THE EXTRACTION AND CHARACTERIZATION OF NATURAL DYE

FROM PAWPAW (CARICA PAPAYA)

BY

CHUKS MOSES CHINEMEREM

REG NO
20151013433

SUBMITTED TO
DEPARTMENT OF POLYMER AND TEXTILE ENGINEERING
SCHOOL OF ENGINEERING AND ENGINEERING TECHNOLOGY
FEDERAL UNIVERSITY OF TECHNOLOGY, OWERRI

IN PARRTIAL FULFILLMENT OF THE REQUIREMENT FOR THE

AWARD OF BACHELOR’S DEGREE IN ENGINEERING (B.ENG)
IN POLYMER AND TEXTILE ENGINEERING

AUGUST, 2021
 CERTIFICATION

This is to certify that the research “The Extraction and Characterization of natural

dye from pawpaw fruits (Carica Papaya)” was carried out by CHUKS MOSES

CHINEMEREM with (Matriculation Number: 20151013433) in partial fulfilment

of the requirements for the award of bachelor’s degree (B.Eng) of the Department

of Polymer and Textile Engineering in the School of Engineering and Engineering

Technology, Federal University of Technology Owerri.

……………………………… ……………….
Engr. Dr. (Mrs) G.C. Date
(Project Supervisor)
Onuegbu

……………………………… ……………….
Engr. Dr. H.C. OBASI Date
(H.O.D)

……………………………. …………….…
External Examiner Date
./

……………………………. ………………
./ CHUKS MOSES C. Date
(Student)

II
 DEDICATION

I dedicate this project to God Almighty, my creator, my strong pillar, my source of

inspiration, wisdom, knowledge and understanding. He has been the source of my

strength throughout this program and on His wings only have I soared.

III
 ACKNOWLEDGEMENT

First and foremost, I would like to express my sincere thanks to God Almighty for
His showers of blessings and provision throughout this research work.

My sincere appreciation also goes to my supervisor Engr. Dr. (Mrs) G.C. Onuegbu
whose contribution and constructive advice has pushed me to explore deeply the
kind of efforts I have exerted to make this work as original as it can be. Thanks to
her, I have experienced true research and my knowledge on the subject matter
(Extraction and Characterization of natural dye from Pawpaw Fruits) has been
broadened. I will never forget your kind gesture ma.

My grateful thanks are also extended to my Head of Department Engr. Dr. H.C.
Obasi for his advices and fatherly love. To my course adviser who has been with
me from the start with patience, love and guidance, thank you sir. To all the
lecturers and lab technologist in the department of polymer and textile engineering
and also to my fellow project members (Nkemjika Mildred, Tukur Mahmud, John
Caleb, Ezekwe Victor, Awuzie Cosmos and Tony) for their support and
contributions to the success of this work. I say may God bless you abundantly.

And, also to Mr. Nti Simeon, the lab technologist at the School of Agriculture and
Agriculture Technology (SAAT), Federal University of Technology, owerri
(FUTO) for his assistance, support and guidance in using all the equipment for the
purpose of this experiment.

Finally, my utmost regard also goes to my parents, Mr. and Mrs. Chuks Maduka
who painstakingly laid the foundation for my education, giving it all it takes. For
being my backbone and my support from day one. My love for you all can never
be quantified. God bless you.

IV
 ABSTRACT

The extraction and characterization of natural dye from pawpaw was studied

during this research. Acid, Alkaline, Ethanol and Aqueous solvents were used in

the extraction of the dye. The dyes extracted were analyzed using Fourier

Transform Infra-Red (FTIR) analysis and Ultra-visible (UV) spectroscopy. By

using the Fourier Transform Infra-Red (FTIR) analysis, the extracted dyes were

subjected to different functional groups at different wavelengths and the Ultra-

visible (UV) spectroscopy shows that the maximum absorbance of the pawpaw

samples were between 420-480 nm in the order of hierarchy Ethanol

Extract<Aqueous Extract<Alkaline Extract<Acidic Extract.

The specific gravity of the dye samples was determined when compared with water

in order to know their solubility state and Alkaline Extract was the highest of all in

the order of Alkaline Extract> Acidic Extract > Ethanol Extract > Aqueous

Extract. The physical properties of the solvents and the substrate affected the dye

extracts as well.

V
 TABLE OF CONTENT

Certification ii

Dedication iii

Acknowledgement iv

Abstract v

Table of content vi

List of tables ix

List of figures x

CHAPER ONE

INTRODUCTION

1.1 Background of study 1

1.2 Natural dye 3
1.2.1 Classification based on origin 3
1.2.2 Classification based on colour 4
1.2.3 Classification based on application 5
1.2.4 Classification based on chemical constitution 7

1.3 Advantages of natural dyes 9

1.4 Disadvantages of natural dyes 10

1.5 Properties of natural dyes 11

1.6 Transmittance of light in a spectrophotometer 12

VI
1.7 Beer-lambert law of absorption 14

1.7.1 Sources of error in Beer’s law 17

1.8 Problem statement 17

1.9 objectives of study 17

1.10 justification of study 18

1.11 scope of study 18

CHAPTER TWO

LITERATURE REVIEW

2.1 Overview 19

2.2 Dye 19

2.3 Fourier Transform Infra-Red (FTIR) 24

2.4 Principle of Infra-Red Spectroscopy 28

2.4.1 Infra-Red Spectroscopy 28

CHAPTER THREE

MATERIAL AND METHODS

3.1 Material 30

3.2 Apparatus 31

3.3 Preparation of materials and solvents 33

VII
3.4 Preparation of the organic solvent 33

3.5 Extraction of the natural dye 33

3.6 Ultraviolet (UV) Visible spectroscopy analysis 34

3.7 Fourier Transform Infra-Red (FTIR) analysis 35

3.8 Determination of specific gravity 35

3.9 Determination of pH 36

CHAPTER FOUR

RESULTS AND DISCUSSION

4.1 Fourier Transform Infra-Red (FTIR) analysis 37

4.2 Ultraviolet (UV) Visible spectroscopy analysis 47

4.3 Specific Gravity 59

4.4 pH 60

CHAPTER FIVE

Conclusion 61

Recommendation 61

Reference 63

VIII
 LIST OF TABLES

Table 4.1: FTIR Analysis of Acidic Extract of pawpaw 37

Table 4.2: FTIR Analysis of Alkaline Extract of pawpaw 40

Table 4.3: FTIR Analysis of Ethanol Extract of pawpaw 42

Table 4.4: FTIR Analysis of Aqueous Extract of pawpaw 45

Table 4.5: The wavelength of Aqueous Extract and Acidic Extract 47

Table 4.6: The wavelength of Aqueous Extract and Alkaline Extract 49

Table 4.7: The wavelength of Aqueous Extract and Ethanol Extract 51

Table 4.8: The Frequency of Aqueous Extract and Acidic Extract 53

Table 4.9: The Frequency of Aqueous Extract and Alkaline Extract 55

Table 4.10: The Frequency of Aqueous Extract and Ethanol Extract 57

Table 4.11: Specific Gravity of the Extracts 59

IX
 LIST OF FIGURES

Figure 4.1: Acidic Extract of pawpaw 38

Figure 4.2: Alkaline Extract of pawpaw 41

Figure 4.3: Ethanol Extract of pawpaw 43

Figure 4.4: Aqueous solvent of pawpaw 46

Figure 4.5: Absorbance verse wavelength of Aqueous and Acidic Extract 48

Figure 4.6: Absorbance verse wavelength of Aqueous and Alkaline Extract 50

Figure 4.7: Absorbance verse wavelength of Aqueous and Ethanol Extract 52

Figure 4.8: Absorbance verse frequency of Aqueous and Acidic Extract 54

Figure 4.9: Absorbance verse frequency of Aqueous and Alkaline Extract 56

Figure 4.10: Absorbance verse frequency of Aqueous and Ethanol Extract 58

X
 CHAPTER ONE

1.0 INTRODUCTION

1.1 Background of study

Dyeing is very old as civilization is. In the nineteenth century, natural dyes were

used in the colouration of textile fabrics. Primitive dyeing techniques includes

sticking plants to fabric or rubbing crushed pigments into cloth. Natural dyes

produce soft shades as compared to synthetic dyes. Dye as an organic compound is

composed of a chromophore (the coloured portion of the dye molecule) and an

auxochrome (which slightly alters the colour). The dye is soluble because of the

presence of auxochrome and is a site for bonding the fiber.

