13C NMR Spectroscopy

• Nuclear Magnetic Resonance (NMR) spectroscopy is a powerful tool for structural characterization of organic molecules
• Molecular biologists also use it to study proteins
• NMR is primarily used for qualitative analysis to determine a compound's structure
• It identifies the types and number of non-equivalent carbons

Qualitative vs Quantitative Analysis

• NMR is mainly for qualitative analysis
• Use for quantitative analysis is less common due to its lower sensitivity
• The focus of study is on 13C NMR

NMR Spectrum Example

• An NMR spectrometer outputs an NMR spectrum, in plural spectra
• 13C spectra for 2-octanone serves as an example
• Signals in the spectrum correspond to the atoms in the 2-octanone.
• Hydrogens do not appear
• The horizontal axis represents chemical shift, measured in parts per million (ppm)
• Carbon chemical shift ranges ~0 to 200 ppm
• Tetramethylsilane (TMS) is an arbitrary zero reference for chemical shift
• Each carbon creates a signal
• Each carbon has a specific atomic environment

How an NMR Works: Energy and Range

• NMR spectroscopy involves energy absorption leading to an energy level change from ground to excited state
• In NMR, the change involves the energy levels of the nucleus
• The energy absorbed is in the radiofrequency range

Nuclear Spin and Magnetic Fields

• Atomic nuclei with odd mass/atomic numbers exhibit nuclear spin and behave like magnets in external magnetic fields.
• 13C and ¹H have nuclear spin, while 12C does not
• In the absence of a magnetic field (B0), the spins of 13C atoms orient randomly, spin states are degenerate
• When nuclei are placed in a magnetic field (B0), interactions between their nuclear spins and the applied magnetic field are quantized
• This quantization is determined by the spin quantum number (I)
• The formula 2I + 1 determines the number of allowed spin states, ranging from +I to -I
• These states correspond to orientations of the nuclear spin in an external magnetic field

Spin States of Carbon

• For carbon (I = ½), there exist two spin states
• These spin states have varying energies, designated +½ and -½.
• One can interpret these states as orientations of the nuclear spin.

NMR Active Nuclei and Their Properties

• Commonly examined nuclei in NMR spectroscopy have I = ½
• Nuclei with spin 0, such as 12C and 16O, are magnetically inactive and not seen on the spectrum
• Nuclei with spin > ½ are magnetically active but have more complex spectra

Properties of Magnetically Active Nuclei

• Element (Atomic Mass / Spin / % Natural Abundance / Receptivity (13C=1.00) / Resonant frequency (MHz) at 2.348 T):
• Hydrogen (1 / 1/2 / 99.985 / 5670 / 100.00)
• Deuterium (2 / 1 / 0.015 / 0.0082 / 15.35)
• Carbon (13 / 1/2 / 1.108 / 1.00 / 25.15)

Resonance Phenomenon

• When oriented nuclei are irradiated with electromagnetic radiation at the correct frequency, the lower energy state absorbs energy and 'flips' to the higher energy state
• This occurs when the nuclei are in resonance with the applied radiation
• NMR detects this 'flip,' and a signal appears in the NMR spectrum

Energy Absorption and Magnetic Fields

• Energy absorption is a quantized process requiring the absorbed energy equals the energy difference between the two states
• Energy difference is a function of the applied magnetic field (B0)

Energy Levels in External Magnetic Field

• The absorption signal's strength is proportional to the population difference between lower and upper energy levels
• Equalized populations result in no net absorption/signal so solution saturation must be avoided
• Anything that increases ΔE will increase the absorption signal's strength by increasing the population difference

Optimal Nuclei for Strong Absorption Signals

• High natural abundance, e.g., ¹H (99.9%) compared to 13C (1.1%)
• Large magnetogyric ratio (γ)
• Specific sensitive effects associated with nuclei
• These are all set and cannot be changed, and there are alternative methods for increasing signal strength

Magnetogyric Ratio and Energy Dependence

• The magnitude of energy level separation varies based on the specific nucleus (e.g., H, C, O) due to differences in their magnetogyric ratio (γ)
• This value is constant for each nucleus and determines its energy reliance on the magnetic field

Frequency Calculation for Resonance

• The frequency required to cause resonance and transition between two states can be found using: ν = (γ/2π) * B0
  • ν = frequency (MHz)
  • B0 = magnetic field (Tesla, T)
  • γ = magnetogyric ratio

Magnetogyric Ratios and Frequencies

• Nucleus (γ /106 (T-1s-1) / ν (MHz)):
  • 1H (267.5 / varies depending on magnetic field)
  • 13C (67.3 / varies depending on magnetic field)
• Note: The value of γ has been divided by 106 so that our final answer for frequency will be in MHz (not Hz).*

NMR Spectrometer and Resonance Frequency

• An NMR has a fixed magnet strength
• Referred to by the frequency required to excite ¹H.
• Exciting 13C requires a different frequency

Key Points

• Resonance frequency changes with magnetic field strength
• Required frequency for one element can be selected for examination due to differing resonant frequencies
• Not all nuclei respond with similar sensitivity in NMR experiments

