Operation Guide for 500MHz Bruker NMR

Transcription

1 Operation Guide for 500MHz Bruker NMR NMR Laboratory University of Massachusetts Lowell Training and Operation Manual By Wendy Gavin NMR Technician 5/2017 Version 5 1

2 A. General Procedures page 3 1) Training Literature and References. page 3 2) Basic Safety.. page 3 3) NMR Basics and Theory.. page 4 4) Magnet Design... page 5 a. Proton NMR spectroscopy.. page 7 b. 13 C NMR spectroscopy. page 8 c. Pulsed fourier transform. page 10 5) Sample Preparation page 10 6) Spinner and Depth Gauge. page 12 B. Proton NMR Experiment Page 13 1) Create New Data Set page 13 2) Loading Sample page 15 3) Locking on Deuterium Signal... page 16 4) Shimming. page 16 5) Acquiring Data. page 19 6) Data Processing( Fourier Transform and automatic phase correction).. page 20 C. 13 C NMR Experiment.. page 20 D. More Data Processing page 21 a. Reference. page 22 b. Integration page 23 c. peak peaking.. page 23 E. Another sample (Time saving Tips when running multiple samples) page 24 F. Log Off page 24 1) Place standard CDCl3 Reference Standard in Magnet. page22 2) Log out of TopSpin. page22 G. FileZilla(FTP for your data) page25 H. MestReNova (NMR Processing software).. page25 1) Open file in MestReNova.. page25 2) Reference Spectrum. page26 3) Phase Correction.. page 27 4) Baseline Correction. page28 5) Peak Picking.. page29 6) Integration. page29 7) Multiplets Analysis page30 8) Change Display Properties.. page32 9) Save Data in pdf. page33 2

3 A. General Procedures 1) Training Literature and References A. 500NMR Operating Guide.pdf B. Basics Steps for 500 MHz NMR.pdf C. MesteNova Manual.pdf 2) Basic Safety NMR lab consists of 4 major items: Magnet, Console, Workstation, and 5 Gauss line. See figure 1 NMR Magnets are always live and always at field. Be sure that nothing ferromagnetic is near them. Nothing within the 5 Gauss line including: Tools Staples Metal jewelry Metal furniture Cell phones IPods USB drives Credit cards/student ID cards Place these items in the basket near the computers. Figure 1 below shows UMass Lowell NMR lab 0G-8. Figure 1 Magnet Console 3

4 Work Station 5 Gauss Line NMRs are superconducting magnets which are relatively safe and have no known side effects. People fitted with cardiac pacemakers and metallic implants should not go near the magnet. The magnet is potentially hazardous due to: 1. The large attractive force it exerts on ferromagnetic objects. 2. The large content of liquid Nitrogen and Helium. A Magnetic Field surrounds the magnet in all directions. Any ferromagnetic objects will be attracted to the magnet, be drawn towards the magnet, and could damage the magnet of a person in the way. The inner zone is within the taped and roped off areas, and the outer zone is safe. The magnet contains two dewars with one containing liquid helium and one containing liquid nitrogen. They are used to keep the magnet core at a very low temperature. Helium and nitrogen are non-toxic gases, however, they can quickly displace breathable air in a closed room. If the magnet quenches, i.e. gases come from top of magnet, please evacuate the room immediately. 3) NMR Basics and Theory NMR is a technique used to analyze many compounds. NMR compliments other structural and analytical techniques. NMR is non-destructive. An NMR experiment consists of 3 steps: a) Place a sample in a static magnetic field. b) Excite nuclei with a radio frequency pulse. c) Measure the frequency of signals emitted by sample. 4

5 These emitted frequencies lead to information about the bonding and arrangement of atoms in the sample. The NMR active nuclei resonate at diferent frequencies called resonance frequencies. The variation in resoannce frequencies is referred to as chemical shift. The resonance frequency is shifted by the effect of neighboring atoms and in particular the extent of magnetic sheilding from local electrons. o o o Nuclei with odd mass or odd atomic number have nuclear spin i.e. H, C, P, F Nucleus is charged particle in motion and behaves like tiny magnet. In applied magnetic field, the nuclei line up spin aligned or opposed. 4) Magnet Design Figure 2 5

