Using Nuclear Magnetic Resonance Spectroscopy to Uncover the Structure of

Thearubigins

Ashley Cooper1
Grade: 12
Math and Science Academy
Ocean Lakes High School

1 Special thanks to both Dr. Jingdong Mao, Professor at ODU, Wenying Chu, his
graduate student, and Joshua Tubb, a student at William and Mary, for providing me with
the opportunity to collaborate on this research with them as well as providing guidance
and various resources throughout the process.

Using Nuclear Magnetic Resonance Spectroscopy to Uncover the Structure of

Thearubigins
Introduction
Up to now, very little has been known about the structures of thearubigins (TRs),
which make up the majority of black tea pigments. Thearubigins are the numerous
compounds formed during the fermentation process used to produce black tea from the
Camellia Sinensis leaves, which are also used to make green tea and oolong tea, and are
often studied as a mixture and refered to as the thearubigin faction of the tea. By
elaborating on the knowledge regarding these compounds, a better understanding of the
characteristics of various brands of black can be provided, allowing for an advancement
in both tea studies regarding health benefits and the general standard of commercial teas
(Gebely, 2013).
So far, multiple techniques such as chromatography and mass spectroscopy have
been utilized in an attempt to uncover the structures of TRs in tea. However, since there
are so many compounds present in the fraction, it is difficult to identify specific
structures. One of the techniques that has only recently been used in tea research is
Nuclear Magnetic Resonance (NMR) Spectroscopy (Ozawa et al., 1996).

How Nuclear Magnetic Resonance (NMR) Spectroscopy Works

NMR Spectroscopy is employed widely for elucidating the structures of molecular
compounds (Kivela, n.d.). 1H and 13C are the two most frequently used nuclei. (Lambert,
Joseph B., & Mazzola, Eugene P., 2003).

Both protons and the carbon-13 nuclei have non-zero spins, which allows for
them to have a magnetic moment, or the small magnetic field generated by the nuclei of
each individual atom (or proton in the case of H-NMR) (Sheffield Hallam University,
n.d.). An external magnetic field is applied on the sample, and the bulk magnetization, or
the net effect of the magnetic moments of all the nuclei in a sample at equilibrium, will
be aligned with the applied magnetic field. When the magnetic moments of the nuclei do
not oppose the applied magnetic field, they are said to be in their lower energy state,
therefore, the majority of the nuclei are initially in their lower energy state (Keeler,
2004).
The bulk magnetization vector can be thought of as having a longitudinal
component, refered to as the z-axis and a transverse component, refered to as the x-y
plane (Reusch, 2013). After the sample has reached equilibrium and produced a constant
bulk magnetization vector, the pulse sequence, a series of radio frequencies equivalent to
the Larmor frequency, the frequency at which the nuclei in a sample precess about the zaxis, is irradiated. Some of the nuclei then absorb the extra energy and flip their spins, to
enter a higher energy state where their magnetic moments oppose the applied magnetic
field (Blauch, 2014). Typically, the bulk magnetization vector is initially in alignment
with the z-axis. Then, when the pulse sequence is issued, ideally at a 90 degree angle with
respect to the z-axis and applied magnetic field, the bulk magneization vector shifts to the
y-axis. After a period of time, a state called relaxation occurs, and the bulk magnetization
vector relaxes back to the lower energy state (Reusch, 2013).
There are two types of relaxation: spin-lattice and spin-spin. Spin-lattice
relaxation occurs as the excited nuclei resonate with all the other nuclei precessing at

Larmor frequency and release energy back into the lattice, or molecular surroundings.
Therefore, this type of relaxation involves interactions between neighboring spins in
order to return to equilibrium. Spin-spin relaxation deals with transverse relaxation, or
any process that returns the x and y magnetizations to their equilibrium postions
(Lambert, Joseph B., & Mazzola, Eugene P., 2003).
When relaxation occurs, the reduction in y magnetization with time is detected as
free induction decay (FID). This signal is represented on a graph with FID Amplitude on
the y-axis and time on the x-axis. The FID time domain can then be converted to
frequency domain as a chemical shift value using a variation of Fourier Transform, a
process of converting time domain to frequency domain. After this, a new spectrum is
produced with chemical shift on the x-axis, and intensity on the y-axis. (Lambert, Joseph
B., & Mazzola, Eugene P., 2003).
Types of NMR
There are two main types of NMR: solution and solid-state. Like the
terminologies imply, solution NMR requires the sample to be dissolved in a
homogeneous solution prior to transfering it to the NMR tube, while in solid-state NMR,
the sample can simply be packed into the specialized rotor and then inserted into the
NMR. Additionally, various pulse sequences (or techniques) exist for both types of
NMR.
Both solution and solid-state NMR have either one-dimensional (1D) or twodimensional (2D) techniques. 1D NMR is the simplest form, studying only the properties
of an individual nucleus without showing additional correlations, while 2D NMR can
provide information on correlations between nuclei.

