UI500NB Spectrometer: Advanced 1D and 2D NMR Experiments
Contents
1. 22 enter go to start After the acquisition is complete save your spectrum WORKUP use Wft to process Do not auto phase the spectrum aph First type phase 180 if necessary to have the irradiated peak in the negative direction then phase manually the NOESY peaks will be in the positive direction and exchange peaks will in the negative direction for small molecules dssh or dssa to view all spectrum in array To view individual spectrum type dS where is the number of the desired spectrum To print all spectra in the array pl all pscale page To print individual spectrum ds pl pscale page or pl pir ppf pscale page add ppf or pir for peak picking and integration 23 10 Setting up the 2D H H ROESY Experiment The ROESY Rotating frame Overhauser Effect Spectroscopy experiment measures NOE in the rotating frame providing information about distance between two protons in space It is generally used for compounds with a molecular weight of 1000 3000 for which NOESY is approximately close to zero ROESY cross peaks are always having opposite sign to the diagonal peaks contrary to any TOCSY or exchange peaks if exists in the ROESY spectrum The following setup assumes you have done steps 1 7 in Setting up the H Experiment If you have not do them now before continuing 1 To load a ROESY experiment jexp11 mf 1 11 wft full pw pw90 ROESY 2 Check parameters nt 32 or more for better signal to n2. M magnetization at time t after a 180 degree inversion pulse Ms magnetization at equilibrium tau the time when the magnetization is zero 7 To obtain the H spectrum used for the following experiments jexp1 optimize d1 as necessary based on the T1 if attempting a good integration d1 5s optimize nt nt 8 ga Then phase reference and save the H spectrum 11 2 Setting up the 2D H H gCOSY Experiment The following setup assumes you have done steps 1 7 in Setting up the H Experiment If you have not do them now before continuing 1 To load a gCOSY experiment jexp3 mf 1 3 gcosy 2 check ni it should be at least 256 for a typical spectral width of 10 ppm but may be smaller for a smaller sw Check d1 1 2 s or the value optimized in expl 1 5T1 pw pw90 np 2048 sw1 sw phase 1 nt 2 or multiple of 2 for better S N ni 128 or 256 if you change np or ni readjust sb at 2 and sb1 ni 2 sw1 if you change np or ni you may need to reset fn and fnl 3 Check time by typing time lt rtn gt this experiment normally runs less than 10 mins for nt 2 Adjust nt if necessary there is no minimum Make sure the sample is not spinning To turn off go to the VNMR Acquisition window LC Lock LC spin off While this window is open make sure that the lock level is gt 60 you should adjust only the gain if possible When you start the experiment with go below make sure that the lock level sta
3. T T T T z T T z T T L T r T T 9 0 8 5 8 0 7 5 7 0 6 5 6 0 5 5 5 0 4 5 3 5 3 0 2 5 2 0 1 5 f2 ppm Figure H 3 H H 2D gCOSY spectrum of Quinine 33 0 5 1 0 15 2 5 3 0 3 5 4 0 4 5 5 0 5 5 6 0 6 5 7 0 75 8 0 8 5 9 0 f1 ppm H 4 H PC NMR 2D HSQC data CDCl The data developed from the 2D H PC HSQC experiment are illustrated in Figure H 4 These data reveal one bond cross correlations between protons and carbons in a two dimensional format Assignments in either domain allow direct assignment of the opposite domain from their cross contours The assignments illustrated in Figure H 4 are based upon data and conclusions obtained from other experiments i e 2D gCOSY and 1D APT carbon data chemical shifts and coupling constant considerations These data are consistent with the structure of Quinine 18 19 45 1 me 23 204 4 d 9 2 wO H 3 3 n8 HB o m 1H 13C HSQC 11 56 22 22 1 75 22 23 ER 15mg in 700uL CDCl Tiso 12 pu oe 6 L 1522814 11 1732789 30 2 27 40 16 3 45 43 48 2664345 ge Lag 3 88 55 97 2 68 57 25 ig E 2 5v35 3 065726 60 comm L7o o a a 13C 90 g 724 101 63 LLILLL7AUP ts H100 3 7 Za 4 91 114 64 4 98 114 63 H EF N 110 t emmen enemman eemnenn eneen eneen eneen teeme 10 E i oi tt E BRL RU H120 23613175 o 35 M sesso zi r130 2
4. ideally under 5 however this is sample and solvent dependent 2 4 Change CHAN back to 0 2 5 Return the cables back to their original positions J5321 J5301 and J5323 J5302 NOTE DO NOT FORCE THE KNOB TO TURN IF YOU FEEL RESISTANCE STOP Otherwise you could break the rod IH match ISN tune Placement of tuning knobs for Carbon Nitrogen and Proton under the probe The largest brassy red knob is for proton and is the only one with tune and match 3 Tune the PC channel 3 1 Disconnect the cable from the carbon filter on the floor see the photo in the next page and connect it to J5321 on the Probe Tune Interface Disconnect the J5312 cable and connect it to J5323 Tune on the Tune Interface 3 2 Change CHAN to 2 by pushing the button on the tune interface labeled Again leave the setting on the right at 9 3 3 Turn the Green knob underneath the probe labeled carbon to tune the channel Note there is only 1 section to the carbon tuning knob Turn this knob carefully in one direction and watch the value on the tune interface readout If this number increases turn the Tune knob in 5 the other direction until a minimum value is reached Continue to adjust the Tune knob until the smallest readout value is achieved ideally under 5 however this is sample and solvent dependent 3 4 Change CHAN back to 0 and return the cables back to their original positions J5321 carbon filter and J5323 J5312 N
