Laboratory Experiments for Nuclear Magnetic Resonance Spectroscopy May 6, 2004

Transcription

1 CONTENTS 1 Contents Laboratory Experiments for Nuclear Magnetic Resonance Spectroscopy May 6, Introduction 3 2 Safety High Magnetic Fields The Attractive Force between Magnetic Objects and the Magnet Disruption of Electronic Circuitry and Magnetic Media Cryogens Hazards Asphyxiation Sample Toxicity Experiments Samples The Bruker Spectrometer Experiment One: Measuring a 1 H Spectrum Spectrometer Hardware Setup for 1 HNMR Acquisition Data Processing Plotting Data Experiment Two: Measuring a π/2 time Acquisition Experiment Three: Measuring a 1 H Spectrum in the presence of a large linear field gradient Acquisition Data Processing Plotting Data Experiment Four: Measuring the spin-lattice relaxation time, T Acquisition Data Processing Plotting Data Experiment Five: Measuring a 13 C Spectrum without 1 H decoupling Spectrometer Hardware Setup for 13 CNMR Acquisition Data Processing Plotting Data Experiment Six: Measuring a 13 C Spectrum with 1 H decoupling Acquisition Data Processing Plotting Data Experiment Seven: Two-Dimensional 1 H- 1 H COSY (correlation spectroscopy) Experiment Spectrometer Hardware Setup for 1 HNMR Acquisition Processing

2 CONTENTS Experiment Eight: Solid-State NMR - Cross Polarization Magic-Angle Spinning Packing the Rotor Spinning the Rotor Experiment A - Setting the Magic Angle Experiment B - 13 C Cross polarization

3 3 1 Introduction The laboratory component of this course is designed to provide students with hands on experience with an NMR spectrometer, and a working understanding behind a few basic and routine experiments. This IS NOT a self-guided laboratory, and this handout alone does not provide enough information for you to perform NMR experiments successfully on your own. For your first time, you will need the assistance of the TA, Nicole Trease to perform the experiments outlined here. You must bring a laboratory notebook and take detailed notes of every step as you work through these experiments with the TA. Once you have mastered the skills and concepts outlined here, you should be able to perform routine NMR measurements on any of the Bruker NMR spectrometers in the CCIC or chemistry department NMR facilities. 2 Safety Before any student enters the NMR laboratory, it is required that he or she understand all the safety issues described below associated with performing NMR experiments. The most common safety risks that one will encounter are (1) the high magnetic fields in the immediate vicinity around the NMR magnet, (2) the large volume of cryogen gases that could be released during a magnet quench, and (3) sample toxicity High Magnetic Fields The Attractive Force between Magnetic Objects and the Magnet The force of attraction between a magnetic object (such as a screwdriver, flashlight, gas cylinder, scissors, pocket knife, pagers, bobby pins, etc...,) and the superconducting magnet are strong enough to have caused