Dyes are molecules dissolved in water to penetrate the fiber immersed into the

formed solution. They are used to colour fibers or yarn before they are produced as

clothes. Dyeing is a means of adding colour to a fiber, yarn and fabric. When a

textile fiber is immersed into a dye bath, the fiber material tends to absorb the

molecules of the dye thereby accumulating the absorbed dye. The textiles that are

dyed vary in their ability to hold colour.

Most of the dye used in leather, paper, plastic, textile, food and pharmaceutical,

automobile industry, etc. are synthetic organic compounds which are classified by

two ways; according to their chemical structure and areas of application. Some of

1
the dyes according to chemical classification are azo dyes, anthraquinone dyes,

acridine dyes, indophenols dyes, rhdomine dyes, fluorone dyes, etc. According to

another method some of the classes of dyes are acid dyes, basic dyes, mordant

dyes, direct dyes, vat dyes, reactive dyes, azoic dyes, sulfur dyes and dispersive

dyes. The dye is soluble because of the presence of auxochrome and is a site for

bonding the fiber.

Dyes are natural or synthetic colourants which used in various industries like pulp,

paper, textile; paint etc. Dyes have high molar absorptivity properties and are

highly visible. Dyes are the important pollutants in water and are highly toxic in

nature to aquatic life.

An experiment was carried out on some dyes which shows that most dyes are

carcinogenic in nature and requires the separation and advanced treatment before

being introduced into the conventional water sources. Some dyes such as Reactive

dyes are most used with azo dyes in combination with different reactive groups.

They bind to textile fibers by formation of covalent bonds. They have favorable

characteristics such as bright colour, easily water soluble, simple application

technique and low energy consumption.

2
1.2 Natural dye

Colouring matter extracted from the roots, stems, leaves or barriers, and flowers of

various plants have various exceptions and are also not substantive (have little or

no colouring power by themselves) except when used in conjunction with

mordants.

Natural dyes are dyes or colourants derived from plants, invertebrates, or minerals.

The majority of natural dyes are vegetable dyes from plant sources and other

biological sources. Natural colours are beautiful to behold.

Natural dyes are classified according to their origin, colour, application and

chemical constituent. The following classifications are discussed in detail below;

1.2.1 Classification based on origin

There are primarily three sources from which natural dyes are extracted, viz.

plants, minerals, and animals.

i. Plants: Different parts of many plants are found potentially rich in natural

dye; parts like root, bark, stem, seeds, and fruit can be used to extract colour.

Some plants may possess more than one colour depending upon from which

part of the plant they are extracted.

ii. Minerals: These colourants are derived from the natural mineral sources.

Mineral colours are produced from purified natural organic compounds.

3
 Some of the important mineral colourant are chrome yellow, iron buff,

nankin yellow, prussian blue, and manganese brown. However, these are

produced in situ making handling of textile stiffer.

iii. Animals: Animals are also rich source of natural dyes. Dyes normally are

being extracted from the dried body of the insects; common examples are

lac, cochineal and chemical structures, botanical names as well as C I

specification of a few popular natural dyes.

1.2.2 Classification based on Colour

Natural dyes are usually categorized on the basis of the colour that they impart to

the fiber substrate.

i. Red: The red colourants are found in the barks or in roots of the plant or

camouflaged in the bodies of the dull grey insects. Few prominent members

are madder (Rubiatinctorum), manjistha (Rubiacordifolia), cochineal

(Coccuscacti) and lac dye (Coccuslacca).

ii. Blue: The Colour Index lists only four blue natural dyes, viz. natural indigo,

sulphonated natural indigo, Kumbh (Manipur) and the flowers of Japanese

‘Tsuykusa’ used mainly for making awobana paper. The only viable choice

among the blue natural dyes is indigo.

4
iii. Orange: Dyes that create reds and yellows can also yield oranges. The

sources for a natural orange dye are barberry, annatto, sweet pepper blood

roots etc.

iv. Green: Plants that yield green dyes are rare. Both woad (Isatistinctoria) and

indigo have been used since ancient times in combination with yellow dyes

to produce green shades. Woollen cloth mordanted with alum and dyed

yellow with dyer’s green weed was over dyed with woad and, later with

indigo, to produce the once-famous Kendal green. Soft olive greens are also

obtained when textiles dyed yellow are treated with iron mordant.

1.2.3 Classification based on application

Natural dyes are also classified based on the method of their application examples

such as mordant dyes, direct dyes, vat dyes, acid dyes and basic dyes, and disperse

dye.

i. Acid dyes: Acid dyes possess either sulphonic or carboxylic groups in

structure and are most suitable for dyeing of wool and silk from acidic

medium. Aftertreatment with tannic acid, known as back tanning improves

the colourfastness, e.g., saffron.

ii. Basic dyes: Basic dyes on ionization develop coloured cations and form

electrovalent bond with the –COOH groups of wool and silk. These are

5
 applied from neutral to mildly acidic pH and shows poor light fastness, e.g.,

berberine.

iii. Direct dyes: Many natural dyes belong to the class of direct dyes. Direct

dyes possess affinity for cellulosic fibres without any pretreatment to dye or

to the fiber. The most common example is turmeric; others are harda,

pomegranate rind, and annatto.

iv. Mordant dyes: Mordant dyes essentially require a mordant for application

because of their lack of affinity for the fiber. These dyes form complexes

with the mordant added. Three types of mordants are used such as: Metallic

mordants such as metal salts of aluminum, copper, tin etc., Tannic mordant

such as tannic acid, e.g., myrobalan and sumach and Oil mordant which

forms complex with main metal mordant.

v. Vat Dyes: Vat dyes show affinity for natural fibres and are water insoluble

and first reduced with sodium hydrosulphide followed by solubilisation with

sodium hydroxide. After application of the dye on the fiber, it is then

converted into parent insoluble form by oxidation to develop the true colour

of the dye, e.g., indigo.

vi. Disperse dyes: Disperse dyes are dyes have relatively low molecular weight,

low aqueous solubility and no strong solubilizing groups. These dyes can be

post-mordanted with chromium, copper or tin salt and are applied on

6
 synthetic fibres at neutral to mild acidic pH. They can also be applied to silk

and wool. One such dye could be Lawsone or henna and many other flavone

as well as anthraquinone dyes.

1.2.4 Classification based on chemical constitution

Natural organic dyes cover a wide range of chemical classes, viz. anthraquinone,

indigoid, naphthoquinone, carotenoid, flavone and flavonol.

i. Flavonols and Anthocyanins dye: Flavonols and anthocyanins are the main

subclasses of flavonoids present in plants, the latter being found only in red

onions (Basak et al., 2012; Kopsell et al., 2004; 2010). The Flavonols and

anthocy have a variety of colours such as orange, red maroon and blue. The

intensity and stability of these subclasses is independent on factors such as

the structure and concentration of dyes, temperature, pH, light intensity,

quality and presence of other pigments together with metal ions, enzymes,

oxygen, ascorbic acid, sugar, sugar metabolites and sulphur oxides

(Markakis.,1982; Francis., 1989; Mazza et al.,1993). In red onions, the

major existing subclasses of flavonoids present are the flavonols and

anthocyanins (Lanzotti., 2006; Rosa et al., 2010).

ii. Carotenoid dyes: Carotenoid Dyes class name is derived from the orange

pigment found in the carrots. In these, the colour is due to the presence of

7
 long conjugated double bond. Some common examples of the carotenoid

structure based dyes are annatto, saffron etc.

iii. Anthraquinone dyes: Most important red dyes are based on the

anthraquinone structure and are obtained both from plants as well as animals

or insects. These are characterized by good fastness to light and on

formation of metal salt complexes develop good wash fastness too.

