Operating DOSY Experiments

These are the operating instructions for conducting a DOSY experiment.  DOSY must be done in manual mode, so IconNMR must be turned off.

Loading the Sample and Engaging the Software:

Type the following commands into the commandline in TopSpin.  Wait for each process to be "finished" or "completed" before moving on.

1.    sx #            load on your sample in the nmr from the carousel in slot #

2.    new            enter in your experiment info. Load current parameters is fine. 

3.    rpar             read in the experiment parameters called PROTON

4.    lock             choose your nmr solvent and lock onto the 2H signal

5.    atma            automatically tune and match probe to 1H nucleus

6.    ro                turns on sample spinning/rotation to 20 Hz

7.    topshim       automatically shims sample according to Topshim protocol

8.    getprosol     loads in solvent specific parameters for experiment

9.    rga               automatically sets receiver gain

10.  zg                 “zero go” runs your experiment

 

To work-up the data you can either:

Analyze using the mouse:

Select the “Processing” tab, then select “Data Processing Guide”.  Follow the steps on the pop-up guide.

Process the data using command line:

●    ef

●    apk

●    abs

 

Setting the Sweep Range

After getting a standard 1H-NMR spectrum, you want to specify a smaller sweep range

12.  zoom in on the area of the spectrum that you want to study (the narrower the better, but leave at least 0.5 ppm on either side of the peaks you are interested in)

13.  select the “set SW to current region button  - the icon is a red downward arrow pointing to a blue range

 

Determining the Parameters for the DOSY Experiment:

The next step is to determine the correct DOSY parameters – these directions will hold for the pulse program: ledbpgp2s1d (there are many other pulse programs that you can use if you prefer, but the settings for those will not be discussed here)

1d in the pulse program name means you will take a 1D NMR spectrum, for hte full DOSY experiment described below you will use the pulse program ledbpgp2s

1.    Type "iexpno" into the commandline, it creates a new experiment in the same folder that you are working in, the experiment number will increase by +1.

2.    Close and reopen your folder in the Browser on TOPSPIN to see the new experiment. Select this new experiment – the program will run the experiment that is selected.

3.    In the NMR spectrum window select the AcquPars tab.  Here you will set the number of scans and datapoints, etc.  The following are suggested parameters.

●    PULPROG = ledbpgp2s1d

●    td= 16384   Should be a multiple of 2x (# FID data points)

●    ns = 32        Number of scans (16 is good for trials)

If you sweep range is narrow td can be decreased...in time domain you only need a 2-3 second range.

4.    Select the icon with a blue square-wave in the AcquPars tab (this allows you to modify the pulse program for the DOSY experiment).  The following are suggested parameters.

●    D1: 1 sec                 often 1 sec (should be >T1*5); 10 sec for nanocrystals

●    D16: 0.0002 sec

●    D20: 0.3500 sec      recommended 150-200 ms or upto 2sec

●    D21: 0.005 sec        recommended 5 ms

●    Under “Gradient channel” change GPNAM6, GPNAM7, and GPNAM8 to SINE.100 from sine.100

●    GPZ6[%] = 2          This value sets the gradient it can range from 2%-95%

●    GPZ7[%] = -17.13  This value cannot be changed

●    GPZ8[%] = -13.17  This value cannot be changed

●    P19 : 800 usec        can be 1100 usec

●    P30 : 850 usec        cam be 1000 usec (I prefer that P30 > P19)

Reference Examples (from Munro in July 2012):

Pyridine in d-toluene : D20=0.150sec, P19=800 usec, P30=850 usec

Oleylamine in d-toluene: D20=0.550sec, P19=800usec, P30=850usec

5.    rga              automatically sets receiver gain

6.    zg                starts acquisition (or select the “play” icon)

7.    Once the run is over you can process the data as described above.

 

In order to do DOSY you want to have take spectra at GPZ6 = 2% and GPZ6 = 95% and have the signal change by a factor of at least 50.  You can adjust D20 and P30 until this result is achieved.  Once you have set your D20, P30, and P19 parameters you are ready to collect a DOSY spectrum.

