Drawing Inspiration from Nature for Solar Energy Conversion

DRAWING INSPIRATION FROM NATURE
FOR SOLAR ENERGY CONVERSION:
MAPPING THE STRUCTURE-FUNCTION RELATIONSHIPS IN
BETALAIN PIGMENTS TO UNDERSTAND NATURAL
LIGHT HARVESTING SYSTEMS
By
NICHOLAS ANDREW TREAT
A dissertation submitted in partial fulfillment of
the requirements for the degree of
DOCTOR OF PHILOSOPHY
WASHINGTON STATE UNIVERSITY
Department of Chemistry
JULY 2016
© Copyright by NICHOLAS ANDREW TREAT, 2016
All Rights Reserved

ii
To the Faculty of Washington State University:
The Members of the Committee appointed to examine the dissertation of NICHOLAS
ANDREW TREAT find it satisfactory and recommend that it be accepted.
_______________________________
Jeanne L. McHale, Ph.D., Chair
_______________________________
John Fellman, Ph.D.
_______________________________
Aurora Clark, Ph.D.
_______________________________
Chulhee Kang, Ph.D.

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Acknowledgement
I would like to acknowledge the generous financial support of the Washington State
University College of Arts and Sciences, especially Dean Daryll DeWald whose help was crucial
in securing the funding to complete my degree, Dr. Kirk Peterson for the many times he
facilitated access across the hall, Dr. Fritz Knorr for helping with the “factory”, all the McHale
Group members past and present for their solidarity and Dr. Jeanne McHale whose support
enabled this project to continue despite overwhelming odds. Of course, the most important
support I received was from my friends and family, who have shown me unwavering support
throughout the many years of my Ph.D. career.

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Dedication
This dissertation is dedicated to my father, whose support has been unwavering, despite
the odds, over the course of my education and career, and whose guidance has been invaluable
on my journey.

v
DRAWING INSPIRATION FROM NATURE
FOR SOLAR ENERGY CONVERSION:
MAPPING THE STRUCTURE-FUNCTION RELATIONSHIPS IN
BETALAIN PIGMENTS TO UNDERSTAND NATURAL
LIGHT HARVESTING SYSTEMS
Abstract
by Nicholas Andrew Treat, Ph. D.
Washington State University
July 2016
Chair: Jeanne L. McHale
Photophysical properties of betalain pigments from beets (Beta vulgaris L.), amaranth
(Amaranthus cruentus) and pitahaya (Hylocerus polyrhizus) were studied in the context of dye-
sensitized solar cells. Utilizing a nature-inspired approach to solar energy production, natural
pigments were extracted, purified and studied to develop a greater understanding of the light
harvesting systems. The highly efficient light harvesting of the betalains, which is due to their
photoprotective role in the plants, far surpasses the metalorganic dyes commonly used in dye-
sensitized solar cells. Evidence of two-electron oxidation, and ease of preparation and abundant
supply make betalain pigments an ideal candidate for a renewable energy solution.
A process for the extraction and purification of mixtures and pure betalains was
developed based on anion exchange chromatography and high performance liquid

vi
chromatography. The ability to perform experiments on samples with single betalain components
allowed for more precise quantification of the feasibility and performance of betalain-based
devices.
Films sensitized with betanin from non-aqueous solution were shown to have negligible
aggregation of betanin on the TiO2 surface despite the high dye loading. This effect was
observed at all ratios of methanol in water from 4% methanol to neat methanol. No other
changes in the solutions were observed.
Differences in the behavior of two dyes, amaranthin and betanin, were used to develop a
map of the structure-function relationships within betalain pigments. Betanin was shown to
aggregate on TiO2 where amaranthin does not. The aggregate formed by betanin greatly
enhanced the performance and stability of the devices. The fluorescence quantum yields of
betalains were shown to also depend slightly on structure, despite the distance between the
additional sugar or acyl groups and chromophore.

vii
Table of contents Page
Acknowledgement .........................................................................................................................iii
Dedication...................................................................................................................................... iv
Abstract........................................................................................................................................... v
Table of contents ...................................................................................................................... vii
Table of Figures ...................................................................................................................... xii
List of Tables ..................................................................................................................... xvi
1 Introduction............................................................................................................................... 1
1.1 Research Goals...................................................................................................... 4
1.2 Specific Aims........................................................................................................ 4
1.2.1 Purify samples of betalain pigments for spectroscopic studies....................... 4
1.2.2 Investigate dye absorption spectral broadening on TiO2 and aggregation...... 4
1.2.3 Quantify the performance of aggregated compared to monomeric
samples........................................................................................................... 5
1.2.4 Compare the behavior of amaranthin to that of betanin.................................. 5
1.3 Background ........................................................................................................... 5
1.3.1 DSSCs ............................................................................................................. 5
1.3.2 Principles......................................................................................................... 5
1.3.3 Characterization .............................................................................................. 8
1.4 Betalains.............................................................................................................. 11
1.4.1 Properties and Sources .................................................................................. 11
1.4.2 Stability: ........................................................................................................ 14
1.4.3 Photophysical Properties:.............................................................................. 16

viii
1.4.4 Comparisons with Anthocyanins .................................................................. 16
1.5 References ........................................................................................................... 19
2 Preparation of Betalain Pigments............................................................................................ 23
2.1 Introduction: Sources and Previous Techniques ................................................. 23
2.2 Preparatory Techniques....................................................................................... 25
2.2.1 Extraction ...................................................................................................... 25
2.2.2 HPLC............................................................................................................. 25
2.2.3 MPLC............................................................................................................ 29
2.2.4 Anion-Exchange Column Chromatography.................................................. 30
2.3 Conclusions......................................................................................................... 32
2.4 References ........................................................................................................... 35
3 Templated Assembly of Betanin Chromophore on TiO2: Aggregation-Enhanced Light
Harvesting and Efficient Electron Injection in a Natural Dye-Sensitized Solar Cell............. 38
3.1 Introduction......................................................................................................... 38
3.2 Experimental Methods ........................................................................................ 41
3.2.1 Purification.................................................................................................... 41
3.2.2 HPLC............................................................................................................. 42
3.2.3 Film preparation and sensitization (TiO2 and ZrO2)..................................... 42
3.2.4 Absorption and fluorescence spectra............................................................. 44
3.2.5 IPCE and APCE ............................................................................................ 44
3.2.6 Solar Cell Construction................................................................................. 45
3.2.7 Numerical methods. ...................................................................................... 45

ix
3.3 Results and Discussion........................................................................................ 46
3.3.1 Absorption Spectra and Numerical Modeling of Betanin on TiO2 and
ZrO2.............................................................................................................. 46
3.3.2 Quantum Efficiency of Photon-to-Current Conversion ................................ 50
3.3.3 Fluorescence of Betanin on TiO2 and ZrO2 .................................................. 53
3.3.4 Power Conversion Efficiency of Dimer-based Solar Cells........................... 54
3.3.5 Theory of Transition Dipole-Dipole Coupling in Betanin Dimers ............... 56
3.4 Conclusions......................................................................................................... 59
3.5 Supporting Information....................................................................................... 61
3.6 References ........................................................................................................... 66
4 Tuning the Aggregation of Betanin on TiO2 Using Non-Aqueous Solvents and
Phosphate Additives................................................................................................................ 72
4.1 Introduction......................................................................................................... 72
4.2.1 Film Preparation............................................................................................ 75
4.2.2 Film Sensitization.......................................................................................... 75
4.2.3 Absorbance Spectroscopy ............................................................................. 75
4.2.4 Solar Cell Measurements .............................................................................. 75
4.3.1 Phosphate ...................................................................................................... 76
4.3.2 Methanol........................................................................................................ 80
4.3.3 Performance of Cells..................................................................................... 84
4.4 Conclusions......................................................................................................... 86

x
4.5 References ........................................................................................................... 87
5 An Amaranthin Based Solar Cell: Comparison of Efficiency and Properties to
Betanin Based Dye Sensitized Solar Cells.............................................................................. 88
5.1 Introduction......................................................................................................... 88
5.2.1 Preparation of Amaranthin Samples ............................................................. 90
5.2.2 Film Preparation............................................................................................ 91
5.2.3 Sensitization of TiO2 films with Amaranthin................................................ 91
5.2.4 Absorbance Spectroscopy ............................................................................. 91
5.2.5 Fluorescence Quantum Yield Measurements................................................ 91
5.2.6 Preparation of Colloidal Silver Nanoparticles .............................................. 92
5.2.7 Surface Enhanced Raman Scattering ............................................................ 92
5.3.1 Comparison of Aggregation Behavior Based on Absorption Spectra........... 92
5.3.2 Solvents and pH Differences......................................................................... 94
5.3.3 Fluorescence.................................................................................................. 96
5.3.4 Raman............................................................................................................ 97
5.3.5 Device Performance ...................................................................................... 98
5.4 Conclusions....................................................................................................... 101
5.5 References ......................................................................................................... 102
6 Summary and Conclusion..................................................................................................... 103
6.1 Future Work ...................................................................................................... 104

xi
6.2 Final Remarks ................................................................................................... 105
7 Appendix A: MATlab Code.................................................................................................. 106
7.1 BuildRC............................................................................................................. 106
7.2 LeeSeung........................................................................................................... 106
7.3 Lee..................................................................................................................... 111
7.4 Dcalc.................................................................................................................. 111
8 Appendix B: HPLC Methods................................................................................................ 112

xii
Table of Figures Page
Figure 1.1: Structure of N3, Ru(4,4’-dicarboxylicacid-2,2’-bipyridine)2(NCS)2(left), and
its tert-butyl ammonium (TBA) salt analogue N719 (right).5
................................. 3
Figure 1.2: Cartoon schematic of a dye sensitized solar cell.5
reprinted with permission
from Hagfeldt, A.; Boschloo, G.; Sun, L.; Kloo, L.; Pettersson, H. Dye-
Sensitized Solar Cells. Chem. Rev. 2010, 6595–6663.
DOI: 10.1021/cr900356p ........................................................................................ 6
Figure 1.3: Energy level schematic for a DSSC in operation showing the various
reactions (arrows) that occur and the associated rate constants.............................. 8
Figure 1.4: Reaction pathways for betacyanin (BcN) and betaxanthin (BxN) formation
from betalamic acid (BtA). ................................................................................... 11
Figure 1.5: Betacyanin structures from the natural sources studied. ............................................ 13
Figure 1.6: Structures of betaxanthins that are relevant to the sources of betalains in this
research. ................................................................................................................ 14
Figure 1.7: Two possible structures proposed for neobetanin: traditional structure shown
in most literature (left XIVa) and structure suggested recently by
Wybraniec et al. (right XIVb)............................................................................... 15
Figure 1.8: The basic structure of anthocyanins is based on anthocyanidin. In
anthocyanidin all of the R groups are hydrogens.................................................. 17
Figure 1.9: Structures of common copigments found throughout the betalain and
anthocyanin species. The binding site to the base dye structure is denoted
by “R”. .................................................................................................................. 18
Figure 1.10: Aggregates of anthocyanins consisting of units of succinylcyanin and
malonylflavone shown schematically and in wire-frame form (B).
Reprinted with permission from Ellestad, G. A. Structure and Chiroptical
Properties of Supramolecular Flower Pigments. Chirality, 2006, 18, 134-
144. DOI: 10.1002/chir.20228.............................................................................. 18
Figure 2.1: Preparatory HPLC of amaranth extract with amaranthin (1) and isoamaranthin
(2) the predominant pigments, but small amount of decarboxylated
betacyanins (3 &4) being present. ........................................................................ 29
Figure 2.2: Schematic of the preparatory process developed in this research.............................. 31

