Solid-State 2H NMR Spectroscopy: Experimental Training and Data Reduction Methods

Structure of HMBHMB is used as a standard for optimizing parameters in Solid state 2H NMR spectroscopy. (NB. Different such standards are used for the each NMR active nucleus)

Figure 2. Structure of HMB (Courtesy DOI: 10.1021/ed2004774)

Setting up the Experiment

Instrument : DRX 500 Sample : Hexamethyl benzene (HMB-d18)

The ProbeThe probe should be identified according to the nucleus of interest and the experiment you need to perform. During opening of the cup of the probe (to insert or to remove samples) you should keep your free hand palm in such a manner so that even.

The SamplesThe sample containing bottle should be opened and vial should be out on the table so that you can insert the sample into the probe coil immediately after opening the cap of the probe. All the other samples which are not in use should be kept away from the working area of the table. Always be careful not to mix samples as they all look alike once you take them out of the bottle. Probe Insertion1. Identify the correct probe by checking the bottom part of the probe.2. Open the brass cap and keep both on the sample table.3. Open sample containing vial and leave it on the table so that sample is ready to

be inserted into the coil.4. Insert the sample into the coil. Make sure that the part of the sample tube

containing the compound is centered in the coil.

5. Once the sample is properly inserted close the cap gently. (Make sure that youclose it in such way that the threading is not affected)

6. Insert the probe into the magnet tight the screws gently.7. Insert the heating filament, the thermocouple and plug in the air flow and the

Radio Frequency (RF) cable.8. Switch the Heater on.9. Type the command edte, if the Temperature window is not opened already. On

the temperature window adjust the Temperature to the desired Temperature and wait for 30 minutes.

Tuning an NMR probe1. Unplug the receiver cable from l/4 box and connect to the back panel of the

amplifier. (Note: Try not to move the l/4 box that much as the welded wires can be loosen)

2. Run the command wobb on your console and click on “Acquire”- “Observe fid window”

3. Reconnect the transmitter end of the probe to the below port of the front panel of bruker amplifier.

Figure 3. Imagine before tuning the NMR probe

4. Go to the magnet and start adjusting the tuning capacitor so that the spectrum is centered at the larmor frequency (when tuning, the green lights in the l/4 box will blink). Also, you can observe the LED indicators in the amplifier itself to monitor the position of the spectrum.

Figure 4. Screen shot of the computer after tuning the NMR probe

5. Once the spectrum is centered type stop and hit enter.6. Reconnect the receiver and the probe cable back to the l/4 box.

Running NMR Experiments - (for information on what each command does, Example zg = start the experiment, please see Appendix II located near the end of the packet)

Experiment #1 - Determination of RF pulse width of 90 degree (p1)1. Go to File--Open Data Sets by Owner to go to your account. Go to File--Open

Data Sets by name to go to your working directory (eg. /u/data/vikram/nmr/hmb/)

2. Change your directory to a experiment number where a similar experiment was performed by typing “re” (exp. #)

3. Copy experimental parameters from that experiment to your current experiment by running the command “edc” and changing the experiment number there to the current experiment number ( eg 111)

4. Check the copied settings using the command “eda”.5. PulProg : Quadecho_300pc6. TD = 81927. NS = 168. DW = 0.5 micro sec9. RG = 2010. After saving this parameters, make sure that the d1,d6 and d7 values are 1.0

sec, 42.0 micro sec and 5.0 micro sec respectively by entering individual parameter name followed by hitting enter button (you can also check all parameters at once by using “edsp” command).

11. Then set p1 to 1.0 microsec and go up to 12.0 microsec in 0.5 microsec increments.

12. Enter “zg” to start the experiment. Click ‘Acquire’ to see what the spectrum is like and to see how long it takes to end.

Experiment #2 - Determination of T1 1. Insert the probe as we discuss above. 2. Set temperature to be 303.2 K and wait 30 min for temperature to be stable.3. Tuning the NMR probe as we discuss above.4. Enter edc and change experiment number.5. Enter eda and changre pulse program: quadir_malli6. Change 1d to 2d, save7. Enter eda and go to vdlist8. Edit vdlist: 10u; 25u; 50u; 1m; 5m; 10m; 20m; 40m; 60m; 80m; 100m; 200m;

400m; 600m; 800m; 950m9. set F1=16, save10. Change parameters: d1=1 s; P1=5.4 us; TD=8k; dw=0.5 us; NS=32 using ased

command13. Enter zg to start the experiment. Click Acquire to see what the spectrum

is like and to see how long it takes to end.

