U .3

fl? .w.

. “How.” . .
3.: 1h

r

 

... .
. “a < . ., F
a“? r .

‘
£5:

.3
3.1,. .

 

I

 

V ‘
.51 1“ mafia
.. 9.1....mh‘q .

» .. xufru

1

“man“

J . A
.r

 

“1335. ...n.. ‘

A man...“

.m

fit.

. 9%. “Imutmhar... armou- ._I

:..F..\

1‘. .It.

, {Ema

THESIS
2

Dan}

This is to certify that the

dissertation entitled

Synthesis and Characterization of Tailor—Made
Additives for Inhibition of Sparingly Soluble

Calcium Salt Crystallization
presented by

Charles 0. Ngowe

has been accepted towards fulfillment
of the requirements for

Ph . D . degreein Chemistry

Major professor

Date /2- $56 22:202.

 

MS U i: an Affirmative Action/Equal Opportunity Institution 0—127"

—~ V<p "v vfi—v'fie a

v“ h—v—bv—F‘

W
Michigan State

University

 

 

 

PLACE lN REIURN Box to remove this checkout from your record.
To AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

6/01 cJCIRCIDataDuopGS-sz

Synthesis and Characterization of Tailor-Made Additives for Inhibition
of Sparingly Soluble Calcium Salt Crystallization

By

CHARLES OWINO NGOWE

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of
DOCTOR OF PHILOSOPHY
Department of Chemistry

2002

ABSTRACT

SYNTHESIS AND CHARACTERIZATION OF TAILOR-MADE ADDITIVES FOR
INHIBITION OF SPARINGLY SOLUBLE CALCIUM SALT CRYSTALLIZATION

By

CHARLES OWINO NGOWE

Tailor-made additives were designed and used to modify the nucleation and
growth kinetics of calcium oxalate crystallizing from aqueous solution. Tailor-made
inhibitors can be used for a variety of purposes that include crystal morphology
engineering, kinetic resolution of racemates. reduction of crystal symmetry. assignment
of absolute configuration of chiral molecules and polar crystals and control of crystal
polymorphism. The molecules of the present study influenced both the morphology and
phase transformation of calcium oxalate crystals.

Calcium oxalate. CanOi. is a sparingly soluble salt widely found in naturc.
Three hydrate forms of the salt are known including the monohydratc, the dihydratc. and
the trihydrate. In the pulp and paper industries, CaC204 is notorious for forming scale

deposits on the equipment. In animals. CanO4 occurs as a crystal in the urinary tract and

constitutes the largest composition in kidney and bladder stones. Clinical management of
kidney stones includes taking citric acid supplements which are inhibitors of calcium salt
crystallization; however, the amount of citric acid that must be consumed is relatively
large (in excess of 2-8 grams per day). Coincidently, large amounts of amino acids
(0.8%) are found in kidney stone matrix, and they too have long been considered as
crystallization inhibitors; however, several studies have proven otherwise. To improve
efficacy of amino acids in this application, we have derivatized several amino acids with
cis-expoxysuccinic acid. Subsequently, the crystallization of calcium oxalates in the
presence of the deri vatized amino acids was studied at ambient temperature. The glycine
and the glutamic derivative selectively inhibited the phase transformation and stabilized
the dihydrate form of calcium oxalate, while others allowed phase transformation to take
place from a metastable calcium oxalate trihydrate. COT, to the thermodynamically
stable monohydrate. COM. The lysine derivative inhibited the nucleation and the crystal
growth of calcium oxalate much better than citric acid although they both had very lllIlC
effect on CanO4 phase transformation. In all cases, the additives modified the crystal

shape of the dihydrate form of calcium oxalate.

DEDICATION

To Joseph Omony, Adhadha Athieno
And Jason and Lucy Greer

iv

ACKNOWLEDGMENTS
I will always be indebted to my advisor Prof. Kris Berglund for making this

possible. He allowed me the freedom to develop as an intellectual at my own pace. and
gave me much needed advise at critical junctions in my academic career. I can always
count on him giving me his honest opinion about any subject. One of the greatest things I
learned from Kris is keeping my mind focused. Thank you for helping me cross this
bridge.

My wife Karen Ngowe aka Lady Love Karen (LLK) deserves many thanks for
believing in me and pushing me beyond my own limits. Withought her moral and
emotional support I could not have done this alone. Thank you for all your
encouragements and being the strength our family that we depend on. I love you a billion
times plus one.

To my children Athieno Lironah N gowe named after my last surviving
grandmother, and Yosef Opoya Ngowe named after the family that raised my great
grandfather, I extend many thanks in your honor. Only hard work and perseverance \\ ill
guarantee you any success in life. Never give up on anything and always believe In
yourself. The extra energy and motivation I needed to complete my academic career was
driven by the love for you both.

My poor mother Dalia Owino, thank you for giving me all the extra help I needed
in order to pert-arm u. .i in sc ool. M siblings always accused you for loving me more
but I believe that you loved us all the same. However, I needed your nurturing lhc must
This historical achievement is in your honor.

I cannot forget my father Owino Wodomony, and my in-laws Barry and Judy

Kanne. Thank you for investing so much of your time and finances on my education. I

would have had greater difficulty in achieving this goal without your presence in my life.
Thank you for everything.

Also thanks to my second advisor Prof. Gary Blanchard for all his guidance and
acceptance in his group as one of his own. Thanks to the many advises that you gave
me, and for listening to all my problems. To the rest of my committee members, Prof.
Malezcka and Prof. Bohran, I enjoyed the support you afforded me.

Last, but not least is those that most certainly need to be thanked are Dr Stephen
Bakiamoh and Dr. Jaycoda Major for all the late nights we spent coaching, supporting.
and teaching one another. Dr. Dilum DunuWila, thanks for all the discussions about
crystallization as well as listening to all my problems. I will always depend on your
support. The rest of the labmates past and present; Parminder, Sri, Mike, Adam, Carinna.
Fang, Al, Anna, Lilli, Javier, DeeDee, Johnny, Matt, Rosanna, and Hasan, thanks for all

your support.

vi

TABLE OF CONTENTS

List of Tables .......................................................................................... ix
List of Figures .......................................................................................... x
List of Schemes .................................................................................... xiv

List of Spectraxy
List of Abbreviations ................................................................................. xvi
Introduction
Chapter 1
BACKGROUND AND LITERATURE REVIEW
1.1 Calcium oxalate crystal growth in kidney tones ........................................ 1
1.1.1 The Supersaturation/ Crystallization Theory of Kidney Stone Formation ...... 4
1.1.2 The Matrix Theory of Kidney Stone Formation .............................................. 5
1.1.3 The Promoter/Inhibitor Theory of Kidney Stone Formation........ ........6
1.2 Calcium Oxalate Crystal Growth in Plants7

1.2.1 Environmental and Economical Impact of Calcium Oxalate deposits... .......... 8

1.2.2 Calcium Oxalate as a Sparingly Soluble Salt9

1.3 Crystallization Kinetics ................................................................... l 1
1.3.1 Supersaturation ......................................................................... 13
1.3.2 Homogeneous Nucleation ............................................................ 14
1.3.3 Heterogeneous Nucleation ........................................................... 15
1.3.4 Crystal Growth Theory .............................................................. 16
1.3.4.1 Burton. Cabrera. and Frank Model ............................................... 17
1.3.4.2 Birth and Spread Model ........................................................... 20
1.3.4.3 Diffusion-controlled growth ................................................ 22
1.3.5 Crystal Shape ........................................................................... 24
1.3.6 Crystal Habit Modification .......................................................... 29

vii

1.3.7 Phase Transformation ................................................................. 32
1.4 References ....................................................................................... 34

Chapter 2

SYNTHESIS of SIMPLE ORGANIC CHELATES DERIVED FROM AMINO ACID
AND EPOXYSUCCINIC ACID FOR INHIBITION OF CALCIUM OXALATE

2.1 Background .............................................................................. 39
2.2 Experimental ............................................................................. 41
2.2.1 Calibration curve .................................................................... 47
2.2.2 Titration. ................................................................................ 47
2.2.3 Nephelometry experiment ......................................................... 48
2.3 Results and Discussion ................................................................ 51
2.3.1 Chelation constants ................................................................. 51
2.3.2 Crystallization kinetics ............................................................. 56
2.4 Conclusions .............................................................................. 61
2.5 References ................................................................................ 62
Chapter 3

THE INFLUENCE OF TAILOR-MADE IMPURITIES OF AMINOACID
DERIVATIVES ON THE MORPHOLOGY AND PHASE TRANSFORMATION OF
CALCIUM OXALATE HYDRATES 64

3.1 Background .............................................................................. 66
3.2 Experimental ............................................................................ 68
3.3 Results and Discussion . .............................................................. 69
3.4 Conclusion ............................................................................... 91
3.5 References .............................................................................. 92
Chapter 4
FUTURE STUDIES
4.1 Introduction ............................................................................ 95
4.2 Proposed Studies ..................................................................... 96
4.2.1 Constant composition method .................................................. 96
4.2.2 Synthesis of amino acid derivatives .......................................... 98
4.3 References ............................................................................... 99

Conclusion 100

viii

Table 1.1

Table 2.1

Table 2.2

LIST OF TABLES

Common precipitates in kidney stone disease
....................................................................................... 2

Calcium Chelation capacity of amino acid derivatives compared to citrate
as determined by calcium-selective electrode (model 97-20 ionplus

electrode).
..................................................................................... 61

Efieczs of inhibitors on calcium oxalate polymorph nucleation and crystal
growth. Experimental conditions: calcium oxalate, and inhibitor
concentrations were all at 0.8 mM, T°, 280°C; pH 5.5; ionic strength,
0.15M.
................................................................................... 66

ix

Figure 1.1

Figure 1.2

Figure 1.3

Figure 1.4

Figure 1.5

Figure 1.6

Figure 1.7

Figure 2.1

Figure 2.2

Figure 2.3

LIST OF FIGURES

Flow chart depicting the different types of nucleation and the
corresponding subcategories.

Development of a growth spiral starting from a screw dislocation (a) to
spiral staircase (c).
..................................................................................... 18

Depicts such a polycrystalline growth by the birth and spread (B+S)
mechanism.
71

Equilibrium crystal shape as described by Wulff’s theorem. In this case

71 (72°
..................................................................................... 25

Hypothetical three-dimensional crystal presenting the three main types of
possible faces: flat (F), step (S) and kink (K) faces.
................................................................................ ._ . . .. 27

Influence of impurity adsorption on the crystal habit, for the case oi adipic
acid. ‘

.................................................................................... 30
Typical solubility profiles and pertinent supersaturation values.

Optical schematic of the spectrometer for nephelometry study.
.................................................................................. 56.

A plot of moles of bound Ca2+ vs moles of titrant (lysine derivatrx c l Data
of bound calcium was from the average of three replicates. Experimental
conditions: 200 ppm calcium concentration, 0.03M NH4C1/O.07.\l NlLOH
buffer rpH=9.5. ionic .< “ength of 0.1M NaCl, 25 °C).
................................................................................ . 58

cha verses lysine derivative concentration. The cha was determined
from the average of three replicates. Experimental conditions: 200 ppm
calcium concentration, 0.03M NH4Cl/O.07M NI-IiOH buffer (pH=9.S.
ionic strength of 0.1M NaCl, 25 °C).
..................................................................................... 60

Figure 2.4

Figure 2.5

Figure 3.1a

Figure 3.1b

Figure 3.2

Figure 3.33

Figure 3.3b

Typical curve of calcium oxalate crystallization using nephelometry.
Experimental conditions: calcium concentration, 0.8 mM, oxalate
concentration, 0.8 mM; T°, 280°C; pH 5.5; ionic strength, 0.15M NaCl.
The clear line represents the smoothed data using Savitsky-Golay
algorithm.
..................................................................................... 63

A typical desupersaturation curve for the system CaC13_2H20 + NazC204.
Experimental conditions: calcium concentration, 0.8 mM, oxalate
concentration, 0.8 mM; T°, 280°C; pH 5.5; ionic strength, 0.15M NaCl.
..................................................................................... 65

Raman spectra of calcium oxalate hydrates after 1 hour in solution. a)
alanine derivative, b) glycine derivative, c) arginine derivative, (1) citrate,
e) glutamic derivative, f) control, g) lysine derivative, h) aspartic
derivative Experimental conditions: calcium concentration, 0.8 mM,
oxalate concentration, 0.8 mM; 1°, 280°C; pH 5.5; ionic strength, 0.15M
NaCl. Concentration of additives, 0.8mM. ‘
..................................................................................... 76

Raman spectra of calcium oxalate hydrates after 24 hours in solution. a)
alanine derivative, b) arginine derivative, c) control, d) glutamic
derivative, e) glycine derivative, f) citrate, g) lysine derivative, h) aspartic
derivative. Experimental conditions: calcium concentration, 0.8 mM.
oxalate concentration, 0.8 mM; ”1°, 280°C; pH 5.5; ionic strength. 0.15M
NaCl. Concentration of additives, 0.8mM.
..................................................................................... 77

ESEM of a typical COD morphology. Experimental conditions: calcium
concentration, 0.8 mM, oxalate concentration, 0.8 mM; '1", 280°C ; pH
5.5; ionic strength, 0.15M NaCl.
........................................................... 79

ESEM of COD formed in the presence of 0.8 mM glycine deri valtyc after
left insolution for 1 hour. Experimental conditions: calcium
concentration, 0.8 mM, oxalate concentration, 0.8 mM; T°, 28.0"(‘. pH
5.5; ionic strength, 0.15M NaCl.
..................................................................................... 80

ESEM of COD formed in the presence of 0.8 mM [3 -alanine derivative

after left insolution for 1 hour. Experimental conditions: calcium
concentration, 0.8 mM, oxalate concentration, 0.8 mM; 1°. 280°C: pH
5.5; ionic strength, 0.15M NaCI.
..................................................................................... 81

xi

Figure 3.3c

Figure 3.3d

Figure 3.4

Figure 3.5

Figure 3.6

Figure 3.7

Figure 3.8

ESEM of COD formed in the presence of 0.8 mM glutamic derivative
after left insolution for 1 hour. Experimental conditions: calcium
cdncentration, 0.8 mM, oxalate concentration, 0.8 mM; T°, 280°C; pH
5.5; ionic strength, 0.15M NaCl.
..................................................................................... 82

ESEM of COD formed in the presence of 0.8 mM arginine derivative after
left in solution for 1 hour. Experimental conditions: calcium
concentration, 0.8 mM, oxalate concentration, 0.8 mM; T°, 280°C; pH
5.5; ionic strength, 0.15M NaCl.
..................................................................................... 83

ESEM of COD formed in the presence of 0.8 mM citrate after left in
solution for lhour. Experimental conditions: calcium concentration, 0.8
mM, oxalate concentration, 0.8 mM; T°, 280°C; pH 5.5; ionic strength,
0.15M NaCl. ‘
..................................................................................... 84

ESEM of COD formed in the presence of 0.8 mM glutamic derivative
after left in solution for 24 hours. Experimental conditions: calcium
concentration, 0.8 mM, oxalate concentration, 0.8 mM; T°, 280°C ; pH
5.5: ionic strength, 0.15M NaCl.
..................................................................................... 85

ESEM of COD formed in the presence of 0.8 mM glycine derivative after
left in solution for 24 hours. Experimental conditions: calcium
concentratim, 0.8 mM, oxalate concentration, 0.8 mM; T°, 280°C ; pH
5.5; ionic strength, 0.15M NaCl.
..................................................................................... 86

