development of novel 2d nmr techniques for mixture analysis
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Development of Novel 2D NMR Techniques for Mixture Analysis
A dissertation submitted to the University of Manchester for the degree of
Master of Science by Chemistry Research in the Faculty of Science and Engineering
2020
Arika Hisatsune
School of Nature Science Department of Chemistry
The University of Manchester
Development of Novel 2D NMR Techniques for Mixture Analysis
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List of Contents
List of Abbreviations and Symbols ………….………………………………………….……………………………….. 4
List of Figures …………………………………………….……….…………………………………………………..………….. 5
Abstract ……………………………………………………....…………………………………………………………………….. 8
Declaration …………………….………………….……………….………………………………………………………………. 8
Copyright Statement ……………………….……….………..……….………………………………………………..……. 9
Acknowledgements ………………………….………….…………………………………………………….…………….. 10
1. Chapter 1 – Introduction ……………………………….…………………………….……………………..… 11
1.1. Thesis overview …………………………………………….………………,………………….……… 11
1.1.1. Understanding NMR ………………….….……………….….…………………………. 11
1.2. Spectral editing ………………………………………………………….……………………………… 14
1.2.1. Introduction …………………………..………….…..…..…….………..……………… 14
1.2.2. Perfect echo (PE) …………………….…………….…..….….……….……….……… 14
1.2.3. Low-pass J-filters (LPF) ……………………….….….…..….………………..……… 16
1.2.4. Zero Quantum Suppression (ZQS) ……………..…...….…………..…..……… 18
1.3. 2D NMR ……………………………………………………………………………………………………... 19
1.3.1. Introduction …………………………….……….….……………………………………… 19
1.3.2. Acquisition of spectrum ………….……………..…………...………………….…… 19
1.3.3. Pulse sequence of TOCSY ………….………….…….……………………………….. 19
1.4. Existing work, 1D DISPEL experiment ………………………………………….…………….. 20
2. Chapter 2 – 1D DISPEL using simulation ………………………..…………………………….…………. 21
2.1. Introduction ……………..……………..…..……………….…………….….…………………………. 21
2.2. Theory of Bloch equation ……………………….…..……….…..…..…………………….……… 21
2.3. DISPEL 1D simulation …….…………..…………..….…………..……………….…………………. 25
2.3.1. Investigation of 1H 90° off-resonance effect ….……….....…..……………. 25
2.3.2. Investigation of 13C 90° off-resonance effect …….….………………………. 25
2.3.3. Investigation of the influence of composite pulses on 13C ………………….
off-resonance effect ……………………………………………………………………. 26
2.3.4. Investigation of the effect of B1 miscalibration ……………………………… 28
3. Chapter 3 – 1D DISPEL experiment ……………………………………………………………………….. 31
Development of Novel 2D NMR Techniques for Mixture Analysis
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3.1. Introduction ………..………………………………..…….…..…..…….……..……………………… 31
3.2. DISPEL 1D experiment ….………………………..….……………….……………..……..………. 31
3.2.1. Investigation of 13C 90° off-resonance effect using propanol ………….. 31
3.2.2. Investigation of effect of B1 miscalibration …………………………………….. 32
3.3. DISPEL 1D spectrum ……………………………………………………………………..………….. 34
3.3.1. 1D DISPEL spectrum of doped 2.5% propanol .………….……….………….. 34
3.3.2. 1D DISPEL spectrum of 100 mM quinine …………………………..….……….. 35
3.3.3. 1D DISPEL spectrum of Q-mix ……….……………………….….……..….……….. 36
4. Chapter 4 – 2D TOCSY-DISPEL experiment …………………………………………………………….. 38
4.1. Introduction …………………….………………..…………………………..………………………….. 38
4.2. 2D TOCSY-DISPEL spectra ….….…………..…………………….…….………….……………….. 38
4.2.1. 2D TOCSY-DISPEL Spectra of doped 2.5% propanol.…….……...…..……. 38
4.2.2. 2D TOCSY-DISPEL spectrum of 100 mM quinine ………….….….…..…….. 40
4.2.3. 2D TOCSY-DISPEL spectrum of Q-mix ……………….....………..…….……….. 41
5. Chapter 5 - Discussion ……………………………………………………..……………………………………. 45
5.1. Conclusions ……………………..…….……….………………..…….…..………………………….…. 45
5.2. Discussion ………………………………………………………………………………………………….. 45
5.3. Future work ……………..……….……..……………..……………….………………………………… 47
6. References ……………..……………………………………………………………………………………………… 48
Word count: 12023
Development of Novel 2D NMR Techniques for Mixture Analysis
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List of Abbreviations and Symbols
1D One-Dimensional
2D Two-Dimensional
AP Anti-Phase
B0 External static magnetic field (tesla)
B1 Radiofrequency magnetic field (tesla)
COSY COrrelational SpectroscopY
CPMG Carr-Purcell-Meiboom-Gill
CW Continuous-Wave
DISPEL Destruction of Interfering Satellites by Perfect Echo Low-pass filtration
F1 (Indirect) dimension
F2 (Direct) dimension
IP In-Phase
J Scalar coupling constant (Hz)
LPF Low-Pass Filter
M Magnetization
NMR Nuclear Magnetic Resonance
PE Perfect Echo
PFG Pulsed Field Gradient
PO Product Operator
RF Radio Frequency
T Temperature (K)
t1 Incremented evolution period (s)
t2 Acquisition time (s)
TOCSY TOtal COrrelation SpectroscopY
ZQC Zero Quantum Coherence
ZQS Zero Quantum Suppression
δ Chemical shift (ppm)
τ Delay period (s)
𝛾 Gyromagnetic ratio
Development of Novel 2D NMR Techniques for Mixture Analysis
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List of Figures
Fig. 1 500 MHz 1H spectrum of chloroform
Fig. 2 The pulse sequence of the spin echo (SE)
Fig. 3 Spinach simulation of spin echo pulse sequence applied to a two-spin system
Fig. 4 The pulse sequence of the Carr-Purcell-Melboom-Gill (CPMG)
Fig. 5 The pulse sequence of the perfect echo (PE)
Fig. 6 Spinach simulation of perfect echo pulse sequence applied to a two-spin system
Fig. 7 The pulse sequence of the one-staged LPF
Fig. 8 Graphical representation of low-pass filter stages applied on AX spin system.
Reproduced from P. Moutzouri et al.
Fig. 9 Graphical representation of Four-stage low-pass filter applied on AX spin
system. Reproduced from P. Moutzouri et al.
Fig. 10 500 MHz zTOCSY spectrum of menthol
Fig. 11 The pulse sequence of TOCSY
Fig. 12 The pulse sequence of DISPEL
Fig. 13 500 MHz 1H NMR spectra of CHCl3 doped with chromium tris-acetylacetonate
in DMSO-d6
Fig. 14 Spinach simulation of a spin echo pulse sequence applied to an 11 single-spin
system
Fig. 15 The effect of the normal 90° 13C pulses on signals obtained for a 13C chemical
shift range of 75000 Hz, using Spinach package
Fig. 16 The effect of the composite 13C pulses on signals obtained for a 13C chemical
shift range of 75000 Hz,using Spinach package
Fig. 17 The effect of the normal and composite 90° 13C pulses on signals obtained for a 13C chemical shift range of 75000 Hz, using Spinach package
Fig. 18 Spinach simulation of DISPEL pulse sequencewith B1 error using normal 90°
pulse
Fig. 19 The effect of the B1 miscalibration on signals obtained using normal pulses for
a 13C chemical shift range of 50000 Hz, using Spinach package
Development of Novel 2D NMR Techniques for Mixture Analysis
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Fig. 20 Spinach simulation of DISPEL pulse sequencewith B1 error using composite 90°
pulse
Fig. 21 The effect of the B1 miscalibration on signals obtained using composite pulses
fora 13C chemical shift range of 50000 Hz, using Spinach package
Fig. 22 The pulse sequence of 1D DISPEL
Fig. 23 The experimental data of the effect of the normal 90° 13C pulses on signals
obtained for a 13C chemical shift range of 80000 Hz
Fig. 24 The experimental data of the effect of the normal 90° 13C pulses on signals
obtained for a 13C chemical shift range of 80000 Hz
Fig. 25 The experimental data of DISPEL pulse sequencewith normal 90° pulse with B1
error
Fig. 26 The experimental data of the effect of the B1 miscalibration on signals obtained
for a 13C chemical shift range of 80000 Hz
Fig. 27 The comparison between the experimental data and the Spinach simulation
data of the effect of the B1 miscalibration on signals obtained for a 13C chemical
shift range of 80000 Hz
Fig. 28 500 MHz 1H spectrum of 2.5 % doped propanol in DMSO-d6
Fig. 29 Expansion of 500 MHz 1H DISPEL spectrum of 100 mM quinine in DMSO-d6
Fig. 30 Expansion of 500 MHz 1H spectrum of Q-mixture in DMSO-d6
Fig. 31 The pulse sequence of 2D TOCSY-DISPEL
Fig. 32 500 MHz 2D TOCSY spectrum of n-propanol doped with chromium tris-
acetylacetonate in DMSO-d6
Fig. 33 500 MHz 2D TOCSY spectra of n-propanol doped with chromium tris-
acetylacetonate in DMSO-d6
Fig. 34 Expansion of 500 MHz 2D spectra marked by a purple square in Fig. 25
Fig. 35 Expansion of 500 MHz 2D spectra 100 mM quinine in DMSO-d6
Fig. 36 Expansion of 500 MHz 2D Q-mix spectra in DMSO-d6
Fig. 37 Expansion of 500 MHz 2D TOCSY spectra of Q-mix in DMSO-d6, region marked
by orange in Fig. 28
Development of Novel 2D NMR Techniques for Mixture Analysis
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Fig. 38 Expansion of 500 MHz 2D TOCSY spectra of Q-mix in DMSO-d6, the region
marked by yellow in Fig. 28
Fig. 39 Expansion of 500 MHz 2D Q-mix spectrum in DMSO-d6
Development of Novel 2D NMR Techniques for Mixture Analysis
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Abstract
The analysis of high dynamic range mixtures by 1H NMR is complicated by the presence of 13C
satellite signals. While satellites caused by long-range couplings are usually buried beneath
homonuclear multiplet structure, one-bond satellites of major mixture components often
overlap with signals from minor components, complicating both quantification and
identification. Broadband 13C decoupling can eliminate, or at least greatly reduce, interference
in 1H NMR from 13C isotopomer signals, but only at the cost of significant sample heating. This
limits spectral resolution, because longer acquisition times will increase this heating. A recent
NMR experiment, DISPEL (Destruction of Interfering Satellite by Perfect Echo Low-pass
filtration), suppresses one-bond 13C satellite signals in 1D 1H spectra without the need for
decoupling. This new approach is generally applicable, and it is possible to concatenate it with
a wide variety of multidimensional NMR methods such as COSY and TOCSY at very low cost in
signal-to-noise ratio, making it significantly easier to analyze the spectra of high dynamic range
mixtures by 1H NMR.