xiii
Figure 2.3: HPLC chromatogram of purified amaranth (a) and that of filtered amaranth
juice (b) monitored at 254 nm............................................................................... 32
Figure 2.4: HPLC chromatogram of purified samples of amaranth (black), pitahaya (red),
beets (green) and a combination of all the sources. The primary
betacyanins are marked as amaranthin (A), betanin (B), phylocactin (C)
and hylocerinin (D)............................................................................................... 33
Figure 3.1: Structure of betanin (left) and 3D representation of optimized geometry
(right) obtained using DFT as described in Ref. 42.............................................. 39
Figure 3.2: Absorption spectra of Bt on A) ZrO2 and B) TiO2 as a function of
sensitization time in a saturated betanin solution at pH 2. A 900s film on
TiO2 was prepared, but was too dark to determine the absorbance...................... 48
Figure 3.3: Results of NMF analysis of Bt/TiO2 at A) low and B) high dye loading. The
films in A) and B) were sensitized for 300s and overnight (21 hours) and
contain 25% and 65% aggregate, respectively. The components are dimer
(green), monomer (blue), TiO2 extinction (cyan), experimental absorbance
(black), and total calculated absorbance (red dashed). ......................................... 49
Figure 3.4: A) IPCE and B) APCE of Bt-based DSSC consisting of 25% aggregate (red),
and 65% aggregate (black).................................................................................... 52
Figure 3.5: Fluorescence spectra, excited at 514.5 nm and corrected for absorptance at
514.5 nm, of Bt on A) TiO2 and B) ZrO2 as a function of sensitization
time. ...................................................................................................................... 52
Figure 3.6: Photocurrent versus photovoltage for several Bt-based dye-sensitized solar
cells. ...................................................................................................................... 54
Figure 3.7: Scanning electron micrographs of TiO2 films showing 3.5µm thickness (left),
7µm thickness (middle) and the nanoparticles (right). ......................................... 61
Figure 3.8: UV-Vis absorption spectra of the sensitized TiO2 film (black) and the
sensitizing solution (red) showing the dramatic blue-shift from 537nm to
482nm. .................................................................................................................. 61
Figure 3.9: (A) HPLC chromatogram, monitored at 525nm, of the extracted pigments of
films sensitized for 10min (red) and 16hours (green) shown with the
sensitizing solution (black) for comparison. (B) Absorption spectra of film
extract (black) and sensitizing solution (red)........................................................ 62
Figure 3.10: Calibration curve of betanin vs. calculated concentration. ...................................... 62

xiv
Figure 3.11: UV-Vis absorption spectra of films sensitized without phosphate (A, Black)
and with 1 mM phosphate (B, red). ...................................................................... 63
Figure 3.12: Unit components from NMF analysis showing the peaks of the aggregate
(green), monomer (blue), TiO2, and the sum of all components (red).................. 63
Figure 3.13: NMF component analysis showing three subsets of the samples used for:
fluorescence measurements (A), Beer’s Law analysis of solutions (B), and
DSSC characterization (C).................................................................................... 64
Figure 3.14: Linear combination of the aggregate and monomer unit components from
the NMF modeling showcasing the representative peak locations for
aggregation amounts in 10% intervals.................................................................. 65
Figure 3.15: Characteristic current-voltage plots for films with no pretreatment (black),
pretreatment with 0.5M hydrochloric acid in ethanol (red), and purified
water (blue). .......................................................................................................... 65
Figure 4.1: The molecular structure of betanin showing the atomic labeling of the various
functional groups. ................................................................................................. 73
Figure 4.2: Speculative structure of betanin dimer on TiO2 (101) surface. Reprinted with
permission from Treat, N. A.; Knorr, F. J.; McHale, J. L., Templated
Assembly of Betanin Chromophore on TiO2: Aggregation-Enhanced
Light-Harvesting and Efficient Electron Injection in a Natural Dye-
Sensitized Solar Cell. J. Phys. Chem. C, 2016, 120 (17), 9122-9131.
Copyright 2016 American Chemical Society. ...................................................... 74
Figure 4.3: Absorbance spectra of betanin films sensitized overnight in with various
amounts of phosphate, 1.0 mM (black), 0.1 mM (blue), 10 mM (red) and
0.0 mM (pink), and 1.0 mM betanin all exhibit blue-shifted absorbance
maxima around 485 nm. ....................................................................................... 77
Figure 4.4: Absorption spectra of films sensitized with 5 mM betanin (black and blue)
and 1 mM betanin (red and magenta) in a buffer of 1 mM phosphate. ................ 78
Figure 4.5: Sequential absorption spectra of betanin films sensitized in 10 mM (a), 1 mM
(b), 0.1 mM (c) and 0.0 mM (d) phosphate taken at 1 minute intervals for
up to 20 minutes, except (a) which were taken at 10 minute intervals for
90 minutes............................................................................................................. 79
Figure 4.6: Absorbance spectra of TiO2 films sensitized with 1 mM betanin in 1 mM
sodium phosphate at pH 3 taken sequentially at regular intervals........................ 80

xv
Figure 4.7: Absorbance spectra of betanin films sensitized for 5 minutes (black) and 10
minutes (red) from methanol (left) and water (right) with otherwise
identical conditions............................................................................................... 81
Figure 4.8: Absorption spectra of films sensitized with betanin solutions containing 4%
(black), 52% (red) and 96% MeOH (green) for 10 minutes and 4% MeOH
for 30 min (blue). .................................................................................................. 82
Figure 4.9: Absorbance spectra of TiO2 films sensitized with betanin in 100% methanol
sequentially from 1 to 40 minutes, with spectra taken at regular intervals........... 83
Figure 4.10: IPCE spectra of betanin solar cells produced with various amounts of
phosphate in the sensitizing solutions................................................................... 84
Figure 4.11: J-V curves of solar cells produced from betanin in methanol (black) and
water (red)............................................................................................................. 85
Figure 5.1: Molecular structures for betanin (left), amaranthin (right) and their aglycone
betanindin (center). ............................................................................................... 89
Figure 5.2: Absorbance spectra of films sensitized with amaranthin from aqueous
solutions of varying pH (a), and from methanol (MeOH) and acetonitrile
(MeCN) (b). .......................................................................................................... 94
Figure 5.3: Absorbance spectra of solutions of amaranthin in methanol and aqueous
solvents show no significant difference, though amaranthin in acetonitrile
(purple) shows a broadening and blue-shift.......................................................... 95
Figure 5.4: Emission spectra of amaranthin (black) and betanin (red) in aqueous solution
with excitation at 514.5 nm normalized to show that the shapes are
identical................................................................................................................. 96
Figure 5.5: Surface enhanced resonance Raman spectra of betanin and amaranthin show
that the chromophore vibrational modes are nearly identical excited at 458
nm. ........................................................................................................................ 97
Figure 5.6: Replicate IPCE spectra of amaranthin based solar cells sensitized from MeOH
(a) and MeCN (b) solutions. ................................................................................. 99
Figure 5.7: A comparison of the J-V curves of amaranthin from aqueous (a) and non-
aqueous solution (b)............................................................................................ 100

xvi
List of Tables Page
Table 3.1: Power conversion efficiency, open circuit voltage Voc, short-circuit current Jsc,
wavelength of maximum absorption for sensitized film λmax, film
thickness, and percent aggregation for each of the solar cells in Figure 3.6. ....... 56
Table 5.1: Energy conversion efficiencies of amaranthin based solar cells sensitized from
acetonitrile (left), methanol (center) and water (right). The efficiency of
cell 1 in methanol was excluded for the calculation of the average. .................. 100
Table 8.1: Binary gradient developed specifically for analysis of amaranth samples................ 112
Table 8.2: Binary gradient developed for analysis of samples from beets. ................................ 112
Table 8.3: Binary gradient developed for preparatory methods of all betalain sources. ............ 112

1
1 Introduction
Over the past few decades, it has become apparent that our over utilization of fossil fuels
for energy production is having a deleterious effect on the global ecosystem. In addition to their
effect on the environment, research indicates that the global stores of fossil fuels are being
depleted. However, the demand for energy is increasing as we depend increasingly on electronics
in our daily lives. These factors combined have led to an emergent global energy crisis.
Therefore, an effective and ecologically conscientious method of energy production is a rapidly
expanding area of interest in chemical and materials research. Of the several ecologically
friendly energy production methods, solar power is perhaps the most promising. Solar radiation
is a dependable source of energy that nature has adapted to harness. Perhaps we can take a lesson
from nature in our pursuit of reliable, cost effective, and ecologically friendly energy.1,2
Plants have adapted to harness solar energy through photosynthesis involving various
photo-reactive chemical species and complex molecular scaffolds. An understanding of the
principle of light harvesting chemicals and structures in these highly adapted organisms could
lay the foundation for an innovative approach to the energy crisis. Of primary interest is the
underlying principle of the structure-function relationship in these unique systems.1,2
Betalains, a family of photo-protective molecules, are found exclusively in plants of the
order Caryophyllales and a few species of fungi. In recent years, research has shown that
betalains offer a unique potential in the field of dye sensitized solar cells (DSSCs). By utilizing
the photochemical oxidation of these pigments, DSSCs have been constructed to perform at an
efficiency of up to 3%.3
While the ultimate implementation of DSSCs would require higher
efficiencies, the rough and non-optimal engineering of these particular cells indicates a very
promising class of solar energy device. In a comparison study, N3 dye was tested, without

2
significant optimization of the cell, and produced a maximum efficiency of 2.6%.4
This
similarity exemplifies the potential of betalain dyes as sensitizers. In other labs, with
optimization, N3 solar cells have performed with efficiencies as high as 9%.5
Additionally, these
small organic molecules offer several specific advantages over traditional ruthenium based
DSSCs. First, these dyes are environmentally friendly and readily abundant. Since they are
produced naturally in plants, they are easily obtained without difficult and costly synthetic
pathways and do not require the use of ruthenium, which is of limited supply. Secondly, the
organic molecular nature of these pigments indicates that possibility of a two electron oxidation
mechanism.6,7
Multiple electron oxidation mechanisms would allow for an increase in the
thermodynamic efficiency determined by the Schockley-Queisser limit.4,8
Third, the wavelength-
dependent incident-photon-to-electron conversion efficiency (IPCE) of DSSCs based on betanin,
from red beet root (Beta vulgaris L.), has been found to approach 100%.9
These measurements
also show good response at red wavelengths which fits well with the solar spectrum. Finally, due
to the allowed nature of their absorption transition, compared to the marginally allowed d-d
transition of metal-based dyes, betalains allow for DSSC construction with thinner
semiconductor films. Thinner films would allow electrons to more effectively be transferred into
the circuit by decreasing their diffusion length in titanium dioxide.5