Take out the probe1. stop the heater2. take out air tube3. take out the heater4. take out the thermocouple5. unscrewed the probe6. open cap7. take out the sample8. store the sample back to labeled sample vial9. close the cap

Transfer and processing NMR Data in your PC (related Appendix; I/II )

1. Transfer the data from the magnet room to a lab computer● Copy the data transfer software from other lab members and paste (user ready

folder) it on convenient location (ex: desktop))of PC.● Double click on the file “WS_FTP32” to initiate the software and you will see the

software initiation window as shown in Figure 5.

Figure 5. Selection of spectrometer

● Select the spectrometer (AMX300/500, as highlighted in the figure 5) from which you would like transfer experimental data.

● The “User ID” and “Password” for each magnet should automatically load.● Click “OK”

Figure 6. File transfer window

● As shown in Figure 6, the column on the left is the local computer “the computer that you are on currently”, where you want the data to go to. (appropriate folder)

● The column on the right contains the files stored in the spectrometer, highlight your file, and click the left arrow “←” to move files over from the spectrometer to the computer you are on, so that you can MATLAB and Origin process this data later.

● You cannot do this for an incomplete experiment (However, if you are more anxious about your data, you can do so, by tentatively storing the data on spectrometer by “tr” command at spectrometer end. I would advise not to use this command frequently).

● The 3 major things to take away from this section are 1) the type of magnet that you ran your experiment on (Ex. amx300, etc.) , 2) The columns on the left and right are very much like how you navigate through Computer folder, for example C://User/EJ/Desktop/WordDocument., and 3) The “ ← ” button is the initiator to move the data over, Ex. (Copy)4) Do not transfer more than 2 folders at one click with amx300

● This is an image of the software , notice the “ ← ” transfer button in the center.

Matlab Processing

The NMR spectrometer generates data in the form of free induction decay. Using Fast Fourier Transforms, the free induction decay, displaying intensity as a function of time, can be transformed into a spectrum of intensity as a function of frequency. This spectrum can then be numerically deconvoluted or depaked to yield spectra corresponding to macroscopically oriented samples. This process is accomplished using MATLAB.

1. Open MATLAB software2. Go to File - New - Script and copy and paste the script given in Appendix I below

Figure 7. Matlab window

After pasting as below save the file with customized name at customized location. This becomes a standard MATLAB code (Driving program file).

Figure 8. Matlab driving program

3. Alternatively get it from your senior (This is only a driving program, which in turn uses background programs stored in C..(location)....\MATLAB\Proc\BrukerConv1” for the experiment (if proper path has been set in Matlab software)

2. Change the file root in the matlab script Change “RawDir/ProcDir” location according to the location of your

experimental data in that computer (prefer to keep both locations same).In “FIDFile=*” line, change ‘*’ as ‘fid’ for 1D experiment and ‘ser’ for 2D

experiment (only 2D experiment contains ‘acqu2’ file).In Endians=’*’ line, change ‘*’ as ‘l’ for ‘amx300’ and ‘b’ for ‘amx500’

Change “Outfile/Specout/Depakeout” names should contain type of experiment, spectrometer, samplename, and temperature as a single name with different extensionsEX: Spin lattice relaxation time (T1) experiment performed on dmpc with 50% water sample with amx 300 spectrometer at 30C can be labelled as below

Outfile: T1amx300_dmpc50pw_30C.fid Specout: T1amx300_dmpc50pw_30C.mat DepakeOut:dpT1amx300_dmpc50pw_30C.mat (with dp prefix)

● check the values of td, swh according to the the file acqu.● To find the “acqu” file, go to Local Disk C: → Experiments → “Your name” →

“experiment number” →. Open the acqu file with wordpad, scroll until you find td and swh and input those values into the MATLAB code.

