Faculty of Science

CHARACTERIZATION OF UV RESISTANT MOLECULES IN TREMBLING ASPEN
TREE (POPULUS TREMULOIDES MICHX.) POWDER
2023 | ABU HARERA NADEEM

B.Sc. HONOURS THESIS – CHEMICAL BIOLOGY

CHARACTERIZATION OF UV RESISTANT MOLECULES IN TREMBLING ASPEN
TREE (POPULUS TREMULOIDES MICHX.) POWDER

by

ABU HARERA NADEEM
A THESIS SUBMITTED IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF

BACHELOR OF SCIENCE (HONS.)
in the
DEPARTMENTS OF BIOLOGICAL SCIENCES
AND PHYSICAL SCIENCES
(Chemical Biology)

This thesis has been accepted as conforming to the required standards by:
Natasha Ramroop Singh (Ph.D.), Thesis Supervisor, Dept. Biological Sciences
Kingsley Donkor (Ph.D.), Co-supervisor, Dept. Physical Sciences
Lyn Baldwin (Ph.D.), External examiner, Dept. Biological Sciences

Dated this 25th day of April, 2023, in Kamloops, British Columbia, Canada
© Abu Harera Nadeem, 2023

ABSTRACT
Commercial sunscreen products offer protection against ultraviolet (UV) radiation. This
type of radiation can cause numerous health effects on all organisms, such as premature aging,
cancer, and decreased immunity against infections. Hence, it is important for organisms to apply
compounds with UV resistant properties, such as sunscreen, before engaging in activities outside
in the Sun. However, the chemical compositions of commercial sunscreens can cause serious
environmental harm. When sunscreen is washed from skin, toxic chemicals can enter waterways
and, for example, can diminish growth of aquatic life. As a solution to this problem, naturally
derived sunscreen can be used, which can be found on the barks of trees such as the Trembling
Aspen tree (Populus tremuloides) in the form of a grey/white powder. Based on this, there
potentially are UV resistant molecules in the powder from Populus tremuloides that have not yet
been elucidated based on literature searches. It was found that there are approximately five
molecules in the powder that absorb UV light and this work has partially characterized some of
these using, CE (Capillary Electrophoresis) IR (Infrared Spectroscopy), and 1H-NMR (Nuclear
Magnetic Resonance). These molecules may have alkenes, alkynes, amines, amides, and
aldehydes, which have the potential to absorb UV radiation—making them UV resistant.
Thesis Supervisor: Natasha Ramroop Singh (Assistant Teaching Professor).

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ACKNOWLEDGEMENTS
I would like to thank Dr. Natasha Ramroop Singh, Dr. Kingsley Donkor, Dr. Lyn Baldwin,
Dr. Louis Gosselin, and the Department of Biology and Chemistry at TRU for their support and
guidance throughout the project. I appreciate the Canada Foundation for Innovation (CFI) for
providing the necessary analytical and spectroscopic instrumentation. I also would like to thank
Isaac Stephens, Michelle Boham, Dr. Dipesh Prema, Dr. Bruno Cinel, and Dr. Don Nelson for
their support. Finally, I wish to acknowledge the TRU Internal Research Fund, for providing the
funding for the consumables in this project.

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TABLE OF CONTENTS
TITLE PAGE………………………………………………………………………………………i
ABSTRACT………………………………………………………………………………………ii
ACKNOWLEDGEMENTS………………………………………………………………………iii
TABLE OF CONTENTS…………………………………………………………………………iv
LIST OF FIGURES……………………………………………………………………………….v
LIST OF TABLES………………………………………………………………………………..vi
INTRODUCTION……………………………………………………………………………....1-8
MATERIALS AND METHODS……………………………………………………………...8-15
RESULTS AND DISCUSSION……………………………………………………………...15-32
CONCLUSION………………………………………………………………………………32-33
LITERATURE CITED……………………………………………………………………….34-39
APPENDIX A………………………………………………………………………………...40-58

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LIST OF FIGURES
Figure 1. Photo of Trembling Aspen’s photosynthetic bark taken at Lac du Bois in Kamloops,
BC, Canada……………………………………………………………………………………….2
Figure 2. Trembling Aspen tree powder appearing grey/white………………………………….8
Figure 3. Sampling site at Lac du Bois, Kamloops, BC, Canada………………………………..9
Figure 4. Flowchart of methods used in study…………………………………………………..15
Figure 5. Microscopic images of the cells within the powder without Toluidine Blue stain (left)
and with the stain (right)…………………………………………………………………………16
Figure 6. Electropherogram of CDCl3 and hexane:ethyl acetate (4:1) mixture blank…………..21
Figure 7. Electropherogram of raw powder dissolved in CDCl3 and heated…………………....22
Figure 8. Electropherogram of 34 mL purified fraction in CDCl3 and hexane:ethyl acetate
(4:1)..……………………………………………………………………………………………..26
Figure 8a. Zoomed electropherogram of 34 mL purified fraction in CDCl3 and hexane:ethyl
acetate (4:1) from 6.75 min to 11.50 min………………………………………………………..27
Figure 9. NMR spectrum of raw powder dissolved in CDCl3 and heated. ……………………..29
Figure 10. Zoomed NMR spectrum between 2.3 ppm to 3.65 ppm of raw powder dissolved in
CDCl3 and heated.………………………………………………………………………………..29
Figure 11. Zoomed NMR spectrum between 3.5 ppm to 4.2 ppm of raw powder dissolved in
CDCl3 and heated.………………………………………………………………………………..30
Figure 12. Zoomed NMR spectrum between 6.9 ppm to 7.8 ppm of raw powder dissolved in
CDCl3 and heated.………………………………………………………………………………..30
Figure 13. Zoomed NMR spectrum between 9.5 ppm to 10.1 ppm of raw powder dissolved in
CDCl3 and heated.………………………………………………………………………………..31
Figure 14. FTIR spectrum of raw powder dissolved in CDCl3, heated, and absorbed onto silica
gel for analysis.…………………………………………………………………………………..32

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LIST OF TABLES
Table 1. Expected solubilities at 25°C for each solvent (n = 9).………………………………...10
Table 2. Parameters for EVOS M5000 Cell Imaging Microscope.……………………………..12
Table 3. Instrument parameters for PA 800 CE.………………………………………………...13
Table 4. Spectrometer parameters of NMR.…………………………………………………….13
Table 5. Instrument parameters of FTIR.………………………………………………………..14
Table 6. Migration distances and retention factors for TLC plate characterizations of the
dissolved powder.………………………………………………………………………………..19
Table 7. Summarized results of CE analysis showing potentially new UV resistant molecules in
purified fractions at specific migration time intervals.…………………......................................20
Table 8. Peaks and chemical shifts seen in NMR spectra of raw powder dissolved in CDCl3 and
heated from Figures 9-13...………………………………………………………………………28
Table 9. Peak area and height of labelled peaks in FTIR spectrum of raw powder dissolved in
CDCl3, heated, and absorbed onto silica gel for analysis.……………………………………….31

