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Crystallization of L-glutathione on iCrystal plates using metal-assisted and microwave-accelerated evaporative crystallization and a mono-mode microwave

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ABSTRACT
Title of Thesis:
CRYSTALLIZATION OF L-GLUTATHIONE ON iCRYSTAL
PLATES USING METAL-ASSISTED AND MICROWAVEACCELERATED EVAPORATIVE CRYSTALLIZATION AND
A MONO-MODE MICROWAVE
Fatmah Alsharari, Master of Science, June 2016
Thesis Advisor:
Kadir Aslan, Ph.D.
Department of Chemistry
Metal-Assisted and Microwave-Accelerated Evaporative Crystallization
(MA-MAEC) is among the latest developments in the manufacture of biologically
based crystalline drug compounds described by the Aslan Research Group. MAMAEC is a technique that has the capacity to produce crystals with controlled
properties within a very short time. To date, the use of MA-MAEC technique was
demonstrated for the rapid crystallization of individual amino acids, small
molecules and lysozyme using conventional microwave ovens.
In this study, a tri-peptide, L-glutathione (GSH) was successfully
crystallized on iCrystal plates via the MA-MAEC technique using a mono-mode
microwave cavity to increase the effect of microwave heating for faster than ever
crystallization of biological molecules as compared to conventional microwave
ovens. The influence of various processing parameters such as solvent,
microwave power level (200 - 1000 Watts), GSH concentration (0.3 - 0.5 g/mL)
and substrate type (poly (methyl methacrylate) and silver nanoparticle films
(SNFs)) was studied in order to help optimize the GSH crystallization process.
Sodium acetate was found to be the best solvent for GSH crystallization because
a higher concentration of GSH could be dissolved in this solvent compared to the
other solvents. In addition, GSH crystals formed at a more rapid rate in sodium
acetate solutions.
The FTIR analysis reveals that crystals resulting from the MA-MAEC
technique are GSH-based. GSH functional groups that were observed in the
FTIR results include the thiol (-SH), a carboxyl group (COOH), carbonyl group
XC=O / 1713 cmí antisymmetric C=O, amine (-NH2/ XNH), and 1397 cmí,
XCOO- /asymmetric, and 1713-1602 cmí, -C-N / stretching. COOí), (asymmetric COOí), (anti-symmetric -&௘ ௘2 and (1280 cmí į2+ LQGLFDWH WKH SUHVHQFH RI D
í&22+ JURXS ZKLOH WKH EDQG assigned to a stretching 1075 cmí -CN vibrational mode.
The parameters that influenced GSH crystallization time were type of
substrate, microwave power level, and the initial concentration of GSH. Initial
GSH crystal formation was observed at approximately 5 minutes for all
conditions. The minimum crystallization time of 40 ±13 minutes was achieved for
the following conditions: 0.50 g/mL of GSH at the highest microwave power level
of 1000 Watts on the SNFs surface.
In addition, the comparison of the initial GSH studies using conventional
microwave heating by the Aslan Research Group and using a mono-mode cavity
and variable power microwave source (1,200 Watts) was carried out by
comparing crystallization time measurements and crystal size data. GSH crystal
size was observed to decrease by 50% or more with increasing concentration for
both microwave systems. There was a notable difference in crystal size between
GSH crystals grown on the iCrystal plates using both microwave systems based
on the magnitude of the mean and standard deviation.
This study provides evidence that the use of mono-mode microwave
cavity affords superior growth of GSH crystals in terms speed and crystal quality
and these results implies that future crystal growth studies should be carried out
using a mono-mode cavity and mono-mode microwave source with variable
power, especially for samples with low availability.
CRYSTALLIZATION OF L-GLUTATHIONE ON iCRYSTAL PLATES USING
METAL-ASSISTED AND MICROWAVE-ACCELERATED EVAPORATIVE
CRYSTALLIZATION AND A MONO-MODE MICROWAVE
by
Fatmah Alsharari
A Thesis Submitted in Partial Fulfillment
Of the Requirements for the Degree
Master of Science
MORGAN STATE UNIVERSITY
June 2016
ProQuest Number: 10189153
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CRYSTALLIZATION OF L-GLUTATHIONE ON iCRYSTAL PLATES USING
METAL-ASSISTED AND MICROWAVE-ACCELERATED EVAPORATIVE
CRYSTALLIZATION AND A MONO-MODE MICROWAVE
by
Fatmah Alsharari
has been approved
June 2016
THESIS COMMITTEE APPROVAL:
_______________________, Chair
Dereje Seifu, Ph.D.
_______________________, Advisor
Kadir Aslan, Ph.D.
_______________________
Birol Ozturk, Ph.D.
_______________________
Pumtiwitt. McCarthy, Ph.D.
_____________________
Yongchao Zhang, Ph.D.
ii
DEDICATION
This work is dedicated to Mrs. Nadyah Alshymi and Mr. Abdulrahman Alsharari
iii
ACKNOWLEDGEMENT
Foremost, I would like to express my sincerest gratitude to my advisor,
Professor .DGLU$VODQIRUKLVFRQWLQXRXVVXSSRUWRIP\0DVWHU¶Vthesis study and
research, and for his patience, motivation, enthusiasm, and immense knowledge.
I appreciate his guidance and support in helping me to successfully conduct this
research and write my thesis.
I would also like to thank the rest of my thesis committee for their
encouragement, and insightful comments: Prof. Pumtiwitt. McCarthy, Prof. Birol
Ozturk, Prof. Dereje Seifu and Prof. Yongchao Zhang.I would also like to thank
my Mother, Nadyah Alshymi who supported me and encouraged me with her
best wishes and love. I am also exceedingly grateful to Mrs. Aysha Zaakan, who
spent much time helping me with my experiments and Mr. Edward Constance
who help me with molecular modeling and simulation studies of GSH. I would
also like to thank my husband, Abdulrahman Alsharari. He was always there for
me and cheered me up and stood by me through the good times and bad.
