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NTDR, 2014 Nils Nyberg NPR, Department of Drug Design and Pharmacology Basic principles of NMR NMR signal origin, properties, detection, and processing

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Outline 10 00 – 10 45 Establishing current knowledge level Nuclear Magnetic Resonance phenomenon Vector model, in and out of the rotating frame 10 45 – 11 00 Short break 11 00 – 11 30 The phase of pulses and signals Effect of different chemical shifts in the vector model Effect of homonuclear coupling in the vector model The spin-echo sequence (homonuclear case) The spin-echo sequence (heteronuclear case) 11 30 – 12 00 Spin-echo exercise 12 15 – 13 15 Lunch NTDR, 2014

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Outline 12 15 – 13 15 Lunch 13 15 – 14 15 Signal processing Window functions Fourier transform Real and imaginary parts Phasing Topspin starter NTDR, 2014

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Establishing current knowledge level Build (sketch) a NMR-instrument! Magnet Probes Amplifiers Receiver ADC Gradients Temperature control Lock Shimming NTDR, 2014

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Establishing current knowledge level Draw a spectrum! Chemical shifts Integrals Phases Coupling constants Line widths life time of signals, shimming, exchange, dynamics NTDR, 2014

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Nuclear Magnetic Resonance phenomenon Nuclear: concerns the nuclei of atoms. Magnetic: uses the magnetic properties of the nuclei. Resonance: physics term describing oscillations. NTDR, 2014

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Resonance A system prefers some frequencies over others… A small energy input at the right frequency will give large oscillations… NTDR, 2014

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The magnetic properties of atomic nuclei Atoms has a spin quantum number, I, and a magnetic quantum number, m = 2×I +1. The magnetic quantum number = the number of different energy levels when the atom is placed in an external magnetic field. Spin I = 0: 12 C, 16 O Spin I = ½: 1 H, 13 C, 15 N, 19 F, 31 P, 77 Se Spin I = 1: 2 H, 14 N Spin I = 1½: 33 S, 35 Cl, 37 Cl NTDR, 2014

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Chemical shifts The energy for a spin ½ nuclei can take two different levels in a magnetic field. The population of the two states is almost equal. A small surplus in the low energy α spin state and slightly fewer atoms in the higher β spin state. Stronger magnetic field = larger energy differences between the states. NTDR, 2014

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Chemical shifts A magnet provides the static field (B 0 ) in the NMR instrument. The rest of the molecule provides a ’local magnetic field’, which is dependent on structure. NTDR, 2014

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Chemical shifts The chemical shifts are expressed on a frequency scale (by convention plotted in reverse direction). To make spectra comparable between instruments, the frequencies are expressed in parts per million [ppm] relative to a reference frequency. Early instruments with electromagnets worked by slowly change the magnetic field. Hence the terms ‘Downfield’ and ‘Upfield’. NTDR, 2014 Less shielded More deshielded Downfield Higher frequency More shielded Less deshielded Upfield Lower frequency

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Vector model (a statistical abstraction…) Unordered collection of ½-spin nuclei, with a magnetic moment (μ). NTDR, 2014

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Vector model Unordered collection of ½-spin nuclei, with a magnetic moment (μ). In an external magnetic field, the magnetic moment starts to precess… NTDR, 2014

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Vector model Unordered collection of ½-spin nuclei, with a magnetic moment (μ). In an external magnetic field, the magnetic moment starts to precess… …and aligns, at an angle of 54.7°, with the external field… NTDR, 2014

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Vector model Unordered collection of ½-spin nuclei, with a magnetic moment (μ). In an external magnetic field, the magnetic moment starts to precess… …and aligns, at an angle of 54.7°, with the external field… …either up (along the field, slightly lower energy) or down (opposite the field, slightly higher energy) according to the Boltzmann distribution. NTDR, 2014

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Boltzmann distribution The distribution of spins in -state relative those in the - state is described by the Boltzmann distribution. The number of spins in each state is almost equal. There is a small surplus in the lower state. Calculate how many spins in total you need to get one extra spin in the low energy state! [ 1 H, 600 MHz, 298 K] NTDR, 2014

