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MONITORING AND CONTROL OF CRYSTALLIZATION
PROCESSES USING ON-LINE SPECTROSCOPIC
METHODS

presented by

Lili Feng

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MONITORING AND CONTROL OF CRYSTALLIZATION
PROCESSES USING ON-LINE SPECTROSCOPIC
NIETHODS

By

Lili Feng

A DISSERTATION

Submitted to
Michigan State University
in partial fulﬁllment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Chemistry
and
Department of Chemical Engineering & Materials Science

2004

ABSTRACT

MONITORING AND CONTROL OF CRYSTALLIZATION
PROCESSES USING ON-LINE SPECTROSCOPIC METHODS

BY

Lili Feng

Proper control is needed for crystallization processes in order to produce crystalline
products with desired properties such as particle size distribution, shape or solid-state
modiﬁcation. Coupled with on line sampling techniques, spectroscopic methods offer
great advantages in crystallization process monitoring and control. This work focuses on
simplifying crystallization process monitoring and control, and gaining fundamental
insights to the crystallization kinetics using Raman, ATR-FT IR and ﬂuorescence
spectroscopic techniques.

Raman spectroscopy was used for in situ characterization of the polymorph form of
acetaminophen and for monitoring of the transformation process in a crystallization
slurry. Thermal history was identiﬁed as the controlling parameter for crystallization of
the metastable polymorph forms. Polymorph transformation kinetics were also obtained
conveniently by on line measurements using Raman spectroscopy combined with
multivariate curve resolution and using ATR-FTIR. Under the conditions studied,
acetaminophen polymorph transformation is limited by the crystallization rate of the
more stable phase, speciﬁcally by the crystal growth rate of form II for the form T to II

transformation, and by nucleation rate of form I for the form 11 to I transformation.

The temperature proﬁle applied during batch cooling crystallization affects the
supersaturation level, which in turn affects the crystal size distribution. ATR-FTIR
interfaced to a LABMAX® automatic reactor system was used in a feedback mode to
control the cooling rate to maintain low supersaturation during crystallization. The
resulting temperature proﬁle corresponds to the optimal operating conditions for the
maximum in the mean crystal size. This approach is a simple and easy alternative to
traditional control based on kinetic models.

Fluorescence and Raman spectroscopic techniques were applied to probe the
thermodynamic and kinetic properties of highly concentrated sucrose solutions with
fructose and glucose as impurities. Such properties include water activity, viscosity and
molecular conformation in local environment of crystallization. The results suggest that
fructose and glucose inhibit crystallization of sucrose by different mechanisms. Viscosity
is not the controlling factor in sucrose crystallization inhibition by glucose and fructose in
highly concentrated sucrose solutions. It was also demonstrated that ﬂuorescence and
Raman spectroscopic techniques can be adapted to monitor and control industrial sucrose

crystallizations at high concentration and high temperature.

ACKNOWLEDGIVIEN TS

I am very grateﬁ11 to my advisor, Dr. Kris A. Berglund for his patient and most valuable
guidance, which helped me to ﬁilﬁll all the requirements for a dual degree in chemistry
and chemical engineering. I also beneﬁted very much from other unique opportunities he
has presented to me.

I wish to express my appreciation to my committee members, Dr. Babak Borhan, Dr.
Gary Blanchard and Dr. Ramani Narayan. Thank you for all your time and support.

I could not have gone this far without the help and support I got along the way. Every
single member of Berglund group I worked with has helped me one way or another.
Some of the cheering conversations I will never forget. My internship supervisors Dr.
David am Ende at Pﬁzer and Dr. Ruairi Omedhra at Eastman Chemical Company guided
my ﬁrst steps into the industrial world with valuable advice and generous encouragement.
Special thanks to all my friends, especially Wei, Hua, Barry and Bonnie, for the joy and
comfort you brought me and for the conﬁdence you gave me. Thank you for brightening
the gloomy days.

I cannot say thank you enough to my family. My parents and sisters have always been
the source for strength and comfort that pull me through every difﬁculty. My husband,
Dehua, I thank you for standing by me and sharing both the exciting and depressing

moments along this journey.

iv

TABLE OF CONTENTS

List of Tables ......................................................................................... viii
List of Figures ......................................................................................... ix
Chapter 1
INTRODUCTION ................................................................................... l
1.1 Crystallization ............................................................................ 1
1.1.1 Supersaturation .................................................................... 2

1.1.2 Nucleatron3
1.1.3 Crystal Growth ....................................................................... 4
1.2 Signiﬁcance ofControl in Crystallization............ . . 5

1.2.1 Polymorphism ...................................................................... 5
1.2.2 Particle Size Drstnbutron6
1.2.3 Inhibition of Crystallization by Impurity ...................................... 8
1.3 Spectroscopic methods for Crystallization Monitoring and Control ............... 8

1.3.1 Raman Spectroscopy for On Line Polymorph Characterization. . . . . . ..9
1.3.2 ATR—FTIR Spectroscopy for On Line Concentration Measurement. . ...11
1.3.3 Probe the Microscopic properties of Crystallization Solution by

Fluorescence ..................................................................... 13

1.3.4 Chemometrics Data Analysis .................................................. 14

1.4 Overview of This Work ................................................................ 14
1.5 References ............................................................................... 15

Chapter 2

CONTROL CRYSTALLIZATION OF ACETAMINOPHEN POLYMORPHS
USING RAMAN SPECTROSCOPY17

2.1 Introduction .............................................................................. 17
2.2 Experimental Section .................................................................. 19
2.2.1 Solubility Measurement of Different Polymorphs ......................... 21

2.2.2 Polymorph Crystallization of Acetaminophen by Cooling. . . . . . . .21

2.2.3 Transformation of Acetaminophen Form T in Slurry ...................... 22

2.3 Results and Discussion ................................................................. 24
2.3.1 Characterization of Acetaminophen Polymorphs ........................... 24
2.3.2 Solubility of Acetaminophen Polymorphs ................................... 28
2.3.3 Effect of Thermal History on Acetaminophen Polymorph

Crystallization ................................................................... 3 1
2.3.4 Effect of Solvent Composition on Acetaminophen Polymorph
Crystallization ................................................................... 33
2.3.5 Transformation of Form T in Slurry .......................................... 34

2.4 Conclusions .............................................................................. 37

2.5 Acknowledgements ..................................................................... 37

2.6 References ............................................................................... 38

Chapter 3

IN-SITU MONITORING OF ACETAMINOPHEN POLYMORPH
TRANSFORMATION USING RAMAN AND ATR-FI‘IR SPECTROSCOPIES...40

3.1 Introduction .............................................................................. 40

3.2 Experimental methods .................................................................. 42

3.2.1 Polymorph Crystallization and Transformation of Acetaminophen. . ....43

3.2.2 Spectral Data Analysis ......................................................... 43

3.3 Results and Discussion ................................................................. 44
3.3.1 In Situ Monitoring of the Crystallization and Transformation of
Acetaminophen Polymorphs by Raman spectroscopy ..................... 45

3.3.2 In Situ Monitoring of Concentration Changes During Crystallization and
Transformation by ATR-FTIR ................................................ 54

3.4 Conclusions ............................................................................ 56

3.5 References ............................................................................... 57

Chapter 4

ATR-FI‘IR FOR DETERMINING OPTIMAL COOLING CURVES FOR BATCH
CRYSTALLIZATION OF SUCCINIC ACID ............................................... 58
4.1 Introduction .............................................................................. 58

4.2 Experimental Section ................................................................... 60

4.2.1 Calibration and Solubility Curve Measurement ............................ 62

vi

4.2.2 Determination of Cooling Proﬁles ............................................ 62

4.3 Results and Discussion ............................................................... 63
4.3.1 Calibration and Solubility Curve .............................................. 63
4.3.2 Determination of Optimal Cooling Proﬁles .................................. 67
4.4 Conclusions ............................................................................. 73
4.5 Acknowledgements .................................................................... 73
4.6 References .............................................................................. 74
Chapter 5
STUDY THE INHIBITION EFFECT OF FRUCTOSE AND GLUCOSE ON
SUCROSE CRYSTALLIZATION ............................................................. 75
5.1 Introduction ............................................................................ 75
5.2 Experimental Section ................................................................. 77
5 .2. 1 Sample Preparation for Steady State Fluorescence
Measurements .................................................................. 77
5.2.2 Steady State Fluorescence Measurement ..................................... 79
5.2.3 Raman Spectra of Pure Sugar Solutions ...................................... 80
5.3 Results and Discussion ................................................................ 80
5.3 1 Emission Properties of Fluorescence Probe Molecules .................... 80
5.3.2 Effect of Glucose and Fructose on Water Activity of Sucrose
Solutions ......................................................................... 83

5.3.3 Effect of Glucose and Fructose on Viscosity of Sucrose Solutions. . .....88

5.3.4 Molecular Conformation Effect of Glucose and Fructose on Sucrose

Chapter 6

Solution ........................................................................... 91

5.4 Conclusions ..................................................................... ‘ ........ 95

5.5 Acknowledgements ............................................................. i ....... 95

5.6 References .............................................................................. 96
CONCLUSIONS AND FUTURE WORK.97
6.1 Conclusions ............................................................................. 97

6.2 Future Work ............................................................................. 99
APPENDICES ..................................................................................... 100

vii

Table 2.1.

Table 5.1

LIST OF TABLES

Effect of thermal history and solvent composition on polymorph of
acetaminophen obtained ﬁ'om unseeded batch crystallization. H-a, H-b

and H-c represent different heating and cooling proﬁles ..................... 31

Composition of Samples for Fluorescence Measurement (Brix represents
total sugar percent by weight.) ................................................... 78

viii

Figure 1.1

Figure 1.2

Figure 1.3

Figure 1.4

Figure 1.5

Figure 2.1

Figure 2.2

Figure 2.3

Figure 2.4

LIST OF FIGURES

Polymorphs of carbon: graphite (left) and Diamond (right)... ...5

Interrelationships of mass balance, nucleation kinetics, crystal growth
kinetics, and crystal size distribution (CSD) during batch
crystallization............................................... .. ...7

Energy level diagram of IR absorbance, Raman scattering (Stokes) and
Rayleigh scattering. Anti-Stokes lines are not shown as their intensities
are usually much less than that of the Stokes lines. So and S; are the
ground electronic state and the ﬁrst excited electronic state respectively.
V0 to V3 represent different vibrational and rotational levels of a
moleculeIO

Schematic of attenuated total reﬂection. The AT R crystal has a much
higher refractive index than that of the sample, thus allow total internal
reﬂection. (91's the incident angle... ...11

Energy level diagram of ﬂuorescence. So to S2 are the dz'ﬂkrent electronic
states. Three vibrational states are also shown at each electronic
state13

Schematic of experimental setup for solution crystallization of
acetaminophen. a) Jacketed crystallizer connected with two circulators;
b) Thermocouple; c) Condenser; d) Glass stirrer; e) Ramon probe coupled
withﬁber optic cable... ...20

Diﬁerent heating and cooling proﬁles applied to the acetaminophen
solution prior to crystallization. H-a: Low heating at40 ~50 °C followed by
rapid cooling to 0°C; H—b: High heating at boiling point of solution
followed by rapid cooling to 0°C; H-c: High heating at boiling point of
solutionfollowed by slower cooling to 0 °C... ...23

Raman spectra for solid sample of acetaminophen polymorph within the
ranges of1150 ~ 1300cm'1 (a) and 1530 ~ 1 700cm“1 (b) ....................... 25

Diﬂerent morphologies observed during solution crystallization of
acetaminophen polymorphs: (a) Form 1 from ethanol; (b) Form I from
ethanol/water; (c) From I from water; (d) From 11 from ethanol/water; (e)
From 11 from ethanol/water; (I) From T from ethanol/water ................. 27

ix

Figure 2.5

Figure 2.6

Figure 2. 7

Figure 2.8

Figure 3.1

Figure 3.2

Figure 3.3

Figure 3.4

Figure 3.5

Figure 3.6

Figure 3. 7

Figure 4.1

Figure 4.2

Solubility curve of acetaminophen form I in an ethanol/H20 solvent
mixture28

Solubility curve of acetaminophen polymorph in ethanolsH20 solvent
mixture ratio of1:3.. .........30

Raman spectra collected during form acetaminophen polymorph T
transformation in slurry in 25% ethanol in water at 0 °C... ....35

Scores plots of principal components for acetaminophen polymorph
transformation. (a) Spectra in PC], PC2 and PC3 3-dimensional space;
(b) Spectra in PC2, PC3 and PC4 3-diemtional space... ...36

Raman spectra collected during crystallization and transformation of
acetaminophen polymorphs in 25% ethanol in water at 0°C. a) Solution
before crystallization, b) Slurry of form T, c) Slurry of form 11,d) Slurry
of form I. The peak at 1326cm is used for normalization... ...46

Crystallization and transformation proﬁle represented by peak height at
123 7cm-I. For the peak height proﬁle from un-normalized spectra, a
factor of 0.00] has been multiplied to bring it to the same scale as the
normalizedproﬁle.........................................................................48

The four principal components extracted from the MCR analysis (left)
compared with Raman spectra collected for acetaminophen polymorph or
solution (right). 51

Scores plot for principal components from MCR analysis of
acetaminophen polymorph crystallization and transformation ............... 52

Relative composition proﬁles calculated from proﬁles of normalized peak
height at 123 7cm PCI and PCZ by MCR analysis. (a) Form T to II
transformation; (b) Form 11 to 1 transformation... .. ...53

IR spectra of acetaminophen in 25 % ethanol in water. The region from
1000 ~1800cm-1 was used to build a calibration model by PLS
regresszon55

Concentration proﬁle by AT R-F T [R compared with the PC3 proﬁle from
MCR analysis of Raman spectra... .. .. . .. ... ...55

Schematic of the experimental setup: LABMAX automatic reactor coupled
with an in situ ATR-FTIRprobe... ...61

ATR-FTIR spectra of succinic acid aqueous solution at 60°C. The two
peaks exploited in peak area ratio are indicated by arrows ................... 65

Figure 4.3

Figure 4.4

Figure 4.5

Figure 4.6

Figure 4. 7

Figure 5.1

Figure 5.2

Figure 5.3

Figure 5. 4.

Figure 5.5

Solubility curve of succinic acid in H20. R* is solubility represented by
the ratio of the peak area of the 18064675ch band to that of the
1671~1494cm'l band in the inﬁared spectrum... .....66

Optimal cooling proﬁles (top) and CSD (bottom) for succicinic acid
crystallization from aqueous solution with diﬁerent amount of seeding.
Initial concentration is 19%(w/w), seed size is 355-425 ,um, and agitation
rate is 400rpm... ...69

Optimal cooling proﬁle (top) and CSD (bottom) for succicinic acid
crystallization from aqueous solution with diﬁrerent seed sizes. Initial
concentration is 19% (w/w), seed amount is 5g, and agitation rate is

Optimal cooling proﬁle (top) and CSD (top) for succinic acid
crystallization from aqueous solution with diﬂerent agitation speeds.
Initial concentration is 19%(w/w), seed amount is 5g, seed size is 355-

Optimal cooling proﬁle (top) and CSD (bottom) for succicinic acid
crystallization from aqueous solution showing the eﬂect of acetic acid
present as an impurity (2% mole basis). Initial concentration is
19%(w/w), seed amount is 5g, seed size is 355-425,wn, and agitation rate

Chemical structures of pyranine (left) and carminic acid (right) ............ 81

Fluorescence spectrum of 20ppm pyranine (top, peak at 441nm is due to
the protonated form, peak at 517nm is due to deprotonated form.) and
30ppm carminic acid (bottom peak at 419nm is due to protonated form,
peak at 591nm is due to deprotonated form.) in sucrose solution at

PIR of pyranine in sucrose solutions at diﬂerent temperature with diﬂerent
amount of fructose and glucose. Brix represents total sugar percent by
weight. Data at other compositions and temperatures show the same
trend, thus not shown here... .....85

PIR of carminic acid in sucrose solutions at dijferent temperature with
diﬁ'erent amount of ﬁuctose and glucose. Brix represents total sugar
percent by weight. Data at other compositions and temperatures show the
same trend, thus not shown here... ...86

Separate eﬂects of fructose and glucose on the PIR of pyranine in sucrose
solution at 90°C (total sugar content is 80%)... ...87

xi

Figure 5.6

Figure 5. 7

Figure 5.8
Figure 5.9

Figure 5.10

Anisotropy of pyranine fluorescence in sucrose solutions at diﬁerent
temperatures with different amounts of fructose and glucose. Brix
represents total sugar percent by weight. Data at other compositions and
temperatures show the same trend, thus not shown

here... ......88
Separate effects of fructose and glucose on the anisotropy r of pyranine in
sucrose solution at 90 °C (total sugar content is 80%)... .....89
Raman spectra of 8 7% fructose solutions at diﬁ'erent temperatures ........ 92
Raman spectra of 8 7% glucose solutions at diﬂerent temperatures ........ 93

Raman spectra of 8 7% tagatose solutions at different temperatures ........ 94

xii

Chapter 1

INTRODUCTION

Crystallization is not only a widely observed phenomenon but also an important
separation and puriﬁcation unit operation in the food, pharmaceutical, agrochernical and
specialty chemical industries. Control of the crystallization process is often needed to
produce a crystalline product with desired properties such as particle size distribution,
shape or solid-state modiﬁcation. These goals can only be achieved successfully by
adequate knowledge of the thermodynamic and kinetic properties of the crystallization
system combined with efﬁcient process monitoring and control. Coupled with ﬁber
optics and employing Chemometrics software, on line spectroscopic methods offer great
advantages in crystallization process control, from non—invasive sampling and fast
turnaround to the possibility of process control based on chemical information.

The goal of this work is to demonstrate the application of a variety of spectroscopic
methods for the monitoring and control of crystallization processes. The emphases are on
the simpliﬁcation of method development procedures and the fundamental insights to the

crystallization kinetics that can be gained by these spectroscopic studies.

1.1 Crystallization

Crystallization is a phase separation process where molecules aggregate together to form

a crystalline phase. The process is governed by both thermodynamic and kinetic

properties of the crystallization system. Control of operational parameters must be based
on proper information of the system, such as the solubility, supersaturation level and

desupersaturation rate and pathway”.

1.1.1 Supersaturation

A supersaturated solution has a greater chemical potential than the corresponding
saturated solution at equilibrium at the same temperature. This difference in chemical
potential is the driving force for crystallization. Supersaturation level and
desupersaturation rate affect the kinetics of nucleation and crystal growth, thus control

the crystallization outcomes. Theoretically, supersaturation is deﬁned asl’2
— / - In a
5 — All RT — 0—,; (1.1)

Where Au is the chemical potential difference between the supersaturated solution and

the saturated solution at a given temperature, a is activity of the supersaturated solution

and (Bis the activity of the saturated solution. For practical applications, activity can

often be approximated by concentration, thus equation 1.1 becomes

Ssz (1.2)

c at:
where C is the concentration of the crystallization species in the supersaturated solution
and C* is the solubility. As IR absorbance is proportional to concentration, it is possible
to use IR spectroscopy with special sampling conﬁgurations to obtain the supersaturation

information during crystallization5 .

Superaturation can be generated by a number of methods such as cooling, evaporating the
solvent, adding anti-solvent or chemical reaction. The purpose is to reduce the solubility

of the system, thus creating the supersaturation.

1.1.2 Nucleation

Crystallization does not necessarily happen in a supersaturated solution. For
crystallization to occur, molecules have to aggregate together and form stable nuclei.
Such step is called nucleation, which is the beginning point of phase separation.
Controlling nucleation step is of practical importance in many crystallization processes,
as it can affect product qualities in terms particle size distribution or solid phase
modiﬁcation.

