ABSTRACT

KOLELL, HANNAH JOY. Hydrophobic Disperse and Polymeric Dyes for the Coloration of UHMWPE Medical Sutures. (Under the direction of Dr. Harold S. Freeman).

Ultra-high molecular weight polyethylene (UHMWPE) has extremely beneficial properties, including being high strength at a relatively low weight. However, its high crystallinity and very hydrophobic nature make coloration from a dyebath difficult. Attempts at surface modification of the polymer to increase dyeability often adversely affect the desired high-performance properties. UHMWPE has applications in the medical field, including as sutures, and for surgical purposes coloration at sufficient visibility levels is of high importance.

Currently, there is one FDA certified dye, D&C Violet 2, that has the ability to dye UHMWPE sutures. Thus, having additional dyes is of interest as long as the resulting color is not red. To achieve this goal, the present study pertained to the modification of various FDA certified dyes to make them suitable for dyeing PE sutures. Specifically, dyes such as C.I. Solvent Yellow 18 and C.I. Solvent Green 3 (D&C Green 6) were modified with C-4 to C-6 alkyl groups and after structure confirmation through HPLC, UV-Vis, NMR, and HR-MS, the resulting 6 dyes were tested for their ability to dye UHMWPE fibers. The dyeing study showed visibly darker fibers from the modified dye structures in comparison to the D&C Violet 2 prototype. Overall the C-4 substituted dyes showed darker coloration of the fibers and in general showed lower dye release levels during extractions, making them the preferred choices for PE sutures. All synthesized dyes were also tested under a modified Ames protocol using strain YG1041 both with and without S9 enzyme activation and none of the dyes showed mutagenicity.

Poly dyes were also examined for PE sutures coloration through mass coloration. Four yellow anthraquinone polymeric dyes and the group of monomer dyes were incorporated into PE films pressed from the corresponding powder. Most of the polymeric dyes afforded no detectable color following extractions, except for the dye X26669-91 from the Weaver Dye Library. On the other hand, the monomeric dyes were more readily extracted from the films, probably because of the lower crystallinity of the polymer. These films would have a higher ability to swell in the extraction media which leads to the removal of the dyes from the polymer matrix.

For UHMWPE fibers 6 new dyes were identified, synthesized and applied in a manner which led to sufficient coloration and small amounts of extracted dye under physiological conditions. The use of polymeric dyes in mass coloration also showed the ability to color PE films through a mass coloration process with little to no dye leaching.

© Copyright 2019 by Hannah Kolell

All Rights Reserved

Hydrophobic Disperse and Polymeric Dyes for the Coloration of UHMWPE Medical Sutures

by Hannah Joy Kolell

A thesis submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Master of Science

Textile Chemistry

Raleigh, North Carolina 2019

APPROVED BY:

______Dr. Harold S. Freeman Dr. Richard Kotek Committee Chair

______Dr. Lisa Parrillo-Chapman Dr. Malgorzata Szymczyk External Member

BIOGRAPHY

Hannah Kolell was born in Menomonee Falls, WI and lived there until she earned her

Bachelor of Science Degree in Biochemistry with a minor in Mathematics from Valparaiso

University in spring of 2017. During her time at Valparaiso she studied abroad in Cambridge,

England. She also participated in a summer REU program at the University of Southern

Mississippi where she worked on polymer synthesis for a medical application. When it came time to decide on her next step, her love for textiles and sewing as well as for chemistry made the textile chemistry field a perfect fit and combination of these two interests. The pursuit of this field brought her to studying for a masters at North Carolina State University at the Wilson

College of Textiles. Under the direction of Dr. Harold Freeman, she developed an interest in dyes and color science. She has enjoyed her time in North Carolina through hiking, beach trips, and exploring the city of Raleigh.

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ACKNOWLEDGMENTS

I would first like to thank Dr. Harold Freeman for his continued guidance and knowledge throughout the project. I would also like to thank Dr. Malgorzata Szymczyk for her expertise in the lab and help with the dye synthesis and purification. I also thank the other members of my committee, Dr. Richard Kotek and Dr. Lisa Chapman for their helpful suggestions, advice, and availability. Dr.Gisela Umbuzeiro, Judy Elson, Birgit Andersen, Dr. Hanna Gracz, and Danielle

Lehman were all essential for laboratory and instrumental studies.

Personally, I would like to thank my fellow graduate students for support and I am so glad to have met and spent time with you all. I cannot say that I would be the chemist that I am today without Megan Cullinan, and I would specifically like to acknowledge her part in the completion of this research. I cannot express enough how thankful I am for the support that I have received from my parents to pursue this as well as from my siblings and friends who took the time to visit me in North Carolina during my time here.

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TABLE OF CONTENTS

LIST OF TABLES ...... vi LIST OF FIGURES ...... vii Chapter 1: Literature Review and Background ...... 1 1.1. Polyethylene ...... 1 1.1.1. Dyeability of Polyethylene ...... 2 1.2. Medical Sutures ...... 5 1.2.1. FDA approved dyes used for sutures ...... 7 1.3. Extraction studies ...... 9 1.4. Mutagenicity and aquatic toxicity studies...... 10 1.5. Proposed dye structures ...... 15 1.5.1. Long alkyl chains for dyeing improvement ...... 16 1.5.2. Target dyes ...... 21 1.6. Polymeric colorants ...... 23 1.6.1 Proposed polymeric dye structures ...... 24 1.6.2. Mass coloration ...... 26 Chapter 2: Experimental ...... 28 2.1. General ...... 28 2.1.1. Materials ...... 28 2.1.2. Instruments ...... 29 2.2 Synthesis of dyes...... 31 2.2.1. Mono-azo dyes ...... 31 2.2.2. Mono-anilino-substituted anthraquinone dyes ...... 32 2.2.3. Di-anilino-substituted anthraquinone dyes ...... 33 2.3. Fiber dyeing experiments ...... 34 2.4. Mass coloration of PE films ...... 35 2.5. Microscopy ...... 35 2.6. Extraction studies ...... 35 2.7. Mutagenetic and toxicity studies ...... 36 Chapter 3: Results and Discussion ...... 37 3.1. Mono-azo and Anthraquinone dyes ...... 37 3.1.1. NMR spectra of dyes ...... 37 3.1.2. Mass Spectrometry of dyes ...... 51 3.1.3. HPLC of crude dye mixtures ...... 55 3.1.4. Absorption Spectra...... 59 3.1.5. LogP estimations ...... 63 3.1.6. Dyed Fiber Images ...... 64 3.1.7. Microscopy images ...... 66 3.1.8. Extraction studies ...... 71

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3.1.9. Mutagenicity data ...... 74 3.2. Poly dyes ...... 76 3.2.1. Extraction studies ...... 77 Chapter 4: Conclusions ...... 85 4.1. Future Work ...... 85 References ...... 87 Appendices ...... 94

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LIST OF TABLES

Table 1.1 Toxicity classes of the European Community ...... 10

Table 1.2 Dye structures, identification numbers, colors, and approximate melting points for the four poly dyes considered for the solution dyeing portion of the project ..... 25

Table 2.1 Chemicals and dyes used for synthesis along with supplier and purity levels...... 28

Table 2.2 HPLC gradient conditions...... 30

Table 2.3 Gradient conditions for LC-MS analysis ...... 30

Table 3.1 Absorption spectral data for dyes 1 through 6 ...... 59

Table 3.2 LogP estimations from EPA Episuites and ChemDraw for synthesized dyes and the C.I. Solvent Violet 13 standard ...... 63

Table 3.3 The mg of dye extracted from the two fibers types corrected to one gram of fiber in both room temperature and heated conditions ...... 72

Table 3.4 Mean and standard deviations of the counts of the revertant colonies for three doses of each dye, negative controls, solvent controls, and a positive control of C.I. Disperse Blue 373 ...... 75

Table 3.5 Absorption values for extraction media from PE films under heated, room temperature and stretched conditions...... 75

Appendix

A Raw colony count data for three dose miniaturized Ames test...... 95

D Raw absorbance data from the second fiber extraction study ...... 101

E First dying study extraction data in units of mg of dye per gram of fiber of the two UHMWPE fiber types tested ...... 101

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LIST OF FIGURES

Figure 1.1 Molecular structure of polyethylene ...... 1

Figure 1.2 Macromolecular orientation of HPPE or UHMWPE and normal PE ...... 2

Figure 1.3 Representative disperse dye structures (C.I. Disperse Orange 5, C.I. Disperse Yellow 3, C.I. Disperse Black 3, and C.I. Disperse Violet 26) ...... 3

Figure 1.4 Molecular structure of polypropylene (PP) ...... 4

Figure 1.5 Molecular structure of C.I. Solvent Violet 13 ...... 5

Figure 1.6 Example structures of color additives in CFR 74 ...... 8

Figure 1.7 Target analogs of D&C Violet 2 (A), Solvent Yellow 18 (B), and D&C Green 6 (C) ...... 15

Figure 1.8 Structure of a super hydrophobic violet dye...... 16

Figure 1.9 Hydrophobic anthraquinone dyes with alkyl chains ranging from methyl to pentyl...... 17

Figure 1.10 Hexyl amino through octyl-amino substituted anthraquinone dye structures...... 18

Figure 1.11 Magenta anthraquinoid dyes substituted with butyl through dodecyl alkyl chains..19

Figure 1.12 Red anthraquinone dye structures with methyl through octyl alkyl substituents .... 19

Figure 1.13 Di-anilino substituted anthraquinone dyes with ethyl through heptyl alkyl chains. 20

Figure 1.14 Structures of dyes 1 and 3 synthesized in this study ...... 21

Figure 1.15 Structure of dyes 2 and 4 synthesized in this study ...... 22

Figure 1.16 Structures of dyes 5 and 6 synthesized in this study ...... 23

Figure 3.1 1H NMR spectrum for dye 1 ...... 39

Figure 3.2 Carbon NMR spectrum for dye 1 ...... 40

Figure 3.3 1H NMR spectrum for dye 2 ...... 41

Figure 3.4 13C NMR spectrum for dye 2 ...... 42

Figure 3.5 1H NMR spectrum for dye 3 ...... 43

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Figure 3.6 13C NMR spectrum for dye 3 ...... 44

Figure 3.7 1H NMR spectrum for dye 4 ...... 45

Figure 3.8 13C NMR spectrum for dye 4 ...... 46

Figure 3.9 1H NMR spectrum for dye 5 ...... 47

Figure 3.10 13C NMR spectrum for dye 5 ...... 48

Figure 3.11 1H NMR spectrum for dye 6 ...... 49

Figure 3.12 13C NMR spectrum for dye 6 ...... 50

Figure 3.13 HR ESI Mass spectrum for dye 1 ...... 51

Figure 3.14 HR ESI mass spectrum for dye 2 ...... 52

Figure 3.15 HR ESI mass spectrum for dye 3 ...... 52

Figure 3.16 HR ESI mass spectrum for dye 4 ...... 53

Figure 3.17 HR ESI mass spectrum for dye 5 ...... 54

Figure 3.18 HR ESI mass spectrum for dye 6 ...... 54

Figure 3.19 HPLC results from first synthesis of dye 1 ...... 56

Figure 3.20 HPLC results from first synthesis of dye 2 ...... 56

Figure 3.21 HPLC results from first synthesis of dye 3 ...... 57

Figure 3.22 HPLC results from first synthesis of dye 4 ...... 57

Figure 3.23 HPLC results from first synthesis of dye 5 ...... 58

Figure 3.24 HPLC results from first synthesis of dye 6 ...... 58

Figure 3.25 Absorption spectra of dye 1 with peak wavelength data ...... 60

Figure 3.26 Absorption spectra of dye 2 with peak wavelength data ...... 60

Figure 3.27 Absorption spectra of dye 3 with peak wavelength data ...... 61

Figure 3.28 Absorption spectra of dye 4 with peak wavelength data ...... 61

Figure 3.29 Absorption spectra of dye 5 with peak wavelength data ...... 62 viii

Figure 3.30 Absorption spectra of dye 6 with peak wavelength data ...... 62

Figure 3.31 Molecular structures of C.I. Solvent Blue 35 (A) and C.I. Solvent Blue 59 (B) ..... 64

Figure 3.32 Commercial 2 and NCSU UHMWPE fibers dyed with dyes 1 and 3. The order of the samples from left to right: dye 3 on Commercial 2 fiber, dye 1 on Commercial 2 fiber, dye 3 on NCSU fiber, dye 1 on NCSU fiber ...... 65

Figure 3.33 Commercial 2 and NCSU UHMWPE fibers dyed with dyes 2 and 4 and C.I. Solvent Violet 13. The order of the samples from left to right: dye 2 on NCSU fiber, dye 2 on Commercial 2 fiber, dye 4 on NCSU fiber, dye 4 on Commercial 2 fiber, C.I. Solvent Violet 13 on Commercial 2 fiber ...... 65