Examples of anthraquinone dyes are madder (extracted from the root and

known as the alizarin), lac dye (animal dye), kermes (insect dye and the

colouring matter is kermesic acid), cochineal (obtained from an insect and

main colouring component is carminic acid).

iv. Indigoid dyes: Two very important natural dyes possess indigoid structure,

namely indigo and tyrian purple. Indigo is the oldest natural dye used by

man. The dye was used pre-historically in India, where it probably

originated. The word is derived from ‘Indican’. It occurs as the glucoside

indicant in the plant Indigoferatinctoria and is known in India as a-

Naphthoquinone dyes. The most prominent member in this class is lawsone

or henna. It is obtained from Lawsoniainermis, cultivated mainly in India

and Egypt. Another similar dye is juglone obtained from the shells of unripe

walnuts.

8
1.3 Advantages of natural dyes

The advantage of natural dyes over synthetic dyes is that synthetic dyes are

synthesized from petrochemical processes and natural dyes are friendly to the

environment. Some dyes like madder are considered as host in tea gardens while

indigo is renewable and biodegradable i.e. the waste residue after extraction is used

as fertilizer for crops thus eliminating disposal problems.

i. Natural dyes can be extracted from by- products of some industries, hence fit

the zero-emission approach. It does not contain harmful chemicals nor

carcinogenic components common to synthetic dyes that is why unlike

synthetic dyes which pollute the environment with toxics, natural dyes are

health hazardous free (Samanta et al., 2009; Asif et al., 2010; Konar et al.,

2011).

ii. Natural dyes contain value-added benefits to our health and are obtained

from renewable sources that. Many natural dyes are UV protective (Chen et

al., 2002, 2007; Zhang et al., 2010), antimicrobial (Gupta., 2004; Mohamed.,

2013; Wangatia et al., 2015) and deoxidizing (Lee et al., 2002; Fenget al.,

2007; Hwang et al., 2008; Lee et al., 2010) agents.

9
iii. Special colouring effects are produced by natural dyes which cannot be

produced by synthetic dyes. The shades produced are usually soft, vibrant,

soothing to the human eye and are polygenetic (Konar et al., 2011; Sanjeeda

et al., 2014).

iv. Natural dyes bleed but do not stain other fabrics, with turmeric being an

exception. Natural dyes are usually moth proof and can replace synthetic

dyes in kids’ garments.

1.4 Disadvantages of natural dyes

i. Cost: A larger amount of natural dyes may be needed in order to dye a

specific amount of fabric as opposed to synthetic dyes. Since that is the case,

using natural dyes is more expensive than synthetic dyes.

ii. Colour Pay-off: Colour pay-off from natural dyes tend to fade quickly. More

so, quality may not be as consistent than what synthetic dyes can deliver.

iii. Availability: Another issue with natural dyes is their availability. It can be

difficult to produce because the availability of raw materials can vary

from season to season, place, and species, whereas synthetic dyes can be

produced in laboratories all year round.

iv. Harmful Effects: Natural dyes can also be harmful to some extent. Logwood

has ingredients, hematein and hematoxylin, that can be have harmful effects

when inhaled, ingested, or absorbed through the skin. Bloodroot, another

10
 natural dye source, can cause irritation and inflammation when inhaled.

More so, natural dyes may need mordants for application. While these

substances help the dye stick to fabrics, they can also be toxic. Example of

mordants used in natural dyes are aluminum, copper, iron, and chrome.

v. Sustainability: While natural dye sources are renewable, sustainability can

still be an issue for natural dyes because producing them require vast areas

of land.

1.5 Properties of natural dyes

Natural dyes have so many properties attached to it, ranging from

antimicrobial/antibacterial property, ultra violet (UV) protection, mosquito

repellent and so on.

i. Antimicrobial/antibacterial property: Antimicrobial properties of natural

dyes are due to the presence of various compounds such as anthraquinones,

flavonoids, tannins, naphthoquinones etc. Naturally dyed textiles can

provide protection against these microorganisms. It is said that antibacterial

and antifungal properties of natural dye are due to its phenolic content.

Phenolic compounds attach on the surface of the textile fabric by forming a

complex. When the fabric comes in contact with microbes, these attached

11
 phenolic compounds hinders the enzyme production in the microbes; thus,

further cell reaction would not take place, and at the end cell dies.

ii. Ultraviolet (UV) protection: Researches has been carried out in other to

enhance the aesthetic as well as UV protection property of the fabric with

the use of natural dyes. UV protection properties of dyed fabric are analyzed

using UPF of the fabric. Ultraviolet protection factor (UPF) indicates the UV

protection properties of the fabric. Application of natural dyes on fabric

significantly enhances the UPF of the fabric. UPF of fabric is affected by the

absorption characteristics of natural dyes. Various kinds of natural dyes

provide protection against microbes as well as UV rays on different kinds of

fabric such as wool, cotton and silk.

1.6 Transmittance of light in a spectrophotometer

The relationship between transmittance and concentration or sample length is

logarithmic one was founded by (Pierre Bouguer in 1729). This is because

molecules nearest to the source light will experience (I) as it was measured using

the spectrophotometer. As light travels through the sample, the value of (I) reduces

in each succeeding layer.

Absorption spectra of chemical species (atoms, molecules, or ions) are generated

when a beam of electromagnetic energy (i.e., light) is passed through a sample, and
12
the chemical species absorbs a portion of the photons of electromagnetic energy

passing through the sample.

A spectrophotometer is an instrument designed to make this measurement. Using

some very well understood electronics, this device effectively “counts” the number

of photons that enters a sample and compares it with the number of photons that

exits a sample. In addition, the instrument is able to take white light and separate it

into its constituent colours (i.e. somewhat like a prism), allowing the user to

examine the absorption of light of individual wavelengths with nearly 1 nm

resolution.

In optics, that portion of physics that deals with the properties of light, the

measurement of the number of photons delivered at a point in a given unit of time

is called the Intensity, I. (Higher intensity could be thought of as “brighter” and

lower intensity could be thought of as “dimmer”; hence high intensity light will be

bright and low intensity light will be dim.) If we measure the intensity of the

beam of light entering our sample (Io) and compare it with the intensity of the beam

of light exiting our sample (I) we can take the ratio I/I o to get an indication of what

fraction of the light entering the sample was found exiting the sample. This ratio is

called the Transmittance:

Transmittance: T=I
Io

13
We can convert this ratio into a percentage by multiplying by 100 to get Percent

Transmittance (%T):

%Transmittance: %T = I x 100
Io
Thus, if the intensity of the light exiting our sample is 76 and the intensity of the

light entering our sample is 100, then the Transmittance would be 0.76 and the %

Transmittance would be 76%, indicating that 76% of the photons entering our

sample are finding their way out of our sample.

1.7 Beer-lambert law of absorption

It was later rediscovered by Johann Heinrich lambert in 1760 that the transmittance

of light varies exponentially as it passes through an absorbing medium which was

later defined in 1852 by August Beer to be:

For our purposes it is mathematically convenient to define a new concept,

Absorbance (A):
I
Absorbance: A = -log10

Io
Beer showed that absorbance is linearly related to concentration because when no

light is absorbed I=Io when 90% of light is absorbed, T= 0.1 and A=1.

14
This technique is used not only by chemists but by scientists of many fields. The

Beer-Lambert law allows you, the scientist, to measure the absorbance of a

particular sample and to deduce the concentration of the solution from that

measurement. In effect, you can as well measure the concentration of a particular

chemical species in a solution as long as you know the species absorbs light of a

particular wavelength.

A l c

Examining the mathematical form of the Beer-Lambert Law, the molar extinction

coefficient or molar absorptivity, is a constant for a given transition metal ion, and

the path length (l) is a constant as long as the same test tube or cuvette is used to

make each absorbance measurement. In effect, when is multiplied by l, the result

is a constant, and the only difference that can give rise to a change in absorbance is

the concentration of the absorbing species. Thus, the Beer-Lambert Law

establishes a linear relationship between absorbance and concentration:

A l c
k

A= k c
m x

Y = mx + b

15
where:

A= absorbance,

ε = experimentally determined constant known as the molar absorptivity (if the

concentration is in molarity) or the extinction coefficient (Lmol-1cm-1),

l = path length of the sample (cm),

c = concentration of the absorbing species (usually in molarity) (molL-1),

k= proportionality constant

For a given sample, absorbance depends on six factors:

i. Concentration

ii. Identity of the absorbing substance

iii. Pathlength

iv. Identity of the solvent

v. Wavelength of light

vi. Temperature

Clearly, not all solution species absorb light in the same way. A more concentrated

solution has more absorbing substance present, resulting in more light being

absorbed for a given species. The longer the pathlength, the more absorbing

16
substance the light will interact with, resulting in greater absorbance. Solutions

with higher absorbance appear darker or more intensely colored than solutions with

lower absorbance. The existence of colored solutions is indicative that not all

wavelengths of light are absorbed equally strongly.