4 2.2 Cryogens Hazards 4 minor to fatal injuries 1 in the past. Therefore, extreme caution must be taken to ensure that no ferromagnetic objects are brought into NMR laboratory. It is best that you empty your pockets of all such objects and leave them at home or in your office. The strength of the magnetic field falls off exponentially with distance from the magnet. For this reason the risks associated with the magnetic field are often underestimated by workers. A metal object held firmly in your hand will be ripped out of your hand when moved only a few centimeters closer to the magnet no matter how tightly you hold on to it! Individuals with a cardiac pacemaker should not work around high field magnets as the pacemaker can move within the chest wall, change mode of operation, or stop working. Patients with metal implants should also exercise extreme caution around high field magnets. In addition to the possible bodily harm that can be caused by loose magnetic objects, considerable damage can occur to the NMR magnet. If a small keychain flashlight enters the bore of the magnet the only means of removing the flashlight is to de-energize the magnet, remove the flashlight, and then re-energize the magnet. The cost of such an operation ranges from $10,000 to $20,000 (US. dollars in 2002), assuming the magnet was not physically damaged during the incident. If the magnet cannot be repaired the cost of replacement can range from $75,000 to $2,000,000, depending on the field strength of the magnet Disruption of Electronic Circuitry and Magnetic Media A number of electronic devices will not function properly, or at all near the NMR magnet. Therefore, the NMR represents a serious health hazard for persons wearing cardiac pacemakers. Less serious and more of an inconvenience, credit cards and other magnetic storage devices will be permanently damaged when brought inside the 10 Gauss stray field lines of the magnet. 2.2 Cryogens Hazards Superconducting magnet quenches are often indicated by a loud noise and can result in the rapid release of cryogen vapor into the laboratory. The signs of a superconducting magnet quench are white clouds of vapor that appear from the magnet, and venting that may cause a loud hissing noise. The area can remain hazardous for a while after the quench occurs depending on the room size and ventilation. Indications that a magnet quench may have just occurred are (1) large amounts of frost and ice on the outside of the magnet or inside the bore, or (2) tilting of the image on CRT or computer monitors near the magnet. There are two hazards that are possible during or immediately after a quench: (1) Asphyxiation, and (2) Frostbite Asphyxiation Cryogens will rapidly boil and convert from a liquid to a gas at room temperature. As the gas warms to the temperature of the surrounding air, it expands. In confined or poorly ventilated areas, the expanding gas will displace oxygen and can cause rapid asphyxiation or death. These gases are colorless, odorless, and tasteless. OSHA specifies that workers cannot be inside a work space that contains less than 19.5% oxygen without supplied air respiratory protection. Below this level, workers start to experience early warning signs of oxygen deficiency. 1. Between 15-19% oxygen workers may feel: (a) A loss of coordination and energy. 1 Recently, a medical worker brought a small oxygen cylinder too close to a patient in an MRI magnet. The cylinder flew out of the worker s hands and into the head of a patient. Sadly, the patient did not survive the impact.

5 2.3 Sample Toxicity 5 (b) An increase in pulse rate and breathing. (c) A sense of euphoria and clumsiness. 2. At oxygen levels between 12-14% the worker s: (a) Breathing becomes much deeper and faster. (b) Judgment becomes impaired. (c) Physical coordination is deteriorated. (d) Lips turn blue. 3. At levels below 12% the worker will become unconscious and eventually die. If you are working in an NMR laboratory and you believe a magnet quench is occurring you should (1) open the door to the magnet room, and prop it open, (2) remove all persons from the room, and (3) exit the building. If you are unable to open the door then break and exit through a window. 2.3 Sample Toxicity There may be toxicity issues associated with handling samples from your research project, and therefore you will need to implement the proper procedures, as outlined in the Materials and Safety Data Sheet (MSDS) for the compound, and the Standard Operating Procedures of your research laboratory. Before running any sample in the NMR facility, we ask that you first provide an MSDS sheet or equivalent to the facility coordinator (Nicole Trease). Because the NMR is a multi-user instrument all traces of your sample must be removed from the NMR lab and sample holders when your experiment is complete. If there is a spill of your sample in the lab, please contact the Chemistry Safety Office, Dr. Prasad, and Prof. Grandinetti, immediately.

6 6 3 Experiments 3.1 Samples The three samples to be used in this experiment are (1) benzene, C 6 H 6 (dissolved in acetone-d6), (2) p- dioxane, C 4 H 8 O 2 (dissolved in C 6 D 6 ), and (3) ethylbenzene, C 6 H 5 CH 2 CH 3 (dissolved in CDCl 3 ). These two samples have already been prepared and placed into 10 mm NMR tubes for your experiments. O O CH 2 CH 3 (1) (2) (3) 3.2 The Bruker Spectrometer First, you must familiarize yourself with the components of the Bruker Spectrometer. Identifying the magnet should be straightforward. If you haven t read the section of this document dealing with magnet safety (section 2) please do so now before proceeding any further in this section. To the left of the magnet you will find the Bruker Console (shown below) which contains the radio frequency transmitters and receiver, as well as the associated digital interface to the SGI computer. You will have no reason to open or interact directly with this piece of hardware. To the left of the Bruker console you will find the Silicon Graphic computer and monitor interfaced to the spectrometer. Through the Bruker XWINNMR software package you will be able to control nearly all aspects of the spectrometer. If not already logged in, log into the SGI computer. See Nicole Trease for the appropriate username and password. Once you are logged into the computer, open a terminal window and execute the XWINNMR program (type XWINNMR). Once XWINNMR is running you should see a window that looks more or less like the one below.