Finally, I would like to extend my thanks to the Saudi Arabian Cultural
Mission (SACM) for providing financial support for my graduate study.
iv
TABLE OF CONTENTS
List of Tables ....................................................................................................... vii
List of Figures ..................................................................................................... viii
CHAPTER 1: INTRODUCTION ............................................................................ 1
CHAPTER 2: LITERATURE REVIEW .................................................................. 4
2.1 Molecular Modeling of Glutathione Crystals ................................................ 4
2.2 Control of Thermal Gradients in Microwave Processing ........................... 10
2.3 Crystallization of Glutathione using Conventional Microwave. .................. 12
CHAPTER 3: MATERIALS AND METHODS ...................................................... 14
3.1 Materials ................................................................................................... 14
3.2 Methods .................................................................................................... 14
3.3 iCrystal Microwave Cavity. ........................................................................ 15
3.4 Characterization of Glutathione Crystals ................................................... 15
CHAPTER 4: RESULTS AND DISCUSSION ..................................................... 17
4.1 Solubility of Glutathione ............................................................................ 17
4.2 Summary of Crystallization Time Results.................................................. 25
4.3 Comparison of crystallization time between iCrystal system and
commercial microwave for GSH...................................................................... 29
4.4 Average Size of Glutathione ..................................................................... 33
v
4.5 Characterization of GSH Crystals by Fourier Transform Infrared
Spectroscopy (FTIR). ...................................................................................... 41
CONCLUSIONS ................................................................................................. 52
REFRENCES ..................................................................................................... 53
vi
List of Tables
Pages
Table 1.Materials Studio study showing the properties of the morphologically
important facets of GSH crystals. ......................................................................... 6
Table 2.Summary of Average crystallization time8. ............................................ 13
Table 3.Summary of Average initial crystallization time 8. ................................... 13
Table 4.Solubility of GSH with different solvent at 50C. ..................................... 17
Table 5.Solubility of GSH with different solvent at room temperature. ................ 18
Table 6.Summary of crystallization time results. The initial crystallization time for
........................................................................................................................... 27
Table 7.Summary of crystallization time for the GSH in iCrystal and conventional
microwave. ......................................................................................................... 30
Table 8. Average size of GSH crystals. .............................................................. 34
Table 9. FTIR for GSH with PMMA and SNFs surface comparing with GSH in
literature. ............................................................................................................ 43
vii
List of Figures
Pages
Figure 1. Structures of glutamic acid, glycine, and cysteine acid.......................... 1
Figure 2.Structre of Glutathione (GSH) ................................................................ 1
Figure 3.Glutathione tripeptide molecule (Left) and Unit cell of GSH (Right) as
constructed in Materials Studio 8.......................................................................... 5
Figure 4.Crystal habit of GSH obtained using crystal morphology in Materials
Studio. .................................................................................................................. 5
Figure 5.Schematic figure showing the main steps in the metal-assisted and
microwave-accelerated evaporative crystallization process (from reference 4). . 10
Figure 6.Computer simulation generated using COMSOL Multiphasic TM
software showing the electric field distribution within the microwave cavity and
the sample temperature distribution after 3 s (from reference 11). ..................... 11
Figure 7.Crystallization behavior of GSH in sodium phosphate at room
temperature function of time (0-20 min).............................................................. 19
Figure 8.Crystallization behavior of GSH in PBS at room temperature function of
time (0-20 min). .................................................................................................. 20
Figure 9.Crystallization behavior of GSH in sodium phosphate at room
temperature function of time (0-20 min).............................................................. 21
Figure 10.Crystallization behavior of GSH in sodium acetate at room temperature
function of time (0-20 min). ................................................................................. 22
Figure 11.Crystallization behavior of GSH in Di water at 50°C a safunction of
time (0-20 min, Microwave Treatment). .............................................................. 23
viii
Figure 12. Crystallization behavior of GSH in PBS at 50 ° C function of time (0-20
min). ................................................................................................................... 24
Figure 13.Crystallization behavior of GSH in Sodium phosphate at 50°C function
of time (0-20 min). .............................................................................................. 24
Figure 14.Crystallization behavior of GSH in sodium acetate at 50°C function of
time (0-20 min). .................................................................................................. 25
Figure 15. Optical images of GSH crystals formed on PMMA platform at
microwave heating. ............................................................................................. 27
Figure 16. Optical images of GSH crystals formed on SNFs platform at
microwave heating. ............................................................................................. 28
Figure 17.Optical images of GSH crystals formed on PMMA platform at
microwave heating. ............................................................................................. 28
Figure 18.Optical images GSH crystals formed on SNFs-PMMA platform at the
MA-MAEC technique. ......................................................................................... 29
Figure 19.Optical images of 0.50 g/mL GSH crystals formed on circular
crystallization platforms with polymer cover at (A) PMMA and (B) SNFs: Silver
Nanoparticle Films with power level 1000 w. ...................................................... 31
Figure 20. Optical images of 0.50 g/mL GSH crystals grown at microwave
heating power 1000 W on SNFs-deposited circular crystallization platforms at the
same time (20 minutes). ..................................................................................... 32
Figure 21. Optical images of 0.50 g/mL GSH crystals grown at microwave
heating at 1000 W in each of the 21 wells of the blank crystallization platforms at
the same time (20 minutes). ............................................................................... 33
ix
Figure 22. Average size of GSH crystals grown from an initial solution of 0.50
g/mL at microwave heating on blank PMMA platforms versus time and power. . 36
Figure23. Average size of GSH crystals grown from an initial solution of 0.50
g/mL at microwave heating on SNFs versus time and power ........................... 37
Figure 24. Average size of GSH crystals grown from an initial solution of
0.40g/mL at microwave heating on blank PMMA platforms versus time and
power. ................................................................................................................. 38
Figure 25. Average size of GSH crystals grown from an initial solution of
0.40g/mL at microwave heating on SNFs versus time and power. ..................... 39
Figure 26. Average size of GSH crystals grown from an initial solution of
0.30g/mL at microwave heating on blank PMMA platforms versus time and power
........................................................................................................................... 40
Figure 27. Average size of GSH crystals grown from an initial solution of 0.30
g/mL
at microwave heating on SNFs versus time and power. ......................... 41
Figure 28. FTIR for GSH crystals spectra grown at power level 200 W.............. 43
Figure 29. FTIR for GSH crystals spectra grown at power level 200 W.............. 44
Figure 30. FTIR for GSH crystals spectra grown at power level 400 W.............. 45
Figure 31. . FTIR for GSH crystals spectra grown at power level 400 W. ........... 46
Figure 32. FTIR for GSH crystals spectra grown at power level 600 W.............. 47
Figure 33. FTIR for GSH crystals spectra grown at power level 600 W.............. 48
Figure 34. FTIR for GSH crystals spectra grown at power level 800 W.............. 49
Figure 35. FTIR for GSH crystals spectra grown at power level 800 W.............. 49
Figure 36. FTIR for GSH crystals spectra grown at power level 1000 W. ........... 50
x
Figure 37. FTIR for GSH crystals spectra grown at power level 1000 W ............ 50
Figure 38. FTIR for GSH found in the literature [16] ............................................. 51
xi
dysfunction, AIDS or pulmonary fibrosis4. It is important to obtain high purity
crystals of GSH that can be used GSH can serve as a model small tripeptide
protein to study rapid crystallization using the Metal-Assisted and MicrowaveAccelerated Evaporation crystallization (MA-MAEC) technique5 . Previous studies
have used this technique for studying the synthesis and crystal growth of small
amino acids such as L-alanine6
6b
, glycine7, L-arginine acetate8 and larger
molecules such as glutathione and lysozyme9 and show promise for improving
crystal purity and potentially influencing reaction kinetics.
