Synthesis of Polycaprolactone Polymers for Bone Tissue Repair · Synthesis of Polycaprolactone...

of 151 /151
Synthesis of Polycaprolactone Polymers for Bone Tissue Repair John Michael Colwell B. App. Sc. (Chemistry) (Hons) A thesis submitted for the degree of Doctor of Philosophy School of Physical and Chemical Sciences Queensland University of Technology December 2006

Embed Size (px)

Transcript of Synthesis of Polycaprolactone Polymers for Bone Tissue Repair · Synthesis of Polycaprolactone...

Synthesis of Polycaprolactone

Polymers for Bone Tissue Repair

John Michael Colwell B. App. Sc. (Chemistry) (Hons)

A thesis submitted for the degree of Doctor of Philosophy

School of Physical and Chemical Sciences

Queensland University of Technology

December 2006


STATEMENT OF ORIGINAL AUTHORSHIP The work contained within this thesis has not been previously submitted for a degree or

diploma at any other higher institution. To the best of my knowledge and belief, the

thesis contains no material previously published or written by another person except

where due reference is made.

John Colwell



I would like to thank my supervisory team: Dr Edeline Wentrup-Byrne, Prof Franois

Schu, and Prof Graeme George, for their help and support throughout my PhD. It was

a pleasure working with all of them and I would especially like to thank Franois for his

hospitality during my stay in Montpellier.

It would have been impossible to conduct the necessary analyses of my materials

without the help of the various technical and research staff in both QUT, and UQ. My

thanks go to: Dr Llew Rintoul, Dr Barry Wood, Dr Greg Cash, Loc Doung, Dr Thor

Bostrom, Pat Stevens, Dr Chris Carvalho, and Karl Jacques.

I would also like to thank everyone at the Laboratoire de Chimie Macromoleculaire,

Universit Montpellier II, for their hospitality during my three month research visit,

April July, 2004.

My thanks also go to Freddy De Filipis and Rackel San Nicolas, two visiting students

from Polytech Montpellier, who helped with the model mineralisation studies.

Last, but not least, I would also like to thank my fellow postgraduate students who have

provided an interesting and supportive work environment during my time at QUT.





J. M. Colwell, E. Wentrup-Byrne, G. George and F. Schu, Synthesis of

polycaprolactone-block-poly(ethylene glycol)-block-polycaprolactone triblock

copolymers using a calcium-based initiator, Macromolecular Chemistry and Physics (in


J. M. Colwell, E. Wentrup-Byrne, G. George and F. Schu, The effect of calcium

residues on the in vitro mineralisation of polycaprolactone, Biomaterials (in


Conference Presentations

Oral Presentations

J.M. Colwell, E. Wentrup-Byrne, and F. Schu, Synthesis of PCL/PEG/PCL Block

Copolymers Using a Calcium-Based Initiating System, 27th Australasian Polymer

Symposium, Adelaide, Australia (November 2004).

Poster Presentations

John Colwell, Edeline Wentrup-Byrne, Graeme George and Franois Schu, Synthesis

of polymeric tissue scaffolds using a biocompatible calcium initiator, 26th Australasian

Polymer Symposium, Noosa, Australia (July 2003).


J.M. Colwell, N. Guerrouani, F. DeFilippis, E. Wentrup-Byrne, A. Mas, G. George, and

F. Schu, Mineralisation of Polycaprolactone Effected by Calcium Initiator Residues, 7th

World Biomaterials Congress, Sydney, Australia (May 2004).

E. Wentrup-Byrne, J.M. Colwell, K.A. George, and F. Schu, Controlled Polymer

Synthesis for Craniofacial Applications, Australian Society for Biomaterials 14th Annual

Conference, Adelaide, Australia (March 2005)



Polycaprolactone (PCL) is a biodegradable synthetic polymer that is currently used in a

number of biomedical applications. A number of concerns have been raised over the

toxicity of initiators commonly employed for the synthesis of PCL. Therefore, more

biocompatible initiators have been studied. The biocompatibility of PCL, itself, is

adequate; however, improved bioactivity is desirable for several applications.

Copolymerisation, and incorporation of bioactive fillers can both be used as ways of

enhancing the bioactivity of PCL. Therefore, the global objective of this project was to

enhance the bioactivity of PCL by copolymerisation of PCL with poly(ethylene glycol)

(PEG) using a biocompatible calcium-based initiator. This calcium-initiator was

expected to leave potentially bioactive calcium-initiator residues in the synthesised


A study of the ring-opening polymerisation of -caprolactone (CL) in the presence of a

poly(ethylene glycol) (PEG) / calcium hydride (CaH2) co-initiation system was

performed. Polymerisation kinetics were monitored by following the degree of

conversion of CL by Fourier transform-Raman (FT-Raman) spectroscopy and 1H

nuclear magnetic resonance spectroscopy (NMR). Resultant PCL-b-PEG-b-PCL

(PCL/PEG/PCL) triblock copolymers were analysed by NMR and gel permeation

chromatography (GPC).

The observed rates of polymerisation for the synthesis of PCL/PEG/PCL triblock

copolymers using the PEG / CaH2 co-initiator were much lower than expected. 1H NMR

and Raman microspectroscopy analysis showed that the concentration of the active

calcium-PEG alkoxide was much lower than the initial feed concentration of PEG. Even

so, the molecular weight of PCL/PEG/PCL triblock copolymers could be predicted from

the CL : PEG feed ratio. This was found to be due to a fast reversible transfer process.

Inductively coupled plasma-atomic emission spectroscopy (ICP-AES) analysis of

solutions containing acid digested, pure PCL/PEG/PCL copolymers showed calcium


concentrations at 77 % of the calcium feed concentration. These calcium-initiator

residues were isolated and their structures confirmed by Fourier transform infrared-

attenuated total reflectance spectroscopy (FTIR-ATR). They were found to be a mixture

of calcium hydroxide (Ca(OH)2) and calcium carbonate (CaCO3).

The effect of calcium-initiator residues on the in vitro mineralisation of PCL/PEG/PCL

triblock copolymers, as well as the same effect on a model calcium-salt-doped PCL

homopolymer system, was studied by immersion in simulated body fluid (SBF). In the

model studied, PCL homopolymer was doped with low concentrations (0.2 2 w / w %

Ca) of Ca(OH)2, or CaCO3. Results from the model study showed calcium phosphate

(CaP) mineral deposition on Ca(OH)2-doped PCL, and not on CaCO3-doped PCL. This

was attributed to the higher solubility of Ca(OH)2, compared to CaCO3. Minimal CaP

deposition was observed on PCL/PEG/PCL triblock copolymers. This was attributed to

the low Ca(OH)2 concentration in these samples. For all mineralised samples in the SBF

studies, the formation of carbonated HAP was observed.

Overall, the synthesis of PCL/PEG/PCL copolymers using the PEG / CaH2 co-initiator

was found to be a suitable method for preparing reproducible materials. The calcium-

based initiator was also found to have potential for increasing the bioactivity of PCL-

based materials.














2.1 Introduction 6

2.1.1 Polyester Synthesis 6

2.1.2 Living Polymerisation 7

2.1.3 Synthesis of Poly(-hydroxy acid)s 9 Initiators / Catalysts for the Synthesis of Poly(-hydroxy acid)s 10 Transesterification Reactions 13 Aggregation Phenomena 14

2.1.4 Methods for Studying the Kinetics of Poly(-hydroxy acid) Synthesis 15 Nuclear Magnetic Resonance Spectroscopy (NMR) 16 Gel Permeation Chromatography (GPC) 16 Dilatometry 17 Infrared Spectrosopy (IR) 17

ix Raman Spectroscopy 18 Methods-of-Choice For This Study 18

2.2 Experimental 19

2.2.1 Materials 19

2.2.2 Methods 19 Syntheses of PCLx/PEG45/PCLy Copolymers 19 Real-Time Monitoring 21 NMR Characterisation of Initiator 22 Headspace Analysis 22

2.2.3 Measurements 22 Nuclear Magnetic Resonance Spectroscopy (NMR) 22 Gel Permeation Chromatography (GPC) 23 FT-Raman Spectroscopy 24 Raman Microspectroscopy 24

2.3 Results and Discussion 26

2.3.1 Polymerisation Scheme 26

2.3.2 NMR Characterisation 28

2.3.3 PCLx/PEG45/PCLy Synthesis 32 Real-Time Monitoring of Polymerisation Kinetics 39 Analysis of Polymerisation Kinetics 41 Activation Energy 49 Aggregation 52

2.3.4 Elucidation of the Active Species Structure 56 1H NMR 56

2.3.5 Raman Microspectroscopy 59

2.3.6 Reversible Exchange 61

2.4 Summary 66


3.1 Introduction 67


3.1.1 Bone Tissue Repair 67

3.1.2 Modification of Polymeric Biomaterials 69

3.1.3 Biocompatibility and Bioactivity Testing 72 Method-of-Choice for This Study 74

3.2 Experimental 75

3.2.1 Materials 75

3.2.2 Methods 76 Calcium-Doping of PCL Homopolymer 76 Isolation of Calcium-Initiator Residues 77 Melt-Pressing of Polymer Films 77 Simulated Body Fluid Study 77 Ca2+ Release Study 78 Calcium Content Analysis 79

3.2.3 Measurements 79 Tensile Testing 79 Differential Scanning Calorimetry (DSC) 80 Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-