Generally, nucleation is categorized as homogeneous or heterogeneous nucleation.
Homogeneous nucleation occurs without the presence of any particles in solution, while
foreign particles (in the case of primary nucleation) or crystals of the crystallizing solute
(in the case of secondary nucleation) can induce heterogeneous nucleation. Even though
nucleation is rarely truly homogeneous, it still has theoretical signiﬁcance in
understanding the thermodynamic correlation with nucleation kinetics. On the other
hand, heterogeneous nucleation is of fundamental and practical impOrtance in many
crystallization systems. Seeding is a case of heterogeneous nucleation and is a common
practice in industry. By introducing particles of the crystallizing solute, nucleation can
be promoted to happen at a much lower supersaturation level than the corresponding
homogeneous nucleation, thus the nucleation kinetics is signiﬁcantly altered. In other

cases, the presence of one solid-state modiﬁcation of a substance may facilitate the

nucleation of another more stable modiﬁcation, thus promote polymorph transformation.

An empirical power law model best describes nucleation ratez.

B = kgW‘M; (AC") (13)
Where B is the nucleation rate, K 'N is the nucleation rate constant, Wis the agitation rate,
MT is the suspension density, and AC is supersaturation level. It is clearly shown that
nucleation kinetics are affected by many process parameters. In order to obtain desired
crystalline product properties, it is necessary to monitor and control relevant operational
conditions to control nucleation kinetics. The study on acetaminophen polymorph

crystallization in this work is a good example of controlling crystal solid-state

modiﬁcation by controlling the nucleation conditions.

1. 1.3 Crystal Growth

Once stable nuclei are formed by nucleation, molecules will continue to integrate into the
nuclei and form bigger particles. This is the crystal growth step. The crystal growth rate
depends on the nature and strength of the interaction between the crystal surface and
solute molecules in the solution. A number of models have been proposed to describe
crystal growth kinetics”. For example, continuous or normal growth models apply to
growth on the kink sites of the crystal edges, while screw dislocation and two-
dimensional nucleation models apply to growth at a very smooth crystal surface.
Operational parameters such as temperature, supersaturation level, solvent and impurity

etc a1 all affect nucleation rate by affecting the interactions at the solid-liquid interface.

1.2 Significance of Control in Crystallization
As both nucleation and crystal growth are kinetic processes that regulate the solid-state
product properties such as particle size distribution and solid phase modiﬁcation, process

monitoring and control by robust analytical methods is often critical for desired product

quality.

1. 2. 1 Polymorphism

Polymorphism is a phenomenon wherein a substance with the same chemical identity can
crystallize into different crystal structures. It is an important issue because different
polyrnorphs have different physical properties such as thermodynamic stability,
solubility, density, melting point, reactivity and other mechanical properties6'lo. Carbon
is a good example of polymorphism. Both graphite and diamond are made of carbon, but
carbon atoms are arranged differently in the solid state. Carbon atoms are in sheets of six
member rings in graphite, while they are in tetrahedral cubes in diamond (Figure 1.1). As

a result, they exhibit very different appearances, hardnesses and are used for different

purposes.

%‘

 

Figure 1.1 Polymorph of carbon: graphite (left) and Diamond (right)

Because different polymorphs have different stabilities, transformation from one
polymorph to another may occur depending on the manufacturing or storage conditions.
This transformation could be beneﬁcial or deleterious depending on the application of the
polymorphic material. Polymorphic materials can often be encountered in many
industries including food, pharmaceutical and chemical, and many examples have shown
that crystallization procedure determines the polymorph form. Efficient control of
desired polymorph forms by controlling the crystallization process is of great importance.
Any successful control would rely on accurate characterization of polymorph forms. The

”'13, including powder X-ray diffraction

traditional characterization methods are off line
(XRD), differential scanning calorimetry (DSC) or solid state NMR, and thus have
problems with sample transfer and preparation. On line vibrational spectroscopic
techniques can detect the different vibration states due to different molecular arrangement

in polymorph material, thus offering the possibility of characterizing and control

polymorph form during crystallization process.

1.2.2 Particle Size Distribution

Another important property of a crystalline product is particle size. For example, small
particles are oﬁen desirable for active pharmaceutical ingredients, because they have a
faster dissolution rate and can be distributed into tablets more evenly. But as
intermediates during manufacturing process, bigger particles may be preferred to
facilitate down stream process efﬁciency such as during ﬁltration and drying.

As both nucleation and crystal growth will consume solute molecules in the solution

phase, their relative rates affect the resulting particle size distribution: higher nucleation

over crystal grth rate results in a large amount of small particles, while higher crystal
growth over nucleation rate results in fewer, larger. Both nucleation and crystal growth
kinetics are very complicated in practice and very hard if not impossible to be accurately
modeled for real industrial batch crystallization processes”. Operational conditions such
as cooling rates or impurity level may vary from batch to batch. Even within the same
batch, factors controlling nucleation and crystal growth kinetics are constantly changing
non-linearly. The diagram in ﬁgure 1.2 represents the complex crystallization events and
their interactions. In general, higher supersaturation favors nucleation more than it does
crystal growth, thus particle size distribution can be adjusted by controlling the

supersaturation level throughout the crystallization process.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

. Growth
kinetics ;
Mass Su ersaturation '
balance P ’ Population CSD
balance
A Nucleation
—’ kinetics —‘_3
Crystal ‘
area

 

 

 

Figure 1.2 Interrelationships of mass balance, nucleation kinetics, crystal growth

kinetics, and crystal size distribution (CSD) during batch crystallization

1.2.3 Inhibition of Crystallization by Impurity

Impurities are widely known to inﬂuence crystallization from solutions for virtually all
compounds. Impurities can affect both nucleation and crystal growth either
thermodynamically or kinetically”. Under thermodynamic control, impurities are
believed to affect the solubility of the target compound (e.g., the common ion effect).
When solubility of the crystallization species is increased by addition of impurity,
supersaturation level is reduced, and crystallization will be hindered or completely
inhibited. Under kinetic control, impurities can inhibit crystallization through different
mechanisms. They can increase solution viscosity signiﬁcantly and slow crystallization
by slowing mass transfer. They can also disrupt the formation of solution aggregates that
are the basis for nucleation by assuming molecular conformations different than the one
in crystalline form. Impurity molecules can also be absorbed to the target molecule as
“tailor made” impurities and disrupt the crystal growth process. It is also possible that

more than one of the aforementioned mechanisms can be operative simultaneously”.

1.3 Spectroscopic Methods for Crystallization Monitoring and Control

The need for process monitor and control by robust analytical methods in pharmaceutical
industry has recently been emphasized by the Process Analytical Technology (PAT)
initiative by F DA”, which proposes the use of non-invasive, on-line techniques to
measure physicochemical properties of drug materials during processing.

Process measurement tools based on spectroscopic techniques such as IR, Raman
ﬂuorescence and NIR offer obvious advantages15 '17. With advances in instrumentation,

modern spectroscopic techniques are compact, versatile and fast. New sampling

technology allows in situ, on line detection. Not only chemical information can be
inferred from the spectroscopic measurement, physical information may also be obtained,
which is particularly valuable in characterization of manufacturing processes and for
reliable prediction of end product properties early in the manufacturing processes. The
development, acceptance and ready access to chemometric data processing techniques
also justiﬁes the application of on line spectroscopic techniques.

Although the scientiﬁc base for PAT is not new, the application of PAT techniques to
crystallization process monitor and control has only received great interest in recent
years. There are still concerns regarding to method simplicity, robustness and relevant
information that can be extracted from spectroscopic measurement. This work aims to
provide some practical examples where process spectroscopic techniques can contribute

to the crystallization process development and control.

I. 3. 1 Raman Spectroscopy for On Line Polymorph Characterization

As a vibrational spectroscopic technique, Raman spectroscopy is useful for determining
the chemical composition and certain physical properties of samples in both qualitative
and quantitative ways. Because Raman scattering and IR absorbance are different
physical processes (Figure 1.3), they have different selection rules for vibrational modes
in a molecule, thus Raman can often provide complimentary information to what IR
provides”. Although IR is widely used in process applications such as monitoring
reaction kinetics or quality control of reaction materials, Raman has only recently been
introduced to industrial process applications thanks to the improvement in optical

ﬁltering design, detector sensitivity, readily available laser source, and most of all, the

ﬁber optic sampling probes. The use of ﬁber optic probes is very desirable for
crystallization process monitoring and control, since analysis of the solid phase property
can be measured on line and in situ without the need of ﬁltration. Since Raman
spectroscopy contains rich molecular structure information in the ﬁngerprint region, it

can be used for the characterization and quantitative analysis of polymorph materials on

 

 

 

 

 

 

 

 

 

 

 

line'm.
51
Virtual State
0.00.0...O.......‘OOOOOOOOOO‘TOOOOOOO
V3
v
A v v:
80 I V0
IR Rayleigh Raman (Stokes)

Figure 1.3 Energy level diagram of IR absorbance, Raman scattering (Stokes) and
Rayleigh scattering. Anti-Stokes lines are not shown as their intensities are usually much
less than that of the Stokes lines. So and S; are the ground electronic state and the ﬁrst
excited electronic state respectively. V0 to V3 represent different vibrational and

rotational levels of a molecule.

10

1.3.2 A TR-FTIR Spectroscopy for On Line Concentration Measurement

Infrared spectroscopy can measure compositions in solids, liquids, gases and many
hetero-phase systems because almost all chemical substances show characteristic patterns
of absorption of infrared light. IR has long been used in process monitoring and is
continually ﬁnding applications in expanding ﬁelds. Attenuated total reﬂection (ATR) is
a very useful sampling technique, which brings the measurement to the sample instead of
transferring the sample to the instrument”. Figure 1.4 shows the schematic of an ATR

device.

Incident IR 9 I Reﬂected IR

 
 
  

ATR Crystal

Sample Tls

   

<— Evanescent
wave

Figure 1.4 Schematic of attenuated total reflection. ATR crystal has a much higher
refractive index than that of the sample, thus allow total internal reflection. 0 is the

incident angle.

11

The infrared radiation is transferred through the ATR crystal - a transmitting medium
with high reﬁactive index. By controlling the incident angle 9 above the critical angle,
total reﬂection occurs within the ATR crystal. When the ATR is in direct contact with a
sample, the electric ﬁeld of the infrared radiation (the evanescent wave) penetrates a short
distance into the sample beyond the ATR surface. This electric ﬁeld interacts with the
absorbing sample and the reﬂected radiation is attenuated at wavenumbers that are

characteristic to the sample. The effective ‘pathlength’ depends on the penetration depth

d

d _ ,1
(”771(st 0- (772 “702)“2

 

(1.4)

Where 7» is the wavelength of the infrared radiation, n1 and 112 are the reﬂective indices
of the ATR crystal and sample, respectively, 9 is the incidence angle. This pathlength is
intrinsically small, and multiple sample-contact reﬂections are commonly used to
increase the observed absorbance due to stronger attenuation.

The small pathlength has proven to be advantageous to monitoring crystallization.
Because the particles in crystallization slurry cannot get into the micron level pathlength,
it is possible to sample the solution phase concentration in situ without the interference

from the solid crystalss.

12

1.3.3 Probing the Microscopic Properties of Crystallization Solution by Fluorescence

Fluorescence is an emission process in which molecules are excited by absorbing UV or
visible light and then relax to the ground state, releasing the excess energy as photons
(Figure 1.5). Intensity change or peak shift in ﬂuorescence spectroscopy can be
correlated to environment change such as pH, temperature and solvent effects.
Polarization anisotropy measurement can also be used to probe the mobility of the
ﬂuorescence species and microscopic viscosity in solution.

The ﬂuorescence spectrometer used in this study was not conﬁgured for on line
measurement. Nonetheless, it is the purpose of this study to show the type of insight that
can be possibly gained in crystallization inhibition. The approach can be modiﬁed easily

to on line application with commercially available ﬁber optic probes.

 

 

E2 1 V1

 

 

 

 

 

 

 

 

 

 

 

 

 

A IC F NR

Figure 1.5 Energy level diagram of ﬂuorescence. E0 to E 2 are the diﬂerent electronic
states. Three vibrational states are also shown at each electronic state. A:Absorption;

IC:InternaI conversion; F :F luorescence; NR:Non-radiative route.

13

1. 3. 4 Chemometrics Data Analysis

One of the key advantages of process spectroscopy is its rich information about the
fundamental chemistry of the process. But this could lead to information overload
without proper data analysis, because spectra data tend to have high degree of
redundancy. Besides, due to a lack of selectivity in the spectrum, it is often hard to ﬁnd a
single parameter to best predict concentration or composition. Chemometric techniques
such as PCA (principal component analysis) or PLS (partial least square) are well suited
for improving the effectiveness of on line spectroscopic methodslnz. They can be used
for extracting information to develop multivariate calibration, process control, data
visualization and clustering patterns. The monitoring of polymorph transformations in
this study demonstrated the substantial capability of Chemometrics-enhanced Raman

spectroscopic method in the industrial crystallization environment.

1.4 Overview of This Work

The next four chapters will cover the application examples of spectroscopic techniques to
the characterization, monitoring and control of crystallization process. Chapter 2 and 3
mainly focus on the application of Raman spectroscopy for polymorph crystallization and
transformation monitoring and control, using acetaminophen as the model compound.
Chapter 4 demonstrates the use of ATR-FT IR for particle size control during
crystallization with an emphasis on developing a simple, non-model based control
strategy. Chapter 5 deals with the critical insights into sucrose crystallization inhibition

by non-sucrose sugars using ﬂuorescence and Raman techniques.

14

1.5
(1)
(2)

(3)

(4)

(5)

(6)

(7)

(8)

(9)

(10)

(11)

References
Mullin, J. W. Crystallization; Butterworth-Heinemann Ltd.: Oxford, 1993.

Myerson, A. S., Handbook of Industrial Crystallization, 2nd Ed. Myerson, A. S,
Butterworth-Heinemann, Boston, 2002.

Tavare, N. S., Industrial Crystallization, Plenum Press, New York and London,
1995.

Sohnel, O.; Garside, J. Precipitation: Basic Principles and Industrial Applications;
Butterworth-Heinemann Ltd: Oxford, 1992.

Dunuwila, D.D.; Berglund, K.A, J. Cryst. Growth, 1997, 179, 185.

McCrone, W. C., Polymorphism In Physics and Chemistry of the Organic Solid
State, Fox, D.; Labes, M.M.; Weissberger, A., Eds., Interscience, New York, 1965,
Vol 11, 726-767.

Bym, S.R.; Pfeiffer, R.R.; Stowell, J.G., Solid-State Chemistry of Drugs, 2“d ed.,
SCCI, Inc., West Lafayette, 1999.

Bym, Stephen; Pfeiffer, Ralph; Ganey, Michael; Hoiberg, Charles; Poochikian,
Guirag, Pharmaceutical Research, 1995, 12(7), 945-54.

Dunitz, Jack D.; Bernstein, Joel, Accounts of Chemical Research, 1995, 28(4), 193-
200.

Gavezzotti, A.; Filippini, G., Journal of the American Chemical Society, 1995,
117(49), 12299-305.

Giron, D., Thermochimica Acta, 1995, 248, 1-59.

(12) Yu, Lian; Reutzel, Susan M.; Stephenson, Gregory A., Pharmaceutical Science &

Technology Today, 1998, 1(3), 118-127.

(13) Threlfall, Terence L., Analyst, 1995, 120(10), 2435-60.

(14)

US. FDA, Draft Guidance for Industry, PAT A Framework for Innovative

Pharmaceutical Manufacturing and Quality Assurance, August 2003.

(15) Taylor, Lynne S., American Pharmaceutical Review, 2001, 4(4), 60,62-64,66-67.

(16)

Adar, Fran; Geiger, Ruth; Noonan, Jon., Applied Spectroscopy Reviews, 1997, 32(1
& 2), 45-101 .

15

(17) Chalmers, J .M., Spectroscopy in Process Analysis, CRC Press, 2000.

(18) Colthup, N.B.; Daly, L.H.; Wiberly, S.E., Introduction to Infrared and Raman
Spectroscopy. 3"d ed., Academic Press, Boston, 1990.

(19) Wang, F., Watcher, J .A.; Antosz, F.J.; Berglund, K. A., Org. Process R & D. 2000,
4(5), 391-395.

(20) Starbuck, C.; Spartalis, A.; Wai, L.; Wang, J .; Fernandez, P.; Lindemann, C. M.;
Zhou, G.X.; Ge, Z., Crystal Growth & Design, 2002, 2 (6), 515-522.

(21) Mirabella, F.M., Internal Reﬂection Spectroscopy, Theory and Applications, Mercel
Dekker, Inc.: New York, 1993.

(22) Miller, C.E., Journal of Chemometrics, 2000, 14, 513-528.

16

Chapter 2

CONTROL OF CRYSTALLIZATION OF ACETAMINOPHEN
POLYMORPHS USING RAMAN SPECTROSCOPY

It is well known that process parameters can affect the nucleation kinetics, which in turn
can affect the polymorph form during batch crystallization. It is critical to identify the
controlling parameters in order to reproducibly crystallize the desired polymorph form.
Acetaminophen is chosen as the model compound for current study. Thermal history,
which often is neglected in organic species crystallization, is found to play a signiﬁcant
role in the outcome of polymorph forms during crystallization of acetaminophen. Two
metastable forms of acetaminophen can crystallize out after the solution is subjected to
high temperature for a prolonged time. High temperature may have changed the solution
structure prior to crystallization thus changed the nucleation kinetics of different
polymorphs. A Raman spectrometer was used on line and in situ to characterize the

polymorph forms and monitor the transformation process

2.1 Introduction

Acetaminophen is a common antipyretic and analgesic pharmaceutical ingredient. It has
three polymorphs and a number of pseudopolymorphs7. The three polymorphs are
monoclinic form I, orthorhombic form 11 and monoclinic form IH. Form 1 is the
thermodynamically stable form at room temperature and is the form crystallized from

solution phase without any control. Form HI has only been crystallized ﬁom melted form

17

I, and it is highly unstable- it has only been stabilized at room temperature in a binary
mixture containing a tablet binder hydroxypropylmethylcellulose'. F orm II has been
reported to undergo plastic deformation upon compression, thus it may facilitate the
tabletting processz. Acetaminophen has also been found to form solvates and adducts
with hydrogen bond acceptors”. Fachaux et a1.3 ’ 4 prepared a dioxane hemisolvate which
shows an improved compaction property after desolvation to monoclinic form. Recent
studiesg’9 found a trihydrate form T can lose water upon drying and become self-
micronized, which may be used to produce small particles of acetaminophen without
milling.

The orthorhombic form 11 and the trihydrate form T are of particular interest because of
their possible processing advantages over the monoclinic form 1. Recently there have
been some studies on the crystallization of the metastable forms from solutions”, which
is the more desirable process than melt crystallization. Yet the processes still need to be
optimized to better control the crystallization of the desired polymorphs. Form II was
crystallized from ethanol using form II seed that was prepared from melt

“"2. It was found that the purity of form H from seeded crystallization

crystallization
highly depends on the quality of the seed, and it is not always easy to obtain a seed with
100% form 11. The yield from this procedure is relatively low because limited amounts
of material can be dissolved in solution prior to crystallization in order to ensure form II.
Form T was only crystallized at small scale (~10ml), and it requires the pre-ﬁltration of

the solution before crystallization is carried out. The yield for form T was also low,

partially due to the low solubility of acetaminophen in H20 at high temperature, partially

l8

due to the low success rate in controlling the crystallization of form T (only 5 out of 40
attempts gave form T).

The goal of current study is to optimize the solution crystallization processes for
acetaminophen form 11 and form T without the need for seeding or pre-ﬁltration of
solution. More importantly, we can identify process parameters that are important to the
crystallization of different polymorph forms by studying the solution crystallization of
acetaminophen using on line Raman spectroscopy. This would improve the general

knowledge of and add one more dimension of control to polymorph crystallization.

