β-carotene from orange peel and carrot waste for cotton dyeing › f748 › b32449c2ccf8... ·...

Visiting adress: Skaraborgsvägen 3 Postal adress: 501 90 Borås Website: www.hb.se/ths

Thesis for the Degree of Master in Science

with a major in Textile Engineering

The Swedish School of Textiles

2014-06-26

Report no. 2014.14.04

Extraction of

β-carotene from orange

peel and carrot waste

for cotton dyeing

Susan Hecker

II Master thesis by Susan Hecker

Master thesis by Susan Hecker III

Description: Thesis submitted for the degree of Master in Science in Textile

Engineering

Title: Extraction of β-carotene from orange peel and carrot waste for cotton dyeing

Author: Susan Hecker

Supervisor: Mats Johansson, Vincent Nierstrasz

Cooperation partners: Brämhults Juice AB, School of Engineering at the

University Borås, Almedahls

Examiner: Anders Persson

The Swedish School of Textiles Report no. 2014.14.04

IV Master thesis by Susan Hecker

Acknowledgement

At the School of Textiles in Borås I would like to thank Maria Björklund and Catrin

Tammjärv who helped me and had always an open ear for all my questions and thoughts.

For the good feedbacks and discussions I want to thank my supervisors Vincent Nierstrasz

and Mats Johansson. Special thanks for the help with the HPLC analysis to Jorge Ferreira

at the Engineer school at the University of Borås.

Thanks to Stina Haglund at Brämhults Juice AB for the allocation of the orange peel and

carrot residue.

Anna Frisk and Karin Eklund at Almedahls made it possible that I could make the Xenon

test there.

And of course I would like to thank my family and friends who supported me during my

whole studies and especially during my master thesis work.

Borås, June 2014

Susan Hecker

Master thesis by Susan Hecker V

Abstract

The further usage of vegetable and plant waste from juice pressing industry as textile dyes is

presented in this thesis. The thesis is focused on β-carotene (C40H56) dyestuff extracted from

orange peel and carrot residue. The three organic solvents; ethyl acetate, petroleum ether

and hexane/acetone (1:1 v/v) were used for the extraction. The analysis of the extract was

done by RP-HPLC with a C18 column. The yield and the purity of the extracted β-carotene

were determined. The highest yield was achieved with petroleum ether whereas the other

two solvents were nearly as good. The highest and purest amount on β-carotene was found

in the extracts of carrots. The dyeing process was continued with β-carotene dyestuff of

orange peel and carrot residue extracted from 27 g of residue on 0,8 g cotton in the dyeing

ratio 1:50. Unmordant and post mordanted bleached and mercerized cotton fabric was dyed.

10% alum of the weight fraction of cotton was used as mordant. Colour measurements (K/S,

C*, L*, a*, b*, h and ΔE) and fastness properties as light- (ISO-Norm B02) and wash fastness

(ISO 105 – C) were tested. Fair light fastness grades were achieved by β-carotene dyestuff

of orange peel residue dyed on cotton fabric. Poorer were the grades for β-carotene dyestuff

of carrot residue for both unmordanted and mordanted samples. The wash fastness couldn’t

achieve reasonable results neither for β-carotene dyestuff from carrots nor orange peel

residue, dyed on cotton fabric.

Keywords: β-carotene, carrot and orange peel residue, organic solvent extraction, cotton

dyeing

VI Master thesis by Susan Hecker

Popular abstract

Brämhults Juice AB a juice pressing company in Borås, Sweden has every year 8100 tons of

orange peel waste and 570 tons of carrots waste from juice pressing that still contains a lot of

natural resources, such pigments and colorants. Using these pigments and colorants further

would increase its efficiency. A market for the further usage would be the food or textile

dyeing industry. The main colorant in carrots and orange peel residues is β-carotene

(C40H56). β-carotene is a water insoluble yellow-orange pigment. Nowadays it is used in the

food industry as food colorant and pro vitamin A precursor. In this paper the further usage of

dying cotton fabric is investigated. Due to solvent extraction β-carotene can be obtained in a

quite pure form from carrots residue and in less pure form from orange peel residue. The

dyed cotton fabric is red-orange for carrot extract. The orange peel extract contains besides

β-carotene also other carotenes; flavonoids, phenolic acids, pectin and waxes. The dyed

cotton is light yellow-orange. The fastness properties to stand washing and light exposure

were tested. The light fastness properties got fair results for β-carotene dyestuff of orange

peel residue dyed on cotton fabric and less fair results for the carrot extract. The wash

fastness properties couldn’t achieve reasonable results.

