REPLACING SYNTHETIC TEXTILE DYES WITH NATURAL

DYES OBTAINED FROM Tagetes erecta (MARIGOLD) AND

OPTIMIZING THE EXTRACTION AND DYEING
CONDITIONS
Dissertation Submitted to University of Mumbai in partial fulfillment for
the award for
Bachelor of science in biotechnology
By
CHAITANYA KARANJKAR
Under the guidance of
DR. VIJAY WAGHMARE
Head of the department
Department of biotechnology
Bhavan’s College, Andheri (W), Mumbai – 400 058

UNIVERSITY OF MUMBAI
Kalina, Santacruz (East), Mumbai, Maharashtra - 400098
March 2020
 CERTIFICATE
This is to certify that the Dissertation entitled “REPLACING SYNTHETIC TEXTILE
DYES WITH NATURAL DYES OBTAINED FROM Tagetes erecta (MARIGOLD) AND
OPTIMIZING THE EXTRACTION AND DYEING CONDITIONS” is a bonafide record
of independent research work done by “KARANJKAR CHAITANYA” under my
supervision and submitted to University of Mumbai in partial fulfillment for the award of the
Degree of Bachelor of Science in Biotechnology.

Signature of the Supervisor Signature of Coordinator

Dr. Vijay N Waghmare Dr. Vijay N Waghmare

Name and Signature of Examiner:

1.) _____________________________

2.) _____________________________

College Seal :

Date:
 DECLARATION
We, “KARANJKAR CHAITANYA” bonafide students of B.Sc. in Biotechnology, in the
Bhavan’s College, Andheri (West) affiliated with University of Mumbai, Mumbai would like
to declare that the dissertation titled “REPLACING SYNTHETIC TEXTILE DYES WITH
NATURAL DYES OBTAINED FROM Tagetes erecta (MARIGOLD) AND
OPTIMIZING THE EXTRACTION AND DYEING CONDITIONS” submitted by us in
partial fulfillment of the requirements for the award of the Degree of Bachelor of Science in
Biotechnology is our original work.

Name & Signature of the candidate(s)

CHAITANYA KARANJKAR

Place:

Date :
 ACKNOWLEDGEMENT
We owe our deep gratitude and acknowledgement to the “University of Mumbai” for giving
us the opportunity to work in a group project.
We greatly appreciate the support received from Bhavan’s College. We would like to take
this opportunity to express our profound gratitude and deep regards to our guide Dr. Vijay
Waghmare (H.O.D) who provided us with insights in expertise that greatly assisted the
research.
This thesis is the culmination of our journey for further career, like climbing a high peak step
by step accompanied with encouragement, hardship, trust & frustration when we found our
names appearing on the cover of this dissertation including colleagues, institute, teachers and
staff members who contributed to accomplish this task.
Special thanks to the laboratory staff of Biotechnology Department Mr. Pitamber Dandekar,
Mr. Mangesh Acharekar, Mr. Amarkumar Maurya & Mr. Vijay Sawant for providing us with
best facilities and guidance.

__________________________________
KARANJKAR CHAITANYA
 INDEX
Sr. No. CONTENTS PAGE No.

1. Introduction 1

2. Review of literature 3

3. Material and Methods 15

4. Results 26

5. Conclusion 38

6. Future Prospects 39

7. Bibliography 42
ABSTRACT:
Dyeing is the application of dyes or pigments on textile materials with the goal of

achieving colour with desired colour fastness. Today most of the textile industries use

synthetic chemical dyes which are carcinogenic and non-biodegradable. This experiment

exploited Marigold flowers for its dyeing and self mordanting properties along with its

antimicrobial effects. Marigold flowers from waste garlands were used as a source for the

colored pigments. The intensity of color in the extracted dye was optimized by using

water and ethanol as solvents at different mass: liquid ratio at different temperatures. An

extraction condition with 1:50 M: L ratio of petals: ethanol at 600 C temperature gave the

maximum colour intensity. Cotton fabric and wool yarn were dyed in the obtained extract

at different temperatures using different mordants such as Potassium dichromate,

Stannous chloride, Potash alum, Copper sulfate, Ferrous sulfate and Tannic acid to obtain

a diversified range of colors. No change in the surface color intensity was observed when

the dyed fabric and yarn were tested for wash and light fastness. Moreover the extracted

dye showed antimicrobial effects and the dyed fabric was fragrant. These findings reveal

that Marigold can serve as a potential source of natural colorant which can be used in

textile industry for dyeing purposes. Other naturally obtained waste such as onion peels,

beetroot skins, dried hibiscus, Asteraceae flowers and dried berries were exploited to

obtain a wide range of colors of natural dyes.

Keywords: Marigold flowers, natural dyes, textile, mordant

1. INTRODUCTION:

The primary source of dye, historically, has been nature, with the dyes being

extracted from animals or plants. Since the mid-19th century, however, humans have

produced artificial dyes to achieve a broader range of colors and to render the dyes

more stable to washing and general use. Different classes of dyes are used for

different types of fiber and at different stages of the textile production process, from

loose fibers through yarn and cloth to complete garments.

Acrylic fibers are dyed with basic dyes, while nylon and protein fibers such

as wool and silk are dyed with acid dyes, and polyester yarn is dyed with disperse

dyes. Cotton is dyed with a range of dye types, including vat dyes, and modern

synthetic reactive and direct dyes. These chemical dyes although economically

advantageous are carcinogenic and have harmful effects on the environment.