ESEM of COD formed in the presence of 0.8 mM alanine derivative after
left in solution for 24 hours. Experimental conditions: calcium
concentration, 0.8 mM, oxalate concentration, 0.8 mM; T°, 280°C : pH
5.5; ionic strength, 0.15M NaCl.
..................................................................................... 89

ESEM of COD formed in the presence of 0.8 mM arginine den vatrvc after
left in solution for 24 hours. Experimental conditions: calcium
concentration, 0.8 mM. oxalate concentration, 0.8 mM; T°, 280°C; pH
5.5; ionic strength, 0.15M NaCl.
..................................................................................... 90

xii

Figure 3.9a

Figure 3%

Figure 3.9c

Figure 3%

Figure 3.10

Figure 3.11

ESEM of COD formed in the presence of 0.8 mM citrate derivative after
left in solution for 24 hours. Experimental conditions: calcium
concentration, 0.8 mM, oxalate concentration, 0.8 mM; T°, 280°C; pH
5.5; ionic strength, 0.15M NaCl.
..................................................................................... 91

ESEM of COD formed in the presence of 0.8 mM lycine derivative after
left in solution for 24 hours. Experimental conditions: calcium
concentration, 0.8 mM. oxalate concentration, 0.8 mM; 1°, 280°C; pH
5.5; ionic strength, 0.15M NaCl.
..................................................................................... 92

ESEM of COD formed in the absence of additives after left in solution for
24 hours. Experimental conditions: calcium concentration, 0.8 mM,
oxalate concentration, 0.8 mM; 'I‘°, 280°C; pH 5.5; ionic strength, 0.15M
NaCl.
..................................................................................... 93

ESEM of COD formed in the presence of 0.8 mM aspartic derivative after
left in solution for 24 hours. Experimental conditions: calcium
concentration, 0.8 mM, oxalate concentration, 0.8 mM; T°, 280°C: pH
5.5; ionic strength, 0.15M NaCl.
..................................................................................... 94

XRD spectra of COD crystal of the control sample, and the samples
formed in the presence of lysine derivative, and citrate after left in solution
for an hour. Experimental conditions: calcium concentration, 0.8 mM.
oxalate concentration, 0.8 mM; T°, 280°C; pH 5.5; ionic strength. 0. 15M
NaCl.
..................................................................................... 95

XRD spectra of COD crystal of the control sample, and the samples
formed in the presence of additives ( glutamic and glycine derivative)
which prevented phase transformation of COD crystals after it Was left in
solution for 24 hours. Experimental conditions: calcium concentration.
0.8 mM, oxalate concentration, 0.8 mM; T°, 280°C; pH 5.5; ionic
strength, 0.15M NaCl.

....................................... 96

xiii

Scheme 2.1

Scheme 2.2

Scheme 2.3

Scheme 2.4

Scheme 2.5

Scheme 2.6

Scheme 3.1

LIST OF SCHEMES

Synthesis of lysine derivative (6-(1,1-Dicarboxy-2-hydroxy-ethylamino)-
2-(1,2-dicarboxy—2-hydroxy-ethylamino)-hexanoic acid).
.. ..................................................................................... 42

Synthesis of arginine derivative (2-(l-Carboxy-4-guanidino—butylamino)-
3-hydroxy-succinic acid).

....................................................................................... 44
Synthesis of glutarrric derivative (2-(1,2-Dicarboxy-ethylamino)-
pentanedioc acid). ’
....................................................................................... 46
Synthesis of aspartic derivative (2-(1,2-Dicarboxy—ethylamino)-3-
hydroxy-succinic acid).
....................................................................................... 48

Synthesis of glycine derivative (2-(Carboxymethyl-amino)-3-hydroxy-
succinic acid).
....................................................................................... 50

Synthesis of ,6 -alanine derivative (2-(2-Carboxy-ethylamino)- 3-hydroxy-
succinic acid).
....................................................................................... 52

Tailor-made additives synthesized by reacting cis-epoxysuccinic acid and
amino acid. See chapter 2 for more details on the synthesis procedure.
....................................................................................... 70

xiv

Spectrum 2.1

Spectrum 2.2

Spectrum 2.3

Spectrum 2.4

Spectrum 2.5

Spectrum 2.6

LIST OF SPECTRA

lH-NMR spectrum of lysine derivative (6-(1,l-Dicarboxy-Z-hydroxy-
ethylamino)-2-(1,2-dicarboxy-2—hydroxy-ethylamino)—hexanoic acid).
....................................................................................... 41

1H-NMR spectrum of arginine derivative (2-(l-Carboxy-4-guanidino-
butylamino)-3-hydroxy-succinic acid).
....................................................................................... 44

lH-NMR spectrum of glutamic derivative (2-(1,2-Dicarboxy-ethylamino)-
pentanedioc acid).
....................................................................................... 46

'H-NMR spectrum of aspartic derivative (2-(1,2-Dicarboxy-ethylamino)-
3-hydroxy-succinic acid).
....................................................................................... 48

1H-NMR spectrum of glycine derivative (2-(Carboxymethyl-aminol-3-
hydroxy-succinic acid).
....................................................................................... 50

1H-NMR spectrum of ,8 -alanine derivative (2-(2-Carboxy-ethylamrnol- 3-
hydroxy-succinic acid).
........................................... 52

XV

>’°°q

H
N

"‘ G-

'10

U: W w wQOU>VJQ

9)

LIST OF ABBREVIATIONS

preexponential factor

Bolzmann’s constant

temperature

free energy change for an aggregate undergoing a phase transition

volume free energy change
surface free energy change

volume-shape factor

characteristic length of the crystallizing solute
molecular volume of the crystallizing solute
area shape factor

interfacial energy per Unit area

growth rate of a crystal face

combination of physical constants of the system including
adsorption, surface diffusion, and growth unit incorporation factors
supersaturation

boundary layer thickness

edge free energy per molecule of the nucleus

the 2D nucleation rate

the height of the layer

the area of the face

surface area of the crystal

diffusion coefficient

bulk concentration

interfacial concentrations

diffusion controlled rate

solute incorporation rate
crystal growth rate

A quantitative measure of the influence of any resistance to growth
represents the ratio of the pseudo-first-order rate coefficient at the
bulk conditions to the mass transfer

the mass fraction of solute in solution

the activity of the impurity

transformation time

growth time

dissolution time

xvi

Chapter 1

INTRODUCTION

BACKGROUND AND LITERATURE REVIEW

1.1 Calcium Oxalate Crystal Growth in Kidney Stones

The major components of urinary stones are calcium oxalate (CaC204) and calcium
phosphate.1 Other compounds contributing to the mineral portion of stones and are listed
in Table 1.1. Kidney stones are mostly mineral in composition with only about 2.5'
percent of the weight of atypical stone due to organic compounds from the urine, known
as the stone matrix.2 Calcium oxalate monohydrate (COM), dihydrate (COD), and
trihydrate (COT) are all found in kidney stones with COM occurring most frequently

followed by COD. 7‘ COT occurs least often, and has been reported in only a few cases}5

 

Chemical Name

Mineral Name

Formula

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

calcium oxalate whewellite CaC204. H20

monohydrate

calcium oxalate dihydrate weddelite CaCzO4. 2H20

calcium oxalate trihydrate CaC204. 3H20

basic calcium hydrogen hydroxyapatite Cas(PO4)3(OH)

phosphate

calcium hydrogen brushite Cal-[PO4.2H20

phosyhate _

tricalcium phosphate whitlockite Ca3(PO4)2

magnesium ammonium struvite Mg(NH4)(PO4).6H30
htflahate hexahydrate

uric acid C5H4N4O3

UI'IC acid dihydrate C5H4N4O3.2H20 l

monosodium urate NaC5H4N403j-120 l

monohydrate '

l-cystine SgC6H12N204

octacalcium phosphate CagHg(PO4)(,.5HgO

magnesium hydrogen newberite MgHPO4.3HgO

phosphate trihydrate )5

Table 1.1

Common precipitates in kidney stone disease3

IQ

Urinary stone formation theories include supersaturation/crystallization, urinary
inhibitors, epitaxy, matrix, and combinations of these processes.2‘°‘7 Urine is a medium
unique in its ability to hold solutes (i.e, calcium, oxalate) in solution. In normal urine, the
concentration of calcium oxalate salt is four times higher than its solubility in water.8
Urinary supersaturation/crystallization is necessary for the formation of stones. As
concentrations of solutes increase, the solubility product is reached, above which
dissolved salts can form nuclei of its solid phase (the metastable zone). These nuclei can
form heterogeneously. that is, on foreign surfaces (other crystals, cellular debris, urinary
casts, epithelial lining. etc).9 Solute levels reaching the formation product and beyond
(the supersaturated zone) may form crystal nuclei homogeneously. Homogenous
nucleation occurs in pure solution and requires more thermodynamic energy in order for
a stable nuclei to form from which crystal growth can take place. Heterogeneous
nucleation is thought to initiate crystal formation. These crystals may attach to the
epithelial lining of uriniferous tubules/collecting ducts and subsequently serve as crystal
growth sites. Small stones can grow and either spontaneously pass or become large and
can lead to obstruction. colic, and/or infection.

Calcium oxalate crystallization from aqueous solution results in the fomiation of
three types of hydrates: the monohydrate, the dihydrate, and the trihydrate. dependin g on
solution conditions. including concentrations of the crystallizing species, the solution
temperature, the pH. the ionic strength, and the concentrations of growth-modifiers.”
These effects include increases or decreases in the following: 1) the numbers of crystals
nucleated per solution volume. 2) the time required for the first crystal nuclei to torm. or

the induction time”, and 3) the rate at which the individual faces of a crystal grow. In

addition, the crystal habit and the crystal phase formed (monohydrate, dihydrate, or
trihydrate, in the case of calcium oxalate)'also depend on solution conditions.
Subsequent agglomeration (the formation of multi-crystal masses) of growing crystals
also depends on the various solution conditions. The effects of several solution

parameters on nucleation, growth, and agglomeration have been reviewed by Finlayson.l2

1.1.1 The Supersaturation/ Crystallization Theory of Kidney Stone Formation

The supersaturation theory of kidney stone formation hypothesizes that calcium
oxalate kidney stone formation is wholly due to an increased supersaturation of calcium
oxalate in the urine.13 This increase is thought to be the result of higher output of calcium
and oxalate ions by the kidneys either because of greater ingestion of these substances or
through some metabolic malfunction in the body. The high supersaturation of calcium
oxalate leads to nucleation of crystals in either free solution or on the luminal walls.

These two modes of nucleation suggest two possible mechanisms for the initiation
of stone growth, the free particle mechanism and the fixed particle mechanism.” Rapidly
growing crystals flowing freely in solution can, after reaching sufficient size, becmnc
trapped somewhere in the urinary tract. forming the embryos of a stone. According to the
free particle theory. other crystals and urinary debris collide with the entrapped cn stal
and stick to it, and the stone continues to grow in this manner. In the fixed partic lt-
mechanism, a crystal nucleates on a luminal wall grows and attracts urinary debris
Accumulation of organic and mineral matter gradually leads to stone formation.
Alternatively, tire crystals ma} agglomerate together freely in the urine solution running
a multi-crystal mass which becomes too large to pass. The agglomerate then acts as a

nidus for stone growth in much the same way as a single crystal.

The supersaturation levels of both stone-forming and non-stone-forming urines
are high enough for crystals to form,14 yet crystals occasionally lead to stone formation in
the former while they pass harrnlessly from the latter. Even though stone-forming urines
often show a greater volume of crystals than that observed in non-stone-forming urine15 ,
at the supersaturation level commonly observed for stone-forming urine, calcium oxalate
growth rates are not fast enough to cause blockage of the urinary tract solely due to
crystal growth. Therefore, the hyperexcretion/supersaturation theory does not fully
explain stone formation. This theory simply suggests one causative factor in the disease
solution conditions conducive to rapid crystal nucleation and growth. The other two
theories, the matrix theory and the promoter/inhibitor theory, attempt to explain how

crystal nucleation, growth, and agglomeration are affected by solution conditions other

than supersaturation.

1.1.2 The Matrix Theory of Kidney Stone Formation

The matrix theory of kidney stone formation hypothesizes that the nucleation of
calcium oxalate crystals is promoted by the existence of a mucous organic matrix
deposited in the urinary tract.'°'l7 This matrix is composed of various organic
macromolecules such as glycosaminoglycans and glycoproteins that are thought to
precipitate from urine. As they precipitate, they form a loose mucous mass in the tract.
This mucous matrix serves as a substrate for crystal nucleation. Crystal nuclei lomi over
the matrix and subsequently grow, causing encrustation and hardening of the f omiin g
kidney stone. The tendency for deposition of the matrix is thought to be one factor that
separates stone-formers from non-stone-formers and is thought to be a function ol the

excretion of macromolecules into the urine from various sources in the body.

The matrix theory lacks credibility in that there is a lack of evidence supporting
the idea of large-scale deposition of the organic macromolecules prior to encrustation. A
more plausible explanation, and one that seems to be more verifiable, is the

promoter/inhibitor theory.

1.1.3 The Promoter/Inhibitor Theory of Kidney Stone Formation

The promoter/inhibitor theory of kidney stone formation is based on the tendency
for calcium oxalate crystals to nucleate and grow in urine at a higher rate for stone-
formers than for non-stone formers.‘8 This is not, however, necessarily due to a greater
calcium oxalate supersaturation in stone-forming urine, as noted above. Rather, there are
certain chemical species in urine that modify the nucleation, growth, and/or
agglomeration of calcium oxalate.

Various substances have been implicated as growth modifiers. including
macromolecular species such as glycosaminoglycans and glycoproteins. The
modification may be inhibitory or promotional depending on the substance present. the
amount present, and the process being considered. Normal urine contains
macromolecules that inhibit crystal growth but promote the nucleation of crystals. 1”
There is apparently a greater concentration of these macromolecules in normal urine than
in stone-forming urine.20 The creation of many small, slowly growing crystals in normal
urine allows excretion before stone formation can occur. Urinary macromolecules may
also inhibit the agglomeration of crystals.21 Enhancement of nucleation by urinary
macromolecules and the apparent increase in the probability of agglomeration due to a
higher concentration of crystals in urine is offset by the inhibition of agglomeration by

urinary macromolecules. Thus, the promoting effect of certain urinary macromolecules

on crystal nucleation is offset by their inhibitory effect on crystal growth and
agglomeration. The net effect is that crystals pass from the urinary tract before stone
formation can occur. The low concentration or lack of these macromolecules in stone-
forming urine is one possible cause of an increased tendency to form kidney stones.

The promoter/inhibitor theory is actually an extension of supersaturation theory in
that high levels of calcium oxalate supersaturation are still required to form crystals and
consequent stones. The overall mechanism of stone formation, whether by a fixed or free
particle mechanism, is still a matter for debate and can only be resolved by experiments
simulating the complete environment of the kidney, including the flow characteristics,
luminal walls, and insoluble debris. The plausibility of the theory can be and has been
tested by simple experiments of solution crystallization in the presence of various
modifiers. Experiments such as these, while intended to explain kidney stone formation.

also contribute to the general understanding of crystal growth.

1.2 Calcium Oxalate Crystal Growth in Plants

A vast array of organisms produce biological minerals, or “biominerals.”
Biominerals encompass a wide spectrum of scale and composition, from macroscopic
structures such as vertebrate bone, composed of apatite, to microscopic silicon ll’UslulCS

2. 3. 4 - -
2 2 2 Biomineralt/ation

of diatoms and subcellular ferrimagnetic particles in bacteria.
fulfills a variety of crucial functions, including important skeletal and protective roles.
The plant kingdom exhibits a varied assortment of mineralized structures formed by cells.
including deposits of silica, calcium oxalate, and calcium carbonate.25 Calcium oxalate

crystals are by far the most prevalent and widely distributed mineral deposits in higher

plants. Calcium is very abundant in the natural environment in which most plants grow.

