spectroscopy

What is NMR?

Nuclear Magnetic Resonance (NMR) spectroscopy is an analytical chemistry technique used to determine the molecular structure of a sample. This is done by analyzing the chemical environment of a selected nucleus. When nuclei are placed into a magnetic field, their magnetic moment (spin) becomes aligned with the magnetic field. NMR uses a pulse of RF (radio frequency) energy to deflect the nuclei. When the energy is removed, the nuclei relax back to their original state and emit an electromagnetic pulse. A coil inside the NMR receives this RF pulse, and a computer transforms the signal into a spectral graph, which can be read by a scientist. Each nucleus relaxation translates into a peak in the NMR spectrum. Its location on the x-axis (chemical shift) and the multiplicity of the peak tells the scientist about its environment. The area of the peak corresponds to the number of nuclei experiencing the same environment.

NMR is a powerful tool in structural characterization. It can be used to determine how all atoms of a particular molecule are interconnected, thereby determining a molecule’s content and purity.

NMR Magnetic Field Pulse Graph

Our NMR variable-temperature, tunable broadband probe can be set to virtually any nucleus that has a non-zero spin, including hydrogen, carbon, silicon, fluorine, chlorine, nitrogen, phosphorous, and more. One-dimensional and two-dimensional NMR experiments provide full characterization of known molecules and full structure elucidation of unknown compounds, such as impurities found in pharmaceutical products or metabolites from the biological breakdown of an active pharmaceutical ingredient (API).

Thymidine is used in the following examples of common NMR experiments.

Typical Outline Of NMR Experiments For Structure Elucidation

1-Dimensional NMR Experiments

¹H NMR

¹H NMR spectroscopy is an analytical technique used to determine the structure of organic compounds by analyzing the magnetic environments of hydrogen atoms (protons) in a molecule.

Key Features:

  • Chemical Shift (δ):
    Measured in ppm, indicating the electronic environment of each proton.
    Common ranges:

    • Alkyl: 0.5–2 ppm
    • Alkenes: 4.5–6.5 ppm
    • Aromatics: 6–8.5 ppm
    • Aldehydes: ~9–10 ppm
    • Carboxylic acids: ~10–13 ppm
  • Integration:
    The area under each peak corresponds to the number of protons it represents.
  • Multiplicity (Splitting Patterns):
    Caused by coupling between non-equivalent neighboring protons (n+1 rule), gives info on proton neighbors:

    • Singlet (no neighbors)
    • Doublet (1 neighbor)
    • Triplet (2 neighbors)
    • etc.
  • Coupling Constant (J):
    Measured in Hz, indicates the strength of spin-spin coupling between adjacent protons.

Applications:

  • Determining molecular structure
  • Identifying impurities
  • Analyzing reaction products
  • Studying dynamic processes (e.g., conformational changes)

 

1H NMR Thymidine

¹³C-NMR

¹³C NMR is a type of nuclear magnetic resonance spectroscopy that focuses on the carbon-13 isotope of carbon atoms. While carbon-12 is the most abundant isotope (about 98.9%), it has no magnetic moment and is NMR-inactive. Carbon-13 makes up about 1.1% of natural carbon and has a nuclear spin (I = 1/2), making it NMR-active and detectable.

Why is ¹³C NMR Important?

¹³C NMR provides detailed information about the carbon framework of organic molecules. It helps in:

  • Determining molecular structure
  • Identifying functional groups
  • Confirming purity and identity
  • Studying carbon environments in polymers, natural products, and pharmaceuticals

 Key Features of ¹³C NMR

1. Chemical Shift (δ)

  • Reported in parts per million (ppm) relative to tetramethylsilane (TMS) at 0 ppm.
  • Typical chemical shift ranges:
    • Alkyl carbons: 0–50 ppm
    • Alkenes/aromatics: 100–150 ppm
    • Carbonyls: 160–220 ppm

2. Signal Intensity

  • Weaker than ¹H NMR signals due to:
    • Lower natural abundance of ¹³C (~1.1%)
    • Lower gyromagnetic ratio
  • Often requires signal averaging and longer acquisition times

3. Proton Decoupling

  • Most ¹³C spectra are recorded with broadband proton decoupling, simplifying the spectrum by removing ¹³C–¹H coupling.
  • This results in singlets for each carbon environment, making interpretation easier.