Figure 3.34 Commercial 2 and NCSU UHMWPE fibers dyed with dyes 5 and 6 and standards C.I. Solvent Blue 59 and C.I. Solvent Blue 35. Order of the samples from top left to bottom right: dye 5 on Commercial 2 fiber, dye 5 on NCSU fiber, dye 6 on Commercial 2 fiber, dye 6 on NCSU fiber, dye Solvent Blue 59 on NCSU fiber, dye Solvent Blue 35 on Commercial 2 fiber ...... 66

Figure 3.35 Dye 1 on Commercial 2 fiber with black thread contrast on the left and dye 1 on NCSU fiber on the right ...... 67

Figure 3.36 Dye 2 on Commercial 2 fiber with white thread contrast on the left and dye 2 on NCSU fiber on the right ...... 67

Figure 3.37 Dye 3 on Commercial 2 fiber with black thread contrast on the left and dye 3 on NCSU fiber on the right ...... 68

Figure 3.38 Dye 4 on Commercial 2 fiber with white thread contrast on the left and dye 4 on NCSU fiber in the image on the right ...... 69

Figure 3.39 Dye 5 on Commercial 2 fiber with white thread contrast on the left and dye 5 on NCSU fiber on the right ...... 70

Figure 3.40 Dye 6 on Commercial 2 fiber with white thread contrast on the left and dye 6 on NCSU fiber on the right ...... 70

Figure 3.41 Solvent violet 13 dye on Commercial 2 fiber with white thread contrast ...... 71

Figure 3.42 Dyes 2 (left) and 1 (right) on Commercial 2 and NCSU fiber after the heated extraction study and additional 5 mon submersion in the extraction medium ...... 73

Figure 3.43 Dyes 3 (left) and 4 (right) on Commercial 2 and NCSU fiber after the heated extraction study and additional 5 mon submersion in the extraction medium ...... 73

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Figure 3.44 Dyes 6 (left) and 5 (right) on Commercial 2 and NCSU fiber after the heated extraction study and additional 5 mon submersion in the extraction medium ...... 73

Figure 3.45 Dyes C.I. Solvent Violet 13 (left), C.I. Solvent Blue 59 (center), and C.I. Solvent Blue 35 (right) on Commercial 2 and NCSU fiber after the heated extraction study and additional 5 mon submersion in the extraction medium ...... 74

Figure 3.46 Anova plotted results for all three doses of the dyes and the standards along with the Tukey-Kramer analysis of means ...... 76

Figure 3.47 PE film colored with polymeric dye X26669-91 at 0.5% dyeing ...... 77

Figure 3.48 PE film cut into strips after dyeing with polymer dye X26669-91. From left to right: after pressing, after room temperature extraction, after 50 ˚C extraction and after being stretched and then subjected to a room temperature extraction ...... 79

Figure 3.49 PE film cut into strips after dyeing with polymer dye X26955-42-2. From left to right: after pressing, after room temperature extraction, after 50 ˚C extraction and after being stretched and then subjected to a room temperature extraction ...... 80

Figure 3.50 PE film cut into strips after dyeing with polymer dye X26669-89. From left to right: after pressing, after room temperature extraction, after 50 ˚C extraction and after being stretched and then subjected to a room temperature extraction ...... 80

Figure 3.51 PE film cut into strips after dyeing with polymer dye X26669-93. From left to right: after pressing, after room temperature extraction, after 50 ˚C extraction and after being stretched and then subjected to a room temperature extraction ...... 81

Figure 3.52 PE film cut into strips after dyeing with dye 1. From left to right: after pressing, after room temperature extraction, after 50 ˚C extraction and after being stretched and then subjected to a room temperature extraction ...... 81

Figure 3.53 PE film cut into strips after dyeing with dye 2. From left to right: after pressing, after room temperature extraction, after 50 ˚C extraction and after being stretched and then subjected to a room temperature extraction ...... 82

Figure 3.54 PE film cut into strips after dyeing with dye 3. From left to right: after pressing, after room temperature extraction, after 50 ˚C extraction and after being stretched and then subjected to a room temperature extraction ...... 82

Figure 3.55 PE film cut into strips after dyeing with dye 4. From left to right: after pressing, after room temperature extraction, after 50 ˚C extraction and after being stretched and then subjected to a room temperature extraction ...... 83

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Figure 3.56 PE film cut into strips after dyeing with dye 5. From left to right: after pressing, after room temperature extraction, after 50 ˚C extraction and after being stretched and then subjected to a room temperature extraction ...... 83

Figure 3.57 PE film cut into strips after dyeing with dye 6. From left to right: after pressing, after room temperature extraction, after 50 ˚C extraction and after being stretched and then subjected to a room temperature extraction ...... 84

Figure 3.58 PE film cut into strips after dyeing with C.I. Violet 13. From left to right: after pressing, after room temperature extraction, after 50 ˚C extraction and after being stretched and then subjected to a room temperature extraction ...... 84

Appendix

B Graphs of dose responses for Ames assay for all six dyes ...... 96

C Absorption spectra for standard dyes used for dye studies of C.I. Solvent Blue 35, C.I. Solvent Blue 59, and C.I. Solvent Violet 13 ...... 99

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CHAPTER 1: Literature Review and Background

1.1. Polyethylene

Polyethylene (PE) (Figure 1.1) is an extremely versatile polymer, with a simple backbone structure that consists of a carbon chain bonded only to hydrogen. The versatility of this material comes from the ability to vary parameters such as the chain length and the crystallinity of the matrix. The chain’s highly non-polar quality mean that it is a highly hydrophobic material. The extent of its hydrophobicity varies due to the properties of a specific sample, but highly crystalline polyethylene becomes problematic for polar solvent based dyeing due to its resistance to swelling in media such as water. Coloration requires hydrophobic dyes or for the material to be mass pigmented in the melt prior to the formation of the fibers.

Ultra high molecular weight polyethylene (UHMWPE) is a term used for the linear polymer polyethylene which has very high molecular weight which can range from 3.5x106 to

7.5x106 g/mol. The fibers are produced through gel spinning which leads to the highly oriented nature of the fibers (Figure 1.2), which could have as high as 85% crystallinity. Polyethylene produced via melt spinning is recorded as having crystallinity less than 60%.Error! Bookmark not defined.

Figure 1.1: Molecular structure of polyethylene.

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Figure 1.2: Macromolecular orientation of HPPE or UHMWPE and normal PE. 1

Increased molecular weight leads to physical and mechanical properties which include chemical inertness, lubricity, abrasion and impact resistance and biocompatibility.1 These desirable characteristics make UHMWPE a suitable material for end uses as varied as towing boats, skydiving equipment, and joint replacements. Dyeing UHMWPE is even more difficult than PE, because of its higher crystallinity and because adding large foreign molecules disrupts the crystallinity which can adversely affect the desired strength and abrasion and impact resistance qualities.

1.1.1. Dyeability of Polyethylene

When dyeing PE in a polar liquid medium like water, disperse dyes will be the most common dye class used. Disperse dyes were developed after the synthesis of the first commercial hydrophobic synthetic fiber, cellulose acetate. The size of these dye molecules usually ranges from 300- 600 amu (Figure 1.3), which is small when compared to the size of the polymer chains. Disperse dyes lack ionizable groups like sulfonic (SO3H) or carboxylic acid (CO2H)

2

groups. (Figure 1.3) This is why disperse dyes have low water solubility and are more favorable for hydrophobic fibers.

C.I. Disperse Orange 5 C.I. Disperse Yellow 3

C.I. Disperse Black 2 C.I. Disperse Violet 26

Figure 1.3: Representative disperse dye structures (C.I. Disperse Orange 5, C.I. Disperse Yellow 3, C.I. Disperse Black 3, and C.I. Disperse Violet 26).

Research into the possibility of dyeing UHMWPE has been heavily focused in the area of modifying the polymer to increase wet-ability which would allow for better dyeing in aqueous media. Areas of focus for polymer modification include plasma and UV treatments2-3, gamma ray irradiation4-7, and electron beam radiation8. Enomoto et al conducted surface modification through the use of an oxygen plasma treatment. This treatment was shown to successfully modify the surface of the nonwoven UHMWPE sample, however, when acid and reactive dyes were examined for dyeing the sample, minimal color change was observed. A more favorable result occurred through the use of a fluorocarbon plasma, but concerns over toxicity would not make this a favorable approach for a commercial application.9

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Polypropylene fabric has been modified by chlorination to improve its dyeability. The results did show increased color strength from the dyed chlorinated fibers in comparison to the dyed non-chlorinated fibers. The wash fastness was rated as moderate for most dyes that were tested. This study did not address the use of highly crystalline UHMWPE fibers or the loss of tensile strength which is very likely with this type of polymer modification.

A growing focus in the chemical and drug industry at large and in the dye industry involves computer modeling of the chemical structures in a manner that could help predict the dyeing properties of the molecules.10-13These cheminformatics techniques allow for the understanding of a dye’s general predicted properties without the need for the time involved in the synthesis of the compound. In relation to extremely hydrophobic polymers, a parameter called LogP is used to describe the hydrophobicity of a molecule. This is a partition coefficient and it stems from a calculation involving the molecule’s distribution between a water and 1- octanol medium. In the case of hydrophobic polymers, it has been found through actual dyeing and comparisons to the LogP calculations, that a LogP of at least 5 is needed in order for sufficient dyeing of polypropylene.14 Figure 1.4 shows the chemical structure of polypropylene

(PP). In the case of UHMWPE, the FDA approved dye C.I. Solvent Violet 13 (Figure 1.5) has been found to have sufficient dyeing ability and has a calculated LogP of 4.35. This calculated value then became the target LogP for the dyes chosen for the present study.

Figure 1.4: Molecular structure of polypropylene (PP).

4

Figure 1.5: Molecular structure of C.I. Solvent Violet 13.

1.2. Medical Sutures

Surgical sutures have been used to hold together tissue that was severed, aiding in the healing process after surgery, and stopping the bleeding from blood vessels, for thousands of years. Evidence has been found of the Egyptians using linen as a suture over 4000 years ago.15

Cat gut has also been used as a suture material for hundreds of years. Up until around the 1950’s cat gut and silk were the most common sutures, but developments from the production of synthetic fibers led to many more materials which could be used for medical purposes.15 In the present day there are two general categories of sutures. Sutures can be absorbable or non- absorbable. Non-absorbable sutures are made from synthetic and inherently colorless materials including silk, polypropylene, polyester, nylon, or ultra high molecular weight polyethylene

(UHMWPE).16 Nonabsorbable means that the polymer will stay in the body permanently, whereas the absorbable sutures are designed to degrade over time within the body. Absorbable sutures can lose their mechanical strength over a series of timescales. When these fibers were first developed, two mon was the standard desired time frame for the target loss of mechanical strength, but then it was extended to three mon for some materials and up to even a year for certain constructions. 17 Both types of sutures can be commonly found in monofilament and braided multifilament constructions. Which material will be made in which construction is

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chosen based on the material’s stiffness, since sutures need high flexibility for proper handling and knotting. Materials that are stiffer require the multi-filament construction. Examples of these materials would be polyester and UHMWPE. 4

Non-absorbable sutures are classified by the United States Pharmacopeia (U.S.P.) into three classes. The first class contains silk or synthetic fibers of monofilament, twisted or brained construction. Class II contains cotton, linen, or coated natural or synthetic fibers where the coating adds thickness to the fiber without adding strength. Class III contains sutures that are made of metal wire made in monofilament or multifilament construction.18 Non-absorbable sutures from these different classes will have a wide range of properties and end uses due to both material and construction differences; however, since most of these materials are inherently colorless there would be no way of visually distinguishing between the wide array of sutures.

This identification issue is one motivation for the need to color the sutures. It is also understood that from the doctor’s perspective the addition of color allows for better visibility and precision.

When polymers are used for medical sutures they often exhibit poor visibility characteristics, because they are often not only colorless, but also highly transparent. This gives the doctor or medical personnel little visual difference between the tissue and the polymer suture.19

Polyethylene sutures specifically are used because they are flexible and have a smooth feel which is gentle on both tissue and gloves. Orthopedic clinicians and cardiovascular surgeons prefer this type of suture due to its knotting properties including the ability to precisely place the knots and the tighter knot security and higher break strength.

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1.2.1. FDA approved dyes used for sutures

Colorants added to sutures have limitations due to the in-body end use. Each must be a compound that is approved by Federal Safety Administrations and its concentration must be below the maximum weight percentage that has been specified by regulating bodies. The FDA regulations that apply to the color additives used in medical devices are laid out in section 721 of the FD&C Act. Color additives used for surgical sutures and/or in the eye area are specifically dealt with in Title 21 of the Code of Federal Regulations (CFR) Part 70.5. The FDA has compiled lists of certified colorants in Parts 73, 74, and 81. Title 21 outlines color additives that are Exempt from Certification20 and Subject to Certification21. The collection of approved dyes for the purpose of coloring medical sutures and/or materials that will be in contact with the eye shows great variability in the properties of the dyes. Certain dyes that can be used in the coloring of contact lenses are water soluble in an initial form and then can be oxidized once inside of the polymer matrix to a water- insoluble form. Due to the great variety in materials used for constructing medical sutures, an equally varied collection of dyes has been approved by the

FDA. Dyes in section CFR Part 74 include dyes FD&C Blue 2 and D&C Green 5 which are both water-soluble sulfonated dyes which will have an affinity for nylon or polyamide structures.