1.7.1 Sources of error in Beer’s law

Errors that can be encountered while using Beer’s law is as follows; use of dirty

cuvettes, incorrect wavelength, determining incorrect range of solutions and poorly

mixed solutions.

1.8 Problem statement

Synthetic dyes are harmful to plants, animals and to the environment at large

because of the toxic chemicals used in the preparation and its non-biodegradable

nature as well but with the use of natural dye which tends to be eco-friendly, less

toxic and easily biodegradable, the processes of dyeing tends to be more

convenient as to health wise.

1.9 Objectives of study

i. Extraction and characterization of dye extracted from pawpaw using acid,

alkaline, and aqueous solution.

17
 ii. Determination of Fourier Transform Infra-red analysis.

iii. Determination of spectrophotometric analysis.

iv. Determination of the specific gravity.

1.10 Justification of study

The dyes gotten from natural sources such as wool, silk, cotton, and linen are used

to dye food substrates, leather, wood, and natural fibers from different parts of

plants. The study providing beneficial information on the extraction and

characteristics of natural dyes from pawpaw (Carica Papaya) and also creates the

awareness of unexplored plant dyes and finally serve as a reference material to

other research works. Natural dye is safe to use because it is environmentally

friendly unlike the synthetic dye which is its opposite.

1.11 Scope of study

The scope of this study involves

i. Extraction of natural dye from pawpaw using aqueous extraction method.

ii. Extraction of natural dye from pawpaw using acid extraction method.

iii. Extraction of natural dye from pawpaw using alkaline extraction method.

18
iv. Determination of micro-chemical analysis of the dye using Fourier

Transform Infrared Spectroscopy (FTIR) and ultraviolet-visible

spectroscopy analysis.

CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 Overview

The literature review covers colour in textile, dyes, natural plant dyes (types of

natural dyes), synthetic dyes, characteristics of natural dyes, mordants, uses of

plant dye in food additives, food colourants and summary of discussion. Dyes are

generally divided into natural dye and synthetic dye.

2.2 Dye

Dyes are indeed revolutionary because it adds colour and a different perspective to

most of the things used in the home or at the workplace. Dyes are formed and used

in different ways. They are produced using chemicals like acids or extracted from

earth and mineral sources. Dyeing with natural pigment is one of the oldest
19
methods of dyeing practiced by the ancient civilization people. This is an act that

has been in existence for a long time and the Egyptian pyramid was painted with

natural colourant (Ferreira et al., 2004).

Dyes of natural origins are great for colour appreciation as any variation in the

concentration of dye, mordant, type of water, soil and climate give variations in

colours. Natural dyes obtained from plants have gained economic advantage over

synthetic dyes because of their non-toxic and biodegradable nature. Researchers’

attention has been shifted to the use of natural dyes for dyeing of textile materials

because of the current eco-consciousness (Bechtold, 2006). Dyes were employed

to get color to textiles, ceramics etc., where color had several ceremonial and

quotidian contexts (M. Ivic, M. Berguer, Magaloni 2008).

Globally, the demand for natural dyes is significantly increasing due to its cheaply

available, widely applicable, and extraction that is carried out by green methods

from natural resources (Siva, 2007; Kadolph, 2008; Jaffer et al., 2019). In the early

age of the maddle plants grown practically everywhere to produce a range of reds.

He made mention that until the 19th century, most the dyes produced were mostly

from vegetable and most rarely from mineral and animals. Natural dyes serve as

food colourant, the food determines the colour based on the food’s degree of

sweetness, flavor or ripeness. Basically, natural dyes have been added to food for

aesthetic purpose in order to increase the desirability of the food, to make it more
20
appetizing and so on. Natural dyes are used for fabric dyeing in which the dyes

used requires a mordant which are metallic salt of iron, copper, aluminum and

others, to serve as a colour fastness to washing, dry cleaning and sunlight (Adu-

Akwaboa 1994:129).

Natural dyes are dyes or colourants derived from plants, minerals or animals. The

majority of natural dyes are vegetable dyes from plant sources like the roots of a

plant, bark, leaves and wood and other biological sources such as fungi

(Wikipedia.org/natural dye). The earliest grouping was according to the

alphabetical order. Later it was shifted to chemical structure based, where grouping

within each group structure class according to colours was carried out. Later on,

these were classified in various other ways, e.g., based on colours, chemical

constitution, application, and origin. Natural dyes are frequently categorized on the

basis of their colour that they impart to the fiber substrate.

Natural dyes are nonhazardous and has wide applicability in pharmaceuticals food,

cosmetics, leather, and in different art of dyeing (Kanchana et al., 2013; Tayade et

al., 2016). Natural dyes of plant, mineral and animal sources are fascinating,

beautiful and sometimes they challenge the wits of researchers and educators. Most

of them produce very colourful effects that are so amazing to behold. Natural

colours are beautiful to behold (Lyon Tex, 1992:732). Colouring matter extracted

from the roots, stems, leaves or barriers, and flowers of various plants have various
21
exceptions and are also not substantive (have little or no colouring power by

themselves) except when used in conjunction with mordants. The beautiful colours

that are created from natural dyes would initially appear vivid, but soon fade. Lack

of colour fastness resulted in the discovery of mordants - substances which aid in

the absorption of dyes (Joseph, 1977).

Natural colourants from plants with antimicrobial properties are being used widely

as both herbal medicines and dye such as the black kohl or green malachite was

used as cosmetic and also to prevent eye disease as well. A large number of plants

and animal sources have been identified for the extraction of colours and their

effective use in textile dyeing and functional finishing, cosmetics, Ph indicator,

Food colouration and several other applications.

The disadvantages associated with usage of natural dyes is the low to medium

fastness properties especially poor light fastness. Almost all natural dyes except

substantive dyes need fixation with mordants such as metal salts which are toxic

and environmentally unfriendly. Natural dyes also have poor reproducibility,

particularly the plant sources since they are affected by environment conditions

like varying planting time, place and species. Other disadvantages include: non-

availability due to difficulty in collection, bulk isolation of dye-stuff,

standardization of dyeing procedure, colour yield and complexity of the dyeing

process. (Samanta and Agarwal, 2009).

22
The knowledge of the natural environment need not be ignored, since it is glaring

for the eyes to see and appreciate; hence the need to explore in depth the extraction

of dyes from natural plants to produce suitable dyes for selected textile fabrics and

also serve as colourants for food.

The synthetic dyes are environmentally toxic and hazardous to health over natural

dyes. The synthetic dye is obtained from the different inorganic metal complexes

whereas, natural dye is obtained from the natural plants, insects and minerals

(Singh et al., 2017; Singh & Singh, 2018; Wangatia, 2015). Since, long time ago,

in the ancient time of human civilization, mineral, plant and animal products were

the main sources of dyes and drugs with their excellent therapeutic properties

(Bhuyan et al., 2016). The synthetic colorants possess various side effects and

radiation hazards (Chengaiah et al., 2010; Samata & Agarwal, 2009). Turmeric, a

naturally occurring yellow dyes which revitalizes the skin, is a powerful antiseptic,

while indigo gives a cooling sensation.

Green methods for plant dye extraction have been studied and classified as

medicinal importance as well as some of these have recently been shown to possess

antimicrobial, antifungal, antioxidant and anticancer activities (Hussein et al.,

1997). Curcuma longa, Punica granatum L, lawsone from Lawsonia inermis L,

have been reported for the antimicrobial, antibacterial and antifungal activities

(Paudel et al., 2011).

23
There is a traditional use of dye yielding plants for medicinal purposes. Plants like

turmeric, saaj, tulsi, amala, khayer, neem, etc. are traditionally used for medicine.

They can be used as anti-oxidant, anti-inflammatory, anti-cancer, anti-viral, anti-

fungal, anti-bacterial, hyperglycemic, anti-diarrheal, anti-spasmodic, skin

disorders, etc. (Chaitanya, 2014; Saivaraj et al., 2018; Prabu & Anabarasan, 2015;

Sakthivel, 2015; Bukhari et al., 2014).