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INTRODUCTION
Trembling Aspen Tree (Populus tremuloides Michx.)
The Trembling Aspen species is deciduous, perennial, can grow up to 25 metres tall, has
the broadest distributional range of all tree species in North America, and is one of the most
genetically diverse plant species known (Lindroth & St. Clair, 2013; Mitton & Grant, 1996;
Government of British Columbia, n.d.; Evert & Eichhorn, 2013). Specifically, the Trembling
Aspen tree grows in Alaska, Canada, and to the South of Mexico (The National Wildlife
Federation, n.d.). In addition, the Trembling Aspen species is abundant in North America and
controls biodiversity by helping to maintain habitats in forests, making them a foundational species
(Endress et al., 2012; Kashian et al., 2007; Zegler et al., 2012; Ellison et al., 2005).
The life cycle of Trembling Aspen trees involves a form of reproduction that uses pollen
and eggs that are in catkins (The National Wildlife Federation, n.d.). This form of reproduction
relies on the wind to carry the pollen to the eggs in the catkins in order to fertilize them (The
National Wildlife Federation, n.d.). Trembling Aspen trees can also reproduce asexually by
shooting new stems from their single root system, creating a clone which is the culmination of all
the stems and their root system (The National Wildlife Federation, n.d.).
Trembling Aspen is also well-known for its characteristically smooth, white bark that often
has a white powder on its surface. Anecdotally, this powder is believed to act as a “sunscreen” for
the tree, especially on the south-facing side of the tree. Like commercial sunscreens, the white
powder may have the potential to absorb UV radiation and emit it back into the environment.
However, commercial sunscreens contain chemicals such as oxybenzone that, when washed from
skin, can enter waterways and reduce the growth of green algae—stressing the need for a less toxic
sunscreen (Califf & Shinkai, 2019). The application of the white powder as a natural sunscreen is
supported by Native and Indigenous people, as they have incorporated this white powder into their
traditions and have used it for protection against UV radiation from the Sun (Plant Watch Nature
Alberta, n.d.; University of Saskatchewan, 2021; Medieval Manor Gardens, n.d.).
Trembling Aspen Tree Bark and Powder
In most trees, photosynthesis occurs primarily in its leaves; however, Trembling Aspen has
photosynthetic bark with has green chlorenchyma cells found beneath the bark’s outer surface (The

1

National Wildlife Federation, n.d.; National Geographic Society, n.d.). Specifically, the bark has
the pigments beta-carotene, pheophytin, chlorophyll-a, chlorophyll-b, and xanthophyll that are
responsible for photosynthesis (University of Colorado Boulder, 2015, Figure 1). The preferred
light for photosynthesis is within the blue and red ranges which are 425-450 nm and 600-700 nm,
respectively (Vernier, 2018). But, since sunlight encompasses other types of light, some UV light
is also absorbed during photosynthesis and can degrade the plant (Valenta et al., 2020).
As Trembling Aspen is a deciduous tree, it is believe that this photosynthetic bark extends
the photosynthetic season of these trees (The National Wildlife Federation, n.d.).
However, this bark is thin and can be easier for UV radiation to penetrate the layers of the
bark such as the periderm or perhaps the living phloem and cause degradation (University of
Minnesota, n.d.; United States Department of Agriculture, n.d.). Due to this, Trembling Aspen
may expend energy and effort to make the white powder in order to protect itself from UV
degradation.

Figure 1. Photo of Trembling Aspen’s photosynthetic bark taken at Lac du Bois in Kamloops,
BC, Canada.
The powder found on Trembling Aspen’s bark has been described as mature bark cells that
are shed from the tree allowing light to penetrate the cork and cambium, eventually reaching the
chlorenchyma (University of Colorado Boulder, 2015). Reports also indicate the powder initial

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colour is orange and composed of conglutinated cells that become white with age (University of
Colorado Boulder, 2015). On the other hand, the powder may primarily be composed of naturally
occurring yeast or lichens (The Survival University, n.d.), or made of anti-microbial compounds
that help to fight off bacteria (St-Pierre et al., 2018).
There are many mechanisms for how the powder could be made in Trembling Aspen trees.
The powder could simply be the accumulation of dead bark cells from the cambium. Or, the
powder could be a secondary metabolite that the tree makes by expending energy. Plants and
humans make a diverse number of secondary metabolites (Torres & Schmidt, 2019; Teoh, 2015).
The powder would be classified as a secondary metabolite instead of a primary metabolite because
primary metabolites are similar in each species, while secondary metabolites are different in every
species and the powder is unique to Trembling Aspen (Byjus, n.d.). Plants make secondary
metabolites, such as phenolics and terpenes, to defend themselves against environmental
constraints and to be competitive within their environment (Guerriero et al., 2018; Teoh, 2015;
Pang et al., 2021).
If considering that the purpose of the powder is to protect Trembling Aspen’s
photosynthetic bark from too much exposure to UV radiation, then it would seem logical for the
plant to make the secondary metabolite as it protects the tree from environmental damage. Vascular
rays and the formation of heartwood are known to produce secondary metabolites in trees (Celedon
& Bohlmann, 2017; Sandved et al., 1993). Some secondary metabolites are made by taking the
carbon from the sugars that are produced during photosynthesis and metabolizing them to make
other compounds, one of which is erythrose-4-phosphate (Sinha et al., 2019). This compound gets
fed into the Shikimic acid pathway and makes aromatic amino acids that can make nitrogencontaining secondary products such as nicotine or polyamines, or phenolic compounds like
flavonoids or simple phenolics (Sinha et al., 2019). However, the secondary metabolites made
from vascular rays or heartwood formation tend to stay within the interior of the bark (Celedon &
Bohlmann, 2017; Barlow, 2005). They perhaps could make their way to the outside of bark through
accumulating in the dead cells that make up the outer layer of the bark.
Photooxidative Stress
Photooxidative stress occurs when the energy dissipation mechanisms and structures that
are used for photosynthesis are not developed yet and, consequently, reactive oxygen species are
3

created since there is a buildup of energy in chloroplasts (Muñoz & Munné-Bosch, 2017). This
type of stress happens in senescing leaves or during cold temperatures (Muñoz & Munné-Bosch,
2017). Since Trembling Aspen is deciduous and sheds its leaves during the winter, the
energetically expensive chemicals that make up the chlorophyll in the leaves are potentially
degraded and lost, so the photosynthetic bark may act to offset the deciduous habit.
Existing Chemicals in Trembling Aspen Bark and Powder
Previous researchers have identified anti-microbial compounds in the powder as flavonoid
structures (St-Pierre et al., 2018). In plants, flavonoids are located in vacuoles of cortical cells and
are made through the phenylpropanoid pathway that forms 4-coumaroyl-CoA from phenylalanine
which then enters the flavonoid pathway (Hassan & Mathesius, 2012; Falcone Ferreyra et al.,
2012). Interestingly, flavonoids have structures that absorb UV radiation such as double bonds, so,
the powder could be gaining UV resistance from antimicrobial compounds.
In addition, salicin—which is a glucoside—has also been isolated from Trembling Aspen,
along with a compound termed ‘populin’ (Pearl & Darling, 1959). Salicin was found to be ohydroxymethylphenyl-O-𝛽-D-glucopyranoside, while synthetic populin was found to be 6benzoylsalicin (Pearl & Darling, 1959). Another glucoside termed ‘tremuloidin’ was also isolated
from the bark of Trembling Aspen and was found to be 2-benzoylsalicin, which is a monobenzoate
of salicin and an isomer of populin (Pearl & Darling, 1959). The structure of salicin has an aromatic
group along with hydroxyl functional groups, of which the former can absorb UV radiation, thus,
it could be possible that this molecule or isomers of it are also responsible for UV resistance.
Other researchers investigating volatiles released from Trembling Aspen bark have
identified 1-hexanol, benzyl alcohol, benzaldehyde, and nonanal (Borden et al., 1998). These
volatiles disrupted the secondary attraction by mountain pine beetles where they kill living pines
(Dendroctonus ponderosae Hopkins). The compound 1-hexanol was able to disrupt secondary
attraction by itself, while the other 3 compounds had to be in binary, ternary, or quaternary
combinations with each other to have a disruption effect (Borden et al., 1998). The structures of
benzyl alcohol, benzaldehyde, and nonanal have double bonds or conjugated pi-bond electron
systems which have the ability to absorb UV radiation, so, it is possible some of these compounds
could be present in Trembling Aspen’s powder.