5. is more robust to poorly calibrated pulses However the cross peaks are modulated by homonuclear proton J coupling which broadens the carbon dimension All cross peaks in HMQC spectrum have the same sign HSQC cross peaks are not affected by homonuclear proton J coupling and has a superior resolution in the carbon dimension The edited gradient enhanced HSQC gHSQC inverts the CH signals leaving these cross peaks negative The CH and CH cross peaks are positive in the 2D spectrum If you work with a well calibrated system gHSQC is preferred 14 4 Setting up the 2D H C gHMBC Experiment The following setup assumes you have done steps 1 7 in Setting up the H Experiment 1 To load a gHMBC experiment jexp5 mf 1 5 ghmbc enter the answers accordingly 2 Check parameters nt should be 4 to 8 times of that necessary for the gHMQC experiment ni should be set between 256 and 512 pw pw90 phase 0 jnxh 8 is the default value the average long range H C coupling constant Typical values are between 5 and 10 If you are missing some correlations you may need to run the experiment again with a different value of jnxh By default j1xh2140 the average one bond H C coupling constant for aliphatic proton carbon pairs aromatic 1 bond H C coupling constants are 170 250 Hz so j1xh 180 if you want to filter out one bond H C aromatic peaks 3 check time adjust nt if necessary It normally takes 1 12 hrs depending on sample co
6. process Note The gradient double quantum filtered phase sensitive COSY gDQCOSY removes the intense singlets and observes only the magnetization associated with double quantum coherence multiplets The diagonal peaks are in absorption mode giving much narrow diagonal peaks compared with gCOSY which has dispersive diagonal peaks Therefore gDQCOSY presents less interference to the cross peaks near the diagonal This experiment reveals germinal and vicinal spin spin coupled pairs and measures the proton proton J coupling constants However its S N is half of that of gCOSY therefore twice as many scans are needed to achieve the same S N as in gCOSY 16 Example Measurement of proton proton J coupling constants 5 Has A 0 0 A 0 8 25 8 15 v 99 Wo 6 5 Hi H QO 6o 95 60 eee 8 9 8 8 8 9 8 8 9 15 H H Fig 4 20 Parts of the one dimensional and phase sensitive COSY H The bottom row shows multiplets from the one dimensional spectrum row shows cross peaks from the COSY spectrum and the top row sho through the top set of responses of each cross peak from Modern NMR Spectroscop i ists y a guide for chemists by J S d Editi Oxford University Press Page 116 nne eat gees 17 6 Setting up the 2D H H TOCSY Experiment The TOCSY mixing time Tm is related to how far the spin spin relay carries throughout the spin system in the range of 60 100ms is typically useful to observe 4 and 5 b
7. save your spectrum WORKUP use Wft to process Do not auto phase the spectrum aph First type phase 180 if necessary to have the irradiated peak in the negative direction then phase manually the ROESY peaks will be in the positive direction and exchange peaks will in the negative direction for small molecules dssh or dssa to view all spectrum in array To view individual spectrum type dS where is the number of the desired spectrum To print all spectra in the array pl Call pscale page To print individual spectrum ds pl pscale page or pl pir ppf pscale page add ppf or pir for peak picking and integration 26 E Guideline for 2D Data Acquisition You can make a good approximation of the number of scans needed for your 2D data acquisition by looking at your 1D H spectrum The H 1D spectrum needs to be acquired under the following conditions 1 pw pw90 2 nt l 3 Check the S N to the smallest peak of interest in your spectrum 4 Check that number versus the suggested numbers below gcosy if the S N gt 50 1 use nt 1 the experiment time is 5 minutes ghmqc if the S N gt 250 1 use nt 1 the experiment time is 15 minutes if the S N gt 100 1 use nt 4 the experiment time is 1hour ghmbc if the S N gt 250 1 use nt 8 the experiment time is 45 minutes It should be 4 to 8 times of that necessary for the gHMQC experiment Note In a 1D experiment if you want to double the S N you need to in
8. 0 HT n Tu 24 5 74 141 95 la m 8 60 147 77 9e S37 Sa F150 T 7 T T T T 7 T T T T M T T T T T M T T X T T T T id T T T r T T 9 5 9 0 8 5 8 0 7 5 7 0 6 5 6 0 5 5 5 0 45 4 0 3 5 3 0 25 2 0 15 1 0 0 5 0 0 f2 ppm Figure H 4 H PC 2D HSQC Spectrum of Quinine 34 H 5 H C 2D HMBC data CDCl The data developed from the 2D HMBC experiment are illustrated in Figure H 5 These data reveal multiple bond cross correlations between protons and carbons in a two dimensional format The assignments illustrated in Figures H 5 are primarily from 3 bond proton to carbon cross correlations The experiment is optimized with a time delay for 8 Hz coupling CJuc coupling Occasionally 2 bond and 4 bond correlations will appear Furthermore the suppression on one bond couplings also appears in the spectra due to non optimal bird pulse suppression Some of the important 3 bond correlations are summarized in the structural illustration r10 r20 r30 r100 Lois L120 Lizo Luo Lum Lio r170 below 85 p A 23 201 2449415 0 2 w10 1H 5 3Y TAA 8 11 1 112 H C HMBC 15mg in 700uL CDCl L 79 710 ZI W 119 41 10 9 89 it 8 10 28 ho 9 9 n TIS 2 379 312 o 2 24 2 33 e 2 12 Kre Zr H t N oes SN TM EM i hi te tes 432 Rc E MIT Um 44206 01424 32 s NX NT wp LN i a 0921 15 910 972 98 T sa e 432 16208 gt g 1618 9 0 8 5 8 0 75 20 65 6 0 25 5 0 45 4 0 3 5
9. 3 0 25 2 0 1 5 1 0 o5 f2 ppm Figure H 5 H PC 2D HMBC Spectrum of Quinine 35 f1 ppm H 6 H H NMR 2D NOE data CDCI3 The results of H H NMR 2D NOE spectroscopic analysis Figures H 6 are consistent with the structure of Quinine These data reveal through space or proximal located spins and are based upon relaxation phenomenon Thus strong NOE observed between protons 2 and 5 and strong NOE observed between protons 9 and 3 suggest that the relative stereochemistry of this compound is as shown 18 6 23 704 24 5 9 2 Q0 5 37758 1i 0 H H 2D NOESY 95 15mg in 700uL CDCl Je er pio r2 0 los 3 0 r3 5 r4 0 r4 5 r5 0 r5 5 IMMUNE ae 6 0 r6 5 7 0 LA a e 8 0 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 05 f2 ppm Figure H 6 H H 2D NOESY Spectrum of Quinine 36 f1 ppm