Some dyes are nominally water soluble and could be potentially used on hydrophobic PET sutures. The only dye that has been found to be suitable for the highly hydrophobic PE sutures is

D&C Violet 2 which is also known as C.I. Disperse Blue 72 and C.I. Solvent Violet 13.

Molecular structures of example dyes from CFR Part 74 are shown in Figure 1.6 with their names and with the types of fibers for which they would have affinity.

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Figure 1.6: Example structures of color additives in CFR 74.

Another method for the coloration of UHMWPE sutures is accomplished by making a multifilament suture consisting of uncolored UHMWPE and a colored, lower molecular weight polyethylene or some other synthetic polymer which can be more easily dyed. This option is only available for certain suture constructions and thus only for specific end uses. This option also only provides limited coloration if a high ratio of UHMWPE is maintained.

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1.3. Extraction studies

For extraction (leanching) studies details pertaining to the appropriate extraction media, conditions, and material surface or weight to-solvent-volume ratio can be found in ISO 10993-12 and ISO 10993-13. The choice of extraction medium, temperature and time should be relevant to the nature of the finished product, purpose of the test, and the physicochemical properties of the materials composing the device and any other known leachable substances or residues.22-23

A variety of polar and non-polar solvents can be selected for an extraction medium in order to simulate in vivo conditions, depending on the specific area of the body that the device will be used in or on the specific nature of the extractable compounds. For ionic or potentially charged leachables such as metal ions, many solutions have been tested and are used to simulate specific areas of the body and the ions that would be found in those regions. Herting et al.24, investigated many body mimicking solutions on stainless steels to test for metal ion leaching.

The solutions used were mostly DI water based with the addition of ionic compounds such as sodium chloride, potassium chloride, sodium bicarbonate, and one with the addition of formaldehyde. Since these ionic solutions will not interact strongly with the hydrophobic nonpolar dyes, these solutions are not optimal for the present extraction study purposes.24

A phosphate buffered saline solution can closely mimic the pH conditions and ionic strength of an in vivo environment. However, this medium is not the ideal choice for the extraction of nonpolar molecules. The addition of organic solvents can lead to a more favorable medium if there is a need for the extraction of nonpolar molecules. A leachables study has been performed with a sample of quinizarin blue in polyether block amide (PBA) polymer and a

Pluronic F87 detergent solution for the extraction. Pluronic F87 is a synthetic block copolymer which functions as a nonionic surfactant.25 This is a more favorable method for bringing

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nonpolar molecules into a polar solution, although it is not a close reproduction of most bodily fluid environments.

For the volume of solvent needed for a study, ISO Part 13 section 4.1.7 recommends at least a ratio of 1 g of sample: 10mL medium and that the volume of medium should be sufficient to immerse the sample. In addition to testing under standard end use conditions (21˚C), a study will be performed at 50˚C.

1.4. Mutagenicity and aquatic toxicity studies

The Ecological and Toxicological Association of the Dyestuffs Manufacturing Industry

(ETAD) was founded in 1974 with the focus of understanding and reducing the potential adverse effects of dyestuffs on the environment, users and consumers and to work with both corporations and the government on topics involving the toxicological impact of their products.26 27 A study conducted by this organization included approximately 4,000 dyes and found that more than 90%

3 of the dyes tested have an LD50 value of about 2x 10 mg/kg. The classes of dyes that were found

27-28 to be the most ecotoxic were the bisazo basic and direct dyes. The LD50 value is the lowest dosage of a compound needed to kill 50% of the population of organisms exposed to the test

29 compound. The higher the LD50 of a material, the less toxic it is. Table 1.1 outlines the toxicity classes according to European Community criteria. 30

Table 1.1: Toxicity classes of the European Community.

퐿퐷50 Range Toxicity Class

퐿퐷50 ≤ 25 Very Toxic

25 < 퐿퐷50 ≤ 200 Toxic

200 < 퐿퐷50 ≤ 2000 Harmful

2000 < 퐿퐷50 Unclassified

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The acute toxicity effects of specific dyes can be understood through the use of traditional or enrichment toxicity tests.31 While toxicity is a major concern, it is not the only concern in relation to health and dyes. Certain chemicals used for imparting color have also been found to be carcinogenic, mutagenic, or teratogenic to various organisms.32-33 Mutations to the

DNA (deoxyribonucleic acid) of an organism are extremely serious because the DNA holds the information necessary for the life of the cell and because when DNA is replicated during cell division any mutations are passed on to the next generation of cells. DNA is composed of four nitrogenous bases: adenine, guanine, cytosine, and thymine. The 64 possible triple combinations of these bases lead to the 64 codons which determine the amino acids that will be used in protein synthesis. This is called the genetic code and there are 20 amino acids and 64 codons with some codons coding for same amino acids. This is said to be the degenerate nature of the code.

However, each codon itself codes for only one amino acid and this means that the code is unambiguous. In addition to the codons for all of the amino acids, there are also start and stop codons, which signal the beginning and end of a specific polypeptide chain.34

Mutations occur spontaneously through the lifecycle of the cell, especially during the replication process, but can also be caused by substances called mutagens or through exposure to

UV light. There are many types of mutations that can occur to a DNA strand, which can vary in widely in severity. Point mutations are changes in the nucleotide sequence through additions, deletions, or substitutions of the bases. This type of mutation occurs quite frequently to DNA, often with minimal effect on the cell’s function. Substitution mutations occur when an incorrect base is inserted for the base that should be in that location. This can possibly lead to no change in the amino acid that the codon codes for, because there are multiple codons that result in the same amino acid. This is called a silent mutation. If the substitution does cause a change in the amino

11

acid, the new protein can either completely change the conformation of the protein and thus change its functionality, or it can have minimal effect on the protein’s ability to function. Some codons are called stop codons which signal to stop the amino acid chain because protein synthesis is complete. If a substitution is made that converts the codon into a stop codon, the protein that the original DNA sequence was coding for can no longer be fully produced. Point mutations can also involve the addition of an extra bases into the chain or the deletion of a base from the chain. They can cause frame shift mutations because the addition or deletion of bases can cause all codon information to be shifted. This can cause all codons after the mutation to lose the information that they once contained. A larger scale mutation that can occur involves the change in the number of chromosomes in the genome. This can include either the deletion or addition of chromosomes and often has extremely serious consequences for the organism. 34

A point mutation caused by a dye molecule may not be expressed immediately, which can make the detection of these effects even more important.35 The potential effects of azo and nitro compounds have been well studied. These compounds can be reduced to potentially carcinogenic amines either inside an organism or in the sediment of aquatic bodies36-37 Benzidine is a known carcinogenic amine and was once used widely in dye synthesis. 38

The most comprehensive and concrete tests for assessing the mutagenicity of chemicals are in vivo experiments. Testing living organisms is time consuming and extremely expensive and can be too focused on only collecting data about lethality. It can also be difficult to definitively apply the data to humans since the tests are performed on other species. In vitro testing has been developed for fast and economical mutagenicity testing and uses bacteria or isolated tissue to screen for mutagens. The salmonella/microsome assay (Ames test) is the most commonly used mutagenicity test for screening pure chemicals and environmental samples.39

12

The test is a type of reverse mutation assay which uses a variety of possible strains of Salmonella typhimurium. There are seven strains of the bacteria that can be used to detect different types of mutations and that vary in their spontaneous reverse frequencies. The strains have been modified so that they do not have the ability to produce the amino acid histidine which is essential for growth. A mutagen can cause a specific reverse mutation in the histidine gene which would allow for the production of histidine and then for the bacteria to grow. This produces a revertant colony and the number of colonies present after the bacteria are exposed to the chemical of interest can indicate the level of mutation when compared to a base count. Generally, if the number of revertant colonies is double the number of base colonies then the compound is considered mutagenic. The number of colonies can be used to classify the chemical more specifically as a non-mutagen, weak mutagen, or a strong mutagen. The test can be performed first at the limit of solubility of the chemical to observe the properties of the most concentrated solution possible, and then the assay can be performed again with various dose concentrations to gain more understanding about the mutagenicity of the chemical as a function of concentration.

Another important addition to the assay is an enzyme that can metabolize the compound to mimic metabolism that occurs in biological systems such as the liver. Some compounds will become mutagenic only after it is metabolized. The rat liver microsomal enzyme system, S9, is used in the Ames assay for this metabolism process.40

Since its first development in 1975 by Ames and colleagues, many modifications and revisions have been made to the original Ames protocol. Reasons for the revisions include the discovery of some types of compounds that were shown to give either false positives or false negatives in the tests, and to reduce the amount of sample, agar, and media needed for the assay.

Many investigations into the miniaturization of the Ames assay have shown the ability to use

13

smaller amounts of sample yet obtain equal or higher sensitivity to the mutagenic activity of the test compounds. The tests make use of a micro suspension technique which leads to the increased sensitivity. The detected increases in sensitivity vary greatly, depending on the types and classes of compounds that are being tested and the conditions of the specific protocols.41-44 Zwarg et al employed the micro suspension in 12 well microplates instead of the traditional plates. This reduction leads to a large reduction in the amount of materials and labor time needed for the tests. The study showed comparable sensitives to previously described and accepted protocols. 39

14

1.5. Proposed dye structures

Modifications to dyes D&C Violet 2, C.I. Solvent Yellow 18, and D&C Green 6, as seen in Figure 1.7, will be used in order to produce dyes in the target LogP range and to make them function as dyes for polyethylene.

A B

C

Figure 1.7: Target analogs of D&C Violet 2 (A), C.I. Solvent Yellow 18 (B), and D&C Green 6 (C).

15

The modifications involve the addition of a four carbon or six carbon length chain as the “R” group in the dye structures. This will increase the hydrophobicity of the dyes which should increase affinity for the hydrophobic UHMWPE fibers.

1.5.1. Long alkyl chains for dyeing improvement

Kim et al conducted dyeing studies after synthesizing mono-azo, bis-azo, and some anthraquinone based dyes with added long alkyl chains, typically ranging from 1 to 10 carbons.46-60 The purpose was to test the dyeability, color strength, tensile strength and general fastness properties on dyed hydrophobic fibers. The addition of super hydrophobic dyes with long alkyl substituents was explored so that neither extrusion dyeing or any modifications to the fiber would need to be made. The azo structures were generally used to product red, orange and yellow colored dyes and generally showed increased ability to dye the hydrophobic fibers. 45-52

No direct and absolute conclusions can be made across all studies, as some showed improved wash fastness as alkyl chain length increases and some showed different chain lengths giving the highest wash fastness. The same was true for color strength. Some studies showed improvement with increased chain length and some showed this trend only to a certain length. A specific mono-azo dye of interest that was studied was the dye shown in Figure 1.8 and was used to dye polypropylene (PP) and UHMWPE. The UHMWPE showed much lower color strength than on

PP. The dye was also found to have poor light fastness on the UHMWPE. 45

Figure 1.8: Structure of a super hydrophobic violet dye.45

16

The study on UHMWPE fibers from Kim et al used the dyes C.I. Disperse Blue 14,

C.I. Solvent Blue 59, C.I. Solvent Blue 35, and C.I. Solvent Blue 14 (Figure 1.9) which are all

1,4 substituted anthraquinone dyes with different length of alkyl substituents ranging from methyl to pentyl.53

C.I. Disperse Blue 14 C.I. Solvent Blue 59

C.I. Solvent Blue 35 C.I. Solvent Blue 14

Figure 1.9: Hydrophobic anthraquinone dyes with alkyl chains ranging from methyl to pentyl.53

Under consistent dyeing conditions, the longest substituted chain (5 carbon chain) showed the highest dyeability as measured by the color strength of the resulting fibers. The pentyl substituted dye was tested for wash fastness, rubbing fastness, and light fastness using the standards KS K ISO 105-C06 A1S, KS K 0650, and KS K ISO 105-B02. Wash fastness and rubbing fastness testing gave a rating of 4-5 and light fastness testing gave a 4 rating.53. In a study involving the dyeing of PP, it was found that the molar extinction coefficient was not affected by the length of the alkyl chain, and that the maximum absorption wavelength was not affected by the chain length.54 Another study with these dyes on PP highlighted the dye uptake of the different chain lengths and found that the increase in chain length led to an increase in dye

17

uptake. 55 All studies highlighted the importance of the surfactant for uniform dye uptake and the studies utilized either N-dodecyl-N,N-(dimethylammonio)butyrate (DDMAB) or didecyldimethylammonium bromide (DDAB).