2.3 Fourier Transform Infra-Red spectroscopy (FTIR):

FTIR (Fourier-transform infrared spectroscopy) has proven a valuable tool for the

characterization and identification of compounds or functional groups present in an

unknown mixture of plants extract. For most common plant compounds, sample

preparation for FTIR analysis involves the various ways. For liquid samples, one

drop of sample is placed between two plates of sodium chloride and it forms thin

film between the plates (Sasidharan et al., 2011).

Infrared (IR) or Fourier transform infrared (FTIR) spectroscopy has a large

application range, from the analysis of small molecules or molecular complexes to

the analysis of cells or tissues. The imaging of tissues is one of the recent

developments of infrared spectroscopy, taking advantage of infrared microscopy

and of the use of synchrotron IR radiation. It is used for the mapping of cellular

24
components (carbohydrates, lipids, proteins) to identify abnormal cells (Levin and

Bhargava 2005; Petibois and De´le´ris 2006). FTIR spectroscopy has also been

increasingly applied to the study of proteins. This concerns the analysis of protein

conformation, protein folding, and of molecular details from protein active sites

during enzyme reactions using reaction-induced FTIR difference spectroscopy

(Siebert and Hildebrandt 2008).

FTIR difference spectroscopy has been widely applied in photosynthesis research

and related areas. This approach gives complementary information to the three-

dimensional structural data obtained by X-ray diffraction (or Nuclear Magnetic

Resonance, NMR). The analysis of active sites in proteins by means of reaction-

induced FTIR difference spectroscopy gives information on minute structural

changes, hydrogen-bonding interactions, and proton transfer reactions, which are

often beyond the sensitivity of X-ray diffraction analyses. Moreover, time-resolved

techniques, with time resolution now up to the femtosecond range (Di Donato et al.

2008) allow structural changes to be observed for protein active sites ‘‘at work’’.

In photosynthesis, this approach has given information of prime interest concerning

the cofactor-protein interactions (Nabedryk 1996; Breton 2001; Berthomieu and

Hienerwadel 2005; Noguchi and Berthomieu 2005), as well as proton transfer

routes in bacterial reaction centers (Nabedryk and Breton 2008). It now plays a

25
central role in the detailed analysis of the oxygen evolving complex of

Photosystem II (Chu et al. 2001; Noguchi 2007; Debus 2008).

The vibration frequency thus depends on the bond strength, with higher

frequencies for triple or double bonds as compared to single bonds. A consequence

of the dependence of stretching mode frequencies on the bond strength is that the

frequencies are very sensitive to the group environment, the electronegativity of

neighboring atoms or groups, or to hydrogen bonding interactions. The

involvement of one of the atoms in a hydrogen bond will induce a weakening of

the bond strength, and thus a frequency downshift of the stretching mode of the

chemical group. The sensitivity of this method is high, and corresponds to changes

in bond length smaller than 0.2 A˚ (Deng and Callender 1999; Barth 2007). For a

carbonyl group, formation of a hydrogen bond induces downshifts up to 20 cm -1.

The inspection of stretching mode frequencies of chemical groups, and specifically

of carbonyl or carboxylic groups in proteins, reveals information on fine structural

details. Formation or disruption of hydrogen bonding interactions is one of those.

The vibration frequency also depends on the mass of the atoms involved in the

vibration. It is possible to specifically alter a vibration frequency by isotope

labeling of one of the atoms involved in the vibration. Substitution of hydrogen by

deuterium has been widely used to identify and analyze groups with exchangeable

protons. Specific isotope labeling (13C, 15

N, 18
O, 2H) is also a very powerful
26
approach to identify the frequency of one specific group in a complex infrared

spectrum (see below).

In summary, the vibrational frequencies of a given chemical group are expected in

specific regions which depend on the type of atoms involved and the type of

chemical bonds. Tables are available for the main chemical groups, as well as for

amino acid side chains (Venyaminov and Kalnin 1990a; Socrates 1994; Barth

2000; Wolpert and Hellwig 2006). Within these vibration regions, the frequencies

of the chemical groups are modulated by the specific environment of the group.

Therefore, to establish a clear relationship between the infrared mode frequency

and the structural properties of a given residue, or when there are no data available

in the tables, it is necessary to perform a detailed IR analysis on simplified model

compounds in different environments (solvents) and/or to analyze experimental

results using theoretical chemistry approaches. Absorption of proteins in solution

While this approach suffers from several limitations, it delivers unique information

by addressing directly the properties of cofactors, amino acids, and water

molecules, with very high sensitivity to structural parameters and electronic

interactions. This justifies the experimental efforts that have been made to optimize

its use and the interpretation of the data.

27
In the following, we briefly present the principles of infrared spectroscopy and

describe the development of experimental approaches to identify and analyze IR

signatures from active sites in proteins by reaction-induced FTIR difference

spectroscopy. We describe the methodology to obtain reliable spectra and

interpretations, and show typical examples of specific information brought by this

technique in the study of photosystems.

2.4 Principle and methodology of infrared spectroscopy and FTIR difference

spectroscopy

2.4.1 Infrared Spectroscopy

Infrared spectroscopy probes the molecular vibrations. Functional groups can be

associated with characteristic infrared absorption bands, which correspond to the

fundamental vibrations of the functional groups (Colthup et al. 1975; Griffith and

de Haseth 1986). For a nonlinear molecule with N atoms, there are 3N-6

vibrational motions of the molecule atoms, or 3N-6 fundamental vibrations or

normal modes. A normal mode of vibration is infrared active (i.e., it absorbs the

incident infrared light) if there is a change in the dipole moment of the molecule

during the course of the vibration. Thus, symmetric vibrations are usually not

detected in infrared. In particular, when a molecule has a center of symmetry, all

28
vibrations which are symmetrical with respect to the center are infrared inactive. In

contrast, the asymmetric vibrations of all molecules are detected. This lack of

selectivity allows us to probe the properties of almost all chemical groups in one

sample, and notably of amino acids and water molecules which can hardly be

observed by other spectroscopic techniques.

Strong infrared absorptions are observed for groups with a permanent dipole (i.e.,

for polar bonds). As such, the carbonyl groups of the polypeptide backbone

contribute largely to the infrared absorption spectra of proteins.

In the mid-infrared region (4,000-1,000 cm-1), two main types of vibrations are

observed: vibrations along chemical bonds, called stretching vibrations (m), which

involve bond-length changes; and vibrations involving changes in bond angles, and

notably bending vibrations (d—in plane, p—out of plane).

The stretching vibrations can be modeled using the harmonic oscillator model in

which a chemical bond is represented by two-point masses linked by a spring.

29
 CHAPTER THREE

3.0 MATERIALS AND METHOD

3.1. Materials

Materials used for the purpose of this experiment are:

i. Pawpaw fruit: The pawpaw fruit used in this research work was purchased

at relief market, Owerri and all equipment used were from the department of

soil science, school of agriculture and agricultural technology (SAAT),

Federal University of Technology, Owerri (FUTO).

ii. Distilled Water: Treated water was purchased at the department of soil

science, school of agriculture and agriculture technology (SAAT), Federal

University of Technology, Owerri (FUTO).

iii. Ethanol: The solvent was gotten from the lab technologist at the department

of soil science, school of agriculture and agriculture technology (SAAT),

Federal University of Technology, Owerri (FUTO).

30
 iv. Sodium Hydroxide: The alkali was gotten from the lab technologist at the

department of soil science, school of agriculture and agriculture technology

(SAAT), Federal University of Technology, Owerri (FUTO).

v. Hydrochloric acid: The acid was gotten from the lab technologist at the

department of soil science, school of agriculture and agriculture technology

(SAAT), Federal University of Technology, Owerri (FUTO).

3.2. Apparatus

i. Knife

ii. Beakers

iii. Grater

iv. Hand grinder (manual)

v. Bucket

vi. Bowl

vii. Ceramic moulter

viii. Filter paper

ix. Weighing balance

x. Spectrophotomer

xi. Measuring cylinder

xii. Pycnometer

xiii. Wash bottle

31
3.3 Preparations of materials and solvents

3.3.1 Preparation of the samples

The pawpaw fruit was peeled, meshed into little fragments and 280g was weighed

for extraction. The ground pawpaw was mixed with 600mls of Hcl, NaOH and

ethanol respectively.