10. Cl3 The Attached Proton Test APT BC data collected with a fully decoupled proton domain is present in Figure H 2 The J modulated PC data exhibit up and down peaks depending upon the numbers of its directly attached protons giving CH and CH up and CH and quaternary carbons down or vice versa A total of 20 carbon peaks were observed consistent with the structure of Quinine 13C APT 15mg in 700uL CDCI 5 7 rd ON E f NOH y N 18 13 HC UL Tuc Sa 18 Il X OS 23 20 N noise from a nearby outside source T v T v T h T hd T Md T Y T v T v T d T T T M T id T v T M T d T v 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 f1 ppm Figure H 2 Carbon 13 spectrum of Quinine 32 700 600 500 r400 r300 r200 r100 ro r 100 r 200 r 300 400 H 3 H H NMR 2D gCOSY data CDCI3 The data developed from the 2D gCOSY experiment are Two aromatic spin systems and one continuously coupled spin system including aliphatic and vinyl protons are observed illustrated in Figure H 3 These data reveal isolated contiguously coupled spin systems Pu M H a IN AMA se H H gCOSY 15mg in 700uL CDCl i o g z oe pe eo BB 42 e al 8 11 _ 11 6 lt e e i d o 18 pat 56 1 9 e aq eo amp e3 12 iB B oe 5 aa 5 6 H B e 4 e a 9 10 9 8 AN sa AAA o 9 Psal E o a i 91920 uc wA S 18 EE 2 2324 CT T T bal T 7 T
11. M C N E 1H 9F Observe Preamp i z Preamp a tl B Please note the carbon cable connected to the filter circled in green at the lower right hand corner NOTE DO NOT FORCE THE KNOB TO TURN IF YOU FEEL RESISTANCE STOP Otherwise you could break the rod 4 Tune the N channel if needed 4 Disconnect the cable from the nitrogen filter on the floor and connect it to J5321 on the Probe Tune Interface Disconnect the J5312 cable and connect it to J5323 Tune on the Tune Interface 4 2 Change CHAN to 3 by pushing the button on the tune interface labeled Again leave the setting on the right at 9 4 3 Turn the knob underneath the probe labeled nitrogen to tune the channel in a similar way as tuning PC channel Note there is also only 1 section to the nitrogen tuning knob 4 4 Change CHAN back to 0 and return the cables back to their original positions J5321 nitrogen filter and J5323 J5312 NOTES Tuning and matching is performed to optimize sensitivity Typically 2D experiments take a long time to acquire and optimization of sensitivity can make the most effective use of the data acquisition time Greater sensitivity means larger signals The instrument is designed to measure C and DN indirectly and these channels normally calibrated by NMR lab staff Consequently the pw90 for the proton is the only pw that requires careful calibration Typical Values on the Tune Interface M
12. M Triphenylphosphate CDCl 5mm NMR tube 5 For 10mm BB probe C and P Sensitivity Standards are in 10mm NMR tube B Probe tuning for the hcn probe on UIS00NB Insert your sample into the magnet bore 1 In the VNMR command line type tune H1 C13 or tune H1 C13 N15 This sets the tuning CHAN 1 to H and CHAN 2 to C and CHAN 3 to PN 2 Tune the H channel 2 1 Disconnect the J5301 cable and connect it to J5321 on the Probe Tune Interface and disconnect the J5302 cable and connect it to J5323 Tune on the Tune Interface H F Observe Preamp ourpur 2 2 Change CHAN to 1 by pushing the bottom button on the tune interface labeled leave the setting on the right at 9 The TUNING INTERFACE readout should turn green and give a reading 2 3 Locate the large shiny brass knob underneath the probe It is labeled Proton and some red 4 color is visible on the knob This proton adjustment knob has a large lower shiny portion and a smaller upper knurled portion The upper portion is for Tuning while the lower portion is for Matching Turn these knobs carefully in one direction and watch the output change on the tune interface readout panel If the readout increases turn the Tune knob the other direction until a minimum value is reached Adjust the Match knob to decrease the value further Continue to turn the tune and match knobs until you reach the smallest number possible
13. R500 and UI600 Contact Dean Olson 4 0564 if you need to use this probe or have any other special requests VT Setup The temperature limits for the hcn probe and for most gradient probes are 20 C to 80 C Use FTS unit to cool the system below room temperature see VT user manual in a separate document As always you will need to be checked out for VT before you can use the FTS unit If you have not been checked out on VT please do the VT training checkout which will allow you to use VT on this instrument For long 2D experiments it is recommended that the experiments are collected at a fixed temperature For example set temp 25 C for room temperature experiments Probe Tuning You must be trained and checked by lab staff before you are allowed to tune the probe Instruction for tuning the probe is given in this document and is also given in a separate handout Shimming You must be trained again on shimming even if you ve done it in the basic NMR training section Due to the design of the experiments running on UISOONB you will collect your spectra mostly without sample spinning therefore you should shim the system both in spinning shimming and none spinning shims following the procedure given in this document more information can be found in the SCS NMR website Scheduling Check the SCS NMR website and the following page for the current rules and regulations TABLE 1 Comparison of Sensitivity Sens for NMR Spectromet
14. Revised 26 June 2012 LZ and DLO UIS500NB Spectrometer Advanced 1D and 2D NMR Experiments With Application to Structure Elucidation of Small Organic Molecules You should have finished basic NMR lab training with NMR staff before proceeding to train on UIS500NB for advanced 1D and 2D NMR experiments The following knowledge and skills are presumed e Tuning and Matching the UISOONB Probe e pw90 Calibration e T1 Determination e Solvent Pre saturation e Decoupling TABLE OF CONTENTS Page Pe Titten ue DTO EAEE E T nad hdasdoameher PENNE conan das dai aab 2 B Probe TUBE ceo Eb ER E E PERDERE dacs 4 C SIITRIHE od eyes ee urere Heck EAVERUR RF E ER On a e di eta eg oe ee 8 D Setup of 1D and 2D NMR Experiments cesses 9 iS TD H SDOCIFUD TuS eee tre eot ei ne tero veu 10 2 2D A H COSY costae COP EDO RUE DE SR 12 3 2D H C gHMQC or gHSQO oiiire 13 4 ID H OSBMBG 255 iiei ot ctp e edo net tested ok 15 5 2D H H gDQCOSY x eeteecesi xvetocgeme davor opere pb pedes 16 6 2D H TOCS Y css esiti poitea Gute o sepp dut crt s 18 T ID H H TOCSY us cciaiecioset tni larder easier sunset Es 19 8 2D H H NOBS Voce yi tattered BH i ass ee alt 21 Oi ID E NOESY oenostsexunadendid Daten tert be adem eia 22 102D HAH ROBSY crosiera ect e qua unita ete 24 TLD He ELROESY ari i sets up oeste stunt te Cu 25 E Guideline for 2D NMR Acquisition sees 27 F Guideline for 2D NMR processing eese 28 G C