Impact of Magnetic Field

• Signal strength enhances with stronger magnetic fields
• Stronger magnetic fields improve spectra resolution

Superconducting Magnets in NMR

• Modern NMR spectrometers use superconducting magnets to achieve strong magnetic fields
• The magnet must be kept cold via immersion in liquid He (-268.9 °C) contained in liquid N2 (-195.8 °C) which is replenished weekly
• NMR instruments typically operate between 100-800 MHz
• Bruker has a world-record-breaking 1.2 GHz NMR
• Most NMR instruments use cryogenic storage
• Small bench top units operate between 60-80 MHz

Sample Preparation

• The sample (~10 – 25 mg) should be dissolved in ~0.5 mL of deuterated solvent
• The liquid sample transfers to an NMR tube, placed in a holder, and lowered into the NMR

Most Commonly Used Solvents

• Chloroform-d1 (CDCl3)
• DMSO-d6
• D₂O
• 13C spectra display solvent peaks of CDCl3 (~77 ppm, triplet peak) and DMSO-d6 (39.52 ppm, septet in a coupled spectrum) which should be ignored

NMR spectrum Plots and Chemical Shift

• NMR spectrum plots signal intensity versus chemical shift
• The x-axis is a relative scale where chemical shift is relative to the reference compound tetramethylsilane (TMS)
• Reference compound tetramethylsilane, TMS, has a chemical shift defined as zero

TMS and Signal Placement

• TMS produces a single, narrow signal (singlet)
• Its protons and carbons are more shielded than carbons and protons in most organic compounds
• Signals from compounds of interest appear on one side of the TMS signal
• For organic compounds:
  • TMS signal is on the right of the spectrum at zero
  • C signals are toward the left with chemical shift increasing positive toward the left
  • The range for carbon signals is typically 0-200 ppm

Shielding and Magnetic Fields

• Electrons possess spin and create ‘local’ magnetic fields which oppose the applied field
• This ‘shields’ the nuclei from the applied magnetic field
• Electron density influences the electronegativity of elements attached to it
• When a carbon atom is attached to an electronegative atom, it gets ‘de-shielded,' causing a shift in the C signal to the left

Local Magnetic Fields

• For a given applied magnetic field, each nucleus of a particular type will experience a slightly different local magnetic field
• This is because electron clouds generate a magnetic field that can either partially cancel or partially augment the applied field
• These small differences correspond to kilohertz frequency differences which causes resonance, creating different positions on the spectrum

Chemical Shifts and Attached Elements

• Chemical shift provides vital information about the atoms attached to a carbon
• Approximate carbon chemical shifts in organic compounds have been charted

Chemical Shift Rules of Thumb

• Double bonds cause a downfield shift (higher ppm) of the signal
• Reduction in the number of H attached to a C causes a downfield shift meaning increased deshielding

Impact of Electronegative Atoms

• Increasing electronegativity of attached groups causes electron density to be pulled away from the carbon atom
• This exposes it to the magnetic field, increasing the chemical shift, causing it to move downfield

Multiple Bonds and Heteroatoms

• Carbons containing multiple bonds and heteroatoms show at 150-210 ppm

Fourier Transform Spectrometer

• NMR spectrometers are pulsed fourier transform (FT) spectrometers with an internal lock on the field, which allows for noise reduction via multiple scans

Pulse Sequences and Relaxation

• Through the center of a selected frequency and excites all the nuclei, short radio frequency pulses create frequencies
• Nuclei return to ground state through free induction decay (FID) containing frequency signals
• Time domain data turns into frequency domain results by fourier transform
• FIDs add together to find peak above the noise
• Nuclei must relax fully between pulse repetitions to avoid the loss of signal intensity where Hydrogen relaxes quickly but 13C can take longer
• The rate of relaxation in carbon nuclei relies primarily on the environment

Problem from Long Delay Times

• Long delay times are often needed for the nuclei of a dilute sample

Compromise from Long Delay Times

• The acquisition and delay has a time compromise

Consequence of Compromise

• Carbon signals shift in peak size, causing the representative number of carbon types to be completely relaxed

Spectrum Coupling

• Coupled spectrum of protons attached to a carbon create problems but can be opportunities
• A carbon with n protons attached will be split into 2nI+1 signals, or n+1.

Simplified Carbon Spectra

• Carbon spectra run decoupled to streamline signals into singlets

Nuclear Overhauser Effect (NOE)

• Increases carbon signal strength
• Proportional to the number of attached protons
• Carbons with no protons show smaller signals
• Carbons with more protons give larger signals because the energy moves from the proton

DEPT Experiment

• Running this experiment varying pulse sequences recovers lost data from the Decoupling Problem
• A DEPT has number referring to the angle of the hydrogen impulse, where the angle gives variable results
  • A 135° angle produces all CH and CH3 angled opposite to CH2

Polarization Transfer from ¹H to ¹³C

• Increased sensitivity due to proton detection
• Makes for a quicker experiment
• Carbons with different number of protons attached show different signals:
  • Odd number of protons attached show CH and CH3 point up
  • Even: CH2 points down
  • No protons attached: C show no signal
    • NOTE: The convention is that CH2 is phased down, but some books show opposite