AES) 80 Flame Atomic Absorption Spectrometry 80 Scanning Electron Microscopy (SEM) 80 Energy-Dispersive X-ray Microanalysis (EDX) 81 Fourier Transform Infrared Spectroscopy-Attenuated Total Reflectance

(FTIR-ATR) 81 Water Contact Angle 81

3.3 Results and Discussion 82

3.3.1 Tensile Properties 83

3.3.2 Thermal Properties 85

3.3.3 Calcium-Initiator Residues 87

3.3.4 Ca2+ Release 90

3.3.5 SBF Study 91 Model Study: Calcium-Doped PCL 91

xi PCLx/PEG45/PCLy Copolymer Study 100

3.4 Summary 107


4.1 Conclusions 108

4.2 Future Research Directions 110





Figure 2.1: Typical nM versus conversion plots for the synthesis of a polymer under

living conditions (----) compared to ideal polycondensation for an X-R-Y-type system

( ). Adapted from Odian, p.852 .....................................................................................8

Figure 2.2: Some poly(-hydroxy acid)s and their respective lactone monomers..........10

Figure 2.3: Tin(II)2-ethylhexanoate / alcohol co-initiated polymerisation of lactones,

adapted from Albertsson and Varma.10 NB: R 4 C......................................................11

Figure 2.4: Intra- and inter-molecular transesterification reactions of a polyester .........13

Figure 2.5: Heating and stirring controllers / devices for real-time kinetic studies by FT-

Raman spectroscopy.........................................................................................................21

Figure 2.6: Proposed polymerisation scheme for the synthesis of PCL/PEG/PCL using a

PEG / CaH2 co-initiator....................................................................................................27

Figure 2.7: Typical 1H NMR spectra of a crude PCLx/PEG45/PCLy polymerisation

mixture (upper) and a pure PCLx/PEG45/PCLy copolymer (lower). Peak assignments are

given in Table 2.3.............................................................................................................31

Figure 2.8: Fraction of theoretical CL content, [CL]t / [CL]0, versus time ([CL] was

determined from 1H NMR using PEG45 as an internal standard) for the synthesis of

PCL50/PEG45/PCL50 under an argon atmosphere in a dry box (note that the point near 0

min was taken two minutes after addition of CL to the pre-heated vessel) .....................32

Figure 2.9: ln [CL]0/[CL]t versus time plot for the synthesis of PCLx/PEG45/PCLy

copolymers; [CL]0 : [OH]0 : [CaH2]0 = 50 : 1 : 0.67 (), and [CL]0 : [OH]0 : [CaH2]0 =

100 : 1 : 0.67 (x) in flame-sealed glass tubes at 70 C. Error bars: 1 S.D., n = 1 3.

Where n = 1, the error bars represent the error inherent in the analysis technique..........34


Figure 2.10: ln [CL]0/[CL]t versus time plot for the synthesis of PCLx/PEG45/PCLy

copolymers; [CL]0 : [OH]0 : [CaH2]0 = 100 : 1 : 3 (), and [CL]0 : [OH]0 : [CaH2]0 = 100

: 1 : 0.67 (x) in flame-sealed glass tubes at 70 C. Error bars: 1 S.D., n = 1 3.

Where n = 1, the error bars represent the error inherent in the analysis technique..........35

Figure 2.11: Conversion versus time plot for [CL]0 : [OH]0 : [CaH2]0 = 100 : 1 : 0.67, at

70 C (); 96 C (); and 128 C () .............................................................................36

Figure 2.12: GPC chromatograms of PCLx/PEG45/PCLy copolymers synthesised at 133

C. Upper: [CL]0 : [OH]0 : [CaH2]0 = 100 : 1 : 3; tmax x 3. Lower: [CL]0 : [OH]0 :

[CaH2]0 = 50 : 1 : 3; tmax x 4.............................................................................................37

Figure 2.13: FT-Raman spectra. Upper: CL. Lower: PCL50/PEG45/PCL50 ..................40

Figure 2.14: Comparison of kinetic curves for the synthesis of PCLx/PEG45/PCLy at 128

C, [CL]0 : [OH]0 : [CaH2]0 = 100 : 1 : 0.67, using FT-Raman monitoring (), and oil

bath heating / 1H NMR analysis () ...............................................................................41

Figure 2.15: Typical first-order kinetic plots, ln [CL]0/[CL]t versus time, for the

synthesis of PCLx/PEG45/PCLy using FT-Raman monitoring. [CL]0 : [OH]0 : [CaH2]0 =

50 : 1 : 0.67 () [CL]0 : [OH]0 : [CaH2]0 = 50 : 1 : 3 (). [CL]0 : [OH]0 : [CaH2]0 = 100

: 1 : 0.67 () [CL]0 : [OH]0 : [CaH2]0 = 100 : 1 : 3 () ...................................................44

Figure 2.16: Typical second-order kinetic plots, 1 / (1 ) versus time, for the synthesis

of PCLx/PEG45/PCLy using FT-Raman monitoring. [CL]0 : [OH]0 : [CaH2]0 = 50 : 1 :

0.67 () [CL]0 : [OH]0 : [CaH2]0 = 50 : 1 : 3 (). [CL]0 : [OH]0 : [CaH2]0 = 100 : 1 :

0.67 () [CL]0 : [OH]0 : [CaH2]0 = 100 : 1 : 3 ()...........................................................44

Figure 2.17: Normalised polymerisation rate versus [CaH2]0 : [OH]0 for [CL]0 : [OH]0 =

50 : 1 at 3 polymerisation temperatures. 70 C (); 106 C (); 133 C () ...................48

Figure 2.18: Arrhenius plots for the synthesis of PCLx/PEG45/PCLy. Upper: [CaH2]0 :

[OH]0 = 0.67 : 1. Lower: [CaH2]0 : [OH]0 = 3 : 1 ...........................................................51

Figure 2.19: ln kobs versus ln [OH]0 for [CaH2]0 : [OH]0 = 0.67 : 1 at 128 C................53


Figure 2.20: ln kobs versus ln [OH]0 for [CaH2]0 : [OH]0 = 3 : 1 at 128 C.....................55

Figure 2.21: Upper: 400MHz 1H NMR spectrum of pure, dry tetraethylene glycol in

benzene-d6. Integration a : b : c = 1.00 : 2.05 : 6.00. Lower: 400MHz 1H NMR

spectrum of tetraethylene glycol heated at 70 C with CaH2 for 24 hrs, benzene-d6

extract. Integration a : b : c = 1.00 : 1.96 : 5.86 ..............................................................58

Figure 2.22: Raman spectra from air, and the headspace of a reaction vessel: [CL]0 :

[OH]0 : [CaH2]0 = 100 : 1 : 3, 133 C; maximum conversion of CL ...............................59

Figure 2.23: Graph of H2 production versus time from the reaction of PEG45 and CaH2

in a vessel sealed with a Teflon key and heated to 130 C (spectra were collected after

cooling to room temperature)...........................................................................................60

Figure 2.24: GPC chromatograms plotted as a function of conversion for the synthesis

of PCLx/PEG45/PCLy, [CL]0 : [OH]0 : [CaH2]0 = 100 : 1 : 0.67 at different

polymerisation temperatures. Upper: 70 C. Lower: 96 C...........................................62

Figure 2.25: nM (1H NMR) versus for [CL]0 : [OH]0 : [CaH2]0 = 100 : 1 : 0.67. (x)

96 C; () 128 C; () theoretical prediction based on a living polymerisation model63

Figure 2.26: nM (1H NMR) versus , for [CL]0 : [OH]0 : [CaH2]0 = 250 : 1 : 0.67. ()

128 C; () theoretical prediction based on a living polymerisation model...................64

Figure 2.27: PDI versus , for [CL]0 : [OH]0 : [CaH2]0 = 100 : 1 : 0.67 at (x) 96 C and

() 128 C.......................................................................................................................64

Figure 2.28: PDI versus , for [CL]0 : [OH]0 : [CaH2]0 = 250 : 1 : 0.67 at 128 C.........65

Figure 3.1: Typical stress-strain plots for melt-pressed PCLx/PEG45/PCLy copolymer

films, and a melt-pressed PCL film..................................................................................84

Figure 3.2: DSC thermograms of melt-pressed PCLx/PEG45/PCLy copolymers, PCL and

PEG ..................................................................................................................................86


Figure 3.3: FTIR-ATR spectra of calcium-initiator residues isolated by filtration. Top

to bottom: 5 X, 5 S, 4 X, 4 S............................................................................................88

Figure 3.4: Relevant calcium-alkoxide, and CaH2 reactions ..........................................89

Figure 3.5: Ca2+ release from Ca(OH)2- and CaCO3-doped PCL samples immersed in

ultrapure water, over a 14 day period...............................................................................90

Figure 3.6: EDX spectrum from CaCO3-doped PCL (0.2 w / w % Ca) after 14 days

immersion in SBF, showing the presence of Cl, in the absence of CaP ..........................92

Figure 3.7: SEM image showing spherical, and plate-like CaP growth on a Ca(OH)2-

doped PCL sample, 1 w / w % Ca, after 14 days immersion in SBF...............................93

Figure 3.8: Advancing and receding water contact angles for PCL, Ca(OH)2- and

CaCO3-doped PCL...........................................................................................................95