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Boltzmann distribution One spin extra in the low energy state! [ 1 H, 600 MHz, 298 K] N β = 12 922 N α = 12 923 Σ = 25 845 NTDR, 2014

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Vector model The ordered collection of spins can be handled from a common origin. The Boltzmann distribution of up- and down-spins, makes a net magnetic vector along the external field (green). An external magnetic field (radio frequency pulse, B 1 ) perpendicular to the first (B 0 ) have two effects: NTDR, 2014

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Vector model The ordered collection of spins can be handled from a common origin. The Boltzmann distribution of up- and down-spins, makes a net magnetic vector along the external field (green). An external magnetic field (radio frequency pulse, B 1 ) perpendicular to the first (B 0 ) have two effects: Creation of phase coherence (‘bunching of spins’) NTDR, 2014

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Vector model The ordered collection of spins can be handled from a common origin. The Boltzmann distribution of up- and down-spins, makes a net magnetic vector along the external field (green). An external magnetic field (radio frequency pulse, B 1 ) perpendicular to the first (B 0 ) have two effects: Creation of phase coherence (‘bunching of spins’) Switch from up- to down-spin (or down- to up- !) NTDR, 2014

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Vector model The resultant magnetic vector is spinning at the precession frequency, which is the same as the frequency of the external magnetic field. The spinning magnetic vector induces a current in the detector coil around the sample. The alternating current is recorded. The detector senses the absolute length of the magnetic vector in the horizontal plane (XY-plane). Cosine curve along y-axis. Sine curve along x-axis. NTDR, 2014

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Vector model The resultant magnetic vector is spinning at the precession frequency, which is the same as the frequency of the external magnetic field. The ‘rotating frame’ reference is used to simplify the model. The coordinate system is spun at the same speed as the vectors the vectors appear as fixed. NTDR, 2014

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Relaxation T 1 -relaxation Exponential recovery of magnetization along B 0 -axis Back to equilibrium populations of up- and down-spin NTDR, 2014

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Relaxation T 1 -relaxation Exponential recovery of magnetization along B 0 -axis Back to equilibrium populations of up- and down-spin T 2 -relaxation Gradual ‘fanning’ out of individual magnetic vector. emission-absorption among spins (changes phase) bad homogeneity of magnetic field NTDR, 2014

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Relaxation T 1 -relaxation Exponential recovery of magnetization along B 0 -axis Back to equilibrium populations of up- and down-spin T 2 -relaxation Gradual ‘fanning’ out of individual magnetic vector. emission-absorption among spins (changes phase) bad homogeneity of magnetic field NTDR, 2014

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Pulsed experiments The basic 1D-FT NMR experiment Pulse (μseconds) Broadband (covers a wide range of frequencies) Acquisition (seconds) Records all frequencies within a preset frequency width Relaxation delay (seconds) To return the magnetization vector close to equilibrium Repeat and add results signals increases linearly with n, while the noise partly cancels out and increases with n ½. NTDR, 2014

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Phase of pulses and signals Basic 1D NMR-experiment: With a 90°-pulse along the x-axis NTDR, 2014

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Phase of pulses and signals Basic 1D NMR-experiment: With a 90°-pulse along the y-axis NTDR, 2014

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Phase of pulses and signals The phase of the pulse gives the phase of the signal… NTDR, 2014

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Phase of pulses and signals X Y X Y NTDR, 2014

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Phase of pulses and signals X Y X Y NTDR, 2014

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Different chemical shifts in the vector model Two signals with different chemical shifts rotates with different speed in the vector model Interpreted as two different frequencies in the spectrum X Y NTDR, 2014

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Different chemical shifts in the vector model Two signals with different chemical shifts rotates with different speed in the vector model Interpreted as two different frequencies in the spectrum X Y NTDR, 2014

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Different chemical shifts in the vector model One of the signals right on the carrier frequency The other resonance will have a different speed X Y NTDR, 2014

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Different chemical shifts in the vector model One of the signals right on the carrier frequency The other resonance will have a different speed X Y NTDR, 2014