3
Unfortunately, betalains suffer from several drawbacks that need to be overcome before
effective implementation of betalain-based DSSCs is feasible. The primary hurdle in betalain
research is stability.10-14
In our lab, one successful solar cell system was studied for a period of a
few months and was functional during daylight hours for the majority of the time. However, the
current produced by the arrangement decreased by approximately 30% over the first few days. It
appears that irreversible photo-degradation occurred and hindered device performance. While
betalain stability is a current topic of research in the food sciences, a systematic approach to
understanding the structural aspects of betalain stability has yet to be performed with regards to
the interfacial chemistry occurring at solution-titanium dioxide surfaces. Furthermore, a study
delineating the relative stability and photophysical properties of different betalain dyes could
improve our understanding of the differing aspects of structure and function of photo-protective
systems in nature.15
Figure 1.1: Structure of N3, Ru(4,4’-dicarboxylicacid-2,2’-bipyridine)2(NCS)2(left), and
its tert-butyl ammonium (TBA) salt analogue N719 (right).5

4
1.1 Research Goals
Overall the main objective of this research is to develop an understanding of structure-
function relationships in betalain pigments and investigate how these relationships can be
harnessed in solar energy conversion technology. By utilizing a spectroscopically focused
research plan, these relationships will be probed in four specific ways.
1.2 Specific Aims
1.2.1 Purify samples of betalain pigments for spectroscopic studies
Previous work on betalain-based DSSCs was significantly hampered by the lack of
experimental control.9
The biggest variable in any natural product based research is purity.
Without a pure sample, a thorough understanding of experimental outcomes cannot be
established. Therefore, this research developed a method to produce purified dye samples which
can then be used to carefully study the behavior of betalains in DSSCs.
1.2.2 Investigate dye absorption spectral broadening on TiO2 and aggregation
Spectral characterization of betanin adsorbed on TiO2 shows a significant broadening of
the absorption spectrum in both the blue and red regions with the most prominent being in the
blue. The overall change produces a spectrum with an absorption maximum near 480 nm, nearly
50 nm blue of the solution phase maximum. Whether this phenomenon is the product of
degradation, multiple species adsorbing or aggregation of the betanin is of great interest due to
the near 100% IPCE achieved at this blue shifted wavelength.9
Based on some preliminary work,
it is believed that this blue-shift is associated with chromophore aggregation on the TiO2, but
other research shows that degradation products would likely produce similar spectra.6
Using

5
purified dye, spectroscopy and HPLC we aimed to determine the cause of the spectral
broadening.
1.2.3 Quantify the performance of aggregated compared to monomeric samples
The broadened spectra seem to produce more efficient solar cells, and quantification of
this enhancement could lead to inspired design of future DSSCs. Therefore, additives commonly
used to inhibit aggregation were used to control the spectral broadening and develop a model for
the mechanism of aggregation.16
1.2.4 Compare the behavior of amaranthin to that of betanin
The small differences in structure between amaranthin and betanin provide a good
starting point to begin mapping structure-function relationships in betanin. Prior to this research,
no studies had been done utilizing amaranthin in a solar cell. We wished to determine what effect
the steric bulk of an additional sugar group has on the behavior of betalains in DSSCs.17
1.3 Background
1.3.1 DSSCs
Dye sensitized solar cells (DSSCs) are a form of photoelectrochemical cell that harnesses
the sun’s energy to produce electricity. Unlike traditional photovoltaic cells, DSSCs utilize a dye
sensitizer to perform the duty of absorbing the light. Photosynthesis is also a
photoelectrochemical process, though much more intricate, and can serve as inspiration in DSSC
design. Photosynthesis, however, converts the solar energy into chemical energy, whereas
DSSCs convert the solar energy directly into electricity.5,18
1.3.2 Principles

6
The essential components of a DSSC are a semiconductor film, dye sensitizer, platinum
or carbon catalyst, and two electrical contacts. Figure 1.2 shows the components of a typical
DSSC. Titanium dioxide is used as the semiconductor and platinum as the cathode catalyst,
though carbon may also be used. These materials are bonded to conductive glass to form the
foundation of the solar cell. Several different electrolytes are used, but the most common to date
is the iodide-triiodide redox couple dissolved in an organic solution.5
The operation of a DSSC can be broken down into three essential steps: light absorption,
dye oxidation/electron injection and dye regeneration. The light absorption step is pretty
straightforward: a photon is absorbed by the sensitizer which causes the molecule to enter into an
electronically excited state. Once in this state, the dye can either inject the excited electron into
the semiconductor, step 2, or it can relax back to the ground state. Reverse electron injection into
the ground state can also occur, which is called recombination. Recombination results in dye
molecules that have returned to their ground state without the electron completing its journey
through the circuit. Electrons that have been injected into the semiconductor, and are not
Figure 1.2: Cartoon schematic of a dye sensitized solar cell.5 reprinted with permission
from Hagfeldt, A.; Boschloo, G.; Sun, L.; Kloo, L.; Pettersson, H. Dye-Sensitized Solar
Cells. Chem. Rev. 2010, 6595–6663. DOI: 10.1021/cr900356p

7
recombined with the dye, are free to move, and are now able to flow through the electrical
circuit. After electron injection the dye molecule is returned to its ground state but in an oxidized
form. The oxidized dye is then reduced by the iodide-triiodide couple in the third and final step.
The platinum catalyst serves the function of regenerating the electrolyte at the cathode and
completing the circuit. Figure 1.3 shows an energy level diagram for the cell and each step. Each
process occurs at a given rate, which is determined by the nature of the dye, semiconductor and
the electrolyte. The maximum voltage for the device is determined by the conduction band and
electrolyte energies and not by the sensitizer. The rate at which the dye undergoes electron
injection is determined by the difference in energy, on an electrochemical scale, between the
conduction band of the semiconductor and the excited state of the dye. A large difference,
therefore higher the driving force, results in a faster rate of injection.5,18
Figure 1.3: Energy level schematic for a DSSC in operation showing the various reactions
(arrows) that occur and the associated rate constants.

8
The most unique component of the solar cell is the dye that is used to sensitize the
titanium dioxide. Many different types of molecules have been used a sensitizers and can range
from organometallics to simple organic dyes. The most efficient dyes that have been researched
recently are ruthenium based organometallic compounds. These metal based dyes have shown up
to 11% total energy conversion efficiency in a laboratory setting.5
Natural pigments, on the other
hand, have only shown efficiencies up to 1% with the exception of betanin which has achieved
3% in our lab. While this efficiency may not directly compare with those shown by other
research labs with organic and metalorganic dyes, it compares well with the efficiency that our
lab has achieved with N3 of 4-5%. In light of this comparison, the natural sensitizers are quite
promising.5,9,17
1.3.3 Characterization
The efficiency of a solar cell is measured in two ways each giving crucial information on
the performance of the device. Most important to this research is the external quantum
efficiency, alternatively known as the incident photon to current conversion efficiency (IPCE).
Most simply put this quantity measures the ratio of the of electrons injected to photonsincident
on the sample:5
IPCE =
𝑁 𝑒
𝑁 𝑝
Equation 1.1
Here Ne and Np are the number of electrons injected into the circuit and the number of photons
incident on the surface respectively. Experimentally, the device is illuminated with
monochromatic light of a known intensity, and the resulting current density is measured. By
scanning over the range of absorption of the device, the wavelength specific conversion
efficiency can be determined by:5

9
𝐼𝑃𝐶𝐸 =
𝐽𝑠𝑐(𝜆)
𝑒Φ(λ)
= 1240
𝐽𝑠𝑐(𝜆)[𝐴 𝑐𝑚−2]
λ(nm)𝑃 𝑖𝑛(λ)[𝑊𝑐𝑚−2]
Equation 1.2
JSC(λ) is the short circuit current when illuminated at wavelength λ, e is the charge of an electron,
Φ(λ) is the incident photon flux. The denominator can be separated into the wavelength λ and
incident power Pin with a constant conversion factor of 1240 with units of V*nm. Essentially the
IPCE can be considered analogous to the spectral response function of the device. Knowing how
a device performs at varying wavelengths can inform the researcher of conditions and variables
that are most favorable to device optimization. 5
The other primary method of device characterization is the total energy conversion
efficiency. The total conversion efficiency can be measured under simulated solar conditions
(AM1.5) and the resulting efficiency is calculated with:5
𝜂 =
𝐽𝑠𝑐 𝑉𝑜𝑐 𝐹𝐹
𝑃 𝑖𝑛
=
𝑃 𝑚𝑎𝑥
𝑃 𝑖𝑛
Equation 1.3
Once again JSC is the short circuit current, VOC is the open circuit voltage, FF is the fill factor, Pin
is the incident light power and Pmax is the maximum power of the device. This method of
characterization is essential for characterizing device performance issues that are correlated to
construction and engineering as well as those caused by the specific sensitizer used.5
Much like the ideal Carnot Cycle governs a heat engine; solar energy conversion is
limited in the maximum conversion efficiency it can achieve by the Schockley-Queisser Limit.
Based upon the physical laws that govern light and its interaction with matter, the maximum
efficiency of solar energy conversion is given by a detailed balance treatment of the conditions
of the device. Many different forms of this calculation are given in the literature; each with
specific modifications for the researchers’ approach to photovoltaic construction. The generally

10
accepted fundamental limit for the maximum energy conversion efficiency for a single junction
semiconductor photovoltaic device is 31%.8
This principle is based on several assumptions:
 The device absorbs all photons which have energy greater than the band gap of the
semiconductor;
 One electron is generated for each photon that is absorbed;
 The spectral response of the device is constant over all absorbed photon energies; and
 The excess energy of the photons above the band gap is lost as heat.
For the most part, these assumptions are appropriate and provide an accurate estimation
of the maximum achievable efficiency, however, for betalains, the proposed oxidation reaction is
a two-electron process.6,7
By doubling the photon to electron ratio, the fundamental maximum
efficiency increases to approximately 41.9%.4
1.4 Betalains
1.4.1 Properties and Sources
Betalains consist of two subclasses of pigments: yellow betaxanthins and magenta
betacyanins, which are derived from the parent molecule betalamic acid. Both classes are forms
of condensation products of amino compounds with betalamic acid. Betacyanins are specifically
the condensation products involving cyclo-dopa derivatives, see Figure 1.3. The purple
betacyanins show the greatest overlap with the solar spectrum compared to the betaxanthins.
Thus, the betacyanins show the most promise in DSSCs. Furthermore; the betacyanins show a
wide range of acylated derivatives, which have varying stability, redox chemistry, and
photophysical properties.19-39

11
Betalains have been studied extensively in the food sciences for the purposes of food
coloring. Many sources of betalains have been identified. The primary sources include: red beets
(Beta vulgaris L.), cactus pears (Optuntia sp.) purple pitahaya (Hylocereus sp.), amaranth
(Amaranthus sp.), and bougainvillea (Bougainvillea sp.).26
Each source has a unique palette of
betalains with varying ratios of betaxanthins to betacyanins. Of particular interest to this research
are the betalains from red beets, amaranth, cactus pears and pitahaya. Figure 1.5 shows the
betacyanin pigments from these sources which are derivatives of betanin (I) and amaranthin
(IV). All of these Betacyanins show different stability characteristics and could be used in lieu of
betanin in DSSCs. Hylocerenin (III), for example has shown increased thermal stability when
the carboxylic acid moiety at the C2 is removed.15
Of the Betaxanthins present in these sources
only indicaxanthin (VIII), portulacaxanthin II and III (IX and X respectively), and vulgaxanthin
I (XI) are in sufficient quantity to be extracted efficiently, see Figure 1.6.26,30,31,33,35,37
Figure 1.4: Reaction pathways for betacyanin (BcN) and betaxanthin (BxN) formation
from betalamic acid (BtA).