Crystallization is considered by scientists as an important tool for
preparing and understanding the structure of a molecule. It is crucial to mention
that crystallization techniques ensure the production of high quality crystals that
are as pure as required by the application. The MA-MAEC technique is a very
beneficial method for crystallization of important compounds. MA-MAEC is a
faster method of crystallizing substances. In addition, it is a selective method for
preparing smaller molecules. Using the MA-MAEC method, a scientist can
rapidly create a glycine model within a few minutes. However, the latter depends
on the desired size of crystals being formed. Most importantly, glycine crystals
appear quite large when prepared on silver nanostructures even in the presence
of moderate heating in a microwave cavity.
The focus of this study involves determining how the MA-MAEC technique
influences the crystallization of GSH. The thesis statement is that the application
of the MA-MAEC technique to the crystallization of GSH using the iCrystal
crystallization plates, a mono-mode microwave cavity and a variable power ±
2
mono-mode microwave source will enhance the crystallization time for more
rapid GSH crystal growth. These results will be compared to GSH crystallization
data
collected
during
conventional
microwave
and
room
temperature
crystallization studies10.
3
Chapter 2: Literature Review
GSH is a very important biomolecule that has many applications as a
therapeutic agent as well as a model system to study fundamental phenomena in
biological activity and crystallization phenomena. The focus of this work is to
investigate a novel way to grow GSH crystals using a microwave processing
technique. This project also investigates how solvent and substrate influence the
GSH crystal growth phenomena. This literature review will focus on the following
aspects of GSH crystallization: 1) Molecular modeling of GSH crystals, 2) the
novel MA-MAEC microwave processing technique and its advantages over
conventional biomolecule crystallization techniques.
2.1 Molecular Modeling of Glutathione Crystals
It is important to understand the crystal structure of GSH on a fundamental
level. Crystal structure simulations were conducted using Materials Studio
Software and the results are shown in Figure 3 (unpublished results from the
Aslan Research Group). GSH belongs to the orthorhombic crystal class with unit
cell parameters a=0.562 nm, b=0.878 nm, and c=2.779 nm and belongs to the
space group P212121. Figure 3 shows a molecular representation of GSH along
with a ball and stick molecular model and how the four tripeptide units are
arranged in the unit cell. The four tripeptide units in each unit cell are bonded
together via hydrogen bonding of the ±SH functional group. Figure 4 displays an
image of a GSH crystal with prominent crystal faces shown with their
corresponding Miller Indices.
4
Table 1 provides a nice summary of important data related to GSH
crystals including dhkl, which is the spacing between successive crystal planes in
families of planes with Miller Indices {hk}.Eatt(total) represents the attractive
energy for specific GSH crystal faces with (200) having the lowest energy of -369
kJ/mol, and the total percent of face area representing the area occupied by a
specific crystal face.
Table 1.Materials Studio study showing the properties of the morphologically
important facets of GSH crystals.
Hkl
Multiplicity
dhkl
Eatt(Total)
kJ/mol
% Total facet
area
{0 0 2}
2
14.01
-65.02
53.12
{0 1 1}
4
8.38
-121.28
27.91
{0 1 2}
4
7.44
-121.33
8.36
{2 0 0}
2
2.81
-369.97
10.61
The first step in the process of crystallization is referred to as nucleation.
It is necessary to create conditions within the mixture so that the molecules can
give rise to the formation of crystals11. The crystallization process relies on both
mass transfer mechanisms and the amount of movement. In other words, the
existence of a higher solute concentration in solution at the saturation
concentration (solubility limit) speeds up the process of saturation. This state is
naturally unstable and that is why nucleation is possible 12.
6
Crystallization is a process whereby solid compounds are purified13 It is a
fundamental technique that chemists and industrialists use to separate
compounds made up of solids that are more soluble in hot water. When the
solute or compound is boiled in a hot solvent, it easily dissolves. Once the
solution cools down after the loss of heat energy, pure compounds in the form of
crystals are recovered in the process. Research has shown that this method
does not eliminate all impurities from crystals. Impurities can be removed by
either
filtration or any other convenient technique 5. The entire crystallization
process relies on a mixture of art and scientific skills. One can find numerous
studies on various crystallization techniques for small laboratories and industrial
applications14.
lament that most modern crystallization methods may fail to meet these
standards even though they are used in large scale15. A case in point is the
milling process that takes place as a post-crystallization technique. It is also
prudent to underscore the fact that supersaturation of liquid mixtures determines
the overall nature of crystals formed13. When mixtures are saturated beyond a
certain limit, the growth of crystals may be hampered significantly. In most
instances, nucleation takes place when liquid mixtures are exposed to extreme
supersaturation. The latter affects sub-processes such as spray drying, highpressure homogenization, solvent shifting, impinging jet crystallization, and
supercritical fluid crystallization16. The above methods often generate fine
crystals. Nevertheless, liquid mixtures exposed to extreme saturation tend to
form solids that are not crystalline at all. In some cases, undesired forms of
7
crystals may be generated when supersaturation takes place. It is only the
polymorph forms that are desired during the process of crystallization6a.
Numerous industrial processes apply evaporative crystallization in the
formation of crystals. Solvent evaporation can be quickened through microwave
irradiation. The latter is a crucial procedure in evaporative crystallization 17. In
other words, crystals are obtained at a faster rate when microwave irradiation is
made as part and parcel of the experimental procedure. Indeed, drug formulation
relies on this method owing to its efficiency. It ensures that the size of particles
are minimized as desired. Modification of the size of the drug also facilitates rapid
dissolution of the component18.
Amino acids are important molecules that performs vital functions in the
structures of most proteins19. Various food industries, pharmaceutical companies
and chemical manufacturers have a high demand for crystallized amino acids,
but it takes a long time to generate alanine crystals with the best characteristics
using conventional evaporative methods. New and improved methods for
crystallization are needed.to obtain enhanced crystals.
The Aslan Research Group at Morgan State University recently described
a novel crystallization technique, called Metal-assisted and MicrowaveAccelerated
Evaporative
Crystallization
(MA-MAEC)5.
In
the
MA-MAEC
technique, metal nanoparticles, such as silver, gold, copper, nickel can be used
for two purposes: 1) as a microwave-transparent medium to create a microwaveinduced temperature gradients between the metal surface and the solvent and 2)
as a selective nucleation site for crystal growth. MAEC refers to a procedure
8
whereby molecules of interest solution is exposed microwave heating to
accelerate the evaporation of the solvent and drive the molecules towards the
cooler metal surface. More specifically, the MA-MAEC technique is a unique
combination of microwave heating and silver nanoparticle films (SNFs), which
generates microwave-induced temperature gradients to enhance the crystal
growth process. The temperature gradient between the solution and the metal
surface arises due to the difference in thermal conductivity. There is an
approximately 620-fold difference in the thermal conductivity of silver (429 W/mK)
and water (0.61 W/mK) and mass transfer of specific molecule such as the
glycine molecule from the warmer solution to the cooler metal nanoparticle
surface is facilitated to achieve thermal equilibration as shown in Figure 5. The
technique also allows for the control of the rate and direction of diffusion which
can promote nucleation and crystal growth by controlling the thermal gradients.