1D Solution NMR
This type of analysis produces a spectrum consisting of chemical shift on the xaxis and intensity on the y-axis.
In an NMR spectrum, the peaks are differentiated by their chemical shift values.
The chemical shift represents the frequency at which a nucleus undergoes relaxation and
can be used to help determine more information about the environment of that nucleus,
including the types of surrounding nuclei and bonding. For example, in 1H NMR,
aromatic compounds typically fall in the range of 6.5-8.5 ppm, while halides have
chemical shift values of between 2.1-4.5 ppm (Lambert, Joseph B., & Mazzola, Eugene
P., 2003).
One of the determining factors of chemical shift is nuclear shielding, or to what
extent the the nucleus is shielded by its surrounding electrons. The electronegativity of
neighboring atoms plays a large role in determining the amount of shielding. If a proton
is bonded to a carbon that is also bonded to an electronegative atom such as oxygen, then
it will be less shielded than a proton on methane, because the electronegative atom will
pull electrons from the hydrogen, deshielding it in the process (Khan Academy, 2014,
July 23). There is an inverse relationship between nuclear shielding and the chemical
shift value, because the shielding lowers the effective magnetic field on the proton or
atom, so that a lower frequency is required to flip the spin of its nucleus (Khan Academy,
2014, July 21). If two or more protons are in the same environment, then they are
chemically equivalent and will have the same chemical shift (Khan Academy, 2014, July
22).

Intensity in a 1H NMR spectrum represents the quantity of chemically equivalent

protons or atoms. The relative number of each type of proton in a sample can be
calculated by taking the integral of the area under each peak in the spectrum. Then, divide
all calculated integral values by the smallest value. If all integers are produced, then each
number represents the number of protons or atoms specific to a certain peak. If not, then
the numbers must be multiplied by a common factor to produce all integers (Khan
Academy, 2014, July 24). This process yields the number of protons specific to each
peak.
In a 1H NMR spectrum, there will be spin-spin splitting or coupling present.
Splitting is the term given to the tendency of the spins of neighboring protons in a sample
to alter the resonance of a proton group, therefore splitting its respective signal into
multiple peaks (Khan Academy, 2014, July 26). This can contribute to knowledge
concerning how many equivalent neighboring protons surround a given proton group
using the n+1 rule, where n is the number of neighboring protons. Using this rule, one
can deduce the number of peaks by adding one to the number of neighboring protons or
use it in reverse to determine the number of neighboring protons by subtracting 1 from
the number of peaks. For example, if a spectrum has a doublet, which consists of two
peaks, then one could conclude that the proton group represented by the doublet has one
neighboring proton (Khan Academy, 2014, July 27). Another factor involved with
splitting is the coupling constant, which refers to the distance between peaks of a signal
(Khan Academy, 2014, July 28). If protons are coupled, then they have the same coupling
constant and the n+1 rule can be applied, however, if they have different coupling
constants, then it produces complex splitting. In this case, only a modified version of the

n+1 rule can be applied, where the signal is split first based on the coupling constant of
one group, then each group produced by that split is then split again based on the
coupling constant of the next group. This process is then repeated for the number of
different chemical shifts present and then one can be added to the resulting number to
show how many peaks to expect from the signal (Khan Academy, 2014, July 28).
Figure 1, shown below, an example of a 1H NMR spectrum is shown. As stated
previously, the height of the peaks represent the intensity of the signal, while the location
of the peaks suggests the identity of the functional group which it represents.