12
Figure 1.5: Betacyanin structures from the natural sources studied.

13
1.4.2 Stability:
Many factors have been identified to affect betalain stability in solution, and degradation
pathways have been proposed. However, observation of these degradation pathways has not been
thoroughly studied and the influence of substitutions on degradation is still not understood.
Certain aspects of betacyanin structural degradation are ambiguous in the literature. For example,
the proposed structure of neobetanin varies within the literature between a zwitterionic structure
with a positive charge on the nitrogen (XIVa), and a structure with the double bond shifted to
form a pyridine ring at the base of the molecule (XIVb), see Figure 1.7.40
The structure of this
degradation product is of interest to our lab because the neobetanin structure XIVb may have
Figure 1.6: Structures of betaxanthins that are relevant to the sources of betalains in this
research.

14
more favorable properties for electron injection into TiO2. Further, it is interesting to note that
none of the literature has been able arrive at a consensus on the absorption maximum of
neobetanin. Some sources cite 450-460 nm and other 470-490. The solution conditions of these
measurements could cause the neobetanin to preferentially exist in one form or the other.
Another interesting aspect of neobetanin behavior is that the addition of a double bond, which
would normally cause the absorption maximum to shift to the red, causes the absorption to shift
significantly blue. We believe that this blue shift is based on a change to the charge transfer
characteristics of the ground to excited state transition and may provide insight into the
underlying photophysics of betalains.
Figure 1.7: Two possible structures proposed for neobetanin: traditional structure
shown in most literature (left XIVa) and structure suggested recently by Wybraniec et
al. (right XIVb).

15
1.4.3 Photophysical Properties:
Recently, a study by Wybraniec et al showed interesting photophysical properties of
indicaxanthin. 41
This study indicated that the fluorescence quantum yield depends on solvent
viscosity. They showed that as the viscosity of the solution increased, so did the quantum yield.
It was proposed that this increase is due to restriction of the internal rotation of the molecule.
This fluorescence increase is consistent with preliminary work performed in our lab on
amaranthin. In combination, these results indicate the potential for tunable photophysical
properties in betalains.42
Restriction of internal rotation can also be achieved by aggregation, and
comparing the fluorescence properties of aggregated dye and monomeric dye should yield
similar results, however, this trend was not observed upon dye aggregation as will be discussed
in Chapter 3.
1.4.4 Comparisons with Anthocyanins
Anthocyanins are the familiar plant pigments that are found in all plants not in the order
Caryophyllales. These pigments are found in everything from berries to roses and their light
absorption spans almost the entire visible spectrum. While anthocyanins are a different class of
pigments, many of their photophysical properties are determined by their copigments, or acyl
groups, which are the same groups found in betalains. Figure 1.8 shows the basic unit of all
anthocyanins, anthocyaninidin, and through functionalization at any of the R positions, unique
pigments are formed. A significant body of research exists on the nature of these pigments, and
parallels can be drawn between the two dye classes since they share a common function and a set
of common building blocks.

16
What is perhaps most interesting to this study is the evidence for highly ordered
aggregates of anthocyanins in nature. Many plants utilize the interactions of covalently bound
acyl and sugar groups to generate unique aggregates of pigment that can drastically alter the
photophysical properties. In fact, it is only through aggregation that plants can produce blue
colors. 43
Many anthocyanins favor intermolecular interactions so strongly that they can only be
observed in dimeric states, even in solution, unless conditions are specifically constructed to de-
aggregate the dyes. As mentioned above these copigments are the same for betalains and
anthocyanins. A few examples of the acyl groups which are found throughout both classes of
natural pigments are shown in Figure 1.9. Figure 1.10 shows one particularly complex aggregate
formation of anthocyanins that even exhibits chiral character. 44,45
Figure 1.8: The basic structure of anthocyanins is based on anthocyanidin. In
anthocyanidin all of the R groups are hydrogens.

17
Figure 1.9: Structures of common copigments found throughout the betalain and
anthocyanin species. The binding site to the base dye structure is denoted by “R”.
Figure 1.10: Aggregates of anthocyanins consisting of units of succinylcyanin and
malonylflavone shown schematically and in wire-frame form (B). Reprinted with
permission from Ellestad, G. A. Structure and Chiroptical Properties of Supramolecular
Flower Pigments. Chirality, 2006, 18, 134-144. DOI: 10.1002/chir.20228

18
1.5 References
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Chem. Bio. 2014, 10, 492-501.
2 Fassioli, F.; Dinshaw, R.; Arpin, P. C.; Scholes, G. D. Photosynthetic Light-Harvesting:
Excitons and Coherence. J. Royal Soc. Interface 2014, 11, 20130901/1-22.
3 Treat, N. A.; Knorr, F. J.; McHale, J. L., Templated Assembly of Betanin Chromophore on
TiO2: Aggregation-Enhanced Light-Harvesting and Efficient Electron Injection in a Natural
Dye-Sensitized Solar Cell. J. Phys. Chem. C 2016, 120 (17), 9122-9131.
4 Hanna, M. C.; Nozik, A. J. Solar Conversion Efficiency of Photovoltaic and Photoelectrolysis
Cells with Carrier Multiplication Absorbers. J. Appl. Phys. 2006, 100, 074510–074510.
5 Hagfeldt, A.; Boschloo, G.; Sun, L.; Kloo, L.; Pettersson, H. Dye-Sensitized Solar
Cells. Chem. Rev. 2010, 110, 6595–6663.
6 Knorr, F. J.; McHale, J. L.; Clark, A. E.; Marchioro, A.; Moser, J.-E. Dynamics of Interfacial
Electron Transfer from Betanin to Nanocrystalline TiO2: The Pursuit of Two-Electron
Injection. J. Phys. Chem. C 2015, 119, 19030-19041.
7 Knorr, F. J.; Malamen, D. J.;McHale, J. L.; Marchioro, A.; Moser, J.-E. Two-Electron Photo-
Oxidation of Betanin on Titanium Dioxide and Potential for Improved Dye-Sensitized Solar
Energy Conversion. Proc. SPIE 9165, Physical Chemistry of Interfaces and Nanomaterials
XIII 2104, 9165-0N.
8 Shockley, W.; Queisser, H. J. Detailed Balance Limit of Efficiency of p-n Junction Solar
Cells. J. Appl. Phys. 1961, 32, 510–510.
9 Sandquist, C.; Mchale, J. L. Improved Efficiency of Betanin-Based Dye-Sensitized Solar
Cells. Journal of Photochemistry and Photobiology A: Chemistry 2011, 221, 90–97.
10 Cai, Y.; Sun, M.; Corke, H. Colorant Properties and Stability of Amaranthus Betacyanin
Pigments. J. Agric. Food Chem. 1998, 46, 4491–4495.
11 Elbe, J. V.; Attoe, E. Oxygen Involvement in Betanine Degradation—Measurement of
Active Oxygen Species and Oxidation Reduction Potentials. Food Chemistry 1984, 16, 49–
67.
12 Reynoso, R.; Garcia, F. A.; Morales, D.; Mejia, E. G. D. Stability of Betalain Pigments from
a Cactacea Fruit. J. Agric. Food Chem. 1997, 121, 2884–2889.

19
13 Schliemann, W.; Strack, D. Intramolecular Stabilization of Acylated Betacyanins.
Phytochemistry 1998, 49, 585–588.
14 Herbach, K. M.; Stintzing, F. C.; Carle, R. Betalain Stability and Degradation: Structural and
Chromatic Aspects. J Food Science 2006, 71, R41-R50.
15 Herbach, K. M.; Stintzing, F. C.; Carle, R. Identification of Heat-Induced Degradation
Products from Purified Betanin, Phyllocactin and Hylocerenin by High-Performance Liquid
Chromatography/Electrospray Ionization Mass Spectrometry. Rapid Commun. Mass
Spectrom. 2006, 19, 2603-2616.
16 Cai, M.; Pan, X.; Lui, W.; Sheng, J.; Fang, X.; Zhang, C.; Huo, Z.; Tian, H.; Xiao, S.; Dai, S.
Multiple Absorption of Tributyl Phosphate Molecule at the Dyed-TiO2/Electrolyte Interface
to Suppress the Charge Recombination in Dye-Sensitized Solar Cell. J. Mater. Chem. A
2013, 1, 4885-4892.
17 Narayan, M. R. Review: Dye Sensitized Solar Cells Based on Natural
Photosensitizers. Renewable and Sustainable Energy Reviews 2012, 16, 208-215.
18 O’Regan, B.; Grätzel, M. A Low-Cost, High-Efficiency Solar Cell Based on Dye-Sensitized
Colloidal Titanium Dioxide Films. Nature 1991, 353, 737-40.
19 Bartoloni, F. H.; Gonçalves, L. C. P.; Rodrigues, A. C. B.; Dörr, F. A.; Pinto, E.; Bastos, E.
L. Photophysics and Hydrolytic Stability of Betalains in Aqueous Trifluoroethanol. Monatsh
Chem 2013, 144, 567–571.
Active Oxygen Species and Oxidation Reduction Potentials. Food Chemistry 1984, 49–67
23 Wybraniec, S.; Starzak, K.; Skopińska, A.; Szaleniec, M.; Słupski, J.; Mitka, K.; Kowalski,
P.; Michałowski, T. Effects of Metal Cations on Betanin Stability in Aqueous-Organic
Solutions. Food Sci Biotechnol 2013, 353–363
24 Moßhammer, M. R.; Rohe, M.; Stintzing, F. C.; Carle, R. Stability of Yellow-Orange Cactus
Pear (Opuntia ficus-indica [L.] Mill. cv. ‘Gialla’) Betalains as Affected by the Juice Matrix
and Selected Food Additives. Eur. Food Res. Technol. 2006, 225, 21–32.