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UV Radiation and its Forms
With exposure to UV radiation being one of the leading causes of human skin cancer and
other human skin diseases, it is important to apply sunscreen before being exposed to the sun
(D’Orazio et al., 2013). UV radiation is light that has photons with high energy compared to visible
light, and is a form of electromagnetic light (Rockett, 2019; U.S. Food and Drug Administration,
2020). There are three types of UV radiation: UVA, UVB, and UVC (U.S. Food and Drug
Administration, 2020). The first form has long wavelengths allowing it to go beyond the Earth’s
ozone layer, something UVB and UVC radiation cannot do as they have shorter wavelengths (U.S.
Food and Drug Administration, 2020). However, some UVB rays get through the ozone layer and
can be absorbed by plants and reach the outer layer of human skin, or the epidermis (U.S. Food
and Drug Administration, 2020). UVA rays go further and can reach the middle portion of the
skin, which is the dermis (U.S. Food and Drug Administration, 2020). All forms of UV radiation
can be emitted by the sun and not only damage skin, but also materials such as plastic.
Mechanism of UV Radiation
If UV radiation encounters skin or other materials, then, through the high-energy photons
in UV radiation, it can break bonds that compose those materials (Rockett, 2019). By breaking
bonds, the material becomes shorter and decreases molecular weight which results in degradation
by lessening strength, ductility, colour, and texture. As for skin, UV radiation causes kinks in
thymine bonds that are present in deoxyribonucleic acid, or DNA. This can lead to mutations or
skin cancer. Hence, why sunscreen is important to use.
Properties of UV Resistant Molecules and Mechanism
To overcome the deleterious effects of UV radiation, certain molecules with UV
resistant properties are used. UV resistance is commonly displayed in sunscreens as they consist
of chemicals that can protect human skin. Molecules that have this property may contain double
bonds aromatic rings, highly electronegative atoms, or metals. For instance, oxybenzone is a
common molecule that is present in most sunscreens whose structure consists of aromatic groups

5

(D’Orazio et al., 2013). A common structural pattern in most UV resistance molecules in
sunscreens is the presence of double bonds. These bonds absorb UV radiation and emit it back to
the environment which protects the material or skin it is coated on as the UV radiation does not
get past the molecule. Metals act in a similar way since they have a lattice structure and share a
cloud of delocalized electrons that interfere with UV light by reflecting it back to the environment
(Omnexus, n.d.; Rockett, 2019; Steeley et al., 2014).
Characterization Techniques
When dealing with unknown molecules, characterization techniques are used to ascertain
the chemical structures and functional groups (Impact Analytical, 2022). Knowing the structure is
the first step to identifying the molecule’s functionality and applicability as a natural sunscreen.
To do this, information from techniques such as Fourier Transform Infrared spectroscopy (FTIR),
Capillary Electrophoresis (CE), and Nuclear Magnetic Resonance spectroscopy (NMR) was
gathered. With this information, deriving the structure of the unknown molecules was similar to
solving a puzzle, with each piece of information validating the placement of a functional group or
a carbon atom.
Fourier Transform Infrared Spectroscopy (FTIR)
In the IR region, there are three subregions which are the near, mid, and far infrared regions.
The near IR region is between 14000-4000 cm-1, while the mid and far IR regions are from 4000400 cm-1 and 400-10 cm-1, respectively (Systems Chemistry, n.d.). FTIR detects frequencies in
these regions that are absorbed by the bonds or groups within molecules (Mathias, 2015).
Absorption in this case refers to the frequency of the absorbed radiation matching the vibrational
frequency of the bond or functional group (Systems Chemistry, n.d.). Typically, when a spectrum
is recorded, the transmittance is observed on the y-axis—not the absorbance. Transmittance is the
percentage of energy that passes through a molecule or bond that is not absorbed (Sigma Aldrich,
n.d.). On the x-axis, wavenumber is recorded, which is the number of wavelengths in a certain
distance. Each bond type vibrates at a specific frequency, hence, FTIR analysis is useful for
determining specific functional groups. However, the way a molecule vibrates—such as symmetry
of stretching, scissoring, rocking, wagging or twisting—can lead to a peak not being observed. For
instance, if a molecule is symmetrical and has one bond, then it will not produce a peak, but, if the
molecule is asymmetrical then it will show a peak.

Capillary Electrophoresis (CE)
When there are multiple molecules that make up a solution, it is important to separate them
and see what the characteristics of each molecule are—which is what capillary electrophoresis
does. This separation technique relies on voltage and an electric field to separate molecules based
on differences in electrophoretic mobilities that are dependent on charge, viscosity of the solvent,
and size (U.S. Food and Drug Administration, 2020). Large amounts of voltage are used in CE
and provide high-performance separations. It is easy to assume that large voltages can cause
heating effects, but due to capillaries having a large surface area to volume ratio they are able to
effectively dissipate heat, mitigating heating effects (U.S. Food and Drug Administration, 2020).
When under an electric field, molecules that can be ionized become charged ions that are either
positive or negative (Sutipatanasomboon, 2021). In CE, there are two electrodes—cathode and
anode—that create an electric force. This force is strong and causes ions to move towards the
opposite charge leading to separation. For instance, a cation would move towards the negatively
charged cathode.
Nuclear Magnetic Resonance Spectroscopy (NMR)
For structure determination, NMR is valuable as it details which atoms are located next to
others. This is but one application of NMR. The technique can be tailored to specific elements,
such as hydrogen or carbon. Hydrogen, or proton-NMR (1H-NMR) is commonly used as 1H is
readily available in the environment, whereas NMR that is specific to carbon uses carbon isotopes,
such as 13C, that are not as abundant as 1H is. Placing a molecule in a magnetic field causes the
nuclei of some atoms to act like small magnets and if radio frequency waves are applied, then the
nuclei will resonate at specific frequencies. From here, the frequencies are measured and form an
NMR spectrum. The higher the radio frequency, the higher the resolution and separation of peaks
are.
Objectives of Study
The purpose of this study was two-fold. The first objective was to develop a purification
and separation technique that could successfully elute potential UV resistant molecules in the
powder. The second goal of the project was to characterize and elucidate the structures of any UV
resistant molecules that were separated from the powder. The characterization part of the work

7

included finding the polarity of the powder, while elucidating the structure comprised of finding
the functional groups present in the UV resistant molecules.
METHODS AND MATERIALS
Sample Collection
The Trembling Aspen Tree powder was collected in June 2022 at a site at Lac Du Bois,
Kamloops, BC, Canada. The site was outside of the protected area—samples were collected
without scraping off significant amounts of bark or other parts of the tree that could cause it to
degrade faster than it typically would. The bark was thin, smooth, and the distance between the
powder and the bark was small. As for the powder, it appeared grey/white, and its texture was not
waxy but was grainy, like sand, and is seen in Figure 2. Also, the powder was loosely held to the
bark and seemed to be a surficial powder that could be removed through manual scraping.