Figure 3.9: FTIR-ATR for Ca(OH)2-doped PCL after 14 days immersion in SBF........98

Figure 3.10: Percentage mass change of PCLx/PEG45/PCLy copolymers as a function of

SBF immersion time ......................................................................................................100

Figure 3.11: Advancing and receding water contact angles for PCL and

PCLx/PEG45/PCLy copolymers ......................................................................................101

Figure 3.12: SEM images (left), EDX spectra (right), showing CaP coverage of non-Si

contaminated SBF-treated samples. Top to bottom: series 4 X; 3, 9, and 14 days. ...104

Figure 3.13: FTIR-ATR spectra from series 4X after different SBF immersion times



LIST OF TABLES Table 2.1: Typical feed ratios and quantities of reagents used for copolymer syntheses20

Table 2.2: Separation range of GPC columns .................................................................23

Table 2.3: 1H NMR (CDCl3) peak assignments for CL and PCLx/PEG45/PCLy.............30

Table 2.4: MWD data for PCLx/PEG45/PCLy copolymers synthesised at 133 C ..........38

Table 2.5: Summary of pseudo-first-order rate constants, kobs, for all reaction conditions

used throughout this study................................................................................................45

Table 2.6: Temperature study of the homo-polymerisation of CL with CaH2 ................47

Table 3.1: Reagent Purities.............................................................................................75

Table 3.2: Composition of Ca(OH)2-doped samples.......................................................76

Table 3.3: Composition of CaCO3-doped samples..........................................................76

Table 3.4: SBF Reagents .................................................................................................78

Table 3.5: PCLx/PEG45/PCLy copolymers selected for bioactivity testing .....................82

Table 3.6: Tensile properties of PCL, and PCLx/PEG45/PCLy copolymer melt-pressed


Table 3.7: Concentration of calcium in PCLx/PEG45/PCLy copolymers synthesised with

the CaH2 / PEG co-initiator..............................................................................................88

Table 3.8: Atomic calcium to phosphorus, Ca / P, and magnesium + calcium to

phosphorus, (Mg + Ca) / P, ratios for Ca(OH)2-doped samples ......................................93

Table 3.9: Atomic calcium to phosphorus, Ca / P, and magnesium + calcium to

phosphorus, (Mg + Ca) / P, ratios for CaCO3-doped samples .........................................94


Table 3.10: Ca / P ratios of CaP minerals129 ...................................................................96

Table 3.11: CaP phase determined from FTIR-ATR spectra of SBF-treated, Ca(OH)2-

doped PCL........................................................................................................................98

Table 3.12: Characteristic infrared frequencies (cm-1) for CaP minerals128,130 ...............99

Table 3.13: Atomic calcium to phosphorus ratio, Ca / P...............................................103

Table 3.14: Atomic magnesium + calcium to phosphorus ratio, (Mg + Ca) / P ...........103

Table 3.15: CaP phase determined from FTIR-ATR analysis of SBF-treated

PCLx/PEG45/PCLy copolymers ......................................................................................106



AR Analytical Reagent

CaP Calcium Phosphate

CL -Caprolactone

DCM Dichloromethane

nDP Number Average Degree of Polymerisation

DSC Differential Scanning Calorimetry

EDX Energy Dispersive X-Ray

FTIR-ATR Fourier Transform Infrared-Attenuated Total Reflectance

GPC Gel Permeation Chromatography

HAP Hydroxyapatite

ICP-AES Inductively Coupled Plasma-Atomic Emission Spectroscopy

nM Number Average Molecular Weight

wM Weight Average Molecular Weight

MWD Molecular Weight Distribution

NMR Nuclear Magnetic Resonance

OCP Octacalcium Phosphate

PCL Polycaprolactone

PDI Polydispersity Index

PEG Poly(ethylene glycol)

PGA Polyglycolide

PLA Polylactide

SBF Simulated Body Fluid

SEM Scanning Electron Microscope

TMS Tetramethyl Silane

UV Ultraviolet

Chapter 1 General Introduction



Polycaprolactone, PCL, is a hydrophobic, slow-degrading synthetic polymer that is

widely studied because of its potential in a wide range of biomedical applications.

Among the most commonly reported are controlled drug delivery systems, and implants

for orthopaedic surgery.1 The PCL-based materials being fabricated in this study are

being targeted at applications concerning bone tissue repair. From a public health

perspective, the importance of bone tissue repair is statistically significant. At the time

of the national health survey of Australia in 1995, there were approximately 100,000

people with fractures and a further 1.8 million people with conditions that could be

attributed to bone injuries.2

Bone is a complex, composite material, composed of 65 % inorganic hydroxyapatites

and 35 % organic matrix (mostly collagen).3 The nature of this inorganic / organic

combination in bone results in strong, load-bearing biological materials. The difficulties

in manufacturing a similarly structured material with the same properties are enormous;

hence, traditionally, metal pins and plates have been used for bone repair and

replacement due to their inherent beneficial properties such as high strength. However,

there are intrinsic problems associated with their use, such as the need for revision

surgery,4 refracture upon removal of the implant,4 wear,5 high thermal and electrical

conductivity, poor tissue bonding, or tissue rejection,6 and issues with permanency in

juvenile patients. Acrylic polymers, especially poly(methyl methacrylate), have been

used as alternative materials to metal implants since World War II.6 They show

enhanced tissue compatibility, and have poor thermal and electrical conductivity.6

However, they too, are permanent implants and hence do not allow for significant

growth or change around implant sites.

In recent years a much greater emphasis has been placed on developing biodegradable

polymers for use as biomedical implants because they offer the possibility of tissue

ingrowth during resorption of the polymer, leaving a repaired wound site exclusive of

Chapter 1 General Introduction


foreign material. Much of this recent interest has been due to the advent of tissue

engineering, a concept that combines the principles of biology and engineering to the

development of functional substitutes for damaged tissue.7 One of the main aims of

tissue engineering is to grow functional tissue substitutes, including complete organs.

To do this, specific substrates, or scaffolds need to be designed such that cell attachment

and maintenance of cell function can be supported.8 For such substrates and scaffolds,

synthetic biodegradable polymers hold several advantages over other materials.

Tailorable mechanical properties and degradation kinetics are two key advantages of

synthetic biodegradable polymers for tissue engineering applications.9 Other advantages

include: ease of fabrication into scaffolds of various shapes and sizes, ability to design

desirable pore morphology, and the ability to functionalise such polymers to induce

tissue ingrowth.9 There are also some limitations to the use of polymers in tissue

engineering. For instance, in load-bearing applications metal materials are still preferred

as many polymers have a low modulus and cannot offer the load-bearing support that

metal materials can. Therefore, low modulus polymers are used in non-load-bearing

applications, or as a component in a metallic device in load-bearing applications, for

example, in a total hip replacement device.

There are a number of synthetic biodegradable polymers presently being used for

various biomedical applications. Such polymers include: poly(-hydroxy acids),10

polyalkanoates,11 polyurethanes,1,12 polyorthoesters,13 polycarbonates,14 or copolymers

of these.15,16 The most widely researched of these in the biomedical field are poly(-

hydroxy acids).17 They were originally employed as resorbable sutures in the 1960s,10

and their application in the biomedical arena has grown to include drug delivery

vehicles,18,19 tissue regeneration scaffolds20,21 and other fixation devices.22 For

biomedical applications there are three main poly(-hydroxy acids): polylactide (PLA),

polyglycolide (PGA) and polycaprolactone (PCL). PLA, PGA and PLA / PGA

copolymers constitute the majority of poly(-hydroxy acids) in use.22 However, there

has been increased interest in the use of PCL in recent times.

Chapter 1 General Introduction


PCLs low degradation rate (up to two years in vivo1) allows for slow tissue ingrowth in

sites, such as bone, where long-term remediation (4 8 weeks22) may be required. With

respect to bone tissue repair, PCL has been used as an injectable bone substitute,23 a

scaffold for bone-tissue engineering,24 and as an in-situ-polymerised material for

maxillofacial applications.25 Although PCL is known to elicit only a rather mild

inflammatory response,26 cell attachment and growth on PCL is limited due to its

intrinsic hydrophobicity.24 Copolymerisation of PCL with hydrophilic blocks such as

poly(ethylene glycol), PEG, has been performed as a means to enhance the

hydrophilicity of the parent, PCL homopolymer.27-30 This approach has led to enhanced

cell attachment, and accelerated hydrolytic degradation. At certain PCL : PEG

compositional ratios, PCL/PEG/PCL triblocks constitute an excellent support for human

endothelial cell adhesion and growth.28 In this study, two PCL/PEG/PCL copolymers

were compared, and the material with a higher PEG content was found to be a better

support for endothelial cell growth. PCL-PEG copolymers have also been shown to

support both human, and rat marrow stromal cell proliferation.31 It was found that the

PCL-PEG copolymers were better at supporting cell proliferation than the PCL

homopolymer itself. This was attributed to the greater hydrophilicity, and lower water

contact angle, of the PEG-containing copolymers. Since, PCL-PEG copolymers are

more hydrophilic than PCL homopolymer, it is expected that they should show a faster

rate of hydrolytic degradation, and this has been shown. Compared to PCL

homopolymer, the degradation of PCL-PEG copolymers has been shown to be

significantly faster, although still rather slow (60 weeks in PBS at 37C for a weight loss

of 7%).31 The major degradation products from the hydrolytic degradation of PCL-PEG

copolymers, have been found to be 6-hydroxyhexanoic acid (a naturally occurring

metabolite1) and a large amount of PEG-rich species,31 (PEG of relatively low molecular

weight is readily excreted through the kidneys).32 Since it is, in practice, not possible to

completely remove catalyst / initiator residues from synthetic polymers,33 the release of

such residues also inevitably occurs during degradation. Therefore, it is of great interest

and importance to synthesise polymers using catalysts / initiators that leave biologically

compatible residues.