● Td2 can only be found in acqu2 (appears simply as TD in the acqu2 file) and you will only have an acqu2 file if you ran a 2D experiment. If the experiment is a 1D experiment, Td2 = 1.

● An example of what the acqu and acqu2 file looks like is found in Appendix I, located near the end of the packet, along with a bolded td, swh, and td2 within the example file.

3. change the value of max, theta and points, ● Max = The max value (line ~21 of code) is determined by the highest peak (or

highest peak region) of Figure 1, once you run the code. You click on Figure 1 and click on the “￮+” (magnifying glass) tool of the figure to zoom in on an area. You pick the most suitable/highest/sharpest peak area, whether negative or positive, (it automatically takes the absolute value) by zooming in on the Figure 1 graph. If it is not obvious to pick an exact number, pick a number within that region of the peak. Take this number and input it into the line “Max =__” in the MATLAB code of the experiment that you ran. You can then press F5 to save and run the experiment with your new max. I think of the graph in Figure 2 being shifted upwards and downwards across the horizontal reference line, , when ┅the Max is changed.

● Theta = You adjust the theta value accordingly to “shift“ the data, closer to the reference line. You do this by picking random numbers to some extent....The “reference line”, the dashed vertical line, , serves as a mirror/symmetry line, ┆and the theta value adjusts the two small “slopes” ( and ) on both sides of ⤵ ⤴the “shoulders” of the graph. Your goal is to “even” out the slopes to have them rest on top of the horizontal dashed reference line, , as a horizontal line, , as ┅ ↭much as possible. Depending on the type of experiment, either T1 or T2, the slopes will look like , or, , respectively, with “ “ being the ⤵ ⤴ ⤴ ⤵┆ ┆ ┆vertical dashed reference/mirror/symmetry line.

● Note : You could have a different graph with different maxes and thetas, try multiple options! I start with the max and then find a suitable “theta range”, that evens out the slopes the best, and from there I see if other maxes would work better.

● Points = “Points” is the easiest of all 3 to change. The points =___ can be any number, and all it does is shift the vertical reference line, , to the left or right. ↑ ┆the # of points and the ref. line will shift to the right, and ↓ the # of points and the ref. line will shift to the left. You want to zoom in on the topmost portion of the peaks on the graph and adjust the points so that these peaks are positioned equidistant from the zero point.

● Note: If you are doing data analysis for a 2D experiment, make sure to have the Brukerconv1 file opened and change the number of “cntr” in that file from 0 to td2-1 for each change in loop number (Ex. In an experiment where TD = 16, the value of cntr will be changed from 0-15)

Data Analysis - Igor (for lipid samples)

In order to determine structural parameters for a lipid sample, it is first necessary to associate the peaks of the depaked spectrum with the various positions along the acyl chain. A Lorentzian function can be fit to the spectrum to integrate the depaked resonances (see figure 12). The areas of the depaked reference spectra are directly proportional to the number of deuterons associated with each resonance. Therefore, by integrating the reference spectrum one can assign carbon indices to each depaked resonance.

1. Process spectra with Matlab program2. Copy the columns into Igor worksheet – depaked data for lipid samples

a. Data > Load Wavesb. Check the “make table” boxc. Click “file” and find your depaked file d. Click “Do it” to load the columns into Igor

Figure 9. Depaked data loaded into Igor table

3. In Igor:a. Windowsb. New graphc. Select columns

4. Compile Macrosa. Windows > Procedure Window

b. #include <Peak Functions>c. #include <Multi-peak fitting 1.3>d. Click “compile” in the lower part of the window

Figure 10. Procedure window5. Run macros

a. Macrosb. Create fit setup panel

6. In fit window (macros > create fit setup panel)a. Choose data axes (Xwave = wave0, Ywave = wave1)b. Graph spectrum

Figure 11. Fit Setup Panel and Spectrum Graph

c. Expand the graph in the area in which you want to assign peaks

Figure 12. Fit Setup Panel and Spectrum Graph after focusing in on the peak area (DMPC sample)

7. Peak Pickinga. Go to man in Fit Setup Panel, and insert new peakb. Set number of peaksc. Delete or insert peaksd. Adjust peak position - you can do this by adjusting the amplitude, position, and linewidth in the Fit Setup Panel or manually with your mouse on the graph itselfe. Click on finish insert