Chapter 1 contains an introduction to the theoretical NMR background necessary for this
thesis.
Chapter 2 contains details of 1D DISPEL simulations, investigating factors that influence the
degree of 13C-1H satellite peak suppression.
Chapter 3 contains experimental 1D DISPEL results, explaining how it can be applied to the
analysis of minor components in high dynamic range mixtures.
Chapter 4 contains experimental 2D DISPEL results, explaining how it can be applied to the
analysis of minor components in the high dynamic range mixtures.
Chapter 5 summarizes the conclusions of the research.
All the experimental and simulation data and parameters for the work described are freely
available at https://dx.doi.org/10.17632/2zvkcp3hjf.1.
Declaration
The author declares that all the work presented in this thesis has been completed at the
premises of The University of Manchester and at home due to COVID-19. Unless stated
otherwise at the beginning of each chapter, no portion of the work referred to in this thesis
Development of Novel 2D NMR Techniques for Mixture Analysis
9
has been submitted in support of an application for another degree or qualification of this or
any other university or other institute of learning.
Copyright Statement
I. The author of this dissertation (including any appendices and/or schedules to this
dissertation) owns certain copyright or related rights in it (the “Copyright”) and
she has given The University of Manchester certain rights to use such Copyright,
including for administrative purposes.
II. Copies of this dissertation, either in full or in extracts and whether in hard or
electronic copy, may be made only in accordance with the Copyright, Designs and
Patents Act 1988 (as amended) and regulations issued under it or, where
appropriate, in accordance with licensing agreements which the University has
from time to time. This page must form part of any such copies made.
III. The ownership of certain Copyright, patents, designs, trademarks and other
intellectual property (the “Intellectual Property”) and any reproductions of
copyright works in the dissertation, for example graphs and tables
(“Reproductions”), which may be described in this dissertation, may not be owned
by the author and may be owned by third parties. Such Intellectual Property and
Reproductions cannot and must not be made available for use without the prior
written permission of the owner(s) of the relevant Intellectual Property and/or
Reproductions.
IV. Further information on the conditions under which disclosure, publication and
commercialisation of this dissertation, the Copyright and any Intellectual Property
and/or Reproductions described in it may take place is available in the University
IP Policy, in any relevant Dissertation restriction declarations deposited in the
University Library, The University Library’s regulations and in The University’s
policy on Presentation of Dissertations
Development of Novel 2D NMR Techniques for Mixture Analysis
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Acknowledgements
I would like to thank my academic advisors, Professor Gareth A. Morris and Professor
Mathias Nilsson for unconditional support and giving me this opportunity to complete my MSc
in Chemistry Research as a member of Manchester NMR Methodology Group. Every day, I saw
myself grow as an NMR spectroscopist and as a researcher. I’d also like to thank Dr Peter Kiraly,
who has always here for me, guiding me throughout my projects. My days were filled with full
of new knowledge and experiments! Thank you for your endless help, guidance and
discussions.
Thank you, Manchester NMR Methodology Group members, for honest opinions,
friendships and discussions (over pints sometimes). I could not have asked for a better group
to spend my MSc year with, and I would not have been here today without you guys!! You guys
made my time in the UK a special one. Thank you to my old supervisors from the University of
Toronto, Professor Andre Simpson and Dr Ronald Soong for supporting me in my
undergraduate year and acknowledging my growth as an NMR spectroscopist at ENC. That
meant a lot. Thank you, Ryan Anthony, for being here through ups and (many more of) downs,
listening to my complaints and always pushing me to be a better person. Without you, I am
not sure if I would be to complete the program. Merci pour tout. I am also thankful for the
School of Chemistry, the University of Manchester for providing a safe work environment and
for EPSRC for the research funding.
Last but not least, a special thanks go to my parents, Toshiyuki and Mika and my siblings,
Miki and Taiki, for unconditional love and support throughout my time in Manchester! You
guys have provided me with an opportunity to go and to live in another country, and always
had faith in me. Thank you for never leaving my side and acknowledging every single small
accomplishment of mine along the way. I LOVE YOU GUYS and ありがとう.
Development of Novel 2D NMR Techniques for Mixture Analysis
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Chapter 1 – Introduction
1.1 Thesis overview
This thesis describes new experiments for the analysis of high dynamic range mixtures
by Nuclear Magnetic Resonance (NMR) spectroscopy. NMR is often used as a non-destructive
and minimum sample preparation analytical method, but problems can arise when it comes to
the analysis of a high dynamic range mixture. Mixture analysis provides rich information and
is important in fields like food chemistry and the pharmaceutical industry but can be
challenging.
The first successful accurate measurements of nuclear magnetic moments using
magnetic resonance absorption were done in 1938 by I. I. Rabi.1 In 1946, the group of F. Bloch
and E. M. Purcell independently demonstrated NMR for condensed matter, and the start of
NMR’s journey as an analytical technique began.2 In 1950, it was discovered that there are
slight changes in the atomic nucleus Larmor frequencies3 due to chemical shifts and spin
couplings. Larmor frequencies is also known as precessional frequencies, which refers to the
rate of precession of the magnetic moment (such as 1H) around the external magnetic field.
Over the past 80 years, NMR spectroscopy has bloomed into a major tool for analytical
chemistry, and it is now routinely used in the fields of chemistry, pharmaceutical science,
biology and medicine.
1.1.1 Understanding NMR
The atomic nucleus4 can be regarded as a spinning charged particle, which generates a
magnetic field. With the application of an external magnetic field (B0), the quantization of spin
energy occurs. The spin movement is controlled by the energy state of the spins, which are
known as 𝛼-spin state and 𝛽-spin state. The𝛼 is the low energy state for nuclei with a positive
gyromagnetic ratio (𝛾).5 The gyromagnetic ratio is a constant for a particular nucleus and is
directly proportional to the strength of the tiny nuclear magnetic moment.
The energy required to induce flipping depends on the strength of the magnetic field
(B0). The frequency of the nuclear transition can be written as
𝑣! = 𝛾𝐵!/2𝜋 (equation 1.1)
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This two-state description is only applicable for those nuclei with spin quantum number of
I=1/2 such as 1H, 13C, 15N, 19F and 31P. Some spins precess about the positive z-axis and some
about the negative z-axis. The total magnetization determines an NMR signal.
The distribution of nuclei in the different energy state is given by the Boltzmann
equation6 "!""#$"%&'#$
= 𝑒#$%/'( = 𝑒#)*/'( (equation 1.2)
where Nupper and Nlower represent the population of nuclei in the upper and lower energy state.
It is important to have an excess number of nuclei in the lower energy state for a signal to be
detected. The small population difference presents a significant sensitivity problem for NMR.
As seen in equations 1.1 and 1.2, the use of a stronger magnetic field will increase the
sensitivity. The application of the magnetic field strength should be uniform across the sample
to avoid achieving the Larmor condition at a range of frequencies, leading to a broader signal.
Relaxation processes return the spin system to thermal equilibrium, in the absence of
perturbing radio frequency (RF) pulses.7,8
The most common form of NMR performed is proton (1H) NMR. In samples with natural
abundance, 99.98% is the isotope 1H.9 Proton NMR spectra of most organic compounds are
characterized by chemical shifts in the range of +14 to -4 ppm and by spin-spin couplings
between protons.10 NMR can be performed in both liquids and solids, but the simplest form is
in liquids and that generally requires a solvent. The solvent used in liquid-state NMR is
deuterated to avoid swamping of the signals, to accurately define 0 ppm and to stabilize
magnetic field strength. Deuterated water (D2O) and deuterated chloroform (CDCl3) are
common solvents.
Chemical shifts, signal integrals and splitting patterns provide rich information on the
structure of the sample being analyzed. The chemical shift9,11,12 determines the position in the
spectrum at which nuclei resonate. The exact values depend on local electron density,
determined by molecular structure, neighbouring functional groups, hybridization, solvent,
and temperature. The stronger a nucleus’ local magnetic field, the higher their chemical shifts
will be (deshielding)3, the higher their chemical shift values will be. The area under the signal9
is determined by the relative numbers of spins contributing to the signal. Multiplet patterns
Development of Novel 2D NMR Techniques for Mixture Analysis
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are determined by the number and magnitude of the scalar couplings experienced by a nucleus
and by the spin quantum numbers of the nuclei to which it is coupled.
The magnitude of splitting is known as the scalar coupling constant (J).13 This J-coupling
does not change with the magnetic field, so it is quoted in Hz and not ppm. Spin-spin
couplings14 are caused by the small magnetic fields that nuclei with I > 0 possess. Due to these
magnetic fields, they influence each other, resulting in the changes in the energy and hence
Larmor frequency of nearby nuclei as they resonate. The scalar coupling is one of the most
important types of coupling and arises from the magnetic interactions between two nuclei that
are communicated through chemical bonds. It can be used to determine the conformations of
chemical species.
Small extra peaks can often be seen on either side of the main peaks on 1H NMR spectra.