15. adjust Z1 and Z2 again 3 3 Adjust Z4 as the same fashion as Z3 step 3 2 that is every change in Z4 must be followed by the optimization of Z1 and Z2 until the highest possible lock level is obtained 3 4 Adjust Z5 The best way is the same as above or you can adjust the Z5 value to maximize the lock signal then check Z1 and Z2 again Repeat steps in step 3 iteratively until the highest possible lock level is obtained If you are satisfied with Z shims turn off the spin by click spin OFF button watch the lock level If the lock level drops more than 5 units you need to shim the none spinning shims X1 Y1 XZ YZ and XY and X2Y2 4 1 Adjust X1 and Y1 iteratively to maximize the lock signal 4 2 Adjust XZ in one direction to decrease the signal intensity about 20 Adjust X1 to maximize the signal It it s better adjust XZ more in the same direction If it becomes worse adjust XZ in the other direction until signal maximized 4 3 Adjust YZ in one direction to decrease the signal intensity about 20 Adjust Yl to maximize the signal as above 4 4 Repeat step 4 1 Note the following is non routine 4 5 Maximize lock level by shimming XY then repeat step 4 1 4 6 Maximize lock level by shimming X2Y2 then repeat step 4 5 4 7 Repeat steps 4 1 to 4 3 4 8 Repeat steps in step 3 shim on Z again with spin ON Finally if you have a good shimmed system there should not be a big difference 1096 in lock leve
16. crease 4 times nt used 27 F 2D Data Processing This section will be expanded later Unless you have been informed of a macro that will do the proper data processing of your spectrum for you you will need to process the data manually Below is a table showing the optimum processing apodizations window functions and the proper processing commands in Vnmr software Experiments Apodization 1 Apodization 2 Processing Process Shortcut gCOSY sb at 2 sb1 ni 2 sw1 wft2d t2dc 1 0 0 1 wft2d phase 1 foldt optional foldt TOCSY gf at 2 gfl ni 2 sw1 wft2da phase 1 2 gHMQC gf at 2 gfl ni 2 sw1 wft2da phase 1 2 gHMBC gf at 2 gfl ni 2 sw1 av av wft2da phase 0 For gCOSY gf at 2 and gfl ni 2 sw1 can be used to enhance S N For plotting you can use cosyplot and hetcorplot Check the appropriate handouts for more information cosyplot can be used on the instrument or the SUNDS If you want to use hetcorplot you need to have acquired a good 1D C spectrum on an instrument other than the uiS00nb You can then plot on the SUNDS To plot using hetcorplot on the SUNDS logon to the ui500nb jexpl load your 1D H fid and process jexp4 load your ghmqc data and process logon to the instrument with your 1D C fid jexp2 load your 1D 13C fid and process jexp4 10 dconi to display 2D data 11 set the window as you want then type hetcorplot and answer the qu
17. d In expl set up parameters for a standard H experiment set the temp parameter if you are going to use VT then enter su If using VT wait until the temperature has reached the set point and your sample has equilibrated Then tune the probe according to the instructions NOTE You should have your parameters for a standard H experiment loaded before tuning the probe or alternatively type command tune H1 or tune 1H C13 or tune 1H C13 N15 as described in probe tuning section on page 4 Optimize the H parameters mainly SW by setting the two cursors 0 5 ppm beyond last proton peak on both sides of spectrum then type the command movesw Re acquire 1D H experiment make sure the parameters are correct Then phase and reference the H spectrum Determine the H pw90 of the sample more details of this operation can be found at the SCS NMR website named 90 Degree Pulse Width Determination mf 1 2 jexp2 wft gain A number will show up in the command line gain the number Array experiment won t run if you have gain n ntz1 type array lt rtn gt NOTE The next four items are the answers to the questions posed by the array macro Parameter to be arrayed pw lt rtn gt Enter number of steps in array 10 lt rtn gt Starting value 20 lt rtn gt an example This number should be set to approximately 4 pw90 from the default setup 2 Array increment 1 lt rtn gt Then type pw 1 5 Re
18. e only acquiring an HMQC spectrum you can narrow the C sw to include only protonated carbons If you are also acquiring an HMBC spectrum the SW should be set for both protonated and quaternary carbons Please also remember that the pw90 of C needs to be calibrated but this has already done on a standard organic sample by NMR staff you can use the default C values Please talk to the NMR staff if you want to calibrate the C data on your particular sample To load a HMQC or HSQC experiment jexp4 mf 1 4 ghmqc or gHSQC enter the answers accordingly Check parameters at should be around 0 2 seconds np 2048 d1 should be set so that a at at d1 lt 0 15 THIS CONDITION MUST BE MET If necessary reduce np i e np np 2 and or increase d1 b d1 at 1 to 1 5 x T1 This is a recommendation If your T1 s are long you can try the default setting of d122 If you set d1 for less than the recommended time you will sacrifice sensitivity However if your sample concentration is adequate the savings in time will more than balance this loss nt should be set according to sample concentration no minimum required 2 or 4 is adequate ni should be set between 256 and 512 phasez1 2 By default j1xh 140 which is the average one bond H C coupling constant for your sample If you do not see a correlation you believe should be visible it may be because the coupling constant for this particular C H bond differs too much from this av