7 3.2 The Bruker Spectrometer 7

8 3.3 Experiment One: Measuring a 1 H Spectrum Experiment One: Measuring a 1 H Spectrum In this experiment we will obtain the 1 H NMR spectrum of samples (1). To obtain this spectrum we will apply a pulse to the sample, immediately followed by acquisition of the free induction decay (FID) from the magnetization precessing in the x-y plane. This experiment is sometimes called the Bloch Decay Experiment. A Fourier transform of the FID will yield the NMR spectrum Spectrometer Hardware Setup for 1 H NMR To perform these experiments you may have to switch the spectrometer to transmitting and detecting at the 1 H NMR frequency. The probe you will be using is a 10 mm solution probe.

9 3.3 Experiment One: Measuring a 1 H Spectrum Acquisition Below are some of the relevant XWINNMR commands and parameters you will need to perform this experiment. The zg pulse sequence will be used in this experiment. Be sure to set the reference of TMS (tetramethylsilane) to 0 ppm. wobb - to tune the probe ii - to initialize the spectrometer edc - for new file name eda - to view all acquisition parameters ased - to view parameters based on pulse program td - number of points in time domain data ns - number of acquisitions for signal averaging d1 - experiment recycle time in seconds p1 - pulse length in microseconds pl1 - power level for pulse in decibels (db). sfo1 - rf resonance frequency for nucleus to be observed (sfo1 = sf + o1) o1 - offset frequency from sf to put nucleus on resonance. a - to view acquisition in progress. zg -(zero and go) to start experiment edcpul - To view the current pulse program edpul - To view pulse program from list of all possible programs.

10 3.3 Experiment One: Measuring a 1 H Spectrum Data Processing bc - baseline correction (removes dc offset) lb - line broadening parameter (for matched filtering) em - apply exponential line broadening to fid ft - apply Fourier transform to fid phase button - enter interactive phasing mode ph0 button - click, hold, and move to apply zeroth order phase correction interactively ph1 button - click, hold, and move to apply first order phase correction interactively save button and return button

11 3.3 Experiment One: Measuring a 1 H Spectrum Plotting Data edo - select plot layout setti - set title for plotting (also available inside xwinplot command). xwp lp - plot parameters (also available inside xwinplot command). xwinplot - get new plotting window

12 3.4 Experiment Two: Measuring a π/2 time Experiment Two: Measuring a π/2 time In this experiment you will use sample (1) and the paropt command to vary the pulse length and find the length that corresponds to a π/2 pulse Acquisition acquire spectrum with approximate π/2 pulse, process, and save. define f1p and f2p (in ppm) to choose the spectral window. f1p f2p ω run paropt and choose p1 variations give smallest pulse length give increment give number of experiments. Try to go up to a 720 pulse rotation. after paropt is complete plot the data. As paropt create a new file with extension 999, you will need to switch back to data block 1 with re this will show last experiment from paropt list ie to increment the experiment number, and create a new file for next data block.

13 3.5 Experiment Three: Measuring a 1 H Spectrum in the presence of a large linear field gradient Experiment Three: Measuring a 1 H Spectrum in the presence of a large linear field gradient In this experiment you will use sample (1) to measure the 1 H spectrum in the presence of a large linear field gradient. To create the field gradient you need to first record the X1 shim current, and then dial in the most extreme X1 shim current possible. Once you have acquired, processed, and plotted the data, set the X1 shim current back to it s original setting Acquisition

14 3.5 Experiment Three: Measuring a 1 H Spectrum in the presence of a large linear field gradient Data Processing

15 3.5 Experiment Three: Measuring a 1 H Spectrum in the presence of a large linear field gradient Plotting Data

16 3.6 Experiment Four: Measuring the spin-lattice relaxation time, T Experiment Four: Measuring the spin-lattice relaxation time, T Acquisition The t1ir pulse sequence is used to perform an inversion recovery T 1 measurement. In addition to the acquisition parameters given in (section 3.3.2) you will also need to use the following parameters: parmode - specify the number of dimensions (2d in this case) l4 - number of delay times to be measured. vdlist - name of file containing recovery delay times (T1-benz).