Chapter 1 General Introduction


A number of catalysts have been employed for the synthesis of PCL-PEG copolymers.

They range from the most commonly employed catalysts for the synthesis of poly(-

hydroxy acids) such as tin(II)2-ethylhexanoate,1,34,35 to biologically compatible initiators

such as calcium36 and zinc.37 Even though tin-based initiators are considered to be some

of the best initiators for the synthesis of poly(-hydroxy acid)s, concerns have been

raised over the potential toxicity of their residues.38,39 The toxicity of various organotin

compounds is well known,40 with the high toxicity of compounds such as tributyl tin

leading to restrictions on their use in anti-fouling paints.41 Tin(II)2-ethylhexanoate is

considered less toxic than organotin compounds and has been approved for use as an

anti-microbial agent by the FDA (United States food and drug administration) for use in

food stuffs.42 However, strict control over the exposure of people to this compound is

required and even though it has been approved for use in minimal quantities in food;

from an industrial perspective, there is a growing awareness about the dangers of using

compounds such as tin(II)2-ethylhexanoate in large scale production.42 Also, where the

materials are employed for biomedical applications it is particularly important to

maintain safe levels of toxic compounds, or more appropriately, exclude them

completely. As a result, there has been a move towards the development of potentially

more biocompatible initiators than those traditionally used for the synthesis of poly(-

hydroxy acid)s. This has resulted in the study of initiators for the synthesis of poly(-

hydroxy acids), based on metals (calcium, magnesium, iron and zinc) that participate in

human metabolism.33 The emergence of new initiation systems has in turn led to the

need to study the mechanism of such polymerisations in depth such that the

polymerisation processes can be exploited to give controlled, reproducible materials.

In this study, a previously described calcium hydride / PEG co-initiating system used by

Li et al.43 and Rashkov et al.44 for the synthesis of PLA/PEG/PLA block copolymers has

been applied to the synthesis of PCL/PEG/PCL block copolymers. The effect of varying

synthetic parameters such as temperature and reactant concentration has been studied in

more detail, as a means to elucidate the mechanism of polymerisation. Chapter 2 will

focus on the synthesis of PCL/PEG/PCL copolymers prepared with a PEG / calcium

hydride co-initiator. The mechanism of polymerisation will be discussed in terms of the

Chapter 1 General Introduction


kinetics of polymerisation, formation of by-products, and resultant PCL/PEG/PCL

copolymer structure.

With the intention of using the synthesised PCL/PEG/PCL copolymers for bone tissue

repair, the effectiveness of these materials at nucleating hydroxyapatite, or other

calcium-phosphate (CaP) mineral phases in vitro has also been studied. By

incorporating a potentially active, biologically compatible, CaP-mineral nucleating agent

(calcium from the calcium-initiator) through a one-pot synthetic process this study not

only removes the problem of residual catalyst / initiator toxicity, it has also turned the

persistence of initiator residues into an advantage. Calcium ions, Ca2+, released from the

polymer matrix (due to the presence of calcium-initiator residues) in media, such as

simulated body fluid (SBF), can cause the nucleation of a CaP mineral phase,45 with the

formation of bone-like apatite (CaP mineral) on artificial materials being an established

condition for bonding to living bone.24 The enhanced water permeability of

PCL/PEG/PCL copolymers in aqueous media, compared to PCL homopolymer,27 should

aid in the diffusion of Ca2+ from the polymer matrix. Ultimately, it is hoped that the rate

of diffusion of Ca2+ from these materials will be sufficient to cause nucleation of a

uniform, bone-bonding or bone-growth-promoting, CaP mineral coating in vivo. A

preliminary investigation into the effect of calcium-initiator residues on the in vitro

(SBF) mineralisation of PCLx/PEG45/PCLy copolymers, synthesised in this study, and

commercially available PCL homopolymer (doped with calcium hydroxide, or calcium

carbonate) is presented in Chapter 3. The use of calcium-doped PCL homopolymer

served as a proof-of-concept for the action of calcium-initiator residues on the

mineralisation of the synthesised copolymers. Diffusion of Ca2+, from the calcium-

doped PCL, in an aqueous environment was also studied as a means to understand the

mechanism of CaP mineral formation on these materials.

Chapter 2 Introduction



2.1 Introduction

Polymer syntheses can be classified through two general phenomena: step-growth

(condensation) and chain-growth (addition) polymerisation. Chain-growth

polymerisation involves the sequential addition of monomers to form a polymeric

species.46 Such examples include the polymerisation of styrene, by both free radical and

anionic mechanisms, and the ring-opening of cyclic monomers, such as trioxane to give

polyoxymethlyene.46 Under certain conditions, this can lead to a living polymerisation,

which will be discussed later. In contrast to chain-growth polymerisation, the

mechanism of step-growth polymerisation involves the random reaction of two, di-

functional molecules that may be any combination of monomers, oligomers or higher

molecular weight intermediates.46 The consequence of this is that polymers of high

molecular weight are only obtainable at high conversion, due to the greater probability

of the reactive ends of larger molecules meeting at this stage than when a large amount

of monomer is present. Some common examples of condensation polymers are: nylon-

6,6, polycarbonate and poly(ethylene terephthalate). Polyesters, which are of particular

interest for this study, can be prepared by either a step-growth or chain-growth


2.1.1 Polyester Synthesis

Step-growth polymerisation to prepare polyesters typically involves reaction between

difunctional monomers of the type X-R-X / Y-R-Y or X-R-Y, where X and Y represent

different functionalities that react to form an ester linkage. These functionalities ( X / Y,

or Y / X) are usually acid chlorides / alcohols or carboxylic acids / alcohols. Therefore,

the by-products of these reactions are respectively hydrochloric acid, or water. Being an

Chapter 2 Introduction


equilibrium reaction, these by-products often need to be removed to achieve high

conversion, hence high molecular weight.47 In contrast to condensation polymerisation,

ring-opening polymerisation (a chain-growth mechanism) can produce polymers of

relatively high molecular weight, without significant by-products, and may yield well-

defined structures if a judicious choice of initiator or catalyst is made.10 Polyester

synthesis by ring-opening polymerisation involves polymerisation of cyclic monomers

(lactones) through an ionic or coordination-insertion mechanism. Ionic polymerisation

may proceed by either a cationic or anionic ring-opening mechanism; however,

polymers of high molecular weight have only been obtained by anionic ring-opening

polymerisation.10 The sensitivity of ionic species to contaminants is the major drawback

of this technique. It is necessary to provide an oxygen-, water- and carbon dioxide-free

environment;48 ensuring that these contaminants are kept below parts-per-million levels.

Similarly to ionic polymerisation, coordination-insertion polymerisation catalysts can

also be poisoned by the introduction of contaminants such as water,10 but are generally

considered more robust than their ionic counterparts. If strict experimental conditions

are observed, then many anionic and coordination-insertion polymerisation catalysts

show the characteristics of a living polymerisation.

2.1.2 Living Polymerisation

The area of living polymerisation has expanded in recent times to meet the demand for

elegant polymers with tailored architectures that supply function through their structure.

Such examples, with respect to biodegradable polymers, include the synthesis of well-

defined copolymers and polymers with controlled architectures for drug delivery


In the ideal living polymerisation, there are only two processes that occur: initiation and

propagation.49 When these are the only two processes that occur, the ideal

characteristics of a living polymerisation can be summarised as below.50,51

Chapter 2 Introduction


1. The polymerisation proceeds until all the monomer has been consumed, with further addition of monomer resulting in continued polymerisation

2. Number average molecular weight ( nM ) is a linear function of conversion

3. The number of polymer molecules (and active centres) is constant, which is independent of conversion

4. Molecular weight can be controlled by the stoichiometry of the reaction 5. Polymers with a narrow molecular weight distribution are produced 6. Block copolymers can be prepared by sequential monomer addition 7. Chain-end functionalised polymers can be prepared in quantitative yields 8. The kinetics of propagation should follow pseudo-first-order behaviour

Figure 2.1 illustrates one of the advantages of living polymerisation. It depicts typical

nM versus conversion plots for polycondensation and living polymerisation, showing

linearity for living polymerisation, and the dependence of high conversion for obtaining

high molecular weight in the case of polycondensation.