Master thesis by Susan Hecker VII

Content List

ACKNOWLEDGEMENT IV

ABSTRACT V

POPULAR ABSTRACT VI

TABLE LIST IX

FIGURE LIST X

LIST OF ABBREVIATIONS XI

1. INTRODUCTION 2

2. LITERATURE REVIEW 4

2.1. Β-CAROTENE 4

2.2. CARROTS AND ORANGE PEEL 7

2.3. EXTRACTION METHODS AND SOLVENT PROPERTIES 8

2.4. DYEING WITH NATURAL SOURCES 10

2.5. MORDANTS 11

2.6. DYE AND DYEING METHOD 12

3. PROBLEM DESCRIPTION 14

3.1. AIM 14

3.2. RESEARCH QUESTION 14

4. MATERIALS AND METHODS 16

4.1. MATERIALS 16

4.2. DRYING OF THE CARROT AND ORANGE PEEL RESIDUE 17

4.3. EXTRACTION METHOD 17

4.4. ANALYSIS OF THE DYESTUFF, PURITY AND YIELD 18

4.5. COTTON DYEING 18

4.6. SPECTROPHOTOMETRIC MEASUREMENTS 20

4.7. FASTNESS PROPERTIES TESTS 21

4.7.1. XENON TEST (ISO-NORM B02) 21

4.7.2. WASH FASTNESS TEST (ISO 105 – C) 21

5. RESULTS AND DISCUSSION 22

5.1. DETERMINATION OF Β-CAROTENE SPECTRUM 22

5.2. RESULTS OF THE RP-HPLC ANALYSIS 23

5.3. MORDANTING AND DYEING 26

5.4. SPECTROPHOTOMETRIC COLOUR MEASUREMENTS 28

5.5. RESULTS OF THE WASH FASTNESS TEST (ISO 105 – C) 30

5.6. RESULTS OF THE XENON TEST (ISO-NORM B02) 31

VIII Master thesis by Susan Hecker

6. CONCLUSIONS 33

7. FUTURE RESEARCH 34

REFERENCES 35

APPENDIX I

APPENDIX I II

AQUEOUS EXTRACTION II

ORGANIC SOLVENTS EXTRACTION WITH ETHANOL III

RESULTS OF THE HPLC ANALYSIS IV

APPENDIX II VI

CHEMICAL STRUCTURES OF THE SOLVENTS USED VI

APPENDIX III VII

VAT DYE VII

APPENDIX IV VIII

HPLC RESULTS VIII

APPENDIX V XII

SPECTROPHOTOMETRIC AND FASTNESS PROPERTY RESULTS XII

Master thesis by Susan Hecker IX

Table List

TABLE 1: TOTAL CAROTENOIDS AND THE CONTENT OF DIFFERENT CAROTENE AND XANTHOPHYLLS IN CARROTS

AND ORANGE PEEL IN MG/G CARROTS/ORANGE PEEL ............................................................................ 7 TABLE 2: SOLVENT PROPERTIES OF THE USED SOLVENTS ............................................................................ 9 TABLE 3: AMOUNTS OF THE DRIED RESIDUES (G) AND SOLVENTS (ML) THAT WERE USED FOR THE HPLC

SAMPLES AND THE DYESTUFF EXTRACTION FOR THE COTTON DYEING. ................................................. 17 TABLE 4: COMPOSITION OF THE USED Β-CAROTENE DYESTUFF OF CARROT AND ORANGE PEEL RESIDUE THAT

WAS USED FOR THE DYEING OF 0,8 G COTTON FABRIC. ....................................................................... 19 TABLE 5: FINAL DYE RECEIPT. AMOUNT PER LITRE WATER AND THE CALCULATED AMOUNT THAT WAS USED FOR