Mass transfer of the solute molecules continues during microwave heating, and
the solute growth units assemble onto the surface of SNFs in a process called
nucleation as shown in step 2 of Figure 5. Crystal growth continues as the
solution evaporates and subsequent crystals grow together until all the solution
crystallizes as shown in Figure 5 step 3
Alabanza et. al., (2013) 20compared such an experiment with another one
carried out at room temperature as a control20. Observations and research
evidence revealed that the experiment carried out at a higher temperature led to
the rapid formation of large crystals. The MA-MAEC method is commonly used
9
because the large crystals that are typically made from this technique are desired
by most industries21.
Different types of crystals tend to compete during the process of
formation. Some of the factors that contribute towards this competition include
reduction of surface free energy, volume free energy, and critical nucleus size
effects. In addition, homogeneous nucleation process is used to JURZ ȕ-glycine
nanocrystal5.
Figure 5.Schematic figure showing the main steps in the metal-assisted and
microwave-accelerated evaporative crystallization process (from reference 5).
2.2 Control of Thermal Gradients in Microwave Processing
The distribution of energy within a microwave cavity can lead to thermal
gradients and non-uniform heating. It is important to take measures to ensure
that uniform heating occurs. In the novel MA-MAEC technique developed by
Aslan et al., multiple samples are heated in a multi-well poly (methyl
10
methacrylate) (PMMA) plate. Homogenous heating can be achieved in the 21
wells during the entire heating cycle because the small size and circular shape of
the PMMA well platform design as well as the electric field distribution in the
microwave cavity promote a more uniform temperature distribution. This
important fact is shown by a simulation (simulation software program name) in
Figure 6. Note that the temperature appears very uniform in most sample
regions. Figure 6 shows the predicted temperature variation and electric field (zcomponent) distribution after 3 sec of microwave heating in a conventional
microwave oven with the current design for the circular PMMA platform. The
temperature variation between all 21 wells of the PMMA platform at different
initial temperatures varied by <0.2C after 3 sec heating13.
k
Figure 6.Computer simulation generated using COMSOL Multiphasic TM
software showing the electric field distribution within the microwave cavity and
the sample temperature distribution after 3 s (from reference 13).
11
2.3 Crystallization of Glutathione using Conventional Microwave.
Crystallization studies with GSH were conducted by Aslan Research
group using the MA-MAEC technique using a conventional microwave oven and
room temperature for PMMA and SNFs coated for GSH concentration ranging
from 300 ± 500 mg/mL10. Table 2 and 3 show the results for all concentrations.
As shown in Tables 2 and 3, no crystallization was observed for either PMMA or
SNFs surfaces using MA-MAEC or room temperature conditions when the GSH
concentration was lower than 200 mg/mL. Comparatively, the duration of
crystallization at microwave and room temperature conditions for the SNFs
substrate was 25-30% faster than for the PMMA substrate. The experimental
outcomes show that 300 mg/ml of GSH in PMMA platform took an average
duration of 628±314 minutes. In comparison, the experimental outcomes also
show that 300 mg/ml of GSH in SNFs coated PMMA platform took 415±38
minutes. As Table 3 shows, the durations of crystallization for SNFS coated
platform and PMMA platform were 108±12 minutes and 130±20 minutes
correspondingly. The characterization of GSH crystals grown on both PMMA and
SNFs substrates using either the MA-MAEC technique or room temperature was
carried out using XRD and FTIR.
12
Table 2.Summary of Average crystallization time10.
Fixed power at 900 W
Average initial
Average initial crystallization
Power Level 1
crystallization time at
time at MW (minutes)
RT (minutes)
Concentration of
PMMA
SNFs
PMMA
SNFs
100 mg/Ml
*
*
*
*
200 mg/mL
*
*
*
*
300 mg/mL
67 ± 40
20 ± 10
17 ± 2.9
13 ± 7.6
400 mg/mL
25 ± 5.0
12 ± 2.9
12 ± 7.7
8.3 ± 2.9
500 mg/mL
23 ± 7.6
8.3 ± 2.9
8.3 ± 2.9
6.6 ± 2.9
glutathione
Table 3.Summary of Average initial crystallization time10.
Fixed power at
Average crystallization
Average crystallization time at
900 W
time
MW (minutes)
Power Level 1
at RT (minutes)
Concentration of
PMMA
SNFs
PMMA
SNFs
100 mg/mL
*
*
*
*
200 mg/mL
*
*
*
*
300 mg/mL
628± 314
415 ± 37.8
130 ± 20.0
108 ± 11.2
400 mg/mL
242± 25.7
227 ± 87.4
86.7 ± 7.64
65.0 ± 8.66
glutathione
13
Chapter 3: Materials and Methods
3.1 Materials
L-Glutathione, N,N-Dimethyl Formamide and phosphate buffered saline (PBS)
pH=7.4 were purchased from Sigma-Aldrich. Sodium acetate trihydrate was
purchased from McMaster-Carr (Elmhurst, IL, USA Ethyl alcohol, which was
bought from Pharmaco-Aaper in Brookfield. Sodium Phosphate Dibasic was
purchased from Fisher Scientific. Deionized water (> 0Ÿ‡FPUHVLVWLYLW\DW
°C) was obtained from a Millipore Direct Q3 system.
Silicone isolators (21 wells, 2.0 mm depth x 4.5 mm diameter) were
purchased from Grace Biolabs (Bend, OR, USA). A 1200W iCrystal system (GAE
Variable Attenuator 3KW CPR284) was purchased. Silver targets and an EMS
150RS sputter coater were purchased from Electron Microscopy Sciences.
3.2 Methods
3.2.1 Preparation of Solvents
The solubility of GSH in various solvents at room temperature and at 50°C
was studied. The solvents used were dimethyl formamide (DMF), ethanol, water,
phosphate buffered saline (PBS) at pH = 7.4, sodium acetate, and sodium
phosphate dibasic. 0.01 g/ml solution of GSH was prepared by weighing 0.01 g
and putting it in 1 ml solvent. To dissolve GSH completely, the solute and the
solvent were shaken vigorously for five minutes. To enhance dissolution of GSH
at room temperature, a Corning magnetic stirrer was used. However, 1ml of DMF
and ethanol did not dissolve 0.01 g of GSH to make a concentration of 0.01 g/ml
despite vigorous shaking for 60 minutes. GSH was weighed and gradual
14
increments of 0.01 g were added to the solvents while shaking vigorously by
means of a Corning stirrer pending complete dissolution of GSH. The visual
appearance of the solute was used to judge the extent of dissolution and the
temperature as determined using thermometer.