6 A sample is placed in a strong magnetic field. A strong Radio frequency (RF) field is also imposed on the sample to excite the nuclei spins into their higher energy state. When this strong RF signal is switched off, the spins return to their lowest state, producing a small amount of radiation. This induces a radio frequency signal in the detector coil which is amplified to display the NMR signal. Currently, there is an inverse probe in the 500NMR. A sample is spun to average the magnetic field variations and the NMR tube imperfections. Probes contain radio frequency coils, tuned at specific frequencies for specific nuclei in a given magnetic field. Probes contain two coils, one inner and one outer. Inverse probes give highest sensitivity y to 1 H nuclei (inner coil). The NMR spectrum is able to provide details about: Types of atoms present in the sample Relative amounts of atoms present in the sample Specific environments of atoms within the sample Purity and composition of sample Structural information about molecule There are a number of important characteristics of the NMR spectrum which allow it to be useful to a wide array of industrial, commercial, and research applications: There are many nuclei which are NMR active. Some of the more common NMR active nuclei are 1 H, 13 C, 19 F, and 31 P. NMR is usually a non-destructive technique. Analysis requires only small amount of material :5-20mg sample sizes. Sample preparation is simple and minimal. 6

7 A. Proton NMR Spectroscopy Chemical shifts ranges from 1-15ppm Integration of peaks leads to number of protons Electronic enviroment leads to different chemical shifts Spin-spin coupling leads to splitting of NMR signals due to Magnetic interaction of neighboring protons. Each proton senses the number of equivalent protons (n) on the carbon atom(s) next to them. Resonance peak is split into n+1 peaks 7

8 Singlet at 1.96ppm Triplet at 1.17ppm Quartet at 4.04ppm 2 Protons 3 Protons 3 Protons B. Carbon-13 NMR Spectroscopy o o Carbon-13 has a nuclear spin (I=1/2) 13 C makes up 1.1% of all naturally occurring carbon 13 C NMR spectroscopy provides that following information: The number of different types of carbon atoms The electronic environment of different types of carbons. The number of neighbors a carbon has 8

9 Differences between 1 H and 13 C No integration of carbon spectra. Wide range (0 to 200ppm) of resonances for common carbon atoms, whereas the typical range for proton NMR is 1-15ppm. C-13 NMR spectroscopy is much less sensitive than proton NMR. C 2 13 C OF ETHYL ACETATE C 3 C 1 C

10 C. Pulse Fourier Transform Excite all proton Nuclei in a molecule at the same time, followed by a mathematical analysis of the comples radio frequencies emitted as they relax back to the equilibrium state. I. A sample is palced in a strong magnetic field II. A strong radio freqeuncy pulse is also imposed on the same to excite some of the nuclear spins into their higher energy state III. When this strong radio frequency signal is switched off, the spins tend to return to their lower state, producing a small amount of radiation IV. The emission of radiation is associated with the spin relaxation of the protons from their excited state. It induces a radio freqeuncy signal in the detector coil which is amplified to a FID signal. FID stands for free induction decay and it is based on time. Using Fourier Transform, this FID signal is changed to a frequency signal. 5) Sample Preparation. A. Quantity For a 1 H spectra, the quantity of sample needed is 5 to 25mg. 13 C spectra are six thousand times less sensitive than 1H spectra, thus a good rule of thumb is to give as much material as will give a saturated solution. B. Clarity Filter all samples into a NMR tube, so that there are no solid particles. You should filter through a small plug of glass wool tightly packed into a Pasteur pipette. C. Deuterated Solvents Samples must be dissolved using solvents that contain deuterium instead of hydrogen. Using deuterated solvents helps to prevent unwanted solvent signals. Second, the frequency of the deuterium ( 2 H) NMR signal locks the magnetic field. It ensures that the magnetic field doesn t change during an NMR experiment. 10

11 D. Sample Volume The amount of volume in the NMR tube should be 4.5cm to 5.5cm (0.5ml to 0.7ml). If you have less volume in the NMR tube, the sample will be hard to shim. See figure 3 below. Figure 3 11