2.2 Experimental Section

Acetaminophen (98% pure) was obtained from Aldrich and used as received. Solvents
used during crystallization were reagent grade ethanol and distilled, deionized water.
Crystallization experiments were carried out in a 1L jacketed crystallizer, equipped with
a condenser, a thermocouple and an anchor-type glass stirrer (Figure 2.1). Stirring speed
was 500rpm. Two circulators were used to provide heating and cooling ﬂuids,
respectively. At the end of heating period, the crystallizer was disconnected from the
heating circulator and the heating ﬂuid was emptied out. The crystallizer was then
connected with the cooling circulator.

A Hololab 5000 Raman spectrometer from Kaiser Optic Systems was used for in situ
monitoring the crystallization and transformation process, as well as the solid-state
characterization of different polymorphs. The spectrometer is equipped with a 785nm

laser and coupled with an immersion probe through ﬁber optic cables.

19

 

 

 

 

 

 

| 6
ha I I
II II I \
<— ‘ To Raman
_ To I spectrometer
crrculators
—>

Figure 2.1. Schematic of experimental setup for solution crystallization of
acetaminophen. a) Jacketed crystallizer connected with two circulators; b) Thermal

couple; c) Condenser; d) Glass stirrer; e) Ramon probe coupled with ﬁber optic cable.

20

2. 2. 1 Solubility Measurement of Different Polymorphs

The solubility of acetaminophen polymorph I was determined by gravimetric analysis.
Mixtures of ethanol and water were used as solvents. The slurry was stirred at
equilibrium for 30 minutes before a small sample of the solution was drawn from the
slurry using a ﬁitted pipette. The wet sample was accurately weighed before it was dried
in the oven to evaporate the solvent, then the dried sample was weighed again and the
solubility was calculated.

To evaluate the relative stability, the solubilities of acetaminophen form I, II and
trihydrate were also determined in the mixture solvent of ethanol:water ratio of 1:3
(based on weight). A small sample of solution was drawn from the slurry at equilibrium
using a ﬁitted pipette. Three samples were taken for each polymorph at —10°C, -5°C,
0°C, 5°C and 10°C. The samples were weighed and dried in oven overnight and then
analyzed by HPLC. Due to the fast transformation of trihydrate polymorph to monoclinic

polymorph at high temperature, no sample was taken for trihydrate at 10°C.

2. 2.2 Polymorph Crystallization of A cetaminophen by Cooling

The crystallization of acetaminophen was carried out in pure water and in mixtures of
ethanol and water. The concentration range of acetaminophen solutions studied were: 1-
3 g/ 100g water in water, 7-28g/ 100g solvent in 25% aqueous ethanol solvent and 20-
80g/ 100g solvent in 50% aqueous ethanol solvent. Crystallization with an initial
concentration higher than the upper limit would result in very thick slurries after

nucleation, thus prevent an efﬁcient mixing even at high stirring rate.

21

Solutions with different initial concentrations were subjected to three different preheating
procedures prior to rapid cooling using the coolant at 0°C (Figure 2.2): a) Low
overheating proﬁle: Solution was heated at 40°C (water as solvent) or 50°C (ethanol and
water mixture as solvent) for 30minutes before cooling; b) Solution was vigorously
reﬂuxed at boiling point for 30 minutes and then rapidly cooled to 0°C at about
6°C/minute; c) Solution was vigorously reﬂuxed at boiling point for 30 minutes and then
cooled to 0°C at about 2°C/minute. The onset of nucleation was detected both visually

and by the Raman spectrometer. The polymorphic form of acetaminophen was also

identiﬁed using Raman spectroscopy in the crystallization slurry.

2. 2.3 Transformation of A cetaminophen Form T in Slurry

The slurry of acetaminophen trihydrate in 25% aqueous ethanol solvent was monitored at
0°C using an immersion Raman probe. The resulting spectra were analyzed by Principal
Component Analysis (PCA) within HoloReact software ﬁ'om Kaiser Optical Systems to

extract the transformation proﬁle.

22

90

 

 

 

 

 

 

mar-e3 annals-swam: tram rum-mmnwi
55 "l
8
O
2
3 4o-
0
ii
Ii
*-
15
-10 -
0 20 40 60 80

Time (minute)

Figure 2. 2. Diﬂrerent heating and cooling proﬁles applied to the acetaminophen solution
prior to crystallization. H-a: Low heating at40 ~50 °C followed by rapid cooling to 0 °C;
H-b: High heating at boiling point of solution followed by rapid cooling to 0°C; H—c:

High heating at boiling point of solution followed by slower cooling to 0 °C.

23

2.3 Results and Discussion

2.3 1 Characterization of A cetaminophen Polymorphs

Form 1, form II and form T were obtained from solution crystallization. The
characterization has been previously reported using offline analytical techniques such as
DSC and XRD8'15. While DSC can differentiate different forms of acetaminophen from
melt crystallization, it often times fails to tell the difference between form I and form II
from solution crystallizationl 1. Moreover, forms H and T from solution crystallization
tend to transform to stable form I rapidly at room temperature, especially when there is
solvent residue left on the crystal. Thus, extra care has to be taken to prevent
transformation during sampling and subsequent sample preparation such as ﬁltration,
drying and sample mounting, which makes it inconvenient to use the ofﬂine
characterization methods as routine characterization tools.

Raman spectroscopy with an immersion probe can be conveniently used to identify the
polymorph form of acetaminophen in the crystallization slurry without any sampling
procedure. Raman spectra for solid samples of different acetaminophen polymorphs are

shown in Figure 2.3.

24

2500

 

2000

 

500

 

 

 

1150 1200 1250 1300
Ramon Shift (cm")

(a)

 

 

 

 

 

1530 1580 1630 1680
Ramon Shift (cm“)

0))

Figure 2. 3. Raman spectra for solid sample of acetaminophen polymorph within ranges

of1150 ~ 1300cm" (a) and 1530 ~ 1 700cm" (b)

25

Signiﬁcant differences can be observed within the ranges of 1150 ~13OOCm-1 and 1530 ~
17000m-1. These differences in Raman characteristics reﬂect different conﬁgurations of
acetaminophen molecules in the crystalline state caused by intermolecular hydrogen
bonding. The slurry of different acetaminophen polymorphs in ethanol and water showed
similar spectroscopic characteristics to those of the corresponding solid sample, thus
different polymorph was identiﬁed in the crystallization slurry using Raman
spectroscopy.

The three polymorphs of acetaminophen also exhibit different morphologies (Figure 2.4).
Thus, optical microscopy was used as a complimentary technique to conﬁrm the
polymorph form of acetaminophen, although it should not be used as the sole technique
for identiﬁcation purpose. The same polymorph can present different morphologies
depending on the solvent system and other crystallization conditions applied, different
polymorphs can adopt the same morphology as well. Besides, microscopy does not give

quantitative information for a sample of mixed polymorphs.

26

 

(d) (e) (0

Figure 2. 4. Diﬁérent morphologies observed during solution crystallization of
acetaminophen polymorphs: (a) Form I from ethanol; (b) Form I ﬁ'om ethanol/water; (c)
F arm I from water; (d) F arm IIﬁ'om ethanol/water; (e) Form 11 from ethanol/water; (ﬂ

Form T from ethanol/water.

27

2. 3.2 Solubility of Acetaminophen Polymorphs

Acetaminophen has only limited solubility in water even at high temperature (Figure 2.5),
thus water is not a good recrystallization solvent for acetaminophen. Its solubility
increases with ethanol percentage, with a maximum solubility in 75% of ethanol in water.
Twenty-ﬁve percent ethanol in water was chosen for the subsequent solubility
measurements for different polymorphs because acetaminophen has relatively high
solubility at high temperature and low solubility at about 0°C. More importantly, all
three modiﬁcations of acetaminophen, forms I, II and T were able to crystallize out in

this solvent by applying different crystallization conditions.

40 r
1—0— 100% ethanol
{+75% ethanol

30 i— -I- 50% ethanol

l- 4- 25% ethanol

Lair—0% ethan-

 

 
  
  
   

Solubility (we/n.

10

 

 

o 20 40
Temperature (°C)

Figure 2. 5. Solubility curve of acetaminophen form I in ethanol/H20 mixture solvent.

28

The solubility of acetaminophen polymorphs in 25% aqueous ethanol can be used to
evaluate their relative thermodynamic stability. During sampling, modiﬁcations H and T
were stirred in slurry at low temperature for 10 ~ 30 minutes before transforming to
polymorph I. In addition, the solubility and the differences in solubility of I, H and T
modiﬁcation are large enough to allow accurate sampling and subsequent analysis by
HPLC.

As discussed by Terry Threlfall” and Wolf Beckmann”, the relative stability of true
polymorphs observed through solubility measurement is independent of the solvent
system used. True polymorphs are the ones with exactly the same chemical identity, e. g,
form I and form 11 of acetaminophen. Thus, the thermodynamic stability of
acetaminophen form I and form 11 can be derived ﬁom their solubility diagram as shown
in Figure 2.6. The monoclinic polymorph I is less soluble than the orthorhombic form II,
thus it is the more stable modiﬁcation within the temperature range of —10°C to 10°C.
While solvates and adducts are considered pseudopolymorphs, their relative solubility
can change depending on the solvent system used, and the stability assessment based on
solubility diagram should be carried out in the desired solvent system. In 25% of ethanol
in water, the trihydrate modiﬁcation T is most soluble, thus least stable above 0°C, but its
solubility decreases rapidly below 0°C and it is enantiotropic with respect to polymorph
II. The transition temperature is estimated to be about -9°C. The stability of polymorph
T is greatly increased at low temperature. Slurry samples of polymorph T in ethanol and
water can be stored at -—lO°C for weeks without inducing transformation. These current
observations agree with the results of the study of the trihydrate form in water by

Peterson et a1.

29

 

 

Concentration (wt%)
to

 

 

 

-1 5 -5 5 1 5
Temperature (°C)

Figure 2. 6. Solubility curve of acetaminophen polymorphs in ethanol:H20 ratio of 1:3.

As the solubility difference between the metastable polymorph and the stable polymorph
decreases with temperature, the driving force of transformation is signiﬁcantly reduced at
temperatures close to 0°C and the metastable polymorphs are more likely to remain in
slurry before transformation occurs. In other words, a low nucleation temperature will
favor the crystallization of the metastable polymorph. Later crystallization experiments
proved the chance of obtaining polymorph II or T is greatly increased when the onset of
crystallization is controlled to occur at temperatures below 10°C. As nucleation is a
kinetically controlled process, in order to crystallize the metastable polymorphs, it is
necessary to adjust the process parameters such as solvent composition and to control the
nucleation temperature to change the nucleation kinetics of different polymorphs of

acetaminophen.

30

2. 3.3 E [feet of Thermal History on Acetaminophen Polymorph Crystallization

The crystallization conditions for obtaining different polymorphs of acetaminophen are
summarized in Table 2.1. Among all the operational parameters studied for the unseeded
batch crystallization of acetaminophen in mixed solvent of ethanol and water, thermal
history of the solution is found to be the most signiﬁcant factor in determining which
polymorph can be crystallized. The three heating and cooling proﬁles described in the
experimental section are represented as H-a, H-b and H-c. Compared to the solutions
heated at temperature much lower than boiling point, solutions that were heated at boiling
point prior to cooling showed a signiﬁcantly lower nucleation temperature or metastable
point temperature. As this nucleation temperature extends below 5°C, the metastable

modiﬁcations H and T have a much better chance to crystallize than polymorph I.

Table 2.1. Effect of thermal history and solvent composition on polymorph of
acetaminophen obtained from unseeded batch crystallization. H-a, H-b and H-c represent

different heating and cooling proﬁles.

 

 

 

 

 

Solvent Initial concentration of Acetaminophen Polymorph
Composition solution
Ethanolﬂzo (g/100g solvent) H-a H-b H-c
1:1 20-80 I II 11
1:3 7-28 1 T 11
0: 1 1-3 I T T

 

 

 

 

 

 

 

31

Similar effects were observed for solution crystallization of vanillin by Hussain etc”,
which showed “high overheating” increases the metastable zone width (MZW) and leads
to the precipitation of metastable form. Although the effect of thermal history on MZW
has been known to aqueous solution crystallization of inorganic substances, vanillin
crystallization has been the only case reported for the effect of thermal history on
polymorph crystallization of organic substances. The mechanism causing this effect is
not known yet. One possible explanation is extensive heating changes the pre-nucleation
solution structure, although no difference in Raman or NMR spectroscopy were observed
for acetaminophen solutions subjected to different heating proﬁles. Another explanation
might be the solid heterogeneous particles in the solution are deactivated or destroyed,
thus the solution becomes more homogenous, which increases the activation energy for
nucleation and the onset of nucleation is “delayed” to the region of high supersaturation.
As high supersaturation favors metastable polymorph formation, there is a better chance
for metastable polymorphs to crystallize out of solution. While the mechanism for this
effect still needs to be investigated, it is worth to note that thermal history might be a very
important parameter to be considered in high throughput polymorph crystallization,
especially for systems with water as the solvent.

Cooling rate also affects the outcome of polymorph for acetaminophen crystallization in a
25% aqueous ethanol solvent. For a solution heated to the boiling point, fast cooling
results in the less stable trihydrate polymorph while slower cooling results in more stable
polymorph B even when nucleation occurs at the same temperature in both cases. This

might be caused by changes in solvent and solute molecular interactions during cooling,

32

fast cooling preserves the interactions at high temperature, while the same type of

interactions may have changed during the course of slow cooling.

2. 3.4 Effect of Solvent Composition on Acetaminophen Polymorph Crystallization

It is well known that solvent plays an important role in crystallization of polymorphs. As
Threlfalll7 pointed out, when crystallization occurs at a temperature far away from the
transition point temperature, polymorph crystallization is under thermodynamic control,
where only the thermodynamically stable polymorph will be crystallized out regardless of
the solvent used. But when crystallization occurs at a temperature close to the transition
point temperature, the solvent can affect the polymorph formed by changing the
crystallization kinetics of each polymorph.

The effects of solvent composition on crystallization of acetaminophen polymorphs are
also included in Table 2.1. For solutions that are not heated to the boiling point,
nucleation tends to occur at temperatures well above 10°C where crystallization is under
thermodynamic control and only the stable polymorph is obtained. While for solutions
that are heated to the boiling point, nucleation can be controlled to occur at temperatures
close to the transition point temperature of polymorph T and H where the composition of
solvent affects which polymorph can be crystallized out. As the percentage of water is
increased from 50% to 100%, the trihydrate polymorph becomes easier to crystallize than
polymorph H. The low solubility of acetaminophen in water reduces the transformation
rate from form T to more stable forms, thus form T is relatively stabilized.

As water content is decreased to 25%, even when the solution is heated to the boiling

point and nucleation is controlled to occur at temperature about 0°C, the trihydrate form

33

could not be obtained at all, and polymorph II was obtained with a very low possibility
(<5%). The increased solubility of acetaminophen in solvent with decreased content of
water promotes the solvent mediated polymorph transformation, which may be too fast
for the metastable form to be observed. Low water content also limits the interaction of

water and acetaminophen molecules, which in turn limits the formation of the trihydate

polymorph.

2. 3. 5 Transformation of Form T in Slurry

When acetaminophen trihydrate crystals are suspended in a 25% ethanol in water slurry,
a solvent mediated transformation occurs (Figure 2.7). Principal component analysis was
applied to the Raman spectra collected during this transformation. The process can be
clearly followed in the scores plot of different principal components (Figure 2.8). At
0°C, form T transforms to form H ﬁrst, then form II transforms to thermodynamically
stable form I. This transformation of form T to form I with form H as an intermediate is
observed for the ﬁrst time. It proves that the system follows the Ostwald’s rule of stages,
which states the system tends to go to the thermodynamically stable form by crystallizing
the least stable polymorph then transforming to the next metastable form until it reaches
the thermodynamically stable form. The intermediate stage with polymorph II can only
be observed with the right process condition, e.g. low temperature. At higher
temperature, the transformation process is too fast for the form H to be observed as
intermediate, which might be the reason it was not observed previously by other

researchers).

34

 

..... ..... CECE? rm

meson—...5 n

it.” 11. i”

        

V

e
\
....

e ....

m ...,A. ,

    

 

Raman Shift (cm'l)

Figure 2.7. Raman spectra collected during form acetaminophen polymorph T

ethanol in water at 0 °C.

transformation in slurry in 25 %

35

  

 

 

 

 

h .m
m. a
o n
m c
m. m.
p w
......... m a
‘\ W m
ssnnrrn nnnnn u ...... 0
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s.. s m S
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. . a .1
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Home m rrrrrrr . lllllllllllll \F m 3
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...m. u .. m. P
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._ "m . .m 1. m.
.. I W. C s
m ....... S “.2. r0! .1". m
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P P 2 C
m .m P.
m... m 2
.l a C
F W. P

 

 

36

2.4 Conclusions

This study shows that process parameters play a signiﬁcant role in the crystallization of
acetaminophen polymorphs. Thermal history coupled with solvent composition is the
primary parameter controlling the polymorph forms. Heating the solution to higher
temperature and cooling the solution down at a faster rate both favor the crystallization of
metastable polymorphs. Higher water content in solvent mixtures of ethanol and water
also favors the trihydrate form T, which has only limited stability at room temperature.
Both metastable form T and form 11 can be reproducibly crystallized ﬁ'om solution by
applying proper crystallization process parameters. Although often times neglected in the
crystallization of organic substances, it is believed that thermal history can have strong
impact on the outcome of polymorph forms.

Raman spectroscopy is also shown to be a convenient tool for on line and in situ
characterization of polymorph forms in slurry. Coupled with chemometric data analysis
methods such as PCA, it is easy to follow a complex polymorph transformation processes
without the need of building a calibration model. This would greatly reduce the amount
of work needed for method development during kinetic study of polymorph

transformation and reduce the project turnover time.

2.5 Acknowledgements

The authors wish to thank Kaiser Optical System for a support of the instrument and

software used in the study.

37

2.6

(1)

(2)

(3)

(4)

(5)

(6)

(7)

(8)

(9)

(10)

(11)

(12)

(13)
(14)

References

Rossi, Alessandra; Savioli, Alessandra; Bini, Marcella; Capsoni, Doretta;
Massarotti, Vincenzo; Bettini, Ruggero; Gazzaniga, Andrea; Sangalli, Maria
Edvige; Giordano, Ferdinando; Thermochimica acta, 2003; 406, 55-67.

Martino, P. Di; Guyot-Herrnann, A.-M.; Conﬂant, P.; Drache, M.; Guyot, J.-C.,
International Journal of Pharmaceutics, 1996, 128(1,2), 1-8.

Fachaux, J. M.; Hermann, A. M. Guyot; Guyot, J. C.; Conﬂant, P.; Drache, M.;
Huvenne, J. P.; Bouche, R., Powder Technology, 1995, 82(2), 123-128.

Fachaux, J. M.; Hermann, A. M. Guyot; Guyot, J. C.; Conﬂant, P.; Drache, M.;
Huvenne, J. P.; Bouche, R., Powder Technology, 1995, 82(2), 129-133.

Vrcelj, Ranko M.; Clark, Nathan 1. 8.; Kennedy, Alan R.; Sheen, David B.;
Shepherd, Evelyn E. A.; Sherwood, John N., Journal of Pharmaceutical Sciences,
2003, 92(10), 2069-2073.

Fabbiani, Francesca P. A.; Allan, David R.; Dawson, Alice; David, William I. F.;
McGregor, Pamela A.; Oswald, Iain D. H.; Parsons, Simon; Pulham, Colin R.,
Chemical Communications (Cambridge, United Kingdom), 2003, 24, 3004-3005.

Parkin, Andrew; Parsons, Simon; Pulham, Colin R., Acta Crystallographica,
Section E: Structure Reports Online, 2002, E58 (12), 1345-1347.

McGregor, Pamela A.; Allan, David R.; Parsons, Simon; Pulham, Colin R., Journal
of Pharmaceutical Sciences, 2002, 91(5), 1308-1311.

Peterson, Matthew L.; McIlroy, David; Shaw, Paul; Mustonen, J. Peter; Oliveira,
Mark; Almarsson, 0111., Crystal Growth & Design, 2003, 3(5), 761-765.

