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Chemistry 315

Experiment 6:
Determination of NMR relaxation times
Overview
The goal of this lab is to measure the longitudinal and transverse relaxation times of a
Quinine/CDCl3 sample. Quinine is a molecule that has a variety of uses in medicine. The sample
is a standard that is kept in the 145 RAL, which is the location of the UIUC NMR lab. There
will be no sample preparation required and the TA will perform the procedure, however you are
expected to come prepared to observe and ask questions. This experiment is designed to provide
insight into the use of NMR spectroscopy to study important chemical properties.

You MUST sign up for a time slot with the TA during the NMR mini lecture. You will perform
the experiment on that day and will only report to your regular lab day to turn in assignments.
We will meet in Noyes Lab 157, and then walk together to the NMR facility.

Required Readings
1. Introduction to Nuclear Magnetic Resonance Spectroscopy. University of Illinois. 2014.
http://www.scs.illinois.edu/nmr/handouts/getting_started/NMR-intro-DLO.pdf
2. Inside an NMR Magnet. Massachusetts Institute of Technology. 2014
http://web.mit.edu/speclab/www/Facility/shim-probe-sample.html
3. What is a Probe? University of Colorado. 2014.
http://chemnmr.colorado.edu/moreinfo/instruments/probe_whatis.html
4. Reich H.J., Relaxation in NMR Spectroscopy. University of Wisconsin. 2014.
http://www.chem.wisc.edu/areas/reich/nmr/08-tech-01-relax.htm

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5. Download MNova according to emailed instructions. Any questions or problems can be
addressed at the mini lecture.

Background
You have likely seen NMR spectroscopy before as a tool for organic structure determination.
While it is true that it is very useful in this setting, NMR spectroscopy can be used for many
other applications as well. These include kinetic rate determination, protein structures and
interactions, temperature studies, and molecular property characterization. In particular, this lab
will focus on molecular property characterization. However, first we must understand the basics
of NMR spectroscopy, and then we can conduct and understand specialized experiments.
Nuclei with non-zero spins align when they are placed in an external magnetic field. When they
align with the field they are said to be in a low energy state, and when they align against the field
they are said to be in a high energy state. A diagram depicting this is shown below in Figure 1.

Figure 1. Energy levels for different spin states are shown. Splitting occurs for nuclei with non-
zero spins. A higher energy indicates an alignment against the magnetic field.

There is a natural equilibrium between lower-energy-state molecules and higher-energy-state


molecules in a strong magnetic field. When a nucleus is exposed to radiation of a suitable
energy, such as from a radiofrequency pulse, it undergoes a transition from the lower-energy-
state to the higher-energy-state. After a relaxation time, these nuclei release the absorbed energy
and return to their lower-energy-state. This relaxation time can be either longitudinal (1 ),
transverse (2 ), or a combination of the two. They are dependent on various system factors and
will be described in detail below. These relaxation times are characteristic of nuclei in different
chemical environments. They can be used to study molecular interactions and dynamics, as well
as be used for characterization. In this lab, we seek new ways of exploring chemical systems
using NMR. For this purpose, we will determine the longitudinal and transverse relaxation times
for a standard sample.
Before relaxation times can be determined, however, the 90o pulse width must be found. It is
normally written as pw90, and represents the length of a radiofrequency pulse, in microseconds,
that produces the maximum emission of energy from a nucleus of interest. An array of pulse

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width values is created to determine the pw360, or the null pulse. At this point, there is zero
emission from the nucleus of interest. A figure illustrating such an experiment is shown below.
The pw90 is simply found by dividing the pw360 by 4.

Figure 2. Pulse width array. The pw360 is determined because it is the easiest to detect, and the
pw90 is calculated from this value.