A study was conducted to extend this work to the synthesis of the hexyl, heptyl, and octyl analogs of the dyes, as shown in Figure 1.10. These dyes were used for the coloration of PP and it was found that even in comparison with the previously synthesized dyes the hexyl length chain gave the highest depth of shade. All dyes performed acceptably in the washing, rubbing and light tests.56

Figure 1.10: Hexyl amino through octyl amino- substituted anthraquinone dye structures. 56

Magenta anthraquinoid dyes with alkyl chains ranging from butyl to dodecyl were synthesized and used in the dyeing of both UHMWPE and PP. The dye structures as shown in

Figure 1.11.

18

Figure 1.11: Magenta anthraquinoid dyes substituted with butyl through dodecyl alkyl chains.58

Increasing alkyl length was found to lead to higher color strengths in both the PP and UHMPE fibers. It was assumed that UHMWPE would show lower depth of shade when compared using the same dye concentrations as the PP fibers due to the higher crystallinity of the UHMWPE.

This study, however, found that the UHMWPE had higher color strength that the PP at every alkyl length. Since the dodecyl length chain had the highest color strength, fibers dyed with that dye were used for wash, rubbing and light fastness tests. Both types of fibers received 4 or 4-5 ratings in all tests.57

A molecule similar to the magenta anthraquinoid dyes used in the previous study were made with the addition of a benzene ring to the alkyl chain which is reported to create a red colored dye molecule. 58 Structures for these dyes are shown in Figure 1.12.

Figure 1.12: Red anthraquinone dye structures with methyl through octyl alkyl substituents. 59

An aim of that study was to use the anthraquinone chromophore instead of an azo chromophore, in order to improve on the poor light fastest that was seen using azo dyes on

UHMWPE. There was an improvement seen in the light fastness from a rating of 2 for the azo

19

dye to a rating of 4 for some of the dyes in this anthraquinone study. The color strength comparisons of the eight dyes showed a great improvement in color strength for the methyl to the ethyl substitution. From the chain lengths of 2 to 7 no significant changes in color strength were seen. The octyl dye showed the lowest color strength, which could be indicative of the point where the molecule started to become too large to easily enter the polymer matrix and so the amount of dye in the fiber decreased. 59

One other study involved the synthesis of six di-substituted anthraquinone dyes which resulted in a blue-green color (Figure 1.13). The alkyl chain lengths of ethyl to heptyl were examined for the dyeing of both PP and high strength PE fibers.

Figure 1.13: Di-anilino substituted anthraquinone dyes with ethyl through heptyl alkyl chains. 59

Results of this study again suggested that the increase in chain length did not affect the absorption maximum, as all dyes were found to have a λmax of 645 nm. For the high strength PE fibers, the increase in chain length increased the color strength up to the C-6 length and the C-7 dye shows similar color strength to the C-6. On the PP fibers no real difference in color strength was seen as a function of chain length. 59

20

1.5.2. Target dyes

The present study pertained to the synthesis of Figure 1.14 dyes as analogs of C.I.

Solvent Yellow 18 having PE affinity.

Figure 1.14: Structures of dyes 1 and 3 synthesized in this study.

Dyes 1 and 3 dyes have a monoazo chromophore and a pyrazolone group as a part of the extended conjugated system as shown in the molecular drawing in Figure 1.14. Dye 1 with the hexyl chain, 3H-pyrazol-3-one, 4-[2-(4-hexylphenyl)diazenyl]-2,4-dihydro-5-methyl-2-phenyl, has the CAS number 98271-76-2. This dye was studied in the early 1960’s for the dyeing of PP61 and for general use as a water insoluble azo dye60. Dye 3, 3H-pyrazol-3-one, 4-[2-(4- butylphenyl)diazenyl]-2,4-dihydro-5-methyl-2-phenyl has the CAS registry number 95700-44-0.

It was studied in the same PP dyeing study61 as dye 1 and was also considered for use in a resin composition for optical devices which would give an anti-dazzle effect. For the optical device study, the structure was used as a precursor and then a metal complex was created and used as an additive in the films to achieve the desired anti-dazzle, anti-glare properties.62 A similar structure includes CAS #461709-04-6, which has a two carbon chain on the benzene ring instead of 4 or 6 carbons as in dye 1 or dye 3. This molecule could only be found in use as a reaction intermediate. The molecule registered as CAS # 16335-61-8 has a methyl group on the benzene ring is considered in antibacterial and antifungal63-64 studies as well as some dye synthesis studies.65-66

21

Figure 1.15: Structures of dyes 2 and 4 synthesized in this study.

Dyes 2 and 4 are substituted with the aniline carbonyl group and also substituted with a hydroxyl group on the same ring of the anthraquinone as shown in the molecular drawing in

Figure 1.15. Dye 4, 1-(4-butylanilino)-4-hydroxyanthraquinone, has been CAS registered with the number 71334-38-8. The applications of the dye mostly include the liquid crystal display application along with a study on printing with ink paste67 and another involved imparting color to optical filters68. The alkyl substituted anthraquinone dyes were found to possess higher photostability than comparable dyes with contained azo-linkages, especially when the alkyl and alkoxy groups were substituted in the para position on the aniline compound.69 CAS 116310-95-

3 is the 1,5 substituted isomer of dye 2. Dye 2 with the 6 carbon chain length has not yet been

CAS registered. The same structure with a five carbon chain instead of four or six has also been considered in liquid crystal display studies. (CAS 71334-39-9)

22

Figure 1.16: Structures of dyes 5 and 6 synthesized in this study.

Dye 5 and 6 structures are di-substituted with the aniline addition at the 1 and 4 positions on the anthraquinone ring as shown in Figure 1.16. Dye 5 (9,10-anthracenedione, 1,4-bis[(4- hexylphenyl)amino]), has a CAS registry number of 155106-61-9 and is included on a patent for heat-sensitive sublimation transfer recording cyan dyes.70 Dye 6 with the four carbon chain length, 9,10-anthracenedione, 1,4-bis[(4-butylphenyl)amino], is registered as 42980-14-3 in the

CAS system. This dye is included in the same heat sensitive transfer patent and other heat transfer related studies.71- 72 This dye was also considered in an in vivo dental study for the staining of the cavity floors. The preestablished use of the dye in an in vivo application adds confidence to the in vivo safety of the molecule. 73

1.6. Polymeric Colorants

Typically, color is imparted on to a polymeric material either through the addition of the colorant at the bulk stage of processing or the dye is imparted on the surface of the material later.

However, the investigation into polymeric colorants allowed for more possible ways of adding color to a polymer. Polymeric colorants can include: a polymer chain that is chemically modified so that it has chromophores as pendant groups, a non-colored polymer which has a chromophore as an end group, a polymer where chromophores are present in the backbone of a copolymer

23

which includes a functionalized dye and other monomers, or where the repeat unit of the polymer itself is a dye. 74 The present study focuses on the use of the last classification which can be called polymeric dyes, which are dyes that contain a polymerizable group so that they can form a polymer. These polymers are soluble in the medium in which they are applied or within the substrate to which they are being added.75 Depending on their application, polymeric colorants can either be applied so that they can easily be washed off of a material due to their large size not penetrating the surface and their high-water solubility76 or if they are integrated inside the polymer matrix, they show improved ability to not migrate in the matrix, be sublimed out, or extracted from the material.77 They also show less toxicity in comparison to the monomolecular versions of the dyes. Polymeric dyes have advantages over pigments in the fact that they are soluble in thermoplastics and they are less abrasive in processing.77 They also have shown fast leveling onto fabric along with excellent light fastness. As the molecular mass of the polymer increased, the fastness properties increased.78

Of the classifications of polymeric colorants, poly dyes have the potential for the highest color strengths due to the fact that each repeat unit is a dye itself. Variability exists with these poly dyes in the length of the polymers that can be produced. They can range from in the oligomer range to a polymer of high molecular weight.77

1.6.1. Proposed poly dye structures

Poly dyes of interest were selected from the Max Weaver Dye Library for testing in various solvents to assess their solubility as well as their melting points. A high melting point was desired so that the dye would not decompose during the mass coloration. The dye types which were selected were chosen for their low solubility in polar and non-polar solvents and for the availability in the Max Weaver Dye Library. The four dyes selected are shown in Table 1.2

24

along with data obtained about the samples in the Weaver dye library and their ID number within the library catalog.

Table 1.2: Dye structure drawings, identification numbers, colors, and approximate melting points for the four poly dyes considered for the solution dyeing portion of the project.

Dye Structure ID Number Color Melting Point (˚C)

X26955-42-2 Orange 260- 270

X26669-93 Orange 209-213

X26669-89 Orange 261-264

25

Table 1.2 continued

X26669-91 Orange 191-198

Dye X26669-89 having the butyl linking chain and dye X26669-91 were used in a solubility test, as representatives of this group of dyes and were found to be sparingly soluble in DMF, ethyl acetate, methanol, and hexane.

1.6.2. Mass coloration

Mass coloration, mass pigmentation, or dope dyeing is an alternative method for the coloration of all man-made fibers both regenerated and synthetic. The process involves the addition of pigments and/or dyes to the solution or melt of the polymer before the polymer has been spun into fibers. This method is economically viable when only a select number of colors are desired, and they are needed in large quantities. Small batches and changing colors often is less common using this process and much more expensive. The method leads to high shade uniformity since a large batch of the polymer and pigment are mixed thoroughly before spinning.

Some synthetic fibers can be heat sensitive and so the normal melt spinning procedure could cause damage to the fiber, whereas the solvent spinning method subjects the polymer to no additional heat outside of the spinning process. The efficiency of the use of the colorants is much higher for this process since practically all of the colorant is incorporated into the final colored fiber, whereas with dyeing the dye bath will always contain unexhausted dye.79 Due to the large size of the polymeric dyes, the option of traditional dyeing will lead to a dye loosely affixed to

26

the outside of the fiber and not through to the core. In this study, mass coloration will involve pressing PE powder and pellets with the chosen polymeric dyes from the Weaver Dye Library at

North Carolina State University (NCSU).

27

CHAPTER 2: Experimental

2.1. General

2.1.1. Materials

Solvents used were obtained from Fisher Scientific. For the synthesis of the dyes, Congo

Red test paper and potassium iodide-starch paper were used, sourced from Fisher Scientific.

Table 2.1 outlines the chemical and dyes used in the study along with information about the supplier and purity levels when appropriate.

Table 2.1: Chemicals and dyes used for synthesis along with supplier and purity levels.

Chemical Purity (%) Supplier 4- Butylaniline 97 TCI Dialuryldimethylammonium bromide 98 TCI Leucoquinizarin 98 TCI Propionic acid 99 TCI Boric acid 99.5 Sigma-Aldrich 1,4- Dihydroxyanthraquinone 95 Alfa Aesar Hexanaline 98 TCI HCl 37 Millipore NaNO2 99.7 Fisher 3-Methyl-1-phenyl-2-pyrazoline-5-one - TCI Na2CO3 - Fisher NaOH 90 Fisher Ethyl alcohol 70 VWR 2-Methoxyethanol 99 Acros Organics Silica gel 60 - Sigma Aldrich 1,5-Dichloroanthraquinone 96 Aldrich Polyethylene pellets - Aldrich Polyethylene powder - Aldrich

Dyes Provider Lot# C.I. Solvent Blue 35 Standard Colors INC. CX-050-0593032 C.I. Solvent Blue 59 Standard Colors INC. 2017-7-1-01065601 C.I. Solvent Violet 13 Standard Colors INC. 2016-10-39-01025980 X26955-42-2 Weaver Dye Library --- X26669-93 Weaver Dye Library ---

28

Table 2.1 continued. X26669-89 Weaver Dye Library --- X26669-91 Weaver Dye Library ---

2.1.2. Instruments

For dyeings, a Datacolor Ahiba Nuance Top Speed Rotary Dyeing Machine was used.

UV- visible (UV-VIS) spectra were recorded using an Agilent Technologies Cary 300 coupled with Cary Win UV software.

A Bransonic-3200 Ultrasonic Cleaner was used to help dissolve the dyes and dispersants in water.

Buchi Switzerland produced the V-850 Vacuum Controller, R210 Rotavapor, and B-491 heating bath, a type of rotary evaporation equipment, which was used for solvent evaporation after dye synthesis.

Melting points were determined on a Fisher-Johns melting point apparatus from Fisher Scientific and are uncorrected.

The plates used for thin layer chromatography were Millipore silica gel plates with UV254 indicator.

For HPLC an Alliance HPLC system, equipped with a Waters 2695 Separation Module and

Waters 2996 Photodiode Array Detector run by the Empower Pro software was used along with the Atlantis C18 (3.0 x 100 mm, 5 m) with a flow rate 1.0 mL/min, at room temperature and with the gradient elution as shown in Table 2.2.