Figure 2. Trembling Aspen tree powder appearing grey/white.
The site was approximately 20 m x 20 m and extended to the far green bushes on the left
of Figure 3. The soil within the site was slightly moist, with the tallest blades of grass reaching up

8

to shin-length. A metal scraper and the back end of a knife were used to scrape the powder off 4
trees into a plastic bag. An issue with this method of sampling was that the powder experience
static effects from the plastic bag, causing it to disperse within the air. As a solution, the bag was
held up to the tree and when the powder was scraped, it fell into the bag. The powder still
experienced static effects, but some of the powder fell into the bag—enough to give a mass of 2.02
grams (g).

Figure 3. Sampling site at Lac du Bois, Kamloops, BC, Canada.
Dissolution Attempts
The powder was attempted to be dissolved with 5 polar and 3 non-polar solvents along
with 1 polar and non-polar mixture. The 6 polar solvents were deionized water, ethyl acetate
(Fisher Scientific Co., ON, Canada), methanol (Fisher Scientific Co., ON, Canada), ethanol (Fisher
Scientific Co., ON, Canada), acetonitrile (Fisher Scientific Co., ON, Canada), and dimethyl
sulfoxide or DMSO (Fisher Scientific Co., ON, Canada). The 3 non-polar solvents attempted were,
hexane (Fisher Scientific Co., ON, Canada), tetrahydrofuran, or THF (Fisher Scientific Co., ON,
Canada), and chloroform-D (Sigma-Aldrich Co LLC, Darmstadt, Germany). Deuterated
chloroform was used instead of unmodified chloroform for consistency since the powder was
going to be analyzed by nuclear magnetic resonance (NMR). Lastly, the polar:non-polar mixture
was composed of methanol, acetonitrile, and THF (1:1:1). For each dissolution attempt, a total
volume of 3 mL of the solvent was poured onto 20 mg of the powder which was in a 5 mL glass

9

vial. All dissolution attempts were done at 25°C and were vortexed for 5 min immediately after
adding the solvent to the powder. The expected solubility for each solvent is shown in Table 1.
Interestingly, chloroform-D has the lowest solubility of all the solvents used, but visibly dissolved
more than any of the other solvents. This discrepancy could be due to chloroform’s polar nature
and tendency to dissolve both polar and non-polar solutes, hence, appearing like it dissolved more
of the powder than other solvents which primarily dissolve either polar or non-polar solutes.
Table 1. Expected solubilities at 25°C for each solvent (n = 9).
Solvent Type

Mass (mg)

Mass of Solvent (g)

Expected Solubility
(mg/100 g)
Methanol
17
2.373
716.4
Ethanol
18
2.367
760.5
Acetonitrile
16
2.358
678.5
Dimethyl sulfoxide
21
3.300
636.4
Ethyl acetate
25
2.706
923.9
Hexane
19
1.977
961.1
THF
20
2.667
749.9
Chloroform-D
20
4.500
444.4
Mixture (1:1:1)
18
2.366
760.8
Note: The mass of the solvent was calculated by multiplying the volume of solvent used and the
density of the solvent at 25°C.
Sample Preparation/Purification
Samples were prepared two different ways. The first sample was prepared by dissolving
the raw powder in CDCl3 and heating it to 40°C to assist with dissolution. This sample was then
filtered using a glass filter. The second method stems from purifying the sample, which had to first
be prepared for flash column chromatography. However, the typical flash column chromatography
method was not suitable for the solvent of choice—which was CDCl3—since it is stronger than
traditional solvents such as methanol (Merck, 2021; Techiescientist, n.d.). Instead, a dry-loading
technique for flash column chromatography was used. To prepare a sample for dry-loading, a
solvent mixture had to be used that gave retention factors of the components of interest that were
between 0.2-0.3 when separated using thin layer chromatography (TLC). Given the retention
factors had to be low, both polar and non-polar mobile phases were used of varying ratios of
hexane, ethyl acetate, THF, ethyl alcohol, 2-propanol, and methanol. This was done because the
polarities of the components of the powder were not clear even after dissolution attempts.

10

Though a majority of the powder dissolved in CDCl3 which is considered to be non-polar, the
nature of CDCl3 causes it to dissolve some polar compounds, making it appear that more of the
sample was dissolved. Since both non-polar and polar components could be dissolved, assuming
the powder consists of polar or non-polar would be accurate. Before applying the sample to TLC
plates, it was purified using a Rotovap which evaporated the methanol that partially dissolved the
sample. Methanol was used instead of deuterated chloroform to conserve resources and prevent
solvent waste since only the mobile phase needed to be optimized. There was approximately
0.5000 g of sample dissolved in 25.0 mL of solvent, filtered, and transferred into a 50 mL round
bottom flask. The sample was subjected to the Rotovap for 30 min and then 3 mL of methanol
were added to the small volume of liquid that remained in the round bottom flask to increase the
volume for TLC spotting. For the other mobile phases, the same procedure was followed. Each
mobile phase had a final volume of 200 mL. The polar solvent mixtures did not give the desired
retention factors, but, a non-polar solvent mixture of hexane and ethyl acetate (4:1) did. The dryloading technique used this mobile phase.
To setup the dry-loading technique, 100 mg of sample was dissolved in 12.5 mL of
deuterated chloroform, heated, filtered, and transferred to a 25 mL round bottom flask that was
placed onto a Rotovap. After evaporation, 2 g of silica gel (Sigma-Aldrich Co LLC, Darmstadt,
Germany) was added to the flask and mixed until the solution had a slush consistency. The solution
was evaporated util the slush consistency turned into the same consistency as silica gel. At this
point, the colour changed from white to yellow. Then, to a 60 mL flash column which had cotton
in the narrow bottom of the column, enough silica gel was added to the column to fill the bottom
up to 5 cm. Then, the yellow silica gel was added onto the white silica gel and 2-3 cm of the mobile
phase was added. To this, 2-5 mm of sand (Fischer Scientific Co., ON, Canada) was added. Once
the dry-loading technique was set, the mobile phase was run through twice and fractions were
collected every 2 mL, giving a total of 34 mL being collected.
Instruments and Parameters
Cell Imaging Microscopy
Before any analytical and spectroscopic analysis was done, the powder was viewed under
an EVOS M5000 (Life Technologies Inc., Burlington, ON, Canada) imaging microscope and

11

stained using Toluidine Blue (Sigma-Aldrich Co LLC, Darmstadt, Germany). The parameters for
the microscope are seen in Table 2.
Table 2. Parameters for EVOS M5000 Cell Imaging Microscope.
Parameters
Illumination
LED light cubes
Contrast
Epifluorescence
Methods
and transmitted
light
Objective Turret 5-position
control
LCD Display
18.5 in. highresolution
Camera
3.2 MP,
monochrome
CMOS camera
(2048 x 1536
pixels)
Pixel resolution
3.45 𝜇m

Capillary Electrophoresis
With the sample purified from flash column chromatography, the purified fractions were
analyzed using a Beckman Coulter P/ACETM MDQ capillary electrophoresis (Beckman Coulter
Inc., Fullerton, CA, USA) instrument that had an ultraviolet detector. Data was acquired and
processed using 32Karat software. In addition, a 60 cm (52 cm to the detector) x 50 μm uncoated
fused-silica capillary was used. The separations and UV detection of the purified fractions were
done by rinsing the capillary with 0.1 M NaOH, 18 MΩ water, and an organic buffer or
background electrolyte composed of 60% acetonitrile and 5 mM of NaOH. The NaOH and
buffer rinses were done for 4.00 min at 20 psi, while the 18 MΩ water rinse was done at 20 psi
for 1.00 min. When injecting the sample, the pressure was 1 psi and was injected for 5.0 sec.
Once the sample was injected, the analysis time was set for 20.0 min at a voltage of 20.0 kV with
a 0.17 min ramp. Between each injection the capillary was washed with 0.1 M NaOH (4.00 min),
followed by 18 MΩ water (1.00 min) then lastly the run buffer (4.00 min). The instrument
parameters are seen in Table 3.