 

Running a DOSY Experiment:

1.    iexpno         This will input your last set of parameters into a new experiment

2.    In the NMR spectrum window select the AcquPars tab.

●    change PULPROG to PULPROG = ledbpgp2s

●    In this tab select the icon with 1,2,3 in the upper left corner (near the blue pulse icon) – this will allow you to change the dimensions of the experiment from 1D to 2D.

●    This will open a dialogue box – select “Change dimension from 1D to 2D” and click “OK”.

●    Back in the  AcquPars tab, you will notice that there are now two boxes (or columns) for TD, labeled TDF1 and TDF2.

TDF1 = 16384        sets the # of points in each spectrum

TDF2 = 16  sets the # of points in the second axis

FnMODE = QF

3.    Type “dosy” into the command line – will bring up a series of dialogue boxes

●    Enter first gradient amplitude: 2

●    Enter final gradient amplitude: 95

●    Enter number of points: 16     -This sets the # of gradients that will run

●    ramp type (l/q/e {linear/squared/exponential}): l

4.    The final dialogue box will read, “Do you want to start acquisition?” - Select OK

 

Processing the Data:

1.    Select the ProcPars tab

SIF2 = 32768

SIF1 = 16

PH_mod (F2) = pk

PH_mod (F1) = no

2.    Select the  “FID” tab. 

3.    xf2              Type the command “xf2” into the command line

4.    abs2            Type this command “abs2” into the command line

5.    setdiffparmType into the command line.

 

Now you need to decide if you want to generate a 2D DOSY plot (where 1 axis is the H-NMR spectrum and the other is the log of the diffusion constant) or if you want to fit the diffusion of upto 3 individual peaks in the NMR spectrum.  You can do both, but you must repeat steps 1-3 to choose the second option.

 

Making a 2D plot

1.    eddosy        Type into the command line and then set the following parameters:

●    Method = exponential

●    ExpVar = Gradient

●    Ndata = 16 (should match the number of gradient points)

●    PC = 10 – Noise sensitivity factor (if too high it won't fit data)

●    Scale = Logarithmic

●    DISPmin = -10

●    DISPmax = -8

2.    Type "dosy2d" into the command line

 

Fitting Diffusion Constants

1.    Click on “Analysis” tab in the main menu bar

2.    Select “T1/T2 Relaxation” - this will open the “NMR Relaxation Guide”

(follow the steps of the NMR Relaxation Guide)

3.    Click on the “Extract” icon

●    A new series of dialogue boxes will be opened chose “Spectrum” and then “Slice Number = 1” then select “OK”.

●    this will open a new window where you can specify up to 3 peaks to analyze (you can select the peaks in step 4)

4.    Click on the “Peak/Ranges” icon

●    You will manually select the ranges, make sure that the bracket is highlighted green (1 icon to the right of the blue integration sign highlighted in yellow). You can define up to 3 regions when this icon is highlighted.

●    Once you have defined each peak, select the “Save As” icon (a disk with a blue letter A) and select the “Export Regions to the Relaxation Module and .ret” option

5.    Click on the “Relaxation Window” icon

●    Enable the “Intensity” option

6.    Click on the “Fitting Function” icon

●    Close the first dialogue box that opens

●    Set the Relaxation Parameters

●    Left limit for baseline correction = 1000

●    Right limit for baseline correction = -1000

●    Number of drift points = 10 (increase from 5)

●    Number of points = 16 (this should match the # of gradients used)

●    Function Type = vargrad

●    List file name = difflist

7.    Select the “Calculate fitting parameters for all data points” icon in the data window (two red arrows on green)

 

To End the Run (or change samples)

12.               ro                    turns off sample spinning/rotation

13.               sx ej               ejects the sample and returns it to the sample holder

14.               restart Icon NMR