Haisa Masao; Kashino Setsuo; Maeda Hironobu, Acta Crystallography, 1974, B30,
2510-2512.

Nichols, Gary; Frampton, Christopher S., Journal of Pharmaceutical Sciences.
1998, 87, 684-693.

Al-Zoubi, N.; Nikolakakis, 1.; Malarnataris, S., Journal of Pharmacy and
Pharmacology, 2001, 54, 325-333.

Sacchetti, M., Journal of Thermal Analysis and Calorimetry, 2001, 63, 345-350.
Szelagiewicz, M.; Marcolli, C.; Cianferani, S.; Hard, A. P.; Vit, A.; Burkhard, A.;

Von Raumer, M.; Hoﬁneier, U. Ch.; Zilian, A.; Francotte, E.; Schenker, R., Journal
of Thermal Analysis and Calorimetry, 1999, 57(1), 23-43.

38

(15) Martino, P. Di; Conﬂant, P.; Drache, M.; Huvenne, J .-P., Guyot-Hermann, A.-M.;
Journal of Thermal Analysis, 1997, 48, 447-458.

(16) Threlfall, Terry, Organic Process Research & Development, 2000, 4, 384-390.
(17) Beckmann, Wolf, Trans IchemE, 1996, 74 (part A), 750-757.
(18) Hussain, Khalid; Thorsen, Gunnar; Malthe-Sorenssen, Dick, International

Symposium on Industrial Crystallization, 14th, Cambridge, United Kingdom, Sept,
1999, 631-643.

39

Chapter 3

IN-SITU MONITOR ACETAMINOPHEN POLYMORPH TRANSFORMATION
USING RAMAN AND ATR-FTIR SPECTROSCOPY

Raman and ATR-FTIR spectroscopic methods were used to in situ monitor the
acetaminophen polymorph crystallization and transformation process. While Raman
spectroscopy probes the polymorph form in the solid state, ATR-FTIR measures solution
phase concentration at the same time. The relevant kinetic information can be obtained
conveniently by such measurements. Under the conditions in this study, acetaminophen
polymorph transformation is limited by the crystallization rate of the more stable phase,
speciﬁcally by crystal growth rate of form 11 for form T to II transformation, and by
nucleation rate of form I for form II to I transformation. As a soft modeling chemometric
‘tool’, multivariate curve resolution was also demonstrated to be well suited for

extracting the crystallization and transformation proﬁle in a complex polymorph system.

3.1 Introduction

The signiﬁcance of polymorphism and polymorph transformation has been realized in
pharmaceutical and specialty chemical industries over recent years”. As different
polymorphs have different thermodynamic stability, transformation ﬁom a metastable
form to a stable form may occur in production processes, thus causing problems with
product quality or in process development. Understanding the kinetics of such
transformation processes can help to establish production conditions to better control the

40

desired polymorphic form”. Solvent mediated polymorph transformation is of practical
importance, as solution crystallization is oftentimes the preferred method for material
puriﬁcation and separation. Study of the proﬁle of solid phase polymorph composition
and solution phase concentration during transformation can help to identify the kinetic
processes that regulate the transformation rate. On line Raman and ATR-FTIR
spectroscopic methods are well suited for this type of study. In comparison to common
offline analytical techniques such as X-ray powder diffraction (XRPD) and differential
scanning calorimetry (DSC), Raman and ATR-FTIR spectroscopy can make
measurements in crystallization slurry in situ, thus do not require sample preparation.
This not only reduces possible sources of error, but also greatly simpliﬁes the
measurement procedure”.
On line spectroscopic methods are especially suitable for monitoring complex chemical
processes by providing rich information on chemical species. To take advantage of the
large amount of information contained in on-line spectroscopy, chemometric analyses are
often desirable to extract the relevant information in a simple and efficient way. For
example, multivariate curve resolution (MCR) is a particularly useful technique in
extracting estimates of pure component spectra and composition proﬁles from spectral
data matricesg.

Acetaminophen was used as the model compound in this study. Solution crystallization
of acetaminophen can produce three forms: Trihydrate form T, orthorhombic form II and

monoclinic form I, with form I being the most stable and form T being the least stable

form under the conditions of this study. The goal is to develop suitable methods for

41

probing the kinetic information during acetaminophen polymorph transformation by

using on line Raman and ATR-FTIR spectroscopic techniques.

3.2 Experimental methods

Acetaminophen (98% pure) was obtained ﬁ'om Aldrich and used as received. Solvents
used during crystallization were reagent grade ethanol and distilled, deionized water.
Crystallization experiments were carried out in a 1L jacketed crystallizer, equipped with
a condenser, a thermocouple and an anchor-type glass stirrer (Figure 2.1). Stirring speed
was 500rpm. Two circulators were used to provide heating and cooling ﬂuids,
respectively. At the end of heating period, the crystallizer was disconnected from the
heating circulator and the heating ﬂuid was emptied out. The crystallizer was then
connected with the cooling circulator.

A Hololab 5000 Rarnan spectrometer from Kaiser Optic Systems was used for in situ
monitoring the crystallization and transformation process, as well as the solid-state
characterization of different polymorphs in slurry. The spectrometer is equipped with a
785nm laser and coupled with an immersion probe through ﬁber optic cables.

The solution concentration proﬁle during crystallization and transformation was
monitored by a ReactIR 1000 FTIR spectrometer from ASI Mettler Toledo. An
attenuated total reﬂection (ATR) probe with diamond as the internal reﬂection element is
attached to the FTIR spectrometer. The system is purged by dry air, and the MCT

detector is cooled with liquid N2.

42

3. 2. 1 Polymorph Crystallization and Transformation of A cetaminophen

The crystallization of acetaminophen was carried out in 25% (by weight) ethanol in
water. The initial concentration of acetaminophen is 9.7g / 100g solvent. In order to
crystallize the metastable trihydrate polymorph T, the solution was vigorously reﬂuxed at

the boiling point for 30 minutes and then rapidly cooled to 0°C at about 6°C/minute.

After crystallization occurred at 0°C the slurry was continually stirred until the
transformation from form T to stable form I was complete. Both Raman and ATR-FTIR
spectra were recorded to in situ monitor the crystallization and transformation process
after the solution was cooled to 0°C, thus the spectra were collected at isothermal

conditions.

3. 2.2 Spectral Data Analysis

The Raman spectra collected during crystallization and transformation were subjected to
a peak height analysis and a multivariate curve resolution (MCR) analysis. The results
from both analyses were compared. The peak height proﬁle at 1237cm-1 after
normalized by peak at 1326cm'l was found to be able represent the transformation
process. The MCR analysis was performed using the HoloReact software package from
Kaiser Optical Systems. MCRlo analysis begins with principal component analysis
(PCA) to transform the original spectra data matrix D into a reproduced data matrix D*
with reduced number of factors (principal components). These factors describe the major
variances in data set, but they either are not physically meaningful or are mixed spectra of
more than one chemical species. The D* matrix is then analyzed by evolutionary factor

analysis (EPA) and alternating least square (ALS) method"). Some realistic boundary

43

conditions are applied to ALS analysis, e.g non-negative values for spectra and
concentration proﬁles. Eventually D* is transformed to SC, which is the product of
matrices of individual “pure” spectra S (the factor matrix) and of concentration proﬁles C
(the score matrix). D* and S have the same rank or dimension. Thus the advantage of
MCR analysis is to provide the concentration proﬁles of “pure” spectra, which are
physically meaningful. Mean centering and normalization were applied to all spectra as
data pretreatment.

Solution concentration changes were monitored by ATR-FT IR spectroscopy. A
calibration model was developed using partial least square (PLS) regression over the
spectral region 1150~18000m'l. The model is then applied to the IR spectra collected
during the crystallization and transformation processes. The purpose is to correlate the
solution phase concentration change with the polymorph transformation in the solid

phase.

3.3 Results and Discussion

Raman spectroscopy with an immersion probe can be conveniently used to identify the
polymorph form of acetaminophen in the crystallization slurry without any sampling
procedure. Raman spectra for solid samples of different acetaminophen polymorphs are
shown in Figure 2.2. Signiﬁcant differences can be observed within the ranges of 1150
~1300cm-1 and 1530 ~ 1700cm-1. These differences in Raman characteristics reﬂect
different conﬁgurations of acetaminophen molecules in the crystalline state caused by
intermolecular hydrogen bonding. The slurry of different acetaminophen polymorphs in

ethanol and water showed similar spectroscopic characteristics to those of the

44

corresponding solid sample, thus different polymorphs were identiﬁed in the

crystallization slurry using Raman.

3. 3. 1 In Situ Monitor Crystallization and Transformation of Acetaminophen

Polymorphs by Raman

When acetaminophen crystals of metastable polymorphs are suspended in a slurry,
solvent mediated transformation occurs. Figure 3.1 shows the Raman spectra collected
during crystallization and transformation. The system follows the Ostwald’s rule of
stages with the least stable form T crystallizing out ﬁrst. It then transforms to the next
metastable form H before it transforms to the stable form I. The knowledge of
transformation kinetics is important to deve10ping a reliable crystallization process.
Raman spectroscopy is a useful tool in such a kinetic study. By following the
transformation process, the transformation rate under different crystallization conditions
can be measured in situ. It is crucial to extract the correct kinetic information by
applying proper spectra analysis. For the acetaminophen polymorph transformation
process, two types of spectra analysis methods were applied and their results were

compared.

45

 

 

1 :1
v— E
El 2
@
:r 0.5 g
2.". E
‘2 51

O v

1100 1150 1200 1250 1300 1350
Raman Shift (cm'l)

Figure 3.1 Raman spectra collected during crystallization and transformation of
acetaminophen polymorph in 25% ethanol in water at 0°C. a) Solution before
crystallization; b) Slurry of form T; c) Slurry of form 11; d) Slurry of form 1. Peak at

I326cm‘] is used for normalization.

46

Raman spectra between 11500m'1~1300cm’l were ﬁrst analyzed by non-chemometric
methods. First, a few common spectra pre-processing techniques were applied including
base line correction, derivatives of original spectra and normalization of spectra. After
the pre-processing of spectra, peak height as well as peak height ratios at different
wavenumbers and peak areas under different regions were calculated over the course of
crystallization and transformation. Although pre-processing of spectra improves the
results to some degree, many transformation proﬁles extracted from the aforementioned
analyses still contain too much noise or have poor shapes, thus they cannot be used for
kinetic study. After using the peak at 1326cml to normalize the spectra, the peak height
at 1237cm'l exhibited a very smooth proﬁle and was used to extract transformation
kinetic (Figure 3.2). All three forms of acetaminophen exhibit peaks at 1326cm”1 with
about the same intensity. Only form I shows a strong peak at 1237cm", while form T
and form H have peaks shifted to different wavenurnber, thus exhibit different intensity at
1237cm'1. Normalization of the spectra with the peak at 1326cm'l greatly enhances the
peak intensity difference at 1237cm'l caused by structural differences in different
polymorphs while minimizing the effect of interferences such as baseline shift. Thus, the
quality of this analysis is greatly improved and it is possible to use such a simple spectra

analysis method to monitor the crystallization and transformation process.

47

2.50

 

 

 

 

 

 

 

2 00 i _‘ karma
Unnormalized
,5, 1.50 ~
:3
a
E 1.00
1:
O
'2 I
g 0.50
n.
0.00 7"';'F .
0.00 5.00 10.00 15.00 20.00
Time (hours)

Figure 3.2 Crystallization and transformation proﬁle represented by peak height at
123 7cm-1. For the peak height proﬁle from un-normalized spectra, a factor of 0. 001 has

been multiplied to bring it to the same scale as the normalized proﬁle.

48

Although it was possible to ﬁnd a good non-chernometric method for spectra analysis in
this case, it took a lot of effort in searching and optimizing this method, even though this
is a relatively simple system with no more than two components present in the solution at
the same time. For a system with more components or with peaks experiencing a high
degree of overlapping, this kind of analysis would be very hard if not impossible. As a
result, it is often necessary to use chemometric methods to extract relevant information
and improve the quality of spectra analysis. MCR is a very useful analysis method,
which can extract “pure” component concentration proﬁle from on-line spectra. Figure
3.3 shows the principal components resolved by MCR after the spectra were normalized
by the peak at 1326cm'l, and ﬁgure 3.4 plots the score (i.e. the so called “concentration”)
proﬁles of different components during the crystallization and transformation process.
The extracted principal component spectra converge very well with the Raman spectra of
solid state samples. PCl peaks at 1177, 1244, 1271 and 1290cm", which are
characteristics in Raman spectrum of trihydrate form T, it has the highest score between a
little after 0 and 2 hours when form T is ﬁrst crystallized out of solution. PC2 peaks at
1168, 1238, 1258 and 1278cm-l, which are due to the thermodynamically stable form I,
thus its highest score appears at the end of the process when all the metastable polymorph
II has transformed to form I. PC4 features peaks at 1171, 1222, 1247 and 1281cm-1,
which belong to the metastable form 11, its highest score appears between 5 and 15 hours
when form 11 is the dominant form in slurry. The third principal component PC3
resembles the spectrum of acetaminophen solution in 25% ethanol in water, but with
more spectral features resolved. It roughly represents the relative concentration proﬁle

during crystallization and transformation. It drops dramatically when nucleation happens

49

and trihydrate form T crystallizes at the beginning of the experiment, a second and a third
drop happen at the transformation from T to H and from H to I respectively due to
decrease in solubility of different forms. Compared to the peak height proﬁling method,
much less effort is needed to reach the optimum analysis results using the chemometric
method.

Both the peak height and the PC proﬁles can be used to calculate the polymorph
composition changes during the transformation. The results ﬁ'om both approaches agree
very well as shown in ﬁgure 3.5. Both transformation processes follow the typical
polymorph transformation proﬁle with induction, acceleration and decay periods.
Nucleation of the more stable form occurs and dominants in the induction period, while
crystal growth mainly happens in the acceleration period. It appears that transformation
rate from form T to form H is limited by crystal growth rate of form H, while the

transformation rate from form H to form I is limited nucleation of the stable form I.

50

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

8 i 2000 1—1

6 . Form T l E’.

I g

4 . l a.

2 . W a
O 1 . i 0

- - 2500 ...

10 , PC2 , Form I E.

G

a

5 » . .5
0 - 1 L 0

6 Solution 2500 '5‘

4 i W i

2 3
0 . . 0

1500

 

Form II

  

Kuwaiti]

 

 

 

 

0

 

 

HON-BO

150 1200 1250 1300 11501200 12501300
Raman shift (cm'l) Raman shift (cm'l)

Figure 3.3. The four pure component spectra extracted from the MCR analysis (left)

compared with Raman spectra collected for acetaminophen polymorph or solution

(right).

5

ﬂ

010 1 . 8190:
ﬂ * aPC2
1 P03 l
0.08 4 ’ ‘
. APC4
O
2 0.06 3
"6
2
O
O
U)

 

 

 

Time (hours)

Figure 3.4 Scores plot for principal components from MCR analysis of acetaminophen

polymorph crystallization and transformation.

52

 

120

 

100 . Peak height

80—
601

—PCI II

Form II (%)

204 T

 

 

 

3 4
Time (hours)

(a)

 

120

100
' so «
60 ~

     

0 Peak height
— P02

40~

Form ii (96)

20*

 

 

 

13 14 15 16
Time (hours)

Figure 3.5 Relative composition proﬁles calculated from proﬁles of normalized peak

height at 123 7cm'l, PCI and PC2 by MCR analysis. (a) Form T to II transformation; (b)

Form 11 to I transformation.

53

3. 3.2 In Situ Monitor Concentration Change During crystallization and

Transformation by A TR-FTIR

In order to characterize the acetaminophen polymorph transformation kinetic processes,
the solution phase concentration change was also monitored by ATR-FTHt spectroscopy.
ATR is a special sampling technique of FTR that is capable of measuring the solution
phase concentration in slurry without the interference ﬁom the solid phase. Another
chemometric method, PLS, was used to develop a calibration model using acetaminophen
solutions with known concentration. Figure 3.6 shows the region subjected to the PLS
analysis. The monitored concentration proﬁle is shown in Figure 3.7. According to
Davey and Cardew, the solvent mediated polymorph transformation process consists of
continuous dissolution of the less stable form and crystallization of the more stable form.
Solution concentration continually drops toward the solubility of the more stable form
early in the process for a dissolution-controlled transformation, while it remains close to
the solubility of the less stable form until the very end for a crystallization-controlled
transformation. It is apparent that both transformation from form T to II and from forrn H
to I are the extreme cases of crystallization-controlled transformation, since the solution
concentration stays constant during the transformation process and only starts dropping

when the less stable form has all transformed (test by Rarnan).

54

0.8
0.7 .
0.6 4
0.5 4
0.4 J
0.3 ~

0.2 1 k

0.1

 

 

Absorbance (AU)

 

 

 

1800 1600 1400 1200 1000

Wavenumber (cm-1)

Figure 3.6 IR spectra of acetaminophen in 25% ethanol in water. The region from 1000

~18000m-1 was used to build calibration model by PLS regression.

 

 

 

 

 

 

 

 

 

 

5 7 0.06

1 Concentration by

1 I502 « 0.05
44 *
2° ~ 0.04
§ 3 » « 0.03 g
s i
c - 0.02
is
O
o « 0.01

1 0.00
o 4 8 12 16 20

Time (hours)

Figure 3. 7. Concentration proﬁle by A TR-F TIR compared with PC2 proﬁle from MCR

analysis of Raman spectra.

55

3.4 Conclusions

Both Raman and ATR-FTm spectroscopies are suitable for on line monitoring of solvent
mediated acetaminophen polymorph transformation processes. These two techniques
provide complimentary information about solid phase polymorph form and solution
phase concentration respectively, which are important in understanding the solvent
mediated transformation pathways and kinetic features of such a process. Under the
conditions studied, the concentration proﬁle measured by ATR-FTIR reveals that
acetaminophen polymorph transformation rate is controlled by the crystallization rate of
the more stable form, while the polymorph composition proﬁles extracted from Raman
spectra identify nucleation and crystal growth are the rate limiting process for form H to I
and form T to II transformation, respectively. Applying chemometric analysis methods
such as MCR to on line spectral data greatly simpliﬁed the method development process

for polymorph transformation monitoring.

56

3.5 References
(1) Bym, S. R., Solid-state Chemistry of Drugs, Academic Press: New York, 1982

(2) Chemburkar, Sanjay R.; et al. Organic Process Research & Development, 2000,
4(5), 413-417.

(3) Vippagunta, S. R.; Brittain, H. G.; Grant, D. J. W., Advanced Drug Delivery
Reviews, 2001, 48(1), 3-26.

(4) Rodriguez-Homedo, Nair; Murphy, Denette., Journal of Pharmaceutical Sciences,
1999, 88(7), 651-660.

(5) Wang, Fang; Wachter, John A.; Antosz, Frederick J .; Berglund, Kris A., Organic
Process Research & Development, 2000, 4(5), 391-395.

(6) Starbuck, Cindy; Spartalis, Angela; Wai, Lawrence; Wang, Jian; Fernandez, Paul;
Lindemann, Christopher M.; Zhou, George X.; Ge, Zhihong, Crystal Growth &
Design, 2002, 2(6), 515-522.

(7) Yu, Lian; Reutzel, Susan M.; Stephenson, Gregory A., Pharmaceutical Science &
Technology Today, 1998, 1(3), 118-127.

(8) Head, Teresa; Rydzak, Jim, American Pharmaceutical Review, 2003, 6(1), 78-80,
82-84.

(9) Miller, Charles E., Journal of Chemometrics, 2000, 14: 513-528.

(10) Schoonover, Jon R; Marx, Rob; Zhang, Shuliang, Applied Spectroscopy, 2003, 57,
154A-170A.