3.2.2. Crystallization of Glutathione
Glutathione powder (0.30, 0.40, or 0.50 g/mL was dissolved in 2.00 mL of
sodium acetate (ph4.6) in a clean glass vial. The crystallization solution was later
extracted using a sterile syringe and slowly filtered back into a clean vial. All
wells of the 21-well circular crystallization platforms were filled with 20 microliters
of GSH solution. The GSH solution was delivered to PMMA or SNFs (20
microliters) and was crystallized at microwave heating at different power levels
(200-1000 W).
3.3 iCrystal Microwave Cavity.
GSH at different concentrations (0.30, 0.40 and 0.50 g/mL) was processed
at 200, 400, 600, 800 and 1000 watts of power using the microwave processing
system from General Applied Engineering (GAE) modified iCrystal microwave
cavity designed by the Aslan Research Group. Initial crystallization was shown to
occur within 5 min of heating at all power levels and with the different
concentrations.
3.4 Characterization of Glutathione Crystals
Optical images of the growth of GSH crystals on circular crystallization
platforms were recorded every 5 min using a Swift Digital M10L. Measurements
of crystal size were also carried out, and the glutathione crystals were calculated
15
with Image J software. Crystal size was determined by using Motic images plus
2.0 software. The infrared spectrum of GSH crystals was obtained using an
Fourier Transform Infrared Spectroscopy (FTIR).
16
CHAPTER 4: Results and Discussion
4.1 Solubility of Glutathione
A variety of solvents were used to determine the best solvent that could be
used for crystallization experiments. The solubility of the glutathione (GSH) was
measured at room temperature and 50°C. Table 4 and 5 show the results.
Sodium acetate, sodium phosphate ethanol, de-ionized (DI) water, DMF, and
Phosphate-Buffered saline (PBS) were prepared at a concentration of 1 M. GSH
was insoluble in ethanol and DMF. The solubility of GSH varied from 0.08 to 0.13
g/mL at room temperature (RT) for de-ionized (DI) water, PBS, sodium
phosphate, and sodium acetate. The lower solubility was observed in DI water
and the maximum solubility was found in sodium acetate. GSH was more soluble
at 50 °C. The solubility ranged from 0.29 g/mL in PBS to 0.50 g/mL in sodium
acetate. Sodium acetate was chosen as the best solvent for subsequent
crystallization experiments.
Table 4.Solubility of GSH with different solvent at 50C.
Solvent
Observations at 50°C
Deionized water
Dissolves up to 0.32 g/Ml
Sodium Acetate (pH 4.6)
Dissolves up to 0.50 g/mL
Sodium Phosphate (pH 9.1)
Dissolves up to 0.35 g/mL
Phosphate-Buffered saline (pH 7.4)
Does not dissolve > 0.29 g/mL
Ethanol
Does not dissolve > 0.01 g/mL
Dimethyl Formamide
Does not dissolve > 0.01 g/mL
17
Table 5.Solubility of GSH with different solvent at room temperature.
Solvent
Observations at RT at RT
Deionized water
Dissolves up to 0.08 g/mL
Sodium Acetate (pH 4.6)
Dissolves up to 0.13 g /mL
Sodium Phosphate (pH 9.1)
Dissolves up to 0.12 g/mL
PBS (pH 7.4)
Dissolves up to 0.09 g/mL
Ethanol
Does not dissolve
Dimethyl Formamide
Does not dissolve
An optical microscope was used to visualize the crystallization behavior of
GSH in DI water, sodium phosphate, PBS, and sodium acetate. The data is
shown in Figures 7-14. No crystals were observed to form in DI water or PBS at
room temperate as shown in Figures 5 and 6. Minimal crystal formation occurred
at room temperature for sodium phosphate at 20 min, and sodium acetate at 15
min as shown in Figures 9 and 10. Microwave treatment caused crystallization
kinetics to increase for all solutions except the DI water. No crystals formed in DI
water at 50°C. The solution played a big role in influencing the time when crystals
formed during microwave treatment at 50°C. PBS formed crystals at 20 min and
sodium phosphate showed crystal formation at 15 min. The best result occurred
for sodium acetate where the GSH crystals formed at 5 min at 50°C.
18
H2O_RT
0 min
5 min
15 min
20 min
10 min
Figure 7.Crystallization behavior of GSH in sodium phosphate at room
temperature function of time (0-20 min).
19
PBS_RT
0 min
5 min
15 min
20 min
10 min
Figure 8.Crystallization behavior of GSH in PBS at room temperature function of
time (0-20 min).
20
NH2PO4_RT
0 min
5 min
15 min
20 min
10 min
Figure 9.Crystallization behavior of GSH in sodium phosphate at room
temperature function of time (0-20 min).
21
CH3COONa_RT
0 min
5 min
15 min
20 min
10 min
Figure 10.Crystallization behavior of GSH in sodium acetate at room temperature
function of time (0-20 min).
22
H2O_MW (50ͼC)
0 min
5 min
15 min
20 min
10 min
Figure 11.Crystallization behavior of GSH in Di water at 50°C as a function of
time (0-20 min, Microwave Treatment).
23
PBS_MW
0 min
5 min
15 min
20 min
10 min
Figure 12. Crystallization behavior of GSH in PBS at 50 ° C as a function of time
(0-20 min).
0 min
5 min
15 min
20 min
10 min
Figure 13.Crystallization behavior of GSH in Sodium phosphate at 50°C as a
function of time (0-20 min).
24
CH3COONa_M
0 min
5 min
15 min
20 min
10 min
Figure 14.Crystallization behavior of GSH in sodium acetate at 50°C as a
function of time (0-20 min).
4.2 Summary of Crystallization Time Results
Table 6 displays the summary data for GSH crystal growth as a function of
microwave power level (200, 400, 600, 800, and 1000 W) using the MA-MAEC
technique for PMMA and SNFs coated platforms. The crystallization time for
initial observation of GSH crystal formation was consistent at 5 minutes for all
conditions of microwave power (200-1000 W), substrate type (PMMA and SNFs),
and initial GSH concentration (0.30-0.50 g/mL). Different times were observed for
complete crystallization of GSH based on the microwave process conditions as
shown in Table 6.
At an initial GSH concentration of 0.30 g/mL with microwave power level
of 200 W, complete crystallization was observed at an average time of 377 ±
25
5.77 minutes for PMMA and 230 ± 5.00 minutes for SNFs. Pictures of GSH
crystals grown under these conditions are shown in Figure 15 and Figure 16.
These pictures are representative of all GSH crystallization time experimental
conditions. In summary, the following observations can be made regarding the
influence microwave power, substrate, and glutathione concentration. As the
microwave power increased from 200-1000 W, the crystallization time decreased
time 377 ± 5.77 min. increasing initial GSH concentration from 0.30 g/mL to 0.50
g/mL resulted in a large decrease in the crystallization time. Crystallization time
decreased from 377 ±5.77 min to 140 ± 30.0 min on GSH concentration
decreased from 0.30 to 0.50 g/mL on PMMA substrate at a power level of 200 W.