1 relaxation occurs when a nucleus in the higher energy state passes precessional energy to the
surrounding material which is referred to as the lattice. The 1 relaxation process is a non-
radiative process, and is possible due to the molecular motion and energy of the lattice which can
interact with the spinning nucleus of the sample. The average amount of time that a nucleus
spends in the higher-energy-state is what we measure as the longitudinal relaxation time. It is
dependent on the identity of the lattice, as well as the magnetogyric ratio of the absorbing
nucleus. Because these processes are all based on molecular motion, 1 is shorter for lattices of
low viscosity and small nuclei with flexible bonds.
1 relaxation can be measured through an inversion-recovery experiment, depicted below in
Figure 3. Essentially, a pulse is applied, which puts the nucleus into the opposite spin state,

and after a delay time, a 2 pulse is applied. The system will naturally undergo relaxation to the
original orientation. However, the intensity of the observed NMR signal is dependent on the
fraction of the molecules still in the excited state.

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Figure 3. Inversion-recovery experiment2. From this figure, it can be seen that various time
constants of relaxation delay produce different intensity values.
An array of delay times is constructed to determine the null time,1 , which is derived in the
following way. In general,
0
= ( ) (Equation 1)
1

represents the magnetic intensity at a certain time. 0 is the initial magnetic field signal.
Equation 1 can be integrated and solved to gain the magnetization expression dependent on time.

= 0 1 (Equation 2)

In an inversion recovery experiment, the effects of 2 are removed because no energy is lost in
the x-y plane, and the following are true:
(0) = (Equation 3)

= (1 2 1 ) = (1 2 ) (Equation 4)

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The system can be modeled as a type of exponential fit using the quality on the right side of the
equation. The exponential constant is related to longitudinal relaxation in the following way:
1
= (Equation 5)
1

Once the data are fit using this equation, 1 can be easily determined. However, Microsoft Excel
does not have a natural function to model this system, so we will use the Solver Add-in. To add
this to Microsoft Excel, go to File, Options, Add-ins, Go (near the bottom where it says Manage
Add-ins), and select Solver Add-in. Press okay, and it will be added to the Data tab in the
Analysis section. Solver requires that we set up a system as shown in Figure 4. Enter in the
appropriate data in the first two columns and enter the formulas with the correct cell references
into the remaining columns. We will minimize the Sum of Square Difference by modifying the B
and F cells. Plot the Formula values as a trendline over your Intensity data points and include
the formula on the plot.

Figure 4. Setup required for use of the Solver tool in Microsoft Excel.

The simplest way to quantify error for this process is to calculate an 2 value. Fortunately, the
sum of difference squared is part of the calculation, so only two more equations are needed. The
variance, Var, is equal to the sum of the difference squared values between the observed
intensity values, , and average intensity value, .
2
= ( ) (Equation 6)

Finally, the 2 value can be calculated through Equation 7.



2 = 1 (Equation 7)

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In contrast to 1 relaxation, 2 relaxation is not as simple to determine because it is dependent on
several effects that broaden NMR lines. 2 values are very small for viscous liquids, but
comparable to 1 for low-viscosity liquids. This prevents their use for high-quality spectra
unless additional compounds are added to increase relaxation times. When two neighboring,
identical nuclei have identical precession rates but different magnetic quantum states, they can
exchange these energy states. This causes the lower-energy-state to become excited and the
higher-energy-state to become relaxed. This decreases longitudinal relaxation time and increases
line broadening, and is referred to as 2 . 2 values decrease as molecular motion decreases. 2
can be determined through the Hahn Spin Echo sequence, shown below in Figure 5.

Figure 5. Cartoon depiction of the Hahn Spin Echo sequence used to determine 2 2.

A 2 pulse is applied, and some spins slow down due to local magnetic field inhomogeneities. A
pulse is then applied, which causes slower spins states to go ahead of the main momentum and
the faster spins to lag. Eventually, spin rephasing occurs which eliminates the differences in spin
states. 2 is measured as the spin echo, which causes the dephasing back to the original spread
of phases. Again, we will look change the delay time to look at the intensity changes for the spin
echo. This follows the same exponential curve as Equation 3. However, the inversion recovery
steps are removed for observation, giving the following equation.