0 0.2 0.4 0.6 0.8 1



Figure 2.1: Typical nM versus conversion plots for the synthesis of a polymer

under living conditions (----) compared to ideal polycondensation for an X-R-Y-

type system ( ). Adapted from Odian, p.852

Chapter 2 Introduction


In practice, not all polymerisation systems show all the living characteristics listed

above. In the case where no irreversible chain-breaking reactions occur they may be

more appropriately described as pseudo-, or quasi-living.49 Deviation from ideal living

characteristics can be caused by a number of phenomena. Such phenomena include,

reversible chain-breaking reactions (chain transfer and termination) and reversible

aggregation.49 Most commonly, for the case of living anionic polymerisation, ionic

aggregates will form, leading to an equilibrium between propagating, polymeric ions and

less reactive, non-propagating aggregates.49 The rate of polymerisation can then be

expressed, including the equilibrium constant (K) for such processes.49

[ ][ ] [ ][ ]( )KMN

kMPkR pppp +==


Equation 2.1

Where kp, [P*], [M] and [Np] are the rate constant of polymerisation, the concentration

of propagating species, the monomer concentration and the total concentration of

polymer molecules in the polymerisation system, respectively. As K approaches zero,

the system can be seen to become more living in nature.53,54 Depending on the nature of

the quasi-living system; ie, aggregative, terminative, or transferative,49 a number of

conditions can influence the magnitude of K. These may include monomer and solvent

type as well as the initiator used for the polymerisation.

2.1.3 Synthesis of Poly(-hydroxy acid)s

Poly(-hydroxy acid)s are generally synthesised by ring-opening polymerisation from

the respective lactone. In most cases, control of the polymerisation is obtained by

choosing the most appropriate initiator, or catalyst.

Chapter 2 Introduction














O *





O *




polycaprolactone polylactide polyglycolide

caprolactone lactide glycolide

Figure 2.2: Some poly(-hydroxy acid)s and their respective lactone monomers Initiators / Catalysts for the Synthesis of Poly(-hydroxy acid)s

Initiator selection plays an important role in the control of polymer syntheses. This, in

turn, can affect both physical and chemical polymer properties, including: crystallinity,

melt and glass transition temperatures, molecular weight, molecular weight distribution,

end groups, sequence distribution, and the presence of residual monomer.55 Of

particular interest for the syntheses of poly(-hydroxy acid)s are initiators / catalysts that

show an anionic, or coordination-insertion ring-opening mechanism, due to their ability

to produce polymers of high molecular weight.10 For the ring-opening polymerisation of

(di)lactones, such as lactide, glycolide and caprolactone; tin- and aluminium-based

initiators are the most widely used.56 In the past, aluminium systems were preferred

where a high degree of control was desired56 because tin-based initiators are good

transesterification catalysts compared to aluminium-based initiators.56,57 However, the

greater hydrolytic stability,56 low cost,58 and the development of tin-based initiators to

provide greater control over macromolecular architecture56 has resulted in their more

widespread use as initiators for the synthesis of poly(-hydroxy acid)s.

Chapter 2 Introduction


Tin(II)2-ethylhexanoate, or stannous octoate (Sn(Oct)2) as it is also known, is the most

commonly employed tin-based initiator for the synthesis of poly(-hydroxy acid)s.58

Ring-opening polymerisation using tin(II)2-ethylhexanoate requires an active hydrogen

compound as co-initiator,10 usually an alcohol. The mechanism of poly(-hydroxy acid)

synthesis using tin(II)2-ethylhexanoate has been extensively studied. The formation of a

tin(II) alkoxide active centre, formed by the reaction of tin(II)2-ethylhexanoate with

added alcohol,58,59 followed by a coordination-insertion, ring-opening polymerisation

mechanism (Figure 2.3) is considered the most likely mechanism of polymerisation.10

The consequence of such a mechanism is that alkoxy end groups from the polymer may

be covalently bound to tin post-synthesis, even after purification, if complete hydrolysis

of the tin-alkoxy bond does not occur. Purification by dissolution and precipitation has

been shown to be inadequate for the complete removal of tin residues in polymers

synthesised using tin(II)2-ethylhexanoate. PLA synthesised by Schwach et al.60 using

tin(II)2-ethylhexanoate as the initiator was shown to contain 306 ppm tin after











Sn(Oct)2 + R1-OH OctSnOR1 + OctH

Tin alkoxide formation:


Figure 2.3: Tin(II)2-ethylhexanoate / alcohol co-initiated polymerisation of

lactones, adapted from Albertsson and Varma.10 NB: R 4 C

Chapter 2 Introduction


Because it is impractical to completely remove all traces of initiator from polymeric

products,33,39 a number of groups have been investigating more biocompatible initiators,

based on metals that participate in human metabolism. Such studies have involved the

use of initiators based on: calcium,33,38,61,62 iron,39 magnesium33 and zinc.42,63 Zinc-

based initiators are highly studied, and include zinc metal, which has been used

industrially in France for the polymerisation of D,L-lactides.60 Zinc salts such as zinc

octoate,64 zinc stearate,42 and zinc lactate42,60 have also been used as initiators for the

ring-opening polymerisation of lactones. Ring-opening polymerisation of D,L-lactide by

zinc metal is known to produce polymers with high molecular weight, and high

conversion ratios, after long polymerisation times (approximately 4 days) at

temperatures about 140 C.63 Zinc / PEG and CaH2 / PEG co-initiation for the ring-

opening polymerisation of L-lactide were compared and both showed good control over

the degree of polymerisation.43 However, some racemisation was noted for the CaH2 /

PEG co-initiator and not for the zinc / PEG co-initiator, leading to a preference for the

zinc-based initiator.

Other calcium-based initiating systems that have been studied for the synthesis of

poly(-hydroxy acid)s include, calcium dimethoxide,65 bis(tetrahydrofuran)calcium

bis[bis(trimethylsilyl)amide] / alcohol co-initiatior,33,38 calcium -diketonate

complexes,62 and calcium acetylacetonate.66 Copolymerisation of glycolide with -

caprolactone and L-lactide using calcium acetylacetonate afforded high conversion, only

after several days at temperatures of 150 and 200 C. Calcium dimethoxide-initiated

polymerisation of -caprolactone and L-lactide in the bulk at 120 C showed quantitative

consumption of monomer after 5 10 min for the case of -caprolactone, but longer

reaction times were required for L-lactide polymerisation using the same initiator. In all

cases polydispersity was greater than 1.25, with initiator efficiency in the range 0.41

0.54, possibly due to aggregation and insolubility of the calcium dimethoxide initiator.

Greater control in the ring-opening polymerisation of -caprolactone and L-lactide has

been obtained with a bis(tetrahydrofuran)calcium bis[bis(trimethylsilyl)amide] / alcohol

co-initiatior. The in situ generated calcium alkoxide formed from the reaction of the co-

initiators during polymerisation helped circumvent the problems of aggregation and

Chapter 2 Introduction


insolubility of pre-made calcium alkoxides. This, in turn, allowed for polymerisation

under mild conditions that proceeded in a living manner. In the synthesis of poly(-

hydroxy acid)s, quasi-living systems are often observed due to the co-existence of

processes such as transesterification. Transesterification Reactions

For the case of the ring-opening polymerisation of poly(-hydroxy acid)s, the ionic

nature of the initiator / propagating species is an important factor for determining the

rate of intra- and inter-molecular transesterification.






































Intramolecular transesterification:

Intermolecular transesterification:



Figure 2.4: Intra- and inter-molecular transesterification reactions of a polyester

As the propagating species moves from anionic towards covalent in nature, the

formation of macrocycles due to intra-molecular transesterification is less prevalent at

early polymerisation times.67 This has been shown experimentally for the synthesis of

PCL using sodium trimethylsilanolate, an anionic initiator, compared to

Chapter 2 Introduction


aluminiumdiethylmethoxide, a pseudoanionic / coordination-insertion-type initiator.68 It

is also important to note that steric hindrance can play a role in the transesterification

process. Where respective initiators have the same propensity to polymerise a particular

monomer, ie, where the kp (actual rate constant of polymerisation) of respective

initiators is the same, greater steric hindrance around the active centre has been shown to

decrease the rate of transesterification.67 One example is the synthesis of PCL using

aluminiumdialkylalkoxide initiators.69 In this case, the selectivity co-efficients for

diisobutyl and diethyl aluminiumdialkylalkoxides differed by a factor of 1.67, with the

more sterically hindered isobutyl initiator showing greater selectivity in producing linear

polymer, rather than cyclic, intra-molecular transesterification products. As well as

transesterification side-reactions, polymerisation systems that utilise anionic and

pseudoanionic / coordination-insertion-type initiators commonly show aggregation

phenomena. Aggregation Phenomena

Evidence for aggregation may be observed through kinetics; specifically, through the

external order in initiator.67 An external order in initiator of less than one is indicative of

aggregation phenomena. Penczek et al.67 have derived a method for determining the

aggregation degree in systems where aggregation dominates. This can be performed by

plotting the left-hand-side of Equation 2.2 against ln[I]0, to obtain a slope of x1 , where x

is the aggregation degree.