29

Table 2.2: HPLC gradient conditions.

Time (min) Methanol 0.1% Formic Acid 15 min 30 min running running 0 0 48 52 3 3 70 30 10 25 70 30 15 30 48 48

Mass spectrometry analysis was carried out on the Thermo Fisher Scientific Exactive Plus MS, a benchtop full-scan Orbitrap mass spectrometer using Heated Electrospray Ionization (HESI).

Samples were dissolved in acetone and analyzed via LC injection into the mass spectrometer at a flow rate of 500 µL/min. The mobile phase used was acetonitrile with 0.1% formic acid (A) and water with 0.1% formic acid (B) and using the gradient shown in Table 2.3. The column was a

Thermo Hypersil Gold 50 x 2.1mm, particle size 1.9 µ. The mass spectrometer was operated in positive ion mode.

Table 2.3: Gradient conditions for LC-MS analysis.

30

NMR spectra were run on a Bruker 700 MHZ NMR instrument and analyzed using Bruker

Topspin software at the NCSU NMR Facility.

Microscopic images of the cross sections of the fibers were obtained using the Nikon Eclipse 50i

POL Polarizing Microscope using the 50x magnifying lens and a Nikon D5 Fi1 color camera.

The PE polymer films were prepared with a Carver Laboratory Press (Model B, Fred S.Carver,

Menomonee Falls, WI), after the polymer was melted in a Stabil-therm gravity oven from Blue

M.

2.2. Synthesis of dyes

2.2.1. Mono-azo dyes

Aniline (0.02 mol) was dissolved in a mixture of 30 mL water and 6 mL 36% HCl in a small beaker and the solution was cooled to 0˚C using an ice bath. NaNO2 (1.355g, 0.02 mol) was added slowly so that the temperature did not rise above 5˚C. The acidity was checked throughout the addition of the NaNO2 using Congo Red test paper and if the solution was no longer acidic then small amounts of HCl solution were added. After 30 min the solution was checked with starch paper to ensure that there was no residual HNO2. The coupler was prepared in a separate beaker with 3-methyl-1-phenyl-2-pyrazoline-5-one (3.449 g, 0.02 mol), NaOH (1.5 g), and

Na2CO3 (1.5 g) combined. The NaOH was added first to the beaker along with 50 mL of water before the Na2CO3 was dissolved while stirring. Then the 3-methyl-1-phenyl-2-pyrazoline-5-one was added, and the solution was cooled to 5˚C in an ice bath. Once cooled, the diazonium salt solution was slowly poured into the coupler solution. A pH of 8 was obtained using either HCl solution or NaOH solution. The reaction was stirred overnight and then heated to 70˚C to dissolve the dye and then cooled to room temperature to reprecipitate the more pure dye.

31

Dye 1: Purity: 100%,Yield 93%; m.p. 74∘ C; UV-Vis. spectrum: (휆max), 399 nm, Rf (hexane: ethyl acetate 4:1): 0.78, 1H NMR: δ 7.95-7.97 ( d, 2H), δ 7.41-7.44 ( m, 2H), δ 7.35-7.36 ( m,

2H), δ 7.26 ( t, 1H), δ 7.20-7.23 ( d, 2H), δ 2.60-2.63 ( m, 2H), δ 2.37 (s, 4H), δ 1.58-1.63 ( m,

2H), δ 1.30-1.32 ( m, 6H), δ 0.88-0.90 (m, 3H), 13C NMR (ppm) 157.9, 148.5, 141.5, 139.0,

138.1, 129.6, 128.9, 118.6, 115.89, 35.5, 31.7, 31.4, 28.9, 22.6, 14.1.

Dye 3: Purity: 100%, Yield 93%; m.p. 86∘ C; UV-Vis. spectrum: (휆max), 399 nm, Rf (hexane: ethyl acetate 4:1): .72, 1H NMR: δ 7.95-7.97 ( d, 2H), δ 7.41-7.44 (m, 2H), δ 7.35-7.36 ( m, 2H),

δ 7.26 ( t, 1H), δ 7.22-7.24 ( d, 2H), δ 2.63 ( m, 2H), δ 2.37 ( s, 4H), δ 1.56-1.60 ( m, 2H), δ 1.36-

1.37 (m , 2H), δ 0.94 ( m, 3H). 13C NMR (ppm) 157.9, 148.5, 141.1, 139.0, 138.1, 129.6, 128.9,

125.1, 118.6, 115.9, 35.2, 33.6, 22.3, 13.9, 11.8.

2.2.2. Mono-amilino substituted anthraquinone dyes

Quinizarin (9.6g, 0.04 mol), leucoquinizarin (2.6 g, 0.01 mol), boric acid (6 g, 0.1 mol), the aniline compound (0.06 mol), and 50 mL of ethanol were added in order into a 150 mL round bottom flask. A stir bar was added and then the round bottom was placed in a water bath set to

79˚C. A condenser was attached, and the reaction was heated and stirred overnight. The mixture was cooled, filtered and the solid was rinsed with ethanol. The filtered product was added to 400 mL of 7% NaOH solution and heated to the boil with stirring. The product was filtered again and rinsed with hot water. This alkaline rinse was used until the TLC of the product showed that the starting material had been removed.

Dye 2: Purity: 75%, Yield 60%; m.p. 70∘ C; UV-Vis. spectrum: (휆max), 571 nm, Rf (hexane: ethyl acetate 4:1): 0.81. 1H NMR: δ 11.79 (m, 2H), δ 8.35-8.39 (m, 2H), δ 7.76-7.82 ( t, 3H), δ

7.55 ( d, 1H), δ 7.18-7.21 ( t, 2H), δ 2.63 ( t, 2H), δ 1.64 ( m, 2H), δ 1.29-1.33 ( m, 4H), δ 0.90 (

32

t, 3H). 13C NMR (ppm) 187.4, 182.7, 158.0, 145.1, 140.33, 135.2, 129.5, 128.2, 126.9, 126.5,

126.3, 124.4, 113.6, 109.9, 35.5, 31.7, 31.5, 29.0, 22.6, 14.1.

Dye 4: Purity: 73%,Yield 75%; m.p. 97∘ C; UV-Vis. spectrum: (휆max), 571 nm, Rf (hexane: ethyl acetate 4:1): 0.67. 1H NMR: δ 11.79 (s, 1H), δ 8.35-8.39 (d, 2H), δ 7.76-7.82 ( m, 2H),

δ7.55-7.57 ( d, 2H), δ7.18-7.22 ( m, 4H), δ 2.62-2.65 (m, 2H), δ 1.57-1.63 ( m, 2H), δ 1.39-1.40 ( m, 2H) δ 0.96 ( m, 3H) . 13C NMR (ppm) 187.4, 182.7, 158.0, 145.1, 140.3, 136.5, 135.2, 132.9,

128.2, 126.9, 126.5, 126.3, 124.4, 113.6, 109.9, 35.2, 33.6, 22.4, 14.0.

2.2.3. Di-anilino-substituted anthraquinone dyes

Synthesis for the symmetrical di-anilino substituted anthraquinone dyes was performed based off of a synthesis procedure outlined by Mills et al.80 The aniline compound (18.06g, 0.1 mol), boric acid (1.41 g, 0.023 mol), leucoquinizarin (2.42 g, 0.01 mol), and propanoic acid (0.8 mL, 0.01 mol) were added to a double necked round bottom flask and refluxed under nitrogen at 160˚C.

For the beginning of the heating, a condenser and trap were attached which could collect the aqueous distillate. Once the water had been removed the solution was stirred at 160˚C for 4h.

The resulting residue was cooled to room temperature and a mixture of 150 mL water and 18 mL conc. HCl was prepared and used to wash the product into a beaker. The mixture was heated to

70˚C and stirred at that temperature for 15 min and then filtered while hot. The crude dye was purified through multiple methyl cellosolve treatments by dissolving the dye in the hot methyl cellosolve and then precipitating the dye by adding water in a drop-wise manner.

Dye 5: Purity: Yield 16%; m.p. 126∘ C; UV-Vis. spectrum: (휆max), 640 nm, Rf (hexane: ethyl acetate 4:1): 0.78, 1H NMR: δ 12.26 (s,1H), δ 8.38-8.39 (d, 2H), δ 7.74-7.76 (d, 2H), δ 7.46 (d,

2H), δ 7.26 (d,2H), δ 7.18-7.19 (d, 2H), δ 2.60-2.63 (m, 2H), δ 1.60-1.63 (m, 2H), δ 1.37-1.40

33

(m, 4H), δ 1.25 (m, 2H), δ 0.94 (t, 3H). 13C NMR(ppm) 183.2, 144.3, 139.8, 137.0, 134.5, 132.5,

129.4, 126.3, 125.0, 124.2, 111.2, 35.4, 31.7, 31.5, 29.0, 22.6, 14.1.

Dye 6: Purity: 72%, Yield 14%; m.p. 130∘ C; UV-Vis. spectrum: (휆max), 640 nm, Rf (hexane: ethyl acetate 4:1): 0.81, 1H NMR: δ 12.27 (s,1H), δ 8.38-8.39 ( d, 2H), δ 7.74-7.75 (d, 2H) δ

7.47 (d, 2H), δ 7.26 (d, 2H), δ 2.62 (m, 2H), δ 1.60-1.61 (m, 2H), δ 1.25-1.38 (m, 2H), δ 0.95

(t, 3H). 13C NMR (ppm): 183.2, 144.3, 139.8, 137.0, 134.5, 129.4, 126.3, 125.0, 124.2, 111.2,

35.1, 33.6, 29.7, 22.3, 22.3, 13.9.

2.3. Fiber dyeing

Dyeing studies were performed using commercial samples of PE. The dye and dispersant mixtures were prepared by adding 0.1g dye and 50 mL tetrahydrofuran into individual 50 mL beakers. DDDMAB (0.8 g, mol) dispersant and 50 mL tetrahydrofuran were mixed and the two solutions were then combined, and the solvent was evaporated. The remaining dye and dispersant mixture was then added to 100 mL of water and sonicated for 2.5 h. Each dispersion was then transferred to 250 mL volumetric flasks and diluted to volume. The fiber was prepared by washing 2 g of the North Carolina State (NCSU) supplied fiber and 1 g of the Commercial 2 fiber with soap and water the day before the dyeing step. For the NCSU supplied fibers, 50 mL of dye solution and 5 drops of acetic acid were added to the dyeing tube with the fiber. For

Commercial Fiber 2, 25 mL of the solution and 3 drops of acetic acid were added. The dyeing conditions were 30 min of ramping up to 130 oC and then 1 h of heating at 130 oC for a 25:1 liquor ratio and a bath containing 1% owf dye and 0.4% owf dispersing agent. After the dyeing process, the dyed fibers were reduction cleared (2% NaOH/ Na2S2O4) and then washed with acetone.

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2.4. Mass coloration of PE films

PE films were solution dyed with the selected poly dyes, the standard of C.I. Solvent

Violet 13 and the six synthesized dyes (dyes 1-6). The polydye (0.05 g) was mixed and ground using a mortar and pestle and combined with 0.1 g powdered PE. The pellet polymer (5 g) was softened in the oven at around 120 ˚C and once soft 0.075 g the powder and dye mixture was added so that 0.5% dyeing could be achieved. The polymer was heated and mixed occasionally for 1 h. Approximately 2 g of the polymer was sectioned off for pressing. The polymer was pressed between two Teflon sheets for 30 sec at approximately 2500 lb of pressure and a temperature of 110˚ C. After cooling slightly, the film was folded and repressed to further mix the system and achieve uniform dye distribution. A heated (60 ˚C) reduction clear (2 g/L NaOH,

2g/L NaS2O4, 5g/L Igepal CO-630 nonionic surfactant) step was performed on the films colored with the monomeric dyes (dyes 1-6) for 10 min per film.

2.5. Microscopy

Compound microscope images of the cross sections of the fibers were obtained by pulling the fibers mixed with a contrast filler fiber (either black filler for the yellow dyes or white trilobal nylon for the purple and green/blue dyes) through a piece of cork. The cork with the circle of fibers pulled through was then cut into thin slices with a razor blade so that the samples could be positioned on a glass slide, viewed through the microscope, and an image captured with the attached Nikon camera system.