= 0 2 = (Equation 8)

By modeling the exponential fit of the graph, 2 is determined. The exponential constant is
related to transverse relaxation in the following way:
1
= (Equation 9)
2

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The determination of T2 from your collected data will be done in MNova. Detailed instructions
on how to do this can be found here:
http://www.scs.illinois.edu/nmr/handouts/general_pdf/T2Determination.pdf

The data file from the NMR will be emailed to you. You should process the data in this file.
You should follow the instructions on the second page of the document. On Step 3 of the
instructions, you need to select Peak Graph. Remember to copy the data from the generated
chart into your lab report as a table. To access the data go to Advanced > Data Analysis > Show
Table. You should copy the data into excel first and then into word so you can properly format
the table. Be sure to include this in the SI of your report.
The graph will need to be edited. The axes of the graph can be formatted in MNova by right
clicking on it and selecting properties. From there, you can change the horizontal and vertical
axis labels. Please do so to reflect what each axis represents. You can also uncheck the boxes in
the Basic tab under Grid to get rid of the gridlines. Below is an example of what your graph
should generally look like. It is suggested that you attempt to plot your data soon after collection
so you have plenty of time to get help if needed!

Title (This is in a text box from word)

Also include the equation for the


trend line from MNova

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Prelaboratory Assignment
1. Describe the following components of an NMR spectrometer and describe why they are
necessary: super-cooled magnet, shim coils, and sample probe.
2. What is pulse width (pw)? What is pw90 and why is it necessary for determining T1 and
T2?
3. Which relaxation time can be found using an inversion recovery experiment? Which two
pulses are used? Briefly explain the experiment.
4. Why is T2 relaxation often referred to as spin-spin relaxation?
5. How do molecular size and viscosity affect T1 and T2?

Experimental Methods
Equipment:
Quinine/CDCl3 sample
UNITY 400 NB High-Resolution FT NMR Spectrometer

Procedure (TA will perform):


Determine the pw90 for the sample:
Before pw90 is determined, one parameter must be changed to prepare for the future
experiments.
gain = 50 (lowers gain to defined value)

Gather an initial, properly shimmed spectrum. Then, move the parameters and change
experiments using the following:
mp(1,2) moves parameters from experiment 1 to experiment 2
jexp2 joins experiment 2

We will use an array of pulse width values in order to determine the true value.
array a macro that requires the following answers to the prompted questions
Parameter to be arrayed: pw pulse width
Enter number of steps in array: 30 array size
Starting value: 0 (This changes for different probes and different instruments.)
Array increment: 5 T1 values are close

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These commands set of the remainder of the experiment.
pw[1]=1 replaces first array element, 27, with the value 1.
da displays current array values for pw
d1=10 first delay, given in seconds (allows equilibration)
ai vp=70 sets absolute intensity mode; places spectrum about half-way up on the display.
ga begins acquisition

Repeat the array experiment with lower number of steps and a smaller increment around the
pw360 to get a more accurate result

We can use the following commands to observe the spectra and quit when we have finished.
dssh view accumulated spectra
pw360 occurs when the peak of interest is null.
(NOTE: if the second spectrum is already positive, reset the array with a smaller starting value.)

Finally, we return to the original experiment and set our determined pw90 value.
jexp1 returns to experiment 1
pw90=(the numeric value for the 360 found above)/4 gives pw90 a value
pw=pw90 sets the instrument pw as the determined pw90

Measure the 1 for the sample peak:


Gather an initial, properly shimmed spectrum. Then, move the parameters, change experiments,
and lower the gain using the following:
mp(1,2) moves parameters from experiment 1 to experiment 2
jexp2 joins experiment 2

Use the macro to set up the experiment.


dot1 sets up the 1 experiment, and prompts for the following:
Enter minimum 1 expected (sec): 5 (a good starting value)
Enter maximum 1 expected: 10 (5 to 10 sec are typical)

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Enter number of transients (nt): 1 (use minimum nt)

Start the experiment.


ga begins acquisition

View the spectra as they are acquired.


dssh displays completed spectra horizontally

Expand around the signal of interest and set a minimum threshold for the line.
ds(#) displays the last spectrum in the array
dll displays listed line frequencies for the expanded region of interest
fp measures the intensity of each line in the dll for the entire array and outputs the data
t1 executes an exponential curve fitting to determine T1 and outputs the data
printon t1 printoff prints the data into a chart

Write down the observed intensity data. Use these data to create a curve in Microsoft Excel
using the Solver tool in order to determine 1 .