Clothing comes into prolonged contact with one’s skin, the largest organ, and so

toxic chemicals are often absorbed into the skin, especially when one’s body is warm

and skin pores have opened to allow perspiration. This absorption has been shown

to cause significant health effects, such as an increase in tumours. They have been

shown to have carcinogens, as they are made with many chemicals. Textile dyes can

also cause allergies such as contact dermatitis and respiratory diseases, allergic

reaction in eyes, skin irritation, and irritation to mucous membrane and the upper

respiratory tract.[3]

During the coloration process, a large percentage of the dye does not bind to the fabric and

is lost to the wastewater stream. Approximately 10-15% dyes are released into the

environment during dyeing process making the effluent highly colored and aesthetically

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unpleasant. The dying process discharges many chemicals through the polluted water, which

results in the death of aquatic life, the ruining of soils and poisoning of drinking water.[13]

Natural dyes are colorants that are derived from minerals, invertebrates and plants.

A majority of these dyes are vegetable dyes, sourced from plants – leaves, flowers,

roots, fruits and wood – and other organic sources like lichens and fungi. These

natural dyes prove to be a better alternative over chemical dyes because; first, the

colours produced by natural dyes and pigments are vibrant. Next, they are not only

biodegradable but nontoxic and non-allergic too. Additionally, natural dyes

neither contain harmful chemicals nor carcinogenic components, common to

artificial or synthetic dyes. Furthermore, the products used in producing natural dyes,

particularly plants, produce no waste, unlike the products used in the synthetic

dyeing process. This is because plants bypass the entire production process it takes

to create synthetic dyes. Another interesting advantage of natural dyes is that they

provide higher UV absorption in the fabrics they are used on.[16]

There are more than 500 dye yielding plants in nature. One such dye yielding plant

species is Tagetes erecta (marigold). Marigold petals contain natural colorants

mainly flavonoids and carotenoids which when extracted at suitable conditions using

a suitable solvent yield a dye which gives a soft hue and soothing shade on fabrics

and yarns. Marigold petals also contain some amount of ‘ellegic acid’ which itself

acts as a morant.[5]

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2. REVIEW OF LITERATURE:

2.1. Tagetes erecta (Marigold)[4]

2.1.1 General Description

It is a herbaceous annual or perennial whose height ranges from 30–110 cm. The root is

cylindrical, pivoting, with a fibrous and shallow branching system. The stem is striated,

sometimes ridge, smooth or slightly with villi, cylindrical, oval and herbaceous to

slightly woody, with resin channels in the bark, which are aromatic when squeezed.

Opposite leaves at the bottom alternate at the top, up to 20 cm long, pinnate, composed

of 11 to 17 leaflets, lanceolate to linear-lanceolate, up to 5 cm long and 1.5 cm wide,

acute to acuminate, serrated to sub-holders, the lower ones of each leaf frequently

setiform (in the form of threads), the superiors are sometimes completely setiform; with

abundant round glands.

The main characteristic of the flowers is that they are grouped in small heads or in

solitary inflorescences, on peduncles up to 15 cm long, they are liguladas of yellow

colors to red. In the flowers of the disc: 150 to 250 in the simple heads, in the doubles it

shows different degrees of transformation in ligules, yellow to orange corollas, of 8 to

10 mm in length. The fruits and seeds are: linear achenes 7 to 10 mm long, smooth or

slightly covered with stiff hairs at the corners. It has a long flowering period extending

throughout the summer and fall. It reproduces easily by seeds.

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2.1.2 Distribution

Although native to Mexico, they are also found in the countries of Central America and

the Caribbean: Belize, Bolivia, Colombia, Costa Rica, Cuba, Ecuador, El Salvador,

Guatemala, Guyana, Honduras, Jamaica, Nicaragua, Panama, Puerto Rico, and

Venezuela.

In their wild form they are found in the Balsas basin and western Mexico and live in

diverse types of ecosystems, such as tropical deciduous forests, thorny forests, cloud

forests and pine-oak forests. In the wild, it is found as a getaway in heavily disturbed

places at altitudes of 800–2300 m.

As an introduced species (cultivated) it can be found in China, India, Zambia, South

Africa and Australia.

2.1.3 Annual production of Tagetes erecta in India

The exact knowledge on the annual production of marigold in India is lacking.

Below is tabular representation of the production of marigold from various states of

India in the year 2015-16.

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 Sr.no State Lose value Cut value Total value

1 Madhya Pradesh 94.00 0.0 94.00

2 Karnataka 87.34 0.0 87.34

3 Gujarat 81.70 0.0 81.70

4 Andhra Pradesh 66.54 0.0 66.54

5 Haryana 61.83 0.0 61.83

6 West Bengal 58.10 0.0 58.10

7 Maharashtra 48.29 0.0 48.29

8 Chattisgarh 30.10 0.0 30.10

9 Tamil Nadu 18.08 0.0 18.08

10 Sikkim 16.05 0.0 16.05

Table 1 : Annual Production of Tagetes erectus in India.

The Table 1 depicts the contribution of the top 10 states of India in the Annual

production of marigold in India in the year 2015-16

2.1.4 Ecology

Marigolds require mild climate of luxuriant growth and profuse flowering. For seeds

germination optimum temperature ranges 180 to 300 C. Soil and planting is carried out

during rainy season winter and summer season hence flowers of marigold can be had almost

throughout the year

Depending on environment, planting of marigold can be done in three season i.e. rainy,

winter and summer and seeds are sown accordingly. Hence, flowers of marigold can be

Page | 5
obtained throughout the year. The seasons of sowing and transplanting of seedling for

obtaining flowers at different seasons of a year are as under :

Flowering season Sowing time Transplanting time

Late rains Mid-June Mid-July

Winter Mid-September Mid-October

Summer Jan-February February- March

2.1.5 Uses of Tagetes erecta :

• The yellow flowers of Tagetes erecta are the source of two food-colorant products:

‘marigold meal’ and ‘marigold extract’.