3.4 Preparation of the organic solvents

3.4.1 Alkaline medium (NaOH)

In the preparation of the alkaline medium used in this research, 80g of sodium

hydroxide was dissolved in water to make a solution of 250ml in the beaker. The

reaction was highly exothermic and proper care were taken while carrying out this

procedure. The solutions used were stirred constantly to avoid caking of the

molecules. Later on, a homogenous solution was achieved and the molecules

dissolved. The temperature of the solution was cooled before transferring it into a

conical flask as the flask cannot withstand the heat.

3.4.2 Acidic medium (Hcl)

32
For acidic medium, 1010ml of water was used to dilute 91g of hydrochloric acid

and then turned into the measuring cylinder.

3.4.3 Alcohol medium (Ethanol)

The ethanol used was not diluted. 600ml was tested for the experiment.

3.5 Extraction of the Natural dye

3.5.1 Acid Extraction (Hcl)

600ml of diluted Hcl was used to extract the dye from the 280g of ground pawpaw.

280g of grounded pawpaw was weighed out first using a weighing balance. The

grounded pawpaw was later transferred to a plastic container where the 600ml of

diluted acid solution was added and then left for extraction to occur. After a day,

the mixture was sieved out and the β-carotene dyestuff extracted was from the

pawpaw. A code name was given in order to identify the natural dye liquid gotten

from this extraction method as (Acidic Extract).

3.5.2 Alkali Extraction (NaOH)

600ml of diluted NAOH was used to extract the dye liquid from the 280g of the

ground pawpaw. 280g of ground pawpaw was weighed out first using a weighing

balance. The measured pawpaw was grounded and later transferred to a plastic

33
container where the 600ml of diluted NaOH solution was added and then left for

extraction to occur. After a day, the mixture was sieved out and the β-carotene

dyestuff extracted was from the pawpaw. A code name was given in order to

identify the natural dye sample gotten from this this extraction method as (Alkaline

Extract).

3.5.3 Solvent Extraction (Ethanol)

In this extraction, 600ml 0f diluted ethanol was used to extract the dye liquid from

the 280g of ground pawpaw. 280g of ground pawpaw was weighed out first using a

weighing balance. The grounded pawpaw was later transferred to a plastic

container where the 600ml ethanol was added and then left for extraction to occur.

After a day, the mixture was sieved out and the β-carotene dyestuff extracted was

from the pawpaw. A code name was given in order to identify the natural dye

sample gotten from this extraction method as (Ethanol Extract).

3.5.4 Aqueous Extraction

This extraction method was used as control sample. In this extraction method, the

pawpaw was broken down by grinding in other to improve the extraction

efficiency. The dye liquid was extracted after grinding by the aid of a sieve and a

code name was given to this extraction method (Aqueous Extract) for easy

identification.

34
3.6 Ultraviolet (UV) visible spectroscopy analysis

At first, water was used to normalize the absorbance value to zero value as water

has no wavelength. Water was used as an initiator. The different pawpaw specimen

of Aqueous Extract, Acidic Extract, Alkaline Extract, Ethanol Extract were put in

the cuvette respectively and consecutively to get the maximum absorbance and

corresponding wavelength. The excess solution and spill on the cuvette were wiped

with the piece of tissue paper to prevent the excess/spill from blocking the passage

of light into the cuvette.

The dye solution of the different extracts was subjected to an ultraviolet (UV)

visible Spectrophometer for absorption spectra analysis. Series of absorbance and

wavelengths were recorded in other to determine the peak absorbance of the

extracted dye sample. A graph of absorbance against wavelength was plotted

afterwards using the respective values of the variables.

3.7 Fourier Transform Infra-Red (FTIR) analysis

Fourier Transform Infrared (FTIR) Analysis is an analytical technique used to

identify organic, polymeric, and in some cases inorganic materials. The extracted

samples of acid, alkaline, ethanol and aqueous solvent were transferred into a

plastic container which was sent to another laboratory, FTIR Springboard Research

lab, Awka for further analysis. FTIR measures the range of wavelengths in the

35
infrared region that are absorbed by a material. The basic theory at work is that the

bonds between elements absorb light at different frequencies.

3.8 Determination of specific gravity

The beaker was washed with distilled water and then dried thoroughly. The beaker

was then filled with the sample (dye solution) of 10mls at room temperature. The

excess solution from the beaker was wiped with the piece of tissue paper and

weighed using a weighing balance. The mass of the samples was recorded and

their respective densities obtained. This procedure was used as an alternative to

measuring with the specific gravity bottle.

3.9 Determination of pH

The pH of the Control sample of pawpaw (Aqueous solution) was determined by

measuring out 10ml of the sample using a pH meter to determine the pH value.

36
 CHAPTER FOUR

4.0 RESULTS AND DISCUSSION

The analysis was carried out using FTIR and spectrometer. The ultra-visible (UV)

spectroscopy result was presented using MS-Excel 2013. The result obtained from

FTIR analysis are represented in the table 1-4 and figure 1-4.

4.1: Fourier Transform Infra-Red (FTIR) Analysis

Fourier Transform Infra-Red (FTIR) analysis are illustrated in the tables and
figures below:

Table 4.1: FTIR Analysis of Acidic extract of pawpaw

37
S/N Wavelength Functional Compounds
(cm-1) group
1 1304.352 H2C=CH Ethene CH2 anti-symmetric stretch

2 1401.954 H2C=CH Ethene CH2 anti-symmetric stretch

3 1602.430 RNH3 10 amine NH stretch

4 1829.457 R-C00 Cyclic ester C0 symmetric stretch

5 2056.940 RC00H Carboxylic acid C0 stretch

6 2451.366 R-C≡N Nitriles CN antisymmetric stretch

7 2566.450 R-C≡N Nitriles CN antisymmetric stretch

8 2787.876 CH2 Methylene CH3 stretch

9 2989.318 R-S-C≡N Thiocyanate SCN antisymmetric

stretch
10 3381.196 R2CH0H 20 alcohol 0H stretch

11 3542.671 R3CH0H 30 alcohol 0H stretch

12 3674.157 R3CH0H 30 alcohol 0H stretch

13 3806.033 R3CH0H 30 alcohol 0H stretch

Figure 4.1: Acidic

Extract of pawpaw
From table 4.1 and figure 4.1, the peak value around 1304.352cm-1 and

1401.954cm-1 were assigned C=C stretching vibration of alkene compound

respectively. The absorbance around 1602.430cm-1 was assigned to NH stretching

vibration of 10 amine compound. The peak around 1829.457cm-1 was assigned to

38
C0 stretching vibration of cyclic ester compound. The absorbance around

2065.940cm-1 was assigned to C00 stretching vibration of carboxylic acid whereas

the absorbance around 2451.366cm-1 and 2566.450cm-1 corresponds to CN anti-

symmetric vibration of nitrile compound. The weak band around 2787.976cm-1

was assigned to C-H stretching vibration of methylene compound. The height

around 2989.318cm-1 was due to SCN stretching vibration of thiocyanate

compound. The broad band around 3381.196cm-1, 3542.671cm-1, 3674.157cm-1

and 3806.033cm-1 were due to 0H stretching vibration of 2 0 & 30 alcoholic

compound respectively.

39
Table 4.2: FTIR Analysis of Alkaline extract of pawpaw

S/N Wavelength Functional Compounds

(cm-1) group
1 1407.004 H2C=CH Ethene CH2 anti-symmetric stretch

2 1609.202 RNH3 10 amine NH stretch

3 2117.062 RC00H Carboxylic acid C0 stretch

4 2257.026 R=C0 Carbonyl compound C00 stretch

5 2530.493 R-C≡N Nitriles CN antisymmetric stretch

6 2905.027 R-S-C≡N Thiocyanate SCN antisymmetric

stretch

7 3072.633 RCH0H 10 alcohol 0H stretch

8 3189.044 RCH0H 10 alcohol 0H stretch

9 3481.109 R2NH 20 amine NH stretch

10 3816.688 R3CH0H 30 alcohol 0H stretch

40
Figure 4.2: Alkaline
Extract of pawpaw
From table 4.2 and figure 4.2, the peak value around 1407.004cm-1 was assigned

to C=C stretching vibration of alkene compound. The absorbance around

1609.202cm-1 was assigned to NH stretching vibration of 10 amine compound. The

absorbance around 2117.062cm-1 was assigned to C00 stretching vibration of

carboxylic acid whereas the absorbance around 2257.026cm-1 was due to C00

stretching vibration of carbonyl compound. The height around 2530.493cm-1

corresponds to CN anti-symmetric vibration of nitrile compound. The broad band

41
around 3072.633cm-1, 3189.044cm-1 and 3816.688cm-1 corresponds to 0H

stretching vibration of 10 & 30 alcoholic compound respectively.