17 3.6 Experiment Four: Measuring the spin-lattice relaxation time, T Data Processing

18 3.6 Experiment Four: Measuring the spin-lattice relaxation time, T Plotting Data

19 3.7 Experiment Five: Measuring a 13 C Spectrum without 1 H decoupling Experiment Five: Measuring a 13 C Spectrum without 1 H decoupling In this experiment we will obtain the 13 C NMR spectrum without 1 H decoupling of samples (2). Be sure to set TMS reference to 0 ppm Spectrometer Hardware Setup for 13 C NMR To perform these experiments you will have to switch the spectrometer from transmitting and detecting at the 1 H NMR frequency to the 13 C NMR frequency.

20 3.7 Experiment Five: Measuring a 13 C Spectrum without 1 H decoupling Acquisition Create a new file using edc and enter filename. run the eda command and choose 13C under nuclei edit to change the channel, etc, oruseedasp Continue using the commands in section 3.3.2

21 3.7 Experiment Five: Measuring a 13 C Spectrum without 1 H decoupling Data Processing

22 3.7 Experiment Five: Measuring a 13 C Spectrum without 1 H decoupling Plotting Data

23 3.8 Experiment Six: Measuring a 13 C Spectrum with 1 H decoupling Experiment Six: Measuring a 13 C Spectrum with 1 H decoupling In this experiment we will obtain the 13 C NMR spectrum with 1 H decoupling of samples (2) and (3). Don t forget to retune the probe with the wobb command when you change from sample (2) to (3) Acquisition The zgdc pulse sequence is used to perform a one pulse and acquire experiment on 13 C with 1 H decoupling. In addition to the acquisition parameters given in (section 3.3.2) you will also need to use the following parameters: pcpd2 - π/2 pulse length on the 1 H decoupling channel pl2 - rf power level for the 1 H decoupling channel pl12 - rf power level for the 1 H decoupling channel sfo2 - rf resonance frequency for 1 H

24 3.8 Experiment Six: Measuring a 13 C Spectrum with 1 H decoupling Data Processing

25 3.8 Experiment Six: Measuring a 13 C Spectrum with 1 H decoupling Plotting Data

26 3.9 Experiment Seven: Two-Dimensional 1 H- 1 H COSY (correlation spectroscopy) Experiment Experiment Seven: Two-Dimensional 1 H- 1 H COSY (correlation spectroscopy) Experiment The COSY sequence is a frequently used to study connectivity between nuclei via the J coupling. A 2D COSY spectrum generated by two 90 degree pulses shows cross peaks if J coupled spins are present. The sample for this experiment will be ethylbenzene in acetone-d Spectrometer Hardware Setup for 1 H NMR Connect the 1 H cable through the prepamp. Tune the probe for 1 H Acquisition 1. Acquire a 1D spectrum of the aliphatic region using the following parameters: (a) Type parmode: choose 1D (b) pulprog= zg (c) TD= 1k (d) NS=8 (e) DW= 500 µs (f) D1 =1s (g) Keep O1 at the middle of -CH2 and -CH3 signals i.e., O1= 650 Hz. 2. Acquire a 2D COSY spectrum of the aliphatic region (a) Type ie: increment experiment # (b) Type parmode: choose 2D (c) pulprog= cosy90 (d) TD= 1k (e) NS=16 (f) DW= 500 µs (g) D1 =1s (h) IN0=1msec (i) TdF1=256 (invoke eda) Processing 1. Type edp: edit process parameters 2. F2=1k 3. F1= type xfb to do double Fourier tranformation 5. plotting: (a) type edo: choose COSY in the layout. (b) use xwinplot to plot and print.