8. Do fit (fit Lorentzian)a. Adjust + readjust depending on how well Igor fit the spectrum, then fit again

9. Save fita. Fit panel save

10. Macrosa. Runmeaftermanualfit

11. Macrosa. PrintPeakParamsb. Outfile area and frequencies

12. Import into Origin filea. Use frequencies to determine SCDb. Use areas to assign carbon positions

1. Sum the areas of all of the peaks2. For each peak, take the area of the peak, divided by the total area, and then multiply the result by 54 (since there are 54 total deuteriums)3. Make assignments based on the number of deuteriums (see Dodd’s paper for more explanation)

General rules of peak assignment for DMPC

Figure 13. DMPC Molecule

1 flexibility(sn1)>flexibility(sn2)2 flexibility(tail)>flexibility(head)3 flexibility(sn1 M)>flexibility(sn2 M)>flexibility(sn1 M-1)4 greater splitting lead to smaller flexibility

* the Sn1 chain is also known as the ‘a’ acyl chain while the Sn2 chain is the ‘b’ acyl chain

The Sn1 chain is more flexible than the Sn2 chain, corresponding to decreased segmental order parameters and thus smaller quadrupolar splittings. The C2 carbons of the acyl chains, closest to the glycerol backbone, are coplanar with the carbonyl double bond as well as the partial double bond character of the ester. This configuration results in the restriction of segmental motion. As a result, segmental order parameters decrease in magnitude from the C2 carbon to the terminal methyl group. See Dodd’s thesis for a more detailed analysis

Data Analysis - Origin (for lipid and HMB samples)

Once the peaks of the NMR spectrum have been associated with specific carbon indices and the frequencies corresponding to each peak recorded, a number of characteristic quantities can be calculated for the lipid bilayer/HMB and compared for various temperatures and osmolyte amounts. For the remainder of the analysis, we use Origin.

1. Open software Originpro 8.2. Import data from Matlab to Origin. Click on the icon “Import Multiple ASCII” on

the toolbar. (it should be beside the print icon) Select the box which says “import each selected file into a new workbook”. Find the experiment data you want to process and import them into origin. (if it is HMB, use not depaked data; if it is lipid, use depaked data)

3. Open a new Workbook (under the “Edit” tab), copy all columns into this new

workbook ( the frequency values for an experiment should be the same for each parameter; just copy one time as the x-axis). After all copy-paste are done, you can delete all separated columns if you want. Give proper name to all columns. The x-axis should be frequency and all other columns should be the parameter we are changing (Ex. delays)

4. Plot a graph for all data. Select all columns (by clicking the blank “slab” above “Long Name, and next to “A(x)”, in the box) and plot them with solid line. The icon for graphing with a solid line is on the bottom-left of Origin.

5. To maximize the graph and change the scale of axis properly. Remember that, for x-axis, it should be from positive value to negative value, from left to right.

6. Restore the graph so you can see the box again. For lipid samples, choose the column of parameter which gives you the best spectrum (always the first one or the last one) and plot a graph. In this graph, you should only have one spectrum. Name it “peak assignment”.

7. In the “peak assignment” graph, assign peaks (Ex. L1, L2, R14 etc.). By right clicking you can find “add text”.

8. For each peak, find the x-value of the maximum and then record this x-value and make a table for all peaks. (you need to use the “Data Reader” tool which is in the toolbar”). These frequencies will be compared to the frequencies obtained in Igor to find the error in the position of each peak

9. If you are doing an HMB analysis, you will need to graph the frequencies against the intensity data for each delay. Therefore, you will have a graph corresponding to each delay. The frequencies of both the left and right peaks for each of these graphs will need to be recorded.

Figure 14. Spectrum for a DMPC sample at a particular temperature in Origin

10. Create a new Workbook and name it as “peak values”. The x-axis column of this table should be the values we give for the parameter we are changing (Ex. delays), the y-axis columns should be the intensity of the peak we assigned.

11. The next steps should be based on what kind of experiments you are doing, thus you make different tables and use different equations to fit and reduce your data.