Similar trends were noted for all power level. The substrate also influenced the
crystallization time. GSH crystallization time was more rapid on SNFs than
PMMA. Similar trends were also noted for the influence of GSH concentration on
crystallization time for the SNFs versus PMMA surface. The minimum observed
crystallization time of 40.0 ± 13.0 min was achieved at the highest microwave
power, the highest glutathione concentration, and on the SNFs substrate as
shown in Figures 17 and 18. It is interesting to note that the variability in
crystallization time data, as indicated by the standard deviation, seems to
increase on the glutathione concentration increases.
26
Table 6.Summary of crystallization time results. The initial crystallization time for
all samples occurred as early as 5 min.
Crystallization time, minutes
Glutathione
Microwave
0.30g/mL
0.40g/mL
0.50g/mL
PMMA
SNFs
PMMA
SNFs
PMMA
SNFs
377±5.77
230±5.00
135±13.2
103±18.9
140±30.0
81.6±37.
Power, W
200
5
400
272±2.88
205±5.00
135±5.00
135±5.00
135±40.0
95.0±5.0
0
600
207±2.88
115±5.00
103±2.88
103±2.88
101±7.63
93.3±5.7
800
190±10.0
145±5.00
108±17.5
108±17.5
113±2.88
76.6±7.6
3
1000
156±15.3
113±2.88
71.6±7.63
71.67.63
73.310.0
40.013.0
380 min
141µm
0 min
5 min
245 min
335min
175 min
360 min
380 min
Figure 15. Optical images of GSH crystals formed on PMMA platform at
microwave heating.
27
141µm
5 min
30min
215 min
220 min
60 min
225 min
105 min
230 min
Figure 16. Optical images of GSH crystals formed on SNFs platform at
microwave heating.
141µm
0 min
5 min
15 min
30 min
40 min
55 min
65 min
70 min
Figure 17.Optical images of GSH crystals formed on PMMA platform at
microwave heating.
28
141µm
0 min
5 min
20 min
30 min
35 min
40 min
25 min
Figure 18.Optical images GSH crystals formed on SNFs platform at the MAMAEC technique.
4.3 Comparison of crystallization time between iCrystal system and
commercial microwave for GSH.
An interesting comparison can be made between conventional microwave
heating10 and heating using the iCrystal system by comparing crystallization time
measurements and crystal size data from this experiment and earlier studies by
Aslan Research Group. The data comparison is shown in Table 7. The initial
crystallization time was faster for iCrystal comparing to the conventional
microwave. For the iCrystal system, the initial crystal growth of GSH occurred at
the same time with all concentrations (0.30-0.50 g/mL) at 5 minutes for PMMA
and SNFs substrates while for the conventional microwave, the initial GSH
crystal growth took longer than 5 minutes to appear with all concentration (0.300.50 g/mL) for PMMA and SNFs.
The GSH crystal size was observed to decrease by 50% or more with
increasing concentration for both the iCrystal and conventional microwave
systems. There was a notable difference in crystal size between GSH crystals
29
grown on the PMMA substrate under conventional and iCrystal conditions based
on the magnitude of the mean and standard deviation. There was much less of a
difference between GSH crystals grown under iCrystal and conventional
microwave conditions on SNFs.
Table 7.Summary of crystallization time for the GSH in iCrystal and conventional
microwave.
Concentration(g/mL)
0.30
0.40
0.50
Conventional iCrystal
microwave
system
PMMA
130±20.0
156±15.3
86.7±7.64
110±5.00
63.3±2.89
73.3±10.0
Conventional
microwave
SNFs
108±11.2
65.0±8.66
46.7±7.64
iCrystal
system
106±7.63
71.6±7.63
40.0±13.0
The effect of microwave exposure time and substrate on GSH
crystallization is shown in Figure 17. The time required for complete GSH
crystallization was 80 min for PMMA and 40 min for SNFs. The microwave
energy seems to interact more strongly with silver and enhance crystallization
kinetics.
30
The average size of the GSH crystals that grew on the PMMA well was 161± 74
Pm and size range varied between 78 to 361 Pm. The average size of the GSH
crystals that grew on the SNFs wells was 199 ± 24 Pm and size range varied
between 80 to 369 Pm. The GSH crystals that formed on the PMMA well had
large variation and size and display a large number of very small crystal. In
contrast, the crystal that formed on the SNFs well exhibited much larger crystals
that more uniform in length and with variable width.
GSH_SNFs_
MW_All Wells
Well-1
Well-2
Well-8
11Well
11
16Well
16
Well-17
12Well
12
18
13Well
13
Well-19
10
14
Well-20
Well-15
21Well21
Figure 20. Optical images of 0.50 g/mL GSH crystals grown at microwave
heating power 1000 W on SNFs-deposited circular crystallization platforms at the
same time (20 minutes).
32
GSH_PMMA_ 141µm
MW_All Wells
Well 1
Well
6
Well-5
Well-2
WellWell
3
Well-3
Well
4
Well
5
Well-7
Well
Well74
Well8-6
Well
Well
9
Well-9
WellWell
10
Well
Well 14
Well 15
Well 19
Well 20
Well-
Well
Well
Well 17
Well
WellWell 18
Figure 21. Optical images of 0.50 g/mL GSH crystals grown at microwave
heating at 1000 W in each of the 21 wells of the blank crystallization
platforms at the same time (20 minutes).
4.4 Average Size of Glutathione
Data for the number of GSH crystals that formed and their size was
measured to provide an estimate of crystal size and nucleation phenomena. This
data is shown in Table 8. There was some scatter in the data that diminished the
appearance of trends. However, it seemed to resemble that more GSH crystals
formed on SNFs substance than PMMA substance. There were no obvious
indications that microwave power on glutathione concentration had a major
importance on GSH crystal size range and number.
33
Table 8. Average size of GSH crystals.
Crystal size, µm
Size Range(min-max),µm
Number of Crystals
0.30g/mL
Power
0.40g/mL
0.50g/mL
PMMA
SNFs
PMMA
SNFs
PMMA
SNFs
25.5-474
30.1-647
27.7-389
17.7-389
23.6-403
40.2-857
147
205
55
194
32
30
34.5-424
30.2-906
30.9-537
21.4-525
20-786
23.6-475
47
71
100
124
39
47
29.0-369
20.1-452
17.0-641
32.4-610
40-616
49-599
74
68
241
155
30
32
23.8-455
26.7-474
18.0-327
41.9-627
26.5-541
42.3-341
216
180
47
123
33
62
21.8-424
23.1-364
16.3-489
21.8-369
25.3-356
25.3-382
163
139
84
236
64
113
level, W
200
400
600
800
1000
The average size of GSH crystals as a function of time that formed during
microwave heating on PMMA and PMMA coated with SNFs substrates were
measured and plotted using sigma plot. The data is shown in Figures 22-27.