THE DYEING OF 0,8 G COTTON FABRIC IS LISTED. ............................................................................... 20 TABLE 6: THREE DIFFERENT CONCENTRATIONS USED FOR THE HPLC-STANDARD CURVE ............................ 23 TABLE 7: Β-CAROTENE CONTENT IN CARROTS AND ORANGE PEEL (MG/G OF CARROTS OR ORANGE PEEL) ...... 25 TABLE 8: COLOUR COORDINATION OF THE DYED COTTON FABRICS WITH CARROT EXTRACT .......................... 28 TABLE 9: COLOUR COORDINATION OF THE DYED COTTON FABRICS WITH ORANGE PEEL EXTRACT ................. 29 TABLE 10: RESULTS OF THE WASH FASTNESS TEST OF UNMORDANTED AND POST MORDANTED (ALUM) DYED

COTTON FABRICS OF ORANGE PEEL AND CARROT EXTRACT................................................................. 30 TABLE 11: RESULTS OF THE XENON LIGHT FASTNESS TEST OF UNMORDANTED AND POST MORDANTED (ALUM)

DYED COTTON FABRICS OF ORANGE PEEL AND CARROT EXTRACT. ....................................................... 31 TABLE 12: Β-CAROTENE CONTENT IN CARROTS AND ORANGE PEEL (ΜG/G) ................................................... IV TABLE 13: DIFFERENT PARAMETERS FOR VAT DYEING ON COTTON FABRIC .................................................. VII TABLE 14: RESULTS OF THE DETERMINATION OF Β-CAROTENE BY HPLC ................................................... VIII TABLE 15: COLOUR COORDINATION OF THE ALL DYED COTTON FABRICS WITH CARROT EXTRACT .................. XII TABLE 16: COLOUR COORDINATION OF THE ALL DYED COTTON FABRICS WITH ORANGE PEEL EXTRACT ......... XII TABLE 17: RESULTS OF THE WASH FASTNESS TEST OF ALL UNMORDANTED AND POST MORDANTED (ALUM)

DYED COTTON FABRICS OF ORANGE PEEL AND CARROT EXTRACT. ...................................................... XIII TABLE 18: RESULTS OF THE LIGHT FASTNESS TEST OF ALL UNMORDANTED AND POST MORDANTED (ALUM)

DYED COTTON FABRICS OF ORANGE PEEL AND CARROT EXTRACT. ...................................................... XIII

X Master thesis by Susan Hecker

Figure List

FIGURE 1: FRUITS AND VEGETABLE WASTE FROM JUICE PRESSING OF BRÄMHULTS JUICE AB IN TONS PER

YEAR. ................................................................................................................................................ 2 FIGURE 2: WASTE HANDLING OF BRÄMHULTS JUICE AB. WASTE IN TONS PER YEAR. ..................................... 3 FIGURE 3: ALL-TRANS Β-CAROTENE ............................................................................................................ 4 FIGURE 4: CAROTENOIDS CLASSIFICATIONS ................................................................................................ 5 FIGURE 5: Β-CAROTENE SYNTHESIS BY WITTIG REACTION ............................................................................ 6 FIGURE 6: 9-CIS-Β-CAROTENE .................................................................................................................... 6 FIGURE 7: DESICCATOR SILICA UNDER VACUUM CONTAINING ORANGE PEEL AND CARROT PIECES ................. 17 FIGURE 8: GRINDING DRIED ORANGE PEEL AND CARROTS IN A MORTAR ....................................................... 17 FIGURE 9: EXTRACTION FROM CARROTS IN PETROLEUM ETHER .................................................................. 17 FIGURE 10: GRAPH OF THE DETERMINED WAVELENGTH OF Β-CAROTENE AT 450 NM. ................................... 22 FIGURE 11: HPLC-STANDARD CURVE OF Β-CAROTENE .............................................................................. 23 FIGURE 12: HPLC GRAPH AT 450 NM SHOWING Β-CAROTENE, Α-CAROTENE AND ZEAXANTHIN EXTRACTED

FROM CARROT RESIDUE .................................................................................................................... 24 FIGURE 13: HPLC GRAPH AT 450 NM SHOWING Β-CAROTENE, Α-CAROTENE AND ZEAXANTHIN EXTRACTED

FROM ORANGE PEEL RESIDUE ........................................................................................................... 24 FIGURE 14: Β-CAROTENE DYESTUFF OF CARROT RESIDUE, DYED ON COTTON, MORDANTED AND UNMORDANTED

....................................................................................................................................................... 27 FIGURE 15: Β-CAROTENE DYESTUFF OF ORANGE PEEL RESIDUE, DYED ON COTTON, MORDANTED AND