These are caused not by homonuclear scalar coupling (JHH) but by heteronuclear scalar
coupling (JCH), and they are called carbon satellite peaks.13,15 Carbon satellite peaks are small
because the natural abundance of the I=1/2 13C isotope is only 1.1%, so couplings to 13C results
in two extra peaks of 0.55% intensity, one-bond 13C coupled to 1H (1JCH ) Hz apart, either side
of the main 12C-1H peak. See Fig. 1 for an example of satellite peaks in 1H NMR.
Figure 1: 500 MHz 1H spectrum of chloroform showing 13C satellite peaks either side of a parent peak.
This spectrum was acquired with 16 scans, in an experiment time of 1 min and 46 s.
Satellite peaks generally show the same homonuclear multiplet structure as parent peak, so if
the parent peak is a triplet, the satellite peaks would also normally be triplets. 13C satellite
peaks cause problems when it comes to the analysis of high dynamic range mixtures because
satellite peaks can mask or be confused with signals of components that are present at low
concentration, making the latter harder to identify and quantify. This thesis describes methods
for removing satellite peaks in 1D simulation and in 2D NMR.
0.55%
Development of Novel 2D NMR Techniques for Mixture Analysis
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1.2 Spectral editing
1.2.1. Introduction
The analysis of chemical mixtures can be hard, not only because mixtures contains more than
one analyte but also due to signal overlap. With overlap, some signals could be masked, making
chemicals that are present at low concentration hard to identify and quantify. Spectral
editing16 is a technique used to suppress certain classes of NMR signals based on their NMR
properties. The perfect echo, low pass J-filters, and zero-quantum suppression are examples
of building blocks used to suppress unwanted signals.
1.2.2. Perfect echo (PE)
The spin echo18 is the single most important building block in the modern NMR. The
pulse sequence is
Figure 2: The pulse sequence of the spin echo (SE).
It contains 90 and 180 pulses shown as thin and thick rectangles with delay (𝜏) in between.
With a simple spin echo, a spin system with homonuclear coupling shows J-modulation as a
function of the time 2𝜏. J-modulation is caused by the effect of the scalar coupling remaining
while the chemical shift is refocused, resulting in distortion of the spectrum. A simulation
package (Spinach)17 can be used to demonstrate J-modulation effects in 1D 1H NMR, see Fig.
3.
Figure 3: Spinach simulation of 1H spin echo pulse sequence applied to a coupled two-spin system,
for 2𝜏 times of (a) 1ms, (b) 10 ms, and (c) 100 ms. 1JHH of 150 Hz.
a) b) c)
Development of Novel 2D NMR Techniques for Mixture Analysis
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With increasing 𝜏, more J-modulation can be seen, affecting the phases of the spectral peaks.
In more complex experiments, it can result in sensitivity loss or missing spectral peaks.
To suppress J-modulation, a Carr-Purcell-Meiboom-Gill (CPMG)14 experiment can be
used. The pulse sequence is
Figure 4: The pulse sequence of the Carr-Purcell-Meiboom-Gill (CPMG) experiment. tau is usually in 𝜇𝑠.
However, it comes with the cost of high RF power deposition, which will cause sample heating,
resulting inter alia in problems such as line-shape distortions and shifts in signals. CPMG can
suppress the modulation arising from couplings between spins with chemical shift differences
∆𝜈 << 1/𝜏.14 However, J-modulation can be also refocused by a method called the “perfect
echo.”18
The perfect echo14,19,20 is used to refocus the effects of both JHH and chemical shifts. The
perfect echo pulse sequence is
Figure 5: The pulse sequence of the perfect echo (PE).
A perfect echo is made by inserting a 90° flip angle orthogonal (with phase y if the initial
excitation pulse has phase x) pulse at the midpoint of a double spin echo. During the spin echo,
the chemical shift evolves during the first delay 𝜏 and is then refocused by the 180° pulse; the
same goes for JHX. However, JHH keeps on evolving even after application of a 180° pulse,
contributing to phase distortion. The distortion is caused by the buildup of anti-phase (AP)
coherence with respect to homonuclear J-coupling. The perfect echo acts as a J-compensated
Development of Novel 2D NMR Techniques for Mixture Analysis
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building block by refocusing the unwanted AP coherences, giving an improvement in spectral
quality. See Fig. 6.
Figure 6: Spinach simulation of 1H perfect spin echo pulse sequence applied to a coupled two-spin system,
for 2𝜏 times of (a) 1 ms, (b) 10 ms, and (c) 100 ms. 1JHH of 150 Hz was used.
This sequence gives spectra that are free of phase anomalies arising from undesired J-
modulation. In the next section, it will be shown that this also provides time to apply a four-
stage 13C low-pass 1JCH filter, allowing the signals of all protons directly attached to 13C to be
suppressed.
1.2.3. Low-pass J-filters (LPF)
A low-pass J-filter20,21 suppresses signals whose scalar coupling constants exceed a
lower limit. Signals with large J-couplings, for example, one-bond 13C satellite signals, which
typically have 1JCH in the range of 115-250 Hz and are to be from the spectrum while long-range
satellite signals, with nJCH of 0-30 Hz, remain unperturbed.
The simplest low-pass JCH filter pulse sequence is shown below. Instead of refocusing
AP coherences with respect to homonuclear J-coupling like the perfect echo, this method
suppresses AP coherences with respect to heteronuclear J. This is a one-stage low-pass filter,
as it only contains one carbon pulse. The tau delay in the pulse sequence is set to 1/(21JCH), of
the order of 4 ms.
Figure 7: The pulse sequence of the one-staged LPF.
The initial 1H 90°x excitation pulse generates transverse magnetization, −Hy. The product
operator is explained the section 2.2. During the τdelay, proton magnetization coupled to 13C
a) b) c)
Development of Novel 2D NMR Techniques for Mixture Analysis
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evolves into −Hycos(πJCHτ), in-phase (IP), and −2HxCzsin(πJCHτ), anti-phase (AP) coherences.
The amount of AP coherence generated is dependent on the coupling constant and the
duration of the τdelay. The 90° pulse on the carbon converts AP coherence into multiple
quantum coherence. Only single-quantum coherence results in a detectable signal; multiple
quantum coherence will not be seen.
A four-stage low-pass JCH filter is used in the DISPEL sequence. The more stages are
used, the greater the opportunity to suppress signals with a wider range of 1JCH. The perfect
echo provides enough time for four pulses. See Fig. 8 and Fig. 9 for how the number of stages
influence a heteronuclear AX spin system suppression. Fig. 8 and Fig. 9 were retrieved from a
paper published in 2017 by P. Moutzouri et al. 13
Figure 8: Graphical representation of (a) one-stage, (b) two-stage, (c) three-stage
low-pass filter stages applied on AX spin system. Reproduced from P. Moutzouri et al.13 The delay τ is set to 4.05 ms.
With a one-stage filter, only a narrow range of 1JCH gives sufficient (>>10:1) suppression, but
with increasing numbers of stages, the range of 1JCH giving good suppression is widened.
Figure 9: Graphical representation of four-stage low-pass filter applied on AX spin system. The blue line is a 10x magnification of the red line. Reproduced from P. Moutzouri et al.13
τ1, τ2, τ3, and τ4 were 3.2, 1.1, 3.95, and 1.56 ms, respectively.
Development of Novel 2D NMR Techniques for Mixture Analysis
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The blue line in Fig. 9 is the red line magnified by ten times to see the oscillation patterns. With
a four-stage low-pass filter, a suppression factor of 70:1 or better is achieved for heteronuclear
couplings from 110 to above 350 Hz.
1.2.4. Zero Quantum Suppression (ZQS)
Many types of coherences can be generated, but only single-quantum coherences can
be detected. Zero-quantum coherence evolves at the frequency which is the difference
between two chemical shifts. When zero quantum coherences are converted into observable
single quantum coherences, they do so with phases which depend on the evolution time. The
result is the appearance of AP dispersive signals, interfering with the spectrum acquired. See
Fig. 10 for an example. The baseline of the Fig. 10a is distorted due to zero-quantum coherence.
(a) (b)
Figure 10: 500 MHz selective 1D 1H TOCSY spectrum of menthol (a) without and (b) with ZQS. Each spectrum was acquired with 8 scans, in an experiment time of 45 s.
In order to get rid of the AP dispersive signals, zero-quantum suppression20,22,23 is used.
This uses a frequency-swept 180° pulse24 applied simultaneously with a field gradient. The field
gradient causes the Larmor frequency to become a function of position in the active volume
of the NMR tube. This ensures that spins at different positions experience the 180° pulse at
different times, which results in different parts of the sample having different zero-quantum
evolution times. The field gradient is turned off at the end of the 180° pulse or else the different
parts of the sample will give a signal at a different Larmor frequency. The signal components
that originate from zero-quantum coherence averaging to zero, giving a spectrum with no AP
dispersive signal interference. The zero-quantum suppression is followed by a further gradient
pulse, which is used to dephase all other orders of coherence. The result is a pulse sequence
element that only allows longitudinal magnetization to survive.
0.7
𝛿 𝐻/𝑝𝑝𝑚" 𝛿 𝐻/𝑝𝑝𝑚
"𝑝𝑝𝑚
3.7 2.9 2.1 1.5 3.7 2.9 2.1 1.5 0.7
a) b)
Development of Novel 2D NMR Techniques for Mixture Analysis
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1.3 2D NMR
1.3.1 Introduction
The principles of 2D NMR spectroscopy were first presented in 1971, and during the 1980s the
approach found wider application. “Two-dimensional” refers to two frequency dimensions,
while 1D methods have only one.25,26 “Pseudo-2D” methods have also been developed for
which the idea of a 2D representation has been adapted to include one frequency and one
‘other’ dimension. Conventional 2D experiments find wide utility in chemical research.