GSH crystal size was determined after a single layer of crystals formed
during solvent evaporation. The figures show the crystallization time for PMMA
34
platform was slower than PMMA coated with SNFs. The rate crystal growth
increasing with increase microwave power. The concentration of glutathione
influence the growth rate and interesting manner at lowest concentration of
glutathione seems to be two reagent where there is repaid crystal growth with the
first 5 minutes follow by as lower growth rate for remain time of the growth .at
lowest power level and lowest glutathione concentration the initial crystal rate
growth was about 10 µm/min. we can measure the initial fast growth rate within
first 5 minutes and slow secondary crystal growth rate with (5 minutes and end of
the crystal growth) based on the data in Figures 26 and 27. This example
compares the growth rate for different GSH concentration and microwave power
level and how to influence the crystal growth rate. At GSH concentration 0.30
µm/min, the initial growth rate was measured as approximately 10 µm/min at the
lowest microwave power level (200 W). The initial crystal growth rate to 30
µm/min at the microwave power between (400-1000 W). The growth rate at 600w
is only slightly higher than microwave power level 200 W, and it is lower than
microwave power level 400 w.
Figure 26 shows that the initial crystal growth rate flowed similar trends
but the initial crystal rate higher microwave power level was 36 µm/min. The
show at initial crystal growth rate was faster than PMMA substrate. The trend
was different for the secondary growth rate. At the highest microwave power
level, the GSH crystal growth rate was 5 µm/min on SNFs compare to 3.8 on
PMMA.
35
As GSH concentration increase there was less different between initial
and secondary crystal growth reagent. The slope which increase crystal growth
rate was more uniform over the time range. Increase the GSH concentration
there is no different between the substrate not influence crystal growth rate as
much because the crystal growth rate was similar to measurement in Figures 22
and 23.
Average size (micrometers)
800
PMMA
PMMA
PMMA
PMMA
PMMA
600
MW-PL200
MW-PL400
MW-PL600
MW-PL800
MW-PL1000
0.50 g/mLGSH PMMA
400
200
0
0
20
40
60
80
Time (min)
Figure 22. Average size of GSH crystals grown from an initial solution of 0.50
g/mL at microwave heating on blank PMMA platforms versus time and power.
36
Average size (micrometers)
500
SNFs
SNFs
SNFs
SNFs
SNFs
400
MW-PL200
MW-PL400
MW-PL600
MW-PL800
MW-PL1000
0.50 g/mL GSH SNFs
300
200
100
0
0
20
40
60
80
Time (min)
Figure23. Average size of GSH crystals grown from an initial solution of 0.50
g/mL at microwave heating on SNFs versus time and power.
37
Average crystal (micrometers)
600
PMMA
PMMA
PMMA
PMMA
PMMA
500
MW-PL200
MW-PL400
MW-PL600
MW-PL800
MW-PL1000
0.40 g/mL Glutathion-PMMA
400
300
200
100
0
0
20
40
60
80
100
Time(min)
Figure 24. Average size of GSH crystals grown from an initial solution of 0.40
g/mL at microwave heating on blank PMMA platforms versus time and power.
38
700
SNFs
SNFs
SNFs
SNFs
SNFs
Average size (micrometers)
600
500
0.40 g/mL GSH SNFs
MW-PL200
MW-PL400
MW-PL600
MW-PL600
MW-PL1000
400
300
200
100
0
0
20
40
60
80
100
Time (min)
Figure 25. Average size of GSH crystals grown from an initial solution of 0.40
g/mL at microwave heating on SNFs versus time and power.
39
Average size (micrometers)
800
PMMA
PMMA
PMMA
PMMA
PMMA
600
MW-PL200
MW-PL400
MW-PL600
MW-PL800
MW-PL1000
0.30 g/mL Glutathione PMMA
20
40
400
200
0
0
60
80
100
Time (min)
Figure 26. Average size of GSH crystals grown from an initial solution of 0.30
g/mL at microwave heating on blank PMMA platforms versus time and power.
40
600
SNFs
SNFs
SNFs
SNFs
SNFs
Average size (micrometers)
500
MW-PL200
MW-PL400
MW-PL600
MW-PL800
MW-PL1000
0.30 g/mL Glutathione SNFs
400
300
200
100
0
0
20
40
60
80
100
Time (min)
Figure 27. Average size of GSH crystals grown from an initial solution of 0.30
g/mL at microwave heating on SNFs versus time and power.
4.5 Characterization of GSH Crystals by Fourier Transform Infrared
Spectroscopy (FTIR).
Figures 28 to 37 show the infrared spectra of GSH crystals after
microwave heating (200-1000 W) using the MA-MAEC technique for both PMMA
and PMMA coated with SNFs for all concentrations. These FTIR spectra can be
compared to FTIR data for GSH from the literature review of GSH. Figure 38
highlights some important FTIR spectrum vibrational modes from the literature
review.
Glutathione peaks were observed in the experimental data for peaks
which occur 2520 cmí1 (symmetric -COOí), 1382 cm
-1
(asymmetric -COOí),
41
1713 cmí1 (ant symmetric -&௘ ௘2DQGFPí1 į2+LQGLFDWHWKHSUHVHQFHRI
Dí&22+JURXSZKLOHWKHEDQGDW1282 cm-1 can be assigned to a stretching -CN vibrational mode. Characteristic -N-H stretching modes were observed at 3344
and 3246 cm-1 were observed in the experimental data. There is a very good
agreement between experimental vibrational features for GSH and GSH features
reported in the literature.
A brief description of the most important vibrational modes that identify
GSH is present next. Peaks which occur 1713±1602 cmí1 (symmetric -COOí),
1397 cmí1 (asymmetric -COOí), 1713 cmí1 (ant symmetric -&௘ ௘2 DQG cmí1 į2+ LQGLFDWH WKH SUHVHQFH RI D í&22+ JURXS ZKLOH WKH EDQG DW cmí1 can be assigned to a stretching -C-N vibrational mode. Characteristic -N-H
stretching modes were observed at 3346 cmí1 and 3250 cmí1.provide evidence
for the presence of a -NH2 group. The characteristic -SH - stretching mode is
clearly seen at 2526 cmí1. The result for the experimental synthesis of GSH
compare very nicely with reported vibrational modes. While there were some
slight differences between the FTIR vibrational and modes in this work compared
to the values from the literature. However, these differences are very slight (less
than 5 cm-1) and within experimental and instrumental error limits. This indicates
that the MA-MAEC synthesis process produced GSH crystals of good quality.
While the crystal growth conditions did not really have an impact on the location
of the major GSH vibrational modes, it is interesting to note that the FTIR spectra
obtained from the GSH samples prepared at 0.40 g/mL consistently displayed the
most distinct patterns with well resolved vibrational modes.