19. erage value If you re run the experiment optimized for the new coupling constant the correlation should appear If you chose to set C parameters manually set the following SW1 sw of C spectrum collected previously in step 1 otherwise use the default value dof tof of C spectrum collected previously in step 1 otherwise use the default value check time adjust nt if necessary It normally take 20 40 min depending sample concentration 13 Make sure the sample is not spinning To turn off go to the VNMR Acquisition window LC Lock LC spin off While this window is open make sure that the lock level is gt 60 you should adjust only the gain if possible When you start the experiment with go below make sure that the lock level stays above 20 enter dps to check the pulse sequence enter go to start After the acquisition is complete save your spectrum WORKUP use wft2da to process use twod to setup proper display window The difference between HMQC and HSQC Both H C HMQC Heteronuclear Multiple Quantum Coherence eg 2LSy and HSQC Heteronuclear Single Quantum Coherence eg 21 S x detect the correlation between directly bonded H and C nuclei via the one bond H and PC J coupling interaction The cross peaks in the 2D spectrum contain both chemical shifts information of the carbon and its directly attached protons providing a correlation map between the coupled spins HMQC has shorter relaxation pathway and
20. eriment jexp12 mf 1 12 wft full pw pw90 ROESY1D ds 2 Expand around first peak to be irradiated inverted Center cursors around desired peak ensuring that the full peak is between the two cursors then LC Select Selects current region between cursors for selective inversion Repeat Select for all desired peaks When completed LC Proceed Calculates shaped pluses and completes ROESY1D setup You will need to answer the following questions Enter reference 90 degrees pulse with usec Enter your determined pw90 from pw90 calibration you ve done early Enter reference power level Enter the tpwr value used in pw90 calibration da Displays array Make sure all your desired peaks have been selected They will appear with name similar to ROESYID 5 67p RF where the numbers refer to the chemical shift of the peak 3 Check nt 64 or 128 or 256 for a better S N d1 normally 2 seconds or longer mix 0 2 200ms or other value as describe in 2D ROESY section 4 Check time adjust nt if necessary 5 Set bs 16 or 32 il y so that the data will be collected for the first FID first arrayed peak in the block size of 16 or 32 then go to the second FID instead of finishing the first FID first 25 arrayed peak then go to the second arrayed peak Such if the S N is good enough you can always stop the experiment Make sure the sample is not spinning enter go to start After the acquisition is complete
21. ers NMR Instrument Probe H DF BC 3p Variable Sens 1 Sens 2 Sens 3 Sens 4 Temperature Range C U400 QUAD 160 1 150 1 150 1 110 1 100 C to 100 C H F Spon 5mm switchable 120 1 150 1 150 1 100 C to 100 C UI400 QUAD 120 1 120 1 130 1 90 1 100 C to 100 C H PF B p disabled U500 QUAD 260 1 270 1 210 1 300 1 100 C to 100 C H F ap VXR500 QUAD PFGZ 260 1 280 1 230 1 200 1 80 C to 100 C H F 3C p H X switchable 300 1 210 1 100 C to 100 C UIS00NB H PC PN PFG Z 750 1 80 1 20 C to 80 C H P X PFG Z 700 1 NA 30 C to 50 C N P BB 10mm NA 550 1 100 C to 100 C UI600 H PC PN PFG X Y Z 1000 1 125 1 20 C to 80 C AutoX PN P and Hor 370 1 475 1 320 1 206 1 80 C to 130 C N IP BB 10mm NA NA 1100 1 550 1 150 C to 150 C 80 1 for 5mm tube VNS750NB H PC PN PFG X Y Z 1350 1 180 1 20 C to 80 C ISN P BB 10mm 1200 1 780 1 150 C to 150 C PC H 5mm 530 1 100 C to 100 C BC H 3 mm 220 1 100 C to 100 C Numbers in indicate sample used see corresponding list at the bottom 1 H Sensitivity Standard 0 1 ethylbenzene CDCl 5mm NMR tube 2 PE Sensitivity Standard 0 05 CF3C6Hs CsD 5mm NMR tube 3 Bc Sensitivity Standard ASTM 40 dioxane C amp D 5mm NMR tube 4 P Sensitivity Standard 0 0485
22. estions as appropriate Q9 ug oub RemM Some useful commands pcon page used to plot 2D spectrum as displayed on the screen dconi used to redisplay 2D after changes e g vs2d fullt sizes the 2D display so that 1D spectra can be plotted on the side and top of the 2D plot vs2d used to rescale the 2D plot used dconi after using this command 28 Supplemental material on processing 2D spectrum 1 At end of experiment set appropriate weighting functions and linear prediction parameters type commands setLP1 sqsinebell 2 If fn parameter now equals 4096 process will be slow then type fn np fn fn 3 Switch f1 and f2 axes make f2 the x axis type trace f2 or trace fI 4 Display full across screen removes error message full 5 Display full ppm scale f 6 Display as contour plot type command dp10 7 Spectrum should be appropriately referenced already but you should confirm this if necessary re reference by putting cursor on appropriate diagonal peak and type assuming CDCl rl 7 26p r11 77d dp10 8 Adjust vertical scale with vs 20 and vs 20 menu buttons or with middle mouse button or manually changing the parameter vs2d so vs2d 100 the lower the number the less noise displayed then redraw 2D with dp10 command 9 Print with full rectangle type full dp10 pcon 10 1 2 page 10 Save data type svf filename 29 G Chemical Shift Referencing H Spec
23. eter Solvent Proton Carbon Nitrogen CDCI 007 001 001 CD2Cly 024 002 001 DMSO Dg 010 002 001 D20 045 001 001 Scheme of the Preamplifier Housing TUNE INTERFACE 1 4 WAVELENGTH BROADBAND 1H 19F PREAMP OBS PREAMP 15 400 MZH OUTPUT Or J5312 J5302 C Shimming For 1D 2D gradient NMR experiments you will be collecting your data without sample spinning Therefore you should shim your sample both in spinning and none spinning shims The following is the basic shim procedure more can be found on the SCS NMR website Again this procedure is written on the bases that you ve trained on shimming previously when you first come to the NMR lab 1 2 3 4 5 Load the system shim map by typing rts hcn on the command line Lock your sample by adjusting z0 value check lockphase value also Shim spinning shims first Z1 to Z5 while turning on the spin spin rate at 20 Hz by clicking spin ON button 3 1 Adjust Z1 then Z2 then Z1 again iteratively After the maximum lock signal level reaches 3 2 Adjust Z3 clockwise to decrease the signal intensity about 20 then adjust Z1 and Z2 iteratively to maximize the signal Continue changing Z3 in the same direction if the lock signal is higher than it was initially and then adjust Z1 and Z2 until reach to maximum It the lock signal is worse adjust Z3 count clockwise to decrease the signal intensity about 20 then
