NMR PRAKTIKUM 1. INTRODUCTION... 2 1.1. Description of a Spectrometer... 2 1.2. Principle of a NMR Experiment... 4 1.2.1. 1D NMR experiment... 4 1.2.2. 2D NMR experiment... 5 2. PRACTICAL PART... 8 2.1. Spectrometer setup... 8 2.1.1. Shimming the magnet and tuning the probe... 8 2.1.1.1 Tuning the Probe... 8 2.1.1.2 2.1.1.3 Shimming the magnet... 8 Check the Quality of the Shims... 9 2.1.2. Calibrate Parameters for 1 H... 9 2.1.2.1 90 degree pulse calibration... 10 2.2. Data acquisition and data processing... 12 2.2.1. 1D spectrum of Aspirin... 12 2.2.1.1 Data acquisition... 12 2.2.1.2 2.2.1.3 Data processing... 12 Data Analysis... 13 2.2.2. T 1 experiment... 14 2.2.2.1 2.2.2.2 Data acquisition... 14 Data analysis and questions... 15 2.2.3. 2D spectrum: COSY (COrrelation SpectroscopY)... 16 2.2.3.1 2.2.3.2 Data acquisition... 17 Data processing... 17 2.2.3.3 Data analysis... 19 1
1. INTRODUCTION The aim of this practical work is to introduce you to simple 1D and 2D NMR of organic molecules. You will learn how to set up the spectrometer and record several NMR spectra of Aspirin (1D and 2D). You will be guided through these experiments. Sample: Aspirin (acetyl salicylic acid) in DMSO-D 6. Figure 1: Aspirin The outline of the practical work is as follows: - Spectrometer set up (including 90 degree pulse calibration) - Acquisition of a 1D NMR spectrum - T 1 relaxation experiment - 2D COSY experiment You will be working on your own spectra to answer questions. Note that some questions (in bold) should be answered during the praktikum. 1.1. Description of a Spectrometer The magnetic field is generated by superconducting electromagnets. The superconducting state is obtained at low temperatures. Therefore, the inner dewar (A) is filled with liquid Helium (boiling point 4 K = -269 C). To slow down the evaporation of the helium, a second cooling bath (B) surrounds the helium vessel (Dewar). This second bath consists of liquid nitrogen (boiling point 77 K = -196 C). The radiofrequency probe (C) is located in the central bore of the magnet and is kept at room temperature. The sample (D) is placed inside this probe for the measurements. The temperature of the sample is precisely adjusted by a precooled stream of nitrogen gas that can be heated to the desired temperature. The broadband direct observe dual channel probe (BBO) The probe consists of a radio frequency (RF) circuit containing a coil for applying RF pulses at a certain nucleus frequency and subsequently detecting the magnetization. In the BBO probe, there are actually 2 coils: one is used for 1 H and 2 H, the other one is used for heteronuclei. The characteristics of a probe RF circuit as a function of an applied RF frequency ω is described by 2
the complex impedance Z = R + i[ωl 1 / (ωc)] where, R = resistance, L = inductance, C = capacity. This circuit has minimal impedance (it resonates) at a frequency ω 0 = 1/(LC) -1/2. In order to efficiently deliver RF energy to the sample and to optimize signal sensitivity, the probe circuit must be tuned so that the resonance frequency ω 0 equals the RF frequency. This is done by adjusting a capacitor, i.e. by changing C. In addition, the impedance of the coil has to be matched to the impedance of the amplifier output (see section 2.1.1.2). The two adjustments are usually called tuning and matching. This is necessary to minimize heat dissipation. A D B C Figure 2: Cutaway diagram of NMR spectrometer (from http://lucas.lakeheadu.ca) 3
Figure 3: Scheme of a NMR spectrometer. The static magnetic field B 0 is created by a superconducting coil (in grey). The RF coil is used for excitation and detection of the magnetic oscillations of the nuclei. Excitation: a frequency synthesizer controlled by the computer produces a pulse of duration τ p of a sinusoidal RF signal with frequency ν RF (= ω RF /2π). The frequency ω RF is set close to the Larmor frequencies of the nuclei of interest. This signal is fed to the RF coil to produce a sinusoidal magnetic field B 1 c o s 2 ( R F p) at the position of the sample. Detection: the different nuclei oscillate with their various Larmor frequencies ν i. This induces a current in the coil that is modulated by these frequencies. This current is detected by the same RF circuit and is demodulated by the frequency ν RF. The resulting frequencies are the chemical shifts δ i = ν i - ν RF The signal is then amplified, converted to digital format (analog to digital conversion, ADC), and finally processed by the computer. The symbol denotes the switching between the excitation and detection functions of the circuit. 1.2. Principle of a NMR Experiment 1.2.1. 