42
Table 9. FTIR for GSH with PMMA and SNFs surface comparing with GSH in
literature.
Functional
group/stretch
Literature
-NH/
3346
3250 cm-1
-SH
2526 cm-1
C=O/
antisymmetric
COO-/
asymmetric
-COOH/(OH)
1713 cm-1
-C-N/
stretching
1075 cm-1
100
1397 cm-1
1280 cm-1
0.30 g/mL
PMMA
3344
3246
cm-1
2520
cm-1
1710
cm-1
1382
cm-1
1284
cm-1
1070
cm-1
0.40 g/mL
SNFs
3344
3246
cm-1
2520
cm-1
1714
1382
cm-1
1285
cm-1
1065
cm-1
PMMA
3345
3251
cm-1
2325
cm-1
1712
cm-1
1395
cm-1
1283
cm-1
1076
cm-1
0.50 g/mL
SNFs
3340
3255
cm-1
2523
cm-1
1715
cm-1
1394
cm-1
1281
cm-1
1074
cm-1
PMMA
3347
3255
cm-1
2523
cm-1
1712
cm-1
1396
cm-1
1285
cm-1
1073
cm-1
SNFs
3340
3249
cm-1
2528
cm-1
1711
cm-1
1393
cm-1
1279
cm-1
1077
cm-1
PMMA-MW-PL200
98
%Transmittance
96
94
92
90
88
86
4000
0.30 g/ml GSH MW-PMMA
0.40 g/ml GSH MW-PMMA
0.50 g/ml GSH MW-PMMA
3000
2000
1000
Wavenumber (cm-1)
Figure 28. FTIR for GSH crystals spectra grown at power level 200 Watts.
43
100
SNFs-MW-PL200
98
%Transmittance
96
94
92
90
88
86
4000
0.30 g/ml GSH MW-SNFs
0.40 g/ml GSH MW-SNFs
0.50 g/ml GSH MW-SNFs
3000
2000
1000
Wavenumber (cm-1)
Figure 29. FTIR for GSH crystals spectra grown at power level 200 Watts.
44
100
SN Fs-M W -PL400
98
% T ransm ittance
96
94
92
90
88
86
4000
0.30 g/m l G S H M W -S N Fs
0.40 g/m l G S H M W -S N Fs
0.50 g/m l G S H M W -S N Fs
3000
2000
1000
-1
W avenum ber (cm )
Figure 30. FTIR for GSH crystals spectra grown at power level 400 Watts.
45
100
PM M A-M W -PL400
98
% T ransm ittance
96
94
92
90
88
86
4000
0.30 g/m l G S H M W -P M M A
0.40 g/m l G S H M W -P M M A
0.50 g/m l G S H M W -P M M A
3000
2000
1000
-1
W avenum ber (cm )
Figure 31. FTIR for GSH crystals spectra grown at power level 400 Watts.
46
100
SN Fs-M W -PL600
% T ranam ittance
95
90
85
80
75
0.30 g/m l G S H M W -S N Fs
0.40 g/m l G S H M W -S N Fs
0.50 g/m l G S H M W -S N Fs
70
4000
3000
2000
1000
-1
W avenum ber (cm )
Figure 32. FTIR for GSH crystals spectra grown at power level 600 Watts.
47
100
PMMA-MW-PL600
98
%Transmittance
96
94
92
90
88
86
4000
Col 5 vs Col 6
Col 5 vs Col 8
Col 5 vs Col 10
3000
2000
1000
Wavenumber (cm-1)
Figure 33. FTIR for GSH crystals spectra grown at power level 600 Watts.
48
110
PMMA-MW-PL800
% Tranamittance
100
90
80
0.30 g/ml GSH MW-PMMA
0.40 g/ml GSH MW-PMMA
0.50 g/ml GSH MW-PMMA
70
4000
3000
2000
1000
Wavenumber (cm-1)
Figure 34. FTIR for GSH crystals spectra grown at power level 800 Watts.
100
SNFs-MW-PL800
% Tranamittance
95
90
85
80
75
70
4000
0.30 g/ml GSH MW-SNFs
0.40 g/ml GSH MW-SNFs
0.50 g/ml GSH MW-SNFs
3000
2000
1000
Wavenumber (cm-1)
Figure 35. FTIR for GSH crystals spectra grown at power level 800 Watts.
49
110
PMMA-MW-PL1000
% Tranamittance
100
90
80
70
60
4000
0.30 g/ml GSH MW-PMMA
0.40 g/ml GSH MW-PMMA
0.50 g/ml GSH MW-PMMA
3000
2000
1000
-1
Wavenumber (cm )
Figure 36. FTIR for GSH crystals spectra grown at power level 1000 Watts.
110
SNFs-MW-PL1000
% Tranamittance
100
90
80
70
60
4000
0.30 g/ml GSH MW-PMMA
0.40 g/ml GSH MW-PMMA
0.50 g/ml GSH MW-PMMA
3000
2000
1000
-1
Wavenumber (cm )
Figure 37. FTIR for GSH crystals spectra grown at power level 1000 Watts.
50
Figure 38. FTIR for GSH found in the literature 22.
51
CONCLOSIONS
Glutathione crystals were successfully grown using or utilizing the MetalAssisted and Microwave-Accelerated Evaporative Crystallization (MA-MAEC)
technique that was pioneered and developed by the Aslan Research Group.
Sodium acetate was found to be the best solvent for glutathione (GSH)
crystallization because a higher concentration of GSH could be dissolved in
sodium acetate (PH 4.6) solutions 0.50 g/mL compared to De-ionized water 0.32
g/mL, sodium phosphate (PH 9.1) 0.35 g/mL, ethanol 0.01 g/mL, PBS (pH 7.4)
0.29 g/mL, and DMF 0.01 g/mL for a temperature of 50°C. In addition, GSH
crystals formed at a more rapid rate in sodium acetate solutions.
The parameters that influenced GSH crystallization time the most were
substrate (SNFs or PMMA), microwave power level (200 - 1000 W), and the
initial concentration of GSH (0.3-0.5 g/mL). Initial GSH crystal formation was
observed at approximately five minutes for all conditions. The minimum
crystallization time of 40 ±13 minutes was achieved for the following
experimental conditions: 0.50 g/mL of GSH at the highest microwave power level
of 1000 Watts on the SNFs surface.
Glutathione crystal analyzed by FTIR in this study showed that the
functional groups for GSH crystals were present and remained unchanged. The
most important GSH functional groups that were observed include a thiol (-SH),
a carboxyl group (COOH), carbonyl group X-C=O / antisymmetric C=O, amine (NH2࣠-NH), and X-COO- asymmetric, and -C-N / stretching.
52
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Biology of Lipids 2013, 1831 (2), 314-326.
2.
Ortega, A. L.; Mena, S.; Estrela, J. M., Glutathione in cancer cell death.
Cancers 2011, 3 (1), 1285-1310.
3.
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