S/N 4.3:
Table Wavelength Functional
FTIR Analysis of EthanolCompounds
extract of pawpaw
(cm-1) group
1 722.9132 C-CI Chloro C-Cl symmetric stretch

2 1376.727 H2C=CH Ethene CH2 anti-symmetric stretch

3 1458.571 H2C=CH Ethene CH2 anti-symmetric stretch

4 1614.779 RNH3 10 amine NH stretch

5 1820.931 R-C00 Cyclic ester C0 symmetric stretch

6 2062.046 RCH0H Carboxylic acid C00 stretch

7 2210.864 RCH0H Carboxylic acid C00 stretch

8 2455.115 R-C≡N Nitriles CN antisymmetric stretch

9 2650.023 CH2 Methylene CH3 stretch

10 2795.836 CH2 Methylene CH3 stretch

11 2970.811 R-S-C≡N Thiocyanate SCN antisymmetric

stretch

12 3244.016 RCH0H 10 alcohol 0H stretch

13 3404.451 R2NH 20 amine NH stretch

14 3632.810 R3CH0H 30 alcohol 0H stretch

14 3807.075 R3CH0H 30 alcohol 0H stretch

42
Figure 4.3: Ethanol
Extract of pawpaw
From table 4.3 and figure 4.3, the peak value around 722.9132cm-1 was assigned

to C-CI stretching vibration of halogenous compound. The absorbance around

1376.727cm-1 and 1458.571cm-1 was assigned to C=C stretching vibration of

alkene compound. The absorbance around 1614.779cm-1 and 3404.451cm-1 was

assigned to NH stretching vibration of 10 and 20 amine compound. The peak value

at 1820.931cm-1 was due to C00 stretching vibration of cyclic ester compound.

The absorbance around 2062.046cm-1 and 2210.864cm-1 was assigned to C00

stretching vibration of carboxylic acid whereas the absorbance around

43
2455.115cm-1 was due to CN anti-symmetric vibration of nitrile compound. The

weak band around 2650.023cm-1 and 2795.836cm-1 were assigned to CH

stretching vibration of methylene compound respectively. The peak at

2970.811cm-1 was assigned to SCN stretching vibration of thiocyanate compound.

The strong band around 3244.451cm-1, 3632.810cm-1 and 3807.075cm-1 were all

assigned to OH stretching vibration of 10 & 30 alcoholic compounds respectively.

44
Table 4.4: FTIR Analysis of Aqueous Extract of pawpaw
S/N Wavelength Functional Compounds
(cm-1) group
1 722.9132 C-CI Chloro C-Cl symmetric stretch

2 1376.727 H2C=CH Ethene CH anti-symmetric stretch

3 1458.571 H2C=CH Ethene CH anti-symmetric stretch

4 1614.779 RNH3 10 amine NH stretch

5 1820.931 R-C00 Cyclic ester C0 symmetric stretch

6 2062.046 RCH0H Carboxylic acid C00 stretch

7 2210.864 RCH0H Carboxylic acid C00 stretch

8 2455.115 R-C≡N Nitriles CN antisymmetric stretch

9 2650.023 CH2 Methylene CH stretch

10 2795.836 CH2 Methylene CH stretch

11 2970.811 R-S-C≡N Thiocyanate SCN antisymmetric

stretch

12 3244.016 RCH0H 10 alcohol 0H stretch

13 3404.451 R2NH 20 amine NH stretch

14 3632.810 R3CH0H 30 alcohol 0H stretch

14 3807.075 R3CH0H 30 alcohol 0H stretch

45
Figure 4.4: Aqueous
Extract of pawpaw
From table 4.4 and figure 4.4, the peak value around 722.9132cm -1 was assigned to

C-CI stretching vibration of halogenous compound. The absorbance around

1376.727cm-1 and 1458.571cm-1 was assigned to C=C stretching vibration of

alkene compound. The absorbance around 1614.779cm-1 and 3404.451cm-1 was

assigned to NH stretching vibration of 10 and 20 amine compound. The peak value

at 1820.931cm-1 was due to C00 stretching vibration of cyclic ester compound. The

absorbance around 2062.046cm-1 and 2210.864cm-1 was assigned to C00 stretching

46
vibration of carboxylic acid whereas the absorbance around 2455.115cm -1 was due

to CN anti-symmetric vibration of nitrile compound. The weak band around

2650.023cm-1 and 2795.836cm-1 were assigned to CH stretching vibration of

methylene compound respectively. The peak at 2970.811cm-1 was assigned to SCN

stretching vibration of thiocyanate compound. The strong band around

3244.451cm-1, 3632.810cm-1 and 3807.075cm-1 were all assigned to OH stretching

vibration of 10 & 30 alcoholic compounds respectively.

4.2 ULTRA-VIOLET (UV) VISIBLE SPECTROSCOPY ANALYSIS

Ultra-visible

spectroscopy results
Table 4.5: The wavelength and absorbance of Aqueous Extract and Acidic
Extract of pawpaw
Aqueous Extract Acidic Extract
P-juice P-Acidic
Wavelength
1 (nm) Absorbance Wavelength (nm) Absorbance

380 0.95 0.850 380 0.811

0.9
ABSORBANE

400 0.860 400 0.776

0.85
420 0.864 420 0.953
0.8

440 0.75 0.866 440 0.926

0.7
460 380 400 0.866440
420 460 460480 500 520 0.890 560
540
WAVELENGH (nm)
480 0.861 480 0.834
Figure 4.5: The plot
500 0.860 500 0.787
of absorbance verse
wavelength of
520 0.856 520 0.725
Aqueous and Acidic
extract of pawpaw 0.840
540 54047 0.661

560 0.823 560 0.619

Table 4.5 and figure 4.5 shows the plot of absorbance using wavelengths of

aqueous extract and acidic extract of pawpaw. The aqueous extract which is the

control sample was compared to the acidic extract. From figure 4.5, the Acidic

extract has greater absorbance than the aqueous extract. From the graph, an

observation was made which showed the maximum absorption of the acidic extract

to be 440nm. While aqueous extract at pH 6.15 exhibited its peak of absorption at

440-460nm. The similarities of the both samples are that they both exhibited strong

absorption at 440nm.

Aqueous Extract Alkaline Extract

Table 4.6: The
Wavelength (nm)
wavelength and Absorbance Wavelength (nm) Absorbance
absorbance of
380
Aqueous Extract and 0.850 380 0.786
Alkaline Extract of
400
pawpaw 0.860 400 0.940

420 0.864 420 0.980

440 0.866 440 0.981

460 0.866 460 0.975

480 0.861 480 0.954

500 0.860 500 0.903

520 0.856 520 0.807

540 0.840 54048 0.680

560 0.823 560 0.587

 P-juice P-Alkalin
1

0.95

0.9
ABSORBANE

0.85

0.8

0.75

0.7
380 400 420 440 460 480 500 520 540 560
WAVELENGH (nm)

Figure 4.6: The plot

of absorbance verse
wavelength of
Table 4.6 and
Aqueous andAlkaline
figure 4.6 shows the plot of absorbance using wavelengths of

extract ofextract
Aqueous pawpaw and Alkaline extract of pawpaw. The aqueous extract which is the

control sample was compared to Alkaline extract. From figure 4.6, The Alkaline

extract of pawpaw has greater absorbance than the aqueous extract. From the

graph, an observation was made which showed a strong absorption which is the

49
 maximum absorption at 420nm in Alkaline extract. While aqueous extract at pH

6.15 exhibited its peak of absorption at 420-440nm. The similarities of the both

samples are they both exhibited strong absorption at 420nm.