001 ]ln[1ln


lnln Ix




Equation 2.2

A = kp(nKa)-1/x, Ka is the equilibrium constant of aggregation, [M]0 and [M]t are

respectively the initial and instantaneous monomer concentrations, [I]0 is the initial

concentration of initiator and t is the polymerisation time. For the complete derivation

of Equation 2.2 see Section

Chapter 2 Introduction


Potential solutions for the problem of aggregation have been investigated using a variety

of approaches. Such methods include the use of polymerisation solvents of varying

polarity and the introduction of specific chelating agents, such as crown ethers and

cryptates to enhance the solubility of ionic species. Aggregation of ionic and

pseudoanionic initiators has been shown to be solvent dependent. For the

polymerisation of -caprolactone, CL, initiated with diethylaluminium ethoxide,70 the

determined equilibrium constant of deaggregation was found to increase with increasing

polarity of the polymerisation solvent. That is, the free initiator / propagating species

was favoured over the aggregated form for increasingly polar solvents. Also, generally

for the case of anionic polymerisation of styrene and dienes, aggregation is negligible in

polar solvents such as tetrahydrofuran and dimethoxyethane for initiator concentrations

of less than 5 x 10-3 M.71 Specific chelating agents, such as crown ethers and cryptates

have been used to both reduce aggregation and alter ionic character. Specific binding of

metal ions to these chelating agents results in the destruction of aggregates.72

Furthermore, the trapping of metal ions by such agents allows for higher dissociation

into free ions.72 It was generally thought that a higher dissociation constant would

increase the rate of polymerisation; however, this is not true for all cases. Instead, the

process is more complex and can be better explained through three main factors: the

interaction of the monomer with the counter-ion, the charge localisation on the anion,

and the polarisability of the monomer.73 These factors, as well as the other previously

mentioned phenomena (transesterification, aggregation), that help to account for the

mechanism of polymerisation manifest themselves in the kinetics of polymerisation.

Hence, kinetic studies are an essential component of any in-depth investigation of

polymerisation mechanisms.

2.1.4 Methods for Studying the Kinetics of Poly(-hydroxy acid) Synthesis

The ring-opening polymerisation of poly(-hydroxy acid)s from lactones has been

studied by a number of techniques, including: nuclear magnetic resonance spectroscopy

Chapter 2 Introduction


(NMR), gel permeation chromatography (GPC), dilatometry, and infrared spectroscopy

(IR). Due to the atmospheric sensitivity of many of the initiators / catalysts employed

for the synthesis of poly(-hydroxy acid)s, and the subsequent difficulty in handling,

monomer conversion is usually evaluated by analysing crude reaction mixtures after

quenching by an appropriate method. Even so, manual sampling techniques are often

laborious and time-consuming leading to a preference for minimising data collection.

So, for further, more complete determination of polymerisation kinetics, a number of

real-time monitoring techniques have also been applied for the synthesis of poly(-

hydroxy acid)s. Nuclear Magnetic Resonance Spectroscopy (NMR)

The ring-strain inherent to lactones gives rise to differing chemical shifts between

monomer and polymer signals in both 1H and 13C NMR. Complete separation of some

monomer signals and the respective signal from the polymer makes kinetic analysis

easier and reduces error associated with overlapping peaks. Along with this, 1H NMR

spectra are directly quantitative,74 allowing determination of the degree of conversion by

a ratio of polymer / total monomer and polymer peak areas. 1H NMR is commonly used

for determination of lactone conversion by manual sampling, but has also been used for

the in situ analysis of the enzyme-catalysed polymerisation of CL.75 In this case, poor

mixing led to the requirement for the sample to be removed from the spectrometer every

7 9 min and shaken. This made the process more arduous and highlights the problem

of poor-mixing for in situ NMR analysis. This problem of poor mixing is especially

prevalent for heterogeneous systems. Gel Permeation Chromatography (GPC)

GPC (with refractive-index and UV detectors) has been used to determine the

conversion of CL with a variety of tin(II)2-ethylhexanoate / ROH co-initiating

systems.58 Monomer conversion was established by peak height comparison to a

standard UV absorption curve of known CL concentrations and then normalising to

Chapter 2 Introduction


injection volumes. As with other manual sampling techniques, the removal of aliquots

and subsequent sample preparation are time-consuming. GPC has the additional

hindrance of chromatographic separation, which often takes tens of minutes. Even so,

one of the advantages of using GPC in such a way is that the molecular weight

distribution of the growing polymer species may also be studied at the same time, giving

some insight into the mechanism of the polymerisation. Dilatometry

Vacuum dilatometers have been used by Penczek et al.76 in their extensive studies of the

polymerisation of lactones. The use of dilatometry has the advantage of allowing real-

time monitoring of conversion. However, special dilatometers are required in order that

kinetics may be studied in a sealed, inert environment. One other major drawback of

dilatometry is that bulk polymerisation cannot be studied by this method due to the very

high increase in viscosity during polymerisation. In contrast, vibrational spectroscopic

methods lend themselves to the study of polymerisation in the bulk. Therefore, they

have more wide-spread applications ranging from laboratory research to industrial-scale

synthesis. Infrared Spectrosopy (IR)

In situ FT-IR has been applied to the real-time monitoring of the synthesis of PLA by

two groups. Both Hillmyer et al.77 and Messman and Storey78 followed the reaction

progress by measuring the reduction in the absorbance of the 1240cm-1 peak (C-O-C

stretch) of D,L-lactide or L-lactide. However, the overlapping polymer C-O-C stretch at

1185 cm-1 limited the use of peak-area quantitation. Therefore, Messman and Storey78

used peak-heights to overcome this problem. The overlapping peaks due to the

inherently broad width of infrared absorption peaks plus the expense of the diamond

ATR probe used in this case seem to be the major disadvantages of this method.

Overall, the data obtained using this method were comparable to other techniques.

Chapter 2 Introduction


However, the complimentary vibrational spectroscopy technique, Raman spectroscopy,

may be better suited to the study of the polymerisation of poly(-hydroxy acid)s. Raman Spectroscopy

Raman spectroscopy offers a number of advantages over IR for the study of the

synthesis of poly(-hydroxy acid)s. Raman spectroscopy permits the use of glass, or

quartz optics as opposed to the infrared-transparent optical materials required by IR.74

This allows for analysis of polymerisation mixtures in sealed-glass vessels (commonly

employed for poly(-hydroxy acid) synthesis), reducing the need for expensive probes

(as used for in situ IR) or other expensive infrared-transparent vessels. Secondly, there

is no rotational broadening of Raman peaks. Raman bands are generally very sharp and

narrow,74 minimising problems associated with overlapping peaks. There are, however,

a few disadvantages associated with using Raman spectroscopy. Raman scattering is a

weak effect, which has the consequence that spectra can be dominated by more efficient

phenomena such as sample fluorescence.46 FT-Raman is one well-recognised method

for overcoming this effect. Fourier transformation provides improved signal to noise

compared to conventional Raman spectroscopic techniques, therefore allowing the use

of longer wavelength light sources. This helps to reduce fluorescence by decreasing the

energy of the incident radiation.46 Methods-of-Choice For This Study

Polymerisation kinetics were monitored by manual sampling using 1H NMR and real-

time monitoring using FT-Raman spectroscopy. 1H was also used to determine

molecular structure and give insight into the polymerisation mechanism. In addition,

molecular weight distributions as a function of conversion were monitored by GPC to

further elucidate the mechanism of polymerisation.

Chapter 2 Experimental


2.2 Experimental

2.2.1 Materials

Argon (high purity) for the dry box was purchased from BOC gases, Australia. The

argon was dried through a commercially available drierite laboratory gas drying unit,

capable of drying gases to 0.005 mg L-1 H2O, purchased from W.A. Hammond Drierite

Co. Ltd, USA. Oxygen was subsequently removed through an OXY-TRAP oxygen

removal column purchased from Alltech Associates inc., USA. Calcium hydride (CaH2)

(99.99%) (Sigma Aldrich, Australia) was stored in a dry box under an argon atmosphere.

-caprolactone, CL, (Sigma Aldrich, Australia) was dried and purified before use by

storage over CaH2 for at least 48 hours, followed by distillation under vacuum at ~ 2 torr

and 90C. It was then sealed under argon and stored in a dry box until required. PEG45,

nM = 2 000, (Sigma Aldrich, Australia) was dried and purified by dissolution /

precipitation in chloroform / diethyl ether, followed by azeotropic distillation with

dichloromethane (DCM). After removal of the DCM, it was dried under vacuum at 100

C for three hours to remove all traces of solvent and water. It was then stored under

argon in a dry box until required. All other materials were of analytical reagent, AR,

grade or better.

2.2.2 Methods Syntheses of PCLx/PEG45/PCLy Copolymers

The syntheses of all polymers were performed in vacuum-sealed, glass tubes equipped

with two 6 x 3 mm Teflon-coated magnetic stirrer bars. All reagents were added to the

tubes in a dry box under argon atmosphere employing standard dry box techniques. All

glassware and other utensils (spatulas, mortar and pestle) were dried overnight in an

oven at 100 C before use. The syntheses were performed in the bulk at three different

temperatures, (oil bath thermocouple reading: 70, 100 and 130 C; actual temperature in

Chapter 2 Experimental


tube: 70, 96 and 128 C respectively), with a summary of the [CL]0 : [PEG]0 feed ratios

and quantities commonly used in Table 2.1. CaH2 was powdered by crushing, using a

mortar and pestle for a period of five minutes before addition to the tube. PEG was then

added, followed by CL and the tube stoppered with a Teflon key, then sealed with

laboratory film. The tubes were then removed from the dry box and taken to a high

vacuum line.