• Marigold meal consists of dried powdered flowers and is used mainly in poultry

feed to enhance the yellow colour of the meat and of the egg-yolks.

• It is used similarly in feed for salmon and crustaceans.

• Marigold extract is a solvent extract of the flowers, used mainly in western Europe

as a yellow to orange food colorant, e.g. in salad dressings, ice cream, dairy products

and other foodstuffs with a high fat content, but also in soft drinks, bakery products,

jams and confectionery.

Page | 6
• Fresh and dry flowers can be used to dye wool, silk and cellulose fibers into shades

of golden-yellow to orange and olive-green to bronze, depending on the mordanting

substances used.

• Tagetes erecta is popular worldwide as an ornamental, in Central America

particularly in cemeteries.

• In Honduras, a water extract of the plants was formerly used to wash corpses

because of the strong fragrance of the essential oil. Hence the Spanish common

name for Tagetes erecta used throughout Mexico and Central America ‘flor de

muerto’, flower of the dead. The Portuguese name ‘cravo de defuntos’, carnation of

the dead, has the same origin.

• The whole herb is considered medicinal with anthelmintic, aromatic, digestive,

diuretic, sedative and stomachic properties. It is used internally to treat indigestion,

colic, severe constipation, dysentery, cough and fever, and externally to treat sores,

ulcers, eczema, sore eyes and rheumatism.

• In East Africa its roots are eaten with oysternut (Telfairia pedata (Sm. ex Sims)

Hook.) to relieve pain in the sexual organs. In Mauritius a decoction of the flowers

is drunk against jaundice.

• Secretions from the roots have insecticidal and nematicidal effects. Sometimes

Tagetes erecta is planted in crop fields as an insect repellent because of its sharp

peculiar smell, although the plant itself is susceptible to insect pests.

• In India it is grown for its essential oil from which an ‘attar’ is produced. The oil is

used in small traces in perfumery to impart floral and ‘apple’ notes. In Gabon the

leaves are occasionally used as a condiment.

• There are various actions of marigold on an individual for which they can be used

those are

Page | 7
 Antimicrobial, Antipyretics, Antioxidant, Antifungal, Antiseptic, Antiviral,

Antibacterial, Anti-inflammatory and many more.

2.1.6 Medicinal properties of Tagetes erecta :

• Quantitatively, the colorants in Tagetes erecta flowers are mainly

quercetagitrin(pigment) and tagetiin(pigment), two glycosides of the flavonoid

quercetagetin, giving strong, beautiful dyes for textiles, but with relatively poor

light fastness.

• Currently most important from an economic point of view are the carotenoid

components of the flowers, among which lutein (xanthophyll) amounts to 64% and

sometimes up to 80%, with smaller amounts of antheraxanthin, zeaxanthin,

cryptoxanthin, β-carotene and about 14 other carotenoids.

• Colorants from marigold are available in a variety of forms. Formulations for

animal feeds usually consist of ground dried flowers, oleoresins, or saponified

oleoresins. Food colorants are available e.g. as purified lutein esters in oil-soluble

or water-dispersible systems, spray-dried emulsions, gum-based emulsions and

emulsifier-based emulsions. They show good stability to heat, light, pH changes

and sulfur dioxide.

• They are susceptible to oxidation, which can be minimized through encapsulation

or the addition of antioxidants such as ethoxyquin, ascorbic acid, tocopherols or

butylated hydroxyanisole and butylated hydroxytoluene.

• In the United States marigold meal and its extracts are approved only as colorants

in poultry feed, but not in human foods (they have not been given FDA-GRAS

Page | 8
 status). However, admission rules for the use of Tagetes extract as oil in foods for

direct human consumption are currently being widened.

• Naturally derived lutein is classified as E161b in the European Union, but marigold

extract has not been assigned an E number and is traded as ‘vegetable extract’. In

the Colour Index it is listed under CI 75135.

• Lutein is thought to be an essential micronutrient for normal vision and it is present

in high concentrations in the eye. It protects the eye by filtering the light and by its

antioxidant properties. Lutein is widely available in foods, e.g. in green leafy

vegetables, eggs and red peppers.

• Samples of the essential oil hydrodistilled from the flowers and leaves of Tagetes

erecta from Hungary were characterized by limonene, α-terpinolene, piperitone and

caryophyllene; the main components of samples from India were: limonene 7%,

(Z)-β-ocimene 42%, dihydrotagetone 14%, cis-tagetone 8%, cis-ocimenone 5% and

trans-ocimenone 6%. The essential oil appears to be a strong fungicide.

• At an average concentration of 2000 ppm it is more active than many commercial

fungicides such as captan and agrosan (potassium chlorate).

• Thiophenes and thiophene derivatives with nematicidal properties are present in the

leaves and roots of Tagetes erecta and other Tagetes species.

2.2. Solvents

2.2.1 Ethanol

Ethanol is considered a universal solvent, as its molecular structure allows for the

dissolving of both polar, hydrophilic and nonpolar, hydrophobic compounds. As

Page | 9
ethanol also has a low boiling point, it is easy to remove from a solution that has been

used to dissolve other compounds, making it a popular extracting agent for botanical

oils and natural dyes. Ethanol is a 2-carbon alcohol. Its molecular formula is

CH3CH2OH. An alternative notation is CH3−CH2−OH, which indicates that the carbon

of a methyl group (CH3−) is attached to the carbon of a methylene group (−CH2–),

which is attached to the oxygen of a hydroxyl group (−OH). It is a

constitutional isomer of dimethyl ether. Absolute ethanol is used as a solvent for

laboratory and industrial applications, where water will react with other chemicals,

and as fuel alcohol. The compound is widely used as a chemical solvent, either for

scientific chemical testing or in synthesis of other organic compounds, and is a vital

substance used across many different kinds of manufacturing industries. Ethanol alone

served the function of both wetting and leveling agent successfully where it is readily

biodegradable and hence this process becomes eco-friendly. This alternative process

gives better environmental effects due to the chemical substitution of toxic auxiliaries

with ethanol. Cost comparison with the traditional dyeing process shows a great

advantage for the ethanol process.