12

Table 3. Instrument parameters for PA 800 CE.
Parameters
Voltage
20.0 kV
Rinse Pressure
20.0 psi
Inject Pressure
1.0 psi
Detector
UV
Wavelength
214 nm
Data Rate
4 Hz
Absorbance
Direct
Signal
Polarity
Normal
Current
300 𝜇A
(maximum)
Cartridge and
25.0°C
Sample Storage
Temperatures
Peak Detection
2
Threshold
Peak Detection
9
Width
1H-NMR

The 1H-NMR spectra were acquired with a Bruker Avance III Ultrashield 500 MHz NMR
spectrometer (Bruker Co., MA, USA) using CDCl3 as the solvent and residual protons as an
internal reference. When pouring samples into the NMR tubes, approximately 1-2 mL were used.
The operating parameters of the NMR are in Table 4.
Table 4. Spectrometer parameters of NMR.
Parameters
1H
Nucleus
Pulse Width
7.00 𝜇s
Power
17.445 W
Note: ‘Observe’ and ‘decouple’ parameters are the same.

13

FTIR
Infrared spectra were obtained using a PerkinElmer Spectrum Two (UATR Two) FTIR
spectrometer (PerkinElmer Analytical and Enterprise Solutions, CT, USA). The samples were only
run in a powder form, which was often done by absorbing a liquid sample onto silica gel. Blank
solutions depended on the matrix of the sample, which was either CDCl 3, hexane:ethyl acetate,
silica gel, or a mixture of some or all. The instrument parameters of the FTIR are in Table 5.
Table 5. Instrument parameters of FTIR.
Abscissa Units
Ordinate Units
Wavenumber Range
Accumulations
Resolution
Crystal
Source
Beamsplitter
Detector

Parameters
Wavenumber
%T (Transmittance)
450-4000 cm-1
4 scans
4 cm-1
Diamond
MIR (8000-30) cm-1
OptKBr (7800-400) cm-1
LiTa03 (15700-370) cm-1

UV-Vis Spectrometry
It should be noted that UV-Vis spectrometry was also performed but did not yield any
useful results despite significant dilution attempts, thus, it was not shown in Figure 4.

14

Figure 4. Flowchart of methods used in study.
RESULTS AND DISCUSSION
Cell Imaging Microscopy
The powder was viewed under a cell imaging microscope to see the cellular structure of
the powder and is seen in Figure 5. From the microscopic images, it can be observed that the cells
of the powder have not been fully stained with Toluidine Blue since the colour is very faint. This
indicates that the walls of the cells comprising the powder are unlikely to be made primarily of
cellulose (which stains purple with Toluidine Blue) or lignin (which stains blue-green with
Toluidine Blue).

15

Figure 5. Microscopic images of the cells within the powder without Toluidine Blue stain (left)
and with the stain (right).
Polarity
From the dissolution attempts, chloroform worked the best as it was able to visibly dissolve
more of the powder than the other solvents used. The polarity of chloroform is non-polar and
slightly polar due to the presence of an electronegative chlorine atom, asymmetry, and a low
dielectric constant (Goyal, 2022; Ohio State University, n.d.; Techiescientist, n.d.). Due to the
complex polarity of chloroform, it was difficult to classify the powder as either polar or non-polar
based on the principles of dissolution. So, information from the other dissolution attempts was
used to get a finer picture of the powder’s polarity. For instance, the powder did not dissolve in
water, but slightly dissolved in methanol and DMSO which are stronger polar solvents than water
as seen with the expected solubilities in Table 1. This property is exhibited by polar oils: insoluble
in water but soluble in stronger polar solvents (O’Lenick, 2008). However, polar oils are expected
to dissolve more in methanol and DMSO than they do in deuterated chloroform since the former
compounds are more polar than the latter. The Populus tremuloides tree powder did not follow
this pattern. This discrepancy may be due to the ability of deuterated chloroform to dissolve nonpolar compounds which may compose a significant amount of the powder. Thus, the powder would
appear to be more dissolved since not only are some of the polar components being dissolved, but
also the non-polar components are too, which methanol and DMSO cannot do.

16

The retention factors obtained (Table 6) with thin layer chromatography plates support the
hypothesis that the powder is like a polar oil. Since the TLC plates had a polar stationary phase
made of silica gel, the mobile phases were created as mixtures of polar and non-polar solvents to
maximize the chances of observing UV resistant molecules in the powder. For mobile phases such
as THF and hexane: 2-propanol (1:1), only one spot was observed at 10.1 cm and 7.5 cm,
respectively, which is close to the migration distance of the methanol standard. This indicates that
no components in the powder were separated when using either mobile phase, or the components
were separated but they travelled off the TLC plate. If the latter reasoning is true, then it would
suggest that the components in the powder were of similar polarity to the mobile phase since they
will have been more attracted to the mobile phase and have travelled with it.
When using the ethyl alcohol:hexane (1:1) mobile phase, three components were separated
with distances of 1.0 cm, 7.8 cm, and 14.0 cm, one of which belongs to the methanol standard
which was the dot at 1.0 cm. The other two components had very large retention factors because
they travelled with the mobile phase instead of being attracted to the polar stationary phase, thus,
these components would be non-polar. Whether the two components were UV resistant molecules
or other molecules in the powder remains unknown. This uncertainty is applicable to the THF and
hexane:2-propanol mobiles phases too.
So, a mobile phase that showed multiple separated components was sought after since it
had a higher likelihood of successfully separating some of the UV resistant molecules. In fact, the
hexane:ethyl acetate mobile phase showed exactly that. The ethyl acetate:hexane (1:1) mobile
phase separated five components in the powder with distances of 2.5 cm, 7.0 cm, 8.0 cm, 9.0 cm,
and 11.8 cm. The first component belongs to the methanol standard which had a migration distance
of 2.0 cm. The components at 7.0 cm, 8.0 cm, 9.0 cm, and 11.8 cm had retention factors of 0.41,
0.47, 0.53, and 0.69. These retention factors are of an intermediate polarity, indicating that the
polarity of the components are between polar and non-polar. Interacting with the stationary phase
equally with the mobile phase will cause separated components to travel half the distance of the
solvent front, so they will not remain close to the bottom of the plate or travel the entire distance
of the plate with the mobile phase. This result supports the classification of the powder as being
between a polar oil and a non-polar compound.