12 E. NMR Tubes The NMR tubes and caps should be clean, dry, and undamaged. They should be rinsed after each use with Acetone, at the minimum, and dried with a blast of nitrogen. Do not place your NMR tube in an oven to dry. I recommend using an NMR tube cleaner and rinsing 3 times each with water, methanol, chloroform, acetone. The NMR magnet uses 5mm NMR tubes. Do not use any other size. Using cheaper NMR tubes will affect the quality of your spectrum. Clean tube immediately after use. F. Label your sample Be sure each sample is clearly identified, but do not use tape that could leave a flap or fall off inside the magnet. Labeling the top part of the NMR tube with a Sharpie marker is acceptable or the NMR tube cap, or try to use different colored NMR tube caps. 6) Spinner and Depth Gauge. Figure 4 The NMR tube must be positioned properly in the spinner before it can be placed into the NMR instrument. The spinner has a gripping mechanism to hold the NMR tube securely in place. See figure 4 above. Before the tube is inserted into the spinner, it should be wiped off with a Kimwipe. Grease or dirt will lead to the spinner no longer working effectively. IF the NMR tube slides easily up and down the spinner, then you should not load your sample using that spinner. Try another spinner or ask the NMR technician. NMR tubes must be positioned at the proper depth in the spinner using the depth gauge. This is done to make sure that your sample is in the detected region of the NMR probe once the spinner is lowered into the NMR instrument. The tube should never exceed the maximum sample depth. If you exceed this depth, the sample or the probe or both could break. The depth gauge should be set for 5mm tube depth. 12

13 The bottom of the NMR tube and the solution meniscus at the top should be equidistant from the center of the detected region. It will be difficult to shim if the NMR tube is positioned improperly. B. Proton NMR Experiment 1.) Data Set I. Login to PC Work Station of the 500MHZ NMR Instrument. a) Enter your user name. It should be your name all in lower case letters. b) Enter your password. Passwords are case sensitive. II. Open the TopSpin TM NMR Software. a) Double click on the icon labeled TopSpin ) Create a dataset to acquire, process, and save the NMR data. Before you acquire any new data, you should always create a new data set in which it can be stored. This will prevent overwriting existing data. i. Click on FILE at tope of screen. Select New from newly opened window. A new window appears for the creation of a new dataset that can be used to acquire, process, and save the NMR data. See figure 5 below. Figure 5 Change NAME of file (-and_ only) Change EXPNO, if needed No need to change Change Solvent if needed Leave Experiment use current params Title the experiment Enter sample name, solvent, date Click OK when finished 13

14 o Change NAME of the file and EXPNO, if needed. Dashes and underlines are acceptable in file names (No special characters!). Do not change PROCONO or DIR. DIR and USER should be set to your parameters already. There should be no need to change solvent and type of experiment. Enter sample name, solvent and date in TITLE. Click on OK when finished. Control Board Lock Gain Lift On/Off Z 2 Z 1 FINE Z 0 STDBY X Y ON AXIS 3.) Loading Sample Introducing the spinner with the NMR tube into the magnet is done on a cushion of air. This is called lift air. Never drop a sample into the magnet with no lift air flowing. NEVER put depth gauge in magnet! 4.) Press the LIFT On/Off Key on the top of control board under sample. Hear the sound of air. (CAUTION: Do not proceed to the next step, if you don t hear the activation of the air sound.) Wait for the sample in the magnet to come-up at the top of the magnet. Take it out. While air is on, drop your sample in the spinner at the top of the magnet. 14