For HMB Experiments: 1. You should now create the workbook that will be used to find the T1 values for

each peak of each delay. The x-axis column of the workbook should be the delays (make sure that they are all written in the same units so that they can be graphed appropriately). For the y-axis columns, you will need to reference the left/right frequencies values of each peak for each delay. For example, for the data corresponding to a 20m delay, you have already recorded the left and right frequencies of the resulting spectrum (either in a separate workbook or in a table located on the graph). Take the left frequency value, find it in the original Matlab data table, and copy the entire row corresponding to that frequency (so that you are copying the data from each delay corresponding to the frequency). Now you will paste (transpose) those numbers into your new workbook. Title the column

“Left 20m” or something that will easily distinguish that data from the data corresponding to other peaks and/or delays. This procedure will need to be completed for each peak for each delay.

2. To find the T1 value corresponding to each peak of each delay, you next need to graph the delays vs. each of the y-axis columns that you just created. Continuing with the example used in the previous point, this graph will be used to find the T1 value for the left peak corresponding to the 20m delay. Create a scatter plot of the data by clicking on the scatter plot icon in the lower left corner of Origin Pro.

3. With the graph created, make the necessary changes to the labels and title of the graph so that you can easily keep track of the graphs created (there will be a lot).

4. We next need to fit a function to the graph: go to “analysis” - “fitting” - “non-linear curve fit” - open dialog

5. You will probably need to create a new function so under “Category”, select “New Category”. Then change the function name to something that you will easily remember.

6. The function you will be using has three parameters. So in the parameters box of the window, type in “M0, T1, Mz” (without quotations)

7. For the actual function, type y = M0*(1-exp(-x/T1))+Mz*exp(-x/T1)8. If you are doing a T2 analysis, the necessary equation is y = M0 + Mz*exp(-x/T2)9. Click “save” and then click “ok” - this should take you back to the original function

screen10.With your function now defined and selected, we need to estimate the

parameters. 11.There should be a tab on the same window that says “parameters”. Once you are

in this tab, estimate the values of M0, T1, and Mz. M0 corresponds to the y-max of a particular data set, T1 corresponds to the x-intercept (or x-min), and Mz corresponds to the y-min.

12.With the parameters set, click on the first iteration icon, followed by the second iteration icon, and then click “fit”

13.A table should print out on your graph with the calculated T1 value and error. 14. In a table containing the delays and left and right frequencies for each delay, also

include columns for the left and right T1 values and the corresponding error values.

15.Once you have obtained the T1 values and errors for each peak and each delay, you will add columns to your workbook that contain various equations in order to calculate SCD (segmental order parameters) and Rlz (related to relaxation) values. Get these equations from a template file.

16.Graph SCD^2 vs. Rlz (including error!) as a scatter plot. Make sure to change the SCD^2 column to an x-axis label and the Rlz Error column to a yEr label (right click on column and go to “set as”)

17.Go to analysis - fitting - fit linear - open dialog - OK

18.The end result should be a graph depicting this linear fit as well as a table from the fitting procedure that states the intercept and slope values and relative errors. You should also include a table (go to graph - new table) that contains the delays as well as the SCD^2, Rlz, and Rlz error values so that the data as well as the graph can be easily analyzed if needed.

*Notes: ● For each graph, you should right click, go to “add text” and record the sample

name, the temperature at which the experiment took place, and the spectrometer used

● If you are working with lipid samples and doing this same type of analysis, you will need to complete two linear analyses; one for each acyl chain

For DMPC Experiments:1. Much of the data reduction process for lipid samples involves inputing data into

equations to find the values of specific parameters. As a result, it is helpful to use a template for these types of analyses in which you can just enter the values that change between each data set without having to create all the tables and graphs again.

2. For lipid experiments, you need to incorporate the frequency values obtained from Igor fitting. These frequency values will be compared with those obtained through Origin (procedure detailed above) to calculate the frequency error value. Aside from the obtaining the Igor frequency values and following the peak assignment procedure, the rest of the data reduction process can be easily gleaned from a DMPC template file. In this file, the results from Matlab, Igor, and Origin will be combined to produce the final desired graphs and values.