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Coupling in the vector model NTDR, 2014 A doublet with two signals The same effect as two different chemical shifts, but usually depicted with the carrier frequency in the middle of the doublet. J = Coupling constant in Hz (Hz = rounds per seconds)

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Spin-echoes in pulse sequences Chemical shifts are refocused NTDR, 2014

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Spin-echoes in pulse sequences Chemical shifts are refocused NTDR, 2014

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Spin-echoes in pulse sequences Chemical shifts are refocused NTDR, 2014

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Spin-echoes in pulse sequences Couplings evolve (if both of the coupled nuclei are inverted) NTDR, 2014

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Spin-echoes in pulse sequences Couplings evolve (if both of the coupled nuclei are inverted) NTDR, 2014

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Spin-echoes in pulse sequences Couplings evolve (if both of the coupled nuclei are inverted) NTDR, 2014

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Spin-echo example Explain the appearance of the normal 1 H spectrum of the hypothetical molecule. NTDR, 2014

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Spin-echo exercise I NTDR, 2014 Explain the appearance of the spin-echo spectrum… Use vector model What delay was used around the 180-degree pulse?

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Spin-echo exercise II Explain the appearance of the spin-echo spectrum with simultaneous 180-pulses at both proton and carbon… Use vector model What delay was used around the 180-degree pulse? NTDR, 2014

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Spin-echo exercise I NTDR, 2014

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Spin-echo exercise II NTDR, 2014

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LUNCH The lunch is served in the cafeteria in building 22 12 15 -13 15 NTDR, 2014

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Outline 12 15 – 13 15 Lunch 13 15 – 14 15 Signal processing Window functions Fourier transform Real and imaginary parts Phasing Topspin starter NTDR, 2014

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Acquisition time The acquisition time is usually ~100 ms – 10 sec depending of type of experiment. The best theoretical resolution in the spectrum is the inverse of the acquisition time (t a ). t a = 10 seconds Δν= 0.1 Hz t a. = 0.1 seconds Δν= 10 Hz NTDR, 2014

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Acquisition time The line width is determined by the acquisition time and the relaxation! Fast relaxation => the signal fades out fast => broad lines long acquisition time will in this case only increase the noise NTDR, 2014

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Spectral width, sampling rate & dwell time Dwell time = Time between sampling points Sampling rate = Number of data points per second Sampling rate = Total no. of data points / acquisition time Dwell time = (Sampling rate) -1 NTDR, 2014

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Spectral width, sampling rate & dwell time Dwell time = Time between sampling points Sampling rate = Number of data points per second Sampling rate = Total no. of data points / acquisition time Dwell time = (Sampling rate) -1 Faster sampling larger spectral width (sw) Spectral width=½ × Sampling rate (according to Nyquist) NTDR, 2014

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Experimental What is the acquisition time (t a ) for the 1D NMR experiment described in this article? SW = 7.2 kHz Sampling rate = 2 × 7.2 kHz = 14400 Hz TD = 32k = 32 × 1024 = 32768 data points Acquisition time; t a = 32768 / 14400 ≈ 2.3 seconds NTDR, 2014

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Sweep width, dwell time and sampling rate The sampling rate must be high enough to determine the frequency of the signal (at least twice per period). NTDR, 2014

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Sweep width, dwell time and sampling rate The sampling rate must be high enough to determine the frequency of the signal (at least twice per period). NTDR, 2014

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Sweep width, dwell time and sampling rate The sampling rate must be high enough to determine the frequency of the signal (at least twice per period). NTDR, 2014

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Relaxation delay NTDR, 2014 After a pulse: The magnetization returns to equilibrium M z increases, M xy decreases Exponentially = fast in the beginning, very slowly in the end Time constant; T = longitudinal relaxation Small molecules, 1 H: 0.5-5 sec, 13 C: 2-60 sec

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Relaxation delay Pulsed NMR! Add several transients! …but what if the recovery is slow and the repetition time too fast? NTDR, 2014

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Relaxation delay Pulsed NMR! Add several transients! …but what if the recovery is slow and the repetition time too fast? Use a small flip angle! NTDR, 2014