2.3. Mordants

2.3.1 Copper Sulphate

Copper (II) sulfate, also known as copper sulphate, are the inorganic compounds with the

chemical formula CuSO4(H2O)x, where x can range from 0 to 5. The pentahydrate (x = 5) is

the most common form. Older names for this compound include blue vitriol, bluestone,

vitriol of copper, and Roman vitriol.

Page | 10
Copper sulphate is used to color protein fibers like wool, silk, etc with natural dyes. It gets

linked chemically with the fibers and creates attachment points which bind with the colorant

from natural dyes creating light and wash fastness colors. It alters the color of the dye to

blue or green. [8]

2.3.2 Ferrous Sulphate

Iron (II) sulfate or ferrous sulfate denotes a range of salts with the formula FeSO₄·xH₂O.

These compounds exist most commonly as the heptahydrate but are known for several

values of x. The hydrated form is used medically to treat iron deficiency, and also for

industrial applications. Ferrous sulphate is used as a color changer. It is a mordant because

the addition of iron increases the light and wash fastness of dyes. It is often used with

cellulose fibers. It makes the cloth slightly hard or brittle. It shifts a color to a deeper, darker

shade. [8]

2.3.3 Potash Alum

Potassium alum, potash alum, or potassium aluminium sulfate is a chemical compound: the

double sulfate of potassium and aluminium, with chemical formula KAl(SO₄)₂. It is

commonly encountered as the dodecahydrate, KAl(SO₄)₂·12H₂O. Potash alum is the

mordant most frequently used by dyes for protein and cellulose fibers. It improves light and

wash fastness of all natural dyes and keeps color clear. It is inexpensive and safe to use. It

is not toxic to humans. [8]

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2.3.4 Potassium Dichromate

Potassium dichromate, K2Cr2O7, is a common inorganic chemical reagent, most commonly

used as an oxidizing agent in various laboratory and industrial applications. As with all

hexavalent chromium compounds, it is acutely and chronically harmful to health. It alters

the color obtained from dye by changing shades of orange to magenta, brown and purple. It

forms a bond with fibers and dyes which help in improving wash and light fastness of the

cloth. [8]

2.3.5 Stannous Chloride

Tin(II) chloride, also known as stannous chloride, is a white crystalline solid with the

formula SnCl₂. It forms a stable dihydrate, but aqueous solutions tend to undergo hydrolysis,

particularly if hot. SnCl₂ is widely used as a reducing agent, and in electrolytic baths for tin-

plating. Stannous chloride is used as mordant for wool and silk. It can absorb both acids and

bases. It helps in improving wash and light fastness. It alters the color obtained from dye to

brightening the color so that red becomes scarlet, yellow becomes even brighter and purple

becomes mauve. [8]

2.3.6 Tannic Acid

Tannic acid is a polyphenolic compound. It is a type of the commercially

available tannins. It acts as a weak acid. Tannic acid is found in the nutgalls formed

by insects on twigs of certain oak trees. It is a common mordant used in the dyeing

process for cellulose fibers such as cotton. It improves light and wash fastness

properties. It reacts with the corrosion products to from a more stable compound. [8]

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2.4. Chemicals for pretreatment

2.4.1 Triton X

Triton X-100 is a commonly used detergent in laboratories. Triton X-100 is widely

used to lyse cells to extract protein or organelles, or to permeabilize the membranes of

living cells. It is mostly used in the field of molecular biology. It is a non-ionic

detergent and helps in cleaning clothes and making the clothes accept the dye. It helps

in the attachment of the dye to the cloth.[3]

2.4.2 Sodium Carbonate

Sodium carbonate is an organic sodium salt and a carbonate salt. Sodium Carbonate is

the disodium salt of carbonic acid with alkalinizing property. Sodium carbonate is also

known as soda ash or washing soda. It is simply used to increase the pH. It reacts with

fibers making a permanent connection that holds the dye to the fiber. It actually

activates the fiber molecules so that they can chemically attack the dye. [3]

2.4.3 Formic Acid

Formic acid, systematically named methanoic acid, is the simplest carboxylic acid,

and has the chemical formula HCOOH. It is an important intermediate in chemical

synthesis and occurs naturally, most notably in some ants. Formic acid is used in the

pretreatment of wool fibers by reducing the pH of the solution. [3]

Page | 13
2.5. Agar cup method

The standard method for assaying different antibiotics in fluids is by the diffusion

assays in which their antibacterial activity is tested against carefully selected indicator

bacterial strains. In the agar cup diffusion assays, the different antibiotics to be used

are added to the agar cups in the agar seeded with the sensitive bacterial species. After

incubation, the diameters of the zones of inhibition are measured.

The agar cup method can also be used for assays of antimicrobial agents in the body

fluids like serum, CSF to determine whether therapeutic levels of antimicrobial agents

have reached the likely sites of infection. This is especially important when

chemotherapy fails to promote patient recovery even when the pathogen was known

to be sensitive to the drug used. Drug concentration are also monitored in the body

fluids to see if an accumulation of the drug is occurring which might lead to potentially

toxic concentrations and cause harmful side effects. The other main reason for the

measurement of antibiotics is to document that oral antibiotics are being absorbed

from the gastrointestinal tracts.

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3. MATERIALS AND METHOD

Materials:

• Flowers- Tagetes erecta (Marigold)

• Substrates - Cotton fabric and wool yarn.