1D NMR experiment The principle of a 1D NMR experiment can be simply described using the vector model. At thermal equilibrium, there is a slight excess of lower energy spins (α) leading to a bulk magnetization of the sample (M). The direction of M defines the z axis (A). When a pulse is applied, a 90 degree pulse along the x axis for instance, then M is rotated toward the x-y plane (B). When the pulse strength is sufficient to align M onto the y axis, then there is an equal amount of α and β states. The magnetization precesses in the x-y plane and returns to the z axis (C). An oscillating signal is recorded over time by the receiver on the y-axis. The oscillations decay with time in an exponential fashion until the magnet returns to equilibrium (see T 1 relaxation time for 1 H nucleus), yielding a signal called free induction decay (FID) (D). The FID can be Fourier transformed to obtain the frequency spectrum (E). 4
Figure 4: Simple 1D NMR experiment. 1.2.2. 2D NMR experiment In a 2D NMR experiment, the signal is recorded as a function of two time variables (as opposed to one in 1D NMR) and subsequently, transformed twice by FT to obtain a spectrum which is a function of two frequencies. A typical 2D NMR experiment always follows the general scheme, called pulse sequence, given below: Figure 5: Principle of a 2D experiment During the preparation time, several pulses are applied to excite the sample generating magnetization of nuclei. In the following time sequence, called evolution, the magnetization evolves during the time t 1. In the next step, called mixing, one or several pulses are used, and the magnetization is eventually transferred to another nuclear spin (this nucleus can either be of the same type as X, in this case we talk about homonuclear 2D, or of a different type, hence the name heteronuclear 2D). After the mixing time, the FID signal is recorded as a function of t 2. 5
The pulse sequence is repeated several times with increasing values of t 1 (indirect detection), and each time the FID is recorded as a function of t 2 (i.e. for each value of t 1, see (a) in figure 6). The FIDs are Fourier transformed to yield a modulated series of F 2 spectra (direct dimension), as shown in (b). For a given value of F 2, C for example, it is possible to extract a data set, which looks exactly like a FID, with the time axis t 1 (see (c)). The same can be done with any value of F 2. Finally, the 2D spectrum (F 1, F 2 ) results from the Fourier transform of these interferograms (d). Figure 6: The operation of a 2D NMR experiment (From NMR: The Toolkit by P. J. Hore et al). During this praktikum you will record a 2D- 1 H COSY spectrum of Aspirin. COSY is a homonuclear 2D technique. The pulse sequence for a COSY experiment is given below: 6
90 x 90 x t 1 t 2 Figure 7: Pulse sequence COSY. Questions: What are the advantages of a COSY experiment over a simple 1D? What do you detect with it? Answers Preparation Evolution Transfert Detection 7
2. PRACTICAL PART 2.1. Spectrometer setup We will use a spectrometer manufactured by the Bruker Company (Bruker AV-250). In the following, all commands are noted in courier italic and are specific for such a Bruker console. The XXX expressions stand for values that need to be adjusted at the console. In addition to the computer keyboard, there is an extra panel to control several functions of the spectrometer like the lock, shims and the sample lift. The assistants will show you how to operate these functions on this panel. 2.1.1. Shimming the magnet and tuning the probe 2.1.1.1 Tuning the Probe To minimize the amount of reflected power from the probe, do the following: Set the display to show the acquired data by left-clicking on acqu on the top menu bar. Start the tuning routine by typing: w Your assistant will adjust the match so that the minimum of the displayed V-shaped curve touches the displayed baseline. And he will adjust the tune so that the dip of the absorption curve displayed is centered in the middle ( = 1 H frequency). Stop the tuning routine by typing: stop 2.1.1.2 Shimming the magnet The magnetic field of the main superconducting magnet is not sufficiently homogeneous for high-resolution NMR spectroscopy. (What happens to the NMR line, if the magnetic field varies across the sample volume?) Therefore, an additional set of small electromagnets are adjusted to produce magnetic fields in all spatial directions and thus to compensate for spatial inhomogeneities of the main static field B 0. This adjustment process is called shimming. An improvement in the adjustment is visualized by an increase of the lock signal level (the amplitude of the DMSO-D 6 signal). As the magnetic field drifts very slightly over time, it is also necessary to stabilize the spectrometer with respect to such time variations. For this purpose, the DMSO-D 6 signal is used as an internal reference and the spectrometer is locked onto this signal. When the sample is introduced into the magnet, the phase and position of the lock signal of the sample have to be adjusted prior to locking. You will be guided through this process. Once the sample is inserted, you should: Read in a previous shim file (a good starting point for the shimming process): rsh Select the file aspirin_qnp.shim in the user folder Lock the sample (onto the solvent DMSO) lock dmso Adjust the lock phase on the shim panel. 8