Table 2.1: Typical feed ratios and quantities of reagents used for copolymer


Desired Copolymer [CL]0 : [OH]0 CL (mL) PEG2000 (mg) CaH2 (mg)*

PCL50/PEG45/PCL50 50:1 0.78 138 4 / 18

PCL100/PEG45/PCL100 100:1 1.56 138 4 / 18

PCL200/PEG45/PCL200 200:1 3.06 138 4 / 18

PCL250/PEG45/PCL250 250:1 3.82 138 4 / 18

* 1.33 / 6 times the amount of CaH2 required to completely react with the number of

hydroxyl groups present in the PEG45, i.e., [CaH2]0 : [OH]0 = 0.67 : 1 and 3 : 1,


After degassing, tubes were flame-sealed under vacuum (0.2 0.3 torr), then completely

immersed in a thermo-stated oil bath (stirrer setting: approximately 700 rpm). Tubes

were removed periodically from the oil bath and frozen at -20 C to give cream coloured

products. Once opened, the reaction mixtures were dissolved in chloroform and

transferred to round-bottomed flasks. The chloroform was evaporated to give crude

products. Samples of the crude products were then taken for analysis by 1H NMR and

GPC. The crude samples were purified by dissolution using chloroform followed by

Chapter 2 Experimental


precipitation in either cold ether or cold ethanol, depending on the molecular weight.

Molecular weights of crude products were estimated by 1H NMR. If the number average

degree of polymerisation, nDP , of PCLx/PEG45/PCLy, (x + y), was found to be less than

100 in total per PEG45, then the copolymer was precipitated in ether, above this nDP ,

copolymers were precipitated in ethanol. After precipitation, the white solid was filtered

and washed, then dried under vacuum to constant mass. 1H NMR and GPC analyses

were performed on the pure copolymers. Real-Time Monitoring

FT-Raman spectroscopy was used for real-time monitoring of polymerisation kinetics.

Samples were prepared in 50 mm high, 12 mm diameter, flat-bottomed, flame-sealed

glass tubes. The preparation of samples was similar to the method described in section A thermo-stated aluminium heating block equipped with a magnetic stirring

device (Figure 2.5), mounted on a moveable stage inside the spectrometer, was

preheated (heating block thermocouple reading: 100, 126 or 130 C; actual temperature

in tube: 106, 128 or 133 C respectively) prior to sample introduction. Spectra were

recorded as soon as the sample was introduced into the preheated heating block. A

macro created using Macro Mania version 5.0 was used to collect spectra every 10 or 15

min over a set period. Work-up of samples was as per section

Figure 2.5: Heating and stirring controllers / devices for real-time kinetic studies by

FT-Raman spectroscopy

Chapter 2 Experimental

22 NMR Characterisation of Initiator

Samples were prepared in a dry box under argon atmosphere using standard dry box

techniques. All glassware and other equipment were pre-dried in an oven overnight at

100 C. CaH2 was powdered by crushing, using a mortar and pestle for a period of five

minutes before adding with other reactants. The initiator was prepared by heating PEG45

and CaH2 in a stoppered flask inside the dry box. Samples were extracted with benzene-

d6 and transferred to 5 mm diameter NMR tubes, which were capped and sealed with

laboratory film. NMR analyses were then performed on a Bruker FT-NMR

spectrometer (400.13 MHz for 1H and 100.61 MHz for 13C). All spectra were referenced

to tetramethyl silane, TMS, using the benzene-d6 solvent residue at 7.16 ppm as an

internal calibration. Headspace Analysis

Raman microspectroscopy analysis was performed on the headspace of flame-sealed

tubes and a Teflon-key-sealed vessel. Both flame-sealed vessels and the Teflon-key-

sealed vessel were individually mounted on their side to a metal plate and set in the

Raman microscope stage. The microscope was focussed onto the under side of the

centre of the top face of the glass tube. The focus was then advanced 400 m into the

headspace of the tube. Spectra were then recorded.

2.2.3 Measurements Nuclear Magnetic Resonance Spectroscopy (NMR)

Samples were prepared at a concentration of approximately 0.5 w / v % for 1H NMR in

CDCl3 using 5 mm diameter tubes. 1H NMR spectra were recorded on a Bruker Avance

FT-NMR spectrometer equipped with a 9.39T magnet equivalent to 400.162 MHz for 1H. 32 scans were taken using a 4.70 s pulse, a dwell time of 100 s, an acquisition

time of 3.27 s, and a relaxation delay time of 1.0 s. The greatest T1 for any of the

Chapter 2 Experimental


samples studied was 840 ms. All spectra were referenced to TMS using the CHCl3

residue at 7.26 ppm as an internal calibration. Gel Permeation Chromatography (GPC) Table 2.2: Separation range of GPC columns

Column Type Effective Molecular Weight Range*

Waters, HR1 100 5 000

Waters, HR3 500 30 000

Waters, HR4 5 000 500 000

Phenomenex, phenogel 5 - 50 100 3 000

Phenomenex, phenogel 5 - 103 1 000 75 000

Phenomenex, phenogel 5 - 104 5 000 500 000


A Waters GPC system equipped with a Waters 1515 isocratic HPLC pump, 200 L

injection loop, column heater and a Waters 2414 refractive index detector (analysis

temperature, 30 C) was used. Due to corrosion problems within this GPC system over

the course of this work, overall it was equipped with one of the three following column

arrangements. The first consisted of three consecutive Waters styragel columns (HR4,

HR3, HR1) operating at 30 C using chloroform as eluent at a flow rate of 0.8 mL min-1.

The second configuration utilised two consecutive Waters styragel columns (HR4, HR3)

operating at 30 C using tetrahydrofuran as eluent at a flow rate of 1 mL min-1. The

solvent was changed to THF to avoid corrosion problems, which may have been caused

by the use of chloroform as eluent. The third configuration consisted of three

consecutive phenomenex, phenogel 5 columns (104 , 103 , 50 ), preceded by a

guard column, operating at 30 C using tetrahydrofuran as eluent at a flow rate of 1 mL

min-1. All systems were calibrated with seven polystyrene standards in the range 1350

Chapter 2 Experimental


450 000 g mol-1. A relative calibration (analyte molecular weight uncorrected to

polystyrene) was used for all configurations, with a third-order polynomial fit employed

for the calibration curve (R2 0.998 in all cases). Due to the switching of solvent, a

correlation between the absolute nM , and the nM obtained by GPC measurements

was made. It was found that there was a linear correlation of absolute molecular weight

as measured by 1H NMR, and molecular weight as measured by GPC for both THF and

chloroform as eluent. These linear correlations were found over a broad molecular

weight range, 4400 13 300 g mol-1 for chloroform as eluent ( HNMRnM 1 = 0.5271 x

GPCnM + 556, R2 = 0.987), and 7400 60 300 g mol-1 for THF as eluent ( HNMRnM 1 =

1.6396 x GPCnM - 7426, R2 = 0.977). Due to the difference in slope for the linear

regressions for chloroform and THF correlations, direct comparison of molecular weight

obtained by GPC using THF as eluent and GPC using chloroform as eluent cannot be

made. Therefore, no direct comparison of data obtained using THF as eluent and data

obtained using chloroform as eluent has been made throughout the rest of this thesis. FT-Raman Spectroscopy

All FT-Raman spectroscopic measurements were carried out on a Perkin-Elmer System

2000 NIR FT-Raman spectrometer, equipped with a diode pumped Nd-YAG laser ( =

1064 nm) as an excitation source and a room temperature InGaAs photoelectric detector.

The backscattered radiation was collected at 180 to the excitation. Typically, spectra

were recorded in the range 200 - 3800 cm-1 at a laser power of 320 mW. 32 co-added

scans were taken for each spectrum with a spectral resolution of 8 cm-1. Grams/32 AI

(6.00) was used for spectral analysis. Raman Microspectroscopy

All Raman microspectroscopy measurements were undertaken using a Renishaw InVia

Raman microscope equipped with a Leica microscope and a frequency-doubled, diode-

Chapter 2 Experimental


pumped Nd-YAG laser ( = 532 nm). Calibration was performed by referencing to the

520.5 cm-1 band of a silicon wafer. Spectra were recorded in the following ranges: 100 -

4300 cm-1 (full scan); 4000 4300 cm-1 (hydrogen-specific scan); 2100 - 2500 cm-1

(nitrogen-specific scan). Spectra were acquired at 100 % laser power (120 mW) using a

long working distance X 50 objective, and 4 spectral accumulations at 60 s per

accumulation. Grams/32 AI (6.00) was used for spectral analysis.

Chapter 2 Results and Discussion


2.3 Results and Discussion

2.3.1 Polymerisation Scheme

The synthesis of PCL using hydroxy-functional and alkoxy initiators has been widely

studied. It is generally accepted that due to its relatively low ring strain, the ring-

opening of CL with such initiators occurs through acyl oxygen bond cleavage, leading to

an alkoxy or hydroxy end-functional propagating species.10

In this study, the initiating species is expected to be a calcium-PEG alkoxide formed by

the reaction of PEG, which contains two terminal primary alcohol groups, with calcium

hydride, CaH2. As calcium prefers to exist as a divalent ion and PEG is a di-functional

molecule, chain extension may occur. For simplicity, the chain extension phenomenon

has been expressed through the use of a * adjacent to the calcium atoms in the

following scheme. Figure 2.6 presents a summary of the proposed polymerisation

scheme for the synthesis of PCL/PEG/PCL using a PEG / CaH2 co-initiator. This

scheme has been based on a living polymerisation model.

In addition, a reversible exchange reaction between alkoxide and hydroxy species,

proposed by Zhong et al.33 for the synthesis of PLA using a

bis(tetrahydrofuran)calciumbis[bis(trimethylsilyl)amide] / alcohol co-initiator, has been

included. The reactivity of the alkoxide towards protic species can be exploited for the

termination of active centres, where the addition of a suitable acid should produce

hydroxy-terminated polymers and the respective calcium-salt of the acid.