• Chemicals –Ethanol, Potash Alum, Copper sulphate, Stannous chloride,

Ferrous sulphate, Potassium dichromate, Tannic acid, Triton-X, Sodium

carbonate, Formic acid.

• Media- Nutrient Agar

• Equipment -Weighing balance, Water bath, Colorimeter, Soxhlet

apparatus, Hot air oven.

3.1 Selection of materials

• Tagetes erecta (Marigold) used for dye extraction was selected on the basis of

literature survey.

• Cotton Fabric and Wool Yarn was selected as substrates for dyeing because both are

natural fibers, cost effective and used abundantly in textile industries.

3.2 Collection of materials

• An orange variety of marigold flowers was procured from a waste pit in the backyard

of a Shiv temple in Andheri. (Refer Fig.1.)

• Cotton Fabric and Wool Yarn were procured from a shop in Andheri West.

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 Figure 1: Collection source of waste Marigold Flowers.

3.3 Processing of materials

a. Preparation of Marigold flowers for dye extraction :

The petals of Tagetes erecta (Marigold) were separated from the calyx and washed

thoroughly with water and cleaned and dried before proceeding for solvent extraction.

Figure 2: Preparation of Marigold Petals

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b. Pretreatment of Cotton fabric and Wool yarn for dyeing :

Pretreatment of cotton fabrics and wool yarn were done by washing it in a solution

containing 0.5g/L Sodium carbonate and 2g/L non-ionic detergent (Triton-X) at 50°C in a

water bath for 30 minutes. The scoured fabrics and yarns were thoroughly washed with tap

water and dried at room temperature. The scoured materials were soaked in clean water for

30 minutes prior to dyeing or mordanting. [3]

Figure 3: Chemicals used in pre-treatment of substrates.

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3.4 Optimization of dyeing conditions

a. Optimization of M:L ratio

• Aqueous Extraction :

In this method water was used as the solvent to extract the dye from flowers. Different Mass:

Liquid ratio of petals: water ranging from 1:10 to 1:50 were prepared and incubated for 24

hours at room temperature for the extraction of dye from the petals into water. Absorbance

was measured to obtain the M: L ratio with the highest color intensity.[7]

Mass of Volume of
M:L ratio petals water
(in grams) (in ml)

Incubate at
1:10 1 10
room
1:20 1 20 temperature
for 24 hours
1:30 1 30 and
1:40 1 40 measure
absorbance
1:50 1 50 at 570nm.
Table 2: Aqueous Extraction of Dyes at Different M:L Ratios.

Figure 4: Aqueous Extraction at different M:L Ratio.

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 • Ethanol Extraction :

In this method ethanol was used as the solvent to extract the dye from flowers. Different

Mass: Liquid ratio of petals: ethanol ranging from 1:10 to 1:50 were prepared and incubated

for 24 hours at room temperature for the extraction of dye from the petals into ethanol.

Absorbance was measured to obtain the M: L ratio with the highest color intensity. [7]

Mass of Volume of
M:L ratio petals ethanol
(in grams) (in ml) Incubate at
room
temperature
1:10 1 10
for 24 hours
1:20 1 20 and measure
absorbance
1:30 1 30 at 570nm
1:40 1 40
1:50 1 50

Table 3: Ethanol Extraction of Dyes at different M:L

Figure 5: Ethanol extraction at different M: L ratio

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c. Optimization of extraction temperature :

The solvent and M: L ratio pair giving the maximum absorbance was selected and incubated

at a temperature range from 300 -800 C for 24 hours in a hot air oven. Absorbance was

measured to obtain the extraction temperature giving the highest color intensity. [7]

Temperature
Solution (in degree celcius) Incubate at
containing respective
petals: 30 temperatures
solvent in a 40 for 24 hours
particular 50 and measure
M:L ratio 60 absorbance at
70 570nm
80
Table 4: Extraction of dye at different temperatures

3.5 Soxhlet Extraction

Soxhlet extraction was carried out to obtain a concentrated form of the dye. The solvent

giving the maximum color intensity was selected. In this method, the weighed quantity of

dried petals (feed) and measured volume of solvent (S) were taken in F/S ratio

corresponding to the optimum M: L ratio. The raw material (dried flower petals) was kept

in thimble of Soxhlet extractor and the solvent was poured in the round bottom flask and a

condenser with high flow rate of water was fitted over it. The extraction was carried for 5

hours at the optimum extraction temperature and the extract thus obtained was used as a

stock for dyeing process.

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 Figure 6: Soxhlet extraction

3.6 Optimization of dyeing conditions

Optimization of dyeing Temperature :

The pretreated cotton fabric and wool yarn were dyed in the stock solution obtained by

soxhlet extraction at a temperatures range of 10o C to 90o C (Boiling point of the solvent

minus 10o C) in a water bath for 5 hours. The substrates dyed at different temperatures were

rated for their color intensity to obtain the temperature giving optimum dyeing of substrates.

Temperature
(in degree celcius)
Stock solution Incubate at
containing 10 respective
pretreated 30 temperatures
cotton yarn and 50 and measure
wool fabric absorbance at
70 570nm
90(only for water as solvent)
Table 5: Dyeing of substrates at different temperatures

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3.7 Comparative study using chemical mordants

Cotton fabric and wool yarn dyed without using any mordant were compared for color

produced on cotton fabrics and wool yarns dyed using different chemical mordants.