1.3.2 Acquisition of spectrum
Generating the second dimension requires data acquisition with a pulse sequence containing
preparation, evolution, mixing and detection periods. The preparation and mixing periods
depend on the nature of the experiment. The simplest form of preparation or mixing period is
a single 90° pulse, which is used to excite transverse magnetizations. The evolution period
provides the key to the generation of the second dimension. The mixing part of the sequence
may transfer magnetization associated with one spin to other spins, for example to spins with
which it is coupled, a process known as coherence transfer.27 Control of the coherence transfer
pathway over the course of a pulse sequence can be achieved by phase cycling and/or field
gradient pulses. While gradient pulses can save time and avoid the need for the signal
addition/subtraction used in phase cycling, phase errors due to chemical shift evolution during
the application of a gradient pulse can cause complications. Therefore, they are often applied
within a spin echo to refocus this evolution. The result of a two-dimensional experiment is a
2D time-domain data set s(t1,t2); the free induction decays observed in t2 are modulated in
phase and/or amplitude as a function of t1, and are doubly Fourier transformed to give a two-
dimensional spectrum S(F1,F2).
1.3.3 Pulse sequence of TOCSY
TOCSY (TOtal Correlation SpectroscopY)26 is a homonuclear experiment; it shows cross-
peaks between spins which are connected by an unbroken chain of couplings, so it is useful for
identifying spins which belong to an extended network of couplings. The pulse sequence of
TOCSY is
Development of Novel 2D NMR Techniques for Mixture Analysis
20
Figure 11: The pulse sequence of Total Correlational SpectroscopY (TOCSY).
The key part of the TOCSY experiment is the period of isotropic mixing or spin locking,
which is shown here as a DIPSI-2 sequence element.26 The spin lock is arranged that only z-
magnetization present at beginning and the end of DIPSI-2 contributes to the spectrum. In a
two-spin system, such a period of isotropic mixing causes the following evolution of z-
magnetization:
𝐼6+,→1/2 [1 + cos(2𝜋𝐽+.𝜏𝑚𝑖𝑥)]𝐼6+, + 1/2[1 − cos(2𝜋𝐽+.𝜏𝑚𝑖𝑥)] 𝐼6.,
− sin(2𝜋𝐽+.𝜏𝑚𝑖𝑥)1/2(2𝐼6+/𝐼6.0 − 2𝐼6+0𝐼6./)
The 𝜏 mix is duration of time during DIPSI-2. The important thing here is that z-magnetization
on spin one is transferred to z-magnetization on spin two, giving rise to cross-peaks in the 2D
spectrum, to an extent that depends on the coupling, and the mixing time. The overall
intensities of the TOCSY cross-peaks depend on the mixing transfer function and the relaxation
that takes place during the isotropic mixing which leads to loss of signal intensity. To get
correlations for smaller couplings, a longer period of mixing is required, thus the loss due to
relaxation will be more severe than when observing correlation through larger couplings.
1.4 Existing work, 1D DISPEL experiment
DISPEL stands for Destruction of Interfering Satellites by Perfect Echo Low-pass filtration. This
work was first done in 2017 in 1D.13 DISPEL contains three components which were
mentioned in Section 1.2: a perfect echo, low-pass filter and zero quantum suppression. The
pulse sequence is
Figure 12: The pulse sequence of the Destruction of Interfering Satellite by Perfect Echo Low-pass filtration (DISPEL).
τ1, τ2, τ3, and τ4 were 3.2, 1.1, 3.95, and 1.56 ms, respectively.
t1 t2
Development of Novel 2D NMR Techniques for Mixture Analysis
21
DISPEL suppresses one-bond satellite peaks with high efficiency at negligible cost in sensitivity.
The effect of DISPEL on a 1D 1H NMR spectrum is shown in Fig. 13. To facilitate quantitative
comparison, the normal 1H spectrum of Fig. 13(a) was obtained using the DISPEL sequence
with the 13C pulses omitted. The one-bond 13C satellite peaks are suppressed with high
efficiency in Fig. 13(b), at negligible cost in signal-to-noise ratio and without the need for
broadband 13C decoupling.
a) b)
Figure 13: 500 MHz 1H NMR spectra of CHCl3 doped with chromium tris-acetylacetonate in DMSO-d6,
(a) without and (b) with the 13C pulses of a 1D DISPEL experiment. One-bond satellite peaks are marked by stars. Each spectrum was acquired with 16 scans, in an experiment time of 1 min and 46 s.
Chapter 2 – 1D DISPEL using simulation
2.1 Introduction
Spinach17 is a MATLAB software library often used for simulation of spin dynamics in all types
of magnetic resonance (NMR, EPR, MRI, etc); it has well-annotated open-source code. Spinach
provides a method to simulate large spin systems quantum mechanically and is a useful tool
to predict the outcomes of experiments in a perfect condition. This chapter contains
simulations done using Spinach and aims to investigate two factors that affect the suppression
of satellite peaks: 13C off-resonance effects and B1 miscalibration. Demonstrations using
important building blocks, the spin echo and perfect echo, were presented in chapter 1 section
2.2.
2.2 Theory of Bloch equations
Before 1966, most chemical applications of NMR used continuous-wave (CW)
spectrometry, which relies on the detection of the steady-state transverse magnetization, as
a function of static magnetic field strength at a fixed frequency. The Bloch equations28 provide
a framework for treating the simultaneous effects of relaxation, RF fields, and resonance offset
(Ω!) for an ensemble of isolated spins-1/2. Offset from resonance is defined by
Ω! = 𝛾𝐵! − 𝜔 (equation 2.1)
𝛿 𝐻"
9.48 9.30 9.12 8.94 8.76 9.48 9.30 9.12 8.94 8.76
𝛿 𝐻"
0.55%
a) b)
Development of Novel 2D NMR Techniques for Mixture Analysis
22
in rad s-1. For simplicity, the Bloch equations follow the magnetization dynamics in a frame of
reference that rotates about the static field direction at the radiofrequency used. The Bloch
equations are a set of three coupled differential equations for the components of the net
nuclear magnetization vector, normalised with respect to the thermal equilibrium z-
magnetization M0. The equations can be written as:29
112G𝑀0𝑀/𝑀,
I=G0 −Ω! 𝜔34 sin𝜙Ω! 0 −𝜔34 cos𝜙
−𝜔34 sin𝜙 𝜔34 cos𝜙 0IG
𝑀0𝑀/𝑀,
I +G–𝑀0 /𝑇.–𝑀/ /𝑇.
(1– 𝑀,)/𝑇+
I
(equation 2.2)
𝜔34 is the nutation frequency of the RF (its amplitude in angular frequency units), and 𝜙 is its
phase. T1 and T2 are time constants used to characterize two types of relaxation – spin-lattice
and spin-spin relaxation that return the spins to equilibrium.8,17,29 The spin-lattice relaxation
re-establishes the Boltzmann distribution of spin states responsible for net z-magnetization,6
and spin-spin relaxation causes loss of the phase coherence between transverse components
of individual spins that is responsible for net transverse magnetization.
The energy of the classical magnetic dipole in a magnetic field is given by:
E =𝜇B0 = 𝜇,Bz (equation 2.3)
where 𝜇 is the magnetic moment, 𝜇, is its z component and the magnetic field B0 is along the
z-axis. The magnetic moment is proportional to its angular momentum, which makes it
𝜇,=𝛾𝐼,.26 𝛾 is the magnetogyric ratio.29 Based on these equations, the quantum mechanical
expression can be obtained by replacing E and Iz by the corresponding quantum mechanical
operators:
𝐻P = 𝛾𝐵!ℏ𝐼6, = ℏ𝜔!𝐼6, (equation 2.4)
where 𝜔! = 𝛾𝐵! is an angular frequency, also known as the Larmor frequency and ℏ is
Planck’s constant divided by 2𝜋. For convenience, energies are often expressed in angular
frequency units in NMR, with ℏ replaced by 1.26
In a CW experiment, the RF field tends to rotate M away from the z-axis while
transverse magnetization is being destroyed by transverse relaxation and spin-lattice
relaxation is regenerating longitudinal magnetization. At a sufficiently long time, the system
settles down into a steady-state, under which these two tendencies are balanced. If the phase
Development of Novel 2D NMR Techniques for Mixture Analysis
23
𝜙 and resonance offset Ω! are both set equal to zero, then the equations for the time
derivatives of the magnetization components My and Mz are: 112𝑀/ = −𝑇. 𝑀/ − 𝜔34𝑀,
#+ (equation 2.5)
112𝑀, = 𝜔34𝑀/ − 𝑇+ 𝑀/(𝑀, − 1)
#+ (equation 2.6)
In the steady-state, both time derivatives vanish and allow one to solve for the two
magnetization components. The steady-state magnetization decreases as T1, T2 and the
nutation frequency (𝜔34)increase.29
However, the Bloch equation assumes that spins do not interact coherently, which is
not the case for many systems for which NMR can provide useful information. NMR, like any
quantum phenomenon, is governed by the time-dependent Schrödinger equation.30 Spins-1/2
have two eigenstates, 𝛼 and 𝛽 : the spin wavefunction (𝜓) is a variable mixture of these.
Quantum mechanics represents the states of systems by wavefunctions (𝜓) and uses
operators to deduce both the values of observable quantities and the evolution of those
wavefunctions. The two eigenfunctions of the operator for the z component of dimensionless
spin angular momentum, Iz, may be given the symbols of 𝛼 and 𝛽 and in Dirac bra-ket notation
|𝛼⟩ and |𝛽⟩:
Iz |α> = ++.|α⟩ Iz |β> = – +
.|β⟩
The state of a single spin-1/2 can be written as a general superposition state:
|𝜓⟩ = U𝐶5𝐶6W (equation 2.7)
where the left-hand side uses the Dirac bra-ket notation for the wavefunction and the right-
hand side tabulates the (complex) coefficients of the spin-1/2 basis states |α> and |β>. If an
operator 𝐴6 acting on a ket |𝜓⟩ yields a result 𝑎|𝜓⟩, then |𝜓⟩ is said to be an eigenvector of 𝐴6
with eigenvalue α.29 If the operator 𝐴6 corresponds to an observable quantity, then the
expectation value obtained from an experimental measurement of this quantity for a system
in the state |𝜓⟩ is given by the scalar product of ⟨𝜓| with 𝐴6|𝜓⟩:
⟨𝐴6⟩=⟨𝜓|𝐴|[𝜓⟩
In practical calculations, we need to know the values Aij of all possible scalar products for
different basis states i and j; these can be written as a matrix representation A, in which the
ij’th element is Aij where Aij =⟨𝜓7|𝐴|[𝜓8⟩.