Ca*n *Ca









*Ca OO











































*CaOR + R'OH *CaOR' + ROH

CaH2 + + 2 H2

+ 2

+ [(x-1) + (y-1)]

+ 2HX+ Ca(X)2


Initiating Species Formation



Reversible Transfer

Figure 2.6: Proposed polymerisation scheme for the synthesis of PCL/PEG/PCL using a PEG / CaH2 co-initiator


Chapter 2 Results and Discussion


2.3.2 NMR Characterisation

1H NMR is commonly used for determination of the degree of conversion for lactone

polymerisations, as well as an aid for the elucidation of polymerisation mechanisms by

end-, and junction-group analysis. 1H NMR was used to determine the degree of

conversion, , of CL for crude polymerisation mixtures as well as the determination of

nM for pure PCLx/PEG45/PCLy copolymers. Equation 2.3 was applied for the

determination of the degree of conversion, where 2.3 is the 1H NMR signal arising

from the methylene group adjacent to the carbonyl of ring-opened CL (see Hc, Table

2.3) and 2.6 is the 1H NMR signal arising from the methylene group adjacent to the

carbonyl of CL (see Hc, Table 2.3). The degree of conversion has been taken as a ratio

of the polymerised CL signal, Hc, integral to the sum of the ring-opened CL, Hc, and

CL, Hc, signal integrals.

( )





Equation 2.3

nM was calculated using PEG45 as an internal standard (Equation 2.4). The following

logic was used to arrive at Equation 2.4. For purified PCLx/PEG45/PCLy copolymers,

from which nM values were calculated, there was assumed to be one PEG molecule per

PCLx/PEG45/PCLy triblock copolymer molecule. Therefore, a ratio of the PEG signal

(see Hd, Table 2.3) to a signal from the PCL segments, in this case Hc, was used to

calculate nM for the PCLx/PEG45/PCLy copolymers. The nM of PEG was known,

nM = 2000 g mol-1, therefore, it was calculated to have a nDP of 45. Since there are

four hydrogens per monomer unit in PEG, the total number of hydrogens per molecule

was calculated to be 180. Therefore, the signal integral for PEG, Hd - 3.6, was set to

represent 180 hydrogens per PCLx/PEG45/PCLy copolymer molecule. Using this

method, the ratio of the signal integral for PEG, and the signal integral for PCL, Hc -

Chapter 2 Results and Discussion


2.3, was used to calculate the relative number of PCL hydrogens per PCLx/PEG45/PCLy

triblock copolymer molecule. Since Hc represents two hydrogens per monomer unit, the

total number of CL repeat units per PEG was calculated by taking a ratio of the signal

integrals of PCL and PEG then multiplying this ratio by the number of PEG hydrogens

per PCLx/PEG45/PCLy copolymer molecule, 180, and dividing by two. Hence, arriving

at: (A2.3 / A3.6 x 180) / 2, which is equal to A2.3 / A3.6 x 90. To calculate the nM of

the PCL segments, the number of repeat units was multiplied by the molecular weight of

each repeat, 114 g mol-1. Finally, to calculate the nM of the PCLx/PEG45/PCLy triblock

copolymers, the nM of the PEG segment, 2000 g mol-1, was added to the calculated

value for the nM of the PCL segments.

( ) 2000114906.33.21 += AAM HNMRn

Equation 2.4

The ratio of the PEG / CL junction-group (Hf, 4.20) to the PEG signal (Hd, 3.60) for

purified copolymers was used as an indication of whether or not all PEG hydroxyl

groups had been consumed. At a ratio 3.60 : 4.20 = 44 : 1, it was assumed that

quantitative consumption of PEG hydroxyl groups had occurred.

Chapter 2 Results and Discussion


Table 2.3: 1H NMR (CDCl3) peak assignments for CL and PCLx/PEG45/PCLy

Signal Position Assignment*































* peak assignments were made according to the relevant literature35

Chapter 2 Results and Discussion






, a.u










Figure 2.7: Typical 1H NMR spectra of a crude PCLx/PEG45/PCLy polymerisation

mixture (upper) and a pure PCLx/PEG45/PCLy copolymer (lower). Peak

assignments are given in Table 2.3

Chapter 2 Results and Discussion


2.3.3 PCLx/PEG45/PCLy Synthesis

In an initial pilot study, the syntheses of PCLx/PEG45/PCLy copolymers were undertaken

in a dry box under an argon atmosphere. This approach was adopted in order to

facilitate the study of the polymerisation kinetics, which were to be monitored by

sampling the polymerisation mixture at set intervals and establishing the degree of

conversion by 1H NMR. This method proved, however, to be problematic. Even

heating of the reaction vessel proved impossible, resulting in monomer condensing on

the cooler, upper portions of the vessel. This affected the concentration of monomer

present in the reaction mixture (Figure 2.8) making reproducibility difficult.







0 50 100 150 200 250 300 350 400 450 500

time, min



of th






Figure 2.8: Fraction of theoretical CL content, [CL]t / [CL]0, versus time ([CL] was

determined from 1H NMR using PEG45 as an internal standard) for the synthesis of

PCL50/PEG45/PCL50 under an argon atmosphere in a dry box (note that the point

near 0 min was taken two minutes after addition of CL to the pre-heated vessel)

The reaction procedure was modified in order to overcome these problems by adopting

synthesis in flame-sealed, glass tubes. This ensured a more reproducible environment

Chapter 2 Results and Discussion


for the polymerisation and also meant that the temperature of the entire reaction vessel

could be kept constant, throughout, by complete immersion in a thermo-stated oil bath.

For the first series of these experiments polymerisation kinetics were monitored at 70

C. This temperature was chosen to ensure both PCL (Tm = 60 C) and PEG (Tm = 55

C) remained molten throughout the polymerisation. The use of this relatively low

temperature is in contrast to other studies of the bulk polymerisation of lactones, where

polymerisation temperatures are generally in the range 100 150 C.10 Such high

temperatures, as well as long reaction times, can lead to transesterification reactions in

the ring-opening polymerisation of lactones.10 Therefore, by keeping the polymerisation

temperature low, it was hoped that competitive processes such as inter- and intra-

molecular transesterification would be minimised.

GPC analysis showed no evidence for the formation of low molecular weight intra-

molecular, cyclic, transesterification products for PCLx/PEG45/PCLy copolymers

synthesised at 70 C. This was indicated by the lack of low molecular weight peaks in

the chromatograms of crude polymerisation mixtures.

The extent of inter-molecular transesterification was determined by analysis of

molecular weight distributions (MWDs) obtained from GPC analysis. Inter-molecular

transesterification has been shown to lead to an increase in polydispersity index

( nw MM ), PDI.10 Since PDIs were generally between 1.1 1.3, for

PCLx/PEG45/PCLy copolymers synthesised at 70 C, it can safely be assumed that inter-

molecular transesterification was not significant for these reaction conditions.

Even though polymerisation at 70 C had beneficial outcomes such as minimal inter-

and intra-molecular transesterification, the rate of propagation was much lower than

expected for a calcium-alkoxide system. A number of days were required to obtain

PCLx/PEG45/PCLy copolymers at 70 C. In contrast, it has been reported that for the

synthesis of PCL using calcium dimethoxide as the initiator at 120 C in the bulk ([CL]0

Chapter 2 Results and Discussion


: [calcium dimethoxide]0 = 100 : 1), complete consumption of CL occurred after 10 min

at an initiator efficiency of 0.54.65 As this polymerisation was performed at a much

higher temperature, the much faster rate of polymerisation could be due to a very high

activation energy for the ring-opening of CL by calcium alkoxides. However, for the

synthesis of PCL using a calcium dimethoxide initiator formed in situ at room

temperature ([CL]0 : [OH]0 = 50 : 1), quantitative consumption of monomer also

occurred after 10 min.33 Even though this polymerisation was performed in THF with

an initiator that was formed in situ, thereby slightly changing the nature of the reaction,

the fast rate of polymerisation at this temperature would suggest that the cause of the

low rate of polymerisation at 70 C is not due to a very high activation energy for the

ring-opening of CL by calcium-alkoxides.







0 50 100 150 200 250

time, hrs

ln ([




Figure 2.9: ln [CL]0/[CL]t versus time plot for the synthesis of PCLx/PEG45/PCLy

copolymers; [CL]0 : [OH]0 : [CaH2]0 = 50 : 1 : 0.67 (), and [CL]0 : [OH]0 : [CaH2]0

= 100 : 1 : 0.67 (x) in flame-sealed glass tubes at 70 C. Error bars: 1 S.D., n = 1

3. Where n = 1, the error bars represent the error inherent in the analysis


Another possible reason for the observed low rate of polymerisation at 70 C could be

the incomplete formation of the calcium-PEG alkoxide initiator. As shown in Figure

Chapter 2 Results and Discussion


2.9, the kinetic curves obtained from the synthesis of PCLx/PEG45/PCLy show

induction periods before reaching a steady-state region. This induction period is

most likely due to the slow formation of the initiator compared to the much faster rate of

propagation. One possibility is that once a critical concentration of initiator has been

formed, the polymerisation proceeds much quicker, finishing before the reaction of PEG

with CaH2 has gone to completion. To increase the rate of calcium-PEG al