Mordanting was carried out by accurately weighing cotton fabrics and wool yarns and the

same were treated with different metal salts as mordants. The mordants used were Ferrous

sulfate, Copper sulfate, Potassium dichromate, Potash alum, Tannic acid and Stannous

chloride. The mordant (2 %) was dissolved in distilled water to make a material to liquor

ratio of 1:40. The samples were dipped into the mordant solution and incubated for 1/2 hour

at temperature range of 80 -85º C. After mordanting, the fabric and yarn samples were taken

out, squeezed and dried at room temperature. The mordanted samples were immediately

used for dyeing. (Refer Fig.7, Fig.8 and Fig.9.)[8]

Figure 7: Mordants for comparative study

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 Figure 8 :Mordanting of substrates in different mordants

Figure 9: Dyeing of substrates pretreated with different mordants.

3.8 Fastness tests

The cotton fabric and wool yarn dyed without using any mordant were tested for light and

wash fastness for 7 days. For light fastness, the materials were kept in daylight for 7

consecutive days and the loss of depth color was at the 7th day. For wash fastness, the

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materials were washed using detergent each day for 7 consecutive days and the loss of depth

color was noted at the 7th day.[9],[14]

3.9 Antimicrobial Assay

The dye obtained from Marigold flowers was tested for its antimicrobial activity using the

agar cup method.

PROCEDURE:

Day 1:

1. Inoculate 0.1 ml of mixed soil culture to 20ml cooled, melted nutrient agar butt, mix

and pour into a sterile plate. Allow it to solidify.

2. Make a well in each plate using a sterile borer. Transfer 0.1ml of the dye into the well

of the plate aseptically.

3. Place the plate in the refrigerator for 30 minutes for pre-diffusion of the antibiotic

solutions.

4. Incubate the plate at 370 C for 24 hours.

Day 2:

1. Measure the diameter of zone of inhibition given by the dye solution and interpret.

3.10 Evaluation of physical properties of the dyed substrates

To check the physical properties of the dyed cotton fabric and wool yarn, epidemiologic

survey was carried out on 25 voluntarily participated candidates. In this survey certain

physical parameters were evaluated-

1. Colour of dyed materials

2. Odour of dyed materials

3. Texture of the dyed materials (Touch test)

Page | 24
And rated accordingly:

1 = Very Poor

2 = Fair

3 = Good

4 = Very Good

5 = Excellent

Page | 25
 4. Results

4.1 Optimization of dyeing conditions

a. Optimization of M:L ratio:

• Aqueous Extraction:

Mass of Volume of
M:L ratio petals water Absorbance
(in grams) (in ml) at 570nm

Incubated
1:10 1 10 0.27
at room
1:20 1 20 temperature 0.14
for 24
1:30 1 30 hours. 0.11
1:40 1 40 0.10
1:50 1 50 0.10

Table 6: Absorbance of extracted dye at different M: L ratios of petals: water.

An M: L ratio of 1:10 petals: water gave the most intensified coloured extract.

Optical densities of dye extracted at different M: L ratios

of petals: water
0.3 0.27
Optical density at 570nm

0.25

0.2
0.14
0.15
0.11 0.1 0.1
0.1

0.05

0
1:10 1:20 1:30 1:40 1:50
M: L ratios

Figure 10 : Graph of optical densities of dye extracted at different M: L ratios of petals:

water

Page | 26
 • Ethanol Extraction:

Mass of Volume of

M:L ratio petals ethanol Absorbance

(in grams) (in ml) at 570nm

1:10 1 10 0.05

1:20 1 20 Incubated at 0.18

1:30 1 30 room 0.21

1:40 1 40 temperature 0.23

1:50 1 50 for 24 hours. 0.28

Table 7 : Absorbance of extracted dye at different M: L ratios of petals: water.

An M: L ratio of 1:50 petals: ethanol gave the most intensified coloured extract.

Optical densities of dye extracted at different M :L ratios

of petals: ethanol
0.35

0.3 0.28
Optical density at 570nm

0.25 0.23
0.21
0.2 0.18

0.15

0.1
0.05
0.05

0
1:10 1:20 1:30 1:40 1:50
M: L ratios

Figure 11: Graph of optical densities of dye extracted at different M: L ratios of petals:
ethanol.

Page | 27
*Note- The best colour intensity was obtained by an M: L ratio of
1:50 of petals: ethanol. Hence further extraction procedure was
carried out using ethanol as a solvent at an M: L ratio of 1:50.*

b. Optimization of extraction Temperature:

Temperature Absorbance at
(in Degree Celsius) 570nm

Solution 30 Incubated at 0.28

containing respective
petals: ethanol 40 temperatures for 24 1.39
at an M:L ratio hours
of 1: 50 50 1.52

60 1.70

70 1.34

80 1.20

Table 8: Absorbance of extracted dye at different temperatures.

An extraction Temperature of 600 C gave the most intensified coloured extract.

Optical densities of dye extracted at different

Temperatures
1.8 1.7
1.52
Optical density at 570nm

1.6
1.39 1.34
1.4
1.2
1.2
1
0.8
0.6
0.4 0.28
0.2
0
30 40 50 60 70 80
Temperature in degree celcius

Figure 12: Graph of Optical densities of dye extracted at different Temperatures.

Page | 28
4.2 Soxhlet extraction

The process of soxhlet extraction was carried out in a soxhlet apparatus using ethanol as a

solvent. The F:S ratio of feed(dried petals): solvent(ethanol) was taken as 1:50. Soxhlation

was carried out for 5 hours at 600 C. The resultant extract obtained was concentrated using

a rotary evaporator and used as a stock solution of dye for further experiments.

Figure 13: Extract from soxhlet apparatus.

4.3 Optimization of dyeing temperature

The pretreated cotton yarn and wool fabric were dyed in the stock solution obtained by

soxhlet extraction at temperatures of 100 C, 300 C, 500 C and 700 C in a water bath for 5 hours

and the following results were obtained. No significant difference in the colour intensity

produced by dyeing of substrates at different temperatures was observed. Yet, cotton fabric

and wool yarn dyed at 700 C showed a slight brighter and intensified yellow-orange colour

compared to the other temperatures.