Fig. 1 (Untitled 1H NMR spectrumfor a protein)

2D Solution NMR
NMR can also be two-dimensional. This type of analysis is more complicated than 1D
NMR, due to the fact that it analyzes the correlations between the different nuclei, rather
than focusing on the signals of an individual nucleus (Lambert, Joseph B., & Mazzola,
Eugene P., 2003). While 1D NMR only undergoes two steps: a preparation period where
the 90 degree pulse is issued and a detection period where the NMR determines the
2 (Untitled 1D NMR spectrum for a protein)

frequency at which relaxation occurs, 2D NMR undergoes two extra periods in between:
evolution and mixing time. Evolution deals with the nuclei precessing freely after the
initial 90 degree pulse for a time T1, while mixing time allows the nuclei to react to a
second 90 degree pulse for time T2. During the detection phase, the NMR then processes
this data to produce the FID signal (Schirra, 1996).
Within 2D NMR, there are different types of correlations which the NMR
machine can be programmed to analyze, either falling under the category of
heteronuclear correlation or homonuclear correlation. Heteronuclear correlation deals
with the interactions between the nuclei of different types of atoms, while homonuclear
correlation refers to the interactions between nuclei of the same types of atoms
(University of California, Davis, n.d.).
The simplest form of 2D NMR is a type of homonuclear correlation called
Correlation Spectroscopy (COSY), which reveals the relationships between protons on
neighboring carbons only. In a COSY spectrum, each dimension represents a 1D H-NMR
spectrum on an axis, as shown in Figure 2. The second dimension in COSY can be
considered to be an identical spectrum, going vertically rather than horizantally
(Wolstenhome-Hogg, 2012). The The diagonal is simply a reference line to keep track of
each proton group by representing them with a contour on the line. However, the
coupling of neighboring protons is shown by the contours outside of the diagonal peak,
which each show the correspondence between two coupled protons. In figure 2, it terms
these contours as cross peaks. By using these cross peaks, one can determine which
protons are attached to adjacent carbons, helping to elucidate the overall structure of
unknown molecules. In Figure 2, an example of a COSY spectrum is shown. As

described above, each axis represents a 1H NMR spectrum, allowing the correlations
between the protons to be determined by finding the cross peaks.

Fig. 2 (Untitled COSY spectrum)

Another common form of 2D NMR is heteronuclear single quantum correlation (HSQC),

which is useful when a 1D NMR spectrum has many overlapping peaks (Lambert, Joseph
B., & Mazzola, Eugene P., 2003). It also has a much higher sensitivity than any carbondetecting experiment, because 1H nuclei are much more abundant than 13C and have a
larger gyromatic ratio. In organic chemistry, this type of analysis is commonly used to
study correlations between 1H and 13C, since they are primary components of most
organic compounds. As shown in Figure 2, the 1D 1H NMR is typically on the horizantal
axis, and the 1D 13C NMR is represented on the vertical axis. Since HSQC studies
heteronuclei, there is no diagonal to place the basic points on, because the spectra on each
axis are different. Thus, the HSQC experiment is slightly different from that of COSY
(Harned, 2009). Although it still inteprets data from only one bond away similar to how
3 (Untitled COSY spectrum)

COSY only shows correlations between adjacently bonded protons, the results yield
information about coupling between protons and their adjacent carbons, revealing their
connectivities (Decator, 2011). Despite the fact that this type of experiment correlates
heteronuclei, it is still based on proton-detection, because this form has a much higher
sensitivity than a carbon-detection experiment would (Parella, 2003). It does this by using
inverse detection, the process by which it detects the chemical shifts of the carbon-13
nuclei by first detecting the chemical shifts of its attached protons (Lambert, Joseph B., &
Mazzola, Eugene P., 2003). Shown below, in Figure 3, is an example of a HSQC
Spectrum. The 1H NMR spectrum is portrayed at thetop on the horizantal axis, while the
13

C NMR spectrum is shown to the left on the vertical axis. Relating these 1-dimensional

spectrum with a 2-dimensional graph allows for an expansion of the knowledge regarding
the linkages between heteronuclei.