Development of Novel 2D NMR Techniques for Mixture Analysis
24
NMR experiments measure transverse magnetization, which corresponds to a net
excess of spins with correlated coefficients such as Cα and Cβ: coherences, typically generated
by applying radiofrequency pulses. They can be tabulated in the form of a density matrix, which
summarises the quantum states of the entire ensemble of the spins, without referring to the
states of individual spins. The density matrix can be regarded as representing a density
operator; for an ensemble of non-interacting spins – 1/2:
𝜌] = ^𝜌55 𝜌56𝜌65 𝜌66_ = `
𝐶5𝐶5 𝐶5𝐶6aaaaaaa
𝐶6𝐶5 𝐶6𝐶6b
The diagonal elements of the spin density operator 𝜌55 and 𝜌66 are the populations of the
states |𝛼⟩ and |𝛽⟩.29,31 The off-diagonal elements 𝜌56 and 𝜌65 are the coherences between
states |𝛼⟩ and |𝛽⟩.
Unfortunately, density matrix analysis rapidly becomes unwieldy as the number of
spins increases. For weakly-coupled spin systems, analysis can be greatly simplified by
decomposing the density operator into a sum of product operators, whose evolution can be
calculated using simple rotations.
For a system of isolated spin −1/2 nuclei, the density operator can be decomposed into
the operators E, Ix, Iy and Iz, where the first is the identity operator and the remainder the x, y
and z components of spin angular momentum. From the vector model, it is easy to see how
these magnetizations transform under the influence of pulses with flip angle 𝛽:
𝐼069)cd 𝐼0𝐼0
69*cd 𝐼0 cos 𝛽 − 𝐼, sin 𝛽
𝐼/69)cd 𝐼/ cos 𝛽 + 𝐼, sin 𝛽 𝐼0
69*cd 𝐼/
𝐼,69)cd 𝐼, cos 𝛽 − 𝐼/ sin 𝛽 𝐼,
69*cd 𝐼, cos 𝛽 + 𝐼0 sin 𝛽
The free precession Hamiltonian is:32
𝐻P = Ω!𝐼, (equation 2.8)
It causes rotation about the z-axis at frequency Ω! . Free precession for a time t causes a
rotation through an angle 𝛼 , where 𝛼 = Ω!𝑡. Only x- and y-magnetization are directly
observable in an NMR experiment; it is the precession of the magnetization in the xy-plane
which gives rise to the free induction signal. For a system of two spins, each spin would have
three operators plus the identity, making a total of 16 combinations. As spins 1 and 2 are
coupled, they generate in-phase and anti-phase magnetization and zero-quantum, single-
Development of Novel 2D NMR Techniques for Mixture Analysis
25
quantum and multiple-quantum coherences. As stated earlier, only single quantum can be
observed.
2.3 DISPEL 1D Simulation
2.3.1 Investigation of 1H 90° off-resonance effect
The spin echo is the single most important building block in modern NMR. This basic building
block is used here to investigate the effect of 1H 90° pulse duration and offset from resonance
on the signals obtained. 11 single spins that are not coupled to each other are placed evenly
across the spectrum to demonstrate the off-resonance effect. Three different 1H 90° pulse
durations, 1, 10 and 50 µs, were used to investigate the effect:
a)
b)
c)
Figure 14: Spinach simulation of a 1H spin echo pulse sequence applied to an 11 single-spin system, with 1H 90° pulse durations of (a) 1µs, (b) 10µs, and (c) 50 µs for a range of 10000 Hz.
The on-resonance signal, at 0 Hz, stays at the same intensity for all three 1H 90° pulse durations.
At pulse durations of 1 µs and 10 µs, the offset has little effect. However, at a pulse duration
of 50 µs, the influence of offset is getting stronger as the spins go further away from resonance,
resulting in increased signal phase changes and decreased intensity. The signal intensity drop
is symmetric with respect to resonance.
2.3.2 Investigation of 13C 90° off-resonance effect
Carbon off-resonance effects can have some influence on the degree of 13C-1H coupled
peak suppression. Here, it is investigated using the DISPEL sequence. As Fig. 9 shows, 13C-1H
coupled peaks at 1JCH of 145 Hz is at great level of signal suppression but not perfectly cancelled.
To demonstrate the effect if 13C resonance offset in DISPEL suppression. To demonstrate the
effect of 13C resonance offset on DISPEL suppression, the 13C chemical shift is varied from -
-5000 0 5000 1H chemical shift (Hz)
a) b) c)
Development of Novel 2D NMR Techniques for Mixture Analysis
26
37500 Hz to 37500 Hz for a 1JCH of 145 Hz in Fig. 15. The ratio of the satellite amplitude with
DISPEL to the original satellite amplitude is shown on the right-hand scale.
Figure 15: The effect of the hard 90° 13C pulses on signals obtained for a 13C chemical shift range of 75000 Hz, using Spinach package. Spin system of AX was used. 1JCH of 150Hz was used.
The 13C-1H coupled peaks change sign from negative to positive around +/- 12500 Hz. Past that
point, the suppression is no longer efficient and is experiencing a lot of off-resonance effects.
For a wide range of chemical shift, the suppression was performed efficiently. As it was for 1H
off-resonance effect, the influence of offset is getting stronger as the spins go further away
from resonance.
It is important to note that a hard 90° pulse is already efficient at suppressing satellite
peaks. Considering a normal 90° pulse’s ability to generate transverse magnetization, it can be
said that it contains ‘in-built’ self-compensation for off-resonance effects. Increased signal
phase changes and intensity are caused by the influence of offset driving the vectors further
towards the traverse plane at the expense of frequency-dependent phase errors. These errors
are approximately a linear function of frequency and can be removed through phase correction
of the spectrum.25
2.3.3 Investigation of the influence of composite pulses on 13C off-resonance effect
Composite pulses33 are made up of a number of conventional pulses which rotate the
magnetization vectors about different axes, sometimes with free precession allowed to occur
in the intervals. They can be used to compensate for off-resonance effects. A carefully chosen
composite of imperfect pulses makes a pulse that is more perfect than just a normal pulse.
Composite pulses can be placed within a pulse sequence in place of a hard pulse; however,
composite pulses take slightly more time than normal pulses. There are many variations of
composite pulses: the composite pulse 10°X – 𝜏 – 100°-X33 is used here. To demonstrate the
13C chemical shift (Hz)
0.1
| | | | | | |
-37500 -25000 -12500 0 12500 25000 37500
0.3 0.2
Development of Novel 2D NMR Techniques for Mixture Analysis
27
effect of 13C resonance offset on DISPEL suppression, the 13C chemical shift is varied from -
37500 Hz to 37500 Hz for a 1JCH of 145 Hz in Fig. 16. The ratio of the satellite amplitude with
DISPEL to the original satellite amplitude is shown on the right-hand scale.
Figure 16: The effect of the composite 13C pulses on signals obtained for a 13C chemical shift range of 75000 Hz, using Spinach package. Spin system of AX was used. 1JCH of 150Hz was used.
The 13C-1H coupled peaks change their sign from negative to positive past +/- 18750 Hz instead
of at +/- 12500 Hz like normal pulse did. A wider range of chemical shift was suppressed
compared to a normal pulse. As it was seen in the 13C off-resonance effect, Fig. 15, the
influence of the offset is getting stronger as the spins go further away from resonance.
The comparison of the 13C off-resonance effect using normal and composite 90° 13C for
a 13C chemical shift range of -37500 Hz to 37500 Hz is shown in Fig. 17. The intensity ratio of
relative satellite amplitude is used as a vertical scale.
Figure 17: The effect of the hard and composite 90° 13C pulses on signals obtained for a 13C chemical shift range of 75000 Hz,
using Spinach package. Spin system of AX was used. 1JCH of 150Hz was used.
-0.05
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
-50000 -40000 -30000 -20000 -10000 0 10000 20000 30000 40000 50000
I cou
pled
/ no
t cou
pled
13C chemical shift (Hz) at 1JCH of 145 HzNormal pulse Composite pulse
13C chemical shift (Hz)
0.1
0.2
-37500 -25000 -12500 0 12500 25000 37500
| | | | | | |
Development of Novel 2D NMR Techniques for Mixture Analysis
28
This result demonstrates that the composite pulse provides a modest increase in 13C
bandwidth. In both pulses, the suppression is no longer adequate beyond some offset.
Composite pulses are especially useful when a wider range of chemical shifts is present, such
as for the analysis of a natural compound that contains alkanes and aromatic functional groups.
With a negligible increase in experimental time, the off-resonance effect can be minimized.
2.3.4 Investigation of the effect of B1 miscalibration
B1 miscalibration has an influence on the degree of 13C-1H coupled peak suppression as
it causes imperfect flip angles, similar to off-resonance effects. B1 inhomogeneity can be easily
compensated by nulling Mz using a composite 90x90y sequence. These composite pulses allow
the magnetization vectors to be placed closer to the transverse plane than a single pulse. Fig.
18 shows the Spinach simulated result of 0%, 10% and 20% errors in B1 for a 13C chemical shift
range of 50000 Hz using hard 90° pulses. A 13C pulse duration of 15 𝜇s was used for these
simulations. The intensity ratio of relative satellite amplitude is shown on the right-hand side.
a)
b)
c)
Figure 18: Spinach simulation of DISPEL pulse sequence with B1 error of (a) 0 %, (b) 10 %, and (c) 20 % using hard 90° pulse. The intensity ratio of relative satellite amplitude is shown on the right-hand side. Duration of 90 degrees 1H pulse was 0.1 𝜇𝑠,
13C pulse was 15 𝜇𝑠, and τ1, τ2, τ3, and τ4 were 3.2, 1.1, 3.95, and 1.56 ms, respectively.