2.6. Extraction studies

Following the dyeing, fastness studies were performed on the fibers and polymer films in physiological conditions both at room temperature and with heating (50˚C). The solvents used for the extraction study were chosen to mimic the environment inside the human body. The

35

solvent mixture contained 21.8 mL 70% ethanol and 30% water, and 17.9 mL hexane. NCSU fibers (1 m) were cut and massed and assigned to a dye number and then Commercial Fiber 2 of similar weight was prepared for the same dye. For the films, approximately 0.25 g of film was cut into two strips and used for the study. The first time that the fibers were added to the solvent mixture there was immediately the visible presence of dye in the hexane layer. The fibers were washed with soap and water with acetone. Then they were placed in a new solvent mixture and capped with rubber stoppers. They were put in a shaking bath at room temperature (21 ˚C) and were left for 24 h. The concentration of dye in solution after 24 h was calculated by performing

UV-Vis analysis on the hexane layer (no dye was present in the ethanol and water layer) to determine the absorbance and then the dye concentration. The same experimental set up was used for a second extraction study except for during the 24 h shaking period the samples were heated to 50˚C for the duration of the shaking. The films were also put through an additional room temperature extraction step after the films had been heated and stretched to increase the orientation of the polymer chains.

2.7. Mutagenic and toxicity studies

The procedure outlined in the Zwag et al. was followed with minor revisions.39 Bacteria strain YG1041 was used, as it has been shown in past studies to have the highest sensitivity, to the type of azo dyes involved with this study.81 The positive control was C.I. Disperse Blue 373 used in a 1:4 ratio which is a concentration of 0.0025 M. This positive control shows mutagenic responses with and without S9.

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CHAPTER 3: Results and Discussion

3.1. Mono-azo and Anthraquinone Dyes

3.1.1. NMR spectra of dyes

1H NMR for dyes 1 and 3 show two doublets very close together at the highest shifts in the area of 7.9 ppm. The aromatic protons are clustered in the 7.2 to 7.3 region. The protons in the alkyl chains gave the lowest shifts (ppm) and are in the same region as the singlet which represents the methyl group and the lone proton which is labeled on the structure as k. The 1H

NMR spectra for dyes 1 and 3 are shown in Figures 3.1 and 3.5.

13C NMR for mono-azo dyes 1 and 3 show peaks for the aromatic and carbons attached to the N-atoms in the range from 115.9 up to the highest peak at 157.9. The alkyl chain and the methyl group are represented by the peaks in the 35.5 to 11.8 ppm range. In this region there are five unique carbon environments in the dye 1 spectra and four unique environments represented in the dye 3 spectra. The 13C NMR spectra for dyes 1 and 3 are shown in Figures 3.2 and 3.6.

1H NMR spectra for dyes 2 and 4 with an OH group attached to the anthraquinone would have a peak around 16 ppm for the proton on the OH group but is out of range in these spectra.

The dye 4 spectrum extends above 13 ppm which then includes the peak for the proton on the amino group. The same aromatic peak patterns are seen for both dyes in the 7.3 to 8.0 ppm regions for the protons on the anthraquinone portion of the structure. The protons on the 6- carbon chain which are located in the middle of that hexyl chain are all in very similar environments and are represented by the multiplet around 1.2 to 1.4 ppm. The 1H NMR spectra for dyes 2 and 4 are shown in Figures 3.3 and 3.7.

13C NMR spectrum for single amine substituted dyes 2 and 4 show peaks in the 187.4 to

109.9 ppm range for the anthraquinone and arene carbons and peaks in the range from 35.5 to

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14.1 ppm for the alkyl chain. The greatest shift is observed for the carbonyl carbons in the anthraquinone system. For the hexyl chain on dye 2, the 5 unique carbon environments are represented by 5 peaks and for the butyl chain on dye 4, 4 unique carbon environments are also represented. The 13C NMR spectra for dyes 2 and 4 are shown in Figures 3.4 and 3.8.

1H NMR spectra for dyes 5 and 6 which are the di-anilino substituted anthraquinones show the peak for the proton on the amine near 12.3 ppm like in dyes 2 and 4. Also as in dyes 2 and 4 the aromatic protons are located in the 7.1 to 8.5 ppm range. The 1H NMR spectra for dyes

5 and 6 are shown in Figures 3.9 and 3.11.

13C NMR spectra for the di-anilino-substituted amino anthraquinone dyes 5 and 6 show less unique carbon environments due to the symmetry of the molecule in comparison to dyes 2 and 4 where one substitution was an OH group. The carbonyl groups in the anthraquinones are now in an identical environment and are represented by the peak at 183.2 ppm. The 1,4 carbons on the anthraquinone are now also in identical environments. The alkyl chains agree with the chemical shifts seen for the respective chain lengths on dyes 2 and 4. The 13C NMR spectra for dyes 5 and 6 are shown in Figures 3.10 and 3.12.

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Figure 3.1: 1H NMR spectra for dye 1.

39

CDCl3

Figure 3.2: 13C NMR spectrum for dye 1.

40

Figure 3.3: 1H NMR spectrum for dye 2.

41

CDCl3

Figure 3.4: 13C NMR spectrum for dye 2.

42

Figure 3.5: 1H NMR spectrum for dye 3.

43

CDCl3

Figure 3.6: 13C NMR spectrum for dye 3.

44

Figure 3.7: 1H NMR spectrum for dye 4.

45

CDCl3

Figure 3.8: 13C NMR spectrum for dye 4.

46

Figure 3.9: 1H NMR spectrum for dye 5.

47

CDCl3

Figure 3.10: 13C NMR spectrum for dye 5.

48

Figure 3.11: 1H NMR spectrum for dye 6.

49

CDCl3

Figure 3.11: 13C NMR spectrum for dye 6.

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3.1.2. Mass spectrometry of dyes

High resolution ESI mass spectrometry was used to analyze all of the synthesized dyes. Dye 1 which has a MW of 362.21, based on the structure C22H26N4O, underwent protonation to give an

[M+H]+ species. The peak in the mass spectrum was m/z 363.22 and is seen in Figure 3.13.

Figure 3.13: HR ESI mass spectrum for dye 1.

Dye 2 has a MW of 399.18 and underwent protonation to give an [M+H]+ species which resulted in a peak at m/z 400.19 as seen in the spectra in Figure 3.14. Two other peaks are seen at m/z

338.34 and m/z 421.16 which give delta m values of 61.84 and 21. The lower peak could be due to the fragmentation of a portion of the alkyl chain but comprises a very small relative abundance.

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Figure 3.14: HR ESI mass spectrum for dye 2.

Dye 3 has a MW of 334.18 and the resulting spectrum in Figure 3.15 showed a single peak at m/z 335.19 which would indicate the [M+H]+ ion.

Figure 3.15: HR ESI mass spectrum for dye 3.

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Dye 4 has a molecular weight of 371.15 and underwent protonation to give an [M+H]+ species.

The resulting spectrum in Figure 3.16 showed a m/z peak at 372.16 in the highest abundance and a smaller peak at 393.13 m/z.

Figure 3.16: HR ESI mass spectrum for dye 4.

Dye 5 has a MW of 558.32 and formed an [M+H]+ species which resulted in the peak at m/z

559.32 seen in Figure 3.17. There was also a peak at 406.32 which has a delta m from the base peak of 153.

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Figure 3.17: HR ESI mass spectrum for dye 5.

Dye 6 has a MW of 502.26 and underwent protonation to form an [M+H]+ species which was seen in the spectra in Figure 3.18 as the base peak at m/z 503.26. Other peaks present included m/z 776.23 and m/z 804.26 which have delta m values of 272.97 and 301 respectively.

Figure 3.18: HR ESI mass spectrum for dye 6.

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3.1.3. HPLC of crude dye mixtures

The HPLC results are shown in Figures 3.19 through 3.24 for each dye (1-6) after the purification steps outlined in the synthesis section. Dye 1, the 6-carbon substituted mono-azo dye gave a single peak at a retention time of 3.732 min. Dye 2, the 6-carbon substituted violet anthraquinone dye gave two peaks. The first had a retention time of 3.400 min and accounted for

25.20 % of the total product and the second peak had a retention time of 3.561 min and accounted for 74.80 %. The UV-Vis analysis of both peaks gave the same curve which would suggest that this is one large peak which represents the target violet dye. Dye 3, the 4-carbon substituted mono-azo dye, gave a single peak at the retention time of 2.738. This is a shift of almost 1 min from the peak arising from the 6-carbon analog. Dye 4, the 4-carbon violet anthraquinone showed two peaks. The first had a retention time of 3.400 min and accounted for

27.34 % of the total product. This corresponds to the violet dye and the retention time for the similar structure with the 6-carbon chain was at the same retention time of 3.400 min. The other peak had a retention time of 10.507 min and corresponded to 72.66 % of the product. This peak represents the di-substituted product, dye 6. The analysis of the crude dye 5 showed two peaks.

The first had a retention time of 4.211 and was 10.47 % of the total and the second peak was at

4.757 min and accounted for 89.53 % of the total. The UV-Vis analysis of the two peaks individually showed that the peak at 4.75 min corresponded to the profile of the mono- substituted anthraquinone and that the small peak at around 3 min which was not profiled in the percentage mixture was actually the di-substituted target. This shows that the first synthesis had a very low yield of the desired product. The HPLC analysis of dye 6 showed three peaks.

According to the UV-Vis of the products, the peak at 10.526 min corresponded to the di- substituted green-blue dye of interest and was 18.44 % of the crude mixture. The peak at 3.395

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min accounted for 72.00 % and the UV Vis corresponded to the mono-substituted profile of dye

2. The last peak is very close to the mono-substituted at a retention time of 3.047 min and accounts for 9.56 % of the crude dye mixture.

Figure 3.19: HPLC results from first synthesis of dye 1.

Figure 3.20: HPLC results from first synthesis of dye 2.

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Figure 3.21: HPLC results from first synthesis of dye 3.

Figure 3.22: HPLC results from first synthesis of dye 4.

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Figure 3.23: HPLC results from first synthesis of dye 5.

Figure 3.24: HPLC results from first synthesis of dye 6.

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3.1.4. Absorption spectra

The visible absorption spectra of dyes 1-6 were recorded in acetone. The results are summarized in Table 3.1 and in Figures 3.25- 3.30. The lambda max was not substantially affected by the change in alkyl length, from butyl to hexyl (dyes 1 vs 3, 2 vs 4, and 5 vs 6). This slight change in the neutral alkyl chains does not affect the conjugated electron-donor/acceptor system and so no bathochromic or hypsochromic shifts are seen. The bathochromic shift seen between dyes 2 and 4 versus dyes 5 and 6 was anticipated due to the addition of another anilino donating group. This structural modification led to the color change from purple to green-blue.

Table 3.1: Absorption spectral data for dyes 1 through 6.

Dye Lambda Max* Emax Color*

1 399 22,600 Yellow

2 571 10,300 Purple

3 399 21,900 Yellow

4 571 10,000 Purple

5 640 14,600 Green-Blue

6 640 14,600 Green-Blue

*in acetone

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Wavelength (nm) Abs ______400.00 0.866

Figure 3.25: Absorption spectrum of dye 1 with peak wavelength data.

Wavelength (nm) Abs ______580.00 0.385

Figure 3.26: Absorption spectrum of dye 2 with peak wavelength data.

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Wavelength (nm) Abs ______399.00 1.027

Figure 3.27: Absorption spectrum of dye 3 with peak wavelength data.

Wavelength (nm) Abs ______582.00 0.406

Figure 3.28: Absorption spectrum of dye 4 with peak wavelength data.

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Wavelength (nm) Abs ______639.00 0.409 403.00 0.189 Figure 3.29: Absorption spectrum of dye 5 with peak wavelength data.

Wavelength (nm) Abs ______639.00 0.428 403.00 0.195 Figure 3.30: Absorption spectrum of dye 6 with peak wavelength data.

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3.1.5. LogP estimations

The expected increases in LogP’s are observed as the longer alkyl chains were substituted onto the analog dye structures. The increase from these estimations is around 1 in LogP for every two carbons that were added. The D&C green analog increases steeply with the lengthening chains because the structure is di-substituted and so two chains are being lengthened. The

Solvent Yellow analog with the butyl substitution is estimated to have a similar partition coefficient as the C.I. Solvent Violet 13 dye which is currently industrially used for the coloration of UHMWPE sutures. This is a good indication that the hydrophobicity levels are similar between the two molecules and the C.I. Solvent Yellow 18 analogs should have reasonable hydrophobicity to be able to also dye UHMWPE fibers. The two tools used to calculate LogP highlight the difficulty which exists with accurate computer estimations of LogP but both tools show the similar trends of increasing hydrophobicity through chain lengthening.

Table 3.2 shows the compilation of the LogP calculations for dyes 1 through 6 as well as C.I.

Solvent Violet 13 from both the EPA Episuites calculation and the ChemDraw calculation.