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Relaxation delay Pulsed NMR! Add several transients! …but what if the recovery is slow and the repetition time too fast? Use a small flip angle! Use the delay to acquire! NTDR, 2014

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Optimum flip angle Optimize the sensitivity with the Ernst angle! For carbons with long T 1 ’sFor high resolution 1 H spectra (aq ≈3×T 1 ) For accurate quantitative measurements! NTDR, 2014

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Processing of spectra Fourier transform (time domain -> frequency domain) NTDR, 2014

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Processing of spectra Fourier transform (time domain -> frequency domain) NTDR, 2014

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Processing of spectra Fourier transform (time domain -> frequency domain) NTDR, 2014

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Processing of spectra Fourier transform (time domain -> frequency domain) NTDR, 2014

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Processing of spectra Window function: Exponential multiplication Line broadening 0.3 Hz Increases apparent T 2 Apodization (‘removal of feet’), end of FID forced to zero. NTDR, 2014

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Processing of spectra Window function: Lorentz-Gauss Line broadening -1.0 Hz, GB = 0.25 Resolution enhancement, trade S/N for better resolved signals NTDR, 2014

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Processing of spectra Window function: Lorentz-Gauss Line broadening -0.3 Hz, GB = 0.5 Resolution enhancement, trade S/N for better resolved signals NTDR, 2014

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Processing of spectra Window function: Traficante Line broadening 0.2 Hz Keep line shape, increase S/N Real and imaginary multiplied with two different functions NTDR, 2014

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Processing of spectra Window function: Sine Sine-bell shape, for data with few points Strong apodization function NTDR, 2014

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Processing of spectra Window function: 90 degree shifted sine Cosine shape Used in the indirect dimension of 2D-data NTDR, 2014

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Processing of spectra Window function: Mixed cosine and sine bell shape Mixture of sine and cosine shape Used in the indirect dimension of 2D-data NTDR, 2014

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Real and imaginary parts Two phase shifted signals detected simultaneously to separate frequencies on either side of the carrier frequency. Quadrature detection NTDR, 2014

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Phasing Fourier transform => two components ‘Real’ and ‘imaginary’ Linear combinations => pure absorption + pure dispersion The base of the dispersion signal is wide (unwanted feature) NTDR, 2014

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Phasing Good phasing NTDR, 2014

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Phasing 0:th order phase correction NTDR, 2014

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Phasing 1:st order phase correction Freq. dep. NTDR, 2014

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Phasing 0:th order + 1:st order phase correction Freq. dep. NTDR, 2014

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Phasing, tips and tricks Reset the phase parameters (PHC0 and PHC1) to zero 1.) Adjust PHC0 on one signal in one end of the spectrum 2.) Adjust PHC1 on signals in the other end… Consider the relative phase (phase errors) of signals… NTDR, 2014

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Topspin in D1 User name: upnmr Password:nmr2013! Contact to license server (50 concurrent licenses) Folder hierarchy: /data/ /nmr/ / = C:/data/ntdr2014/nmr/ NTDR, 2014

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Topspin basics 1.Prepare data directory Make a directory named ‘ data ’ in C:\ Make a directory named ‘ NTDR2014 ’ in C:\data Make a directory named ‘ nmr ’ in the ‘ NTDR2014 ’-folder 2.Download dataset http://drug.ku.dk/research/npr/nmr/ntdr2014/ Download ‘ Exercise3.zip ’ to ‘ C:\data\NTDR2014\nmr ’ Unzip 3.Start Topspin 3.1 Right click in browser pane and select “Add New Data Dir..”. Add “ C:\ ” 4.Fourier transform [ft] and phase. NTDR, 2014

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Topspin basics They try to be more like an apple… NTDR, 2014

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1 Signals from coupled protons that are close together (in Hz) can show distorted patterns. When ν >> J, the spectra is said to be first-order. Non-first-order.

1 Signals from coupled protons that are close together (in Hz) can show distorted patterns. When ν >> J, the spectra is said to be first-order. Non-first-order.

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