4 (Untitled HSQC spectrum)

The third type of NMR experiment that will be useful for analysis of thearubigins
is called heteronuclear multiple bond correlation (HMBC). This type of experiment is
similar to HSQC in that it relates heteronuclei (typically 1H and 13C), however, this
technique allows for the analysis of connectivities across multiple bonds instead of just
one bond (Decator, 2011). Although it is less sensitive than HSQC, it provides some
unique advantages, such as the ability to detect carbons that are not bonded to any
protons (quaternary carbons, carbonyls, etc.). HSQC is unable to detect these types of
carbon, since it uses proton detection, but knowledge regarding these types of
connectivities allows for the determination of couplings between these groups with other
carbon groups and protons as well (Lambert, Joseph B., & Mazzola, Eugene P., 2003).
The analysis process of HMBC is similar to that of HSQC with respect to how the CNMR spectra and the H-NMR spectra are represented on the graph. The main difference
falls under their ability to detect protonless carbons (Vasavi et al., 2011). Shown below is
an example of a HMBC spectrum. As stated previously, the way in which this type of
spectrum is analyzed is similar to that of HSQC, and shows information regarding the
chemical connectivities of the compound.

Fig. 4 (Untitled HMBC spectrum)

Solid State NMR

Solid State NMR is used, because it provides unique insight regarding structural
knowledge, such as spatial and dynamic information based on the anisotropic spin
interactions (Spiess, 2012). It also allows researchers to study compounds which are
generally insoluble.
It varies from solution NMR in that it requires different conditions in order for the
sample to produce meaningful results. Since the sample is not dissolved in any type of
solvent, the molecules are fixed in position rather than randomly tumbling as they do in
liquids. Because of this, the applied magnetic field must be even stronger than it would
for solution NMR to eliminate unwanted coupling effects. Additionally, in solids, the
chemical shielding anisotropy is determined by molecular orientation with respect to the
applied magenetic field rather than having a single average value to represent a peak. In
order to alleviate these complications, magic angle spinning (MAS) can be applied. MAS
involves a rotor spinning at a faster rate than shielding anisotropies at a 55.44 degree
angle with respect to the applied magnetic field. Magic angle with high speed spinning
mimics the molecular tumbling process that occurs in solution, to allow for an average
shielding values to be calculated, thus producing peaks rather than broad lumps (Lambert,
Joseph B., & Mazzola, Eugene P., 2003). Due to the high rates of shielding anisotropies
of some nuclei, such as protons, most solid-state NMRs are more limited in which nuclei
they can analyze, because most cannot spin at a fast enough rate (Schurko, 2013).
These quick rates of protons can actually prove to be an advantage in NMR
analysis though. In addition to MAS, cross polarization is usually applied to observe

dilute spins (13C) with low abundance and long relaxation (Hediger et al., n.d.). This
technique transfers magnetizationfrom abundant spins (1H) to dilute spins (13C) to
enhance S/N and reduce waiting time between successive experiments, making the NMR
analysis process considerably faster (Lambert, Joseph B., & Mazzola, Eugene P., 2003).
Cross polarization may also be implemented with dipolar dephasing. This method
produces a delay after the acquisition phase, and since carbons with strong dipolar
coupling decay faster than carbons with weak coupling, it removes signals from nulcei
bonded to rigid H and produces spectra with only non-protanated C (quaternary and
carbonyl carbons) and mobile groups such as methyl carbons present (Facey, 2015). Then
protanated C spectra can be obtained by subtracting the dipolar dephasing spectrum from
the full spectrum if their pulse sequence timings are the same.
The preparation process for samples for solid state also differs from that of
solution NMR, since it doesnt require any type of solvent. Therefore, the sample is
simply packed into a specialized NMR rotor, then inserted into the machine itself.

Importance of Black Tea

Not only is black tea one of the most widely consumed beverages in the world,
but claims have commonly been made regarding the ability of the antioxidants found in
the tea to prevent various diseases (Menet et al., 2004). Additionally, the extracts from
teas, including black tea, are marketed as dietary supplements (Li et al., 2013). Therefore,
knowing the chemical make up of this tea would prove to be beneficial in nutritional and
medical areas, as well as general quality control of commercial black tea.
Thearubigins and Other Polyphenols in Black Tea