Adjust the field on the shim panel. Lock the sample on the shim panel. Adjust the main shims using the shim panel according to the following protocol: 2.1.1.3 Check the Quality of the Shims Z 1 Z 2 Z 3 Z 1 Z 2 Z 4 The shape of the signal from the methyl protons of the sample is a good indicator for the quality of the shimming, in terms of symmetry. Check the methyl signal by recording a simple 1D spectrum centered on the methyl frequency (around 2.2 ppm). The aromatic signal can be used to check the coupling. Pulse program used: If p1=1 μs, the z-magnetization only rotates about 15-25, but this is already sufficient to detect the huge methyl signal. Go to the directory Aspirin : Make sure that you are in experiment one: re 1 Go to the acquisition window: a Set 1 H pulse to 1μs: p1 1 Acquire 1D spectrum on the methyl signal: zg Process (Fourier Transform) efp Correct the baseline offset of the time domain: abs Process (automatic phase correction) apk Start manual phasing mode by left-clicking on the Process Phase button. Adjust the zero-order phase manually by moving the mouse up and down in the spectrum window while keeping the button pressed with the left mouse button. Once you are satisfied, left-click on store in the top menu-bar. Check that the lineshape of the methyl signal has a nice symmetric Lorentzian shape, else shim again (as in section 2.1.1.2), 2.1.2. Calibrate Parameters for 1 H 9
The duration of the 1 H 90 pulse has to be optimized, so that a pulse with a defined power rotates the proton magnetization by exactly ninety degrees. Pulse program used: 2.1.2.1 90 degree pulse calibration Many NMR experiments, including the COSY experiment you will perform, use 90 degree pulse in their pulse sequences. If you want to obtain optimal data, you will need to calibrate the 90 degree pulse to maximize the performance. Calibrating the 90 degree pulse means to find the exact pulse length that will lead to magnetization in the x-y plane. You will look at the 180 degree pulse value, which should correspond to a null signal, and then simply divide it by two to obtain the 90 degree pulse value. z 90º pulse M x y z 180º pulse x M y Figure 8: Finding the right 90 pulse consists in finding the right pulse duration to get a maximal signal or to adjust 180 pulse length to get zero signal. 10
Intensity 90 360 Pulse angle 180 Figure 9: 1 H signal recorded with different pulse widths. (from http://www.dur.ac.uk/resources/ssnmr). Go to the acquisition window: a Set 1 H offset o1p to its value: o1p 6 Set 1 H pulse length p1 to 12 μs: p1 12 Acquire 1D spectrum, but keeping former corretions: zgb Change p1 to get a null signal in the real and imaginary part of the signal. Question: Note down the 90 pulse length p1. To which flip angle would a pulse length of 1 μs correspond? Answer 11
2.2. Data acquisition and data processing 2.2.1. 1D spectrum of Aspirin 2.2.1.1 Data acquisition Create a new folder for your recording spectra: edc And name it according to your grouf XY Aspirin XY Set 1 H offset o1p to its value: o1p 6 Set 30 ( 1 H) pulse length p1 to its value (see section 2.1.2.1): p1 XXX Set the spectral width to 16 ppm: sw 16 Set the number of point to acquire to 16k: td 16k Set number of scans: ns 16 Set number of dummy scans: ds 2 Acquire 1D spectrum: zg Visualize the time domain signal (the FID ) in the acquisition window. 2.2.1.2 Data processing The data processing usually consists of the following steps: a baseline correction, a multiplication by a window function (apodization), the Fourier transformation, and a phase correction: Baseline correction: in our case, the FID usually needs a baseline. Apodization - Window function: Modification of FID Apodization is the process of multiplying the FID by a mathematical function (window function) prior to Fourier Transform. There are several types of functions, and they have different effects on the final spectrum. This means that through the use of window functions, the user can change the results observed on the frequency spectrum. Fourier transform Phase correction: phase differences between the emitter and receiver are