57

Chapter 4

ATR-FTIR FOR DETERMINING OPTIMAL COOLING CURVES FOR

BATCH CRYSTALLIZATION OF SUCCINIC ACID”

* Crystal Growth & Design, 2002, 2, 449-452.

The temperature proﬁle applied during batch cooling crystallization affects the
supersaturation level, which in turn affects the crystal size distribution. It is possible, in
principle, to calculate the optimal cooling proﬁle; however, the nucleation and grth
kinetics are rarely known to the degree of accuracy necessary for this calculation. The
current study demonstrates an alternative approach to determination of the optimal
cooling proﬁle without any prior knowledge of kinetic data or subsequent modeling. An
attenuated total reﬂectance-Fourier transform infrared (ATR-FTIR) spectrometer was
used to monitor the supersaturation level during batch cooling crystallization. The ATR-
FTHK was interfaced to a LABMAX® automatic reactor system that was used in a
feedback mode to control the cooling rate so that supersaturation level remained close to
the solubility throughout the cooling process. The resulting temperature proﬁle
corresponds to the optimal operating conditions for the maximum in the mean crystal

size.

4.1 Introduction

Batch crystallization is a widely practiced unit operation in specialty chemical industry

such as pharmaceutical and agrochemical industry. An optimized crystallization process

58

is often required to produce a desired crystal size distribution with good reproducibility
from batch to batch”. In case of batch cooling crystallization, it is well known that the
cooling rate affects supersaturation proﬁle and has strong inﬂuence on the crystal size

distribution.3

In order to grow bigger particles, the rate at which supersaturation is
generated by cooling should match the rate of desupersaturation by crystal growth
throughout the course of the batch cooling crystallization. Nucleation should be
suppressed by keeping the supersaturation level close to solubility. However, the
determination of the most appropriate cooling proﬁle in industrial crystallization remains
a challenge since nucleation and crystal growth kinetic data for a particular system are
often not available. Moreover, even when tentative kinetic models are available for a
system, the parameters are often highly sensitive to operation conditions such as agitation
speed and fail to accurately predict crystallization process when operation conditions
change.4

In the present work a method to obtain the optimized cooling proﬁle using ATR-FT IR
spectroscopy is presented. The method does not require kinetic models thereby reducing
possible errors caused by inaccurate kinetic expressions. ATR-FT H1 has been shown
previously to be an excellent technique for in situ monitoring supersaturation in a
crystallizing slurry.5'7 ATR-FTIR was coupled with a LABMAX® automatic reactor

system to obtain optimal cooling proﬁles in a fast, easy and ﬂexible way. The succinic

acid-water system was chosen to demonstrate the practical aspects of this approach.

59

4.2 Experimental Section

Succinic acid was purchased ﬁ'om Aldrich. All aqueous solutions of succinic acid were
prepared with with deionized water. The schematic of the experimental setup is shown in
Figure 4.1. All cooling crystallizations of succinic acid were conducted in the l-L glass
reactor of the Mettler-Toledo LABMAX® System. Two electronic units, the thermostat
unit and the controller unit, control the reactor. The thermostat unit controls the reactor
and jacket temperature based on the feedback from the temperature sensors in the reactor
and in the jacket. Temperature can be controlled either by specifying an endpoint
temperature or by keeping a predetermined temperature difference between the reactor
and the jacket. The thermostat unit also controls the stirrer speed. The controller unit
controls external sensors and devices such as pH electrode, pump and balance, which are
not included in this study. In situ measurement of supersaturation was performed using
ASI-Reacthl 000 spectrometer equipped with an immersion probe equipped with a
diamond internal reﬂection element. The probe was purged with dry air.

The communication between the REACTIR and the LABMAX® is provided by an
interface that transmits values such as reactor temperature and supersaturation level. This
feature is particularly convenient for the present study since it allows real time feedback
control based on the supersaturation value measured by the Reactm. The LABMAX®
can decrease the cooling rate when the supersaturation level is close to the preselected
upper limit or increase the cooling rate when the supersaturation level is close to the

solubility.

60

 

> llllll

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

. Thermometer
V ' ' REACTIR
Thermal urnt 1000 modules
Controller unit ‘
a
9 V
LabMax PC REACTIR PC

 

 

 

 

 

 

 

i 1

 

LabMax-REACTIR serial interface

Figure 4.1 Schematic of the experimental setup: LABMAX automatic reactor coupled

with in situ A T R-F T IR probe.

61

4. 2. 1 Calibration and Solubility Curve Measurement

The calibration curve and operating solubility curve can be combined and obtained ﬁom
a simple experiment described as following. Succinic acid in excess of solubility was
mixed with water and kept at temperatures ranging from 10 to 60°C. At each
temperature, the slurry was stirred for about 1 hour until equilibrium was reached, then
10 spectra were recorded in situ. Different slurry samples were used to make sure the
solid concentration does not affect the solution phase IR signal. Spectra were analyzed

and appropriate features were used to build the calibration and solubility curve.

4. 2.2 Determination of Cooling Proﬁles

A 19% (w/w) (total weight of the solution was 520g) succinic acid aqueous solution was
kept at 60°C for 10 minutes to make sure the solid was dissolved. The solution was
cooled at 2°C/minute until it reached the solubility according to the ReactIR reading.
Seed crystals were introduced followed by immediate measurement of supersaturation
level and temperature. The LABMAX® controlled the cooling rate of the reactor by
adjusting the jacket temperature throughout the course of crystallization so that cooling
was as fast as possible following the solubility limit closely. After the slurry temperature
reached 10°C, the crystallization was ended and the crystals were ﬁltered and dried. The
crystal size distribution of the resulted particles was analyzed using a set of Tyler RX-86

sieves.

62

4.3 Results and Discussion

4. 3. 1 Calibration and Solubility Curve

The common procedure involved in the measurement of supersaturation during

'0 A set of solutions with known

crystallization was carried out in two steps.8'
concentration and temperature were prepared. Ht spectra were taken and various IR
bands were used to build the calibration curve. As recognized by Lewiner and many
others,""8 for ATR-FTIR to be used in an industrial context the calibration procedure
should be fast, efﬁcient and easy. Since crystallization is operated within the metastable
zone, supersaturation level or how far away the solution phase is beyond the solubility
limit is the key information for crystallization process control. With these considerations
in mind, the band ratioing procedure used by Dunuwila and coworkers.5'7 was employed.
The approach simpliﬁes the calibration and solubility measurement by combining them
into one step, requires the minimum amount of measurements, and has good accuracy.

Figure 4.2 shows typical IR spectra of aqueous succinic acid solution at different
concentration at 60°C. A number of IR features are available for use in calibration. The
band ratioing technique minimized the effects caused by instrumental drift. Such
instrumental drift is likely to be associated with IR source instability as well as
conﬁguration changes in the optical conduits. Different peak height ratios and peak area
ratios were tested and it was determined that peak area ratio (R) of 1806~1675cm~1 over
167 l~1494cm-1 gave the best result in terms of reproducibility and accuracy. The reason

could be that more features are hidden under the seemingly single peak at l710cm-l, and

the peak area reﬂects the complexity better than peak intensity at a speciﬁc wavelength.

63

_ Peak area under 1806 ~1675 cm ’1 (4.1)

R _
Peak area under 1671 ~ 1494 cm ‘1

 

Since this R is a function of both temperature and concentration, it implicitly reﬂects the
supersaturation level during crystallization. No further correlation between this R and the
absolute concentration is necessary. Thus R as deﬁned in Equation. 1 was used directly
in the calibration and solubility curve development that follows. A PLS calibration was
also tried for calibration. Although a higher order of precision could be obtained during
one run, this advantage was lost from run to run due to the aforementioned instrumental
drift.

Figure 4.3 shows the solubility curve of succinic acid in terms of peak area ratio R. The
small error bars substantiate the reproducibility of the approach. The supersaturation

level (S) can then be expressed as

S: R(T) (4.2)
R"(7’)

Where R (T) is the real time peak intensity ratio at temperature T, R*(T) is the peak

intensity ratio at solubility at the same temperature T.

64

 

(L7

(16

(15-

  

0J1-

Absorbance

l

0.2— v<C=0>

0&3-

   

 

 

 

 

OJ r . r
1700 1500 1300 1100

Wavenumber (cm")

Figure 4.2 AT R-F TIR spectra of succinic acid aqueous solution at 60°C. The two peaks

exploited in peak area ratio are indicated by arrows.

65

 

 

 

 

 

10 20 (I) 40 50 60
Tarpermle (C)

Figure 4.3 Solubility curve of succinic acid in H20. R“ is solubility represented by ratio
of the peak area of the I806~1675cm'I band to that of the I67I~1494cm” band in the

infrared spectrum.

66

4. 3.2 Determination of Optimal Cooling Proﬁles

The optimal cooling proﬁle was chosen based on the criterion to maximize the product
weight mean size by suppressing the supersaturation level low enough to encourage
crystal grth over nucleation. In the controlled cooling crystallizations of succinic acid
studied, the initial solution conditions and other operational parameters were kept
constant unless otherwise speciﬁed. The values used were 19% (w/w) initial
concentration (total weight of solution is 520g), agitation rate of 400rpm, seed size of
355-425um, and seed mass of 0.5g.

Figure 4.4 shows the effect of seed amount on cooling proﬁle. Faster cooling can be
applied with more seeds due to the presence of a greater seed surface area available for
crystal growth. Excessive seeding results in a decrease in the mean particle size, which
has been also observed by Lewiner et a1.9 While excessive seeding can result in reduced
ﬁnal mean particle due simply the lack of sufﬁcient solute resources in solution to grow,
the excess crystals present can also cause secondary nucleation. As noted by Lewiner et
al.9, even under a controlled low level of supersaturation, secondary nucleation is not as
effectively suppressed as primary nucleation. Since secondary nucleation depends on
magma density, more seeding may promote secondary nucleation and lead to a smaller
crystal size distribution.

Figure 4.5 shows that the cooling proﬁle varies with the size of seed applied when the
same amount of seed in mass is used. When seed size increases the number of particles
decrease as does the overall surface area available for growth. This results in a slower
overall mass deposition rate, which requires a slower cooling rate and longer

crystallization time.

67

Agitation rate is another important factor that affects the cooling proﬁle as shown in
Figure 4.6. Higher agitation rates improve mass transfer which should increase crystal
growth. An agitation rate of 300rpm was not enough to suspend the seeds and product
crystals during crystallization. Inhomogeneous slurry conditions reduce the efﬁciency of
seeding and cause severe agglomeration. Comparison of the mean particle size with
different agitation rates also reveals that excessive agitation causes more secondary
nucleation and reduction in crystal size distribution. The effect of agitation is therefore a
compromise between mass transfer, particle suspension, and secondary nucleation.

The cooling proﬁle is also affected by the purity of solution. Acetic acid is a tailor made
impurity that reduces the crystal growth rate of succinic acid. One g (2mol%) of acetic
acid was added to solution and reduced the crystal grth rate which required the
slowing the cooling process signiﬁcantly in order to maintain constant supersaturation as
shown in Figure 4.7. It is known that impurities are often adsorbed selectively on to
different crystal faces and retard their growth rate. In an industrial context,
crystallization is often operated in the presence many different impurities, hence it is
important to be able to develop the optimal cooling proﬁle according to the impurity
proﬁle of the raw material. The present approach is capable of identifying the optimal
cooling curve when impurities are present. As expected, the crystal size distribution

wasn’t affected as long as the supersaturation was kept at a constant low level.

68

Temperature (C)

 

 

 

 

0 8 : +59 seed
; +0.59 seed
()5 +0059 seed

Weight (%)
9
¢

9
N

 

 

 

 

O 02 0.4 0.6 0.8 1 1.2
CrystalSlze(mm)

Figure 4.4 Optimal cooling proﬁles (top) and CSD (bottom) for succicinic acid
crystallization ﬁom aqueous solution with diﬁhrent amount of seeding. Initial

concentration is 19%(w/w), seed size is 355-425 ,urn, and agitation rate is 400rpm.

69

     
 

 

 

 

 

 

   
 
 
   
   

 

 

 

Q 45
§
*5 35 “
E 25 4 1; 5006001111
,2 ll —Seed size
1 355-4251m
15 i WSeedsize
l l <180un
5 A T b _ _-—_.--._._ _ ._ _ __
0 100 200 300
Time (ninute)
1
f—a—seedsizesoe
0-3 “ ' 600um
S5 —6—Seed size
2.70.6 - 3554251111
'5. t—A—Seed size
30.4 4 <1801rn
; .
0.2 1
0 g L. ... ..

 

 

0 0.2 0.4 0.6 0.8 1 1.2
Crystal Size (m)

Figure 4.5 Optimal cooling proﬁle (top) and CSD (bottom) for succicinic acid

crystallization ﬁom aqueous solution with dijferent seed sizes. Initial concentration is

19% (w/w), seed amount is 5g, and agitation rate is 400rpm.

70

Temperature (C)

 

 

O 1 00 200 300 400

 

 

 

 

Time (minute)
1
o 8 . 31316006111
3 I —e—400rpm
e. 0.6 - j
1:" l—A—300rpm
.9
0
g 0.4 151
0.2 -
0 ~ .

 

 

 

o 0.2 0.4 0.6 0.8 1 1.2
CrystalSize (mm)

Figure 4.6 Optimal cooling proﬁle (top) and CSD (top) for succinic acid crystallization
ﬁ‘om aqueous solution with diﬂerent agitation speeds. Initial concentration is 19%(w/w),

seed amount is 5g, seed size is 355-425 ,um.

71

  
 

 

 

 

 

 

 
 
    

 

 

..454
o
E l
B35 '1
e I
E25 7 _.———_—__
.2 i—1g Acetic acid
1
15 L r—NoAceticacid
5 , 4 ..__-,-____-_ﬁ-,.
0 50 100 150 200 250
Tims(rrlnute)
1 _
0,3 j—a—ingeiicacid
:5 l . .
v ‘ -6—Nvoet1cacrd
E 0.6 l
9 -
é’ 0.4 «
0.2
0 a — —

 

0 0.2 0.4 0.6 0.8 1 1.2
Crystal Size (m)

Figure 4. 7 Optimal cooling proﬁle (top) and CSD (bottom) for succicinic acid
crystallization from aqueous solution showing the eﬂect of acetic acid present as an
impurity (2% mole basis). Initial concentration is 19%(w/w), seed amount is 5g, seed

size is 355 -425 ,um, and agitation rate is 400rpm.

72

4.4 Conclusions

The experimental results for cooling crystallization of succinic acid demonstrate the
capability of the ATR-FTm technique in crystallization process control and design. The
calibration procedure is straightforward and efﬁcient, which is preferred by industrial
users. By coupling an ATR-FTIR with a LABMAX® automatic reactor system optimal,
the optimal cooling curves for crystallization under different operation condition can be
easily obtained by in situ feedback control of the supersaturation level close to solubility.
No single optimal cooling proﬁle works under different operational conditions, and it is
critical that the technique used for optimal cooling proﬁle determination must be easy,
fast and ﬂexible. The approach presented here does not rely on kinetic models and
precludes the error caused by inaccurate kinetic data. Since kinetic data are usually not
known in industrial applications, this method is suggested as a simple tool in process
design and control of crystallization processes when a cooling policy needs to be

determined.

4.5 Acknowledgements
The authors wish to thank ASI Mettler-Toledo for a support of the instruments used in
the study. Additional support ﬁom the Center for New Plant Products and Processes at

Michigan State University is also appreciated.

73

4.6

(1).

(2).
(3).
(4)-
(5).

(6).
(7)-

(8).
(9).
(10).

References
Goren, H.; Hammond, R. B.; Lai,x.; Mougin, P.; Roberts, K. J .; Savelli, N.;
Thomas, A.; White, G.; Williams, H. L.; Wilkinson, D.; Baker, M.; Dale, D.; Erk,
P.; Latharn, D.; Merriﬁeld, D.; Oliver, R.; Roberts, D.; Wood, W.; Ford, L.; Hoyle,
W., Eds.; Pilot Plants and Scale-up of Chemical Processes H; Special publication
236; Royal Society of Chemistry: Cambrige, 1999; pp40-61.
Tanguy, D.; Marchal, P. Trans IchemE 1996, vol. 74, Part A, 715-722.
Mullin, J. W. Cyrstallization, 3Id ed.; Butterworth-Heinemann: Oxford, 1997.
Bohlin, M., Rasmuson, A., C. Can. J. Chem. Eng. 1992, 70, 120-126.

Dunuwila, D. D.; Carroll, L., B.; Berglund, K., A. J. Cryst. Growth 1994, 137, 561-
568.

Dunuwila, D. D.; Berglund, K., A. J. Cryst. Growth 1997, 179, 185-193.

Dunuwila, D. D., An Investigation of ,the Feasibility of Using in situ ATR F TIR
Spectroscopy in the Measurement of Crystallization Phenomena for Research and
Development of Batch Crystallization Process, Dissertation, Department of
Chemical Engineering, Michigan State University, 1996.

Lewiner, F.; Fevotte, G.; Klein, J. P.; Puel, F. Chem. Eng. Sci. 2001, 56, 2069-2084.
Lewiner, F.; Fevotte, G.; Klein, J. P.; Puel, F. J. Cryst. Growth 2001, 226, 348-362.

Togkalidou, T.; Fujiwara, M. J. Cryst. Growth 2001, 231, 534-543.

74

Chapter 5

STUDY OF THE INHIBITION EFFECT OF FRUCTOSE AND GLUCOSE ON
SUCROSE CRYSTALLIZATION*

*Study using Raman spectroscopy was conducted by Javier Falcon.
The inhibition of sucrose crystallization by non-sucrose sugars is of particular interest to
product development in the food industry. To investigate the possible mechanisms of this
inhibition, ﬂuorescence and Raman spectroscopic techniques were applied to investigate
the thermodynamic and kinetic properties of sucrose solutions with fructose and glucose
as impurities. When the overall sugar content is kept the same, both ﬁ'uctose and glucose
decrease the viscosity of sucrose solutions, while glucose increases the water activity and
ﬁ'uctose decreases the water activity. Fructose takes on an open chain form, a molecular
conformation different than the one in crystalline form, at a much higher concentration
than glucose does. It is proposed that fructose and glucose inhibit crystallization of
sucrose in different ways. It is also demonstrated that ﬂuorescence and Raman
spectroscopic techniques can be adapted to monitor and control industrial sucrose

crystallizations at high concentration and high temperature.

5.1 Introduction

The crystallization of sucrose is important in the processing and storage of food
products.1 While a great deal of research has been performed on bulk crystallization of
sucrose that is practiced in the production of sucrose from cane and beets, less attention

has been devoted to crystallization conditions that exist during food processing. One of

75

the main differences in conditions is the intentional addition of non-sucrose sugars (in
particular fructose and glucose) to sucrose solutions to inhibit crystallization in many
food products.

Impurities are widely known to inﬂuence crystallization from solutions for virtually all
compounds. While several research groups have studied the effect of ﬁ'uctose and

“5 no study has been done on their effect on

glucose on the crystal growth of sucrose,
nucleation, which is an equally important aspect in the crystallization process. Impurities
can affect nucleation either thermodynamically or kinetically. Under thermodynamic
control, impurities are believed to affect the solubility of the target compound (e.g. the
common ion effect). Under kinetic control, impurities can alter mass transfer related
physical properties such as viscosity, or they can disrupt the formation of solution
aggregates that are the basis for nucleation by assuming molecular conformations
different than the one in crystalline form, or by absorbing to the target molecule as “tailor
made” impurities. It is also possible that more than one of the aforementioned
mechanisms can be operative simultaneously.

In the present work the inhibition of sucrose crystallization by ﬁuctose and glucose was
studied by ﬂuorescence and Raman spectroscopic methods. These two spectroscopic
techniques can be used to investigate the solution properties such as water activity,
viscosity and molecular conformation, all of which can affect crystallization processes
signiﬁcantly. Both techniques can be applied to process monitor and quality control at

industrial environment. Insights on the mechanisms of sucrose crystallization inhibition

are expected to guide new food formulation development.