Table 4.7: The

wavelength and
absorbanceAqueous
of Extract Ethanol Extract
Aqueous Extract and
Ethanol Extract(nm)
Wavelength of Absorbance Wavelength (nm) Absorbance
pawpaw
380 0.850 380 0.283

400 0.860 400 0.456

420 0.864 420 0.475

440 0.866 440 0.346

460 0.866 460 0.123

480 0.861 480 -0.074

500 0.860 400 0.477

520 0.856 410 0.500

540 0.840 405 0.516

560 0.823 415 0.490

50
 P-juice P-Etoh
0.9

0.8

0.7
ABSORBANE

0.6

0.5

0.4

0.3

0.2
380 400 420 440 460 480 500 520 540 560
WAVELENGH (nm)

Figure 4.7: The plot

of absorbance verse
wavelength of

Table 4.7 and figure 4.7 shows the plot of absorbance using wavelengths of

aqueous extract and Ethanol extract of pawpaw. The aqueous extract of pH value

6.15 showed the maximum absorption to be 420-440nm. The peak of absorption of

the Ethanol extract was noted at 420nm. The difference in the absorbance of the

corresponding measured wavelength of aqueous extract was little when compared

to the Ethanol extract which revealed larger difference in absorbance of the

51
different corresponding wavelength. The similarities of the both samples are that

they both exhibited strong absorption at 420nm.

Table 4.8: The

Frequency and
Aqueous
absorbance of Extract Acidic Extract

Frequency (m/s) Absorbance Frequency (m/s) Absorbance

789473.68 0.850 789473.68 0.811

750000.00 0.860 750000.00 0.776

714285.71 0.864 714285.71 0.953

681818.18 0.866 681818.18 0.926

652173.91 0.866 652173.91 0.890

625000.00 0.861 625000.00 0.834

600000.00 0.860 600000.00 0.787

576923.08 0.856 576923.08 0.725

555555.56 0.840 555555.56 0.661

535714.29 0.823 535714.29 0.619

52
 P-juice P-acidic
1.2

0.8
ABSORBANCE

0.6

0.4

0.2

0
520000 570000 620000 670000 720000 770000 820000

FREQUENCY (m/s)

Figure 4.8: The plot

of frequency verse
wavelength of
Table 4.8 and figure 4.8 shows that for every corresponding frequency of the

absorption of dye molecules the resultant absorbance was recorded and this

resulted to a higher absorbance of pawpaw Acidic extract than pawpaw aqueous

extract. This showed that the pawpaw acidic extraction has greater absorbance than

the Aqueous extract which was recorded to have a pH value of 6.15. It was also

observed that the maximum absorbance of pawpaw Acidic extract had a frequency

of 714285.71 while the aqueous extract had a frequency of 714285.71-681818.18

during its absorption peak.

53
Table 4.9: The
Frequency and
absorbance of
Aqueous Extract Alkaline Extract
Aqueous Extract and
Alkaline Extract
Frequency (m/s) of Absorbance Frequency (m/s) Absorbance
pawpaw
789473.68 0.850 789473.68 0.786

750000.00 0.860 750000.00 0.940

714285.71 0.864 714285.71 0.980

681818.18 0.866 681818.18 0.981

652173.91 0.866 652173.91 0.975

625000.00 0.861 625000.00 0.954

600000.00 0.860 600000.00 0.903

576923.08 0,856 576923.08 0.807

555555.56 0.840 555555.56 0.680

535714.29 0.823 535714.29 0.587

54
 P-juice P-alkaline
1.2

0.8
ABSORBANCE

0.6

0.4

0.2

0
520000 570000 620000 670000 720000 770000 820000

FREQUENCY (m/s)

Figure 4.9: The plot

of frequency verse
wavelength of
Table 4.9 and figure 4.9 shows that at the same frequencies, the Aqueous extract of

the pawpaw sample exhibited more absorbance when compared to the Alkaline

extract. The Aqueous extract showed little absorbance reduction across the

respective frequencies. The Aqueous extract had a frequency of 714285.71-

681818.18 during its absorption peak while the Alkaline extract had a frequency of

714285.71 during the maximum absorbance of the extracted dye molecules.

Similarities where also noted which revealed that the frequency of the dye

molecule decreased with decreasing absorbance.

55
Table 4.10: The
Frequency and
Aqueous Extract Ethanol Extract
absorbance of
Aqueous
FrequencyExtract
(m/s) andAbsorbance Frequency (m/s) Absorbance
Ethanol Extract of
789473.68
pawpaw 0.850 789473.68 0.283

750000.00 0.860 750000.00 0.456

714285.71 0.864 714285.71 0.475

681818.18 0.866 681818.18 0.346

652173.91 0.866 652173.91 0.123

625000.00 0.861 625000.00 -0.074

600000.00 0.860 600000.00 0.477

576923.08 0.856 576923.08 0.500

555555.56 0.840 555555.56 0.516

535714.29 0.823 535714.29 0.490

P-juice P-ethanol
1

0.8
ABSORBANCE

0.6

0.4

0.2

0
520000 570000 620000 670000 720000 770000 820000
-0.2

FREQUENCY (m/s)

56
Figure 4.10: The plot
of frequency verse
wavelength of
Table 4.10 and figure 4.10 shows that at the same frequencies, the aqueous extract

of the pawpaw sample exhibited more absorbance when compared to the Ethanol

extract. Aqueous extract showed little absorbance reduction across the respective

frequencies. aqueous extract had a frequency of 714285.71-681818.18 during

maximum absorption of dye molecules while Ethanol extract had a frequency of

714285.71during its maximum absorption of the extracted dye molecules. Ethanol

extract exhibited a great deal of absorbance reduction with decreasing frequency as

shown in the graph.

4.3 Specific Gravity

Volume of water= 10mls

Density= Mass/volume

Specific Gravity= Density of the sample/Density of water (according to Greek

mathematician Archimedes, 250 B.C)

57
Density of water= 1gcm-1

Table 4.11: Specific Gravity of the Extracts

Samples Mass (g) Specific gravity

Aqueous Extract 10.193 1.01

Ethanol Extract 18.881 1.88

Alkaline Extract 20.218 2.02

Acidic Extract 20.112 2.01

The specific gravity of the extracts as shown in table 4.11 indicates that the

Alkaline Extracts has the highest density of dye stuff and also higher mass than the

rest of the extraction process. This test was carried out to determine the physical

properties of the effective dye solution of all the samples obtained from the

different extraction methods.

4.4 pH

The pH of the Aqueous Extract was recorded to be 6.15 which shows that the

Aqueous Extract was slightly acidic. This test was carried out to determine the

physical properties of the effective dye solution of the Aqueous Extract in

comparison to the other known samples.

58
 CHAPTER FIVE

CONCLUSION

The research work conducted shows that the extraction of dye from pawpaw was

possible using the three methods. Hydrochloric acid (Hcl), sodium hydroxide

(NaOH), ethanol, and water were used as extraction methods in the extraction of

natural dye from pawpaw and the methods used were observed to have their

59
maximum absorbance at wavelength to be within the range of 420nm-480nm. The

Fourier Transform Infra-Red (FTIR) analysis allowed the identification of

functional groups which are the characteristic substituent of the anthocyanins

group. The specific gravity of the dye samples were determined when compared

with water in order to know their solubility state of which Alkaline Extract was the

highest of all and also the physical properties of the solvents as well as the

substrate affected the dye extracts.

RECOMMENDATION

Natural dyes are dyes gotten from natural sources of plants, leaves etc. They are

better used than synthetic dyes which are toxic to the environmental. The processes

involved in the preparation of the dye is not easy but in turn it is easily degradable

compared to the synthetic dye.

The major outcome of exploring plant dyes and their application on textiles and

food is to improve the socio- economic and artistic development of the nation. In

view of the results of the study the following are recommended for consideration.

i. The universities around the world should incorporate a unique course on the

study on extraction and characterization of natural dyes.

60
 ii. Non-Governmental Organizations, public and private agencies involved in

youth employment scheme and skill development training can engage their

trainees in the cultivation of herbal plants for sustainable extraction of dyes.

iii. Natural dyes and pigments in place of conventional art materials for

examination project works to encourage schools to research into plant

material for course work. A policy on this would create room in the creative

arts for students to produce their own dyes from environmental resources

and thereby reduce educational costs.

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