often responsible for funny spectral lineshapes (so-called non-pure-absorptive). This can be corrected by a phase correction of order zero (constant) and or one (linear within the spectral width). In this part you will observe the influence of the window functions. Data processing Process (Fourier Transform): ft Process (automatic phase correction): apk Process (baseline correction): abs Manually phase the spectrum as described for the methyl signal and store ( disc-symbol) when you are satisfied. Then return Process Spec.: window function check manual Ok!. Use exponential function and Gaussian function and change LB (line broadening factor). Once you are satisfied, left-click on the disc-symbol in the top menu-bar to store. 12
Print 3 different spectra: one without any correction, one with the exponential function (em), and one with the Gaussian function (gm). To create a printout: open plot software: click on plot To focus on the aromatic region: Edit XMin/XMax: 8.2, 7.0 adjust YMin/YMax: e.g. -5e5, 1e7 print: file print note LB, GB and window function on the printout For the next printout: go back to xwinnmr Process (apodization): winfunc Change LB or GB or both then store Reprocess: efp (or gfp) Then in the printing window update your data Questions: Describe the effect of the window function on the spectrum. What do you observe when changing the Line Broadening factor (LB)? Answers: 2.2.1.3 Data Analysis Question: Assign peaks on the aspirin spectrum: aromatics, methyl, dmso? Answers: 13
2.2.2. T 1 experiment When a pulse is applied the magnetization vector M precesses about the field B 1 and its axis (i.e. for 90 x pulse, M precesses about the x axis), until the pulse is turned off. Right after the pulse, the spins are equally populating the energy levels. Once the pulse is turned off, the redistribution of the spin states to equilibrium (Boltzmann distribution, i.e. a slight excess of low energy spin) starts. The net magnetization M, will return to +z axis (along B 0 filed); this is called relaxation. It is characterized by a time constant T 1 called the longitudinal or spin lattice relaxation time. To return to equilibrium, energy is exchanged between the spin and its surroundings, hence the name spin-lattice relaxation. The magnitude of T 1 depends on the nuclei, the state in which the molecule is, temperature, viscosity of solvent It often follows an exponential curve with equation: M z (t) = M z (0)*(1-2e -t/t1 ) One of the experiments used to determine T 1 is called inversion recovery. The pulse sequence for this experiment is given below: 180 x 90 x τ 2.2.2.1 Data acquisition Figure 10: Inversion recovery pulse sequence. The experiment consists in applying this sequence with different time t between the pulses. If τ is very short then the magnetization (aligned with the z axis after the 180 x pulse) is aligned with the x axis, therefore giving a spectrum with negative intensities. With larger values of τ, we observe an inversion from negative spectra to positive spectra. Go to Aspirin: Change Experiment: re 2 Make new Dataset edc Set 1 H offset: o1p 7 Set 90 ( 1 H) pulse: p1 XXX Set the spectral width to 16 ppm: sw 16 Set the number of points to acquire: td 16k Set the number of points in the indirect dimension: 1 td 8 Set number of scans: ns 8 Acquire 2D spectrum: zgb 14
Visualize the time domain signal (the FID ) in the acquisition window See τ increments for the T1 time analysis: click on AcquPars Scroll down until you find vd-list E to open the list print via nmr250printer. 2.2.2.2 Data analysis and questions The data can be collected and plotted, intensity vs. τ. The curve obtained is fitted by an exponential to determine T 1. Here you will collect data to determine T 1 for the methyl protons and for one of the aromatic protons (Aryl-H). Process data: Read first series: rser1 Fourier transform + em efp Adjust Phase manually Save the phasecorrections! disk nd And return to previous menu return Go back to 2D-experiment Cyclobutan / Square Process 2D-experiment xf2 Automated baseline correction in 2. Dimension abs2 Click on Analyse Choose T1/T2 Click on FID use FID Click on Peak/Ranges define the region manually Export peaks using disk A Click on Relaxation switches 1D data to relaxation analysis Pick data point arrow down Fit the relaxation for all peaks click > fit for current peak Click on report (i) Print the report Questions 1. Use vector model to show the effect of pulse on the magnetization vector in this experiment (180 pulse τ -90 pulse). 2. Why is τ not chosen linear? 3. What happens if the 180 pulse if not calibrated properly? 4. Note down the intensities and time. Plot Intensity vs. τ and fit with exponential curve using Excel or Origin. Determine T 1. Answers 15