Despite the popularity of black tea as a common beverage, much about its
composition remains unknown. The Camelia sinesis plant is used to make green tea,
oolong tea, and black tea, but in order to produce black tea, the leaves must undergo a
fermentation process (Ohno et al., 2011). During this fermentation process, the
gallocatechins present in the unfermented tea leaves are condensed into theaflavins and
thearubigins during an oxidation reaction catalyzed by polyphenol oxidases. The specific
structures of both the gallocatechins and the theaflavins have been elaborated on
thouroughly, but, as stated previously, thearubigin structures remain a relative mystery.
This thearubigin fraction is accountable for 60-70% of the weight of dry tea leaves,
however, information regarding the identity of the molecules within the mixture has been
extremely limited (Kuhnert et al., 2010). So far, a few methods have been used to study
thearubigins, such as multiple types of mass spectroscopy, high perfomance liquid
chromotography (HPLC), and recently, NMR, each adding and confirming information
regarding thearubigin structure. In 2010, roughly 100 compounds found in thearubigins
had been identified using NMR, 35 of which were able to be confirmed with mass
spectroscopy in a study done by Nikolai Kuhnert (Kuhnert et al., 2010). In one study
involving NMR analysis, the most abundant signals matched that of theanine, caffeine,
theaflavin gallate, and thearubigin 3, 3 gallate. The same study also yielded results
agreeable with the structure predicted by Kuhnert (Ohno et al., 2011).
The Extraction Methods
In order to study the thearubigins, they must first be isolated from the other
mixtures in the tea. Therefore, previous studies have used different extraction techniques
to accomplish this. In one study, ethyl acetate and chloroform were used as extracting

solvents, then once the thearubigin faction was isolated, it was redissolved in boiling
water and then freeze dried to produce a fluffy light brown powder as the final product.
This method had a yield of 8%, which is relatively high in comparison to most of the
other methods. The main drawback, however, was the time required, since it takes nearly
two days to complete one sample (Kuhnert et al., 2010). Another study used CHCl3 to
decaffenate the tea and extract the solution, then freeze dried the product. The resulting
tea extract was then dissolved in 50% aqueous MeOH and transferred into a Solka-floc
cellulose column before being eluted with excess MeOH and Me2CO to produce a 7%
yield of a dark brown powder as a final product. The main drawback of this method is
that it is intended to produce Theatricin A, so it is unclear where exactly the thearubigin
faction is produced (Davis et al., 1997). An additional method for extraction was
suggested in another study, which used methanol to elute the tea prior to freeze drying the
mixture and using High-Speed Countercurrent Chromatography (HSCC) to complete the
extraction. This had an 11% yield of a solid final product, which is the highest out of all
the reviewed methods, however, it requires the use of an HSCC (Degenhardt et al., 2000).

Procedure
Prior to NMR analysis of the thearubigins, the samples must be extracted from
black tea. To ensure full coverage of the tea leaves cultivated in different environments,
samples will be obtained across multiple continents, the specific brands being: Upton Tea
Imports (Africa), Arbor Teas (Asia), American Classic (North America), Holmfirth Tea
(Europe), SerendipiTea (South America), and DainTree Tea (Australia). After reviewing

multiple extraction methods, it was decided that a slightly altered version of the Kuhnert
method would produce the best results for this experiment.
First, freshly ground black tea leaves (3.2 g) were added to an Erlenmeyer flask with 80
mL deionized water and kept boiling for 10 min. The flask contents were filtered through
a Whatman No. 4 filter paper to remove the leaves, and the remaining brew allowed to
cool to room temperature. Caffeine sufficient to achieve 20 mM was added to the brew,
stirred to ensure dissolution, allowed to stand at 4C for 2 h, and centrifuged at 23 300 g
for 20 min. The resulting precipitate was recovered and suspended in boiling water, and
partitioned against aliquots of ethyl acetate until no further color was extracted. The
aqueous phase (bottom layer) was partitioned at 80C against two volumes of
chloroform, the decaffeinated liquid (top layer) was freeze-dried. The freeze-dried
material (TR fraction) was stored at 20C until required and reconstituted as required for
the analysis. TRs were obtained as orange to light brown fluffy powders for each type of
black tea mentioned previously. Once the final samples were obtained, they were
analyzed using solution NMR (1H NMR, COSY, HSQC, HMBC) dissolved in 1:1
mixture of D2O:acetone-dand solid-state NMR (Multi-CP and Multi-CP with Dipolar
Dephasing).

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