15 5.) Press the LIFT On/Off key again on control board and the sample is gently lowered into the magnet on a cushion of air. Look for Green light to the right of DOWN on BSMS Control Board. 6.) Locking on the Deuterium Signal of the Solvent of the o All superconducting magnets run down slowly over time. This is called magnetic drift. The deuterium ( 2 H) NMR signal is used to ensure the magnetic field doesn t change during the NMR experiment. The frequency of the 2 H NMR from the solvent deuterium locks the magnetic field. Once locked, the magnetic field cannot drift even for long-term acquisitions. o The lock-level corresponds to the height of the lock ( 2 H) NMR signal. A narrow (sharp) lock peak will be taller (i.e. larger lock level) and a broad lock signal will be shorter (i.e. lower lock level). Therefore, the lock-level is an indicator of how broad or sharp the peal of the 2 H solvent signal is. 1) lockdisp [lockdisp=lock display] A Lock display window opens with the lock signal visible in the lock display window. See Figure 6 below. 2) Type lock. A new window appears with a list of solvents. Click on the solvent of your sample, and click on OK in this window. Lock signal will be locked. Wait for lock: finished to appear in the status line at bottom of screen. Figure 6 The lock-level should be between the 3 rd and 4 th line of the lock window. If it is not, you should try locking again (type in lock and choose your solvent) 15

16 7.) Shimming To achieve good resolution, the magnetic field must be homogeneous. However, the sample distorts the magnetic field. The shims are coils of wire that surround the sample probe, and they allow one to re-shape the magnetic field. Good shimming means achieving an optimal, homogeneous magnetic field. Good shimming is the key to getting a good, highresolution NMR spectrum. Increasing lock-level is indicative of a narrower lock peak, so we adjust the shims to achieve the highest possible lock-level. This will correspond to the best possible line shape. Common reasons for poor shims are: o Not loading a good starting shim set o low quality NMR tube o Insufficient volume of solution o Improperly positioned NMR tube o Unusual nature of the sample (i.e. highly concentrated) For each sample, you need to optimize shims. Every sample has unique magnetic susceptibility which is a measure of how well a sample can accommodate magnetic field lines. Shims compensate for these heterogeneities to make the field as homogeneous as possible. Low quality NMR tubes have varying thicknesses, and the tube itself may have a heterogeneous composition. A good NMR tube will have unvarying chemical composition, will be perfectly cylindrical, and will have a perfectly rounded bottom. During shimming, the currents in the shim coils are adjusted so the small magnetic field gradients produced cancel the inhomogeneity of the main magnetic field. 16

17 a. Type topshim. This will take a few minutes. Don t worry about where the lock signal ends up. If you receive an error message, type topshim again and see if it can shim the second time. If it still fails, your sample may be too concentrated. b. Manual Shimming a. Shim on ON-AXIS Shims [Axial Shims]. b. Press ON-AXIS key located at lower-left corner of control key board. c. Press the Z 1 button and maximize lock level position by turning the big knob on the control panel counterclockwise. Use the LOCK GAIN button to lower the lock signal if the lock signal goes too high off the screen. You can also press the FINE button on the bottom of the control key when you first start to shim. Once your shims are becoming constant, you can re-press the FINE key. d. Once the Z1 shims are constant, press the Z 2 button and maximize lock level. Go back to Z 1 and Z 2, if necessary, to attain higher lock level. e. Repeat this procedure until you do not see any changes in the lock signal position. c. Shim on OFF-AXIS Shims [Radial shims](only if not spinning) I. Press the X button and then the Z 0 button. Maximize the position of the lock signal using big knob on the control panel. II. Press Y button and then Z 0 button. Maximize lock signal position. d. Press stdby key at bottom of control key board. 17

18 8.) Acquiring NMR data 1. Type rpar PROTON all. [rpar: read parameters]. Proton must be capitalized. Select and read a predefined parameter set. 2. Type getprosol. This command loads probe-specific acquisition parameters including pulse widths and pulse powers. This gets probehead and solvent dependent parameters. 3. Type ii. [ii = initialize interfaces] This interfaces PC work station with the NMR console. 4. Type rga. [rga: automatic receiver gain adjustment] This sets up the pulse program. A NMR experiment involves exciting the sample with a precisely defined sequence of radio frequency (rf) pulses. It is a set of pulses interspersed with delays. 5. Type ns. [ns: number of scans] a) The accumulation of a number of transients (scans) will improve the quality of the spectrum. The number of acquisitions is specified with the parameter NS. b) NS should be in multiples of 8 to accommodate standard phase cycling which is incorporated into pulse programs. c) A new window appears with NS=16 for a proton NMR. Change value if necessary. d) Press return key. e) Type expt. This estimates the experiment time. 6. Type zg. [zg: zero and go, data acquisition.] This begins the acquisition of data. a. Halt = halt current acquisition and save the FIDs till this point b. Stop = stop current acquisition and do NOT save the FID. c. Go = add more scans to your current data. If you need more scans than you have already run, typing in go and return will run the same number of scans again and add to your FIDs. 18