A required element for plant growth and development, calcium plays many important
roles, for example, as a structural component of cell walls, a signal in various
physiological and developmental pathways.24‘2° Nonetheless, cytosolic free calcium
must be restricted to levels of 7M or less, because higher concentrations interfere with a
variety of crucial cell processes, including calcium-dependent signaling, phosphate-based

energy metabolism. and microskeletal dynamics.” 27' 28

In addition, most plants, unlike
animals, do not have well-developed excretory systems to dispose of excess calcium.
Instead, higher plants appear to modulate differences between the natural abundance of
environmental calcium and the very low levels required for cytosolic free calcium by
controlling the distribution of calcium within the cell.29'3°'3l The cell wall and the
vacuole provide major sinks for calcium in plants. In water, calcium oxalate provides a
relatively insoluble, metabolically inactive salt for calcium sequestration. Calcium
oxalate thus provides a high-capacity repository for calcium, and plants may accumulate
this salt in substantial amounts. up to 80% of their dry weight or 90% of total

32.33

calcium. The extent of calcium partitioning into calcium oxalate varies among

different taxonomic groups of plants.

1.2.1 Environmental and Economic Impact of Calcium Oxalate deposits

A good source of cellulose fibers (pulp) for making paper are trees. although
alternative fibers like straw. hemp, and kenaf are used whenever possible. The trees are
usually either ground up by mechanical force or soaked in chemicals to get more purified
fibers. Traditionally. the bleaching of pulp has been effected through sequential reactions
involving chlorine and sodium hydroxide. The early stages remove lignin; while the final

stages brighten the pulp. Classes of bleaching chemicals include strong oxidizing agents

(C12, 03, NaOCl, C102, 02, and H202), and alkali (NaOH). Chlorine, chlorine dioxide
and ozone work best when they are run in acidic conditions at pHs that range from 1.5 to
4. The presence of oxalic acid and divalent ions in plant makes it attractive to bleach in
acidic condition which insures ion exchange between hydrogen ions and the divalent
ions, thus, minimizes the chances of forming insoluble salt calcium oxalate according to
the following reaction:

Ca2+ + c,of' —> Cac,o,(s)

Due to recent regulations passsed by the EPA to limit the use of chlorinated compounds.
alternative routes are being implemented. The major problem with these alternative
bleaching chemicals is that they favor formation of calcium oxalate, which forms scale

deposits on the equipment of bleaching plants.

1.2.2 Calcium Oxalate as a Sparingly Soluble Salt

Calcium oxalate monohydrate (COM) is a sparingly soluble salt with a solubility
product of 2.00 x 10‘0 molezlliter2 at 25 0C.34 COM precipitates from solution at low
concentrations. The other kidney stone precipitates listed in Table 1.1 are also spaiingl y
soluble salts (except for uric acid, uric acid monohydrate and l-cystine) and are also
likely to precipitate from a solution containing relatively low concentrations of their
respective constituent ions. Such a description is typical of sparingly soluble salts in
general. Precipitates of these salts from low concentration solutions occur in vanous
circumstances including industrial water systems and in the aforementioned biological
phenomena. In particular. precipitates of the sparingly soluble salts calcium carbonate.
CaCO3, and calcium sulfate dihydrate, CaSOi‘2H20 (gypsum), are often seen as unwanted

scale in boilers, reactors. and cooling water systems. This scaling reduces heat transfer

coefficients and often leads to obstructions in piping. The removal of scale from process
surfaces and the treatment of raw water to reduce its scaling tendency is done at
considerable expense to industry. In order to develop cost-effective means of reducing
scale as well as managing kidney stone treatment, it is desirable to search for compounds
that inhibit formation of sparingly soluble calcium salt. Inhibitors of calcium salts are

typically studied by considering the crystallization characteristics of the calcium salt.

10

1.3 Crystallization Kinetics

The control of crystallization is of fundamental importance in many biological
and industrial processes; for instance in biomineralization, specific mnineral polymorphs
are selectively crystallized. while in pulp mills calcium oxalate forms deposits on the
equipment of the bleach p am. for example in filtrate tanks an on washing filter wires.
Controlled crystallization can be achieved on?y through an understanding of the
crystallization at the melecular level.

Crystallization involves two disunct processes that are driven by supersaturation
of the solute in the solvent; nucleation and crystal growth. There are two types of
nucleation; primary and secondary nucleation, as illustrated in the Figure 1.1. In primary
nucleation, no crystals are involved in the nucleation, whereas secondary nucleation is
induced by crystal seeds. The main requirement of primary nucleation is supersaturation
and it is the form of nucleation usually associated with the precipitation of calcium salt.
In supersaturated solutions. solute units such as atoms. molecules or ions aggregate
together to form a cluster or embryo. Such processes require energy. depending upon the
surface/interfacial tension of the interface, to expel the solvent molecules. Therefore. the
energy barriers associated with the cluster formation control nucleation kinetics as well as
crystal habit. ‘ Once the cluster has reached a certain critical size, it becomes stable and

. 5
serves as a nucleus for further growth or dissolves.3‘

11

 

 

 

 

HOMOGENEOUS HETEROGENEOUS

 

 

 

 

 

Figure 1.1

The different types of nucleation and the corresponding subcategories“

1.3.1 Supersaturation

Understanding of the driving force for crystallization is essential for
characterizing the kinetics as well as to regulate the parameters that control the crystal
growth. The most important parameter that defines the crystallization process is
supersaturation. Supersaturation is defined as the amount of solute in excess of the
equilibrium solubility that is present in solution at a given temperature and is the driving
force for the crystallization. The process of crystallization can be divided into two parts:
nucleation and growth. Nucleation is energetically more demanding than crystal growth
because there is an energy barrier associated with formation of stable nuclei.
Consequently, there are supersaturation regions in which crystal growth proceeds while
nucleation is suppressed.3°‘37‘38

The driving force for nucleation and growth is the difference in chemical

potential of the solute in a supersaturated solution, ,ul , and in a saturated solution. ,um .

For an anhydrous solute crystallizing from a binary solution, the driving force may be

written as A11 2 #1 - ,u Since, it = ,u" + RTln a , then

Ag = RT ln (ad/am ) = RT ln (yl'Cn/yqum) and the supersaturation a can be expressed in
dimensionless terms by a = A/l/RT = ln (all/am ) = ln (yl'Cl/yfw ).

If the ratio of the activity coefficient, 7 / y. = constant in the given concentration

_ regime, then the supersaturation becomes 0' = ln (C,/C¢,q )= ln (5‘ +1) where C is the
concentration of the crystallizing substance in the supersaturated solution and

Sr = [(Cl -ch )/Cm]. Normally. S‘. is taken to be much less than unity as a

13

consequence 0' = ln (5‘~ + 1) E [(C, - C“, )/ch ] .35

There are various methods to created supersaturation which include (a) solvent
removal (evaporation or freezing). (b) addition of different salts with ions that participate
in precipitation, and (c) dissolution of metastable solid phases. Supersaturation can also
be created by methods that regulate the solute solubility, such as temperature change, pH

change, and addition of a solvent that lowers the solubility of the solute.

1.3.2 Homogeneous Nucleation

The classical theory of nucleation based on the work by Gibbs,39 Volmer,40 and
others, assumes that clusters are formed in solution by an addition mechanism that
continues until a critical size is reached. The rate of nuclei formation (Bo) by this

mechanism is given by an Arrhenius type expression

 

AG
BozACXp( “0] (1.1)

where A is the pre-exponential factor and has a theoretical value of 1030 nuclei/cm‘s. k is
the Bolzmann‘s constant. and T is temperature. The free energy change for an aggregate
undergoing a phase transition. AG, = AG\ + 21Gs , where AG\. is the volume free energy
change associated with the phase transition (a negative quantity). and AG‘ is the surface
free energy change associated with the formation of the aggregate (a positive quantity).

For homogeneous or heterogeneous nucleation

21G‘ =—0tr3i(T/Uln(c/s) (1.2)
where or is the volume-shape factor, r is the characteristic length. and ti is the molecular

volume of the crystallizing solute. and ln(c/s) is the supersaturation ratio. For

homogeneous nucleation

14

21Gs = Bray12 (1.3)
where 13 is the area shape factor and y is the interfacial energy per unit area between the

crystallization medium,1, and the nucleating cluster, 2. Due to the competition between
volume and surface terms, AG0 passes through a maximum at a certain value of r‘. The

value of r' can be obtained by minimizing the free energy function with respect to the

characteristic length.

d(AG )/dr = -30tr21<T/Dln (c/s)+ ziiry12 = 0 (1.4)
The critical radius r* of the cluster is given by:

r‘ = zflqu2
30tr<T In (c/s)
For spherical clusters, (1:47:13 and 13:41:. Therefore,

(1.5)

 

2071::

r =-——- (1.6)
(T ln (c/s)
Considering these geometric factors. the rate for homogeneous nucleation of spherical

cluster is

—16 2 3‘1
B0 = Aexp 1m Y“ , (1.7)

3(KT)BID[E]-

The equation above predicts that nucleation can be controlled experimentally by the

 

following parameters: molecular or ionic transport, viscosity. supersaturation. solubility.

solid-liquid interfacial tension, and temperature.

1.3.3 Heterogeneous Nucleation

Heterogeneous nucleation occurs at an interface, e.g foreign bodies or the walls of the

retaining vessel. The critical supersaturation and the activation energy are considerably

15

lower than homogeneous nucleation. The rate of heterogeneous nucleation has the same
form as that describing homogeneous nucleation above (1.6). Generally, any adsorption
of foreign particles onto the nucleus decreases interfacial tension.41 Hence, an increase of
nucleation rate is also observed. Impurities are also known to decrease the nucleation
rate.42 A review of experimental work on heterogeneous nucleation is given by Tunbull

and Vonnegut.43

1.3.4 Crystal Growth Theory

Once the nucleation step has been achieved, nuclei grow into macroscopic
crystals. This stage of the crystallization process is known as crystal growth. Crystal
growth can be seen as a succession of events as proposed by Bennema:44 a) transport of
species from the bulk solution to a site at the crystal surface, b) adsorption of the growth
unit to a site at the crystal surface, c) surface diffusion from the impingement site to a
growth site, and. d) incorporation into the crystal lattice. Desolvation and/or solvent
adsorption can take place in steps b-d. The rate-limiting step can be any of the above
depending on the growth conditions such as the supersaturation, temperature. additives or
solvent, and hydrodynamics of the system.

A few theories attempting to explain the growth of single crystals from solution
have been proposed. Growth can be divided into two regimes, bulk solution mass
transfer-limited growth and surface-integration limited growth.45 Bulk solution mass
transfer-limited growth theories presume that matter is deposited continuously on a
crystal face at a rate proportional to the difference in concentration between the point of
the deposition and the bulk of the solution, while surface integration theories are more

complex.46 The goals of crystal growth theories are to determine the source of steps and

16

the rate-controlling step for the crystal growth. If diffusion of solute from bulk solution
to the crystal surface is rate limiting, grthh is controlled by mass transfer. However, if
incorporation into a crystal lattice is the slowest process, growth is controlled by surface-
integration.

Depending on the roughness of the crystal surface, layer growth or continuous
growth may result. Continuous growth model applies when the crystal-solution interface
at the molecular level has many kink sites. If the interface is smooth, growth proceeds
through a layer growth model: screw dislocation and two-dimensional nucleation.
Details of the derivation of these models have received considerable attention in the
literature.47
1.3.4.1 Burton, Cabrera, and Frank Model (Screw Dislocation Model)

The spiral growth mechanism or screw dislocation model was first described by
Burton, Cabrera, and Frank (BCF), who developed their theory for growth from the
vapor, but theorized that a similar treatment would be applicable for growth from liquul
solution.48 Bennema modified this theory for crystals growing from aqueous SOlUlltln.w
The screw dislocation emerges on a crystal face provides a step or a sequence of steps

over the surface for the addition of growth units (Figure. 1.2).

17

1 tag.

(a) (b) l (c) 1

Figure 1.2

Development of a growth spiral starting from a screw dislocation (a) to spiral staircase
50
(C)

18

The growth rate of a crystal face may be described by the following equation:
R = A(oz/oa )tanh (ca/o) (1.9)

where R is the growth rate of a crystal face, and A is a combination of physical constants
of the system including adsorption, surface diffusion, and growth unit incorporation
factors. At low supersaturations, where 0‘ << Ga , tanh (oa / 0') —-> 1 , and the equation
reduces to a form in which the growth is proportional to the supersaturation to the second
power.

R=Ao°/oa ' (1.10)
This is known as the parabolic rate law. At high relative supersaturations, where
0 >> 68 , tanh (Cu /0) —> Gd /0 and the BCF equation reduces to

R = A6, (1.11)
growth is linear with supersaturation.

The limitation of the BCF model is that, while it holds for growth from the vapor.

it is difficult to quantify the model in growth from solutions due to the more complex
nature of the systems. In solutions. sometimes the diffusion from the bulk solution to the

interface may be the rate limiting step. For this case, the Chemov model is well known.5 '

The growth rate is given by

R=f{GZ/(l+kln(5/y0))} (1.12)

where 5 is the boundary layer thickness. and k depends on the diffusion coefficient. the
kink density and the step height. At low 0, the equation behaves similarly to the BCF

model. However, the growth rate decreases with an increase in 5.

19

1.3.4.2 Birth And Spread Model (Two-Dimensional Growth Of Surface Nuclei)
Another theory of crystal growth is the birth and spread model described by
Hillig.52 As opposed to the BCF theory, this theory assumes that growth develops from
surface nucleation that can occur at the edges, comers and on the faces of a crystal, as
illustrated in Figure 1.3. Adsorbed growth units join an existing surface nucleus or form
a new nucleus which then grows through the addition of other growth units. In further
steps, 2D nucleus spreads across the surface. When a complete layer has formed, the
crystal has grown by one monomolecular layer. The activation free energy for creating
the 2D nucleus, if the nucleus is square, is AG = 4A2/KBT In B , where it is the edge free
energy per molecule of the nucleus. For the mononuclear model where the whole crystal

face is covered before the next nucleus forms, the growth rate of the face is R = J 3dS,

where J2 is the 2D nucleation rate, d is the height of the layer, and S the area of the face.
In cases where several nuclei spread on the face at the same time, a polynuclear
mechanism predicts a relationship between the face growth rate, and the supersaturation
o, as
R=AoS/°exp(—B/0') (1.13)
At low relative supersaturations, the exponential term will dominate giving a region
analogous to the parabolic region of the BCF curve. At high relative supersaturations. the

exponential term approaches 1, leaving growth rate, R, proportional to 0'5” ; this is

analogous to the linear region of the BCF curve.

 

 

Figure 1.3

Depicts such a polycrystalline growth by the birth and spread (B+S) mechanisms“:

1.3.4.3 Diffusion-Controlled Growth

In the previous sections, we have dealt with the case where growth is controlled
by surface integration, and the rate-limiting step was incorporation of a growth unit into
the crystal lattice. Now-we address a crystal growth rate for the case where the
nucleation is quick in comparison to the time needed for the solute to diffuse to the
surface, namely, diffusion-controlled growth. It is this model that is used in correlating
data for industrial crystallization processes.

The basis of the diffusion-controlled growth is that solute diffuses from regions of
higher concentration and is then incorporated into the crystal. On the assumption that

there is a thin stagnant film of liquid adjacent to the growing crystal face, the rate of mass
. dm 47
increase of the crystals ( -—d——- ) can be expressed as follows.