With 0 % error, the 13C-1H coupled peaks change their sign from negative to positive past +/-
12500 Hz. With 10 % and 20 % error, the heights of 13C-1H coupled peaks are bigger than they
are with 0 % error on resonance and throughout the range.
-25000 -12500 0 12500 25000
13C chemical shift (Hz)
0.06 0.02
| | | | |
0.12 0.06 0.02
0.18 0.12 0.06 0.02
Development of Novel 2D NMR Techniques for Mixture Analysis
29
The comparison of processed B1 miscalibration data is shown in Fig. 19. The intensity
ratio of relative satellite amplitude is used as a vertical scale.
Figure 19: The effect of the B1 miscalibration on signals obtained using hard pulses for a 13C chemical shift range
of 50000 Hz, using Spinach package. The intensity ratio of relative satellite amplitude is shown on the right-hand side. Duration of 90 degrees 1H pulse was 0.1 𝜇𝑠, 13C pulse was 15 𝜇𝑠, and τ1, τ2, τ3, and τ4 were 3.2, 1.1, 3.95, and 1.56 ms,
respectively.
Fig. 19 demonstrates that with greater errors in calibration, less suppression will be achieved
on resonance and throughout the offset range. With calibration errors, the satellite peaks
would be 3 times taller than when they are without errors at -/+ 25000 Hz.
Composite pulses were used to demonstrate the compensation with B1 error. See Fig.
20. The intensity ratio of relative satellite amplitude is used as a vertical scale.
a)
b)
c)
Figure 20: Spinach simulation of DISPEL pulse sequence with B1 error of (a) 0 %, (b) 10 %, and (c) 20 % using composite 90° pulse Duration of 90 degrees 1H pulse was 0.1 𝜇𝑠, 13C
pulse was 15 𝜇𝑠, and τ1, τ2, τ3, and τ4 were 3.2, 1.1, 3.95, and 1.56 ms, respectively.
The highest amplitude of a 13C-1H coupled peak is approximately 1 % of the uncoupled peak
when there is no error in B1. Compared to hard pulses, composite pulses achieved a steady
suppression over a wider range of chemical shift. The comparison of processed B1
-0.020
0.020.040.060.08
0.10.120.140.160.18
0.2
-30000 -20000 -10000 0 10000 20000 30000
I cou
pled
/ no
tco
uple
d
13C chemical shift (Hz) at 1JCH of 145 Hz0% error 10% error 20% error
13C chemical shift (Hz)
0.01
-25000 -12500 0 12500 25000
| | | | |
0.04 0.01
0.07 0.04 0.01
Development of Novel 2D NMR Techniques for Mixture Analysis
30
miscalibration data is shown in Fig. 21. The intensity ratio of relative satellite amplitude is used
as a vertical scale.
Figure 21: The effect of the B1 miscalibration on signals obtained using composite pulses for a 13C chemical shift range of
50000 Hz, using Spinach package. The intensity ratio of relative satellite amplitude is shown on the right-hand side. Duration of 90 degrees 1H pulse was 0.1 𝜇𝑠, 13C pulse was 15 𝜇𝑠, and τ1, τ2, τ3, and τ4 were 3.2, 1.1, 3.95, and 1.56 ms, respectively.
The figure demonstrates that with 20 % error in B1, the 13C-1H coupled peaks on resonance are
nine times as higher as they are with 0 % error at a chemical shift of -/+ 25000 Hz. With 10%
and 20% errors, the suppression around on-resonance is poorer when compared to hard 90°
pulses. The errors are well compensated by the multiple stages of the 1JCH filter, so suppression
is still more than adequate, as it was for hard pulses.
-0.02
0
0.02
0.04
0.06
0.08
0.1
-30000 -20000 -10000 0 10000 20000 30000
I cou
pled
/ no
t cou
pled
13C chemical shift (Hz) at 1JCH of 145 Hz0% error 10% error 20% error
Development of Novel 2D NMR Techniques for Mixture Analysis
31
Chapter 3 – 1D DISPEL experiment
3.1 Introduction
An experimental investigation of the DISPEL sequence without a z-filter was performed using
three samples: propanol sample, quinine sample and Q-mix sample. The propanol sample is
used to illustrate that DISPEL works; quinine is used to see if a sequence works efficiently on a
complicated chemical structure; Q-mix is used to see if a mixture of similar compounds can be
identified using the sequence. The z-filter is removed from the DISPEL sequence on Fig. 12 to
increase sensitivity by cutting off the duration of Z-filter. The z-filter element is not needed if
the acceptable multiplets can be acquired. The pulse sequence used for the following
experiments was
Figure 22: The pulse sequence of 1D DISPEL without z- and ZQS-filters.
3.2 DISPEL 1D experiment
3.2.1 Investigation of 13C 90° off-resonance effect using propanol
The carbon off-resonance effects can have a great influence on the degree of 13C-1H coupled
peak suppression. Fig. 15 showed the simulated data, but the experimental data have noise,
so the high S/N ratio is required for satellite peaks to be detected. The simulated and the
experimental figures look different because there are three signals in the experimental data,
a parent peak and a satellite peak on either side. The satellite peaks are small because the
natural abundance of 13C is 1.1%. Fig. 23 shows the experimental data of 13C 90° off-resonance
effect on methoxy signal of Q-mixture sample (quinine, quinidine and cinchonidine mixture)
from -40000 Hz to 40000 Hz with respect to on-resonance for the methoxy signal.
Figure 23: The experimental data of effect of the hard 90° 13C pulses on signals obtained for a 13C chemical shift range of
80000 Hz on 500 MHz. Spectrum was acquired with 128 scans and an experimental time of 2 h 36 min.
-40000 0 40000
13C chemical shift (Hz)
0.6
Development of Novel 2D NMR Techniques for Mixture Analysis
32
The intensity ratio at 1 is when there is no suppression. The methoxy signal has 1JCH of 145 Hz,
the same 1JCH value as in the simulation. At +/- 40000 Hz, the satellite peaks can be seen clearly,
but the satellite peaks are suppressed by about 40% even at +/- 40000 Hz. The influence of the
offset is getting stronger as the spins go further away from resonance. Fig. 24 shows
comparison between simulated and experimental data for 13C satellite intensity ratio. The
intensity ratio of 1 is when there is no suppression.
Figure 24: The experimental data of the effect of the hard 90° 13C pulses on signals obtained for a 13C chemical shift range of
80000 Hz
The height of satellite peaks differed from chemical shift to chemical shift and were not seen
to be completely symmetrical, as they were in simulation. At 40000 Hz, the experimental
suppression was worse than in simulation. However, the general trend seen in experimental
data matched with simulation: the edges of the ranges have poorer suppression. Unlike
simulation, experimental data has noise causing little ups and downs but even at the edges of
the range, DISPEL worked efficiently. The DISPEL sequence is pleasingly robust with respect to
resonance offset.
3.2.2 Investigation of the effect of B1 miscalibration using propanol
B1 miscalibration has a great influence on the degree of the satellite peak suppression as it
causes imperfect flip angles. Fig. 18 showed the Spinach simulated result of 0%, 10% and 20%
errors in B1 over a wide range of the 13C chemical shift and Fig. 25 shows the experimental data.
The intensity ratio of 1 is when there is no suppression.
-0.1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
-50000 -40000 -30000 -20000 -10000 0 10000 20000 30000 40000 50000
Inte
nsity
ratio
13C chemical shift (Hz) at CH coupling of 145 HzExperimental Simulation
Development of Novel 2D NMR Techniques for Mixture Analysis
33
a)
b)
c)
Figure 25: Experimental data of DISPEL pulse sequence with hard 90° pulse with B1 error of (a) 0 %, (b) 10 %, and (c) 20 %.
It is clear that the satellite peaks are getting taller with an increase in B1 error. Comparison for
processed B1 miscalibration data is shown in Fig. 26. The intensity ratio of 1 is when there is no
suppression.
Figure 26: The experimental data of the effect of the B1 miscalibration on signals obtained for a 13C chemical shift range of
80000 Hz.
The height of satellite peaks differed from chemical shift to chemical shift again with B1 error.
The general trend seemed to follow the simulation. The comparison between simulation and
the experimental data is shown in Fig. 27.
-0.1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
-50000 -40000 -30000 -20000 -10000 0 10000 20000 30000 40000 50000
Inte
nsity
ratio
13C chemical shift (Hz) at CH coupling of 145 HzExperiment - 0 % error Experiment - 10 % error Experiment - 20 % error
-40000 0 40000
13C chemical shift (Hz)
0.6
0.6
0.7
Development of Novel 2D NMR Techniques for Mixture Analysis
34
Figure 27: The comparison between the experimental data and the Spinach simulation data of the effect of the B1
miscalibration on signals obtained for a 13C chemical shift range of 80000 Hz.
Overall, the experimental data had worse suppression than the simulation data, which was
expected. However, the highest intensity ratio was 0.7, so DISPEL is pleasingly robust with
respect to B1 error.
3.3 DISPEL 1D spectrum
3.3.1 1D spectrum of doped 2.5% propanol
This is the structure of propanol:
This sample was made of 2.5% propanol doped with chromium(lll) acetylacetate, in DMSO-d6.
1D 1H spectra, with and without 13C pulses had been acquired. See Fig. 28.
-0.1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
-50000 -40000 -30000 -20000 -10000 0 10000 20000 30000 40000 50000
Inte
nsity
ratio
13C chemical shift (Hz) at CH coupling of 145 HzExperiment - 0 % error Experiment - 10 % error Experiment - 20 % error
Spinach - 0% error Spinach - 10 % error Spinach - 20% error
Development of Novel 2D NMR Techniques for Mixture Analysis
35
a)
b)
c)
Figure 28: 500 MHz 1H spectrum of n-propanol doped with chromium tris-acetylacetonate in DMSO-d6. (a) without DISPEL (b) without and (c) with 13C pulses the satellite peaks are marked by red arrows.