UNMORDANTED ................................................................................................................................ 27 FIGURE 16: HPLC FROM CARROT EXTRACT IN AQUEOUS SOLUTION AT 450 NM ............................................ IV FIGURE 17: HPLC FROM ORANGE PEEL EXTRACT IN AQUEOUS SOLUTION AT 450 NM .................................... IV FIGURE 18: HPLC FROM CARROT EXTRACT IN 80 % AQUEOUS ETHANOL SOLUTION AT 450 NM ..................... IV FIGURE 19: HPLC FROM ORANGE PEEL IN 80 % AQUEOUS ETHANOL SOLUTION AT 450 NM............................ V FIGURE 20: HPLC GRAPH OF SAMPLE 1 Β-CAROTENE FROM ORANGE PEEL, EXTRACTED IN ETHYL ACETATE VIII FIGURE 21: HPLC GRAPH OF SAMPLE 7 Β-CAROTENE FROM ORANGE PEEL, EXTRACTED IN ETHYL ACETATE .. IX FIGURE 22: HPLC GRAPH OF SAMPLE 3 Β-CAROTENE FROM ORANGE PEEL, EXTRACTED IN HEXANE/ACETONE

........................................................................................................................................................ IX FIGURE 23: HPLC GRAPH OF SAMPLE 9 Β-CAROTENE FROM ORANGE PEEL, EXTRACTED IN HEXANE/ACETONE

........................................................................................................................................................ IX FIGURE 24: HPLC GRAPH OF SAMPLE 2 Β-CAROTENE FROM ORANGE PEEL, EXTRACTED IN PETROLEUM ETHER

........................................................................................................................................................ IX FIGURE 25: HPLC GRAPH OF SAMPLE 8 Β-CAROTENE FROM ORANGE PEEL, EXTRACTED IN PETROLEUM ETHER

......................................................................................................................................................... X FIGURE 26: HPLC GRAPH OF SAMPLE 4 Β-CAROTENE FROM CARROT, EXTRACTED IN ETHYL ACETATE ............ X FIGURE 27: HPLC GRAPH OF SAMPLE 10 Β-CAROTENE FROM CARROT, EXTRACTED IN HEXANE/ACETONE....... X FIGURE 28: HPLC GRAPH OF SAMPLE 6 Β-CAROTENE FROM CARROT, EXTRACTED IN HEXANE/ACETONE......... X FIGURE 29: HPLC GRAPH OF SAMPLE 12 Β-CAROTENE FROM CARROT, EXTRACTED IN HEXANE/ACETONE...... XI FIGURE 30: HPLC GRAPH OF SAMPLE 5 Β-CAROTENE FROM CARROT, EXTRACTED IN PETROLEUM ETHER ...... XI FIGURE 31: HPLC GRAPH OF SAMPLE 11 Β-CAROTENE FROM CARROT, EXTRACTED IN PETROLEUM ETHER .... XI

Master thesis by Susan Hecker XI

List of Abbreviations

Abbreviation Unit Definition

Å Ångström

a*

Red-green b*

Blue-yellow

C*

Chroma FTIR

Fourier transform infrared spectroscopy

h Hue HPLC

High Performance Liquid Chromatography

K/S

Kubelka Munk, colour strength or colour uptake L*

Lightness

NaOH

Sodium hydroxide Owf

Of the weight fraction

RP-HPLC

Reverse-Phase High Performance Liquid Chromatography

TLC

Thin Layer Chromatography UV

Ultra violet

v

Volume

V Volt

ΔE

Colour difference between uncoloured and coloured fabric

μg Microgram λ max

Maximal absorption

2 Master thesis by Susan Hecker

1. Introduction

Plant material like fruits, berries, roots, barks, vegetables, leaves etc. have been used for

textile dyeing over hundreds of years (Cardon, 2007). With the industrialisation and the

invention of synthetic dyes, natural dyes disappeared almost entirely in the textile industry

(Cardon, 2007, Hardman and Pinhey, 2009). With their well-known structures synthetic dyes

became easier in handling, higher in process safety and better in reproducibility compared to

natural dyes and so a success for dye houses. Nevertheless different researchers

investigated in the recent years again in new methods on dyeing with natural material and

their extracts. The used extraction methods are mostly organic-solvent extraction (Saleh et

al., 2013, Yi and Yoo, 2010). The article by Saleh et al. (2013) shows great potential of

dyeing cotton fabric with β-carotene containing dyestuff extracts, extracted from banana

leaves by organic solvent extraction. The results evidenced high tensile strength, high colour

strength, and high fastness properties for the dyed cotton fabrics.