20
25 Herbach, K. M.; Maier, C.; Stintzing, F. C.; Carle, R. Effects of Processing and Storage on
Juice Colour and Betacyanin Stability of Purple Pitaya (Hylocereus polyrhizus) Juice. Eur
Food Res Technol 2006, 224, 649–658.
26 Mabry, T.; Taylor, A.; Turner, B. The Betacyanins and Their Distribution.
27 Kanner, J.; Harel, S.; Granit, R. Betalains: A New Class of Dietary Cationized
Antioxidants. J. Agric. Food Chem. 2001, 49, 5178–5185.
28 Kugler, F.; Stintzing, F. C.; Carle, R. Identification of Betalains from Petioles of Differently
Colored Swiss Chard (Beta vulgaris L. ssp. cicla [L.] Alef. Cv. Bright Lights) by High-
Performance Liquid Chromatography−Electrospray Ionization Mass Spectrometry J. Agric.
Food Chem. 2004, 52, 2975–2981.
29 Wybraniec, S.; Mizrahi, Y. Generation of Decarboxylated and Dehydrogenated Betacyanins
in Thermally Treated Purified Fruit Extract from Purple Pitaya (Hylocereus polyrhizus)
Monitored by LC-MS/MS. J. Agric. Food Chem. 2005, 53, 6704–6712.
30 Stintzing, F. C.; Schieber, A.; Carle, R. Identification of Betalains from Yellow Beet (Beta
vulgaris L.) and Cactus Pear [ Opuntia ficus-indica (L.) Mill.] by High-Performance Liquid
Chromatography−Electrospray Ionization Mass Spectrometry. J. Agric. Food Chem. 2002,
50, 2302–2307.
31 Stintzing, F. C.; Schieber, A.; Carle, R. Betacyanins in Fruits from Red-Purple Pitaya,
Hylocereus polyrhizus (Weber) Britton & Rose. Food Chemistry 2002, 77, 101–106.
32 Wybraniec, S.; Mizrahi, Y. Fruit Flesh Betacyanin Pigments in Hylocereus Cacti. J. Agric.
Food Chem. 2002, 50, 6086–6089.
33 Cai, Y.; Sun, M.; Corke, H. HPLC Characterization of Betalains from Plants in the
Amaranthaceae. Journal of Chromatographic Science 2005, 43, 454–460.
34 Cai, Y.-Z.; Sun, M.; Corke, H. Characterization and Application of Betalain Pigments from
Plants of the Amaranthaceae. Trends in Food Science & Technology 2005, 16, 370–376.
35 Sun, M.; Corke, H. Identification and Distribution of Simple and Acylated Betacyanins in the
Amaranthaceae. J. Agric. Food Chem. 2001, 49, 1971–1978.
36 Heuer, S.; Wray, V.; Metzger, J. W.; Strack, D. Betacyanins from flowers of Gomphrena
globosa. Phytochemistry 1992, 31, 1801–1807.

21
37 Kugler, F.; Stintzing, F. C.; Carle, R. Characterisation of Betalain Patterns of Differently
Coloured Inflorescences from Gomphrena Globosa L. and Bougainvillea sp. by HPLC–
DAD–ESI–MS. Anal Bioanal Chem 2006, 387, 637–648.
38 Stintzing, F. C.; Carle, R. Functional Properties of Anthocyanins and Betalains in Plants,
Food, and in Human Nutrition. Trends in Food Science & Technology 2004, 15, 19–38.
39 Gandía-Herrero, F.; Escribano, J.; García-Carmona, F. Structural Implications on Color,
Fluorescence, and Antiradical Activity in Betalains. Planta 2010, 232, 449–460.
40 Wybraniec, S.; Starzak, K.; Skopińska, A.; Nemzer, B.; Pietrzkowski, Z.; Michałowski, T.
Studies on Nonenzymatic Oxidation Mechanisms in Neobetanin, Betanin, and
Decarboxylated Betanins. J. Agric. Food Chem. 2013, 61, 6465–6476.
41 Wendel, M.; Szot, D.; Starzak, K.; Tuwalska, D.; Prukala, D.; Pedzinski, T.; Sikorski, M.;
Wybraniec, S.; Burdzinski, G. Photophysical Properties of Indicaxanthin in Aqueous and
Alcoholic Solutions. Dyes and Pigments 2015, 113, 634–639.
42 Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 3rd
ed.; Plenum Press: New York
2006.
43 Socaciu, C.; Food Colorants: Chemical and Functional Properties; CRC Press LLC Taylor
and Francis 2007.
44 Ellestad, G. A. Structure and Chiroptical Properties of Supramolecular Flower Pigments.
Chirality 2006, 18, 134-144.
45 Fernandes, A.; Bras, N. F.; Mateus, N.; de Freitas, V. A Study of Anthocyanin Self-
Association by NMR Spectra. New J. Chem. 2015, 39, 2602-2611.

22
2 Preparation of Betalain Pigments
2.1 Introduction: Sources and Previous Techniques
Betalains are natural photoprotective pigments found in plants in the order
Caryophyllales. The pigments are all based on Schiff’s base condensations with betalamic acid
and can be separated into two subclasses: betacyanins and betaxanthins as shown in Figure 1.4.
As discussed previously, there are many different structures of betalains based on the amine
source for the condensation, but the primary distinction is that betacyanins are condensation
products of betalamic acid and a cyclo-dopa derivative. All betacyanins are derivatives of
betanidin, which is referred to as the aglycone. Other betacyanins have glucose or other sugar
moieties at either the C5 or C6 position as well as an array of additional acyl groups attached to
these. Figure 1.5 shows a few the betacyanin pigments. Betaxanthins are a much more diverse set
of pigments as they are any non-cyclo-dopa amine derived condensation product with betalamic
acid. The range of betaxanthins found in natural sources comprises the entire spectrum of amino
acids and similar structures. Only a few betaxanthins are expressed in significant quantities in the
sources used in this research and their structures are shown in Figure 1.6. Despite their
occurrence in nature, betaxanthins have shown significant instability and have proven difficult to
isolate. Therefore, betacyanins will be the primary focus of this research.1-23
The stability of the betacyanins is dependent on many factors and is not very well
understood. While food science research has identified factors that influence the stability of these
pigments, their primary focus was on matrix elements.14
Some of this research, however,

23
indicates that the degree and nature of acylation can affect stability.8,13
A study on the stability of
amaranthin derivatives in acidic solution showed that certain acyl groups significantly increased
pigment retention over several hours.13
Another study showed that decarboxylation of
hylocerenin, a betacyanin from pitahaya, increased the thermal stability of the compound to
temperatures approaching 100˚C.15
For practical reasons, the stability of extracted pigments is of
great interest for DSSC applications, and so methods for isolation of these compounds were
developed in order to elucidate these structure-function properties.
A significant amount of research has been done in the field of betacyanin extraction and
purification, and many different methods have been developed over the 40+ years of inquiry.15,21
The methods used in this research were developed off of an assortment of recent publications by
Carle et al. (references 15-18) and Wybraniec et al (reference 26). Since betacyanins are quite
hydrophilic and have multiple charge centers (three carboxylic acid groups, two nitrogen
heteroatoms and plenty of hydroxyl groups), development of a separation technique was a
difficult proposal. These two research groups have developed techniques which combine high
performance liquid chromatography and solid phase extraction to produce ultra-pure pigment
samples as well as a more bulk material process involving ion-exchange chromatography to
produce mixtures of betacyanins removed from other matrix components.22
This research uses
these previously developed methods as a starting point for our purification process.

24
2.2 Preparatory Techniques
2.2.1 Extraction
Raw materials were purchased (beets and pitahaya) from local supermarkets, or harvested
from our personal gardens (amaranth) for use in this study. All of the raw materials were
processed in a similar manner and will be discussed simultaneously with exceptions noted. Raw
materials were juiced in a macerating juicer to produce a colored liquid. The pulp by-product
was re-processed by soaking in 60% ethanolic solution then passed through the juicer again. Raw
juice was centrifuged for 20 minutes at 10,000 rpm then filtered through three consecutive filters,
Whatman #4, #1, #42 respectively. The filtered juice was then diluted with ethanol to a total
concentration of 60% ethanol and allowed to sit at -20˚C overnight to denature proteins. The
supernatant was decanted and filtered through a series of glass microfiber filters at 2.7µm
(GF/D), 1.6µm (GF/A), 0.4µm and 0.2µm. Once filtered the juice was adjusted to pH 3.5 for
further purification. For samples intended for HPLC or MPLC purification, ethanol was removed
via rotary evaporation at 30˚C and reduced pressure.
2.2.2 HPLC
Reversed phase high performance liquid chromatography (RP-HPLC) is the standard
separatory method to begin with for method development. In RP-HPLC the stationary phase is
hydrophobic and a moderate degree of hydrophobicity is required for analyte retention. The
analytes are then eluted from the column by using a mixture of aqueous and non-aqueous
solvents as the mobile phase.24
For the purposes of this discussion the mobile phase will be
limited to two components labeled A for aqueous and B for organic. A mixture of the two

25
components is used to tune the retention of the analytes to the desired level. The simplest form of
mixture is called an isocratic elution which maintains a constant ratio of A and B for the duration
of the experiment. Each analyte is eluted in order of increasing hydrophobicity based on its
partitioning between mobile and stationary phase. Isocratic experiments can be useful if the
analytes have similar hydrophobicity, but are not very effective at separating components over a
wide range of character. In this instance a binary gradient is used, whereby the amount of B is
increased over time to cause the more highly retained analytes to elute sooner. By using a binary
gradient samples with components comprising a wide range of hydrophobic character can be
efficiently separated in a short period of time. Solvent selection also plays a crucial role in tuning
the retention characteristics of the analytes. The aqueous phase typically consists of a buffer to
maintain a pH that produces the most ideal (hydrophobic) conditions of the analyte. For example,
analytes that are acidic in nature will be separated using a low-pH buffer whereas those that are
basic will use a high-pH buffer. The organic phase is typically chosen based on the protonation
consideration of the analyte. There are two primary solvents used in most RP-HPLC methods:
methanol (MeOH) and acetonitrile (MeCN). The main distinction between these two solvents is
the presence of a hydrogen atom on MeOH that can be involved in chemistry with the analytes.
If pH is a significant factor in the efficiency of separation, methanol can change the behavior of
organic acids and should be avoided. In this scenario MeCN is ideal because it will have little
effect on the proton activity in the solvent, and minimize detrimental effects due to pH changes.
It is worth noting, however, that cost and scaling-up can promote use of MeOH over MeCN in
certain circumstances, but additional controls to the pH may be necessary. Other organic solvents