Table 3.2. LogP estimations from EPA Episuites and ChemDraw for synthesized dyes and the Solvent Violet 13 standard. Dye Carbons In Episuite ChemDraw Chain LogKow LogP

C.I. Solvent Violet 13 1 6.24 3.98

Dye 4- Solvent 13 analog 4 7.71 5.23

Dye 2- Solvent 13 analog 6 8.69 6.07

Dye 6- D&C Green 6 analog 4 11.63 8.83

Dye 5- D&C Green 6 analog 6 13.60 10.5

Dye 3- Solvent Yellow 18 analog 4 6.57 5.32

Dye 1- Solvent Yellow 18 analog 6 7.55 6.16

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3.1.6. Dyed fiber images

Visual assessments of the dyed fibers showed a lower depth of shade for the C.I. Solvent

Violet 13 dyed fibers in comparison the depth of shade of all of the synthesized dyes. Dyes 2 and

4, analogs of the standard, gave a much deeper purple color signifying more dye uptake (Figure

3.33). Dyes 1 and 3 gave bright yellow colors on all fibers which again appear deeper in color than the standard (Figure 3.32). Dyes 5 and 6 gave a slightly lighter depth of shade than the other synthesized dyes, but a comparable depth to the standard (Figure 3.34). Between the C-4 and C-6 analogs of the dye structures the butyl substituted dyes (dyes 3, 4, and 6) all gave visibly darker depths than the hexyl substituted dyes. C.I. Solvent Blue 59 and C.I. Solvent Blue 35 contain di- substituted amino groups with shorter alkyl chains and gave uneven dyeings with region that are still lighter in color and other portions which dyed sufficiently (Figure 3.33). The molecular structures of C.I. Solvent Blue 59 and C.I. Solvent Blue 35 which were used in the dyeing study are shown in Figure 3.31.

A B

Figure 3.31: Molecular structures of C.I. Solvent Blue 35 (A) and C.I. Solvent Blue 59 (B).

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Commercial 2 Fiber NCSU Fiber

Figure 3.32: Commercial 2 and NCSU UHMWPE fibers dyed with dyes 1 and 3. The order of the samples from left to right: dye 3 on Commercial 2 fiber, dye 1 on Commercial 2 fiber, dye 3 on NCSU fiber, dye 1 on NCSU fiber.

Dye 2 Dye 4 D&C Violet 2

Figure 3.33: Commercial 2 and NCSU UHMWPE fibers dyed with dyes 2 and 4 and C.I. Solvent Violet 13. The order of the samples from left to right: dye 2 on NCSU fiber, dye 2 on Commercial 2 fiber, dye 4 on NCSU fiber, dye 4 Commercial 2 fiber, C.I. Solvent Violet 13 on Commercial 2 fiber.

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Dye 5 Dye 6

C.I. Solvent Blue 59 C.I. Solvent Blue 35

Figure 3.34: Commercial 2 and NCSU UHMWPE fibers dyed with dyes 5 and 6 and standards C.I. Solvent Blue 59 and C.I. Solvent Blue 35. Order of the samples from top left to bottom right: dye 5 on Commercial 2 fiber, dye 5 on NCSU fiber, dye 6 on Commercial 2 fiber, dye 6 on NCSU fiber, dye Solvent Blue 59 on NCSU fiber, dye Solvent Blue 35 on Commercial 2 fiber.

3.1.7. Microscopy Images

The 6-carbon substituted mono-azo dye was applied to both the Commercial 2 fibers and the NCSU fibers and are shown in Figure 3.35. Dark filler contrast fiber is seen in the corners of the Commercial 2 fiber image. The individual fibers in both images show color throughout the fiber which is indicative of good penetration of the dye.

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Figure 3.35: Dye 1 on Commercial 2 fiber with black thread contrast on the left and dye 1 on NCSU fiber on the right. The 6-carbon chain that was mono-anilino substituted anthraquinone is shown after dyeing both Commercial 2 and NCSU fibers in Figure 3.36. The color rendering is not accurate in these images, especially in the image of the NCSU fiber where the auto white setting made the fibers appear more pink than the true color. However, these images do show dye penetration into the center of the fibers. The white trilobal nylon contrast is seen in the image of the Commercial

2 fibers on the bottom left corner.

Figure 3.36: Dye 2 on Commercial 2 fiber with white thread contrast on the left and dye 2 on NCSU fiber on the right. The 4-carbon substituted mono-azo dyed fibers are shown in Figure 3.37 on both fiber types. Again, the dark filler contrast was used and is seen in the images. The reduction of light

67

allowed through because of the dark filler could be contributing to some of the darker appearing fibers which are near the filler. The images of the cross sections of the fibers show color throughout the fiber. This indicates that the dye penetrated the polymer matrix all the way through the fiber and not just on the surface.

Figure 3.37: Dye 3 on Commercial 2 fiber with black thread contrast on the left and dye 3 on NCSU fiber on the right. Dye 4 the anthraquinone analog of C.I. Solvent Violet 13 with a hexyl alkyl chain is shown in Figure 3.38 on both Commercial 2 fibers on top and the NCSU fiber on bottom. The white trilobal nylon contrast material is seen in both images. Again, color rendering has been altered due to the auto white setting. The images show high dye penetration into both types of fibers.

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Figure 3.38: Dye 4 on Commercial 2 fiber with white thread contrast on the left and dye 4 on NCSU fiber in the image on the right. Images from the di anilino-substituted anthraquinone dye on the two types of fibers

(Figure 3.39) shows greater variation in the depth of the shades of the fibers than the other dyes showed. However, each individual fiber does show consistent color throughout the cross section which indicated that the dye did still penetrate the whole fiber and not just the surface. The dyeing process should be optimized to ensure even dyeing between the fibers for a more even and dark color. Again, color constancy was not preserved in these images due to the whiteness correction functions. The fibers in the images appear more blue or purple than the actual color of the fibers when not under microscope lighting.

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Figure 3.39: Dye 5 on Commercial 2 fiber with white thread contrast on the left and dye 5 on NCSU fiber on the right. The 4-carbon double substituted anthraquinone dyed fibers as shown in Figure 3.40 show similar results as the six carbon dye with the variety in depths of shades of the fibers, but high dye penetration through the cross sections of the individual fibers. The white contrast trilobal nylon is seen in both images as the contrast fiber.

Figure 3.40: Dye 6 on Commercial 2 fiber with white thread contrast on the left and dye 6 on NCSU fiber on the right. The C.I. Solvent Violet 13 dye which was used on the Commercial 2 fiber is monosubstituted with a methyl group instead of the four or six carbons like dyes 2 and 4. The image of the cross sections of these dyes (Figure 3.41) shows less consistency throughout the fibers. It shows less level uptake of the dye into the whole cross section of the polymer matrix.

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The less hydrophobic dye may not have as much ability to consistently, fully penetrate the polymer matrix.

Figure 3.41: C.I. Solvent Violet 13 dye on Commercial 2 fiber with white thread contrast.

3.1.8. Extraction studies

UV-Vis analysis on the extraction solvent was performed on the hexane layer, since no dye could be detected in the polar ethanol/ water layer. The absorption results were below 0.1 for all samples both heated and at room temperature for all dyes besides the mono-azo dyes 1 and 3.

The low absorption readings increased the error associated with those values and indicates the generally very low levels of dye which were extracted over the course of the study.

In order to compare the two dyeing studies and to account for the weight variation between each fiber sample that was used, a conversion was made so the numbers shown in Table

3.3 and Appendix D are in the units of mg dye extracted per 1 g of fiber. This amount of fiber from Commercial 2 would correspond to around 67 m of suture material which is commonly sold in 30-in lengths. This conversion means that from the highest extraction value observed about 4 mg of dye would be extracted from approximately 88 full suture strings (45 µg per suture).

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From the results of the extraction as shown in Table 3.3., The azo dyes (1 and 3) consistently had the highest amount of dye that was extracted from the fiber. The di-substituted anthraquinone molecules (dyes 5, 6, C.I. Solvent Blue 59, and C.I. Solvent Blue 35) consistently showed the lowest extraction levels, especially in the heated samples. The 4-carbon substituted dyes (dyes 6, 4, and 3) showed, in a majority of cases, a slightly lower amount of extracted dye in the solvent solution than the hexyl substituted dyes. The heating of the fiber during the extraction at 50˚ C for 24 h had a larger influence on the mono-azo dyes and mono- anilino substituted dyes than it did on the di-substituted ones. The larger size of the di-anilino substituted molecules could mean that the added energy to the system from the heat was still not enough to extract the molecules from the polymer matrix. Figures 3.42 through 3.45 show the UHMWPE fibers after the heated extraction study and an extended time of over 5 mon of being submerged in the extraction solution.

Table 3.3: The mg of dye extracted from the two fibers types corrected to one gram of fiber in both room temperature and heated conditions. Room Temperature Heated Study Carbons Commercial NCSU Commercial NCSU Dye in Chain 2 Fiber Fiber 2 Fiber Fiber 5 12 0.53 0.29 0.85 0.62 6 10 0.29 0.16 0.43 0.60 C.I. Solvent Blue59 2 - 0.10 - 0.24 C.I. Solvent Blue 35 4 0 - 0.29 - 2 12 0.48 0.69 1.27 3.99 4 10 0.47 0.51 1.17 2.69 C.I. Solvent Violet 13 7 0.41 - 0.83 - 1 6 0.50 1.42 1.66 3.87 3 4 0.59 0.63 1.50 3.13

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Figure 3.42: Dyes 2 (left) and 1 (right) on Commercial 2 and NCSU fiber after the heated extraction study and additional 5 mon submersion in the extraction medium.

Figure 3.43: Dyes 3 (left) and 4 (right) on Commercial 2 and NCSU fiber after the heated extraction study and additional 5 mon submersion in the extraction medium.

Figure 3.44: Dyes 6 (left) and 5 (right) on Commercial 2 and NCSU fiber after the heated extraction study and additional 5 mon submersion in the extraction medium.

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Figure 3.45: Dyes C.I. Solvent Violet 13 (left), C.I. Solvent Blue 59 (center), and C.I. Solvent Blue 35 (right) on Commercial 2 and NCSU fiber after the heated extraction study and additional 5 mon submersion in the extraction medium.

3.1.9. Mutagenicity data

The dose responses for the modified Ames procedure showed that dyes 1 through 6 in concentrations ranging from their limit of solubility, 1.5 times diluted, and 3 times diluted did not show signs of being mutagens when compared to the background count of revertant colonies from controls where no dye was present. The dyes were tested both with S9 to determine the effects of metabolism on the dyes, and without S9 to determine their properties before metabolism. All dyes tested negative at all concentrations under both conditions, as none showed at least twice the number of revertant colonies verses the background count. Data for the mean revertant colony count as well as the standard deviation for the counts for all tested dyes as well as the controls are shown in Table 3.4. The Tukey plot shown in Figure 3.46 next to the Anova graph shows the statistical comparisons of the averages of the colony counts from all of the dyes tested and the controls. The positive control, C.I. Disperse Blue 373, is represented by the highest circles which show no overlap with the control which indicates that they are statistically different values. All other dyes show statistical similarity to the control number of revertant colonies.

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Table 3.4: Mean and standard deviations of the counts of the revertant colonies for three doses of each dye, negative controls, solvent controls, and a positive control C.I. Disperse Blue 373. Without S9 With S9 Dilution Sample Factor Mean SD Mean SD

Negative Control 0 21.3 4.0 19.1 7.5 Solvent Control 0 22.9 7.9 22.6 6.6 C.I. Disperse Blue 373 1:4 51.8 7.6 97.8 11.7 1 1 24.0 4.5 22.0 4.3 1 1.5 27.0 3.2 21.8 7.1 1 3 29.3 5.1 24.5 3.3 2 1 26.5 3.3 20.5 3.7 2 1.5 24.3 6.8 23.8 6.1 2 3 30.0 5.4 17.3 2.3 3 1 28.3 6.0 11.5 0.7 3 1.5 24.8 3.6 27.7 7.5 3 3 29.8 1.5 19.5 3.4 4 1 23.8 4.0 17.0 9.6 4 1.5 24.3 5.0 19.3 8.1 4 3 17.8 4.3 26.5 5.4 5 1 18.3 7.1 25.8 8.7 5 1.5 21.3 3.3 20.5 10.0 5 3 21.3 4.3 16.5 6.2 6 1 24.5 5.7 16.5 4.4 6 1.5 25.5 6.5 26.0 9.8 6 3 25.8 4.1 21.8 5.0

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Figure 3.46: Anova plotted results for all three doses of the dyes and the standards along with the Tukey-Kramer analysis of means. Since azo dyes have been identified in general as having a higher possibility of being a mutagen, dye 3 was subjected to a single dose miniaturized Ames test with bacteria strain TA97 as well. This strain has increased ability for detecting mutagens which intercalate into the DNA strand. This preliminary single dose response showed no mutagenicity from dye 3.

3.2. Poly dyes

Mass coloration of the polymer films was successful for all dye types. The proper grinding of the dye and PE powder led to even dye distribution. Figure 3.47 shows a representative PE film which has been dyed to 0.5% dyeing with the polymeric dye X26669-91.

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Figure 3.47: PE film colored with polymeric dye X26669-91 at 0.5% dyeing.