76

5.2 Experimental Section

The sucrose, glucose and ﬁ'uctose were provided by the Kellogg Company, Battle Creek,
MI and used as received. Pyranine and carminic acid were obtained from Sigma.
Distilled, deionized water was used in all the measurements. A F luorolog-2 from Jobin-
Yvon Inc. was used for all the steady state ﬂuorescence measurement. A Hololab 1000
series from Kaiser Optical Systems Inc. equipped with 632.8nm laser was used for the
collection of Raman spectra. Solution temperatures were regulated using a CN76000

temperature controller (Omega Engineering, Inc., Stamford, CT).

5. 2. 1 Sample Preparation for Steady State Fluorescence Measurements

Pyranine and carminic acid were chosen as the ﬂuorescence probe molecules. Different
amounts of sucrose, glucose, ﬁ'uctose and water were carefully weighted out according to
Table 5.1, then lml of pyranine or carminic acid stock solution was added to each of the
mixture sugar solution to get 20ppm pyranine or 30ppm carminic acid. The sample was
brieﬂy heated to the boiling point to dissolve all sugar solids, about 3ml of this clear
solution was transferred to a quartz cuvette in a temperature controlled sample holder in
the ﬂuorescence spectrometer. Ten to ﬁfteen minutes were allowed for the sample to
reach equilibrium temperature before a measurement was carried out. The predetermined
temperatures were every 10°C interval between 70°C and 110°C. Each sample was
freshly made up at each temperature to minimize the effect of sucrose hydrolysis at high
temperatures. A 4 factor experimental design was applied to optimize the number of

replicate runs for each sample at each temperature.

77

Table 5.1. Composition of Samples for ﬂuorescence Measurement (Brix represents total

 

 

 

 

 

 

 

 

 

 

sugar percent by weight.)

BRIX SUCROSE (9) FRUCTOSE (g) GLUCOSE (9) Water (9)
87 47 0 40 1 2
87 47 20 20 1 2
87 47 40 0 1 2

83.5 43.5 0 40 1 5.5

83.5 43.5 20 20 15.5

83.5 43.5 40 0 1 5.5
80 40 O 40 1 9
80 40 20 20 1 9
80 40 40 0 1 9

 

 

 

 

 

 

 

78

5. 2.2 Steady State Fluorescence Measurement

Steady state ﬂuorescence spectra were recorded for all the samples in the study of water
activity in sucrose solutions. An excitation of 342nm was used for pyranine, with
emission spectra collected at 400-650nm. An excitation of 310nm was used for carminic
acid, with the emission spectra collected at 350-750nm.

Pyranine was used as the probe molecule in anisotropy measurements. Two polarization
ﬁlms were used before and after the sample chamber. Polarization emission spectra were
collected with afore mentioned parameters. Anisotropy (r) was calculated by the peak
intensity at 441nm with the ﬁlms in parallel position (In) and in perpendicular position
(Ii) as in Equation 1.

: Ill—1.1.
III+2I_L (5.1)

Viscosity (n) is correlated with anisotropy by equation 2.
r 0

RT
—=1+ g
r 77V

2' (5.2)

 

Where 1] is viscosity, T is the temperature of solution, to is the anisotropy with no
rotation, Rg is the gas constant, V is the volume of rotating unit and 1: is ﬂuorescence

lifetime. An increase in viscosity 1] would cause an increase in anisotropy r.

79

5. 2.3 Raman Spectra of Pure Sugar Solutions

Aqueous solutions of glucose, fructose and tagatose were prepared in lOO-mL three-
necked round bottom ﬂasks in concentrations ranging from 80 to 87 weight percent using
increments of 1.75 weight percent. For each speciﬁc solution at the speciﬁed
concentrations Raman spectra were obtained at 70, 80, 90, 100 and 110°C.

Individual, “fresh” solutions were heated to above 110°C in order to dissolve all the
crystalline material, using a heating rate of approximately 15 C/min. Once the solutions
became clear, the temperature was lowered to 110°C and the solutions were allowed to
equilibrate for ten minutes before a Raman spectrum was acquired. Next, the solutions
were rapidly cooled to the closest lower temperature, allowed to equilibrate and a Raman

spectrum was acquired. This cooling procedure was repeated at all temperatures.

5.3 Results and Discussion

5.3 1 Emission Properties of Fluorescence Probe Molecules

Pyranine and carminic acid were used as the ﬂuorescence molecules to probe the water
activity in sucrose solutions,7 and their chemical structures are shown in Figure 5.1. The
common characteristic for these two molecules is the equilibrium between protonated
form and deprotonated form when dissolved in water, the deprotonated form dominates
when there is enough water to interact with the probe molecule, while the protonated
form dominates when there isn’t enough water to interact with the probe molecule. Their
emission properties in sucrose aqueous solutions are shown in Figure 5 .2. When excited

at 342nm, pyranine emits light at 517nm due to the deprotonated form and at 441nm due

80

to the protonated form. More water molecules interacting with pyranine results in higher
peak intensity at 517nm or lower peak intensity at 441nm. Thus the peak intensity ratio
(PIR) between 517nm and 441nm indicates the water activity sensed by pyranine.
Similarly, when excited at 310nm, carminic acid emits light at 591nm due to the
deprontonated form and at 419nm due to the protonated form. The PHI between 591nm

and 419nm indicates the water activity sensed by carminic acid.

 

013033 OH

Figure 5.1 Chemical structures of pyranine (left) and carminic acid (right).

81

 

7.E+04
6.E+04 -
5.E+04 i
4.E+04 -
3.E+04 ..
2.E+04 —
1.E+04 ~

0.E+00 ,
400 500 600
Wavelength (nm)

Excitation 342nm

CPS

 

 

 

 

2.E+05

 

Excitation 310nm
1.E+05 -

8.E+04 -~
a:

CP

4.E+04 -

 

 

 

0.E+OO . .
350 450 550 650 750

Wavelength (nm)
Figure 5.2 Fluorescence spectrum of 20ppm pyranine (top, peak at 441nm is due to the
protonated form, peak at 51 7nm is due to deprotonatedform.) and 30ppm carminic acid

(bottom peak at 419nm is due to protonatedform, peak at 591 nm is due to deprotonated

form.) in sucrose solution at 80 °C.

82

Both pyranine and carminic acid are used to investigate water activity in sucrose solution
in similar way, but these two molecules are probing different phases of the sucrose
solution. The carminic acid molecule has a glucosyl unit, which renders it the ability to
closely interact with sucrose molecules and sense the water availability in the vicinity of
sucrose molecules”. In contrast, pyranine has no structure similarity with sucrose
molecule, thus stays in the bulk water phase in sucrose solution and senses the water

availability in bulk water phase.

5. 3.2 Effect of Glucose and Fructose on Water Activity of Sucrose Solutions

The coupled effect of glucose and fructose on water activity in sucrose solutions was
measured by using pyranine as the probe molecule is shown in Figure 5.3. A decrease in
PIR is observed with increasing amount of ﬁ'uctose and decreasing amount of glucose
within temperature range 70~110°C and total solid concentration of 80~87%. The
decreased Pm indicates a decrease in the amount of free water in bulk water phase. This
observation is corroborated by the PR measurement using carminic acid (Figure 5.4)
under the same conditions. With carminic acid as the probe molecule, Pm increases with
increased amount of fructose and decreased amount of glucose. Because of the structural
similarity between carminic acid and sucrose, it interacts closely with the sucrose. The
equilibrium reaction between the protonated and deprontonated carminic acid is affected
by the water availability in the vicinity of sucrose instead of in the bulk water phase.
Thus increasing PR for carminic acid indicates that sucrose is highly solvated by water
with increasing amount of ﬁ'uctose and decreasing amount of glucose in sucrose

solutions.

83

Figure 5.5 shows the separate effect of fructose and glucose on PR of pyranine in
sucrose solution. Comparing to the pure 80% sucrose solution at 90°C, the PR of 40%
sucrose with 40% fructose binary mixture is decreased, and the PIR of 40% sucrose with
40% glucose is increased.

Combined data in ﬁgure 5.3~5.5 conﬁrm that fructose and glucose have opposite effects
on water activity in sucrose solutions, ﬁ'uctose decreases water activity and glucose
increases water activity. This conclusion is no surprise considering the solubility is in the
order of fructose>sucrose>glucose. With fructose in the solution more water molecules
are kept in the hydration shell of solute molecules, while with glucose in the solution
more water molecules are kept in the bulk water phase. According to crystallization
theory, increased water activity corresponds to lower supersaturation, which should leads
to the inhibition of nucleation. While this theory may explain the inhibition effect of
glucose on sucrose crystallization, it does not explain the inhibition effect of fructose on
sucrose crystallization. It appears that ﬁ'uctose and glucose could be working under
different mechanisms when inhibiting the crystallization of sucrose. At least it is
appropriate to conclude that thermodynamic water activity alone doesn’t explain the

inhibition effect by both fructose and glucose in sucrose crystallization.

84

 

0.70

 

 

 

J T 80 Brix 900 7”
[183.5 Brix 11OC ’
050 I ‘ 083.5 Brix 90C
1 A835 Brix 70C
__!_3_7 E'JQOQ. .
0.50 y "
PIR m ‘
0.40 ¥ B
. i
0.30 1
I
I
0.20
0 20 40 Fructose (%)
4O 20 0 Glucose (%)

Figure 5.3 PIR of pyranine in sucrose solutions at diﬂerent temperature with diﬁerent
amount of fructose and glucose. Brix represents total sugar percent by weight. Data at

other compositions and temperatures show the same trend, thus not shown here.

85

 

1.10 a, - 2 ,
CI 83.5 Brix 110C ‘

A 80 Brix 900 - E
O 83.5 Brix 900 I

I 87 Brix 900

0.90 ~ 1 A835 Brix 7OC

PIR A
El

IH—le-E—I

 

 

 

0.70 «i I
A
A
A
0.50
0 20 40 Fructose (%)
4O 20 0 Glucose (%)

Figure 5.4 PIR of carminic acid in sucrose solutions at difﬁrent temperature with
diﬂerent amount of fructose and glucose. Brix represents total sugar percent by weight.

Data at other compositions and temperatures show the same trend, thus not shown here.

86

0.45

 

 

0.4 ~
PIR

 

0.35 4 —

 

 

0.3 -

 

 

 

 

 

 

 

 

 

 

 

 

 

ff ’90 o‘"° r§\ ﬁle
it Q) x
s» as»

00 $9 0
«°\ «(E 11$

Figure 5.5 Separate eﬂects of fructose and glucose on the PIR of pyranine in sucrose

solution at 90 °C (total sugar content is 80%).

87

5. 3.3 Effect of Glucose and Fructose on Viscosity of Sucrose Solutions

The coupled effect of increasing fructose concentration and decreasing glucose
concentration is shown in ﬁgure 5.6. Anisotropy of pyranine in sucrose solution
increases with increasing amount of fructose and decreasing amount of glucose. Figure
5.7 shows the separate effect of ﬁ'uctose and glucose in 80% sugar solutions at 90°C.
Both fructose and glucose decrease the anisotropy of pyranine in sucrose solution, which
indicate that both fructose and glucose decrease the viscosity of sucrose solutions under

the condition of this study. Glucose is more effective than fructose.

 

 

 

 

 

I [83”.5FBﬁx707C'",
0-32 ” n 87 Brix soc ,
O 83.5 Brix 900 A
0.28 g A 80 Brix 900 f I
r ___,. _.
0.24 -+ 1.5 s
0.20 -~ f A
‘ E
0.16 4 I E
CI
0.12
0 20 40 Fructose (%)
40 20 0 Glucose (%)

Figure 5.6 Anisotropy of pyranine in sucrose solutions at diﬂerent temperature with
diﬂerent amount of fructose and glucose. Brix represents total sugar percent by weight.

Data at other compositions and temperatures show the same trend, thus not shown here.

88

0.24

 

 

0.22 -

 

0.2 -

0.18

 

0.16 —

 

 

 

 

 

 

 

 

 

0.14 4

Figure 5. 7 Separate ejfects of ﬁuctose and glucose on the anisotropy r of pyranine in

sucrose solution at 90 °C (total sugar content is 80%).

This conclusion is contradictory to the long held beliefs in industrial sugar crystallization.
Fructose has long been considered to increase the viscosity of sucrose and cause
inhibition of sucrose crystallization by viscosity effects. Only a handful of studies on the
direct viscosity measurement for sucrose/glucose/fructose system can be found in the

literature. Zumalacarregui de Cardenas et al.8 and Smythe9 found that the viscosity of

89

sucrose solutions increases with the concentration of glucose and/or fructose. A closer
look at their experimental conditions revealed that Zumalacarregui de Cardenas et a1.
kept the same sucrose/water ratio and added in glucose or fructose, while Smythe kept
the same supersaturation level for sucrose and added in invert sugar. In both cases, the
overall solid content is increased as glucose and/or fructose is added, which is the real
cause for viscosity increase.

Mazurkiewicz et a1.lo showed that the viscosity of solutions with the same mass
concentration is in the order of pure disaccharide > disaccharide-monosaccharide mixture
> pure monosaccharide. Mathlouthi et a1.ll and Vazquez Una et a1.12 measured the
kinematic and intrinsic viscosity of pure solution of sucrose, glucose and fructose. Both
groups showed that the viscosity of pure glucose or fructose is always lower than that of
the pure sucrose solution at comparative mass concentration. It is worth noting that
normally the viscosity of solution has a great dependence on molecular weight when
comparing the same type of chemicals species. These literature data proved our
conclusion that both ﬁ'uctose and glucose reduce the viscosity of sucrose solutions when
the overall solid content is kept the constant.

A reduced viscosity should promote crystallization by increasing the mass transfer
efﬁciency. In fact both glucose and fructose inhibit the crystallization in sucrose
solutions despite their reduction effect on viscosity. This observation leads us to believe
that viscosity is not the controlling factor in inhibition of sucrose crystallization by

ﬁ'uctose or glucose.

90

5. 3.4 Molecular Conformation Effect of Glucose and Fructose on Sucrose Solution

When dissolved in water, ﬁ'uctose and glucose can exist in isomeric forms with molecular
conformations different than that in crystalline form. Molecules in these conformations
have to go through conformational change in order to be integrated in crystal lattice. This
process takes energy and time, and the conditions in industrial sucrose crystallization
may not allow this process to occur, thus impede the nucleation and crystal growth.
Raman spectroscopy allows us to probe the molecular structure and conformation of
different sugars in solution and make a correlation with the inhibition ability of ﬁ'uctose
and glucose. Figure 5.8 and 5.9 shows the Raman spectra of 87% fructose and 87%
glucose in water at different temperature. A peak at 17306m'l is of particular interest to
us. It shows up in the ﬁ'uctose solution at high temperature, but not in glucose solution at
any temperature. Literature search conﬁrmed that this peak is ﬁ'om the carbonyl group in
the open chain form of fructose”. While glucose also has the open chain form, its
concentration is too low to be detected by Raman spectroscopy. This open chain form
can’t be integrated into crystalline form, which may play a role in sucrose crystallization
inhibition by fructose. The relatively large amount of ﬁ'uctofuranose form due to
complex mutarotation in ﬁ'uctose solution may also play a role at slowing down the
crystallization process, since only the fructopyranose form can be integrated into the
crystal lattice. Raman spectroscopy thus can be used to predict the inhibition ability of
sugar by probing their conformations in solution.

Another reducing sugar, tagatose, is expected to be a good inhibitor to sucrose

crystallization. The Raman spectra of tagatose (Figure 5.10) also show the peak at

1730cm", which indicates that the concentration of its open chain form at high

91

temperature is signiﬁcant. It is possible that tagatose and fructose inhibit sucrose
crystallization by the same mechanism: disrupting the aggregation process by assuming

uncrystallizable conformations.

 

r'\
f \
r" \
'I ,4" ‘\.~ ‘1'
'-"—-r. {I , Cr).
' ‘- " ‘. o
-\__ 51% 110 q
\_~:—,Hl
\‘. xx

 

 

17300111.] 70°C f3~§

 

Intensity

 

 

 

f} ////

 

 

500 1000 1500 2000 2500 3000 3500
Raman Shift (cm")

Figure 5 .8 Raman spectra of 8 7% ﬁuctose solutions at diﬂerent temperature

92

 

Intensity

 

 

 

 

 

 

lll///

1730cm'1

 

 

500 1000 1500 2000 2500 3000 3500
Raman Shift (cm'l)

Figure 5. 9 Raman spectra of 8 7% glucose solutions at different temperature

93

 

Intensity

 

 

 

 

 

I/ ////

 

-1
l730cm Q:

 

500 1000 1500 2000 2500 3000 3500
Raman Shift (cm'l)

Figure 5.10 Raman spectra of 8 7% tagatose solutions at diﬁ'erent temperature

94

5.4 Conclusions

We demonstrated that both ﬂuorescence and Raman spectroscopy can be used to
investigate sugar crystallization at conditions relevant to industrial food processing.
Fluorescence spectroscopy enables us to probe both thermodynamic and kinetic
properties of solutions such as water activity and viscosity on a microscopic scale.
Raman provides structural information on a molecular level.

These techniques can be used not only to monitor the sucrose crystallization process, but
also to aid the study of the inhibition mechanism by non-sucrose sugars. Our initial study
suggests that fructose and glucose may be working under different mechanisms when
inhibiting the crystallization of sucrose. Glucose is more effective at increasing the water
activity of sucrose solution, thus lowering the driving force for crystallization-
supersaturation, while ﬁ'uctose tends to assume molecular conformations different than
the one in crystalline form, thus interrupt the aggregation process during nucleation.
Contrary to the industrial believe, viscosity may not be a signiﬁcant factor in inhibiting

sucrose crystallization by either fructose or glucose according to this study.

5.5 Acknowledgements

The authors wish to thank Kellogg Company for ﬁnancial support of this study. Tony
Gualtieri from Kellogg Company helped with experimental design and data analysis.
Help with ﬂuorescence instrumentation from Dr. Gary Blanchard of the Department of

Chemistry at Michigan State University is also appreciated.

95

5.6

(1).
(2).

(3).

(4)-

(5).
(6).
(7).
(8).

(9).
(10).

(11).

(12).

(13).

(14).

References
Hartel, R. W. Crystallization in Foods, Aspen Publishing: Gaithersburg, 2001.

Hartel, R. W.; Shastry, A. V. Critical Review in Food Science and Nutrition, 1991,
1(1), 49-112.

Sgaldino, G.; Aquilano, D.; Vaccari, G.; Mantovani, G. Cryst. Res. T echnol., 1997,
32(8), 1057-1065.

Sgaldino, G.; Aquilano, D.; Vaccari, G.; Mantovani, G. J. Cryst. Growth, 1998,
192, 290-299.

Dohl, B.; Follner, H. Cryst. Res. T echnol., 1992, 27(1), 3-12.
Wang, B. G.; Krafczyk, S.; Follner, H. J. Cryst. Growth, 2000, 219, 67-74.
Chakraborty, Reena; Berglund, Kris A. J. Cryst. Growth, 1992, 125(1-2), 81-96.

Zumalacarregui de Cardenas, Lourdes; Ruiz Cruz, Carlos; Guerra Deben, Jorge.
Centro Azucar, 1988, 15(3), 57-66.

Smythe, B.M. Aust. J. Chem, 1967, 20, 1097-111.

Mazurkiewicz, Jozef; Nowotny-Rozanska, Maria. Polish Journal of Food and
Nutrition Sciences, 1998, 7(2), 171-180.

Mathlouthi, M.; Hutteau, F.; Angiboust, J. F. Food Chemistry, 1996, 56(3), 215-
221.

Vazquez Una, G.; Chenlo Romero, F.; Alvarez Dacosta, E.; Moreira Martinez, R.;
Pardo Calvo, P. J. Chem. Eng. Data, 1994, 39, 87-89.

Yaylayan, V. A.; Ismail, A. A. Journal of Carbohydrate Chemistry, 1992, 11(2),
149-58.