17

When the same mobile phase was used but with a majority of non-polar hexane (4:1), more
compounds were separated with distances of 4.2 cm, 5.0 cm, 5.2 cm, 6.0 cm, 6.5 cm, 7.5 cm, 9.5
cm, 10.5 cm, 11.0 cm, and 12.0 cm. Of these distances, the 7.5 cm compound is the methanol
standard which travelled 7.8 cm. The components at 9.5 cm, 10.5 cm, and 11.0 cm exhibit the
same intermediate polarity as the components in the 1:1 ethyl acetate:hexane mobile phase since
they have close retention factors. However, the compounds that travelled 4.2 cm, 5.0 cm, 5.2 cm,
6.0 cm, and 6.5 cm have low retention factors of 0.25, 0.29, 0.31, 0.35, and 0.38, respectively.
Low retention factors suggest that the components are polar since they are interacting closely with
the polar stationary phase. On the other hand, the component at 12.0 cm had a large retention factor
of 0.71. A large retention factor implies that the component is non-polar.
The combination of polar, intermediate polarity, and non-polar compounds in the powder
is expected and supports the powder being between a polar oil and a non-polar compound. Since
the powder has compounds with a wide range of polarities, it supports the explanation of deuterated
chloroform dissolving more of the powder than methanol and DMSO. Deuterated chloroform has
an intermediate polarity so it can dissolve a wide range of compounds and since the components
comprising the powder that were separated with TLC had broad polarities, CDCl3 would be able
to dissolve these components—giving the appearance of a more dissolved solution than the
strongly polar solvents methanol and DMSO could.

18

Table 6. Migration distances and retention factors for TLC plate characterizations of the
dissolved powder.
TLC Plate Mobile
Phase

Powder
Migration
Distances (cm)

Standard
Migration
Distance (cm)

4:1 hexane:ethyl
acetate

4.2; 5.0; 5.2;
6.0; 6.5; 7.5;
9.5; 10.5; 11.0;
12.0

7.8

Solvent
Front
Distance
(cm)
17.0

Powder
Retention
Factors

Standard
Retention
Factor
0.46

15.5

0.25; 0.29;
0.31; 0.35;
0.38; 0.44;
0.56; 0.62;
0.65; 0.71
0.07; 0.52;
0.93
0.15; 0.41;
0.47; 0.53;
0.69
0.48

1:1 ethyl
alcohol:hexane
1:1 ethyl
acetate:hexane

1.0; 7.8; 14.0

1.0

15.0

2.5; 7.0; 8.0;
9.0; 11.8

2.0

17.0

1:1 hexane: 2propanol
THF

7.5

7.8

10.1

12.0

13.0

0.78

0.92

0.07
0.12

0.50

UV Detection and Number of Potential UV Molecules in Powder
Before subjecting the purified fractions of the sample to spectroscopic techniques and
analytical instruments, the principle of UV resistance that this work is based on had to be tested.
A common technique to test for UV resistance would be UV-Vis spectrometry, as any molecules
that absorb UV light will show up as a broad spectrum. However, when the purified samples were
run by UV-Vis spectrometry, they showed up as narrow peaks instead of broad ones. Typically,
this issue is fixed by dilution, but in this case, not even diluting the purified samples by 105X made
the peaks broad. The narrow peaks could be due to the matrix of the samples masking any UV
resistant molecules, hence, the narrow peaks might belong to components in the solvent mixture.
To overcome the issue of the solvent masking the UV resistant molecules, a separation technique
with a UV detector needed to be used as it would further separate the UV resistant molecules from
the solvent components while detecting if the molecules absorb UV light. So, CE was chosen.
There were 17 purified fractions that were run on CE, each with 500 𝜇L of solution, along
with 3 blanks that were CDCl3, hexane:ethyl acetate (4:1), and a mixture of CDCl3 and
hexane:ethyl acetate (4:1). The identification of potential UV resistant molecules in the purified
fractions are summarized in Table 7.

19

Table 7. Summarized results of CE analysis showing potentially new UV resistant molecules in
purified fractions at specific migration time intervals.
Fraction Volume (mL)

Migration time (min)

A

2

0-2
X

2-4
X

B

4

X

C

6

D

4-6

6-8
X

8-10
X

10-12
X

12-14
X

14-16
X

16-18
X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

8

X

X

X

X

X

X

X

X

E
F

10
12

X
X

X
X

X

X
X

X
X

X
X

X
X

X
X

X
X

G
H

14
16

X
X

X
X

X

X
X

X
X

X
X

X
X

X
X

X
X

I

18

X

X

X

X

X

X

X

X

J

20

X

X

X

X

X

X

X

X

K

22

X

X

X

L
M

24
26

X
X

X
X

X

N

28

X

X

O
P

30
32

X
X

X
X

Q

34

X

X

X

X

X

X

X

X

X
X

X
X

X

X
X

X
X

X
X

X

X

X

X

X

X

X
X

X
X

X
X

X
X

X
X

X
X

X

X

X

X

X

Note: ‘
’ indicates the presence of a new peak from a potential UV resistant molecule, while
‘X’ indicates the absence of a peak from a potential UV resistant molecule.
CE Electropherogram Discussion
Starting with the blank samples, Figure A1 shows the CDCl3 blank. The large peak at 3.5
min with a peak area of 686386 belongs to a major impurity—such as water—in the CDCl3 solvent
mixture since it is the strongest, and the other smaller peaks on the right side of Figure A1 may be
other contaminants or noise. The latter is more reasonable based on the absence of baseline
resolution for peaks on the right side of Figure A1.
Figures A2 and A2(a) shows the hexane:ethyl acetate (4:1) blank. Since this blank is a
combination of two compounds along with any contaminants from both solvents, the
electropherogram is expected to contain many peaks, which is seen. The peak that belongs to the
major impurity in the hexane:ethyl acetate solvent mixture may be the large peak at 10.8 min that

20

has a peak area of 118317. The other peaks are a mixture of both, impurities from the hexane and
ethyl acetate stock solution and background noise.
Figure 6 shows the CDCl3 and hexane:ethyl acetate (4:1) blank mixture. What is expected
in the electropherogram from this mixture is that the main peaks in Figures A1 and A2 for CDCl3
and hexane:ethyl acetate (4:1) should be present—but they were not. The only peak that was of
significance in Figure 6 was the peak at 3.5 min that had a peak area of 16152. Since this peak has
a close migration time to the impurity peak in Figure A1, it is reasonable to conclude that these
peaks belong to the same impurity. Interestingly, there is another peak that is being masked by the
impurity peak and has a peak area of 19715, which could belong to impurities seen in Figure A2
that are co-migrating with other contaminants. The peak areas for both peaks in Figure 6 are lower
than their associated peaks in Figures A1 and A2 potentially due to background noise or signal
suppression.

Figure 6. Electropherogram of CDCl3 and hexane:ethyl acetate (4:1) mixture blank.

21

Figure 7 shows the raw powder dissolved in CDCl3 and heated. The large peak at 4.0 min
(peak area = 2040504) most likely belongs to the impurity peak in the CDCl3 blank, but it could
be co-migrating with a UV resistant molecule. The peak at 11.4 min (peak area = 46059) could be
a potential UV molecule. Since the peak is relatively larger than the other peaks in the
electropherogram, it might be one of the main UV resistant molecules in the powder. The other
peaks could also belong to UV resistant molecules, but the sample concentration may be too dilute,
or the molecules could have degraded significantly making their peaks very small.