26
can be used as long as they are miscible with water; however, MeOH and MeCN are the main
choices for starting method development.
Additional measures may be necessary to generate the proper hydrophobic environment
for analyte retention when the species have hard to neutralize charge centers. In these cases, an
ion-pairing agent can be used. Typically, a perfluorinated organic acid is used as an ion pairing
agent for positively charged species and a tertiary amine for negatively charged species. These
ion-pairing agents are added to the mobile phase and associate strongly with the analytes to
produce a greater hydrophobic character, and increase retention on the stationary phase. In some
cases the addition of ion-pairing agents can significantly enhance the separation of similar
species. In addition to ion-pairing agents, chiral-pairing agents can be used to enhance
separations of chiral molecules.
For our research with beets, amaranth and pitahaya, RP-HPLC methods were developed
on a C18 stationary phase using a binary gradient with an anionic ion-pairing agent and MeCN.
The first hurdle was to achieve hydrophobicity in the betacyanins. The carboxylic acid groups
were protonated by using an acidic buffer with a pH from 2-3 based on acetic or formic acid. It
was found that formic acid performed ideally by producing a stable buffer at pH 2 whereas the
acetic acid produced a buffer at approximately pH 3. While the initial studies used acetic acid,
for the later studies, I settled on formic acid because of the lower pH and lower boiling point.
Despite the higher purity product that HPLC garners, this method required less technical
development, especially with the large body of literature that has contributed to the methodology
of HPLC to betalain isolation. Some minor adjustments to the mobile phase and gradient were
necessary for use with the specific column we obtained from Phenomenex, which consisted of

27
superficially porous particles compared to the fully porous particles used in most previous
betalain research. The primary components of the mobile phase were 0.1% (V/V) trifluoroacetic
acid (TFA), as the ion-pairing agent, and 1% (V/V) formic acid (FA) in the aqueous phase and
pure acetonitrile (MeCN) as the organic phase. These components were chosen based on the
evidence that perfluorinated acids significantly enhanced the retention characteristics of betalains
in C18 RP-HPLC as discussed previously.
Originally, a simple binary gradient was developed using 10% MeCN and 90% aqueous
(5% acetic acid), which produced good results for betanin from beets, but could not resolve
amaranthin. The amaranthin was not retained at all. For betanin, the retention was sufficient to
isolate the primary pigment, though analysis at 254 nm showed significant residual impurities.
Thus, a better gradient or mobile phase was required. Then I tried a mobile phase consisting of
0.25% TFA as the aqueous component. The switch to TFA did not in itself alleviate the issue,
and the B% had to be reduced to 7%. The high concentration of TFA was a concern given its
high acidity, and so a combined aqueous phase of FA and TFA was developed. The weaker
acidity of the formic acid creates a buffer to keep the mobile phase at pH 2 while the TFA
generates the added retention characteristics discussed above. Being an organic acid, FA should
not produce significant salty deposits on/in the instrument and its high volatility decreases the
amount of residual acid in the final product. Despite the high volatility of TFA, I suspect that the
majority exists as the salt in the eluent and remains in the product despite low pressure rotary
evaporation.

28
2.2.3 MPLC
Previous work in the McHale lab had focused on medium pressure liquid
chromatography (MPLC) as the purification process. The previous researchers used an isocratic
flow scheme with 80% MeOH and 20% purified water on a reversed phase C18 column. This
method provided minimal purification, and needed to be performed multiple times to produce
semi-pure dye. In retrospect, it is clear that the mobile phase was too polar and the betacyanin
was eluted with the solvent front while the less hydrophilic betaxanthins were partially retained.
While the resulting pigment could be purified to be almost betaxanthin free, it did not allow for
separation from sugars and salts that would also elute with the solvent front.
Figure 2.1: Preparatory HPLC of amaranth extract with amaranthin (1) and
isoamaranthin (2) the predominant pigments, but small amount of decarboxylated
betacyanins (3 &4) being present.

29
With the help of the HPLC methods, an appropriate MPLC method could be developed
for the amaranth samples. Similar to the HPLC method, an acidic buffer with TFA was used, and
the organic phase was chosen to be methanol for environmental and cost concerns. It was found
that an isocratic mixture of 7% MeOH and 93% aqueous buffer provided sufficient retention of
the amaranthin to be isolated. The main advantage of the MPLC technique over that of HPLC is
that it allowed for significant amounts of secondary pigments to be purified. In particular, one
secondary betacyanin from amaranthin was isolated using this procedure. While MPLC allowed
for a significant scale increase over HPLC, it was ultimately determined that the most cost
effective mass production scheme was ion-exchange chromatography as discussed below. It is
worth noting, however, that MPLC provides isolated betalain compounds whereas the ion-
exchange method provides mixtures of betacyanins and could be used in future research to
evaluate secondary betacyanin pigments.
2.2.4 Anion-Exchange Column Chromatography
For large scale production, the HPLC system in our lab is insufficient given the small
injection volume and long run time. Therefore, a bulk processing procedure was developed based
on an anion exchange protocol, shown schematically in Figure 2.2. By using Sephadex DEAE-
A25 the betacyanins can be separated from the betaxanthins and proteins. The raw solution is
adjusted to pH 3.5 with 100 mM sodium phosphate buffer. 10-15 g of Sephadex DEAE-A25 is
prepared by forming a slurry in 500 mL of pH 3.5 buffer and allowing to swell for a minimum of
5 hours. After equilibrating with three 500 mL aliquots of pH 3.5 buffer, the raw beet/amaranth
juice is added to the column until the stationary phase has been saturated with pigment (a small
amount of red pigment begins eluting). Following loading, the column is rinsed with a minimum

30
of 5 volumes of pH 3.5 buffer, or until the eluent runs clear with no residual yellow color. To
desorb the pigment, the column is washed with pH 7.5 100 mM sodium phosphate buffer.
Following exchange chromatography, the product is desalted with solid phase extraction on a
C18 cartridge. The cartridge is prepped with five volumes of methanol, and then equilibrated
with 3 volumes of TFA/FA buffer. The pigment solution is brought to a pH of 2 before being
applied to the cartridge. Once the cartridge is loaded it is rinsed with 10 volumes of TFA/FA
buffer or until eluent runs clear. Betanin/amaranthin is eluted with pure methanol. The resultant
solution is dried under reduced pressure at 30˚C.
As validation of this method to produce purified mixtures of betacyanins, HPLC analysis
was conducted on the purified powder and compared with the micro filtered juice. By monitoring
the chromatogram at 254 nm we were able to see all components that would be contaminates of
Figure 2.2: Schematic of the preparatory process developed in this research.
Juice beets
Denature proteins
Microfilter
Acidify: pH 3.5
Anion Exchange: DEAE-A25
pH 3.5 PO4
Load Elute pH 7.5
<pH 2
SPE
MeOH 0.1% TFA/1%FA
Load Elute MeOH
Dry @ 30˚C
Filter
#4 #1 #42/5
GF/D GF/A 0.44µm 0.22µm

31
our sample. Figure 2.3 shows two chromatograms that evidence the efficacy of our anion
exchange protocol. The purified amaranth consists of only one species and no residual absorbing
contaminate. The filtered juice, on the other hand, has so many components that it is difficult to
tell which peak corresponds to the amaranthin. The retention times on these two chromatograms
do not exactly match because they were run with different protocols as discussed above. Similar
quality analysis procedures were run on the samples from beets and pitahaya and the purified
sample chromatograms are show in Figure 2.4 below.
2.3 Conclusions
The preparation of isolated betacyanin compounds has led to significant increases in the
control of experimental variables in exploring the photophysical properties of betalains. The
three primary preparatory techniques discussed here allow for gram-scale quantities of betanin,
amaranthin and other betacyanins to be produced from their raw material sources. In their
Figure 2.3: HPLC chromatogram of purified amaranth (a) and that of filtered amaranth
juice (b) monitored at 254 nm.

32
powdered form, these pigments are stable indefinitely, and can be used in many different studies.
It is likely that the impure nature of the pigments used in previous research has led to
irreproducibility in the data. Careful control of sample purity is essential for laying the ground
work for mapping the structure-function relationships in betalain pigments and constitutes a great
achievement toward understanding their fundamental properties.
Although we were unable to use mass spectrometry to positively identify the individual
betacyanins present in our sources, we can compare the relative retention rates of the betacyanins
using HPLC. Figure 2.4 shows HPLC chromatograms of the betacyanin mixtures obtained from
the anion exchange preparation of amaranth, beets and pitahaya. By comparing with literature,
the primary components could be identified as amaranthin, betanin, phylocactin and hylocerinin.
Figure 2.4: HPLC chromatogram of purified samples of amaranth (black), pitahaya (red),
beets (green) and a combination of all the sources (blue). The primary betacyanins are
marked as amaranthin (A), betanin (B), phylocactin (C) and hylocerinin (D).

33
The betacyanins from amaranth and beets were used in further studies as discussed below.
Unfortunately, time constraints did not allow for further study of the pigments from pitahaya.

34
2.4 References
1 Wybraniec, S.; Mizrahi, Y. Fruit Flesh Betacyanin Pigments in Hylocereus Cacti. J. Agric.
Food Chem. 2002, 50, 6086–6089.
2 Cai, Y.; Sun, M.; Corke, H. HPLC Characterization of Betalains from Plants in the
Amaranthaceae. Journal of Chromatographic Science 2005, 43, 454–460.
3 Cai, Y.-Z.; Sun, M.; Corke, H. Characterization and Application of Betalain Pigments from
Plants of the Amaranthaceae. Trends in Food Science & Technology 2005, 16, 370–376.
4 Sun, M.; Corke, H. Identification and Distribution of Simple and Acylated Betacyanins in the
Amaranthaceae. J. Agric. Food Chem. 2001, 49, 1971–1978.
5 Heuer, S.; Wray, V.; Metzger, J. W.; Strack, D. Betacyanins from Flowers of Gomphrena
globosa. Phytochemistry 1992, 31, 1801–1807.
6 Kugler, F.; Stintzing, F. C.; Carle, R. Characterisation of Betalain Patterns of Differently
Coloured Inflorescences from Gomphrena globosa L. and Bougainvillea sp. by HPLC–
DAD–ESI–MS n. Anal Bioanal Chem 2006, 387, 637–648.
7 Stintzing, F. C.; Carle, R. Functional Properties of Anthocyanins and Betalains in Plants,
Food, and in Human Nutrition. Trends in Food Science & Technology 2004, 15, 19–38.
8 Gandía-Herrero, F.; Escribano, J.; García-Carmona, F. Structural Implications on Color,
Fluorescence, and Antiradical Activity in Betalains. Planta 2010, 232, 449–460.
9 Bartoloni, F. H.; Gonçalves, L. C. P.; Rodrigues, A. C. B.; Dörr, F. A.; Pinto, E.; Bastos, E. L.
Photophysics and Hydrolytic Stability of Betalains in Aqueous Trifluoroethanol. Monatsh
Chem 2013, 113, 567–571
Active Oxygen Species and Oxidation Reduction Potentials. Food Chemistry, 1984, 16, 49–
67.