24. hem now before continuing 1 To load a NOESY experiment jexp9 mf 1 9 wft full pw pw90 NOESY 2 Check parameters nt 32 or more for better signal to noise ratio ni 200 or more for a typical spectral width of 10 ppm but may be smaller for a smaller sw e g ni200 swizsw d1 2 seconds or longer phasez1 2 mix 0 25 0 5 250 500 ms the mixing time for the NOE build up The mixing time is correlated to the molecular weight of the molecule Generally 300 ms is adequate for MW gt 1000 and 500 ms for MW 1000 If a mixing time is too short it will not allow the NOE to buildup if a mixing time is too long it results in spin diffusion and you will get feak NOE peaks Sometimes a couple of NOESY experiments are collected for different mixing time mix 0 5 500ms is the default value 3 Check time adjust nt if necessary Make sure the sample is not spinning enter go _ to start After the acquisition is complete save your spectrum WORKUP use wft2da to process 21 9 Setting up the 1D H H NOESY Experiment 1D H H NOESY is a one dimensional version of 2D NOESY By selectively irradiating one proton with soft shaped pulse only its NOE to other protons can be detected This 1D experiment has advantages over 2D NOESY experiment by eliminating potential overlaps to provide an effective and clean NOE spectrum The following setup assumes you have done steps 1 7 in Setting up the H Experiment If you have n
25. hemical Shift References score Sere cra re ER ERE NR ETRAS SERE HEUS 30 H Structure Elucidation Quinine as an example 31 A Introduction to UISOONB Spectrometer 1 2 3 4 5 6 Spectrometer A Varian Agilent Inova 500 MHz spectrometer a three channel multinuclear solution FT NMR instrument with Z Pulse Field Gradient PFG capability All channels have waveform generators with pulse shaping capability Probe The default probe on this instrument is the indirect detection triple resonance 5mm H C N Z gradient probe hcn It is optimized for the observation of H ONLY and H detected 2D NMR experiments which means that it is best used for 1D IH and NOE difference 1D 2D H H COSY TOCSY NOESY ROESY and 2D H C or H N HMQC and HMBC and any other H observed experiments C sensitivity is so low 80 1 that running C observed experiments on this instrument is strongly discouraged In fact the APT and HETCOR experiments have been disabled on this instrument The standard C 1D experiment should be used solely for the purpose of setting the C parameters in experiments such as HMQC and HMBC A 5mm H P X Z gradient probe hpx is available as a backup and for experiments requiring different combinations of nuclei than H C PN A 10mm broadband probe PBN2 p is also available for this instrument However most experiments requiring this probe are normally done on the U500 VX
26. ift Referencing Schemes for N Compound o NH3 5 CH3NO gt Amines 49 429 NH 0 380 NH4NO3 21 359 NH4CI 39 341 Amides 119 261 CH3CN 243 137 Nitriles 258 122 Pyridine 319 61 Imines 343 37 NH4NO 376 4 CH3NO 380 0 Nitrates 388 8 30 H Structure Elucidation Quinine as an Example Quinine OH Chemical Formula C99H54N505 Exact Mass 324 18 Molecular Weight 324 42 H 1 H NMR proton data CDCI3 The results of H NMR spectroscopic analysis Figure H 1 are consistent with the structure of Quinine A total of 23 protons were observed examined by integration with a long T recovery delay The OH proton was missing in the spectrum due to its broadness including 5 aromatic protons 3 vinyl protons and 15 aliphatic protons The aliphatic protons was further separated to 4 methine 4 methylene groups and 1 methyl group as drawn from logic of further work shown below 15mg in 700uL CDCI 230 7220 210 L200 190 z 180 e p 170 Rs AQH 160 150 ue S Me e 140 Lt L L30 WA Sr 22 r120 r110 100 r90 18 fso r70 r60 Lso 40 TMS L3o e aee yg Bro b sob non Tt E mper SS EA L eo gE oI e rT 10 s E Rz ng B ER ee 2 i loo T T T T T T T T T T T T T T T T T T T T T T 9 0 8 5 8 0 75 7 0 6 5 6 0 5 9 5 0 4 5 4 0 25 3 0 2 5 2 0 1 5 1 0 0 5 0 0 0 5 fi ppm 1H Figure H 1 Proton Spectrum of Quinine H 2 C NMR carbon data CD
27. ls between sample spinning spin 20 Hz and none spinning spin 0 You may want to collect a quick 1D proton spectrum to inspect the quality of the shims D Setting up 1D 2D NMR experiments for structure elucidation of small organic molecules NOTE These instructions assume that you will be collecting a full data set for structure elucidation of small organic molecules including both HMQC inverse HETCOR or HSQC and HMBC inverse long range HETCOR on the same sample Instructions are also given for a 1D 2D TOCSY NOESY or ROESY data collection The experiment library will be as follows and you will have much less trouble if you always follow a routine such as this in terms of your data collection expl exp2 exp3 exp4 exp5 H spectrum H pw90 calibration or optional C spectrum with parameters for gHMQC and or gHMBC 2D H H gCOSY 2D H C gHMQC or 2D H C gHSQC 2D H C gHMBC The following experiments are optional but sometimes necessary exp6 exp7 exp8 exp9 exp10 expll expl12 2D H H gDQCOSY 2p H H TOCSY 1D H H TOCSY 2D H H NOESY 1D H H NOESY1D 2D H H ROESY 1D H H ROESYID 1 2 3 5 2 5 1 Setting up the H Experiment Insert your sample in the spectrometer Lock and shim the sample NOTE You can lock and shim while the sample is equilibrating if the temperature change is lt 5 However you should touch up the shims once the sample has equilibrate