4. DEPT Experiments (Distortionless Enhancement by Polarization Transfer)

  • Used to differentiate between CH₃, CH₂, CH, and quaternary carbons:
    • DEPT-45: Shows CH₃, CH₂, and CH
    • DEPT-90: Shows only CH
    • DEPT-135: CH₃ and CH (positive), CH₂ (negative), quaternary (not shown)

Applications of ¹³C NMR

  • Organic synthesis: Confirm structure of synthesized compounds
  • Natural products chemistry: Identify unknown molecules
  • Pharmaceuticals: Analyze active pharmaceutical ingredients (APIs) and excipients
  • Polymer science: Study monomer units and branching
  • Metabolomics and isotopic labeling: Use ¹³C-labeled compounds to track biochemical pathways

Limitations of ¹³C NMR

  • Low sensitivity: Requires larger sample size or longer acquisition
  • Signal overlap: Less common than in ¹H NMR, but possible in large, complex molecules
  • Cost: Requires high-field NMR instruments and maintenance

 Enhancements and Techniques

  • Cryoprobes: Increase sensitivity by cooling the probe
  • 2D NMR: Techniques like HSQC and HMBC correlate ¹³C with ¹H to assign carbons more effectively
  • Solid-State ¹³C NMR: Used for analyzing insoluble or crystalline samples (e.g., polymers, materials)

Carbon-13 NMR is a necessary step in full structural characterization. Usually, 13C-NMR alone does not provide enough information to assign the carbons in the molecule. Two-dimensional techniques are often necessary to assign all carbons with confidence.

13C NMR Thymidine 1

DEPT-45, 90, and 135

Distortionless Enhancement of Polarization Transfer (DEPT) experiments help assign carbon peaks by determining the number of hydrogens attached to each carbon. In complex molecules, DEPT and HSQC together are useful for confirming both carbon and proton assignments. There are 3 main DEPT experiments. Acquiring a carbon spectrum and several DEPT experiments will provide the number of hydrogens attached to each carbon atom.

13C NMR DEPT Thymidine

2-Dimensional NMR Experiments

¹H-¹H COSY

1H–1H Correlation Spectroscopy (COSY) shows the correlation between hydrogens which are coupled to each other in the 1H NMR spectrum.  In general, this indicates which hydrogen atoms are adjacent to another group of hydrogens. The 1H spectrum is plotted on both axes (2D). While 2-bond and 3-bond 1H-1H coupling is easily visible by COSY, long range coupling can also be observed with long acquisition times. The cross-peaks that are symmetric to the diagonal show the COSY correlations.

NMR COSY Thymidine 1

¹H-¹³C HSQC

1H–13C Heteronuclear Single Quantum Coherence (HSQC) Spectroscopy shows which hydrogens are directly attached to which carbon atoms. The 1H spectrum is shown on the top axis and the 13C spectrum is shown on the left axis. Only 1-bond correlations are observed by HSQC.

NMR HMBC Thymidine ALL

The experiments explained above are the most popular. For a detailed lesson on assigning protons and carbons in Thymidine by NMR, check out our blog. We also perform many more 1-dimensional and 2-dimensional experiments not discussed above, including Nuclear Overhauser Effect (1D NOE and 2D NOESY through-space correlations), multi-nuclear HSQC and HMBC, and more.

At Emery Pharma, we offer comprehensive NMR spectroscopy services to support both R&D and cGMP-compliant studies. Our state-of-the-art instrumentation enables structural elucidation, impurity profiling, quantitative analysis, and even bioanalysis using NMR. Whether you require a simple ¹H-NMR or ¹³C-NMR with rapid turnaround for early-stage research, or fully documented studies for regulatory submissions—including quantitation of impurities—our expert team is equipped to deliver precise, reliable results tailored to your needs.