Brämhults Juice AB, Borås has 8100 tons of orange peel waste and 570 tons of carrots

waste from juice pressing each year (Figure 1). In this state after the juice pressing the

vegetable and fruit residues still contain a lot of natural resources such as colorants and

pigments (Brämhults Juice AB, 2014). A further use of the waste could be the textile dyeing

industry (Guinot et al., 2007, Saleh et al., 2013). With extraction and purification of the

pigments, dyes comparable to synthetically textile dyes can be achieved.

Figure 1: Fruits and vegetable waste from juice pressing of Brämhults Juice AB in tons per year.

8100

570 630

Orange peel

Carrots

Others

Master thesis by Susan Hecker 3

β-carotene is a natural yellow-red pigment. Its chemical formula is C40H56. Its sources are

mainly plants, vegetables and fruits with a yellow-red colour as carrots and oranges. It is an

important precursor for vitamin A since it cannot be synthesised in the human body. The

intake has to be over the diet (Schlieper, 2005). As colorant it is used in the food industry

under the numbers E160 a-f (Domke et al., 2004).

The total amount of residue of the juice pressing of all vegetables and fruits from Brämhults

Juice is 9300 tons per year.

Figure 2: Waste handling of Brämhults Juice AB. Waste in tons per year.

This is composed of 8300 tons that is used for the biogas production and fodder, 500 tons for

fodder and 500 tons for waste disposal (Figure 2) (Brämhults Juice AB, 2014). A suitable

extraction method of β-carotene from orange peel and carrot residue would gain an

additional value to their waste. The further use of the residues can be economically beneficial

for both the juice pressing and the textile dyeing industry. Synthetically produced β-carotene

and the extracted β-carotene from natural sources have the same chemical structure and are

therefore comparative with each other.

8300

500 500

Biogas production and fodder

Fodder

Waste disposal


2. Literature review

The following literature review gives a look into the physical and chemical properties of β-

carotene, its synthesis and uses today. It gives an overview on the existing research in the

field of extraction of β-carotene and other natural pigments and their appliance in different

dyeing methods. It is shown how much β-carotene other researchers could extract from

orange peel and carrots. An investigation on different solvents and their suitability for β-

carotene extraction was done. Different dyeing methods used for water insoluble dyes and

on cotton and the usage of mordants is described as well.

2.1. β-carotene

β-carotene is a natural yellow-red coloured pigment with the chemical structure C40H56,

(Figure 3). It occurs mainly in plants, fruit and vegetables. It belongs to the group of

carotenes that together with xanthophyll belongs to the upper-level grouping of carotenoids

(Bergmann, 2004). Chemically β-carotene is classified as tetraterpene (Koskinen, 2012).

Carotenoids are divided in oxygen containing molecules (xantophylls) and non-oxygen

containing molecules (carotene) (Domke et al., 2004), Figure 4. The lack of hydroxyl groups

makes β-carotene hydrophobic. Due to its two cyclic rings at each end of the molecule chain,

β-carotene is a dicyclic compound, composed of 8 isoprene-units (C5H8). The high amount of

conjugated double bonds is called chromophor and is responsible for the colour impression

(Bergmann, 2004). β-carotene absorbs light of the wavelength 450 nm of the visible part of

the spectrum (Bauernfeind, 1981).

Figure 3: All-trans β-carotene

In the nature carotenoids have an indispensable protective role for chlorophyll and the

human eyes by absorbing and dissipating excessive light energy that would damage them


(Campbell, 2003). β-carotene is the most common carotene (Schlieper, 2005). β-carotene is

very sensitive to light, heat and oxygen. It can change its chemical structure due to oxidation,

degradation or isomerization. The latter doesn’t have any effect on the colour impression of

β-carotene because the double bonds do not break (Liaaen-Jensen, 1989). The handling of

carrots and orange peel residue for the β-carotene pigment extraction and the later dyeing

process therefore has to be handled with care. Too high exposures to light, heat and oxygen

have to be avoided. The storing is recommended under frozen conditions. According to Qian

et al. (2012) the β-carotene stability against degradation is higher at a pH between 4-8.

Natural β-carotene occurs in trans- and cis-isomers whereas synthetically produced β-

carotene is mostly all-trans-form, due to its higher absorbance for the human body

(Bundesinstitut für Risikobewertung, 2013).