1

QflzDA 9—C— (1.14)
dt dx

where A is the surface area of the crystal, and D is the diffusion coefficient. The

concentration versus position through the boundary layer can be written as

 

d — .
dx 5
where C and Cl are the bulk and interfacial concentrations, respectively. Substituting liq.
(1.15) into Eq. (1.14) yields
dm.
dtt=kd(C-Ci) (1.16)

where kd is a coefficient of mass transfer by diffusion, D/S.

The rate of solute integration into the crystal surface can be approximated by the relation

22

fl=r,A(C,—c‘)‘ (1.17)
dt

where i is between 1 and 2, kr is the rate constant for the surface reaction integration
process, and Ci is the interfacial concentration. Equation 1.17 is normally not applied
because it involves the interfacial concentrations that are difficult to measure. Hence, it
is usually more convenient to eliminate the C, term by considering concentration driving
force, (C-C‘). A general equation for crystallization based on this overall driving force

can be written as

9%zkgAAC‘ (1.18)

where kg is an overall growth coefficient and the g is the crystal growth order.

When k d >> kr , the crystal growth rate will be diffusion controlled and k g = k,. On the

other hand, when kr << k d , the crystal growth rate will be controlled by the rate of solute
A incorporation into the crystal.37

If a chemical reaction is usedto produce the insoluble species, the rate of reaction
can be the rate-limiting step. A quantitative measure of the influence of resistance to

growth is made through the concept of the effectiveness factor, TL .53 The effectiseiiess

factor can be defined as

growth rate observed at the interface

c growth rate when interface is exposed to bulk solution

 

which is a function of the Damkdhler number, Da.

‘1. = (1—nCDa)r (1.20)

23

where r is the order of the surface integration process.

Da=kr(C—C°“ )"1 (1—m)k;' (1.21)
where Da represents the ratio of the pseudo-first-order rate coefficient at the bulk
conditions to the mass transfer, and (1) is the mass fraction of solute in solution. When Da

is large (11c —> Da") the growth is diffusion controlled, and when Da is small, the

growth is surface integration controlled (11c —) 1).

1.3.5 Crystal Shape

The external appearance of a crystal is the crystal habit, a property that can be
controlled either thermodynamically or kinetically. Crystals grown at a very slow growth
rate are usually thermodynamically controlled. In 1878, Gibbs proposed that the total
free energy of a crystal in equilibrium with its surrounding at constant temperature and

pressure would be a minimum for a given volume, i.e.:

dZAnyn = ZyndAn =0 (1.22)

where All is the area of the nth face. In 1901 Wulff stated that crystal faces would grow
at rates proportional to their respective surface energies, where the equilibrium shape is

determined by the ratio of the distance from the face, hn, to the specific surface energies.

—1 .54 A schematic representation of this equrlibnum shape IS shown in Figure 1.4.
Yrs

24

 

hr

 

 

 

 

\ /

Figure 1.4

Equilibrium crystal shape as described by Wulff’s theorem. In this case 7, < y: .55

25

Essentially, an equilibrium shape crystal grows to maintain geometric similarity.
Hartman and Perdok developed morphological theory that related bond energies to
internal structures of crystal morphologys6 They theorized that crystal growth is
controlled by the formation of strong bonds between crystallizing particles called
periodic bond chains (PBC). Growth layers of the periodic bond chains form three
different crystal faces as shown in figure 1.5. The F-face (flat) is the elementary faces
that grow slice after slice and is parallel to at least two PBC vectors. The S-face
(stepped) parallel to at least one PBC vector. The K-faces. (kinked) not parallel to any
PBC vector, which needs no nucleation for growth. The rougher S- and K-faces grow
very quickly, and it is rarely observed. On the other hand, the growth velocity of the F-

face is very slow. Thus. the crystal habit is usually dominated by the F-face.

 

Figure 1.5

Hypothetical three-dimensional crystal presenting the three main types of
possible faces: flat (F), step (S) and kink (K) faces.57

27

1.3.6 Crystal Habit Modification

The observed habit of crystals grown from solution is often quite different from
the prediction by the models mentioned previously. For crystals grown in solution, their
shape will depend on kinetic factors which are affected by crystal defects, surface
toughening, solvent type, supersaturation, temperature, impurities in the solvent and other
solution conditions. These environmental conditions affect the growth of a given face.
Some faces grow very fast and have little or no effect on the growth form; the ones that
have most influence are the slow-growing faces. The most important technique used
today for influencing habit modification is the addition of impurities, which preferentially
adsorb on a specific crystal face. The first documented example of crystal habit
modification was described in 1783 by Rome’ de L’Isle.58 Urine was added to a saturated
solution of NaCl, which resulted in changing the crystal habit from cubic to octahedral.
The effect of organic impurities on the growth of inorganic crystals from aqueous
solution was studied by Buckley as well.59 In addition, the influence of impurities and
solvents on crystallization has been discussed extensively by D.L. Klug.45

Impurities which modify crystal habit fall into the following categories: ions.
either anions or cations, ionic surfactants, either anionic or cationic, nonionic surfactants
like polymers, and chemical binding complexes. These impurities all have propensity to
adsorb on a specific crystal surface.°° The specific surface energy, y, that results (ruin the

adsorption of 1" atoms (or ions) per unit area is given by Gibbs"l as

—dy=l“i<Tln(a2) (1.25)
where a2 is the activity of the impurity. If crystal growth was mainly dependent on

thermodynamic factors, then impurity adsorption should increase the growth rate of a

28

face because adsorption decreases the edge free energy. However, impurities affect
kinetic factors much more than thermodynamic factors by inhibiting crystal growth.
When only a few kinks are blocked on the surface, growth rate can be slowed down by
several orders of magnitude."2 Therefore, additives or impurities may be very active at
low concentrations. In general, their efficiency depends on their concentration.
However, an increase in supersaturation reduces their efficiency.63 According to Mullin
et al.,64 impurity ions in the vicinity of the surface will reduce the effective surface
supersaturation. retard diffusion, and hinder the aggregation of growth units.

Ionic surfactant impurities are routinely used in industry to control crystal
morphology during crystallization. As shown in Figure 1.6, anionic surfactants will
adsorb on the negatively charged surfaces of adipic acid and limits their growth, giving
plate-like crystals, while anionic surfactants will adsorb on the positively charged
surfaces of adipic acid crystals and limit their growth, giving needle-like crystals.

Tailor-made impurities are an important class of impurities (additives) which are
designed to interact in very specific ways with selected faces of crystalline material.
Basic strategies for designing crystallization additives are (i) mimicking the solute
molecule (e.g., chemical groups or moieties) such that they can readily adsorb at growth
sites on the crystal.65 (ii) introducing steric hindrance to disrupt subsequent growth
processes a. the affected faces,°° and (iii) establishing chemical differences at the

interface of growing crystal.67

    

 

 

" ~ Cationic

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Anionic ,I ‘.
Surfactant .” \‘Surfactant
\
“ G)
- 69
+
: Retardation of growth on :
Prism Faces I 7 Basis Faces
—. - - ® '-
- 69_ e.)
4.
+
6 e Plates
9
Needles

Figure 1.6

Influence of impurity adsorption on the crystal habit. for the case of adipic acid.“

30,

1.3.7 Phase Transformation

Phase transformations occur through four schemes: solid state, melt mediation.
interface mediation, and solution mediation. Phase changes between solid structures
involve rearrangement of molecules or atoms in the metastable solid such that the
complete transformation occurs in the solid state.68 From a kinetic point of view, solid
state phase transformation is slower in comparison to other schemes due to the large
activation energy of nucleation and growth required. However, phase transformation
through the melt. interface, and solution can reduce the total activation energy for a
complete polymorphic conversion to take place. The driving force of these
transformations is the difference in chemical potential between the polymorphs. Such
transfomiations are an inevitable consequence of the thermodynamic driving force
toward minimizing the free energy of the system.

Kinetic factors involved in phase transformations include (i) nucleation and
growth of the more stable form. (ii) dissolution, evaporation or melting of the less stable

. . . . . t). 70
form, and (111) mass transfer in the medium.°

This concept is illustrated in figure 1.7
for solution-mediated phase transformation for two crystalline phases. The relative
supersaturation, 0, defines a driving force for nucleation of each phase and crystal
growth on existing surfaces. The rates of nucleation and crystal growth of each phase
depend on the magnitudes of specific rate constants, interfacial energies, free energies of

formation, solute diffusivity. and mixing intensity. The transformation time is limited by

the dissolution of phase 1 crystals at their maximum dissolution rate. tn. and the rate of
growth of phase 2 crystal at their maximum growth rate, tG . Thus, the transformation

can be represented as the sum of the growth and dissolution times, t = t(i + I“. If

31

I'D >> tG , the system can be said to be dissolution controlled and similarly if tD << To the

system is growth controlled.

 

Solute concentration

 

 

 

Terri peio ture

Figure 1.7

Typical solubility profiles and pertinent supersaturation values.7|

33

1.4 REFERENCES

1Watts, R. W. Journal Of The Royal College Of Physicians Of London 1973, 7, 161-174.
2Boyce, w. H. Amer. J. Med. 1968, 45, 673-683.

3 Rose, G. A. Urinary stones .' clinical and laboratory aspects; Lancaster, 1982.
4Heijnen, w. M. M. J. Cryst. Growth 1982, 57, 216-232.

5Heijnen, W.; Jellinghaus, W.; Klee, W. E. Urol. Res. 1985, 13, 281-283.
°Fleisch, H. Kidney International 1978, 13, 361-371.

7Robertson, w. (3.; Peacock, M.; Marshall, R. W.; Marshall, D. H.; Nordin, B. E. New
England Journal Of Medicine 1976, 294, 249-252.

°Coe, F. L.; Parks, J. H. Nephrolithiasis : pathogenesis and treatment; 2nd ed.; Year
Book Medical Publishers: Chicago, 1988.

9Coe, F. L.; Favus, M. J. Disorders of bone and mineral metabolism; Raven Press: New
York, 1992. -

10Finlayson, B. Kidney International 1978, 13, 344-360.

llWalton, A. G. Theformation and properties of precipitates; lnterscience Publishers:
New York, 1967.

”Finlayson, B. Kidney International 1978, 13, 344-360.

l3Robertson, W. G.; Peacock, M.; Marshall. R. W.; Marshall. D. H.: Nordin. B. E. New
England Journal Of Medicine 1976, 294, 249-252.

”Robertson, w. 0.; Peacock. M.; Nordin, B. 13. Clinical Science 1968. 34. 579-594.
”Robertson. w. (3.; Peacock, M.; Nordin, B. E. Clinical Science 1971. 40, 365-374.
16Boyce, w. H. Amer. J. Med. 1968, 45, 673-683.

”Haiison, P. 0; Rose, G. A. Lancet 1979. 1, 1000—1002.

1°Fleisch, H. Kidney International 1978, 13, 361-371.

34

”Drach, G. W.; Sarig, s.; Randolph, A. 1).; Thorson, s. Urological Research 1982, 10,
165-168.

20Robertson, w. (3.; Scurr, D. 3.; Bridge, c. M. J. Cryst. Growth 1981, 53, 182-194.
2'P.yaii, R., Hamett, R., and Marshall, v., J. Urology, 1986, 135,174.
22Anderson. H.C., Clin.0rthop. Relat. Res, 1995, 314, 266—280

23Li, c.w., and Volcani,B.E., Trans. R. Soc. Lond. B, 1984., 304, 519-528

24Bazylinski, D.A., Garratt-Reed, A.J., and Frankel, R.B., Microsc. Res. Tech., 1994,
27. 289-401.

25Amott, H.J., and Pautard, F.G.E., Calcification in plants. In Biological Calcification:
Cellular and Molecular Aspects. H. Schraer, ed (New York: Appleton-Century-Crofts).
1970, 375-44( '

26Bush, D.S., Annu. Rev. Plant Physiol. Plant Mol. Biol, 1995, 46, 95—122.
27Kretsinger, R.H., Evolution of the informational role of calcium in eukaryotes. In
Calcium-Binding Proteins and Calcium Function, RH. Wasserman, R.A. Corradino. E.
Carafoli, RH. Kresinger, D.H. MacLennan, and EL. Siege], eds (New York: North
Holland), 1977, 63-72.

28Hepier, P.K. , Cell Calcium, 1994, 16, 322.

29Ciarkson, D.T., Plant Cell Environ, 1984, 7, 449.

' 30Kinzei, H., Flora, 1989, 182, 99.

3Leigh, RA, and Tomos, A.D., Trans. R. Soc. Land. 3., 1993, 341, 75.

”Zindler-Frank, E., Pflanzenphysiol, 1975, 77,80.
33Pinks, New Phytol., 1991, 119. 33.

34Nancollas, G. and Gardner, G., J. Crystal Growth, 1974, 21, 267.

3 5 C. Phillip and Wankart, Rate Controlled Separations; Elsevuer Applied Science
Publishing Co., Inc., New York, 1990 Chapter 3.

3°Nyvlt, J .; Sohnel, O.: Matuchova. M.; Broul, M. The Kinetics of Industrial
Crystallization;_Elsevier: New York, 1985.

35

37Mn11in,l. w. Crystallization; Butterworth—Heinemann Ltd: Oxford, 1993.

38Sohnel, 0.; Garside, J. Precipitation: Basic Principles and Industrial Applications;
Butterwonh-Heinemann Ltd: Oxford, 1992.

39Gibbs, J .W. , Collected Works, Vol. I, Thermodynamics, Yale University Press: New
Haven, 1948.

40Volmer, M. , Kinetic Der Phasenbildung, Steinkopff, Leipzig, 1939.
4'Mutaftschiev, 8., Critical Rev. Solid State Sci., 1976, 6, 157.

42Lacmann, R., and Stranski, I. N., Growth and Perfection of Crystals, Roberts. B.W.
Doremus, R. H., and Tumbull, D., Wiley, New York, 1958, 427.

43rurn1nt11,1).. and Vonnegut, 13., Ind. Eng. Chem, 1952, 44, 1292.
“Bennema,P., J. Cnsralorowtlt, 1967, 1, 278.

45Myerson, A. S., Handbook of Industrial Crystallization, Edt. Myerson, A. S.
Butterworth-Heinemann, Boston, 1993.

46Noyes, A.A. and Whitney. W.R., J. American Chemical Society. 1897, 19, 930.
47Myerson, A. S.. Handbook of Industrial Crystallization, Edt. Myerson, A. S.
Butterworth-Ht'nemam. Boston, 1993. Mullin, J. W., Crystallization, Buttemotth-
Heinemann Ltd. Oxford, 1993, Chemov, A. A., Contemp. Phys. 1989. 30, 252.
Chemov, A. A., Modem C rystallograplty III: Crystal Growth, Springer-Verlag: Berlin.
1980, Bennema, P.. J. C l'_\‘.S‘lal Growth, 1984, 69, 182.
48Burton,W.K.;C't1brer'c1.N;Frank,F.C.,Philos. Trans, 1951. 4243. 299.

49Bennema,P., J. Crystal Grout/l. 1984, 69, 182, Bennema. P.. J. Crystal (ll'llu‘l/l.
1967, l, 225.

50Mullin,J. W., Crystallization, Butterworth-Heinemann, Oxford. 1993. Chap.» (1.
SlChernov,A.A.. sot. Phys. Usp.. 19.61, 4. 129.

32Hil1ig,w.,Acta Meet., 1966. 14. 1968.

53Garsitle,J.,nnt1 Tavure. N.S.. Chem. Eng. Sci., 1981, 40,1485.