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Figure 14 : Dyeing of Substrates at 10oC Figure 15: Dyeing of substrates at 30oC

Figure 16: Dyeing of Substrates at 50oC Figure 17: Dyeing of Substrates at 70oC

Page | 30
4.4 Comparative study using different chemical mordants

Mordants are used to impart different colours to the fabric and to improve their fastness

properties. The mordants used in the experiment were Ferrous sulphate, Potassium

dichromate, Copper sulphate, Tannic acid, Potash alum and Stannous chloride. Various

shades were obtained on cotton fabrics and wool yarn from a single dye by making use of 6

different mordants. Figure 18 shows cotton fabric and wool yarn dyed with the stock

solution of the dye without using any mordant. Figures 19, 20, 21, 22, 23 and 24 show the

dyed samples pre-mordanted with mordants such as Ferrous suplate, Potassium dichromate,

Copper sulphate, Tannic acid, Potash alum and Stannous chloride respectively.

Figure 18: Colouring Effect Of Dye (No Mordant)

Page | 31
 Figure 19: Ferrous Sulphate as Mordant Figure 20: Potassiun Dichromate as Mordant.

Figure 21: Copper Sulphate as Mordant. Figure 22: Tannic Acid as Mordant.

Figure 23: Potash alum as Mordant.. Figure 24: Stannous Chloride as Mordant..

Page | 32
 4.5 Fastness Tests

The cotton fabric and wool yarn dyed without using any mordant were tested for light and

wash fastness for 7 days. For light fastness, the materials were kept in daylight for 7

consecutive days and the loss of depth color was noted at the 7th day. For wash fastness, the

materials were washed using detergent each day for 7 consecutive days and the loss of depth

color was noted at the 7th day. The following results were obtained which are shown in

figures 26, 28 for light and wash fastness tests respectively. Figure 25 and 27 represent day

0 for comparison for light and wash fastness tests respectively.

Light Fastness :

Figure 25: Day 0 of Light Fastness Test. Figure 26: Day 7 of Light Fastness Test.

Page | 33
 Wash Fastness Test:

Figure 27: Day 0 of Wash Fastness Test. Figure 28: Day 7 of Wash Fastness Test

4.6 Antimicrobial assay

The dye obtained from Marigold flowers was tested for its antimicrobial activity using the

agar cup method.

Figure 29 shows the result for the antimicrobial assay of the dye tested using the agar cup

method. The dye showed antimicrobial effect against the soil microbes with a zone of

inhibition of 1.6 centimeters.

Page | 34
 Figure 29: Antimicrobial effect of dye obtained from Tagetes erecta.

4.7 Evaluation of physical properties of the dyed substrates

To check the physical properties of the dyed cotton fabric and wool yarn, epidemiologic

survey was carried out on 25 voluntarily participated candidates and physical properties

were rated accordingly-

1= Very Poor

2= Fair

3= Good

4= Very Good

5= Excellent

Page | 35
A] Colour of the substrates dyed with dye obtained from Tagetes erecta was found

Excellent as graph shows maximum percentage around at 68%.

25 responses A. Colour of the dyed substrates

18 17(68%)
16
14
12
10
8(32%)
8
6
4
2
0( 0%) 0(0%) 0(0%)
0
1 2 3 4 5

Figure 30 : Graph of Evaluation of Colour of the dyed substrates.

B] Odour of the substrates dyed with dye obtained from Tagetes erecta was found Very

good as graph shows maximum percentage around at 72%.

25 responses B. Odour of the dyed substrates

20
18(72%)
18
16
14
12
10
8
6
4(16%)
4 3(12%)
2
0(0%) 0(0%)
0
1 2 3 4 5

Figure 31 : Graph of Evaluation of Odour of the dyed substrates

Page | 36
C] Texture of the substrates dyed with dye obtained from Tagetes erecta was found

Excellent as graph shows maximum percentage around at 68%.

25 responses C. Texture of the dyed substrates

18 17(68%)
16
14
12
10
8(32%)
8
6
4
2
0( 0%) 0(0%) 0(0%)
0
1 2 3 4 5

Figure 32 : Graph of Evaluation of Texture of the dyed substrates

Page | 37
5. CONCLUSION
The present work successfully carried out the extraction of natural dye from Tagetes erecta

and optimized the extraction and dyeing conditions. The maximum dye extraction was

observed at 60 degree celcius using ethanol as the solvent at an M: L ratio of 1: 50. The

dye obtained showed antimicrobial effects against soil micro-organisms. The brightest

colour of the dyed cotton fabric and wool yarn was obtained at 700 C. No change in the

surface color intensity was observed when the dyed fabric and yarn were tested for wash

and light fastness. The colour, odour and texture of the dyed substrates were found to be

Excellent, Very good and Excellent respectively. The obtained results have shown that the

dyeing potential of the marigold flower could be huge which can be used as a source of

textile dyeing. The various color shades can be obtained using safe and ecofriendly

mordants. The dye extracted from marigold petals can be a better alternative to the

carcinogenic synthetic dyes.