Figure 4: Carotenoids classifications

The β-carotene synthesis is either produced by employing a Wittig reaction or a Grignard

reaction (Koskinen, 2012). The following reaction (Figure 5) is by Wittig. It shows a trans-

selective Wittig olefination of aldehydes II—synthesis of β-carotene from a dialdehyde.

Carotenoids

Carotene

(hydrophobic)

β-carotene α-carotene

Lycopene etc.

Xantophylls

(hydrophillic)

Lutein Zeaxanthin

etc.


Figure 5: β-carotene synthesis by Wittig reaction

As vitamin A precursor, synthetically produced β-carotene is especially important for the use

in the food industry as completion to the natural, in the food existing β-carotene. As food

additive with the purpose of a food colorant it is known under the numbers E160 a-f. (Domke

et al., 2004)

Figure 6: 9-cis-β-carotene


2.2. Carrots and Orange peel

Carrots and orange peels are a rich source for carotenoids. Carotenoids, especially β-

carotene is present in both of them and is responsible for their yellow-orange colour. Main

compounds in carrots and orange peel are β-carotene, α-carotene, lutein and zeaxanthin

(Table 1), (Heinonen, 1990, Wang et al., 2008, Curl and Bailey, 1956). Additionally orange

peel contains flavonoids, phenolic acids, pectin and waxes (Wang et al., 2008). The

compounds in orange peels extracts are by some researches tested on their UV-protective

properties and antimicrobial activity (Hou et al., 2013, Yi and Yoo, 2010). The total

carotenoids content and the amount of some xantophylls and carotenes of carrots and

orange peel are listed below.

Table 1: Total carotenoids and the content of different carotene and xanthophylls in carrots and orange peel in mg/g carrots/orange peel

Carrots Article Orange peel Article

Total

carotenoids 0,16 – 0,38

Mustafa et al.

(2012) 0,45 Wang et al. (2008)

Carotenes (hydrophobic carotenoids)

β-carotene 0,046 – 0,10 Heinonen

(1990) 0,05 – 0,056

Dumbravă et al.

(2010), Wang et al.

(2008)

α -carotene 0,022 – 0,049 Heinonen

(1990) 0,017 – 0,019

Curl and Bailey

(1956)

Xanthophylls (hydrophilic carotenoids)

Lutein 0,0011 – 0,0056 Heinonen

(1990) 0,029 Wang et al. (2008)

Zeaxanthin 0,0574 Curl and Bailey

(1956) 0,027 Wang et al. (2008)


2.3. Extraction methods and solvent properties

In literature different extraction methods for β-carotene (carotenes) from orange peel, carrots

and other fruits, vegetables and plants are described. The most common method is the

organic solvent extraction. It was described by several different researches for the extraction

of β-carotene from orange peel (Ghazi, 1999), (Dumbravă et al., 2010) and carrots (Biswas

et al., 2011), (Fikselova et al., 2008), (Livny et al., 2003), (Marx et al., 2000), but also from

other vegetables and plants as tomato, paprika (Levy et al., 1995) and from the algae

Dunaliella salina (Marchal et al., 2013), (Mojaat et al., 2008).

Several other extraction methods for β-carotene were researched. So were investigations on

supercritical fluid extraction of β-carotene done by Kaur et al. (2012), Chandra and Nair

(1997) and Benelli et al. (2010) for carrots and the latter one for orange peel. “Ultrasound

assisted extraction of β-carotene from Spirulina platensis”, a cyanobacteria was researched

by Dey and Rathod (2013). A microwave-assisted extraction of β-carotene from carrots was

done by Hiranvarachat et al. (2013) and the effect of enzymes on carotene extraction in

carrots was investigated by Jaramillo‐Flores et al. (2005).

The “Relative solubility, stability, and absorptivity of lutein and β-carotene in organic solvents

were tested by Craft and Soares (1992). The solubility of both carotenoids (β-carotene and

lutein) was best in tetrahydrofuran, whereas the least solubility for lutein was in hexane and

for β-carotene methanol and acetonitrile. Organic solvents can be classified due to their

chemical structure, according their solubility in water they are grouped into polar and non-

polar solvents (Smallwood, 1996). A good solubility for β-carotene was shown by some

organic solvents of the category hydrocarbons (same chemically classification as β-

carotene), ethers, esters, chlorinated solvents and ketones (Craft and Soares, 1992). For the

selection of a suitable solvent for β-carotene extraction a low boiling point is advantageous.