19 9.) Data Processing 1. TopSpin Interface. Below shows a typical TopSpin window. 2. Click on the to see you spectrum. This switches between FID and Spectrum. See figure 7A and Figure 7B below. Figure 7A-FID scan in Time Figure 7B-Spectrum in Frequency 19

20 3. Type efp. [ efp=exponential Multiplication(e), Fourier-transform (ft), and phasecorrection (pk)]. 4. Type apk. [apk= automated phase-correction] 5. At this point, your data is saved and complete, and you can load your next sample or exit TopSpin following step 11. You can process your data using MestRenova and not be charged. 10.) 13 C NMR Experiment a) Type rpar C13CPD all. C13CPD must be capitalized. b) Type getprosol. This gets probehead and solvent dependent parameters c) Type ii. [ii=initialize interface] This interfaces PC work station with the NMR console. d) Type rga and return. [rga:automatic receiver gain adjustment] e) Type ns and return. [ns:number of scans] A new window appears with NS=1024. It would take an hour to run 1024 scans. It is best to keep in multiples of 8. f) Type zg. [zg:zero and go, data acquisition.] g) Type efp. Click on to see full spectrum. h) Type apk 11.) More Data processing a. Useful buttons: 20

21 Click to see full spectrum b. Reference Calibration 1. Click on the following and the window below will appear. 2. Position the red cursor at the reference peak. 3. Left click at that peak. The following data box will appear 21

22 4. Enter the correct frequency of reference peak. 5. Click OK c. Peak Peaking 1. Automatic Peak peaking. Choose Analysis/Peak Picking from the upper tool bar. This will automatically pick peaks. 2. Manual Peak Peaking. I. Select the Peak Peaking Icon II. The tab bar of the actice data window will be replaced by the toolbar shown below. III. Select the peak picking manually icon. A red line will apppear. IV. Put the red cursor line at the desired peak and click the left mouse button. The peak label will appear at the top of the data window. V. Repeat step 4 for each peak. VI. Click on the green button to leave Define peaks mode. 22

23 VII. Click on the button to save the peaks and exit the peak picking window. d. Integration. A. If you want manual integration, click on the icon. The tab bar of the active data window will be replaced by the toolbar shown below. B. To define integral regions interactively i. Click on the [define integral region interactively] (button turns green) ii. Put the red cursor line at one edge of a peak. iii. Left-click-hold and drag the cursor line to the other edge of the peak. iv. Repeat steps 2 and 3 for all peaks. v. Click the green button to leave define region mode[button turns grey]. vi. Click on the button to save the integration and exit the integration window. 23

24 12.) Another Sample in same solvent and same NMR experiment A. Hit the lift On/Of button on the BSMS control board, and load your next sample. Hit the lift On/Off button again. B. Choose file/new and name the new sample. C. If repeating the same NMR experiment, i.e. proton, you do not need to type rpar PROTON all, getprosol, or ii, since we have already done that with the first sample. D. If using the same solvent, the NMR should find the lock again(i.e. same lock level as previous experiment) without typing in lock. Check the lock display. If not, Type lock and return. Choose the solvent. E. Shim the sample using Z 1, Z 2,(If non-spin: XZ 0, YZ 0 ). Each sample will distort the magnetic field differently depending on concentration, NRM tube quality, amount of solvent, etc. Therefore, each sample should be shimmed. F. Type rga. G. Type zg H. Type efp, I. Type apk. 13.) Log Off A. Place standard sample in magnet. 1. When the scans finished, you NMR spectrum is complete. It is already saved. You can now remove your sample from the magnet and replace it with the standard sampleusing Lift On/Off button. 2. You should lock the signal for the standard sample. Type lock and choose CDCl 3. It is very important that the magnet is left locked. Be sure the Lock On/Off button on the BSMS control board is lit solid green, not flashing! Shimming the reference would be the optimal way the leave the NMR. If another user is waiting, they can just load their sample instead of putting back in the reference standard. 3. Next, you can exit from TopSpin. I. Go to FILE and choose EXIT. A new window appears to be sure you want to close TopSpin. II. Click OK. 4. Be sure to logout from your account. 5. Go to and choose LOG OFF. 6. A new window appears asking if you are sure you want to log off. 7. Choose OK. This ends your charged time on the NMR. 24