Appendix I Standard Matlab driving program script

**********************************************************************************k=1024; % Use power of 2 for fft.%-----------------------------------------------------------------------------% Set relevant acquisition parameters (see file acqus in Bruker data directory)%-----------------------------------------------------------------------------

td=8*k %32768; % Number of data points collected in the time domain (complex). Choose it from "acqus"

td2=1; % Number of relaxation experiments (As a second dimension). Use Choose it from "acqu2s"

% td2=1 for a 1-D experiment (eg. Quadrupolar Echo). % Note: need to look at TD in "acqu2s" if a 2D data setSWH=1000000; % The spectral width in Hz.

Choose it from "acqus"

%-----------------------------------------------------------------------------% Set user defined parameters%----------------------------------------------------------------------------- LB=50; % For exponential foot chopping (apodization).SI=64*k; % The number of elements in the real spectrum (must be a power of 2 for fft). STSI=32*k; % Specify size of strip transform (reduce number of points in % in the output spectrum).STSR=8*k; % Starting point for strip transform.Max=42; % Choose the maximum value from the interpolated Fid.IF=1; % Interp factor; need to be careful because information will be % lost from the Fid if IF is chosen too big (use power of 2).theta=-109; % set angle (degrees) for phasing.Points=-15; % Parameter for minor shifts prior to depaking (in points).Is_bin='n'; % to spectrum output in matlab binary (use .mat extension).%-----------------------------------------------------------------------------% Set filenames and directories%-----------------------------------------------------------------------------

% Set the directory of the raw data file (give the entire path)% Use forward slash for unix, backslash for windows, and a colon for the mac

RawDir='F:\KJM\KJMNew\2012experiments\UG teaching\Suchi\amx300hmb\115';

% Set the directory that you want your files to be saved intoProcDir='F:\KJM\KJMNew\2012experiments\UG teaching\Suchi\amx300hmb\115'; % Specify the base name of the free induction decay output files (ascii).Outfile='T1amx300_hmb_exp115_Suchi.fid';FidFile='ser'; % Use 'ser' for 2-D and 'fid' for 1-D.Endians='l'; % use big- or little-endians (byte format).SpecOut='T1amx300_hmb_exp115_Suchi.mat'; %save spectrum in multiple column format ([field inten]) as asciiDepakeOut='dpT1amx300_hmb_exp115_Suchi.mat'; %Give filename of depaked spectrum.

%-----------------------------------------------------------------------------%-----------------------------------------------------------------------------% Do not modify below this point.%-----------------------------------------------------------------------------%-----------------------------------------------------------------------------

%-----------------------------------------------------------------------------% Run data conversion and Fourier Transformation.%-----------------------------------------------------------------------------ticCurrDir=pwd;figure(1);clf;figure(2);clf;figure(3);clf;figure(4);clfFid=BrukerConv1(FidFile,Endians,td,td2,RawDir,ProcDir,Outfile);

% following to give zero baseline:Fid(Max)=0.5*Fid(Max);

cd(ProcDir)[Freq,Spectra,Fid_Apod,t]=Fourier2(Fid,td,SI,IF,theta,Max,SWH,LB,Points,STSI,STSR);packif (Is_bin(1) == 'Y') | (Is_bin(1) == 'y') RealBin(SpecOut,Freq,Spectra);else RealAscii(SpecOut,Freq,Spectra); endclear Freq Spectra%-----------------------------------------------------------------------------

% Do some depaking.%-----------------------------------------------------------------------------

[FreqDep,SpecDep]=Depake(t,Fid_Apod,SI,IF,Points,SWH);packif (Is_bin(1) == 'Y') | (Is_bin(1) == 'y') RealBin(DepakeOut,FreqDep,SpecDep);else RealAscii(DepakeOut,FreqDep,SpecDep);endelap=toc;elap/60cd(CurrDir)axis([-100 100 -8E8 8E8]);