Rasimas, J.P.; Berglund, K.A.; Blanchard, G.J., Journal of Physical Chemistry,
1996, 100 (42), 17034-17040.

96

Chapter 6

CONCLUSIONS AND FUTURE WORK

6.1 Conclusions

This dissertation focuses on the study of monitoring and control of crystallization
processes using spectroscopic methods, including Raman, ATR-FTIR and ﬂuorescence.
The goals are to optimize the crystallization process for desired crystal size distribution
or polymorph form and to understand the kinetics during various crystallization processes
with the help from aforementioned spectroscopic methods.

In chapters 2 and 3, Raman spectroscopy was used as a convenient process development
tool for on line and in situ characterization of acetaminophen polymorph forms in
slurries. Metastable polymorphs of acetaminophen were crystallized reproducibly by
pre-heating the solution at higher temperature and cooling the solution down at a faster
rate. Higher water content in mixtures of ethanol and water also favors the trihydrate
form, which has only limited stability at room temperature. Both Raman and ATR-FTR
spectroscopies were employed to monitor the solvent mediated acetaminophen
polymorph transformation process. Complimentary information about solid phase
polymorph form and solution phase concentration suggest that crystallization of the more
stable form of acetaminophen polymorph is rate limiting during transformation, with
nucleation limiting the form H to I transformation and crystal grth limiting the form T

to II transformation, respectively.

97

Applying chemometric data analysis methods such as MCR allowed us to quantitatively
follow a complex polymorph transformation processes without the need of building a
calibration model.

ATR-FTIR coupled with a LABMAX® automatic reactor system used to optimize
cooling curves by in situ feedback control of the supersaturation level was discussed in
chapter 4. A straightforward and efﬁcient calibration procedure was developed to obtain
optimal cooling proﬁles in an easy, fast and ﬂexible way, which is preferred by industrial
users. This approach does not rely on kinetic models and precludes the error caused by
inaccurate kinetic data, thus is useful in determining the cooling policy in situations
where kinetic data are not available. The succinic acid crystals resulting from the
controlled process possess a larger mean size with more uniform size distribution than
those from the uncontrolled case.

Fluorescence and Raman spectroscopies were used to investigate sucrose crystallization
at conditions relevant to industrial food processing in chapter 5. Water activity and
viscosity probed by ﬂuorescence spectroscopy together with molecular structure probed
by Raman spectroscopy suggest that fructose and glucose may be working under different
mechanisms when inhibiting the crystallization of sucrose. Glucose is more effective at
increasing the water activity of sucrose solution, thus lowering the driving force for
crystallization-supersaturation, while fructose tends to assume molecular conformations
different than the one in crystalline form, thus interrupt the aggregation process during
nucleation. Contrary to the industrial believe, viscosity may not be a signiﬁcant factor in

inhibiting sucrose crystallization by either fructose or glucose according to this study.

98

6.2 Future Work

Spectroscopic methods have shown great advantage in on line monitoring and control of
crystallization processes. Their potential is yet to be realized in crystallizations in real
industrial environment, e.g., the crystallization species may be a more complex
polymorphic system, or the spectra may experience more interference from impurities in
raw material. Methods developed in this study can be tested in such environment in
future studies. Besides the crystallization process monitoring and control, the possibility
of using these spectroscopic methods should also be explored for down stream processes
such as ﬁltration and drying.

Combined with multivariate curve resolution (MCR), Raman spectroscopic method
provided both selectivity and sensitivity for acetaminophen polymorph transformation
process analysis. One possible study is to test the reproducibility of such analysis. With
good reproducibility, these type of methods can be used in real time monitoring and
quantitative analysis.

The mechanisms of controlling acetaminophen polymorph crystallization were proposed
in chapter 2. More experiments need to be carried out to conﬁrm how thermal history
and solvent composition affect the crystallization process. Other analytical techniques
such as solution NMR or photon correlation spectroscopy (PCS) may be used to measure

solution structure change after different thermal treatments.

99

APPENDICES

100

Table A.l. Data for ﬁgure 2.5. Solubility curve of acetaminophen form I in ethanol/H20

mixture solvent.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Temperature ( C ) O 10 20 3O 40 50
Solubility in 0% ethanol (wt%) 0.7 0.9 1.3 1.7 2.4 3.3
Temperature ( C ) 0 10 25 3O 41 50
Solubility in 25% ethanol (wt%) 2.4 4.0 6.1 7.6 11.8 16.8
Temperature ( C ) 0 10 20 3O 41 50
Solubility in 50% ethanol (wt%) 7.5 10.6 14.4 18.7 25 30.5
Temperature ( C ) 0 10 20 30 42 49
Solubility in 75% ethanol (wt%) 14.3 15.9 19.7 25.4 32.3 36.1
Temperature ( C ) 0 10 20 30 40 50
Solubility in 100% ethanol (wt%) 9.6 10.7 13.3 16.9 21.6 25.7

 

101

 

Table A2. Data for Figure 2.6. Solubility curve of acetaminophen polymorphs in

ethanole20=1:3..

 

Solubility Solubility Solubility
Temperature forI Standard for H Standard for T Standard

( C ) (wt%) error (wt%) error (wt%) error

 

 

 

 

 

-10 1.01 0.017 1.20 0.082 1.12 0.044
-5 1.34 0.023 1.55 0.077 1.75 0.038
0 1.79 0.011 2.06 0.041 2.50 0.132
5 2.28 0.026 2.75 0.03 3.80 0.076
10 2.94 0.025 3.47 0.007 N/A N/A

 

 

 

 

 

 

 

 

 

102

Table A3. Data for Figure 3.2. Crystallization and transformation proﬁle represented by
peak height at 1237cm". For the peak height proﬁle from un-normalized spectra, a factor

of 0.001 has been multiplied to bring it to the same scale as the normalized proﬁle.

 

Peak Height at Peak Height at
Peak Height at 1237cm-1 un- Peak Height at 1237cm-1 un-
Time 1237cm-1 normalized Time 1237cm-1 normalized
(Hour) Normalized (x0.001) (Hour) Normalized (x0.001)

 

0.00 0.75 1.57 6.15 0.12 0.35
0.07 0.75 2.09 6.48 0.11 0.33
0.13 0.74 1.99 6.82 0.12 0.34
0.20 0.74 2.02 7.40 0.11 0.32
0.32 0.72 1.91 7.73 0.11 0.31
0.38 0.4-4 0.56 8.07 0.12 0.30
0.45 0.41 0.45 8.65 0.11 0.30
0.57 0.42 0.56 8.98 0.12 0.28
0.63 0.41 0.56 9.32 0.12 0.27
0.70 0.42 0.58 9.90 0.11 0.29
0.82 0.42 0.58 10.23 0.12 0.26
0.88 0.40 0.57 10.57 0.12 0.26
0.95 0.41 0.58 11.15 0.12 0.27
1.07 0.40 0.61 l 1.48 0.12 0.26
1.13 0.40 0.61 11.82 0.12 0.25
1.20 0.39 0.61 12.40 0.11 0.24
1.32 0.38 0.61 12.73 0.12 0.25
1.38 0.39 0.62 13.07 0.13 0.25
1.45 0.38 0.63 13.65 0.13 0.27
1.57 0.39 0.61 13.98 0.15 0.28
1.63 0.39 0.66 14.32 0.19 0.30
1.70 0.38 0.64 14.90 0.34 0.39
1.82 0.39 0.61 15.23 0.53 0.55
1.88 0.39 0.61 15.57 0.80 0.91
1.95 0.39 0.63 16.15 0.86 0.87
2.40 0.38 0.66 16.48 0.85 0.88
2.73 0.36 0.66 16.82 0.84 0.88
3.07 0.34 0.62 17.40 0.85 0.91
3.65 0.28 0.54 17.73 0.85 0.86
3.98 0.23 0.51 18.07 0.83 0.85
4.32 0.19 0.45 18.65 0.84 0.82
4.90 0.12 0.41 18.98 0.84 0.80
5.23 0.12 0.41 19.32 0.84 0.78
5.57 0.12 0.40 19.65 0.84 0.80

 

103

Table A.4. Data for Figure 3.4. Scores for principal components from MCR analysis of

acetaminophen polymorph crystallization and transformation.

 

Time

(Hours)

0.00
0.07
0.13
0.20
0.27
0.33
0.40
0.47
0.53
0.60
0.67
0.73
0.80
0.87
0.93
1 .00
1 .07
1 .13
1 .20
1 .27
1 .33
1 .40
1 .47
1 .53
1 .60
1 .67
1 .73
1 .80
1 .87
1 .93
2.07
2.40
2.73
3.07
3.40
3.73
4.07
4.40
4.73
5.07
5.40
5.73

PC1 PC2 PC3 PC4

1.87E-02 3.55502 9.33E-02 3.64E-02
1.62E-02 3.57E-02 8.99E-02 4.06E-02
1.67E-02 3.56E-02 8.93E-02 3.98E-02
1.88E-02 3.52E-02 8.74E-02 3.77E-02
1.93E-02 3545-02 8.61 E-02 3.88E-02
8.99E-02 1.99E-02 2.90E-02 6.89E-03
1.01E-01 1.72E-02 2.12E-02 4.10E-03
9.88E-02 1.46E-02 2.295-02 4.73E-03
1.01E-01 1.55E-02 2.29E-02 3.65E-03
1.00E-01 1.56E-02 2.41 E-02 5.87E-03
9.73E-02 1.44E-02 2.325-02 6.78E-03
9.83E-02 1.44E-02 2.37E-02 6.73E-03
9.52E-02 1.43E-02 2.57E-02 5.64E-03
9.71 502 1.43E-02 2.42E-02 6.17E-03
9.90E-02 1.44E-02 2.27E-02 6.35E-03
1.00E-01 1.44E-02 2.31 E-02 5.45E-03
9.96E-02 1.39E-02 2.35E-02 3.89E-03
9.87E-02 1.36E-02 2.45E-02 5.04E-03
9.98E-02 1.35E-02 2.31E-02 4.44E-03
9.86E-02 1.35E-02 2.28E-02 4.22E-03
9.71E-02 1.33E-02 2.27E-02 6.03E-03
9.97E-02 1.31 E-02 2.355-02 4.165-03
9.95E-02 1.35E-02 2.38E-02 4.82E-03
1.00E-01 1.355-02 2.32E-02 4.585-03
1.00E-01 1.35E-02 2.14E-02 4.41 E-03
9.90E-02 1.30E-02 2.16E-02 5.29E-03
9.90E-02 1.28E-02 2.28E-02 5.35E-03
9.83E-02 1.32E-02 2.15E—02 5.53E-03
9.74E-02 1.38E-02 2.25E-02 5.505-03
9.73E-02 1.29E-02 2.18E-02 5.71 E-03
9.64E-02 1.34E-02 2.23E-02 6.47E-03
9.45E-02 1.31E-02 2.09E-02 7.21 E-03
9.08E-02 1.26E-02 1.85E-02 9.24E-03
7.74E-02 1.19E-02 1.77E-02 1.57E-02
6.72E-02 1.09E-02 1.61 E-02 1.96E-02
5.05E-02 9.26E-03 1.295-02 2.73E-02
3.41 E-02 7.49E-03 1.10E-02 3.33E-02
1.89E—02 5.50E-03 7.75E-03 4.08E-02
7.69E-03 3.51 E-03 3.59E-03 4.66E-02
4.90E-03 2.61 E-03 3.055-03 4.795-02
4.80E-03 2.62E-03 4.05E-03 4.73E-02
4.80E-03 2.62E-03 3.35E-03 4.76E-02

Time

(Hours)

6.07

6.40

6.73

7.07

7.40

7.73

8.07

8.40

8.73

9.07

9.40

9.73

10.07
10.40
10.73
1 1 .07
1 1.40
1 1.73
12.07
12.40
12.73
13.07
13.40
13.73
14.07
14.40
14.73
15.07
15.40
15.73
16.07
16.40
16.73
17.07
17.40
17.73
18.07
18.40
18.73
19.07
19.40
19.65

104

PC1 PC2 PC3 PC4

5.385-03 2.87E-03 2.43E-03 4.81 E-02
4.95E-03 2.63E-03 2.97E-03 4.73E-02
5.08E-03 2.71 E-03 2.83E-03 4.73E-02
4.64E-03 2.46E-03 3.545-03 4.73E-02
4.89E-03 2.77E-03 2.66E-03 4.77E-02
4.52E-03 2.51 E-03 2.86E-03 4.71 E-02
5.03E-03 2.86E-03 2.50E-03 4.765-02
6.14E-03 2.73E-03 2.97E-03 4.68E-02
4.62E-03 2.48E-03 4.20E-03 4.71E-02
4.76E-03 2.58E-03 4.12E-03 4.71E-02
5.58E-03 2.73E-03 2.06E-03 4.795-02
5.54E-03 2.89E-03 1.76E-03 4.865-02
5.265-03 2.59E-03 3.53E-03 4.775-02
4.94E-03 2.64E-03 4.52E-03 4.68E-02
5.33E-03 2.97E-03 2.48E-03 4.81E-02
4.91 E-03 2.60E-03 3.93E-03 4.70E-02
5.47E-03 3.06E-03 1.99E-03 4.78E-02
5.60E-03 2.99E-03 1.78E-03 4.87E-02
4.95E-03 2.63E-03 4.72E-03 4.68E-02
5.21 E-03 2.57E-03 3.29E-03 4.78E-02
5.01E-03 2.73E-03 3.04E-03 4.795—02
5155-03 3.21 E-03 3.65E-03 4.73E-02
5.10E-03 3.43E-03 2.69E-03 4.78E-02
5.18E-03 4.31 E-03 4.34E-03 4.66E-02
4.61 E-03 6.34E-03 2.92E-03 4.72E-02
5.16E-03 8.83E-03 2.82E-03 4.81E-02
4.66E-03 1.43E-02 4.11E-03 4.67E-02
4.52E-03 2.47E-02 3.86E-03 4.48E-02
3.02E-03 4.38E-02 5.64E-03 4.32E-02
3.20E-03 5.40E-02 3.71 E-03 4.13E-02
3.43E-03 5.42E-02 3.79E-03 4.13E-02
2.69E-03 5.49E-02 4.28E-03 4.01 E-02
3.12E-03 5.47E-02 4.55E-03 4.00E-02
2.79E-03 5.53E-02 4.23E-03 3.93E-02
3.90E-03 5.51 E-02 2.32E-03 4.12E-02
2.53E-03 5.54E-02 3.47E-03 4.04E-02
2.62E-03 5.47E-02 4.78E-03 3.98E-02
3.14E-03 5.48E-02 3.32E-03 4.05E-02
3.21E-03 5.47E-02 4.17E-03 4.02E-02
3.42E-03 5.50E-02 3.48E-03 4.06E-02
2.77E-03 5.38E-02 4.36E-03 4.05E-02
3.13E-03 5.46E-02 3.90E-03 4.02E-02

Table A5. Data for Figure 3.5. Relative composition of form H calculated from proﬁles
of normalized peak height at 1237cm'1, PC1 and PC2 by MCR analysis during form T to

II transformation (left) and form 11 to I transformation (right).

 

 

Time By peak height By PC1 Time By peak height By PC2
(hourS) (%) (%) (H0118) (%) (%)
1.12 0.0 1.5 12.98 99.7 99.7
1.17 4.6 0.2 13.07 99.8 99.8
1.22 0.4 0.4 13.15 100.0 100.0
1.27 0.2 2.6 13.23 99.4 98.9
1.32 2.9 1.9 13.32 99.7 99.4
1.37 2.6 0.3 13.40 99.9 99.4
1.42 2.7 0.0 13.48 98.9 98.5
1.47 3.6 1.6 13.57 98.5 98.3
1.52 0.1 2.5 13.65 99.3 98.7
1.57 1.3 3.9 13.67 97.6 97.7
1.62 1.8 3.6 13.82 97.2 96.5
1.67 2.8 2.3 13.90 96.6 96.3
1.72 4.4 3.0 13.98 95.9 95.8
1.77 3.2 2.5 14.07 94.5 93.8
1.82 0.8 2.9 14.15 92.8 92.9
1.87 3.8 3.8 14.23 92.9 92.4
1.92 7.0 4.2 14.32 90.3 90.0
1.97 2.4 5.6 14.40 89.1 89.0
2.15 1.4 3.3 14.48 87.8 87.6
2.48 10.3 6.4 14.57 83.6 83.9
2.73 1 1.1 10.9 14.65 80.3 80.7
2.98 19.4 17.9 14.73 78.2 78.5
3.23 23.5 25.6 14.82 73.9 73.3
3.48 35.8 39.8 14.90 70.4 69.8
3.73 50.3 52.4 14.98 64.9 64.5
3.98 60.8 65.8 15.07 58.2 58.4
4.23 73.9 78.6 15.15 52.9 52.5
4.48 87.3 88.1 15.23 43.7 44.3
4.73 95.1 96.7 15.32 34.2 34.2
4.98 98.6 100.0 15.40 21.0 21.5
5.23 97.3 99.6 15.48 10.2 10.8
5.48 97.7 99.9 15.57 5.6 5.1
5.73 100.0 99.7 15.65 3.1 3.0
5.98 98.4 99.4 15.73 0.7 1.7
15.82 0.5 0.4
15.90 0.0 0.0
15.98 1.4 0.9

 

105

Table A.6. Data for Figure 3.7. Concentration proﬁle by ATR-FTIR compared with PC2

proﬁle from MCR analysis of Raman spectra.

 

 

Time Time Concentration
ﬂours) PC2 (hours) (wt%)
0.00 0.0325 0.00 5.40
0.10 0.0324 0.33 5.31
0.20 0.0322 0.42 3.58
0.32 0.0320 0.50 3.06
0.33 0.0199 0.58 2.88
0.42 0.0166 0.67 2.79
0.58 0.0158 0.75 2.73
0.75 0.0152 0.83 2.69
0.88 0.0152 0.88 2.66
1.08 0.0145 1.00 2.64
1.25 0.0144 1.08 2.61
1.50 0.0143 1.17 2.58
1.83 0.0142 1.25 2.57
2.07 0.0141 1.33 2.55
2.40 0.0138 1.42 2.55
2.57 0.0132 1.50 2.54
2.73 0.0132 1.58 2.53
2.90 0.0131 1.67 2.53
3.07 0.0121 1.75 2.52
3.23 0.0120 1.83 2.52
3.40 0.0109 1.92 2.53
3.57 0.0101 2.00 2.52
3.73 0.0089 2.08 2.53
3.90 0.0077 2.17 2.51
4.07 0.0068 2.25 2.53
4.23 0.0055 3.00 2.52
4.40 0.0045 3.50 2.49
4.57 0.0033 4.00 2.51
4.73 0.0023 4.50 2.49
4.90 0.0017 4.75 2.45
5.57 0.0013 4.83 2.38
6.57 0.0017 4.92 2.32
7.57 0.0011 5.00 2.26
8.57 0.0015 5.25 2.18
9.57 0.0017 5.50 2.11
10.57 0.0016 5.75 2.03
11.57 0.0012 6.00 2.01
12.57 0.0013 6.25 1.95
12.65 0.0014 6.50 1.97
12.73 0.0015 6.75 1.95

 

106

with PC2 proﬁle from MCR analysis of Raman spectra.

Table A.6 (contd.). Data for Figure 3.7. Concentration proﬁle by ATR-FTIR compared

 

 

Time Time Concentration
(hours) PC2 (hours) (wt%)
12.82 0.0017 7.75 1.95
12.90 0.0015 8.75 1.96
12.98 0.0020 9.75 1.94
13.23 0.0024 10.75 1.95
13.48 0.0026 11.75 1.96
13.73 0.0030 12.75 1.96
13.98 0.0038 13.75 1.94
14.23 0.0055 14.75 1.95
14.48 0.0078 15.83 1.95
14.73 0.0121 16.00 1.89
14.98 0.0188 16.08 1.86
15.23 0.0284 16.17 1.83
15.48 0.0444 16.25 1.82
15.90 0.0495 16.50 1.77
16.32 0.0498 17.00 1.77
16.40 0.0496 18.00 1.78
17.40 0.0498 19.00 1.77
18.40 0.0494 20.00 1.77
19.40 0.0485
19.65 0.0493

 

107

Table A7 Data for Figure 4.3. Solubility curve of succinic acid in H20. R* is solubility
represented by ratio of the peak area of the 1806~1675cm'l band to that of the

1671~1494cm'l band in the infrared spectrum.