35
13 Schliemann, W.; Strack, D. Intramolecular Stabilization of Acylated
Betacyanins. Phytochemistry 1998, 49, 585–588.
14 Wybraniec, S.; Starzak, K.; Skopińska, A.; Szaleniec, M.; Słupski, J.; Mitka, K.; Kowalski,
P.; Michałowski, T. Effects of Metal Cations on Betanin Stability in Aqueous-Organic
Solutions. Food Sci Biotechnol 2013, 22, 353–363.
15 Herbach, K. M.; Stintzing, F. C.; Carle, R. Identification of Heat-Induced Degradation
Products from Purified Betanin, Phyllocactin and Hylocerenin by High-Performance Liquid
Chromatography/Electrospray Ionization Mass Spectrometry. Rapid Commun. Mass
Spectrom. 2006, 19, 1822–1822.
16 Herbach, K. M.; Stintzing, F. C.; Carle, R. Betalain Stability and Degradation˗˗Structural and
Chromatic Aspects. J Food Science, 2006, 71, R41-R50.
17 Moßhammer, M. R.; Rohe, M.; Stintzing, F. C.; Carle, R. Stability of Yellow-Orange Cactus
Pear (Opuntia ficus-indica [L.] Mill. cv. ‘Gialla’) Betalains as Affected by the Juice Matrix
and Selected Food Additives. Eur. Food Res. Technol. 2006, 225, 21–32.
18 Herbach, K. M.; Maier, C.; Stintzing, F. C.; Carle, R. Effects of Processing and Storage on
Juice Colour and Betacyanin Stability of Purple Pitaya (Hylocereus polyrhizus) Juice. Eur.
Food Res. Technol. 2006, 224, 649–658.
19 Mabry, T.; Taylor, A.; Turner, B. The Betacyanins and Their Distribution.
20 Kanner, J.; Harel, S.; Granit, R. Betalains: A New Class of Dietary Cationized
Antioxidants. J. Agric. Food Chem. 2001, 49, 5178–5185.
21 Kugler, F.; Stintzing, F. C.; Carle, R. Identification of Betalains from Petioles of Differently
Colored Swiss Chard (Beta vulgaris L. ssp. cicla [L.] Alef. Cv. Bright Lights) by High-
Performance Liquid Chromatography−Electrospray Ionization Mass Spectrometry J. Agric.
Food Chem. 2004, 52, 2975–2981.
22 Wybraniec, S.; Mizrahi, Y. Generation of Decarboxylated and Dehydrogenated Betacyanins
in Thermally Treated Purified Fruit Extract from Purple Pitaya (Hylocereus polyrhizus)
Monitored by LC-MS/MS. J. Agric. Food Chem. 2005, 53, 6704–6712.
23 Stintzing, F. C.; Schieber, A.; Carle, R. Identification of Betalains from Yellow Beet (Beta
vulgaris L.) and Cactus Pear [Opuntia ficus-indica (L.) Mill.] by High-Performance Liquid

36
Chromatography−Electrospray Ionization Mass Spectrometry. J. Agric. Food Chem. 2002,
50, 2302–2307.
24 Wybraniec, S.; Mizrahi, Y. Influence of Perfluorinated Carboxylic Acids on Ion-Pair
Reversed-Phase High-Performance Liquid Chromatographic Separation of Betacyanins and
17-Decarboxy-Betacyanins. Journal of Chromatography A 2004, 1029, 97–101.

37
3 Templated Assembly of Betanin Chromophore on TiO2: Aggregation-
Enhanced Light Harvesting and Efficient Electron Injection in a
Natural Dye-Sensitized Solar Cell
Reprinted and adapted with permission from Treat, N. A.; Knorr, F. J.; McHale, J. L.,
Templated Assembly of Betanin Chromophore on TiO2: Aggregation-Enhanced Light-
Harvesting and Efficient Electron Injection in a Natural Dye-Sensitized Solar Cell. J. Phys.
Chem. C, 2016, 120 (17), 9122-9131. DOI:10.1021/acs.jpcc.6b02532
3.1 Introduction
Ongoing efforts to optimize solar energy conversion are frequently inspired by Nature,
which uses finely tuned assemblies of chromophores to harness sunlight.1,2
In photosynthetic
organisms, light-harvesting complexes of chlorophyll and bacteriochlorophyll derivatives are
optimized to collect and funnel solar energy to reaction centers where charge separation takes
place. TiO2-based dye-sensitized solar cells (DSSCs), on the other hand, depend on adsorbed
dyes which perform both the light-harvesting and interfacial charge-separation steps.3,4,5
While
self-assembly of dyes on the metal oxide surface is prevalent owing to their high surface
density,6,7
aggregation of dyes on TiO2 is frequently reported to lower the rate and yield of
electron injection, resulting in lower photocurrents.8,9,10,11,12
Consequently, high-performing
DSSCs often employ spacer molecules,13,14,15,16
competitively binding molecules such as
organophosphates,17
functionalized dyes with steric hindrance,18,19,20
or surface treatments21
to
prevent dye aggregation. The reduced photocurrents that result from sensitizer aggregation are

38
variously attributed to decreased excited state lifetime, self-quenching, attenuation of light by a
thicker dye layer, and weak electronic coupling of dyes to TiO2 as a result of greater distance
from the surface.
On the other hand, dye aggregation can be beneficial to solar energy conversion in that it
is often accompanied by spectral broadening which can improve the overlap of the device optical
absorption with the solar spectrum. Structured biomimetic chromophore assemblies are being
pursued with the goal of exploiting energy transfer in solar energy conversion,22,23,24,25,26
and
there is additional incentive to replace costly synthetic sensitizers with natural pigments.27,28,29
Unfortunately, none of these biomimetic approaches have resulted in energy conversion
efficiencies that rival those of DSSCs using monomeric organic or metallorganic sensitizers.
Figure 3.1: Structure of betanin (left) and 3D representation of optimized geometry
(right) obtained using DFT as described in Ref. 42.

39
Better understanding of the influence of aggregation on TiO2-based solar energy conversion
should permit the enhanced light harvesting of dye assemblies to be exploited without
diminished yield of photoelectrical conversion.
Recently, we have used betanin (Bt) extracted from beet root as a sensitizer and achieved
power conversion efficiencies up to 2.7% and high incident photon-to-current conversion
efficiency (IPCE).30
Bt (Figure 3.1) is a light-harvesting plant pigment belonging to the betalain
family, the pigments of which serve photoprotective and anti-oxidant roles in plants of the order
Caryophyllales.31,32
Betalain pigments can be separated into two subclasses of molecules based
on Schiff’s base condensation with betalamic acid and a nitrogen hetero atom. The simpler
subclass of betalains is the betaxanthins which are condensation products with various amino
acids and amines. Betacyanins, including betanin, are more specifically the condensation
products of cyclo-dopa derivatives with betalamic acid. While betalamic acid and the
betaxanthins exhibit a strong absorption in the blue, with molar absorptivity ε = 45,000 M-1
cm-1
at 470-500 nm, betacyanins absorb in the green, ε = 65,000 M-1
cm-1
at 525-535 nm.33,34
Like
their cousins the anthocyanin plant pigments, betalains have been explored as natural sensitizers
in DSSCs by our group30,35,36
and others.37,38,39
Though anthocyanins are known to self-assemble
in solution and in vivo,40, 41
we are not aware of any studies pointing to aggregation of betanin or
other betalains in vivo or in vitro. However, we have noted previously that the absorption
spectrum of Bt on TiO2 is considerably broader than that of aqueous Bt, and have speculated that
dye aggregation may be responsible for enhanced light-harvesting.36
Though previously we have
considered that preferential absorption of betaxanthins on TiO2 might have contributed to the
blue shift, in the present work HPLC analysis confirms the absence of betaxanthins or other

40
yellow pigments. As shown in Figure 3.1, the optimized ground state geometry of Bt as obtained
by a DFT calculation is decidedly nonplanar, which would inhibit intermolecular interactions via
π-stacking. However, the presence of three carboxylic acid functions and two nitrogen
heteroatoms on Bt provides ample opportunity for intermolecular interactions though hydrogen-
bond formation, perhaps assisted by templated adsorption on the TiO2 surface.
We have reported femtosecond and nanosecond transient absorption spectroscopy of Bt-
sensitized TiO2 along with spectroelectrochemical measurements and DFT calculations,
revealing that excited state Bt undergoes a two-electron, one-proton oxidation.42,43
In the course
of investigating the electrochemical oxidation of Bt on TiO2 as a function of dye-loading, we
observed spectral changes suggestive of aggregation. In this work, we use internal and external
quantum efficiencies for electron collection, fluorescence measurements, and numerical
modeling to understand the evolution of quantum efficiency and light-harvesting as a function of
dye-loading. We report here a record high power conversion efficiency, 3%, for a natural-dye
based DSSC, sensitized with aggregated Bt. Evidence from numerical modeling and theory will
be shown to lead to the conclusion that an excitonically coupled dimer of Bt forms on the surface
of TiO2, leading to enhanced light harvesting, electron injection, and energy conversion
compared to analogous DSSCs containing monomeric Bt.
3.2 Experimental Methods
3.2.1 Purification
Betanin was extracted from red beet (Beta vulgaris L.) via a modified procedure from
Ref. 44. Red beet root was crushed and juiced by a macerating juicer. The juice was mixed 1:1

41
with ethanol, and filtered to remove proteins. This mixture was adjusted to pH 4 with HCl.
Betanin was isolated, on a Sephadex DEAE A25 column at pH 4, and washed with a 0.05 M pH
4 acetate buffer. Betanin was desorbed from the column with a 0.05 M pH 7 phosphate buffer.
All excess salts in the eluent were removed and the product further purified by solid phase
extraction on a Phenomenex Strata C18 SPE column and washed with five volumes of water
acidified with HCl to a pH of 2. The betanin was desorbed with methanol acidified with a small
amount of HCl and dried under reduced pressure at 30 ºC. Purity was confirmed by analytical
HPLC.44
3.2.2 HPLC
Analytical HPLC was performed via a modified method using a binary gradient with
solution A (0.25% aqueous trifluoroacetic acid) and solution B (acetonitrile). The gradient is as
follows: 0-1min 7.2% B, then ramped to 10% B at 5 min, then to 15% B at 7 min, followed by a
rinse of 100% B at 8 min for 1 min, then equilibrated for 2 min at 7.2% B.45,46,47
3.2.3 Film preparation and sensitization (TiO2 and ZrO2)
Films of TiO2 were prepared on a conductive substrate, fluoride doped tin oxide (FTO),
for DSSC construction, while films of TiO2 and ZrO2 for absorption and emission spectroscopy
were prepared on glass microscope slides. For DSSC construction, a blocking layer of titanium
dioxide was prepared by heating FTO coated slides (Tech15, Hartford Glass) in a solution of
dilute 0.1 M TiCl4 to 60°C for 30min. The coated films were dried in an oven at 100°C for
30min before being heated in a muffle furnace to 500°C for 30 min. Films of titanium dioxide
were prepared by the doctor blade method with Dyesol 18NR-T paste on FTO glass slides. The
film consists of 20 nm nanoparticles of TiO2 in the anatase phase. Typical SEM images of the