************************************************************************************

Section: ＂Ｐｒｏｃｅｓｓｉｎｇ Ｄａｔａ＂● Here is an example of what the acqu file looks like once you locate and open it.● Please scroll down to see what it is exactly that you’re looking for (TD and SWH)● I did not include the acqu2 file, it is nearly the same, except the ONLY data that you get

from that file is TD2, not TD and SWH

##TITLE= Parameter file, UXNMR Version 941001.4##JCAMPDX= 5.0##DATATYPE= UXNMR Parameter Values##ORIGIN= UXNMR, Bruker Analytische Messtechnik GmbH##OWNER= root$$ Sun Feb 15 01:48:55 1998$$ File: /u/data/malli/nmr/60%peg1500/101//acqu##$AQ_mod= 1##$AUNM= <au_zg>##$BF1= 46.0722##$BF2= 300.13##$BF3= 300.13##$BF4= 500.13##$BF5= 500.13##$BF6= 500.13##$BF7= 500.13##$BF8= 500.13##$BYTORDA= 0##$CNST= (0..31)

1 1 145 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1##$CPDPRG= <>##$CPDPRG1= <mlev>##$CPDPRG2= <mlev>##$CPDPRG3= <mlev>##$CPDPRG4= <mlev>##$CPDPRG5= <mlev>##$CPDPRG6= <mlev>##$CPDPRG7= <mlev>##$CPDPRG8= <mlev>##$CPDPRGB= <>##$CPDPRGT= <>##$D= (0..31)0 0.6 0 0 0 0 4e-05 5e-06 7.55e-05 0 0 0 0 0 0 0.2 0 0 0 0 0 0 0 0 0 00 0 0 0 0 0##$DATE= 0##$DBL= (0..7)120 120 120 120 120 120 120 120##$DBP= (0..7)150 150 150 150 150 150 150 150##$DBP07= 0##$DBPNAM0= <>##$DBPNAM1= <>##$DBPNAM2= <>##$DBPNAM3= <>##$DBPNAM4= <>##$DBPNAM5= <>##$DBPNAM6= <>##$DBPNAM7= <>##$DBPOFFS= (0..7)0 0 0 0 0 0 0 0##$DE= 4.5##$DECBNUC= <off>##$DECIM= 1##$DECNUC= <off>##$DECSTAT= 4##$DIGMOD= 0##$DIGTYP= 3##$DL= (0..7)10 120 120 120 120 120 120 120##$DP= (0..7)

150 150 150 150 150 150 150 150##$DP07= 0##$DPNAME0= <>##$DPNAME1= <>##$DPNAME2= <>##$DPNAME3= <>##$DPNAME4= <>##$DPNAME5= <>##$DPNAME6= <>##$DPNAME7= <>##$DPOFFS= (0..7)0 0 0 0 0 0 0 0##$DR= 12##$DS= 0##$DSLIST= <SSSSSSSSSSSSSSS>##$DSPFIRM= 0##$DSPFVS= 0##$DTYPA= 0##$EXP= <>##$F1LIST= <111111111111111>##$F2LIST= <222222222222222>##$F3LIST= <333333333333333>##$FCUCHAN= (0..9)0 0 0 0 0 0 0 0 0 0##$FL1= 90##$FL2= 90##$FL3= 90##$FL4= 90##$FOV= 20##$FQ1LIST= <freqlist>##$FQ2LIST= <freqlist>##$FQ3LIST= <freqlist>##$FQ4LIST= <freqlist>##$FQ5LIST= <freqlist>##$FQ6LIST= <freqlist>##$FQ7LIST= <freqlist>##$FQ8LIST= <freqlist>##$FS= (0..7)83 83 83 83 83 83 83 83##$FTLPGN= 0##$FW= 1250000

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150 150 150 150 150 150 150 150##$TP07= 0##$TPNAME0= <>##$TPNAME1= <>##$TPNAME2= <>##$TPNAME3= <>##$TPNAME4= <>##$TPNAME5= <>##$TPNAME6= <>##$TPNAME7= <>##$TPOFFS= (0..7)0 0 0 0 0 0 0 0##$TUNHIN= 0##$TUNHOUT= 0##$TUNXOUT= 0##$USERA1= <user>##$USERA2= <user>##$USERA3= <user>##$USERA4= <user>##$USERA5= <user>##$V9= 5##$VCLIST= <cpmg14_malli>##$VD= 0##$VDLIST= <DDDDDDDDDDDDDDD>##$VPLIST= <PPPPPPPPPPPPPPP>##$VTLIST= <TTTTTTTTTTTTTTT>##$WBST= 1024##$WBSW= 4##$WS= (0..7)83 83 83 83 83 83 83 83##$XGAIN= (0..3)0 0 0 0##$XL= 3##$YL= 3##$YMAX_a= 0##$YMIN_a= 0##$ZL1= 120##$ZL2= 120##$ZL3= 120##$ZL4= 120##END=