 

 

Temperature ( C ) Solubility R* Standand error
10 0.606 0.0030
15 0.607 0.0029
20 0.615 0.0030
25 0.629 0.0030
30 0.651 0.0031
35 0.680 0.0032
40 0.716 0.0033
45 0.758 0.0035
50 0.808 0.0038
55 0.865 0.0042
60 0.929 0.0047

 

108

Table A8. Data for Figure 4.4. Optimal cooling proﬁles for succicinic acid
crystallization with different amount of seeding. Initial concentration is 19%(w/w), seed

size is 355-425um, and agitation rate is 400rpm.

 

 

 

Time Temperature ( °C )
(minutes) 0.05 g seed 0.5g seed 5 g seed
1 50.2 50.2 50.2

1 1 49.6 49.7 49.8
21 49.5 49.1 49.2
31 49.5 48.6 48.7
41 48.8 48.0 48.0
51 48.3 47.5 47.3
61 47.8 47.0 46.4
71 47.2 46.3 45.3
81 46.7 45.5 44.0
86 46.3 45.0 43.0
91 46.0 44.3 42.0
96 45.7 43.3 40.5
101 45.4 42.3 38.3
106 45.0 41.3 36.0
1 11 44.6 40.0 33.0
1 16 44.1 37.8 29.8
121 43.5 35.5 26.6
126 42.9 33.3 23.1
131 42.2 31.0 17.1
136 41.2 28.7 13.0
141 40.2 26.3 9.5
146 39.3 24.0

151 38.1 21.5

156 35.8 18.4

161 33.6 14.5

166 31.3 10.5

171 29.0

176 26.7

181 24.3

186 21.9

191 19.5

196 17.0

201 14.5

206 12.7

21 1 1 1.4

214 9.7

 

 

109

Table A.9. Data for Figure 4.5. Optimal cooling proﬁles for succicinic acid
crystallization with different seed sizes. Initial concentration is 19% (w/w), seed amount

is 5g, and agitation rate is 400rpm.

 

 

 

Temperature CC )
Time Seed size Seed size seed size
(minutes) 500-600um 355-4251]!!! <180um
1 50.7 50.2 50.3
11 50.0 49.7 49.7
21 49.5 49.1 49.3
31 49.5 48.6 48.7
41 49.5 48.0 48.1
51 49.5 47.5 47.5
61 49.3 47.0 47.0
71 48.7 46.3 46.3
81 48.2 45.5 45.7
91 47.7 44.3 44.9
101 47.5 42.3 44.1
111 47.3 40.0 42.8
121 46.8 35.5 40.8
126 46.5 33.3 39.8
131 46.3 31.0 37.6
136 46.0 28.7 35.4
141 45.7 26.3 33.1
146 45.4 24.0 30.8
151 45.1 21.5 28.5
156 44.8 18.4 26.1
161 44.5 14.5 23.7
166 44.3 10.5 21.4
171 43.9 19.0
176 43.5 16.5
181 43.0 13.6
186 42.5 10.1
191 41.8
201 39.8
211 37.9
221 36.0
231 34.0
241 29.4
251 24.6
261 19.8
271 12.7
275 9.9

 

 

110

Table A. 1 0. Data for Figure 4.6. Optimal cooling proﬁles for succicinic acid

crystallization with different agitation speeds. Initial concentration is 19%(w/w), seed

amount is 5g, seed size is 355-425um.

 

 

 

Time Tempersture (°C)
(minutes) 300rpm 400rpm 600mm
1 50.2 50.2 50.2
1 1 50.4 49.7 50.0
21 50.2 49.1 49.7
31 50.0 48.6 49.2
41 49.8 48.0 48.6
51 49.7 47.5 48.0
61 49.5 47.0 47.3
71 48.8 46.3 46.4
81 48.6 45.5 45.3
91 48.3 44.3 44.0
101 47.8 42.3 42.0
11 1 47.3 40.0 40.0
121 46.8 35.5 35.4
131 46.3 31.0 30.6
141 45.9 26.3 23.8
151 45.5 21.5 12.2
161 44.5 14.5
166 44.2 10.5
171 43.8
181 43.0
191 42.3
201 41.6
21 1 40.9
221 40.2
231 39.5
241 38.5
251 37.5
261 36.0
271 34.2
281 31.2
291 26.8
301 22.3
31 1 17.9
321 13.7
329 9.6

 

 

111

Table A.11. Data for Figure 4.7. Optimal cooling proﬁles for succicinic acid

crystallization showing the effect of acetic acid present as an impurity (2% mole basis).

Initial concentration is 19%(w/w), seed amount is 5g, seed size is 355-425um, and

 

 

 

agitation rate is 400rpm.
, Temperature (°C)
Time
(minutes) With acetic acid Without acetic acid
1 50.0 50.2
11 49.3 49.7
21 48.8 49.1
31 48.3 48.6
41 47.7 48.0
51 47.2 47.5
61 46.7 47.0
71 46.1 46.3
81 45.6 45.5
91 45.1 44.3
96 44.8 43.3
101 44.5 42.3
106 44.2 41.3
111 43.8 40.0
116 43.4 37.8
121 42.8 35.5
126 42.2 33.3
131 41.6 31.0
136 40.6 28.7
141 39.6 26.3
146 38.7 24.0
151 37.7 21.5
156 36.8 18.4
161 35.8 14.5
166 33.9 10.5
171 31.7
176 29.4
181 27.0
186 24.6
191 22.2
196 19.8
201 17.4
211 13.9
216 12.4
224 10.0

 

 

112

Table A.12. Data for Figure 4.4. CSD of succicinic acid with different amount of

seeding. Initial concentration is 19%(w/w), seed size is 355-425um, and agitation rate is

400rpm.

 

 

 

Crystal size Median crystal CSD (wt%)
Sieve # range (m) 31260111“) 5g seed 0.5g seed 0.05g seed

16 >1.18 1.18 0 76.6 63.1

18 1~1.18 1.09 1.7 16.4 13.1

20 0.85~1 0.925 33.7 3.1 7.6

25 0.71~0.85 0.78 45.1 2.1 5.4

30 0.6~0.71 0.655 13.4 0.9 2.4

35 0.5~0.6 0.55 41.3 0.5 1.5

40 0.425~0.5 0.463 12.4 0.1 0.8 1
80 O.18~0.425 0.303 0.6 0.1 5.1 '
Pan 00.425 0.09 0 0 1

 

Table A.13. Data for Figure 4.5. CSD of succicinic acid with different seed sizes. Initial

concentration is 19% (w/w), seed amount is 5g, and agitation rate is 400rpm.

 

 

 

Median CSD (wt%)
Crystal size 9178131 size seed size seed size seed size
Sieve # range (mm) (mm) 500-600um 355-425um <180um
16 >1.18 1.18 57.9 76.6 1.2
18 1~1.18 1.09 19.2 16.4 30.2
20 0.85~1 0.925 10.1 3.1 51.3
25 O.71~0.85 0.78 4.8 2.1 10.7
30 0.6~0.71 0.655 2 0.9 1.6
35 0.5~0.6 0.55 1.2 0.5 1.2
40 0.425~0.5 0.463 0.7 0.1 0.5
80 0.18~0.425 0.303 3.4 0.1 2.4
Pan 0~0.425 0.09 0.9 0 1

 

113

Table A.14. Data for Figure 4.6. CSD of succicinic acid with different agitation speeds.

Initial concentration is 19%(w/w), seed amount is 5g, seed size is 355-42511m.

 

 

 

Crystal size Median crystal CSD (wt%)
Sieve # range (m) size (mm) 600rpm 400mm 300rpm

16 >1.18 1.18 32.6 76.6 80
18 1~1.18 1.09 41.5 16.4 6.5
20 0.85~1 0.925 16 3.1 3.2
25 0.71~0.85 0.78 4.7 2.1 2
30 0.6~0.71 0.655 1.7 0.9 0.9
35 0.5~0.6 0.55 1 0.5 0.7
40 0.425~0.5 0.463 0.4 0.1 0.5
80 0.18~0.425 0.303 1.7 0.1 3
Pan 0~0.425 0.09 0.2 0 3.4

 

Table A.15. Data for Figure 4.7. CSD of succicinic acid showing the effect of acetic acid
present as an impurity (2% mole basis). Initial concentration is 19%(w/w), seed amount

is 5 g, seed size is 355-425um, and agitation rate is 400rpm.

 

 

 

Median CSD (wt%)
crystal SIZC crystal S126 With acetic Witout
Sieve # 1”31189011110 (mm) acid acetic acid
16 >1.18 1.18 64.2 76.6
18 1~1.18 1.09 29.5 16.4
20 0.85~1 0.925 3.2 3.1
25 0.71~0.85 0.78 1.4 2.1
30 0.6~0.71 0.655 0.6 0.9
35 0.5~0.6 0.55 0.6 0.5
40 0.425~0.5 0.463 0.2 0.1
Pan 0~0.425 0.2125 0.2 0.1

 

114

Table A.16 Data for Figure 5.3. Peak intensity ratio (PIR) at 517nm over 441nm of
pyranine in sucrose solutions at different temperature with different amount of fructose

and glucose. Brix represents total sugar percent by weight.

 

 

Fructose Glucose Sucrose Peak intensity
RUN # (wt%) (wt%) (wt%) BRIX Temperature (°C) ratio PIR

1 0 40 47 87 110 0.349
2 20 20 40 80 110 0.533
3 0 40 43.5 83.5 90 0.437
4 40 0 40 80 90 0.446
5 20 20 40 80 70 0.475
6 20 20 43.5 83.5 90 0.352
7 0 40 47 87 70 0.315
8 0 40 47 87 110 0.342
9 20 20 40 80 70 0.471
10 20 20 43.5 83.5 110 0.403
11 20 20 40 80 90 0.514
12 40 0 47 87 90 0.245
13 40 0 47 87 110 0.299
14 20 20 43.5 83.5 90 0.377
15 20 20 47 87 70 0.246
16 40 0 43.5 83.5 70 0.319
17 40 0 43.5 83.5 90 0.335
18 0 40 40 80 90 0.590
19 30 10 41.75 81.75 80 0.396
20 0 40 41.75 81.75 80 0.508
21 30 10 45.25 85.25 100 0.336
22 20 20 43.5 83.5 70 0.342
23 30 10 41.75 81.75 100 0.428
24 20 20 47 87 1 10 0.367
25 40 0 47 87 70 0.239
26 0 40 40 80 110 0.605
27 0 40 40 80 110 0.619
28 40 0 47 87 70 0.242
29 0 40 40 80 110 0.553
30 0 40 40 80 70 0.552
31 20 20 47 87 90 0.283
32 20 20 47 87 70 0.263
33 0 40 47 87 90 0.321
34 20 20 40 80 70 0.481
35 0 40 40 80 70 0.549
36 30 10 45.25 85.25 80 0.299
37 20 20 40 80 110 0.536
38 40 0 40 80 110 0.473

 

115

Table A.16 (contd) Data for Figure 5.3. Peak intensity ratio (PIR) at 517nm over 441nm
of pyranine in sucrose solutions at different temperature with different amount of fructose

and glucose. Brix represents total sugar percent by weight.

 

Fructose Glucose Sucrose Peak intensity
RUN # (wt%) (wt%) (wt%) BRIX Temperature (° C) ratio PIR

39 0 40 47 87 70 0.286
40 0 40 47 87 1 10 0.310
41 0 40 43.5 83.5 110 0.458
42 40 0 40 80 70 0.417
43 40 0 40 80 70 0.416
44 0 40 47 87 70 0.287
45 20 20 43.5 83.5 1 10 0.403
46 20 20 47 87 70 0.272
47 0 40 43.5 83.5 70 0.403
48 0 40 40 80 90 0.602
49 40 0 40 80 70 0.379
50 40 0 43.5 83.5 110 0.352
51 40 0 43.5 83.5 110 0.340
52 0 40 43.5 83.5 90 0.390
53 40 0 43.5 83.5 90 0.325
54 40 0 40 80 1 10 0.469
55 40 0 47 87 70 0.225
56 40 0 40 80 1 10 0.483
57 40 0 47 87 1 10 0.340
58 20 20 47 87 110 0.308
59 0 40 40 80 70 0.550
60 40 0 47 87 1 10 0.328

 

116

Table A.17 Data for Figure 5.4. Peak intensity ratio (PIR) at 591nm over 419nm of
carminic acid in sucrose solutions at different temperature with different amount of

ﬁ'uctose and glucose. Brix represents total sugar percent by weight.

 

 

Fructose Glucose Sucrose Temperature Peak intensity ratio
RUN # (wt%) (wt%) (wt%) BRD( (°C) PIR

1 0 40 47 87 1 10 0.773
2 20 20 40 80 1 10 1.242
3 0 40 43.5 83.5 90 0.673
4 40 0 40 80 90 0.918
5 20 20 40 80 70 0.628
6 20 20 43.5 83.5 90 0.730
7 0 40 47 87 70 0.554
8 0 40 47 87 1 10 0.856
9 20 20 40 80 70 0.717
10 20 20 43.5 83.5 110 1.027
1 1 20 20 40 80 90 0.827
12 40 0 47 87 90 0.781
13 40 0 47 87 1 10 0.936
14 20 20 43.5 83.5 90 0.751
15 20 20 47 87 70 0.643
16 40 0 43.5 83.5 70 0.662
17 40 0 43.5 83.5 90 0.838
18 0 40 40 80 90 0.678
19 30 10 41.75 81 .75 80 0.779
20 0 40 41 .75 81.75 80 0.683
21 30 10 45.25 85.25 100 0.934
22 20 20 43.5 83.5 70 0.614
23 30 10 41 .75 81 .75 100 0.941
24 20 20 47 87 1 10 0.974
25 40 0 47 87 70 0.601
26 0 40 40 80 1 10 0.859
27 0 40 40 80 1 10 0.864
28 4O 0 47 87 70 0.606
29 0 40 40 80 1 10 0.688
30 0 40 40 80 70 0.586
31 20 20 47 87 90 0.687
32 20 20 47 87 70 0.558
33 0 40 47 87 90 0.618
34 20 20 40 80 70 0.638
35 0 40 40 80 70 0.547
36 30 10 45.25 85.25 80 0.721
37 20 20 40 80 1 10 1.084
38 40 0 40 80 1 10 1.050

 

117

Table A.17(contd) Data for Figure 5.4. Peak intensity ratio (PIR) at 591nm over 419nm
of carminic acid in sucrose solutions at different temperature with different amount of

ﬁ'uctose and glucose. Brix represents total sugar percent by weight.

 

 

Fructose Glucose Sucrose Temperature Peak intensity ratio
RUN # (wt%) (wt%) (wt%) BRD( (°C) PIR
39 0 40 47 87 70 0.566
40 0 40 47 87 1 10 0.744
41 0 40 43.5 83.5 110 0.798
42 40 0 40 80 70 0.720
43 40 0 40 80 70 0.646
44 0 40 47 87 70 0.585
45 20 20 43.5 83.5 110 1.025
46 20 20 47 87 70 0.576
47 0 40 43.5 83.5 70 0.522
48 0 40 40 80 90 0.617
49 40 0 40 80 70 0.749
50 40 0 43.5 83.5 110 1.049
51 40 0 43.5 83.5 110 0.973
52 0 40 43.5 83.5 90 0.606
53 40 0 43.5 83.5 90 0.886
54 40 0 40 80 1 10 1.062
55 40 0 47 87 70 0.668
56 40 0 40 80 1 10 1.182
57 40 0 47 87 1 10 0.836
58 20 20 47 87 110 1.015
59 0 40 40 80 70 0.576
60 40 0 47 87 1 10 0.780
r2* 20 20 40 80 1 10 1.059
r9* 20 20 40 80 70 0.590
r13* 40 0 47 87 110 0.758
r15* 20 20 47 87 70 0.544
r29* 0 40 40 80 1 10 0.705
r37* 20 20 40 80 1 10 0.986
r52* 0 40 43.5 83.5 90 0.627
r55* 40 0 47 87 70 0.584
r57 * 40 0 47 87 110 0.988

 

*runs are repeated in addition to the original experiemtal design

118

 

Table A.18 Data for Figure 5.6. Anisotropy of pyranine in sucrose solutions at different

temperature with different amount of fructose and glucose. Brix represents total sugar

 

 

percent by weight.
RUN # Fructose Glucose Sucrose BRIX Temperature Anisotropy
(wt%) (wt%) (wt%) C’C) r

1 0 40 47 87 1 10 0.176
2 20 20 40 80 1 10 0.133
3 0 40 43.5 83.5 90 0.196
4 40 0 40 80 90 0.198
5 20 20 40 80 70 0.238
6 20 20 43.5 83.5 90 0.227
7 0 40 47 87 70 0.270
8 0 40 47 87 1 10 0.180
9 20 20 40 80 70 0.237
10 20 20 43.5 83.5 110 0.162
1 1 20 20 40 80 90 0.180
12 40 0 47 87 90 0.286
13 40 0 47 87 1 10 0.225
14 20 20 43.5 83.5 90 0.217
15 20 20 47 87 70 0.302
16 40 0 43.5 83.5 70 0.294
17 40 0 43.5 83.5 90 0.242
18 0 40 40 80 90 0.163
19 30 10 41 .75 81.75 80 0.234
20 0 40 41 .75 81 .75 80 0.198
21 30 10 45.25 85.25 100 0.221
22 20 20 43.5 83.5 70 0.270
23 30 10 41.75 81.75 100 0.178
24 20 20 47 87 1 10 0.206
25 40 0 47 87 70 0.321
26 0 40 40 80 1 10 0.122
27 0 40 40 80 1 10 0.120
28 40 0 47 87 70 0.323
29 0 40 40 80 1 10 0.128
30 0 40 40 80 70 0.21 1
31 20 20 47 87 90 0.267
32 20 20 47 87 70 0.304
33 0 40 47 87 90 0.235
34 20 20 40 80 70 0.235
35 0 40 40 80 70 0.209
36 30 10 45.25 85.25 80 0.272
37 20 20 40 80 110 0.133
38 40 0 40 80 1 10 0.150

 

119

Table A.18 (contd) Data for Figure 5.6. Anisotropy of pyranine in sucrose solutions at

different temperature with different amount of fructose and glucose. Brix represents total

 

 

sugar percent by weight.
RUN # Fructose Glucose Sucrose BRIX Temperature Anisotropy
(wt%) (wt%) (wt%) (° C) r
39 0 40 47 87 70 0.275
40 0 40 47 87 1 10 0.194
41 0 40 43.5 83.5 110 0.143
42 40 0 40 80 70 0.256
43 40 0 40 80 70 0.254
44 0 40 47 87 70 0.274
45 20 20 43.5 83.5 110 0.164
46 20 20 47 87 70 0.304
47 0 40 43.5 83.5 70 0.244
48 0 40 40 80 90 0.160
49 40 0 40 80 70 0.265
50 40 0 43.5 83.5 110 0.185
51 40 0 43.5 83.5 110 0.185
52 0 40 43.5 83.5 90 0.211
53 40 0 43.5 83.5 90 0.242
54 40 0 40 80 1 10 0.148
55 40 0 47 87 70 0.326
56 40 0 40 80 1 10 0.145
57 40 0 47 87 1 10 0.233
58 20 20 47 87 1 10 0.204
59 0 40 40 80 70 0.206
60 40 0 47 87 1 10 0.225

 

120

   

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