28. ncentration Make sure the sample is not spinning enter dps to check the pulse sequence enter go to start After the acquisition is complete save your spectrum WORKUP use wft2da to process use twod to setup proper display window 15 5 Setting up the 2D H H gDQCOSY Experiment The following setup assumes you have done steps 1 7 in Setting up the H Experiment If you have not do them now before continuing 1 Toload a gDQCOSY experiment jexp6 mp 1 6 gDQCOSY 2 check ni it should be at least 256 for a typical spectral width of 10 ppm but may be smaller for a smaller sw Check diz1 2 s or the value optimized in expl pw pw90 np 4096 nt 4 or multiples of 4 for greater S N sw1 sw phasez1 2 ni 200 or 256 or more if you change np or ni readjust sb at 2 and sb1 ni swl and sbs at 2 and sbs1 ni swl squared sine bell with 90 degree shift if you change np or ni you may need to reset fn and fnl 3 Check time this experiment normally runs less than 20 mins for ntz4 Adjust nt if necessary Make sure the sample is not spinning To turn off go to the VNMR Acquisition window LC Lock LC spin off While this window is open make sure that the lock level is gt 60 you should adjust only the gain if possible When you start the experiment with go below make sure that the lock level stays above 20 enter go to start After the acquisition is complete save your spectrum WORKUP use wft2da to
29. ng enter go _ to start 19 After the acquisition is complete save your spectrum WORKUP use Wft to process Do not auto phase the spectrum aph First type phase 180 if necessary to have the irradiated peak in the positive direction then phase manually the TOCSY peaks will be in the same positive direction dssh or dssa to view all spectrum in array To view individual spectrum type dS where is the number of the desired spectrum To print all spectra in the array pl all pscale page To print individual spectrum ds pl pscale page or pl pir ppf pscale page add ppf or pir for peak picking and integration 20 8 Setting up the 2D H H NOESY Experiment The 2D H H NOESY experiment measures NOE Nuclear Overhauser Enhancement between protons within a molecule providing information about distance between two protons in space The stronger the NOE is the closer of the two nuclei locate Sign of NOE cross peaks 1 For small organic molecule all NOE cross peaks have the opposite phase to the diagonal peaks in contrast the cross peaks from chemical or conformational exchange are in phase with the diagonal 2 For large molecules all NOE cross peaks and the exchange cross peaks are in phase with the diagonal In such case ROESY experiment can be perform to distinguish between them if needed The following setup assumes you have done steps 1 7 in Setting up the H Experiment If you have not do t
30. oise ratio ni 200 or more for a typical spectral width of 10 ppm but may be smaller for a smaller sw e g n1 200 sw1 sw d1 2 seconds or longer phasez1 2 mixz0 1 0 3 100 300 ms the mixing time for the ROE build up The mixing time is correlated to the molecular weight of the molecule Long mixing time is needed for a small molecule Generally 100 150ms is adequate for MW 400 2000 and 200 300 ms if you want to observe longer mixing time ROE for MW lt 400 300 500 ms for long mixing time is generally used All ROE cross peaks are in the opposite sign of the diagonal peaks while TOCSY or exchange peaks are in phase with the diagonal mix 0 2 200ms is the default value 3 Check time adjust nt if necessary Make sure the sample is not spinning enter go _ to start After the acquisition is complete save your spectrum WORKUP use wft2da to process 24 11 Setting up the 1D H H ROESY Experiment ID ROESYID is a one dimensional version of 2D ROESY Rotating frame Overhauser Effect Spectroscopy experiment By selectively irradiating one proton with soft shaped pulse only its ROE to other protons can be detected This 1D experiment has advantages over 2D ROESY experiment by eliminating potential overlaps to provide an effective and clean ROE spectrum The following setup assumes you have done steps 1 7 in Setting up the H Experiment If you have not do them now before continuing 1 To load a 1D ROESY exp
31. ond coupling correlations Its default value is 80 ms for a small molecule of size less than 1000 da The larger the molecule is the shorter of the mixing time The following setup assumes you have done steps 1 7 in Setting up the H Experiment If you have not do them now before continuing 1 To load a TOCSY experiment jexp7 mp 1 7 TOCSY 2 Check ni it should be at least 256 for a typical spectral width of 10 ppm but may be smallerfor a smaller sw Check d1 1 2 s or the value optimized in expl pw pw90 np 4096 or smaller e g 2048 nt 2 or multiples of 2 for greater S N SW1zsw phasez1 2 ni 200 or 256 or more mix 0 08 80 ms or other value as described above if you change np or ni readjust sb at 2 and sb1 ni 2 sw1 if you change np or ni you may need to reset fn and fnl 3 Check time this experiment normally runs less than 20 mins for nt 2 Adjust nt if necessary Make sure the sample is not spinning To turn off go to the VNMR Acquisition window LC Lock LC spin off While this window is open make sure that the lock level is gt 60 you should adjust only the gain if possible When you start the experiment with go below make sure that the lock level stays above 20 enter QgO to start After the acquisition is complete save your spectrum WORKUP use wft2da to process 18 7 Setting up the 1D H H TOCSY Experiment 1D TOCSYID is a one dimensional version of 2D TOCSY By selectively ir
32. ot do them now before continuing 1 Toload a 1D NOESY experiment jexp10 mf 1 10 wft full pw pw90 NOESY1D ds 2 Expand around first peak to be irradiated inverted Center cursors around desired peak ensuring that the full peak is between the two cursors then LC Select Selects current region between cursors for selective inversion Repeat Select for all desired peaks When completed LC Proceed Calculates shaped pluses and completes NOESY 1D setup You will need to answer the following questions Enter reference 90 degrees pulse with usec Enter your determined pw90 from pw90 calibration you ve done early Enter reference power level Enter the tpwr value used in pw90 calibration da Displays array Make sure all your desired peaks have been selected They will appear with name similar to NOESY 1D_5_67p RF where the numbers refer to the chemical shift of the peak 3 Check nt 64 or 128 or 256 for a better S N d1 normally 2 seconds or longer mix 0 5 500ms or other value as describe in 2D NOESY section 4 Check time adjust nt if necessary 5 Set bs 16 or 32 il y so that the data will be collected for the first FID first arrayed peak in the block size of 16 or 32 then go to the second FID instead of finishing the first FID first arrayed peak then go to the second arrayed peak Such if the S N is good enough you can always stop the experiment Make sure the sample is not spinning