54wtt1ff, G; 2 Kristallogr.. 1901. 34.449

36

5 5 N ielsen, A. E. Kinetics of precipitation; Pergamon Press; Oxford, New York,, 1964.
5°Hartman, P.; Perdok, w. G. Acta Cryst. 1955, 8, 525-9.

57Elwell, D.; Scheel, H. J. Crystal growth from high-temperature solutions; Academic
Press: London ; New York, 1975.

58Rome de L’Isle, Cristallographie, 2nd edition, Paris, 1783, 379.
”Buckley, 11.13., Crystal GrowthLWiley, New York, 1951.

60Adamson, A.w.. Physical Chemistry ofsttrfaces, 4th Edition, Wiley-lnterscience. New
York, 1982.

61Gibbs, J.W., Collected Works, Longmans, Green, New York, 1928.
62Burrill, K.A., J. Crystal Growth, 1972, 12, 239.
63Boistelle, R. and Astier, J.P., J. Crytal Growth, 1988, 90, 14.

“Mullin, J .W. and Leci, C.L., Eng. Prog. Symp.: Nucleation Phenomena, 3“, Joint
Meeting AIChE and IMIQ, Denver, Colorado. August 1970, Paper 3921.

65Addadi, L.. BerkovitCh-Yellin, Z., Weissbuch, I.. Angewandte Chemie Int., 1985. 24.
466.

“Berkovitch-Yellin. z. J. Am. Chem. Soc., 1985, 107, 8239.

67Weisinger-Lewin. Y., Frolow, F., MCMullan, R. K, J. Ant. Chem. Soc. 1985. I I l. 1035.

68Bamf0rd, CH. and Tipper. C.F.H., Comprehesive chemical kinetics, Amsterdnrtt.
Elsevier, 1980, vol. 22.

69Cardew, P. T. and Davey, R.l. Proc. R. soc. Lond., 1985, A398, 415.
70Sato, K., J. Phys. D: Appl. Phys. , 1993, 26, B77.

7'Thompson. R.W. andDixon. A.G. Proc. R. Soc. Lond., 1987, A413. 369-38l

37

Chapter 2

SYNTHESIS OF SIMPLE ORGANIC CHELATES DERIVED FROM AMINO
ACID A1\' ) EPOXYSUCCINIC ACID FOR INHIBITION OF CALCIUM
OXALATE CRYSTAL GROWTH

The crystallization of calcium oxalate in the presence of different additives was
studied at ambient temperature. Citric acid, aliphatic amino acids, basic amino acids, and
acidic amino acids deri vatized with cis-epoxysuccinic acid were studied. Particle
formation during the crystallization of calcium oxalate was monitored by nephelometry.
The Savitsky-Golay algorithm was used to smooth the desupersaturation data and the
data were subsequently used to estimate nucleation and growth kinetics. Induction times
were determined and used as an indicator for the abilities of the additives as nucleation
inhibitors. The percentage inhibition produced by the additives was calculated from the
slopes of the crystallization curve as [1—(5, /S( )rlOO]. The most effective inhibitor of both
nucleation and crystal growth was the lysine derivative, leading to an inhibition of 98%.
followed by citric acid. The aliphatic amino acid and the basic amino acid det‘iVamcs
inhibited less than 50% crystal growth. The Chelation strengths of the amino ac1t1
derivatives were determined by calcium ion selective electrode. The experimentallx
determined Chelation constants were statistically equal to that of citrate. which lL‘.1tl\ us 111
believe that the inhibition properties of the lysine derivative as well as citrate are

independent of their Chelation properties.

38

2.1 INTRODUCTION

An answer to current environmental concerns is a detergent that biodegrades to
C02 and H20 and in addition to being non-toxic, is inexpensive to produce. Compounds
with polycarboxylic acid groups make good ligands for calcium. Amino acids make
good starting material for preparation of polycarboxylic acids because it contains a
primary amine group which be attached to other molecules. These compounds could then
be applied as detergent additives, as well as inhibitors of scale deposits, and kidney stone
formation.

In the pulp and paper industries, calcium oxalate (CaCan) is notorious for
forming scale deposits on the equipments.l Scale deposit in water-containing systems
such as pipelines and air conditioning systems is another major area of concern. Water is
recycled in many cooling water systems; however, this practice increases scaling
tendencies. Therefore. an effective inhibitor of scale forming salts which also addresses
the environmental concems is a sensible approach to controlling scale deposits and steel
corrosion.

In animals, CanOs occurs as a crystal in the urinary tract and constitutes the
largest composition in kidney and bladder stones.2 Calcium oxalate crystallizes 111 three
hydrates: the monohydrate. the dihydrate, and the trihydrate. depending on solutton
conditions. The monohydrate phase is the main component of the most abundant type of
renal calculi that is also the most difficult to treat and the most poorly understootl.‘ One
form of clinical management of kidney stones is oral administration of citric acid

supplements, which are inhibitors of calcium salt crystallization. Unfortunately. the

39

amount of citrate that must be consumed for effect is relatively large (in excess of grams
per day, 2 to 8 grams/day).4

The factors that determine calcium salt formation include supersaturation and the
resulting crystallization kinetics.5 When the solubility product of a calcium salt is
exceeded, nucleation and crystal growth takes place, leading to stone formation or scale
deposits.6 An effective inhibitor delays or prevents crystal growth. Amino acids were
modified with cis-epoxysuccinic acid to create polycarboxylates with the intention of
finding inhibitory compounds that were effective at concentrations less than citric acid.
Subsequently, the crystallization of calcium oxalates in the presence of the derivatized
amino acids was studied at ambient temperature. In addition, the Chelation properties of

the derivatives were also determined.

40

2.2 EXPERIMENTAL METHODS

Scheme 2.1 Synthesis of the lysine derivative (2,6-Bis-(1,2-dicarboxy-2-hydroxy-
ethylamino)-hexanoic acid)

0 O
H2N H2N’ O
M + HO OH
OH O
Lysine cis-epoxysuccinic acid
85 °C H20
4hrs, pH = 3
O O OH
HO
0*; HO OH
0 N NH
0 \/\/1:
OH O OH

2,6-Bis-(1 ,2-dicarboxy-Z-hydroxy-ethylamino)-
hexanoic acid

L-Lysine dihydrochloride. 4.1502g (0.01894 moles). was added to 5 ml. oi
deionized water containing 5.0034 (0.03788 moles) of cis-epoxysuccinic acid. "I he
reaction mixture was refluxed at 80°C for 4 hours under nitrogen, then allowed to cool to
room temperature. Ethanol was added to the solution until two immiscible llklllld\
formed. The top liquid ethanol/water azeotrope was decanted off and the remaining
filtrate was washed repeatedly with ethanol followed by acetone to remove uni'eactcd

epoxysuccinic acid. The product was characterized by lH NMR in D30. The NMR was

41

recorded using a 300-MHz Varian INOVA spectrometer. Mass Spectrometry analysis
was attempted but the results were inconclusive. Consequently, only the NMR was used
as the definitive metixod of choice.

Lysine derivative ‘H-NMR (300 MHz, D20 @ 40 °C): (ppm) = 1.400 (m, 211); 1.628 (m.

2H); 1.840 (m, 2H); 2.923 (m. 2H): 3.793 (m. 1H); 4.660 (dd, 1H); and 4.662 (dd. 1H).

36w o_o:nxo;-Ao:_Es_>£o->xc.6>;-m->15:55-“_ Timed» 9.1.3:...5 2:, ,_ .: 5:527. xZZi.

_.m Ezbooam

 

 

 

 

 

Scheme 2.2 Synthesis of arginine derivative (2-( 1-Carboxy-4-guanidino-
butylamino)-3-hydroxy-succinic acid)

0 1'31 0
4 hrs pH: 3 H2N N HO
ArginineO Cis- -epoxysuccinic acid H
2-(1-Carboxy-4-guanidinobutyiamino)-
3-hydroxy-succinic acid
L-arginine hydrochloride, 2.39g (0.01136 moles), was added to 5 mL of deionized
water containing 1.5g (0.01136 moles) of cis-epoxysuccinic acid. The reaction mixture
was refluxed at 80 0C for 4 hours under nitrogen atmosphere and allowed to cool to room
temperature. Ethanol was added into the solution until two immiscible liquid formed.
The top liquid ethanol/water azeotrope was decanted off and the remaining filtrate is
repeatedly washed with ethanol followed by acetone to remove unreacted epoxysuccinic
acid. The product was characterized by 1H NMR in D30. The NMR was recorded using
a 300-MHz Van'an INOVA spectrometer. Mass Spectrometry analysis was attempted but
the results were inconclusive. Consequently, only the NMR was used as the definitive
method of choice.

Arginine derivative 1H-NMR (300 MHz. D30 @ 40 UC): (ppm) = 1.560 (m. 2H). 1.820

(m, 2H): 3.138 (m. 2H); 3.724 (m. 1H): 4.752 (dd. 1H): and 4.791 (dd. 1H).

44

35s o_:_oo_a->xc._ac-mgcEE:_>.::_-c:_1:_:_&+->.f:_:C-_:1. 9.1.31.3: 2.5.x“... _: 52.327. 52.2-2.

Nth. 528on

min O.N m.n o.m m.m om. m.w O.m

11: 1 1) 3111.1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

45

Scheme 2.3 Synthesis of glutamic derivative (2-(l,2-Dicarboxy-ethylamino)-
pentanedioc acid)

- 0
HO
NH2 0 O NH O
HO ' OH + V _, f
M HO 01-1 HO HO OH
0 o o 0 OH
0

Glutamic acid Cis-epoxysuccinic acid
2-(1 ,2-Dicarboxy-2-hydroxy-ethylamino)-
pentanedioic acid

L-glutamic acid (1.67g, 0.01136 moles) in water was titrated with 1M NaOHto
pH 9.7 which corresponds the pKa of the a -arnino group. Epoxysuccinic acid (1.50g,
0.01136 moles) was added, upon which the pH dropped to less than 4. Base was added to
return the pH to 9.7. The mixture was heated in an oil bath at 80 °C for 4 hours in a flask
attached to a reflux condenser. After allowing the reaction mixture to cool to room
temperature, ethanol was added into the solution until two immiscible liquids formed.
The supernatant was decanted off and the filtrate was repeatedly washed with ethanol.
The amorphous sample was crystallized by titrating it to =pH 4 with concentrated HCl.
A Buchner filter funnel with medium frit was used to isolate the crystallized product. To
remove any impurities such as tartaric acid or epoxysuccinic acid, the amino derivative
was washed in methanol. The unreacted amino acid was removed by dissolving the
sample in water which resulted in crystallization of the amino acid. After drying. the
product was characterized by 1H NMR. Mass Spectrometry analysis was attempted but
the results were inconclusive. Consequently, only the NMR was used as the definitive

method of choice.

46

Glutamic derivative lH-NMR (300 MHz, D20 @ 25 °C): 2.007 (m, 2H-C(4’)); 2.343 (m,
2H-C(3’)): 3.530 (m, 1H-C(2’)); 3.756 and 3.802 (dd, 1H-C(2)): 4.333 and 4.416 (dd, H-

C(3)).

47

86m oofiocficoonEEa73.???_:U_Q-m. _ Ymv 953:2. BEEEw .3 E2303 522-2.

mum EEUomm

 

 

 

 

 

 

 

48

Scheme 2.4 Synthesis of aspartic derivative (2-(1,2-Dicarboxy-ethylamino)-3-
hydroxy-succinic acid)

0
0 o 0 HO
MOH V 0 NH 0
HO ; + HO OH ——>
NH2 0 0 HO HO OH
Aspartic acid EpOXYSUCClniC aCid O
HO

2-(1 ,2-Dicarboxy-ethylamino)-
3-hydroxy-succinic acid

L—aspartic acid (1.51g, 0.01136 moles) in water was titrated with 1M NaOH to pH
9.8 which corresponds the pKa of the at -amino group. Epoxysuccinic acid (1.50g.
0.01136 moles) was added, upon which the pH dropped to less than 4. Base was added to
raise the pH to 9.8. The mixture was heated in an oil bath at 80 °C for 4 hours in a flask
attached to a reflux condenser. After allowing the reaction mixture to cool to room
temperature, ethanol was added into the solution until two immiscible liquids formed.
The supernatant was decanted off and the filtrate was repeatedly washed with ethanol.
The amorphous sample was crystallized by titrating it to sz 4 with concentrated HC 1.
A Buchner filter funnel with medium frit was used to isolate the crystallized product. To
remove any impurities such as tartaric acid or epoxysuccinic acid, the amino dern mm
was washed in methanol. The unreacted amino acid was removed by dissolvin g the
samplein water which resulted in crystallization of the amino acid. After drying. the
product was characterized by IH NMR in DzO. Mass Spectrometry anal ysis was
attempted but the results were inconclusive. Consequently, only the NMR was used as

the definitive method of choice.

49

Aspartic derivative 'H-NMR (300 MHz, D20 @ 25 °C): 2.656 and 2.715 (m, 2H-

C(3’)); 3.605 (m, 1H-C(2’)); 3.920 (d, 1H-C(2)); and 4.410 (d, lH-C(3)).

50

 

 

 

w. o r .1 t i at w.l u .I.\. . woJ ill: ill a . m -J . d u . .JpJ .. I
35. 5.533 .3863 m 32:57:: 5.5: .2 r :15... 1. .3 3:...7. .2 :5: .7 zEZ 2.

Wm 85.58am

 

 

 

 

51

Scheme 2.5 Synthesis of glycine derivative (2-(Carboxymethyl-amino)-3-hydroxy-
succinic acid)

OH
O
O O HO OH

O
H N
2 \JLOH , HOVOH , O W
Glycine O VOH

Cis-epoxysaccinic acid 0

2-(CarboxymethyI-amino)-
3-hydroxy-succinic acid

Glycine acid (0.85g, 0.01136 moles .. in water was titrated with l M NaOH to pH
9.6 which corresponds the pKa of the a -an11no group. Epoxysuccinic acid (1.50g.
0.01136 moles) was added, upon which the pH dropped to less than 4. Base was added to
raise the pH 9.6. The mixture was heated in an oil bath at 80 °C for 4 hours in a flask
attached to a reflux condenser. After allowing the reaction mixture to cool to room
temperature, ethanol was added into the solution until two immiscible liquid formed. The
supernatant was decanted off and the filtrate was repeatedly washed with ethanol. The
amorphous sample was crystallized by titrating it to sz 4 with concentrated HC 1. A
Buchner filter funnel with medium frit was used to isolate the crystallized product. To
remove any impurities such as tar1...:ic acit; ;_1r epoxysuccinic acid. the amino dcerullVC
was washed in methanol. The unreacted amino acid was removed by dissolving the
sample in water which resulted in crystallization of the amino acid. After drying the
product was characterized by 1H NMR in D20. Mass Spectrometry analysis was
’ attempted but the results were inconclusive. Consequently, only the N MR was used as
the definitive method of choice.
Glycine derivative lH-NMR (300 MHz, D30 @ 25 0C): 3.571 (s. 2H—Ct2‘)); 3.992 (dd,

lH-C(2)); and 4.526 (dd, 1H-C(3)).

AEom o_:_oo:m-zxo€»;-m-3:ETEEoExxGLSUYNv 03:22.8: 2.52% E E2527. 522-2.