The 1D spectrum without DISPEL showed four sets of satellite peaks and those are also seen
in DISPEL without 13C pulses. The satellite peaks are marked by red arrows. Fig 28(a) and 28(b)
are expected to be similar. These satellites are suppressed efficiently in Fig. 28(c). This shows
that 1D DISPEL works on a doped chemical like propanol even without a z-filter or ZQS filter.
3.3.2 1D DISPEL spectrum of 100 mM quinine
This is the structure of quinine:
Unlike propanol, quinine is a complicated chemical that contains aromatic rings and many
other functional groups, with a wide range of 13C chemical shift. 99% quinine was used here
without further purification. The 8.36 ppm to 9.0 ppm region of the 1D DISPEL 1H quinine
spectrum with and without 13C pulses is shown in Fig. 29. Satellite peak of interest is marked
𝛿 𝐻" /ppm
Development of Novel 2D NMR Techniques for Mixture Analysis
36
by a red arrow. A peak that overlaps with one of the satellite peaks is marked by a green arrow.
The overlapped peak is an impurity in the sample.
a)
b)
Figure 29: Expansion of 500 MHz 1H DISPEL spectrum of 100 mM quinine in DMSO-d6 (a) without and (b) with without 13C pulses. Each spectrum was acquired with 16 scans.
DISPEL spectra with an experiment time of 2 min and 11 s.
Another peak around 8.45 ppm is another satellite peak, which was suppressed efficiently
using the DISPEL sequence. This region shows where DISPEL is useful. The satellite peak around
8.8 ppm is close to the non-satellite peak, making identification and quantification challenging.
With satellite peaks removed, the peaks from chemical compound can now be identified.
3.3.3 1D DISPEL spectrum of Q-mix
The Q-mixture solution was made up of 23.5 mM of cinchonidine, 35.3 mM of quinine and 29.4
mM of quinidine in DMSO-d6. Here are the structures of cinchonidine, quinine and quinidine:
The structure of all three compounds is similar so they are expected to have overlapped signals
in a 1D 1H NMR spectrum. The 7.7 to 9.0 ppm region of the 1D 1H Q-mix spectrum with and
without 13C pulses is shown in Fig. 30. Satellite peaks of interest are marked by red arrows.
Peaks that overlap with one of the satellite peaks are marked by green arrows.
𝛿 𝐻" /ppm
Development of Novel 2D NMR Techniques for Mixture Analysis
37
a)
b)
Figure 30: Expansion of 500 MHz 1H spectrum of Q-mixture in DMSO-d6 (a) without and (b) with 13C pulses. Each spectrum was acquired with 16 scans. A 1H conventional spectrum was acquired with an experimental time of 1 min 8 s
and DISPEL spectrum with an experiment time of 4 min.
The green peak A was also present in the quinine spectra, but is present in higher
concentration, suggesting that the impurity present in quinine may be cinchonidine. Unlike in
the quinine sample, the cinchonidine peak is dominant here, merging a non-satellite peak with
one of the satellite peaks. This spectrum also demonstrates a case where the suppression of
satellite peaks is useful. Because one of the satellite peaks is almost masked by the dominant
peak from cinchonidine, it is easy to mistake another satellite as a non-satellite peak,
complicating the analysis of the spectrum acquired.
𝛿 𝐻" /ppm
𝐴 𝐵
Development of Novel 2D NMR Techniques for Mixture Analysis
38
Chapter 4 – 2D TOCSY-DISPEL experiment
4.1 Introduction
The DISPEL sequence is shown to work in 1D in the previous chapter; the pulse sequence used
there is concatenated here with a 2D TOCSY sequence. A 180° carbon pulse in the middle of t1
evolution is used to refocus the effect of couplings to carbon-13 in the indirect dimension. The
minimum phase cycle of this variant of 2D TOCSY is 8 steps, and sufficient suppression of
carbon satellites can be achieved without further phase cycling of the DISPEL part. Instead of
phase cycling, gradients31 are used for the DISPEL part. The gradient pulses can be used to
select particular coherence transfer pathways and selection using gradients offers some
advantages and disadvantages when compared to selection using phase cycling. The gradient
pulses are introduced into the pulse sequence in such a way that only the wanted signals are
refocused and observed in each experiment. Therefore, unlike phase cycling, there is no
reliance on the subtraction of unwanted signals, thus, it is expected to have reduced t1-noise.
However, switching on and off a gradient pulse induces currents called eddy currents34 in
nearby conductors. These induced currents generate magnetic fields that perturb the NMR
spectrum in high-resolution NMR probes.
Figure 31:The pulse sequence of 2D TOCSY-DISPEL. τ1, τ2, τ3, and τ4 were 3.2, 1.1, 3.95, and 1.56 ms, respectively.
4.2 2D TOCSY-DISPEL spectrum
4.2.1 2D TOCSY-DISPEL of 2.5% propanol
As seen in 1D DISPEL of 2.5 % propanol in Fig. 28, there are 8 one-bond satellite peaks in this
sample. 2D TOCSY, 2D TOCSY-DISPEL spectra with and without 13C pulses were acquired. See
Fig. 32 for the 2D TOCSY spectrum. The satellite peaks are marked using red arrows.
t1 t2
gt3 gt1 gt3 gt2 gt4 gt4 gt5 gt5
Development of Novel 2D NMR Techniques for Mixture Analysis
39
Figure 32: 500 MHz 2D TOCSY spectrum of n-propanol doped with chromium tris-acetylacetonate in DMSO-d6. The experiment was acquired with 8 scans and 512 increments in an experiment time of 8 h 53 min. The satellites are
marked by the red arrows.
All 8 satellite peaks (marked with red arrows) were clearly seen in the 2D TOCSY spectrum as
expected. However, the t1-noise showed up as streaks. The t1-noise is caused by instrumental
instabilities, giving (pseudo-)random variations of FID amplitudes during the data acquisition,
which then introduces random noise-like peaks into the spectrum obtained by Fourier
transforming the acquired data. See Fig. 33 for 2D TOCSY-DISPEL spectra with and without 13C
pulses.
a) b)
𝛿 𝐻" 𝛿 𝐻"
𝛿𝐻 "
0.6
1.8
3.0
4.2
0.6
1.8
3.0
4.2
0.6
1.8
3.0
4.2
𝛿𝐻 "
/ppm
4.2 3.0 1.8 0.6 4.2 3.0 1.8 0.6
4.2 3.0 1.8 0.6
𝛿𝐻 "
/ppm
𝛿 𝐻" /ppm
𝛿 𝐻" /ppm
𝛿 𝐻" /ppm
𝛿𝐻 "
/ppm
Development of Novel 2D NMR Techniques for Mixture Analysis
40
Figure 33: 500 MHz 2D TOCSY-DISPEL spectra of n-propanol doped with chromium tris-acetylacetonate in DMSO-d6, (a) without and (b) with 13C pulses. Each experiment was acquired with 8 scans and 512 increments in
an experiment time of 9 h 27 min. The satellite peaks are marked by red arrows.
The 2D TOCSY-DISPEL spectrum of 2.5% doped propanol shows 8 one-bond satellite peaks, as
marked by red arrows on Fig. 33(b). The 2D TOCSY-DISPEL was expected to give similar
spectrum as convectional 2D TOCSY, however, the t1-noise showed up more severely. With 13C
pulses, the satellite peaks were efficiently suppressed, as seen in Fig. 33(b). To take a look at
these spectra closely, part of the spectrum marked by a purple box is shown expanded in Fig.
34.
a) b)
Figure 34: Expansion of 500 MHz 2D TOCSY-DISPEL spectra marked by a purple square in Fig. 33. (a) without and (b) with 13C pulses. Each experiment was acquired with 8 scans and 512 increments
in an experiment time of 9 h 27 min. The diagonal satellite peaks are marked by red arrows.
The diagonal satellite peaks are marked by red arrows. There are other peaks that are not
marked called cross-peaks. The t1-noise creates the technical challenge of this 2D spectra,
however, the DISPEL suppressed satellite peaks successfully as seen in Fig. 33(b) and 34(b). The
DISPEL worked effectively not only in 1D but in 2D NMR on 2.5 % propanol.
4.2.2 2D TOCSY-DISPEL spectra of 100 mM quinine
As it is seen in Fig. 29, the satellite peaks overlap with a non-satellite peak. The 2D TOCSY-
DISPEL with and without 13C pulses was acquired. The region of interest is expanded here to
see how effectively the DISPEL has worked. The satellite peaks are marked by red arrows and
the non-satellite peak is marked by green arrows.
𝛿 𝐻" /ppm
𝛿 𝐻" /ppm
𝛿𝐻 "
/ppm
𝛿𝐻 "
/ppm
Development of Novel 2D NMR Techniques for Mixture Analysis
41
a) b)
Figure 35: Expansion of 500 MHz 2D TOCSY-DISPEL spectra of 100 mM quinine in DMSO-d6, (a) without and (b) with 13C pulses. Each experiment was acquired with 8 scans and 512 increments in an experiment time of
12 h 33 min. The satellite peaks are marked by red arrows and the non-satellite peak is marked by green arrows.
The satellite peaks are separated in 2D better than they were in 1D and can be distinguished
from one another. Therefore, this specific area did not need to use DISPEL to help identifying
the peak from quinine in 2D NMR. Nonetheless, the satellite peaks are suppressed; however,
Fig. 35(a) shows a problem - the satellite peaks are not identical in 2D spectrum. There is no
splitting visible in one of the satellites, which may be noise leading to the two doublet
components not being resolved. This could be due to perfect echo being imperfect due to
concatenation with TOCSY sequence. During the TOCSY period, the spins are still evolving, thus,
may not be in the condition that perfect echo is set to work. The t1 noise is seen as a streak
again, too.