33. places first array element 20 with the value 5 for setting up the correct phase da Displays current arrayed values for pw d1 10 ai vsadj vp 70 Sets absolute intensity mode and adjust the peak hight places spectrum about half way up on the display ga Make sure gain a number do not use auto gain 10 As the spectra accumulate use dssh dssl to view them You can terminate the experiment with aa when you have determined the pw360 where the signal is near zero NOTE if the second spectrum is already positive reset the array with a smaller starting value After you determine the pw360 of your sample jexp1 pw90 the numeric value for the 360 found above 4 e g pw90 pw360 4 pw pw90 ga 6 Quick determination of the T for the sample more details of this operation can be found in the SCS website named T1 Measurement NOTE this step is optional For most small molecules 1 5 seconds of delay d1 is normal You could use 2 0 2 5 seconds for optimal signal to noise mp 1 2 jexp2 gainzthe number found previously doti lt rtn gt NOTE The next three items are the answers to the questions posed by the dot1 macro Minimum T1 expected 0 5 lt rtn gt Maximum T1 expected 5 lt rtn gt Number of scans 1 lt rtn gt ga As the spectra accumulate use dssh to view them You can terminate the experiment with aa when you have determined the T of interest Ti 1 443 x T null Derived from M2M 1 2e 7T 5
34. radiating one proton with soft shaped pulse only its spin spin correlation to other protons can be detected This 1D experiment has advantages over 2D experiment by eliminating potential overlaps to provide an effective and clean TOCSY spectrum The following setup assumes you have done steps 1 7 in Setting up the H Experiment If you have not do them now before continuing 1 To load a 1D TOCSY experiment jexp8 mf 1 8 wft full pw pw90 TOCSY1D ds 2 Expand around first peak to be irradiated inverted Center cursors around desired peak ensuring that the full peak is between the two cursors then LC Select Selects current region between cursors for selective inversion Repeat Select for all desired peaks When completed LC Proceed Calculates shaped pluses and completes TOCSY1D setup You will need to answer the following questions Enter reference 90 degrees pulse with usec Enter your determined pw90 from pw90 calibration you ve done early Enter reference power level Enter the tpwr value used in pw90 calibration da Displays array Make sure all your desired peaks have been selected They will appear with name similar to TOCSYID 5 67p RF where the numbers refer to the chemical shift of the peak 3 Check nt 16 or 32 for a better S N d1 normally 2 seconds or longer mix 0 08 80ms or other value as describe in 2D TOCSY section 4 Check time adjust nt if necessary Make sure the sample is not spinni
35. trum You can use either an internal chemical shifts standard e g TMS 0 ppm or a residual H resonance from the deuterated solvent e g 7 26 ppm for CDCl3 Less commonly you can use a capillary containing a standard to avoid any chemical shifts changes due to solvent conditions such as pH and concentration etc B Spectrum The commonly used HB chemical shift standard is 15 v v BF3 OEt2 in CDCl and is referenced to 0 0 ppm When you use the standard parameter loaded from the manual bar the BF OEt in CDCl will appeared at 0 0 ppm PC Spectrum Commonly a residual PC resonance from the deuterated solvent is used as the reference PF Spectrum The chemical shift standard for F is neat CFCls which is referenced to 0 0 ppm which giving TFA at 73 6 ppm or 0 05 Ce6HsCF3 in CeD at 62 9 ppm P Spectrum 85 phosphoric acid H3PO4 in D2O is used giving a reference peak at 0 0 ppm or 0 0485M triphenyl phosphate in CDCl is reference at 17 9 ppm with respect to the phosphoric acid PN Spectrum Different referencing schemes have been in common use as reported in literature Setting the chemical shift of nitromethane at 0 0 ppm results in most compounds have negative values of PN resonances The chemical shift of liquid ammonia used for bio samples is 380 ppm away from nitromethane used for organic compounds Here is the table listing the N Chemical shifts expressed with respect to both reference compounds Chemical Sh
36. ys above 20 enter go to start After the acquisition is complete save your spectrum WORKUP use wft2d to process Note The gradient gCOSY experiment provides homonuclear chemical shift correlation information via the J coupling interaction revealing 2 bond germinal and 3 bond vicinal spin spin coupled pairs Sensitivity of this experiment is not normally an issue it is acquired and processed in absolute value magnitude mode and usually requires no more than a few minutes to acquire Other COSY type experiments are given later in the document the gradient double quantum filtered phase sensitive COSY experiment gDQCOSY provides information about the adjacent two spin systems TOCSY experiment has a very good S N ratio revealing the spin spin correlation throughout the spin systems in a molecule TOCSY1D ID version of 2D TOCYS giving a clean one spin spin system at a time 12 3 Setting up the 2D H C gHMQC or gHSQC Experiment The following setup assumes you have done steps 1 7 in Setting up the H Experiment If you have not do them now before continuing You should have an optimized H spectrum setup in expl before proceeding with the setup below 1 2 3 4 5 6 Optional jexp2 and set up parameters for a standard C experiment nt 1 or 4 should be sufficient to see the solvent peak Acquire a spectrum with ga then phase and reference Check SW and change with movesw if desired Remember if you ar
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