Wm Ezboomm

 

 

 

 

 

53

Scheme 2.6 Synthesis of fl -alanine derivative (2-(2-Carboxy-ethylamino)-3-
hydroxy-succinic acid)

0
OH
NH2 0 0 O O HO
VL __, JV OH
OH + HO OH HO n
B—alanine O O .
Cis—epoxysuccinic acid 2-(2-Carboxy-ethylamino)-

3-hydroxy—succinic acid

B-alanine acid (1.01g, 0.01136 moles) in water was titrated with 1M NaOH to pH
9.7 which corresponds to the pKa of a -amino group. Epoxysuccinic acid (1.50g.
0.01136 moles) was added, upon which the pH dropped to less than 4. Base was added to
raise the pH to 9.7. The mixture was heated in an oil bath at 80 0C for 4 hours in a flask
attached to a reflux condenser. After allowing the reaction mixture to cool down to room
temperature, ethanol was added into the solution until two immiscible liquids formed.
The supernatant was decanted off and the filtrate was repeatedly washed with ethanol.
The amorphous sample was crystallized by titrating it to =pH 4 with concentrated HC 1.
A Buchner filter funnel with medium frit was used to isolate the crystallized product. To
remove any impurities such as tartaric acid or epoxysuccinic acid, the amino derivative
was washed in methanol. The unreacted amino acid was removed by dissolving the
sample in water which resulted in crystallization of the amino acid. After drying. the
product was characterized by 1H NMR in D20. Mass Spectrometry analysis was
attempted but the results were inconclusive. Consequently, only the NMR was used as
the definitive method of choice.
fl-Alanine derivative 1H-NMR (300 MHz, D20 @ 25°C): 2.919 (m. 2H-C(3’)l: 3.4335

(m, lH-C(2’)); 4.36 (m, lH-C(2)): and 4.72 (m, lH-C(3)).

54.

axon oE_oo:w-»xo€>:-m-3c.EflxfiotxéoEQNYmv 6323.333 ocESTu Co 63627. 522-2.

9m :55on

 

 

 

 

55

2.2.1 CALIBRATION CURVE

Calcium chloride dihydrate was purchased from Fisher Scientific and used as
received to prepare a stock solution of 0.01M CaCl2-2HC1 (1000 ppm, hardness CaCO3)
with distilled deionized'water and 0.03M NHaCl/0.07M NH40H buffer (pH=9.5, ionic
strength of 0.1M, Columbus Chemical Industries, Inc). Standard solutions ranging from
200 ppm to 10 ppm were prepared by dilution of the stock solution with the buffer. A
plot of the logarithm of calcium ion concentration versus the normalized p0tentia1 was
constructed prior to each titration.7'8 This calibration was necessary to minimize the
effect of signal fluctuations due to solution condition (ionic strength, pH, etc.).
Potentiometric measc‘ements were crnducted with a calcium-selective electrode obtained
from Orion Research, Inc (model 97-20 ionplus electrode).

2.2.2 TITRATION

The electrode was immersed in 50 mL of 200 ppm CaC03 solution at 25"(‘ and
the meter reading was taken while the solution was being stirred by magnet.9 The
calcium binding agent solution was added in small increments and the equilibrium-tree
calcium ion concentration was measured. When the meter reading indicated less than 10
ppm present in solution, the titration was stopped. The data collected were normalized.

and from the calibration curve the concentration of the free calcium ion was obtained

56

2.2.3 NEPHELOMETRY EXPERIMENT

A SPEX Fluorolog 1681 equipped with a 150 W xenon lamp as the light source
and a PMT detector was employed for nephelometry, see figure 1. Light from the
excitation spectrophotometer was focused onto the sample. The scattered light from the
sample was reflected to the front-face collection port in the sample compartment module
and collected by the emission spectrophotometer and directed to the PMT detector. The
nucleation and crystal growth was monitored photometrically at 550 nm because this
wavelength provided a better scattering signal to noise ratio compared to the other
wavelengths. In addition, it is in the region of the xenon lamp output profile that has a
flat baseline.

Experiments were performed in a 40 mL quartz cuvette, and the supersaturated
solutions were prepared by mixing equal volumes of calcium chloride and sodium citrate
stock solutions. The aqueous stock solutions of 5 mM calcium chloride dihydrate and
7 sodium oxalate were buffered at pH 5.5 with 9 mM MES (2-[N-Morpholino]
ethanesulfonic acid) and brought to an ionic strength of 0.15M with sodium chloride.
The MES has no Chelation properties. A pH of 5.5 was chosen because it is the pH value

frequently observed in the moming urine of calcium stone-formers.”

An aliquot ol 6 mL
of 5 mM calcium chloride solution was transferred in a sample cell followed h} 24 mL of
9 mM MES buffered solution. While stirring the sample solution with a Teflon-cm ered
magnetic bar, 6 mL of the 5 mM sodium oxalate solution Was added quickly. Readings

' of the light scattering were recorded every second using a PMT detector During the

inhibition study, 6 mL of 5 mM of the inhibitor was transferred in a sample cell. followed

by 6 mL aliquot of 5 mM calcium chloride solution, and 18 mL of the buffered solution.

57

 
  
  

    

(
Xenon Lamp

PMT

  

Excitation Sample Emission Detector

compartment

Figure 2.1

Optical Schematic of the spectrometer for nephelometry study

58

 

As before, the reactant was added rapidly, and the scattered light intensity readings
recorded. Aqueous solution was removed by suction filtration, and the isolated calcium

oxalate precipitate was analyzed using Raman.

2.3 RESULTS AND CONCLUSION

2.3.1 CHELATION CONSTANTS

The solution conditions including ionic strength, temperature, and pH and cell
variables such as depth of electrode immersion and stirring rate affect the electrode
response. The random fluctuation of the cell potential values was corrected by
normalization of the data to a ratio. The r2 regression analysis was determined to be
around 0.9984 :1: 0.0004.

Both the samples and the standards were prepared in a similar way which made it
possible to determine the quantity of free calcium by reading directly from the calibration
curve. The amount of bound calcium (mmol) was calculated by subtracting the free
calcium from the total. Figure 2.2 shows that the bound calcium increased as a function
of the ligand added. At low ligand concentration, we see a linear relationship between
added chelating agent and amount of calcium bound. For the lysine derivative, a
deviation from linear curve was observed near the equivalence point, and more so at
higher ligand concentrations. The stability constant or Chelation constant of 1 to 1

organic chelate with calcium can be written as follows

K = [Ca2L("'2)-:I
[“1”]

59

0.20

 

0.18 —
I
0.16— g Q

0.14 A I

0.12 — §
i

0.08 -

t—o--—i

0.06 -

_ i

Bound Calcium ion (mmol)

 

 

0.00 O 1 i i 1 1
0.00 0.05 0.10 0.15 0.20 0.25

Titrant (mmols)

 

Figure 2.2

A plot of moles of bound Ca2+ vs moles of titrant (lysine derivative). Data of bound
calcium was from the average of three replicates. Experimental conditions: 200 ppm
calcium concentration, 0.03M NHsCl/0.07M NH40H buffer (pH=9.5, ionic strength of
0.1M NaCl, 25 °C).

60

where L represents the chelating agent and n is the anionic charge on the chelator. Figure
2.3 shows a plot of logK versus titrant concentration which has three unique regions. In
the region before the equivalence point, the chelation factor increases slowly as a
function of titrant concentration. Around the equivalence point, an inflection is observed.
The chelation factor after the equivalence point appears to be more constant as a function
of titrant. The average chelation factor was obtained by taking the mean average of the
chelation factor after the equivalence point. The results have been summarized in table
2.1. EDTA is reported to have a calcium chelation value of about 7.0, which exceeds the
chelation strengths 0; our compounds.11 However, these compounds would make good
substitutes for citrate ions because their chelating strengths are statistically similar.
According to Crutchfield. an efficient binding agent must have locha Z 5.12

Among the compounds studied, only lysine derivative comes close to meeting this

criterion.

61

 

 

 

 

 

7 - --
6 ..
8 O
x 5 —
Q.
i
4 _ i
i _L ' l I no
3- g i} t
2 . 1 T 1
0.0e+0 4.09-4 809-4 129-3 1 .69-3
LoglL]
F1gfidf€23

cha verses lysine derivative concentration. The pKCa was determined from the axcragc
of three replicates. Experimental conditions: 200 ppm calcium concentration. 0.03M
NH4C1/0.07M NHtOH buffer (pH=9.5, ionic strength of 0.1M NaCl, 25 °C).

 

 

 

 

 

 

 

 

 

 

 

 

COMPOUNDS ch2,

Citric Acid 3.48 :0.- 0.40
Glycine derivative 3.60 t 0.42
'B-Alanine derivative 3.41 1 0.26
MLysine derivative 4.12 :1; 0.09
lArginine derivative 3.28 i 0.18
Aspartic derivative 3.54 1 0.30
Glutamic derivative 3.61 z 0.41

Table 2.1

Calcium chelation capacity of amino acid derivatives compared to citrate as determined
by calcium-selective electrode (model 97-20 ionplus electrode).

63

2.3.2 CRYSTALLIZATION KINETICS

Scattered radiant power (I)SC is empirically related to the concentration of suspended

particles by the following equation
(I) = (I) K m (2.1)

5C a sc
where K,-r is an experimentally obtained constant, (Do is the incident radiant power, and m
is the mass of studied particle. In nephelometry, the size and shape of the

particles have a large effect on the radiant power of scattering.13 Hence, nephelometry is
an appropriate tool for monitoring nucleation as well as crystal growth. In order to obtain
precise and accurate results, factors such as pH, temperature, concentration of reagents,
ionic strength, and data acquisition time must be carefully reproduced. Figure 2.4 shows
a typical experimental result wherein the amount of particles formed increases with time
until a maximum was reached. As new particles are formed and grow, the signal
intensity increases. Concurrently, noise signal increases due to multiple light scattering
from particles crossing the light path more than once. As a consequence, the Savitsky-
Golay algorithm was used to smooth the data from which all the kinetic parameters were

recovered.

64.

      
 

400000 -

           
  

( f | »
{?o%'$i'z‘3 “Wt
'01-“ I!» (‘1!‘00.

.6 (VP- ‘: 3".
kl 'p' ’. ilk-I
\‘.‘ -7 ‘0;

.1 {I'd 9.118 r
1 .". ‘ T
0100..

\

V
D
2.
c'.
‘e
o

  
  

300000 -

0 Raw data
- Smoothed data

Signal Intensity (a.u)

 

 

I I I ' I I I I I l I I I

I T
0 200 400 600 800 1000 1200 1400 1600
Time (s)

Figure 2.4

Typical curve of calcium oxalate crystallization using nephelometry. Experimental
conditions: calcium concentration, 0.8 mM, oxalate concentration, 0.8 mM; T”. 28.0“C:
pH 5.5; ionic strength. 0.15M NaCl. The clear line represents the smoothed data using
Savitsky-Golay algorithm.

65

The scattered radiant power is a parameter that corresponds to the particle concentration
and increases due to the mass accumulatiOn of the solutes. Thus, supersaturation can be
alternatively defined in terms of the radiant power obtained at any other concentration

and at the steady state region (i.e. at the end of the experiment), (DSC and ‘Cbsc,

respectively. The relative supersaturation is defined by

 

t

(I)
S=l— “ 2.6)
(I) (

51‘

Figure 2.5 is a typical desupersaturation curve. The induction period which corresponds
to the time between the addition of oxalate and the moment at which a change in optical
density is observed was taken as the time corresponding to the intersection of a tangent
line with the steepest part of the experimental function with time axis. The length of the
induction per 1 can ' . mode ited by .mpurities which promote or inhibit nucleation.
We studied the effect of amino acid derivatives on induction period and the results w ere
compared to t, in the absence and the presence of citrate. A summary of the induction
period for the calcium oxalate crystal growth is summarized in table 2.2. None ot the
amino acid derivatives significantly prolonged the induction period except the 1) sine
derivative. The inhibition power of lysine derivative on nucleation of calcium oxalate is
about 3 times greater than that of citrate, and can prolong the on set of calcium m.tl.tte

crystals 5 times longer relative to the control.

66

Supersaturation (S)

 

T

 

it,

I I I ' I ' I ' I 1 I ' I ' I ' I ' I

200 400 600 800 1000 1200 1400 1600 1800 2000

Time (seconds)

Figure 2.5

A typical desupersaturation curve tor the system CaCl3 2H2O + Na2C204 Experimental
conditions: calcium concentration, 0.8 mM, oxalate concentration, 0.8 mM; T". 2801‘:
pH 5.5; ionic strength, 0.15M NaCl.

67

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Additives IInduction time Growth
(8) Inhibitors
ti i 50 %I=(l-Si-SC) x
100

Control 340
Citrate 640 84
1 j me derivative 1800 98
Aspartic 430
derivative
Glutamic 3 40
derivative
Glycine derivative 460 28
B'aiaml‘e - 450 11
derivative
3’3““ 350 41

en vative

Table 2.2

Effects of inhibitors on calcium oxalate nucleation and crystal growth.
Experimental conditions: calcium oxalate, and inhibitor concentrations were all at (1.8
mM, To, 280°C; pH 5.5; ionic strength, 0.15M.

68

 

The percentage of inhibition determined from the turbidity slopes of the crystal
growth curve in the presence (8,) and absence (Sc) of additives were calculated as

[1— (S,/SC )1’100].l4 At pH 5.5, the inhibitory effect of the lysine derivative and citric acid

with regard to the crystallization of CaC204 indicated 98% and 84% inhibition,

respectively, while the glycine derivative, ,6 -alanine derivative, and arginine derivative

inhibition strength was only marginal (see table 2.2). On the other hand, aspartic and

glutamic derivative both appear to have no effect on crystal growth.

2.4 CONCLUSION

Nephelometry technique is a fast and simple method to study the inhibitory effect
of various substances on calcium oxalate crystal growth. From the induction times alone.
it was possible to study nucleation effects, and in addition, we can extract growth kinetic
information from the slope of the curve. Lysine derivative by far was the most effective
nucleation and growth inhibitor followed by citrate. It is difficult to tell if citrate and
lysine derivative exert their inhibitory effect through ion pairing or not. Based on the
chelation constant data it does appear that ion sequestration alone does not explain why
these compounds make good inhibitors of nucleation and crystal growth. Studyin g the
morphological and phase transformation effect on calcium oxalate crystals is the
direction we plan to take next to understand the mechanism of inhibitory effect ot the

amino acid derivatives and citrate.

69,

2.5 REFRENCES

1Elsander, A.; Ek, M.; Gellerstedt, G. Tappi Journal 2000, 83, 73-77.
2Watts, R. W. Journal Of The Royal College Of Physicians Of London 1973, 7, 161-74.

3Peacock. M.; Schneider, H.-J. Urolithiasis .' etiology, diagnosis; Springer-Verlag: Berlin
; New York, 1985.

4McDonaid. M. W.; Stoller, M. L. Geriatrics 1997, 52, 38-40, 49-52, 55-6.

5West, E. 8.; Todd, W. R. Textbook of biochemistry, 3d ed.; Macmillan: New York..
1961.

6Cerini, C.; Geider, S.: Dussol, B.; Hennequin, C.; Daudon, M.; Veesler, S.; Nitsche, S.;
Boistelle, R.; Berthezene, P.; Dupuy, P.; Vazi, A.; Berland, Y.; Dagorn, J. C.; Verdier, J.
M. Kidney International 1999, 55, 1776-86.

7Rechnitz, G. A.; Lin, Z.-F. Anal. Chem. 1968, 40, 696-9.

8Nagarajan, M. K.: Paine. H. L. JAOCS, J. Am. Oil Chem. soc. 1984, 61, 1475-8.

9Biay, J. A.; Ryland. J. H. Anal. Lett. 1971, 4, 653-63.

l0Berg, C.; Tiselius. H. G. European Urology 1986, 12, 59-61.

“Chang, D. M. JAocs, J. Am. Oil Chem. soc. 1983, 60, 618-22.