42
resulting films are shown in Figure 3.7, along with side views which show the film thicknesses.
3.5 μm thick films were prepared with a 50% dilution of the TiO2 paste with ethanol, 7μm films
were prepared by using undiluted paste, 14 μm films were prepared by 2 successive coatings of
paste. The films were placed in a drying oven at 100°C for 1hour to smooth then heated in a
muffle furnace to 500°C for 30 min. Some of the films were selected for an additional treatment
with 0.1 M TiCl4 after cooling, as in Ref 30. Prior to sensitization films were equilibrated either
in purified water or in 0.5 M hydrochloric acid in ethanol.
For absorption and fluorescence measurements, films of TiO2 and ZrO2 were prepared by
doctor-blading directly onto glass slides. TiO2 films for spectroscopy were 3.5 μm, leading to
sufficient transmission for accurate absorption measurement. Owing to lower tendency of Bt to
adsorb on ZrO2 compared to TiO2, 7 μm thick ZrO2 films were used in order to obtain sufficient
dye loading for spectroscopy measurements. Zirconium dioxide films were prepared from Zr-
Nanoxide Z/SP paste purchased from Solaronix. Prior to sensitization, films for fluorescence
measurements were equilibrated in a solution of 0.25% aqueous trifluoroacetic acid. Since the
fluorescence measurements (described below) were done using front face emission and corrected
for absorptance, the film thickness is not critical to the comparison of fluorescence of Bt on TiO2
and ZrO2. Bt-sensitized ZrO2 films were used only for absorption and emission, not for
fabrication of dye-sensitized solar cells.
Films were sensitized in solutions with varying amounts of betanin powder dissolved in
25 mL of purified water and the pH was adjusted by dropwise addition of aqueous 1 M
hydrochloric acid. Films were sensitized for varying times to produce a range of aggregation. To
produce films with very low amounts of aggregation, 1 mM sodium phosphate was used in place

43
of purified water to prepare the betanin sensitizing solution. Phosphate ions are known to bind
strongly to TiO2 in contact with aqueous solutions,48
thus we expected competitive binding
would limit self-assembly in the presence of sodium phosphate.
3.2.4 Absorption and fluorescence spectra
Fluorescence spectra of Bt-sensitized TiO2 and ZrO2 films were excited using a Lexel
95L-UV laser operating at 514.5 nm, using a 45° front facing geometry. The incident laser power
was attenuated to 20 ± 2μW using a neutral density filter. Elastically scattered laser radiation was
rejected with a holographic notch filter. Emission from 400-900 nm was detected with a
thermoelectrically cooled CCD camera. All spectra were taken within 10 min of removal of films
from the sensitizing solution. Films were otherwise kept in the dark to minimize photo-
degradation of betanin.
Absorption spectra were taken on a Shimadzu UV-2550 spectrometer before and after
fluorescence measurements to check for dye degradation. Fluorescence spectra were scaled by
the absorptance (1−10−A
, where A is the absorbance of the film at the excitation wavelength) to
correct for the variations in the percent of light absorbed for different samples.
3.2.5 IPCE and APCE
Incident photon-to-current conversion efficiency was recorded as a function of
wavelength, IPCE(λ), as described in a previous publication.30
Adsorbed photon-to-current
conversion efficiency APCE(λ) was obtained from IPCE(λ) by dividing by the absorptance.
IPCE(λ)=
Jsc(𝜆)
eΦ(𝜆)
Equation 3.1
APCE(λ)=
IPCE(λ)
1−10−A(λ) Equation 3.2

44
η=
JscVocFF
Pin
=
Pmax
Pin
Equation 3.3
In the above, Jsc is the short-circuit current, Φ(λ) is the incident photon flux at wavelength λ, A(λ)
is the absorbance at wavelength λ, η is the total energy conversion efficiency, Voc is the open
circuit voltage, FF is the fill factor, and Pin is the incident radiation power.
3.2.6 Solar Cell Construction.
Platinum counter electrodes were prepared by heating an FTO glass slide to 40˚C and
adding 6 drops of platinum hexachloride dissolved in isopropyl alcohol. Once dry, the films were
sintered at 450˚C for 30 min. Solar cells were constructed by sandwiching the sensitized TiO2
film and the platinum electrode, and then sealed on 3 sides with hot-melt glue. On the fourth side
a drop of 0.50 M lithium iodide and 0.05 M iodine dissolved in acetonitrile was applied and
allowed to fill the gap between electrodes. Hot melt glue was then applied to the remaining side.
The films were tested via the method published in Ref. 30.
3.2.7 Numerical methods.
Non-negative matrix factorization (NMF) was used to determine the number of
components and their spectra for Bt-sensitized films as a function of dye-loading. NMF is a self-
modeling data interpretation method that resolves a data set into its individual components. The
structure of the data must be a linear combination of the components, which in the present case
are spectra of distinct forms of Bt on the TiO2 surface and the background spectrum of TiO2.
The component spectra and their weights are required to have positive values. In this case, the
absorbance of the betanin sensitized TiO2 films is found to be the linear combination of three
components as discussed below. To apply this method, we construct an 801 x 92 data matrix, D,

45
whose 92 columns are the measured absorbance spectra of the samples and whose 801 rows are
the wavelengths (measured every 0.5 nm). Using the algorithm of Lee and Seung49
, D is
factored into smaller all-positive matrices: R, an 801 x 3 matrix which contains the normalized
spectra of the three individual components in its columns and C, a 3 x 92 matrix which contains
the concentration and magnitude information; i.e., D = RC. The spectra in the R matrix are
normalized as part of the algorithm, so it is not possible to separate the magnitude of the molar
absorptivity from the concentration in the C matrix. The number of components was determined
to be three, based on an examination of the residuals of the model of the data and the
reproducibility of the components. The residuals are the difference between the measured data
and the modeled D matrix, equal to RC calculated using the factored R and C matrices. If not
enough components are used, then the data cannot be accurately reproduced, if too many
components are used, then experimental noise is mistaken for a component.
3.3 Results and Discussion
3.3.1 Absorption Spectra and Numerical Modeling of Betanin on TiO2 and ZrO2
Figure 3.2A and B show the absorption spectrum of Bt adsorbed on films of
nanocrystalline ZrO2 and TiO2, respectively, as a function of sensitization time. The absorption
spectrum of the sensitizing solution is shown in Figure 3.8, compared to that of a sensitized TiO2
film at high dye-loading. For low dye-loading, the absorption spectrum of Bt on TiO2 resembles
that of the aqueous solution with an absorption maximum near 530 nm. Films exposed to the
sensitizing solution for longer times are seen to exhibit blue-shifted absorption maxima and
spectral broadening. Previous evidence showed that the absorption spectrum of Bt on ZrO2 did

46
not exhibit as significant a blue-shift and was therefore used to model a film with higher
monomer character. Another factor that we considered was that the blue-shift resulted from a
degradation of the betanin chromophore to a yellow oxidation product.50
This
degradation/oxidation product was observed during time-resolved absorption experiments. We
have previously shown that Bt/ZrO2 films are stable to photooxidation owing to the more
negative conduction band potential of ZrO2 compared to TiO2.42,43
Though Bt appears to absorb
less strongly on ZrO2 than on TiO2, it is clear that in both cases longer sensitization times lead to
a blue shift that accompanies the increased absorbance of the film. Thus the spectral changes
observed at high dye-loading are not the result of photodegradation of the dye. To confirm that
degradation was not the source of the blue-shift, Bt was desorbed from the TiO2 films and the
extract submitted to HPLC analysis. As shown in Figure 3.9, HPLC analysis of solutions of
material desorbed from the films confirms that betanin is the only component giving rise to any
visible light absorption. Similarly, the absorption spectrum of the extract following dye
desorption strongly resembles that of the original sensitizing solution. These results strongly
indicate that betanin undergoes reversible templated aggregation on the surface of TiO2 and ZrO2
films. It should also be noted that repeated efforts to observe Bt aggregation in aqueous solution
were not successful: no deviations from Beer’s law were observed in the absorption spectrum at
the highest achievable Bt concentrations, Figure 3.10. We conclude that intermolecular
interactions of closely packed molecules affect the absorption properties of the dye on the
surface of nanocrystalline TiO2 and ZrO2 at high dye loadings. For similar staining times Bt dye
loading is higher and aggregation is more readily observed on TiO2 than on ZrO2. Further

47
confirmation of dye aggregation was obtained by using phosphate ions as co-absorbents.48
As
shown in Figure 3.8, these inhibit the spectral changes otherwise observed at high dye loadings.
We considered the possibility that spectral shifts and inhomogeneous broadening might
result from a distribution of binding sites which perturb the transition frequency, versus the
formation of discrete aggregates with shifted spectra. We therefore used nonnegative matrix
factorization (NMF) to discern the number of components and their resolved spectra which
contribute to the total absorption spectrum.51
Ninety-two UV-visible absorption spectra of
betanin on TiO2 and ZrO2 films and in solution were compiled and analyzed by NMF. Initial
attempts to analyze the data of sensitized films considered a variable number of components and
revealed that three components were sufficient to account for the total spectrum. Note that the
NMF analysis included a series of spectra: 37 sensitized TiO2 and 19 sensitized ZrO2 films, plus
Figure 3.2: Absorption spectra of Bt on A) ZrO2 and B) TiO2 as a function of sensitization
time in a saturated betanin solution at pH 2. A 900s film on TiO2 was prepared, but was too
dark to determine the absorbance.

48
20 bare films and 16 solution phase spectra. The spectral decomposition and residuals for the
entire series is shown in Supporting Information, Figure 3.12-Figure 3.14. The NMF method
cannot separate each component spectrum into the amount and relative intensity of each
contribution. Rather, it provides a basis set of three spectra for which each experimental
spectrum can be expressed as a linear combination thereof.
Figure 3.3 displays the results of NMF analysis for a film containing a relatively low
(Figure 3.3A) and high (Figure 3.3B) contribution of aggregate to the total absorption of Bt on
TiO2. The three individual components are accounted for as the absorption/scattering of TiO2,
and the absorption of monomer and aggregated forms of Bt. The data in Figure 3.3A are for a
sample for which NMF reveals 75% monomer and 25% aggregate contributions, while Figure
3.3B corresponds to 35% monomer and 65% aggregate. As seen below, the aggregate spectrum
Figure 3.3: Results of NMF analysis of Bt/TiO2 at A) low and B) high dye loading. The
films in A) and B) were sensitized for 300s and overnight (21 hours) and contain 25%
and 65% aggregate, respectively. The components are dimer (green), monomer (blue),
TiO2 extinction (cyan), experimental absorbance (black), and total calculated
absorbance (red dashed).

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