Page | 38
6. FUTURE PROSPECTS
In the present scenario, the growing concerns among the communities globally against the

use of azo and benzidine synthetic dyes due to their carcinogenic, non-biodegradable nature

and hazardous effects on environment and human health, re-established the needs of natural

dyes to human society in terms of packaging and daily use products. With increase in

awareness for eco-friendly materials from sustainable resources, natural dyes attracted

researchers in traditional and diversified applications to develop effective eco-friendly and

cleaner process technologies. Natural dyeing is gradually making its way in the global

market and the production of naturally dyed eco-friendly textiles itself is a boon to save the

environment from hazardous synthetic dyes. However, the color derived from raw plant

materials is known to be very sensitive to the food processing conditions but, in general,

eco-friendly criterion paid safely to reconsideration of technological parameters, with more

attention to their effects on color stability, is therefore advisable and could be promisable

alternate to artificial colorants. Furthermore, the fast moving inexpensive synthetic dyes

stand as a big question before natural dyers. But, the non-toxic, non-carcinogenic, bio-

degradable and eco-friendly characteristics of naturally derived colorants made its own way

to reach the hearts of conscious consumers for healthy lifestyle, and can be achieved on a

higher cost. Hence, the applications of bio-colorants to textile substrates shall be helpful to

entrepreneurs to take up this venture which have good potential and bright future in a

number of applied sectors: leather, textiles and clothings, cosmetics, food, pharmaceutical,

and paint industries etc.[15]

Naturally derived pigments are available in nature with various hues and tones, currently

exploited for the coloring of textile and food materials, and other several other biomedical

applications. New sources of biomass biased pigments need to be available in sufficient

quantities for stability during processing and storing for large-scale cultivation, industrial

Page | 39
extraction, formulations, harvesting and storage and, application of biotechnological tools

including cell and tissue cultures, genetic engineering, promoted by experts as a

replacement for conventional growing techniques. Modem consumer’s demand for novel

eco-materials tend to the expansion in bio-colorant list towards forthcoming future. Evenly,

recent advances have been performed in the development and applications of natural

colorants covering different aspects such as identification of new sources, formulations,

extraction, purification and stability techniques. In spite of enthusiastic studies discussed

the data for the socio-economic viability of natural dye production and applications at

commercial scale for sustainable utilization of bio-resources, related to hygiene and eco-

safety which have a great future scope for the discovery of relatively better and more stable

natural pigments that may have wider industrial applications. More experimental

implementations should be focused to adopt novel technologies for making natural

colorants as a compatible as well as eco-safe alternative with synthetic colorants in different

spheres of our life to make a greener world.[6]

Nature has a multitude of natural dye providing plants, some with self mordanting

properties which will prove to be a better alternative over chemical dyes. In this experiment,

naturally obtained biodegradable waste such as dried hibiscus and Astereceae flowers;

onion and beetroot skin; and dried bulba berries were also exploited to obtain a wide range

of colour dyes. (Refer Fig.27.)[5],[12]

Page | 40
Figure 33: Substrates dyed with natural dyes obtained from biodegradable waste.

Page | 41
7. BIBLIOGRAPHY
1] Ashish Kumar, Samantha and Adwaita Konar. (2011). Dyeing of Textiles with Natural Dyes.

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2] Bruno Lellis et al. (2019). Effects of textile dyes on health and the environment and bio

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3] Chandan Kumar Jha, Ratan Kumar, Venkat Kumar S. And Devi Rajeshwari V. (2015).

Extraction of natural dye from marigold flower ( Tagetes erecta) and dyeing of fabric and

yarns: A focus on colorimetric analysis and fastness properties. Der Pharmacia Lettre, 7

(1):185-195.

4] Dixit Priyanka, Tripathi Shalini and Navneet Kumar Verma. (2013). A brief study on

marigold (Tagetes species): A review. International Research Journal of Pharmacy, 41.

5] Dumitrescu I., Visileanu E., and Marilena M. (2004). Natural Dyes obtained from plants

and vegetable wastes, 51, pp. 121-129.

6] G.W. Taylor. (1986). Natural dyes in textile applications. Review of Progress in Coloratoon

and Related Topics, volume 16, pp. 53-61.

7] Kiran Sachan and VP Kapoor. (2007). Optimization of extraction and dyeing conditions for

traditional turmeric dye. Indian Journal of Traditional Knowledge. 6(2).

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8] Md Moniruzzaman, Moni Mondal and Md. Hossain. (2018). The influence of mordants and

mordanting techniques on ecofriendly dyeing of cotton fabric by extracted used tea. 9. 111-

117.

9] Mohammad Raipur Rashid and Farid Ahmed. (2012). Study on Color Fastness to Wash and

Rubbing, Whiteness Index and Color Efficiency(K/S Value) of Weft Knitted Fabrics Made

From Conventional Ring and Compact Spun Yarn. 2. 1-10.

10] Ploysai Ohama, Nattida Tumpat. (2014). Textile Dyeing with Natural Dye from Sappan

Tree (Caesalpinia sappan Linn.) Extract. International Journal of Materials and Textile

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11] Rattanaphol Mongkholrattanasit, Jiŕi Kryštůfek, Jakub Wiener and Jarmila Studničlovà.

(2010). Dyeing and fastness properties of natural dye extracted from eucalyptus leaves using

padding techniques. Fibres Polym, volume 11, pp. 346- 350.

12] Redwan Jihad. ( 2014). Dyeing of Silk Using Natural Dyes Extracted From Local Plants.

International journal of Scientific and Engineering Research, Volume 5, Issue 11.

13] Rumini Devi Saini. (2017). Textile Organic Dyes: Polluting effects and Elimination

Methods from Textile Waste Water. International Journal of Chemical Engineering Research,

Volume 9, Number 1, pp. 121-136.

14] Shahnaz Khattak, Shabana Rafique, Tanveer Hussain and Baseer Ahmed. (2014).

Optimization of fastness and tensile properties of cotton fabric dyed with natural extracts of

Marigold flower( Tagetes erecta) by pad-steam method. Life science Journal. 11. 52-60.

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15] S.J Kadolp. (2008). Natural dyes: a traditional craft experiencing new attention. Delta

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16] Tassew Alemayehu, Zenebesh Teklemariam. (2014). Application Of Natural Dyes On

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