”Crutchfield. M. M. J. Am. on Chem. soc. 1978,55, 58-65.

l3Ingle, J. D.; Crouch. S. R. Spectrochemical analysis: Prentice Hall: Englewood Cliffs.
N.J., 1988.

l4stttor, D. J. Br. J. Urol. 1975, 47, 585-7.

70

Chapter 3

THE INFLUENCE OF TAILOR-MADE IMPURITIES OF AMINOACID
DERIVATIVES ON THE MORPHOLOGY AND PHASE TRANSFORMATION
OF CALCIUM OXALATE HYDRATES

The crystallization of calcium oxalate in the presence of different additives was
studied at ambient temperature. Tailor-made amino acid derivatives were used as
modifiers and phase transformation modulators. Phase transformation of calcium oxalate
in the presence of additives was analyzed by Raman spectroscopy, x-ray powder
diffraction (XRD), and environmental scanning electron microscopy (ESEM). All the
additives except the glycine and glutamic acid derivatives allowed a phase transformation
to take place from a i 'etastable calcium oxalate dihydrate, COD, to the
thermodynamically stable monohydrate, COM. The glycine and the glutamic acid
derivatives selectively inhibited phase transformation from taking place, while the

E — alanine and arginine derivatives converted all COD phase to the COM phase. In all

cases, the additives modified the crystal shape of the dihydrate form of calcium oxalate.

71

HO OH OH
0 NH 0
H0

0

Glycine derivative or
2-(CarboxymethyI-amino)-
3-hydroxy-suocinic acid

0 O OH
HO
OH HO OH
O H
N NH
0 MVI
0H 0 0H

Lysine derivative or

2,6-Bis-(1 ,2-dicarboxy-2-nydroxy-ethylamino)-
hexanou acid

0
HO
0 NH 0
HO HO OH
0
H0

Aspartic derivative or
2-(1 ,2-Dicarboxy-ethylamino)-
3-hydroxy-succinic acid

0
0H0

HO N
H

OH
OH

O
B—Alanine derivative or
2-(2-Carboxy-ethylamino)-
3-hydroxy-succinic acid

0

ll
H2N N H0

H

0

OH
OH

O
Arginine derivative or
2-(1 -Carboxy-4-guanidino-butylamino)-
3-hydroxy-suocinic acid

0
H0
NH 0
H0 HO OH
0 0H
0

Glutamic derivative or
2-(1 ,2-Dicarboxy-2-hydroxy-ethytaminc)-
pentanedioic acid

Scheme 3.1

, Tailor—made additives synthesized by reacting cis-epoxysuccinic acid and amino acid.
See chapter 2 for more details on the synthesis procedure.

3.1 INTRODUCTION

In the pulp and paper industries, calcium oxalate (CaC204) forms scale deposits
on the equipment,1 while in animals, CaC204 occurs as a crystalline material in the
urinary tract and is the majority constituents in kidney and bladder stones.2 Calcium
oxalate crystallization results in formation of three types of hydrates: the monohydrate,
the dihydrate, and the trihydrate, depending on solution conditions. The monohydrate
phase constitutes the main component of the most abundant type of renal calculi that is
also the most difficult to treat and the most poorly understood.3 The factors that
determine calcium oxalate formation include supersaturation, crystallization kinetics.
urinary inhibitors, and epitaxy.4'5 In normal urine, the concentration of calcium oxalate is
four times higher than its solubility in water.6 This high concentration can be attributed
to the presence of natural inhibitors, which allows such high calcium oxalate.
concentration in vivo. When the solubility product of calcium oxalate is exceeded.
nucleation and crystal growth of calcium oxalate takes place, leading to stone formation.7

Treatment of kidney stones has improved dramatically with the introduction of
extracorporeal shock wave lithotripsy (ESWL). However, stone prevention is a much
less invasive and a more cost-effective approach to patient management. Therefore.
calcium oxalate crystallization studies play an important role in urolithiasis research and
clinical management. The interest in calcium oxalate crystal growth is oriented to
suppress it. Citrate is commonly used as an inhibitor of urinary stone formation.h and
works by controlling the average size of calcium oxalate crystals as well as nucleation

time, thus it prevents retention in the ducts and the eventual development of kidney

73

stones.9 For healthy patients, it is also thought that calcium and oxalate ions are
encapsulated in a protective colloid that keeps the precipitate in colloidal suspension.'0
The important conditions for crystal growth are concentrations of the crystallizing
species, the solution temperature, the pH, the ionic strength, and the concentrations
growth-modifiers.ll The most important technique used today for influencing crystal
growth and the crystallization process is the addition of impurities In this study we have
performed experiments in the presence and absence of tailor-made additives (glycine
derivative, ,8 -alanine derivative, lysine derivative, arginine derivative. aspartic
derivative, and glutamic derivative), and an additive with biological relevance, citrate.
The focus of the study was on the habit modification and phase transformation effects of
the additives. We have addressed the synthesis procedure of these compounds
elsewhere.12 Amino acid derivatives (scheme 3.1) were chosen for their opposite nature
in steric hindrance, carboxylate composition, and size. There are numerous techniques
which can be applied to study the crystal phases. Our method of choice are, Raman
spectroscopy (RS), environmental scanning electron microscopy (ESEM), and x-ray

pOWder diffraction (XRD).‘3-l4-15.l6

74

3.2 EXPERIMENTAL METHODS

Experiments were performed in a 250 mL volumetric flask, and the supersaturated
solutions were prepared by mixing equal volumes of calcium chloride and sodium nitrate
stock solutions. The aqueous stock solutions of 5 mM calcium chloride dihydrate and
sodium oxalate were buffered at pH 5.5 with 9 mM MES (2-[N-Morpholino]
ethanesulfonic acid) and brought to an ionic strength of 0.15M with sodium chloride.

The MES has no chelation properties. A pH of 5.5 was chosen because it is the pH value
frequently observed in the morning urine of calcium stone-formers.l7 An aliquot of 25
mL of calcium chloride dihydrate was transferred in a sample cell followed by 25 mL of
9 mM MES buffered solution or 5 mM solutions tailor-made additives prepared in
buffered solution. While stirring the sample solution with a teflon-covered magnetic bar,
25 mL of the control solution (sodium oxalate) was added quickly. Once the mixing was
done, the magnetic stirrer was stopped to allow the crystals to grow without breakage.
Aqueous solution was removed by suction filtration to isolate the crystals. and the Raman
spectra of the calcium oxalate precipitate was obtained.

Raman spectra were recorded using a HoloProbe Process Raman Analyzer
(Kaiser Optical Systems) equipped with a GaAlAs diode laser. The power of the incident
785 nm laser'beam was about 100 mW on the sample’s surface. The Raman scattered
laser light input signal was collimated, then passed through a notch filter (SuperNotch-
Plus“), and focused on the spectrograph entrance slit. The diffracted light from the
spectrograph was transmitted to the charge coupled detector (CCD) by a volume
holographic transmission grating (HoloPlexTM grating). The CCD chip was 1024x 128

EEV MPP with pixel size of 26 a m and an operating temperature set at -40”C.

75

Photomicrographs of the crystals were obtained with environmental scanning
electron microscopy (ESEM). Subsequently, the powder X-ray diffraction analysis was

performed on the finely powdered samples with the use of the CuKa (40 kV and 20 mA)

radiation with a scanning speed of 0.030 29/min.

3.3 RESULTS AND DISCUSSIONS

In a crystallization process, where the formation of several phases is possible. the
Ostwald-Lussac empirical rule states that the phase with the highest solubility is
kinetically favored.18 The solubility products of calcium oxalate hydrates at room
temperature are reported as follows: COT (4.81 x 10'9 moleszlliterz), COD (2.82 x 10")
molesz/literz), and COM (2.00 x 10‘0 moleszlliterz).'9' 20’ 2‘ Since impurities have great
influence on crystal size, shape, and purity, 22we investigated the selective effect of the
additives on the habit ttodific;.tion a; i the phase transformation of CaC204 using Raman
spectroscopy, x-ray powder diffraction, and environmental scanning electron microscopy.

The characteristic Raman bands due to the C=O symmetric stretching are observed
around 1463 cm"l for the COM and at 1473 cm" in the case of COD.23 When the
crystallized samples were left in solution for about an hour, then the supernatant w as
decanted off. Raman spectra of the wet crystalline material was obtained, and 111.1. the
dihydrate form of calcium oxalate was detected as shown in figure 3.1a. Whereas. the
Raman spectra of calcium oxalate crystals left in aqueous solution for 24 hours 111 the
presence of tailor-made additives, confirmed that a change in phase took place exec pt tor
the samples crystallized in the presence of the glycine and glutamic derivatives. I‘lgllfc

. 3.1b indicates the presence of the COM and the COD in different proportions for the

76

control sample as well as samples crystallized in the presence of the additives. The
Raman spectra indicate that the crystals grown in the presence of these tailor additives:

fl — alanine and arginine derivatives transformed completely from the COD crystal phase

to the COM crystal phase after 24 hours.

77

 

1e+5

 

 

 

 

Qe+4
Be+4
7e+4
3; 6e+4
g 5e+4
(I)
C
.9 4e+4
E
73 3e+4
C
.9
(D 2e+4
1e+4
Oe+0 3* 1 1
1440 1460 1480 1500

Raman Shift (cm'1)

Figure 3.18

Raman spectra of calcium oxalate hydrates after 1 hour in solution. a) alanine derivative.
b) glycine derivative, c) arginine derivative. (1) citrate, e) glutamic derivative. f) control.
g) lysine derivative, h) aspartic derivative Experimental conditions: calcium
concentration, 0.8 mM, oxalate concentration, 0.8 mM; T0, 28.0”C: pH 5.5; ionic
strength, 0.15M NaCl. Concentration of additives, 0.8mM.

78

 

1.3e+5
1.29+5 -
1.1e+5 -
1.0e+5 -
9.0e-1-4 —
8.0e+4 4
7.0e+4 -
6064-4 4
5.0e+4 .4
4.0e-1-4 -
3.0e+4 -\I
2.0e+4 -1
1.0e+4 — ‘
0.0e+0 ” 1‘

1400 1420 1440 1460 1480 1500 1520 1540

Raman Shift (cm’1)

Signal Intensity (a.u)

 

 

 

 

 

 

Figure 3.1b

Raman spectra of calcium oxalate hydrates after 24 hours in solution. a) alanine
derivative, b) arginine derivative, c) control, d) glutamic derivative, e) glycine dern attve.
f) citrate, g) lysine derivative, h) aspartic derivative. Experimental conditions: calcium
concentration, 0.8 mM, oxalate concentration,0.8 mM; To, 280°C; pH 5.5: ionic
strength, 0.15M NaCl. Concentration of additives, 0.8mM.

79.

The morphology of calcium oxalate hydrates were observed by ESEM. Figure 3.2 is
a typical micrograph for COD prepared without additives. The crystal shape is
tetrahedron. 11: the presence of tailor-made additives, some of the crystal faces were
modified. Figures 3.3A-3.4D are micrographs of COD crystals which were crystallized

in the presence of glycine, B-alanine, glutamic, and arginine derivatives. The crystal

faces appear to have been modified from the typical tetraheron shape to a needle-like
shape. As for samples crystallized in the presence citrate, we see a change in all the
crystal faces for the crystals affected as shown in figure 3.5. However, the crystal shape
appears more plate-like.

When the crystallization of calcium oxalate was allowed to take place in solution for
24 hours in the presence and absence of additives, several interesting results were
obtained. Phase transformation from the COD to COM took place in all cases except
samples crystallized in the presence of glutamic and glycine derivatives. These additives
prevented any phase transformation from taking place as shown in figure 3.6.
Interestingly, only the crystals grown in the presence of the glutamic derivative preserved
the original crystal habit of COD. On the other hand, the glycine derivative allowed habit
modification to take place from the need-like shape to the tetrahedron like shape. Figure
3.3a and 3.7 indicates the effect of glycine derivative on the crystal shape after an hour.
and 24 hours in solution respectively. The B-alanine and arginine derivative allowed

complete phase transformation to take place from the COD to the thermodynamically

stable COM.

80

88mm '2

.q ”41“
CONYRL04 TIF 101/09/1814_e¢

 

Figure 3.2

ESEM of a typical COD morphology. Experimental conditions: calcium concentration.
0.8 mM, oxalate concentration, 0.8 mM; '1'", 280°C; pH 5.5: ionic strength. 0.15M \aCl.

81

r' m

A...

‘ sew 750x GSED ' to 13mm“ .- 91
Michigan State Untversrtg GLVCHEGZIIF 101/09/1815

 

Figure 3.3a

ESEM of COD formed in the presence of 0.8 mM glycine derivative after left insolution
for 1 hour. Experimental conditions: calcium concentration, 0.8 mM. oxalate
concentration, 0.8 mM; T“. 280°C: pH 5.5; ionic strength. 0.15M NaCl.

r5111.—
.313 ._.......... ._ ..
:E: 7.7mm I.’ Hi
HLFHCHJBJ TIF 1131/09/1816-2

39 iv 12 13850
Michigan State University

 

Figure 3.3b

ESEM of COD formed in the presence of 0.8 mM B-alanine derivative after left

insolution for 1 hour. Experimental conditions: calcium concentration, 0.8 mM. oxalate
concentration, 0.8 mM; ”1", 280°C; pH 5.5: ionic strength, 0.15M NaCl.

83

 

Figure 3.3c

ESEM of COD formed in the presence of 0.8 mM glutamic derivative after left lllxtilullOn
for 1 hour. Experimental conditions: calcium concentration, 0.8 mM. oxalate
concentration, 0.8 mM; T“. 280°C; pH 5.5; ionic strength, 0.15M NaCl.

84

III-Ill-IIIII 25 rm

0 k0 ZBBBX GSED
Michigan State UniverSitg 8R610004 TIF

 

Figure 3.3d

ESEM of COD formed in the presence of 0.8 mM arginine derivative after left in solution
for 1 hour. Experimental conditions: calcium concentration, 0.8 mM, oxalate
concentration, 0.8 mM; T“. 28.0"C; pH 5.5: ionic strength, 0.15M NaCl.

85

 

Figure 3.4

ESEM of COD formed in the presence of 0.8 mM citrate after left in solution for lhour.
Experimental conditions: calcium concentration, 0.8 mM, oxalate concentration 11 \ 111M:
T°, 280°C; pH 5.5; ionic strength, 0.15M NaCl.

86

gait-.1 11
Michigan 8

66x GSED
bate Universitg

 

Figure 3.5
ESEM of COD formed in the presence of 0.8 mM glutamic derivative after left in

solution for 24 hours Experimental conditions: calcium concentration, 0.8 mM. o\:1|alc
concentration, 0.8 "7.5.1; T . 280°C: pH 5.5: ionic strength, 0.15M NaCl.

87

 

Figure 3.6

ESEM of COD formed in the presence of 0.8 mM glycine derivative after left in solution
for 24 hours. Experimental conditions: calcium concentration. 0.8 mM. oxalate
concentration, 0.8 mM; T“. 280°C; pH 5.5; ionic strength, 0.15M NaCl.

88

The ESEM image shown in figures 3.8 and 3.9 indicates a typical morphology of COM.
As for the cry .1118 01 .tined it. the presence and absence of the following additives (lysine
derivative, aspartic derivative, and citrate), the ESEM image revealed a mixture COD and
COM crystals. See figures 3.10A-3.10D. The COM crystals are much smaller in size
compared to the COD.