The solvent has to be removed by rotary evaporator after the extraction to achieve pure β-

carotene, with a low boiling point the thermal sensitive β-carotene can be preserved.

Two articles used the organic solvent mixture of acetone and hexane (1:1 v/v) for the

“Extraction of β-carotene from orange peel” (Ghazi, 1999) and from carrot juices (Marx et al.,

2000). Ghazi (1999) concluded that the organic solvent mixture of acetone and hexane

achieves the highest extracted yield of β-carotene. Biswas et al. (2011) extracted amongst

others β-carotene from carrots with four different organic solvents; acetone, diethyl ether,


acetonitrile and methanol. With acetone the highest yield was achieved followed by diethyl

ether. In another study β-carotene was extracted from orange (Citrus sinensis L.) fruits peel

extracts by three different organic solvents; ethanol, benzene and petroleum ether. Only in

the petroleum ether extract, β-carotene could be identified. The amount in the ethanol and

benzene extract was too little (Dumbravă et al., 2010). Ishida and Chapman (2009)

compared ethyl lactate as an environmentally friendly solvent with the, for the food industry

commonly used solvents ethyl acetate and ethanol for the carotenoid extraction. It was

mentioned that ethyl acetate is a good solvent for β-carotene and lutein and less good for all-

trans isomer of lycopene. That could help to achieve a higher purity of the extracted β-

carotene. Therefore ethyl acetate was chosen instead of ethyl lactate, which was slightly

better in the extraction of β-carotene.

The following table are the solvents that were used in this thesis for the β-carotene extraction

from orange peel and carrot residue. They are listed after their solvent classification. The

solubility of β-carotene (mg/L) and the stability after 10 days in % of the initial absorbance of

β-carotene at λmax (Craft and Soares, 1992), values of the boiling point (°C) and the polarity

of the solvents are given (Smallwood, 1996).

Table 2: Solvent properties of the used solvents

1 (Craft and Soares, 1992); 2 (Smallwood, 1996)

Solvent

Solubility of β-carotene mg/L

1

Stability after 10 days % of initial absorbance of β-carotene at λmax.

1 Boiling pt. °C

2 Polarity (H2O=100)

2

Article that uses the solvent

Hydrocarbons

Petroleum ether - - 36 0,9

(Dumbravă et al., 2010)

Hexane 600 92 69 0,9 (Ghazi, 1999, Marx et al., 2000)

Esters

Ethyl acetate 500 05 77 6,02

(Ishida and Chapman, 2009)

Ketones

Acetone 200 93 56 20,6 (Ghazi, 1999, Marx et al., 2000)


2.4. Dyeing with natural sources

There are different researches in the field of textile dying with natural plant and vegetable

extracts. Some of them are based on the extraction of pigments from orange peel and

carrots but are not specialized on the extraction of β-carotene. The extraction methods used

are depending on the solubility properties of the natural extract are mostly either aqueous

extraction or organic solvent extraction.

Hou et al. (2013) and Yi and Yoo (2010) investigated UV-protective properties and

antimicrobial activity of orange peel extracted dye. They used water and organic solvent

extraction to achieve dyestuff for wool and cotton fabric. Hou et al. (2013) analysed the water

extracted main colorant compounds in the extracted dyestuff as phenolic colorants and

pectin by UV-visible spectroscopy and FTIR (Fourier transform infrared spectroscopy). A

direct dyeing was done on wool with pre-, one-bath and post-mordanting. As mordants alum

and ferrous sulphate were used to achieve good colour, wash and acceptable light fastness

properties. Yi and Yoo (2010) identified flavonoids as the main colorant extracted by ethanol.

They concluded that the best dyeing conditions for cotton with orange peel dyestuff were a

pH of 3 at 60°C for 60 min, with a dye concentration of 800% owf.