27 3.9 Experiment Seven: Two-Dimensional 1 H- 1 H COSY (correlation spectroscopy) Experiment 27

28 3.10 Experiment Eight: Solid-State NMR - Cross Polarization Magic-Angle Spinning Experiment Eight: Solid-State NMR - Cross Polarization Magic-Angle Spinning Sample: Glycine (for 13 C measurement) and KBr (for 81 Br measurement). Probe: A solids probe that uses 4mm rotors. Duration: Approximately 2 hrs Packing the Rotor 1. Grind the solid to a fine powder. 2. Pack the rotor evenly and tightly. Leave enough space at the top for the cap. 3. Seal the rotor. There should not be any gap. 4. Mark the bottom rim half moon using a sharpie Spinning the Rotor 1. Insert the rotor into the probe with the cap side up. 2. Press insert on the spin rate controller located at the console. 3. Press spin rate and use the arrow to set the rate to 8 khz. 4. Press go. If the rotor does not spin: check the connections. bearing, drive and optical detector cable. 5. To use manual control: toggle auto/manual, and slowly adjust the Bearing and then Drive pressures until you reach the target speeds listed below. Bearing Drive Spin Rate 500 mb 100 mb khz 1000 mb 400 mb 5-6 khz 1500 mb 750 mb 8 khz 6. If the rotor still does not spin or spinning is erratic, check sample packing (stop spinning and repack the rotor).

29 3.10 Experiment Eight: Solid-State NMR - Cross Polarization Magic-Angle Spinning Experiment A - Setting the Magic Angle Magic angle is set by observing the FID of 81 Br resonance in KBr (one can also observe the 79 Br resonance). 1 H (π/2) x y spin lock Decoupling Relaxation Delay 13 C Mixing Acquisition Relaxation Delay contact time Acquisition 1. Insert the glycine sample and spin it at 8 khz. 2. Create a new file for acquiring the 81 Br and 13 C spectra. 3. type edasp and change nucleus to 81 Br 4. tune the probe to 81 Br frequency. 5. Setup experiment parameters. pulprog=zg TD =1k DW=10us Pl1=21 db p1=6us D1=1s O1= Use gs command 7. While watching the FID on screen turn the magic angle stick located on the probe and maximize the spinning side bands on the screen. (If the signal is not on resonance, acquire a spectrum and use utilities and o1 on the sidebar to put the signal on resonance). Processing None necessary.

30 3.10 Experiment Eight: Solid-State NMR - Cross Polarization Magic-Angle Spinning Experiment B - 13 C Cross polarization The polarization from an abundant spin (e.g., 1H) is transferred to a dilute spin such as 13C to enhance the S/N. Acquisition Find the 90 pulse on the 1 H resonance. 1. Make sure the hardware connections are correct. 2. Connect the 1 H cable through the preamp. 3. Create a new file for 1 H experiments. pulprog=zg TD =1k DW=5us Pl1=10 db P1=6us D1=3s NS=4 O1= Follow the Experiment two section on the Lab notes. Note the 90 pulse length and the O1 values. Cross Polarization 1. switch to 13 C file. 2. connect the 1 H cable through the band pass filter. 3. type edasp and change nucleus to 13 C. 4. tune the probe. 5. Setup experiment parameters. pulprog=cp TD =4k DW=10us Pl1=21 db (power level on the 13 C channel) 2 P3=6us (90 pulse on the 1 H channel) Pl2=10 (power level on the 1 H channel) Pl12=10 (decoupling power level) 3 2 The X channel power level is varied to match with the 1H channel power level. Note the 1H power level is set to a constant value corresponding to a pulse length of 6us. 3 The decoupling power level is also adjusted.

31 3.10 Experiment Eight: Solid-State NMR - Cross Polarization Magic-Angle Spinning 31 P15=3 msec (contact pulse) D1=3 s NS=16 O1=15000 ( 13 C offset) O2=2700 ( 1 H offset) Processing Fourier transform and plot the data.