Figure 7. Electropherogram of raw powder dissolved in CDCl3 and heated.
In Figure A3, an electropherogram of the 2 mL purified fraction is shown. The large peaks
at 4.5 min (peak areas = 32164; 15379; 6970) could belong to potential UV resistant molecules
that are co-migrating with each other. Due to the background noise and lack of baseline

22

resolution—which could be due to the high concentration of mobile phase—it is difficult to be
sure which peaks may belong to UV resistant molecules and which are from the blanks.
The separated components in the 4 mL purified fraction are seen in Figure A4. Between
Figures A3 and A4, there is more separation and the shoulder present at 4.8 min (peak area = 6970)
in Figure A3 is separated with acceptable resolution in Figure A4 and can be seen as two peaks
instead of one. These peaks can also belong to UV resistant molecules in the powder as they
disappear in the later purified extractions. If the two peaks did belong to the matrix components,
then they would be in all the electropherograms and not a select few since the matrix is the same
for all of the purified fractions. The two peaks to the left of the electropherogram in Figure A3
were detected in Figure A4 at 1.5 min (peak area = 1234) and 1.8 min (peak area = 685). These
two peaks are likely impurities from the blanks since they were not detected in Figure 7, which
should have detected all the potential UV resistant molecules present in the powder.
The electropherogram of the 6 mL purified fraction is in Figure A5. The peak at 1.5 min
that was present started to disappear. The same trend is seen with the peak at 4.8 min A new peak
appeared at the end of the run at 17.5 min (peak area = 1929) which is seen in the Figure A1 blank
and could be an impurity.
In Figure A6, the 8 mL purified fraction is seen. The peak at 1.5 min in Figure A5
disappeared, supporting how that peak might be an impurity since it may have been fully removed
as it does not reappear in any of the later electropherograms. The peak at 5.0 min (peak area =
14959) does appear in Figure A5 and in some of the later electropherograms, thus, it could be a
residual contaminant from the matrix solution. The peak at 17.5 min in Figure A5 is disappearing
in Figure A6, also suggesting that it might be an impurity. New peaks appeared between 16.0 min
to 17.0 min (peak areas = 599, 874, and 1498) which could be UV resistant molecules, but it is
difficult to make this claim based on their abundance.
The 10 mL purified fraction is shown in Figure A7. The peak at 1.5 min in Figure A4
reappeared in Figure A7 at the same migration time with a peak area of 4359. Two new peaks
appeared at 6.1 min (peak areas = 6278 and 18518), along with two smaller peaks at 11.5 min
(peak areas = 1249 and 1343). These peaks are difficult to interpret as they are small and could be
residual contaminants from the mobile phase mixture. There also seems to be an additional peak

23

at 17.0 min, where in Figure A6 there was only one peak. This second peak could be from the
mobile phase or due to background noise.
Figure A8 shows the 12 mL purified fraction electropherogram. The peak at 1.5 min is still
present and shows up as two peaks (peak areas = 1342 and 3273). What is most likely is that these
peaks are residual contaminants peak based on their broad peak shape. Also, there is a new
potential UV resistant molecule peak in Figure A8 and 5.3 min (peak area = 40228) as this peak is
narrow and relatively large. At the end of the electropherogram there are multiple peaks which
could be from background noise or instability of the matrix solution.
Figure A9 shows the 14 mL purified fraction’s electropherogram. This electropherogram
looks similar to Figure A8, but without the expected background noise peaks. The peak at 5.3 min
that is present in Figure A8 disappeared but there are two peaks (peak areas = 2418 and 621) that
should be ignored since they are small and likely impurities.
Figure A10 shows the electropherogram of the 16 mL purified fraction. The peaks are
similar to the ones in Figure A9, but there appears to be a new peak at 5.3 min (peak area = 8226)
which could be another UV resistant molecule since it is not seen in the blanks.
Figure A11 shows the electropherogram of the 18 mL purified fraction. There is a new
peak at 13.7 min (peak area = 12552) that is too small so it is not a potential molecule but instead
is a contaminant. However, the peak at 5.6 min (peak area = 12552) could be a potential UV
resistant molecule and may be the molecule in Figure A10 that showed up approximately at the
same migration time, or it could be another peak that co-migrated.
Figure A12 shows the electropherogram of the 20 mL purified fraction. The shoulder peak
at 4.5 min (peak area = 2186) has gotten closer to the largest peak and is likely to be an impurity
that co-migrated with the large peak and has now slightly separated. In addition, a new peak
appeared at 10.2 min (peak area = 1389), and is relatively larger than the other potential UV
resistant molecule peaks, so, it is likely that this peak belongs to a UV resistant molecule.
Figure A13 shows the electropherogram of the 22 mL purified fraction. The impurity
shoulder peaks are still present in Figure A13, but there is a new peak at 6.3 min (peak area =
17747) and it could be a UV resistant molecule. At 15.0 min, two peaks appeared (peak areas =

24

170 and 9081) which are relatively small and reappear in the later figures, so they are residual
contaminants from the matrix solution.
Figure A14 depicts the electropherogram of the 24 mL purified fraction. The peaks at 1.5
min, reappeared, which supports how they might be residual contaminants that occasionally get
into the fractions since there is no pattern to the appearance.
Figure A15 depicts the electropherogram for the 26 mL purified fraction. This
electropherogram has many new peaks that were not seen in the other figures. New smaller peaks
appeared at 6.3 min (peak area = 1116), 7.1 min (peak area = 1719), 7.6 min (peak area = 4174),
10.4 min (peak areas = 20884 and 31830), 11.3 min (peak area = 574), 11.5 min (peak area = 2470)
and 12.5 min (peak area = 1875). Of these peaks, the ones at 10.4 min are likely to be UV resistant
molecules since their peaks are relatively larger than others. Also, the peak at 4.4 min (peak area
= 327597) could also be a UV resistant molecule as it has not appeared in any of the prior
electropherograms and is large.
Figure A16 shows the 28 mL purified fraction’s electropherogram. The 10.5 min peak is
small so it is an impurity from the matrix solution. There is also a peak at 16.1 min (peak area =
1003) that based on its size it likely to be an impurity.
Figure A17 shows the electropherogram of the 30 mL. There is a peak that appeared at 1.9
min (peak area = 3165) and is a contaminant since it is broad.
Figure A18 shows the electropherogram for the 32 mL purified fraction. A peak appeared
at 8.7 min (peak area = 876), and since it is small it is an impurity. The peak at 4.7 min (peak area
= 14532) is likely to be a potential UV resistant molecule since its size is relatively larger than
other peaks.
Figure 8 shows the 34 mL purified fraction’s electropherogram. There is a new peak at 6.0
min (peak area = 35708), and since it is large it could be a UV resistant molecule. Upon zooming
in on this peak as seen in Figure 8a, there are two doublet peaks that indicate the presence of 4 UV
resistant molecules. The peak at 8.9 min has a peak area of 536 and is thus a contaminant. Also, a
new peak appeared at 14.5 min (peak area = 4794). This peak could be a molecule with UV
resistance since the size is larger than other peaks, and the peak showed up as narrow.

25

Figure 8. Electropherogram of 34 mL purified fraction in CDCl3 and hexane:ethyl acetate (4:1).