Finally, we return to the original experiment.


jexp1 returns to experiment 1

Measure the 2 for the sample peak:


First, gather an initial, properly shimmed spectrum. Then, move the parameters and change
experiments using the following:
mp(1,2) moves parameters from experiment 1 to experiment 2
jexp2 joins experiment 2

Set up the system for a 2 experiment


doT2 loads the pulse sequence for a 2 experiment

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T2setup sets up the 2 experiment, and prompts for the following:
Enter minimum 2 expected (sec): 1 (0.1 1 sec is good to start)
Enter maximum 2 expected: 8 (5 to 10 sec are typical)
Enter experiment time (hr): 0.2 (will be slightly different than actual time)
ga begins acquisition

Expand around the signal of interest and set a minimum threshold for the line.
ds(#) displays the last spectrum in the array
dll displays listed line frequencies for the expanded region of interest
fp measures the intensity of each line in the dll for the entire array and outputs the data
t2 executes an exponential curve fitting to determine T2 and outputs the data
printon t2 printoff prints data into a chart

Write down the observed intensity data. Use these data to create an exponential curve in
Microsoft Excel to determine 2 .

Finally, we return to the original experiment.


jexp1 returns to experiment 1

For your Laboratory Report:


Tables: include the observed time and intensity values used to determine both relaxation time
constants. These should make up the two tables that you need in the body of the report.
Simply report pw90. Make sure that you tabulate all the data that you use in producing your
graphs.
Plots: use Microsoft Excel for the T1 plot and MNova for the T2 plot. Be sure to label axes
and title the graphs in a descriptive manner. The graphs should be easy to read. You should
have two plots: observed intensity vs time for both time constants. Plot the formula values
from solver over the observed intensity as a black trendline on the T1 graph and display the
formula with the correct constants on the plot using a text box, as well as the calculated R2
value. For the T2 graph use a text box to display the exponential trend line on the graph. You
dont need to modify the MNova graph to make it look like the excel graph but be sure you
have all of the required labels.

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Calculations: show how you calculated the following,
pw90
1
2
Attach your original copy of your lab notebook in the supplemental information (SI) section
of your report. Also in the SI include the table from Excel Solver that you used to obtain T1
and the Table from MNova that you used to obtain T2.

For the discussion section, make sure you include answers to the questions below, doing so
in a discussion format (i.e. DO NOT put Question 1, then Answer 1, etc.). This is required
in addition to normal discussion procedures. The idea is not to only answer the questions
below but answer them as support for your normal discussion. The chemical engineering
question should be placed according to the lab format guidelines which can be found on
Compass. Warning: Be sure to cite ALL of your sources appropriately. Including
information that comes from an outside source and is not cited will result in point deductions.

Questions

1. What is your pw90 and what effect would it have on the data collected if it were different?
2. What are your relaxation constants? Based on the properties of Quinine, are they what you
would expect?
3. You used two different software programs to process the acquired NMR data. Which gave you
a better understanding of the trends in the data? Why? (There is not only one right answer to
this question, I want you to think critically about the data processing methods used.)
4. Quantify sources of error. What are some common sources of error in NMR spectroscopy in
general?

Chemical Engineering Student Question:


1. NMR spectroscopy, though a very useful technique, is plagued with many issues such as
high costs and low sensitivity. Despite these drawbacks, NMR spectroscopy is frequently

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used in many areas of chemistry. NMR is also used in industry. Discuss an example of an
industrial application of NMR spectroscopy that stands out as particularly interesting
and/or useful to you.

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