4.2.3 2D TOCSY-DISPEL spectra of Q-mix
The Q-mix is made out of three chemical compounds: quinine, quinidine and cinchonidine. As
they were shown in Section 3.3.3, they are similar in molecular shape. The quinine and
quinidine are stereoisomers, whereas cinchonidine does not have methoxy. The full spectrum
of the 2D TOCSY-DISPEL was acquired using hard and composite pulses for the carbon channel.
The region of interest in Fig. 30 is shown here to see if the satellites were successfully
suppressed in 2D TOCSY-DISPEL.
8.36 8.64 8.96
8.96 8.64 8.36 8.96 8.64 8.36
8.36 8.64 8.96
𝛿𝐻 "
/ppm
𝛿𝐻 "
/ppm
𝛿 𝐻" /ppm
𝛿 𝐻" /ppm
Development of Novel 2D NMR Techniques for Mixture Analysis
42
a) b)
Figure 36: Expansion of 500 MHz 2D TOCSY-DISPEL spectra of Q-mix in DMSO-d6, (a) without and (b) with 13C pulses. Each experiment was acquired with 16 scans and
512 increments in an experiment time of 25 h.
The two regions of interests are marked by orange and yellow squares. To examine these
spectra closely, the portion of the spectrum marked by orange and yellow boxes is shown
expanded in Fig. 37 and in Fig. 38, respectively.
a) b)
Figure 37: Expansion of 500 MHz 2D TOCSY-DISPEL spectra of Q-mix in DMSO-d6, marked by orange square in Fig. 36. (a) without and (b) with 13C pulses. Each experiment was acquired with 16 scans and 512 increments in an experiment time
of 25 h. The satellite peak marked by red arrow is overlapping with the cinchonidine signal marked by a green arrow.
The satellite peak is now removed and this cinchonidine peak is fully resolved. Another satellite
peak seen at the right top corner of the left spectrum was suppressed with the use of DISPEL.
7.70 8.20 8.70
7.70 8.20 8.70
8.70 8.20 7.70 8.70 8.20 7.70
8.40 8.68 8.92
8.40 8.68 8.92
8.92 8.68 8.40
8.92 8.68 8.40
𝛿𝐻 "
/ppm
𝛿
𝐻 "/p
pm
𝛿 𝐻" /ppm
𝛿 𝐻" /ppm
𝛿𝐻 "
/ppm
𝛿𝐻 "
/ppm
𝛿 𝐻" /ppm
𝛿 𝐻" /ppm
Development of Novel 2D NMR Techniques for Mixture Analysis
43
a) b)
Figure 38: Expansion of 500 MHz 2D TOCSY-DISPEL spectra of Q-mix in DMSO-d6, marked by yellow square in Fig. 36 (a) without and (b) with 13C pulses. Each experiment was acquired with 16 scans
and 512 increments in an experiment time of 25 h. The satellite peak of interest is marked by red arrow.
The satellite peak of interest is marked by red arrow and that satellite peak and the peak one
around 7.5 ppm is not symmetrical. However, this is due to those satellite peaks having
different parent peaks. The composite pulses were used in 2D TOCSY-DISPEL to reduce off
resonance effect. See Fig. 39.
a) b)
Figure 39: Expansion of 500 MHz 2D TOCSY spectra of Q-mix in DMSO-d6, with (a) hard and (b) composite 13C pulses. Each experiment was acquired with 16 scans and 512 increments in an experiment time of 25 h.
7.54 7.68 7.82
7.54 7.68 7.82 7.82 7.68 7.54
7.82 7.68 7.54
7.70 8.20 8.70
7.70 8.20 8.70
8.70 8.20 7.70 8.70 8.20 7.70
𝛿 𝐻" /ppm 𝛿 𝐻" /ppm
𝛿𝐻 "
/ppm
𝛿
𝐻 "/p
pm
𝛿𝐻 "
/ppm
𝛿𝐻 "
/ppm
𝛿 𝐻" /ppm
𝛿 𝐻" /ppm
Development of Novel 2D NMR Techniques for Mixture Analysis
44
For the Q-mixture sample, the hard pulses were able suppress satellite peaks and showed
almost no difference with the spectrum that used composite pulses. The use of composite
pulses is to overcome off-resonance effects but was not required for Q-mixture.
Development of Novel 2D NMR Techniques for Mixture Analysis
45
Chapter 5 – Discussion
5.1 Conclusions
In summary, the DISPEL sequence worked on a simple, a complicated and mixture of samples
not only in 1D but also in 2D NMR.
Chapter 2 investigated two factors that affect the suppression of 13C-1H coupled peaks: 13C off-
resonance effects and B1 miscalibration. Simulations demonstrate that suppression over a
wider range of 13C offset can be obtained with the use of composite pulses, and even with B1
errors, efficient suppression can be achieved for a wide range of chemical shift.
Chapter 3 showed that DISPEL spectra without 13C pulses were similar to 1D 1H NMR spectra.
The satellite peaks are removed by DISPEL, helping with identifying and distinguishing non-
satellite and satellite peaks. The experimental data suggested that the suppression is sufficient
with a normal B1 level and even with significant error in B1.
Chapter 4 used three samples to show that the DISPEL works when concatenated with 2D
TOCSY. However, the t1-noise is seen as streaks, making cross-peaks harder to identify. The
composite pulses were also used; however, the sample is not affected by the off-resonance
effect enough for them to be useful.
5.2 Discussion
Composite pulses were used to overcome the 13C off-resonance and B1 miscalibration
effects, but there are other ways to overcome off-resonance effects. Off-resonance effects can
be dealt with by using higher power transmitters that are able to excite over wider bandwidths.
However, it is hard for the probe to sustain such high powers without being damaged, and
sample heating can occur. Therefore, the use of composite pulses to overcome off-resonance
effects is a better approach.33 But there is a limitation in composite pulses as well – that the
sequences which provides the best compensation tend to be the longest and most complex,
always longer than the simple pulses so may not be suitable for use within all the pulse
sequence.24 There is sufficient time in a DISPEL-TOCSY sequence to implement simple
composite pulses, but to implement something more complicated for better compensation of
off-resonance effects would be challenging. Also, composite pulses that have been designed
for a particular initial magnetization state may not perform well, or may give unexpected
results, so when applied to other states. 180° composite pulses can be designed specifically for
Development of Novel 2D NMR Techniques for Mixture Analysis
46
inversion or for refocusing, in which they act on longitudinal and transverse magnetization
respectively.24 A sequence such as 90y180x90y can provide offset compensation when used as
a refocusing pulse, but it introduces errors in phase that may be harmful to the overall
performance of an experiment. Thus, it is important to check whether a compensated pulse
sequence will actually show improvements over the uncompensated sequence.24 There is
another way to reduce B1 miscalibration, which is to use adiabatic pulses – these are 180° pulse
and give very effective simultaneous compensation for resonance offset and miscalibration,
but swept-frequency 90° pulses do not.35 However, DISPEL does not contain 180° pulses in 13C,
so this cannot be applied in 2D TOCSY-DISPEL.
The graphical figures in chapter 3 show that the general trend seen in experimental
data matched with simulation: the edges of the offset range have poorer suppression. The
experimental data were slightly different from the simulation, as it is expected. This is likely to
be due B1 inhomogeneity, and possibly also to the transient errors in phase and amplitudes
that occur at the starts and ends of pulses, known as phase glitch.36 B1 errors can be partially
compensated by composite pulses, but as can be seen in Fig. 26, this is less effective off
resonance. The phase glitch problem causes phase errors arising from current switching phase
transients in the transmitter coil at both leading and tailing edges of the RF pulse. The idea of
“glitches” came from Ellett et al.37 and Mehring and Waugh.38,39 The phase glitch problem can
be reduced by using longer, lower power pulses, but this would defeat the purpose of
improving performance off resonance.
The t1-noise was one of the problems seen in 2D TOCSY-DISPEL spectra, making cross-
peaks harder to identify. The t1-noise is not a problem that is seen only in DISPEL-TOCSY but
occurs in all nD NMR spectra. In case of the NOESY, co-addition of multiple spectra has been
reported to significantly reduced the t1-noise compared to conventional acquisition with the
same total acquisition time and resolution.40 One factor increasing t1-noise can be long data
acquisition time. Shortening the acquisition time and acquiring many short experiments, then
adding them together can give a reduction in t1-noise. This method could be applied to the 2D
DISPEL-TOCSY sequence. This sequence requires a minimum of 8 phase cycling steps, so 8 scans
per increment would be required. However, this method would only be advantageous when
the number of scans perincrement required to obtain adequate signal-to noise ratio exceeds
the number needed to complete the pulse sequence's phase cycle.40 Breaking a single long 2D
Development of Novel 2D NMR Techniques for Mixture Analysis
47
acquisition into multiple shorter ones will cost additional disk space and time to process, but
this problem is fairly easy to be solved.
Another method for suppressing t1-noise is called reference deconvolution.41 This is a
powerful processing method for removing t1-noise that affect all peaks in a spectrum in the
same way. The application of reference deconvolution to 2D COSY NMR spectrum has been
illustrated on a tetracyclic orthoamide in deuteriochloroform using TMS as the reference
signal.41 This method improved the spectrum quality, giving a much cleaner and more
informative spectrum. Reference deconvolution could also be applied in 2D DISPEL-TOCSY. The
spectrum of interest and the reference signal are acquired simultaneously, and application of
processing method can be applied. One downside of this method is lower S/N ratio, as the
noise from the experimental reference signal is included in the correction function and
therefore gets convolved into the corrected spectrum.42
5.3 Future work
DISPEL offers a way to deal with some complications in mixture analysis, however, it can be
improved. The t1-noise was seen, as expected in 2D spectra. The t1-noise was more noticeable
because the vertical scale was increased to show the 13C satellites, which are only 0.55% of the
parent peaks. t1-noise is a problem in any 2D experiment looking at such small signals, but it
complicates the cross-peak identification and hinders the identification of the structures of the
unknown chemicals. Reduction of t1-noise is an area to look into further. Also, application of
DISPEL in other 2D method could improve analysis of high dynamic range mixtures.
Development of Novel 2D NMR Techniques for Mixture Analysis
48
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