26

Figure 8a. Zoomed electropherogram of 34 mL purified fraction in CDCl3 and hexane:ethyl
acetate (4:1) from 6.75 min to 11.50 min.
Structure Elucidation
When analyzing the purified fractions on NMR and FTIR, useful results were not obtained
as the spectra appeared either exactly like the blank, or messy making it difficult to interpret. This
may be due to the degradation of the UV resistant molecules, but it is more likely that the purified
fractions were too dilute and contained the molecules in concentrations that were lower than the
limits of detections for the spectroscopic techniques. However, when the raw powder that was
dissolved in CDCl3 and heated was run, spectra were obtained that differed from the blanks and
new peaks were visible. This sample was not as dilute as the purified fractions, supporting the

27

reasoning behind why the molecules in the purified fractions were not appearing. Table 8 shows
the peaks and chemical shifts that were seen in the NMR spectra from Figures 9-13. The potential
identities of the peaks seen in Table 8 were the peaks that fit the most with what was expected of
a molecule showing UV resistance, hence, where peaks could be classified as alkene or alkyne
bonds then they were classified as such. The other peaks fit with what is expected of each other,
for instance, the peaks identified as –NH bonds fit with the amide classification. Despite this
information, a structure could not be accurately created as the purified fractions needed to be run
to determine whether the peaks are from functional groups that are on molecules, or if they are
from remnants of bark or other contaminants in the raw powder.
Table 8. Peaks and chemical shifts seen in NMR spectra of raw powder dissolved in CDCl3 and
heated from Figures 9-13.
Chemical Shift of Peak (ppm)
1.3141
2.3792
2.4418
3.6517
4.0805

Peak Splitting
Potential Identity of Peak
Singlet
Water
Triplet
Alkyne or –NH (primary)
Doublet of triplets
Alkyne or –NH (primary)
Triplet
X—C—H (where X = O)
Doublet of triplets (low
Impurity
resolution)
4.1635
Multiplet
impurity
7.0683
Singlet
Alkene
7.2896
Singlet
CHCl3
7.4971
Singlet
Amide Proton
9.8071
Triplet
Aldehyde Proton
Note: Potential identities of peaks were gathered using chemical shift data from ref. 3
(Chemistry Steps, 2020).

28

Figure 9. NMR spectrum of raw powder dissolved in CDCl3 and heated.

Figure 10. Zoomed NMR spectrum between 2.3 ppm to 3.65 ppm of raw powder dissolved in
CDCl3 and heated.

29

Figure 11. Zoomed NMR spectrum between 3.5 ppm to 4.2 ppm of raw powder dissolved in
CDCl3 and heated.

Figure 12. Zoomed NMR spectrum between 6.9 ppm to 7.8 ppm of raw powder dissolved in
CDCl3 and heated.
30

Figure 13. Zoomed NMR spectrum between 9.5 ppm to 10.1 ppm of raw powder dissolved in
CDCl3 and heated.
The FTIR analysis of the raw powder dissolved in CDCl3, heated, and absorbed onto silica
gel did not yield any significant results, as the detected peaks belong to the blanks, as seen in Table
9. However, the small peaks at 3000 cm-1 and 2100 cm-1 could be the amine nitrogen and the
alkyne, respectively .20 Due to these peaks being very small, it suggests that the sample is too dilute
to be reliably detected on the FTIR, thus, this classification should not be taken as concrete.
Table 9. Peak area and height of labelled peaks in FTIR spectrum of raw powder dissolved in
CDCl3, heated, and absorbed onto silica gel for analysis.
Peak Number
1
2
3
4
5

Wavenumber (cm-1)
1060.24
903.55
799.43
725.32
649.94

% Transmittance
69.93
36.12
90.2
28.54
72.38

Identity of Peak
Silica gel
Silica gel
CDCl3
Silica gel
CDCl3

31

Figure 14. FTIR spectrum of raw powder dissolved in CDCl3, heated, and absorbed onto silica
gel for analysis.
CONCLUSION
Through sample collection, purification using flash column chromatography, and
analytical and spectroscopic analysis of purified fractions and raw Trembling Aspen powder, this
work has set the foundation for determining potential structures of UV resistant molecules in
Trembling Aspen tree powder. The work found that there are approximately five molecules in the
powder that absorb UV light, and that these molecules may have a range of functional groups such
as alkenes, alkynes, amines, amides, and aldehydes, which are typical of UV resistant molecules
found in commercial sunscreens. The multiple structures in the powder may have isomeric forms
too, which could explain the reappearance of some peaks in the purified fractions as they may have
slightly different polarities and interactions with the flash chromatography column. This work
strived to act as a preliminary body of information that furthers the goal of characterizing all UV
resistant molecules present in the Trembling Aspen tree powder.

32

Future Work
Future work entails doing two types of analysis: MALDI-MS (matrix assisted laser
desorption ionization-mass spectrometry) and X-ray crystallography. The first type would allow
for the masses of the UV resistant molecules to be acquired, which would narrow down how many
atoms are present in each structure and the maximum number of elements in each (Broad Institute,
2010). The second type would allow for a clear identification of the structure of the UV resistant
molecules which, using NMR spectra, would allow for the determination of atoms in 3D space and
where each atom is connected. In addition, future work could include comparing the powder made
on the shady side of Trembling Aspen during different seasons with the powder on the sunny side
to see if there is a difference in the structures of UV resistant molecules in the powders.

33

LITERATURE CITED
Barlow, P. (2005). From cambium to early cell differentiation within the secondary vascular
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APPENDIX A

Figure A1. Electropherogram of CDCl3 blank.

40

Figure A2. Electropherogram of hexane:ethyl acetate (4:1) blank (horizontal axis was cut off at
15 min since the remaining 5 min was baseline resolution).

41

Figure A2(a). Zoomed electropherogram of hexane:ethyl acetate (4:1) blank.

42

Figure A3. Electropherogram of 2 mL purified fraction in CDCl3 and hexane:ethyl acetate (4:1).

43

Figure A4. Electropherogram of 4 mL purified fraction in CDCl3 and hexane:ethyl acetate (4:1).

44

Figure A5. Electropherogram of 6 mL purified fraction in CDCl3 and hexane:ethyl acetate (4:1).

45

Figure A6. Electropherogram of 8 mL purified fraction in CDCl3 and hexane:ethyl acetate (4:1).

46

Figure A7. Electropherogram of 10 mL purified fraction in CDCl3 and hexane:ethyl acetate
(4:1).

47

Figure A8. Electropherogram of 12 mL purified fraction in CDCl3 and hexane:ethyl acetate
(4:1).

48

Figure A9. Electropherogram of 14 mL purified fraction in CDCl3 and hexane:ethyl acetate
(4:1).

49

Figure A10. Electropherogram of 16 mL purified fraction in CDCl3 and hexane:ethyl acetate
(4:1).

50

Figure A11. Electropherogram of 18 mL purified fraction in CDCl3 and hexane:ethyl acetate
(4:1).

51

Figure A12. Electropherogram of 20 mL purified fraction in CDCl3 and hexane:ethyl acetate
(4:1).

52

Figure A13. Electropherogram of 22 mL purified fraction in CDCl3 and hexane:ethyl acetate
(4:1).

53

Figure A14. Electropherogram of 24 mL purified fraction in CDCl3 and hexane:ethyl acetate
(4:1).

54

Figure A15. Electropherogram of 26 mL purified fraction in CDCl3 and hexane:ethyl acetate
(4:1).

55

Figure A16. Electropherogram of 28 mL purified fraction in CDCl3 and hexane:ethyl acetate
(4:1).

56

Figure A17. Electropherogram of 30 mL purified fraction in CDCl3 and hexane:ethyl acetate
(4:1).

57

Figure A18. Electropherogram of 32 mL purified fraction in CDCl3 and hexane:ethyl acetate
(4:1).

58