Appendix II Section: “Running NMR experiments”

● In this appendix you will find a list of the most common/useful commands that you will use for running experiments. These commands get typed into the machine in the magnet room.

Most useful commands● edc Change experiment from one to another with every setting the same.● edsp check settings● wobb start the tuning process● expt estimate the experiment time● tr transform the spectrum we get this point● stop stop the process● eda acquisition parameters● TD data collect● NS number of trials of experiment● zg start the experiment● halt stop the experiment and store the data till that moment.● re return to older experiment data

References:

A. Abragam, The Principles of Nuclear Magnetism, Oxford University Press, London, 1961.

K. J. Mallikarjunaiah and R. Damle, Solid-state NMR and its implications in molecular dynamics, Lambert Academic Publishing, Saarbrücken, 2011.

J.J. Kinnun et al, dx.doi.org/10.1021/ed2004774.

Appendix III Useful Unix Commands:

man -k SEARCHNAME Read the online manual about commands.

man COMMAND With "-k" you can search for keywords in the short description of the registered commands.

lsls -als -lls -al PATH

Display the entries in the actual directory."-a" also, but including the hidden files (hidden: all file / folder -names which starts with a "." dot)."-l" show the entries in the long form with date, size and acls (see at chmod).You can specify also a different PATH.

cdcd PATH

Move yourself through the filesystem (change directory).Without any parameter cd will bring you to your HOMEFOLDER.

mkdir FOLDERNAME Create a new folder.

rmdir FOLDERNAME Delete a empty folder.

cp ORIGINAL NEWFILE Copy files, from one to another location.

mv OLDPATHNAME NEWPATHNAME

Move a file or folder to an other location;or rename a file or folder.

rm FILE1 Remove (delete) ‘FILE1’ file.

rm –r DIRln -s ORIGINALNAME ALIASNAME

Remove (delete) ‘DIR’ directoriy.Create a (symbolic) link of a file or folder.

chmod ugo+r FILE_or_FOLDER_NAMEchmod go-rwx FILE_or_FOLDER_NAME

Change the acl (access rights) of a file or folder.- rwx rwx rwx = 3 groups of "rwx" for "u" user "g" group and "o" for the rest of the world. For each group you can specify the right for "r" read, "w" write or "x" execute access. At a folder the "x" means the right to go inside the folder. The first item (here "-") show a "d" for a directory, or "l" for a link.With "ugo+r" you allow everybody to read this.With "go-rwx" you disallow any access for everybody except yourself ("u" acls not touched).

tar -cf TARFILE.tar PATH1 PATH2tar -tf TARFILE.tartar -xf TARFILE.tartar -cf - PATH1 PATH2 | gzip > TARFILE.tar.gzgzip -c -d TARFILE.tar.gz | tar -tf -gzip -c -d TARFILE.tar.gz | tar -xf -

With tar you can copy files and folders (with subfolders) into one file ".tar". The parameter "f" is needed to specify the following TARFILE."c" will copy all FILES / FOLDERS into the TARFILE."t" will show you the packaged file names."x" will extract the TARFILE."z" will compress the TARFILE into ".tar.gz" format; this parameter is not available on Sun, but here is also a solution for Sun systems shown.

tar -czf TARFILE.tar.gz PATH1 PATH2tar -tzf TARFILE.tar.gztar -xzf TARFILE.tar.gz

gzip FILENAMEgunzip FILENAME.gz

This are the most common compression programs. The source file will be replace!gzip for ".gz" files.compress for ".Z" files.zip for ".zip" files.

compress FILENAMEuncompress FILENAME.Z

zip FILENAMEunzip FILENAME.zip

df –m Disk space used and available information in megabytes