2.2.3. 2D spectrum: COSY (COrrelation SpectroscopY) 2D COSY is one of the simplest and most common 2D experiments performed in NMR. All 2D NMR experiments are used to correlate two atoms or nuclei of the same molecule, based on interactions which can be through-bond (J-Coupling) or through space (see NOESY technique). As we have seen before COSY is a homonuclear 1 H 2D experiment. Therefore, it is a good technique to determine through-bond interactions between 1 H nuclei. A well known technique to determine J-coupling in molecules is to look at the splitting pattern of a given proton. With good resolution and some patience, one could determine which proton is coupled with the others. A short example is given below. Figure 11: Example of J-coupling (adapted from http://www.cem.msu.edu/~reusch/virtualtext/spectrpy/nmr/nmr1.htm#nmr1). However, it is quite clear that this technique has its limits: the splitting pattern can quickly become very complex! 16
The COSY experiment is a more convenient method to establish J-coupling. 2.2.3.1 Data acquisition Go to the directory "Aspirin": Load experiment re 3 Set 1 H offset: o1p 5 Set 90 ( 1 H) pulse: p1 XXX Set the spectral width in F2 dimension to 10 ppm: sw 11 Set the spectral width in F1 dimension to 10 ppm: 1 sw 11 Set the number of points to acquire: td 2k Set the number of points in the indirect dimension: 1 td 256 Set number of scans: ns 4 Dummy scans ds 4 Acquire 2D spectrum: zg Visualize the time domain signal (the FID ) in the acquisition window 2.2.3.2 Data processing First, you will compare the 1 st increment with the 1D spectrum of aspirin: read first increment: rser 1 apply square sine window: qsin baseline correction: bc Fourier-transform: fp to store it as process #2: wrp 2 Multi display mode:.md Load second file from the Aspirin folder if necessary, adjust intensity of 2nd and/or 1st exp. Go back to single experiment with return Questions: Describe your observations in this comparison! What is similar? What is different? Answers 17
Then you will compare 1 st increment to 2 th increment: Back to 2D mode read second increment: rser 2 apply square sine window: qfp (includes baseline correction and Fourier-transformation) Store it as process #3: wrp 3 Multi display mode:.md Load second file from the temp folder if necessary, adjust intensity of 2nd and/or 1st exp. Questions: Describe your observations in this comparison! What is similar? What is different? Answers Then you will compare 1 st increment to 9 th increment: Back to 2D mode read second increment: rser 9 apply square sine window: qsin baseline correction: bc Fourier-transform: ft to store it as process #4: wrp 4 Multi display mode:.md Load second file from the temp folder if necessary, adjust intensity of 2nd and/or 1st exp. Questions: Describe your observations in this comparison! What is similar? What is different? Answers 18
Here, you will look at the 2D spectrum processed in the direct dimension only: Back to 2D to process 2D experiment only in F2 (direct dim.): xf2 adjust threshold, so that noise vanishes choose 1D trace:.md click on: scan rows/colums if necessary, adjust intensity of the projection! using: +/ step you can change the column! Questions: What are the two dimensions in the displayed spectrum? What do you need to do, in order to obtain a proper cosy spectrum? Processing the entire 2D: to process 2D experiment in both dimensions: baseline correction in F1: baseline correction in F2: Zoom on aromatic region External Projektion Normalize the spectra on the solvent open plot software: mark spectrum with left mouse Edit adjust XMin/XMax and YMin/YMax Close 1D/2D-Edit adjust intensity (*2 etc.) Close for printing: xfb abs1 abs2 plot CTRL P 2.2.3.3 Data analysis How to read a COSY spectrum? 19
Figure 12: Schematic COSY spectrum for two coupled spins A and B (from http://www.oci.uzh.ch/) The two frequency domains are called the direct (F2) and the indirect (F1) frequency domains (see 1.2.2). In a homonuclear 2D spectrum (such as 1H-COSY), the diagonal contains the onedimensional spectrum. Off-diagonal peaks at the frequency F 2 =Ω A, F 1 =Ω B are called cross-peaks, and they indicate that the spins with frequencies Ω A and Ω B are coupled. In the next figure, a COSY spectrum of butanone is shown. 20
Figure 13: 2D 1H COSY of butanone (from http://www.uncp.edu) From this very simple spectrum, we can see that H a and H b are connected by a cross peak (labeled H a -H b on spectrum) because they are coupled, and H c does not show any cross peaks because it is not coupled to any other protons. Questions: Connect the signals and explain the couplings for you aspirin COSY spectrum. 21