Powder XRD was used to confirm the morphology of calcium oxalate crystals
prepared in the presence and absence of additives. The XRD characteristic reflections of
COD crystals which were crystallized for an hour in the presence of the following
additives: glycine derivative, 13 — alanine derivative, arginine derivative, glutamic
derivative, and aspartic derivative, confirmed the ESEM image that the morphology faces
of the crystallized COD are similar. Figure 3.11 shows the XRD spectra of COD crystals
in the absence and presence of citrate and the lysine derivative. The patterns falling at
the following position (14.7, 24.3, 10.1, 38.2 and 40.6) in terms of 26 , had similar peaks;
however, not all the peaks where common in the diffractograms. This is not surprisin g
because the ESEM data for these samples indicate that the sample crystallized in the
presence of citrate resulted in modified faces, while lysine derivative gave mixtures of
products with different crystal habit. Consequently, the diffractograms reflects a mixture
of x-ray diffraction pattern resulting from all the crystal faces.

The XRD of the crystals isolated after 24 hours in the presence of 6 — alanine and

the arginine derivative, confirmed that a complete phase transformation from C01) to
COM took place. From the XRD data shown in figure 3.12, it appears that the glycine

derivative modified the COD crystal face because the characteristic reflections of the

89

crystal faces for the control COD crystal does not match that of the COD crystallized in

the presence of glycii. : derivative.

90

 

' 3e.kv 756x GSED '
Michigan State University

Figure 3.7

ESEM of COD formed in the presence of 0.8 mM alanine derivative after left in solution
for 24 hours. Experimental conditions: calcium concentration, 0.8 mM. oxalate
concentration, 0.8 mM; T°, 280°C; pH 5.5; ionic strength, 0.15M NaCl.

91

REED

ate Universitg

 

Figure 3.8

ESEM of COD formed in the presence of 0.8 mM arginine derivative after left in solution
for 24 hours. Experimental conditions: calcium concentration, 0.8 mM. oxalate
' concentration, 0.8 mM; T”, 280°C: pH 5.5: ionic strength. 0.15M NaCl.

92

 

P m
_&JM~~— ' ' .“- .--mi.-h7:~
28.k0

559x

SED
Michigan State Universitg

Figure 3.9a

ESEM of COD formed in the presence of 0.8 mM citrate derivative after left in solution
for 24 hours. Experimental conditions: calcium concentration. 0.8 mM, oxalate
concentration, 0.8 mM; To, 280°C: pH 5.5; ionic strength, 0.15M NaCl.

93

 

‘7 k . , 4.. ,. it. ‘ _ I . .. .-
26.kV [3 EU 10.3mm 3.1
I

T
Michigan L\"SE1013E11.TIF 181/11/118245

Figure 3%

ESEM of COD formed in the presence of 0.8 mM lycine derivative after left in solution
for 24 hours. Experimental conditions: calcium concentration, 0.8 mM. oxalate
concentration, 0.8 mM; T". 280°C; pH 5.5: ionic strength. 0.15M NaCl.

94

.-sj£
-.kU 906x GSED
chigan State University

 

Figure 3.9C

ESEM of COD formed in the absence of additives after left in solution for 24 hours.
Experimental conditions: calcium concentration, 0.8 mM, oxalate concentration. 1) 8 mM;
To. 280°C: pH 5.5: ionic strength, 0.15M NaCl.

95

 

.55'1.‘ —

. '1' t: . _

. 3 2..» ‘12? re...
2E1.kU 008x . G? _‘
Michiga tat. Uni rsitg

Figure 3.9d

ESEM of COD formed in the presence of 0.8 mM aspartic derivative after left 111 sttlullnn
for 24 hours. Experimental conditions: calcium concentration. 0.8 mM. oxalate
concentration, 0.8 mM; '1". 28.0”C: pH 5.5: ionic strength, 0.15M NaCl.

96

Signal Intensity (a.u)

1 000
800
600
400

200

6000
5000
4000
3000
2000
1000

2000
1 600
1 200

800

400

- Lysine derivative

 

 

 

 

I
.1
q
-
a
uni

Citrate

 

nlnlnltlnlnl

 

 

 

 

 

 

 

H ' I ‘ I
- Control
' I ‘ I ' I r I ' I
10 20 30 40 50 60

2. Theta (degree observed)

Figure 3.10

XRD spectra of COD crystal of the control sample, and the samples formed in the
presence of lysine derivative. and citrate after left in solution for an hour. Expen mental
conditions: calcium concentration, 0.8 mM, oxalate concentration. 0.8 mM; T". 28.0“C;
pH 5.5; ionic strength, 0.15M NaCl.

97

 

a.u)

V

Signal Intensity

1200 n

 

 

 

 

 

l
800 1 Glycine derivative
400 —

0 1 1 1 . 1 v 1 ' 1
1600 10 20 30 4o 50 60
120°: Glutamic derivative

800 —
400 -

0 1 i ' T ' I T l
1600
1200 1 Control

800
400

0 1‘ 1 . 1 . 1 ' T ‘

10 20 30 40 50 60

2 Theta (degree observed)

Figure 3.11

XRD spectra of COD crystal of the control sample, and the samples formed in the
presence of additives ( glutamic and glycine derivative) which prevented phase
transformation of COD crystals after it was left in solution for 24 hours. Experimental
conditions: calcium concentration, 0.8 mM, oxalate concentration. 0.8 mM; T“. 28.0“C;
pH 5.5;

ionic strength, 0.15M NaCl.

98

 

3.4 CONCLUSION

In this study, we used tailor-made additives, which preferentially influenced
specific cryst... faces. .. be ob. erved habit of crystals grown from solutions was
unpredictable. No formation of calcium oxalate trihydrate was observed. All the
additives modified the crystal habit of the COD in a similar manor. Some of these
additives behaved either as phase transformation inhibitors or as promoters. There are
two possible explanations for the observed phenomena: surface adsorption, and/or lattice
incorporation. Regardless of how the additives influenced the crystallization process.
habit modification as well as phase transformation of the calcium oxalate crystal hydrates
was expressed quite well. However, we can rule out lattice insertion because these
molecules are much bigger in size compared to the oxalate molecule. In addition. the
XRD data showed no evidence of diffraction patterns completely different from that of
the control sample.

The interpretation of the morphological results based on preferential adsorption of
the additives has some legitimacy. We tend to believe that the morphological
modification observed is mainly driven by electrostatic interactions of the additix es with
either oxalate ions and/or calcium ions. It has been reported that surfactants conriol
morphological and phase changes in calcium oxalate crystals through preferentiai
adsorption.” 25 Similar results have been reported for other organic molecules 11 1111 high
negative charge density. such as di- and tricarboxylic acids.26 Although further
investigation is needed to prove this, it is safe to say that some sort of specific interaction

is taking place with the surface of the calcium oxalate crystal faces.

99

3.5 REFRENCES

‘Eisander. A.; Ek, M.; Gellerstedt, G. Tappi Journal 2000, 83, 7377.

2Watts, R. W. Joumal Uf 7711 Royal College Of Physicians Of London 1973, 7, 161—74.
3Resnick, M. I. .Urolithiasis; W.B. Saunders: Philadelphia, 2000.

4McDonaid, M. W.; Stotzer. M L. Ger. ttrics 1997,52, 38-40, 49-52, 55-6.

5West, E. 3.; Todd, W. R. Textbook of biochemistry, 3d ed.; Macmillan: New York,,
1961.

6Coe,1=. L.; Parks. 1. H. Hospital Practice (Office Edition) 1988,23, 185-9, 193-5. 199-
200.

7Cerini, C.; Geider, S.: Dussol, B.; Hennequin, C.; Daudon, M.; Veesler, S.; Nitsche. S.:
Boistelle, R.; Berthezene, P.; Dupuy, P.: Vazi, A.; Berland, Y.; Dagorn, J. C.; Verdier. J.
M. KIDNEY INTERNATIONAL 1999, 55, 1776-86.

8Harrison, T. R. Principles of internal medicine, 4th ed.; Blakiston Division McGraw-
Hill: New York,. 1962.

9Chapiin, A. J. Journal ()fCli'ni'cal Pathology 1977, 30, 800-11.

"’1. Butt, J. Urol..l952. 67. 450.

llFinlayson, B. Kidney International 1978, 13, 344-60.

l2C.O. Ngowe and K.A. Berglund. see chapter 2.

l3Byrn, s. R.: Gray. 0.; Pfeiffer. R. R.; Frye, J. J. Pharm. Sci. 1985. 74. 565-8.

l4Stout, G. H.: Jensen. L. H. X-ray structure determination; a practical guide; Macmillan:
New York, 1968.

l5Curtin, D. Y.; Bym. S. R. J. Amer. Chem. Soc. 1969, 91, 6102-6.

16Kontoyannis. C. 0.: Bouropoulos. N. C.: Koutsoukos. P. 0. Appl. Spectrost: 1997. 5].
1205-1209.

l7Berg, C.; Tiselius. H. G. European Urology 1986, 12, 59-61.

18Ostwald,W. Phys. Chem. 1897, 22, 289-330.

100

 

19Nancoiias, G. 11.; Gardner, G. L. J. Cryst. Growth 1974, 21, 267-76.

20Babic-Ivancic, V.; Fueredi-Milhofer, H.; Purgaric, B.; Bmicevic, N.; Despotovic. Z. J.
Cryst. Growth 1985, 71, 655-63.

21Tomazic, B.: Nancollas, G. H. J. Cryst. Growth 19799 46. 355-61-

22Myerson, A. S. Handbook of industrial crystallization; Butterworth-Heinemann:
Boston, 1993. Chapter 3.

2381nppey,r. A. J. Mol. Struct. 1980, 65, 61-70.

24Addadi, L.; Berkovitch-Yellin, Z.; Weissbuch, 1.; Van Mil, J.; Shimon, L. J. W.; Lahav.
M.; Leiserowitz, L. Angew. Chem. 1985, 97, 476-96.

25Tunik, L.; Addadi, L.; Garti, N.; Fueredi-Milhofer, H. J. Cryst. Growth 1996, 167. 748-
755.

26Cody, A. M.; Cody, R. D. J. Cryst. Growth 1994, 135, 235-45.

101

 

FUTURE STUDIES
4.1 INTRODUCTION

The research conducted in chapters 1 and 2 involved the use of a batch
crystallization technique in investigations of the crystallization of calcium oxalate. This
technique involved the use of a glass beaker or flask, into which the calcium and oxalate
solutions are added all at once and the combined solutions were mixed using a magnetic
stirrer. The crystallization was initiated spontaneously. As the crystals grew, calcium and
oxalate ions disappeared from solution decreasing the relative supersaturation driving
force. A crystallization rate was measured by nephelometry. Since the relative
supersaturation falls throughout the experiment, the measured rate or the morphology of
the crystals is not characteristic of any one value of relative supersaturation. It is
important to know that the manner and time scale over which the supersaturation is
brought about will affect the different crystallization processes in different ways. the
crystal size distribution. crystal phase and habit modification.1

The prediction and control of the growth habit of crystals are utmost importance
for chemical, petrochemical, food, environmental, and pharmaceutical industries. In
chapter 1, it was demonstrated how tailor-made amino acid derivatives can be Used as
modifiers and phase transformation modulators. The two additives from the aliphatic
group (glycine and ,6 -alanine derivative) both modified the crystal shape of calcium
oxalate, however only the glycine derivative prevented phase transformation from taking
. place. The basic amino acids used (lysine and arginine derivative) allowed habit
modification and phase transformation of calcium oxalate hydrates to take place. 11 w as

also demonstrated how derivatives of acidic amino acids (aspartic and glutamic

102

 

derivative) affected the crystallization of calcium oxalate hydrate. The glutamic
derivative behaved as an inhibitor of phase transformation while aspartic derivative
promoted it. In addition, they both promoted habit modification. We learned from
chapter 2 that the most effective inhibitor of both nucleation and crystal growth was the
lysine derivative, leading to an inhibition of 98%, followed by the citric acid. However,
the aliphatic amino acids and the basic amino acid derivatives inhibited less than 50%
crystal growth.

Although the lysine derivative appears to be a promising compound which could
be used for controlling kidney stones, it still requires more studies to be conducted. It is
generally believed that a supersaturation decay method such as batch crystallization does
not represent the conditions within the kidney. In order to mimic the physiological
condition for the purpose of evaluating inhibitors, constant composition method is the

technique of choice.

4.2 PROPOSED STUDIES

4.2.1 CONSTANT COMPOSITION METHOD

The constant composition method used in the measurement of calcium oxalate
crystal growth rates was developed by Sheehan and Nancollas.2 This method represents
the condition within the kidney compared to the supersaturation decay method.l (‘aleium
and oxalate solutions and crystal seeds are added to a stirred crystallization vessel at the
beginning of the experiment and concentrated calcium and oxalate solutions are titrated
into the vessel throughout the experiment. Instead of following the depletion of an ion
from solution, the amounts of concentrated calcium and oxalate solutions titrated to

maintain a constant supersaturation during crystallization are recorded. The calcium

103

 

concentration is monitored using a calcium-specific electrode, with the measurement of
oxalate with the assumption of equimolar co-precipitation of the oxalate ion. The
constant composition method allows for the measurement of crystal growth rates at
constant solution conditions, particula. y constant supersaturation. Using this method to
test the six inhibitors synthesized would give a better insight on their potentials as

modifiers, and inhibitors of crystal growth and phase transformation.

4.2.2 SYNTHESIS OF AMINO ACID DERIVATIVES

Only 6 of the 20 amino acids were derivatized. An attempt to derivatize the rest
of the amino acid was successful, but limited by the purification process of the product.
The synthesis procedure for making the following amino acid derivatives (valine, leucine.
isoleucine, methionine, serine, threonine, cysteine, asparagines, and glutamine ) involved
adding equimolar amount of starting reagents (amino acid and cis-epoxysuccinic acid)
and raising the pH of the solution to the pKa value of the amino acid. The unwanted side
reaction was formation of tartaric acid was unavoidable. The purification procedure that
was devised for aspartic and glutamic derivative did not work well for the other amino
acids. A better synthetic route which avoids formation of tartaric acid would be of great
interest. Secondly, vigorous testing of these inhibitors as potential therapeutic agents for

prevention and/or treatement of urolithiasis.

104

 

4.3 REFRENCES

‘ Mullin, J. W. Crystallization, 3rd ed.; Butterworth-Heinemann: Oxford ; Boston, 1993.

3 Sheehan, M. 13.; Nancollas, G. H. Invest. Urol. 1980, 17, 446-50.

3 Khan, S. R. Calcium oxalate ill biological systems; CRC Press: Boca Raton, Fla., 1995.

105

 

CONCLUSION

SYNTHESIS AND CHARACTERIZATION OF TAILOR-MADE ADDITIVES
FOR INHIBITION OF SPARINGLY SOLUBLE CALCIUM SALT
CRYSTALLIZATION

The control of crystals and the crystallization process from solutions using a
structurally similar or tailor-made additives has been demonstrated. Our original goal
was to inhibit calcium oxalate crystal growth using the amino acid derivatives. To our
surprise, only lysine derivative was an effective inhibitor of both nucleation and crystal
growth. Since calcium oxalate exist in more than one phase, we then focused our
attention on the effect of the amino acid derivatives on the phase transformations of
calcium oxalate hydrates. Glycine and glutamic derivatives selectively inhibited phase
transformation from taking place, while alanine and arginine derivative promoted phase
transformation. The only common effect that was observed in the amino acid deny .111st
is their ability to modify the crystal faces of calcium oxalate.

Although these tailor-made additives were carefully designed, what we could not
predict was the effect it would have on nucleation, growth, morphology, and phase
transformation. As it has been stated many times in the literature, the effect of additixes
on crystal growth and morphology still remains a matter of mix and match. How ex ct.
tailor-made additives cannot be discounted as effective method of controlling

crystallization process When the right match has been determined.

106