25 11) MestReNova. Review MestReNova Manual.pdf A. FileZilla. Bring your data from the NMR workstation computer to another computer. 1) Go to the computer station at the bench in the NMR room. 2) DoubleClick on the FileZilla client on top left of desktop. B. In host line, type in IP address of the 250 NMR located on the main screen, C. Type your user name. D. Type your password from the NMR work station. E. Type Port 22. F. Click on Quickconnect. G. Data on left side is data on that computer. Data on the right side is from the 500 NMR. 1. Under remote site on right, you see /usr/people/your name 2. Double click on the / 3. Scroll down under filename on right to find the opt. Double click on opt. 4. Under filename, double click on topspin. 5. Under filename, double click on data. 6. Look for your name and double click. 7. Under filename, double click on NMR. 8. On right side, find your filename, under My Computer. 9. Drag file folder from right side of filezilla to your folder on the left side using the mouse. 10. You can select more than one filename by holding down Ctrl and shift. 11. Wait for transfer to be complete 12. Close FileZilla when data transfer is complete. C. Open MestReNova software by double clicking on icon. 1) To open your data, just click on the folders, shown below, or go to File/Open menu. 1. Navigate through the data to find the FID file for your experiment. (or 2i or 2r). i. Look under name of experiment for experiment number file. ii. Double click on experiment number. iii. Choose FID from list of experiments. 25

26 2. Visualize your Spectrum 3. Reference Spectrum A. You can calibrate your spectrum by selecting Analysis/Reference on the main menu or by clicking on the toolbar button. B. Zoom-in if necessary or bring the mouse over the reference peak. The following box appears 26

27 C. You can type a new value for that peak or click on the button to get a list of solvent chemical shifts. 4. Phase Correction A. Automatic Phase Correction- Choose Automatic Phase Correction 27

28 B. Manual Phase Correction- Manually phase the spectrum. A new dialog box opens Left click and hold the left mouse button while scrolling the mouse up and down to phase the largest peak. Right click and hold mouse down while scrolling to phase all the other peaks. 5. Baseline Correction Baseline Correction should be performed before integration. 28

29 6. Peak Peaking a) Select Analysis/Peak Picking on the menu bar or click on the Peak Peaking icon,, on the toolbar. You can choose automatic or Peak by Peak. If you choose Peak by Peak, use the mouse to click over each peak of interest. b) Choose Manual Threshold to tell the program to peak picks after a certain height only. 7. Integration a) You can get integration by choosing Analysis/Integration, or by choosing the Integration icon,. You can choose automatic or manual integration. b) If choosing Manual integration, select manual, than you can click and drag your mouse to select the area to be integrated. The first integral will be normalized to 1, and all the others will be referenced relative to the first. To change or move anything, just use the mouse. 29

30 c) The integral editor will be displayed by double clicking on any integral curve. You can adjust the normalized value, if necessary. See box shown below. 6) Multiplets Analysis A. Automatic multiplet analysis can be carried out to clicking on the Multiplets icon, or by using Analysis/Multiplets Analysis/Automatic. B. The multiplets data can be listed by using Report Multiplets. An example is shown below. 30

31 C. You will be able to set the multiplet parameters by double clicking on the magenta square containing multiplet information. 31

32 The Multiplet Manager dialog box will be displayed, and you can modify the name, class, color, limits, delete, change integral or report the multiplets. 7) To change the Display Properties A. Right click on the spectrum and choose Properties in the context window. 32

33 B. A lot of display properties can be customized.(i.e. get rids of peak labels, title, grid lines, etc) 8. Finally, you will be able to save or export to PDF. 33