After the films were cut and used for the heated and room temperature extraction studies, the films were heated with a heat gun and manually stretched. Stretching the films was an attempt to orient the polymer chains to lead to less swelling of the films and thus a lower level of dye extracted from the films. The stretching of the films also led to a thinner width. This led to a higher surface area when a comparable mass of film was used for the stretching study

3.2.1. Extraction Studies

For the extraction studies with the solution dyed PE films, films were prepared using

D&C Violet 2, and the four previous listed poly dyes from the Weaver Dye Library. Most of the films colored with the polymeric dyes showed no visible coloration of the extraction solution and any leached dye would be below the limit of detection of the UV-VIS instrument. This was predicted due to the larger size of the poly dye molecules. It was predicted that the molecules would be too large to leave the polymer matrix and that their high level of hydrophobicity would lead to a high level of integration into the PE film. For the polymeric dyes the only time when any detectible dye was in the extraction medium was for dye X26669-89 when it was heated, and

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for dye X26669-91 from the stretched film and from the heated extraction medium study. Dye -

91 contains a benzene ring in the linkage between the repeat units and that total of 8 additional carbons is the largest carbon chain link between the repeat units used in this study. Figures 3.48 through 3.51 are images of the PE film samples dyed with the four polymeric dyes. The samples depicted include the film after pressing, after the room temperature extraction study, after the heated extraction medium study, and after being stretched and then subjected to a room temperature extraction study.

The absorption values seen from the stretched films were only lower than the first 20˚ C study in three cases. Since the films of not highly oriented PE allowed for a larger amount of swelling in the extraction medium in the heated 24-h and room temperature 24 h studies, the large majority of the dye left the film and went into the extraction medium.

Many values between the heated and room temperature studies seem close in size for dyes 1-6, because almost all of the dye was extracted from the film in both cases. When the absorption values for dyes 1- 6 were used to calculate the mg of dye which had been extracted, it was found that approximately one third of dyes 5 and 6 were extracted, two thirds of dyes 2 and

4 were extracted, and for dye 1 about 90% of the dye was extracted and for dye 3 between 60 to

70% of the dye was extracted. D&C Violet 2 had around 60% dye extracted across all studies.

This high extraction level in the lower molecular weight PE with a low level of orientation in the polymer chains emphasizes the importance of a highly crystalline nonpolar environment for these dyes. For a matrix that can allow a higher amount of swelling, the dyes can be removed from the polymer film at a consistently high level. Figures 3.52 through 3.58 depict the PE film strips dyed with dyes 1-6 as well as C.I. Solvent Violet 13. The strips shown include the film

78

before extraction, after room temperature extraction, 50 ˚C extraction, and after being stretched and then subjected to a room temperature extraction.

Table 3.5: Absorption values from the solvent extraction media from PE films at 50 ˚C, 20 ˚C, and stretched film conditions. Heated Dye 20˚C Stretched 50˚ C C.I. Solvent Violet 13 1.065 0.731 1.087 1 3.336 2.848 3.25 2 0.894 0.783 1.15 3 2.134 2.438 2.565 4 0.83 0.894 1.066 5 0.532 0.577 0.703 6 0.265 0.275 0.295 X26669-91 0 0.019 0.07 X26669-93 0 0 0 X26955-42-2 0 0 0 X26669-89 0 0 0.022

Figure 3.48: PE film cut into strips after dyeing with polymer dye X26669-91. From left to right: after pressing, after room temperature extraction, after 50 ˚C extraction and after being stretched and then subjected to a room temperature extraction.

79

Figure 3.49: PE film cut into strips after dyeing with polymer dye X26955-42-2. From left to right: after pressing, after room temperature extraction, after 50 ˚C extraction and after being stretched and then subjected to a room temperature extraction.

Figure 3.50: PE film cut into strips and dyed with polymer dye X26669-89. From left to right: after pressing, after room temperature extraction, after 50 ˚C extraction and after being stretched and then subjected to a room temperature extraction.

80

Figure 3.51: PE film cut into strips after dyeing with polymer dye X26669-93. From left to right: after pressing, after room temperature extraction, after 50 ˚C extraction and after being stretched and then subjected to a room temperature extraction.

Figure 3.52: PE film cut into strips after dyeing with dye 1. From left to right: after pressing, after room temperature extraction, after 50 ˚C extraction and after being stretched and then subjected to a room temperature extraction.

81

Figure 3.53: PE film cut into strips after dyeing with dye 2. From left to right: after pressing, after room temperature extraction, after 50 ˚C extraction and after being stretched and then subjected to a room temperature extraction.

Figure 3.54: PE film cut into strips after dyeing with dye 3. From left to right: after pressing, after room temperature extraction, after 50 ˚C extraction and after being stretched and then subjected to a room temperature extraction.

82

Figure 3.55: PE film cut into strips after dyeing with dye 4. From left to right: after pressing, after room temperature extraction, after 50 ˚C extraction and after being stretched and then subjected to a room temperature extraction.

Figure 3.56: PE film cut into strips after dyeing with dye 5. From left to right: after pressing, after room temperature extraction, after 50 ˚C extraction and after being stretched and then subjected to a room temperature extraction.

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Figure 3.57: PE film cut into strips after dyeing with dye 6. From left to right: after pressing, after room temperature extraction, after 50 ˚C extraction and after being stretched and then subjected to a room temperature extraction.

Figure 3.58: PE film cut into strips after dyeing with C.I. Violet 13. From left to right: after pressing, after room temperature extraction, after 50 ˚C extraction and after being stretched and then subjected to a room temperature extraction.

84

CHAPTER 4: Conclusions

The results of this study indicate that the six target dyes were synthesized and were suitable for dyeing UHMWPE fibers. All six dyes showed similar or improved color depth on PE fibers in comparison to C.I. Solvent Violet 13 dye which is currently used to dye PE sutures. While increased hydrophobicity increased depth of color on the fibers, there was an upper limit as to the length of alkyl chains incorporated into the prepared dyes. The leaching behavior of dyed PE fibers using standard media showed, in most cases, there was little to no color in the extract. The di-anilino substituted anthraquinone dyes gave the lowest extract concentrations and the mono-azo dyes generally the highest. Mutagenicity testing of the synthesized dyes via a miniaturized Ames procedure indicated that the dyes were non-mutagenic across a series of dose levels.

The mass coloration of the PE films using poly dyes and the monomeric dyes showed that sufficient coloration could be achieved and that in most cases no dye was extracted from the films. In the case of the poly dye with the longest carbon chain linker between repeat units only a very small amount of dye could be extracted.

4.1. Future Work

Future work in this study would include:

1. Further optimization of dyeing procedure for di-anilino substituted anthraquinone dyes 5

and 6 for maximum shade depth on PE fibers.

2. TOF-SIMS could be performed on the dyed fibers for more quantitative dyeing level

information.

3. Ames mutagenicity testing with more Salmonella strains on dyes 1-6.

85

4. Perform the Ames test on the polymeric dyes included in this study.

5. Testing of polymeric dyes in UHMWPE fibers instead of PE films.

6. Carbon dioxide dyeing could be implemented in conjunction with dyes 1-6 to possibly

lead to even higher dyeing efficiency without the water-based process.

86

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APPENDICES

94

Appendix A. Raw colony count data for three dose miniaturized Ames test.

Sample Concentration Number of Revertant Colonies Negative Control 0 23 23 28 22 18 16 23 17 Solvent Control 0 19 27 19 32 18 36 15 25 15 11 32 26 C.I. Disperse Blue 373 0.0025 41 59 53 54 h1 1 0.0694 29 25 18 24 h1 1.5 0.0463 24 28 25 31 h1 3 0.0231 24 26 35 32 h2 1 0.14 27 24 24 31 h2 1.5 0.0933 22 18 23 34 h2 3 0.0466 24 35 34 27 h3 1 0.1954 25 37 27 24 h3 1.5 0.1303 24 28 20 27 h3 3 0.0651 29 31 31 28 h4 1 0.1145 26 19 28 22 h4 1.5 0.0763 31 20 21 25 h4 3 0.0382 16 23 13 19 h5 1 0.0406 17 26 12 h5 1.5 0.0271 25 18 23 19 h5 3 0.01353 22 18 27 18 h6 1 0.0876 27 28 27 16 h6 1.5 0.0584 31 17 24 30 h6 3 0.0292 22 31 23 27 C.I. Disperse Blue 373 S 0.0025 81 100 102 108 h1 1S 0.0694 28 22 20 18 h1 1.5S 0.0463 27 12 21 27 h1 3S 0.0231 22 22 29 25 h2 1S 0.14 23 24 16 19 h2 1.5S 0.0933 28 19 30 18 h2 3S 0.0466 16 20 16 h3 1S 0.1954 11 12 h3 1.5S 0.1303 32 19 32 h3 3S 0.0651 19 15 23 21 h4 1S 0.1145 20 29 7 12 h4 1.5S 0.0763 22 29 16 10 h4 3S 0.0382 34 22 23 27

95

h5 1S 0.0406 28 20 18 37 h5 1.5S 0.0271 13 27 31 11 h5 3S 0.01353 13 19 10 24 h6 1S 0.0876 11 19 15 21 h6 1.5S 0.0584 13 35 32 24 h6 3S 0.0292 25 26 21 15 Negative Control S 0 14 23 23 28 14 29 11 11 Solvent Control S 0 31 30 19 19 13 11 26 19 24 25 31 23

Appendix B. Graphs of dose responses for Ames assay for all six dyes.

DYE 1

HK1/-S9 Solvent and Negative Control 373 Positive Control HK1/+S9

60

50

40

30

20

REVERTANT COLONIESREVERTANT 10

0 0 0.5 1 1.5 2 2.5 3 3.5 DILUTION FACTOR

96

DYE 2

HK2/-S9 Solvent and Negative Control 373 Positive Control HK2/+S9

60

50

40

30

20 REVERTANT COLONIESREVERTANT 10

0 0 0.5 1 1.5 2 2.5 3 3.5 DILUTION FACTOR

DYE 3

HK3/-S9 Solvent and Negative Control 373 Positive Control HK3/+S9

60

50

40

30

20

REVERTANT COLONIESREVERTANT 10

0 0 0.5 1 1.5 2 2.5 3 3.5 DILUTION FACTOR

97

DYE 4

HK4/-S9 Solvent and Negative Control 373 Positive Control HK4/+S9

60

50

40

30

20 REVERTANT COLONIESREVERTANT 10

0 0 0.5 1 1.5 2 2.5 3 3.5 DILUTION FACTOR

DYE 5

HK5/-S9 Solvent and Negative Control 373 Positive Control HK5/+S9

60

50

40

30

20 REVERTANT COLONIESREVERTANT 10

0 0 0.5 1 1.5 2 2.5 3 3.5 DILUTION FACTOR

98

DYE 6

HK6/-S9 Solvent and Negative Control 373 Positive Control HK6/+S9

60

50

40

30

20 REVERTANT COLONIESREVERTANT 10

0 0 0.5 1 1.5 2 2.5 3 3.5 DILUTION FACTOR

Appendix C. Absorption spectra for standard dyes used for dye studies of C.I. Solvent Blue 35,

C.I. Solvent Blue 59, and C.I. Solvent Violet 13.

Sample Name: Solvent Blue 35 Peak Table Peak Style Peaks Peak Threshold 0.0100 Range 700.00nm to 350.00nm

Wavelength (nm) Abs ______642.00 0.941 594.00 0.792

99

Sample Name: Solvent Blue 59 Peak Table Peak Style Peaks Peak Threshold 0.0100 Range 700.00nm to 350.00nm

Wavelength (nm) Abs ______639.00 0.856 593.00 0.763

Sample Name: Solvent Violet 13 Peak Table Peak Style Peaks Peak Threshold 0.0100 Range 700.00nm to 350.00nm

Wavelength (nm) Abs ______577.00 0.659

Appendix D. Raw absorbance data from the second fiber extraction study.

100

Absorbance Room Sample Temperature Heated 1 0.288 0.759 1A 0.103 0.312 2 0.064 0.444 2A 0.05 0.142 3 0.175 0.833 3A 0.171 0.39 4 0.059 0.345 4A 0.054 0.141 5 0.048 0.070 5A 0.088 0.089 6 0.019 0.077 6A 0.035 0.053 13A 0.055 0.115 59 0.022 0.058 35A 0 0.054

Appendix E. First dyeing study extraction data in units of mg of dye per gram of fiber of the two

UHMWPE fiber types tested.

Room Temperature Heated Study Number of Carbons in Atex NCSU Atex NCSU Dye Chain Fiber Fiber Fiber Fiber 5 12 4.06 0.34 2.36 1.14 6 10 1.45 0.49 4.06 0.77 59 2 6.70 0.12 0.00 0.55 35 4 0.00 - 2.37 - 2 12 1.17 1.45 2.94 7.10 4 10 1.12 0.33 2.38 6.66 1 6 1.90 2.16 0.00 6.34 3 4 12.34 2.23 12.34 9.28

101