With focus on by-product or waste further usage, dyestuff extracts of carrots and banana

leaves were analysed. Under aqueous extraction flavonoid aglycones were extracted from

carrot by-products and dyed on pre-mordanted hemp fibres. Analysis of the vegetable

colorant was done by TLC (Thin layer chromatography) and HPLC (High performance layer

chromatography). Two solvents for extraction were applied on banana leaves. In the alkaline

extract luteolin and apigenin and in the acetone extract chlorophyll a, and b and β-carotene

were analysed with HPLC. With ferrous sulphate, copper sulphate and potassium dichromate

premordanted cotton fabric was dyed in a closed bath for 5 min. at 56°C. The results showed

high tensile strength, high colour strength, and high fastness properties. (Guinot et al., 2007,

Saleh et al., 2013)

There are different investigations from the University of Innsbruck, Austria on the topic of

natural textile dyes from plant extracts. In all their articles aqueous extraction is applied to


assure an environmental friendly extraction process. In this way the residues of the

extraction can be further used as animal food or combustion. They registered the extraction

method for a patent. The plant materials used are amongst others Canadian golden rod,

onion skin, dyer´s weed and nutshells. The extracted dyes are analysed by a diode array

spectrophotometer. The dyestuff compounds analysed are flavonoids, tannins et al. Solid

dyestuff was produced from the extract due to the better handling and storing for the dye

houses. Exhaust dyeing was the employed dyeing method. The substrates used were linen,

polyamide and wool. To bind the dyestuff better to the fibres mordanting with alum, ferrous

sulphate and ferrous chloride was applied. (Bechtold et al., 2007, Leitner et al., 2012,

Wallner and Wenisch, 2011)

2.5. Mordants

Dye extracts of natural sources often have none or little amount of functional groups. Due to

that, their fastness properties are weaker. To overcome that disadvantage mordants are

often used to fix the colorant to the textile substrate. Mordants are metal or mineral salts that

function as a bond between the textile substrate and the dyestuff (Flint, 2008). There are

many different mordants used to help natural dyestuffs binding to a textile substrate. Some

have only the function of fixing the dyestuff to the textile substrate (alum) whereas others can

also influence or change the colour impression and saturation of the dyed textile (ferrous

sulphate), depending on the nature and concentration of the mordant (Cardon, 2007). Most

of them are heavy metal salts such as copper sulphate, ferrous sulphate and potassium

dichromate others are alum, stannous chloride and sodium chloride. Among them alum,

potassium aluminium sulphate (KAl(SO4)2 ×12H2O)and ferrous sulphate (FeSO4

×7H2O)

are very common used mordants due to their lower environmental pollution (Hou et al., 2013,

Bechtold et al., 2007, Choo and Lee, 2002, Meksi et al., 2012). There are different methods

when the mordant is added to the dye bath. Pre-, simultaneous- or post-mordanting. In pre-

mordanting, the to dyeing fabric is mordanted before the actual dyeing in a separated

mordanting bath. In simultaneously mordanting the mordant is added to the dye bath. This

brings the risk that the dyestuff and the mordant bind together before either of them has

attached to the fibre but its quick and saving resources as water and energy. The post-

mordant method is used to fixate the colours permanently and intensifies them. It is done in a

separate mordant bath after the dyeing process (Flint, 2008). Usually mordanting is done on

wool or other animal fibres. Cotton mordanting is rarely found in literature. One article that


was investigating in the mordanting of cotton fabric was by Ali et al. (2009). They used pre-

and post-mordanting of cotton fabric in a mordant-bath ratio of 1:15 (goods/liquid). The

mordants used were alum and ferrous sulphate. The best results were achieved for post-

mordanted cotton fabric with alum in a concentration of 10% (owf).

2.6. Dye and dyeing method

The difference between natural dyes to synthetic dyes is that natural dyes often don´t consist

of a single colouring molecule. Often they consist of several different colorants that belong to

different chemical groups (Cardon, 2007). A good example therefore is the orange peel.

Besides hydrocarbons (were β-carotene belongs to) orange peel consists of diole,

monoether diole, diether diole and polyole (Curl and Bailey, 1956). This and the changing

amount of the various compounds due to seasonal deviations make the repeatability of those

dyes more difficult compared to synthetic dyes. The classifications of textile dyes are either

done by their chemical structure and properties or by the fibre that will be dyed with it. The

most prominent property of the β-carotene is its water insolubility. This was the factor after

what the dye method was chosen. The other factors were the used fabric material, cotton. As

water insoluble textile dyes vat, sulphur and acid disperse dyes for polyester and metal

complex dyes are known. The substrates dyed with them are mostly cellulosic and polyester

fibres. Reactive dyeing is the most common dyeing method for cellulosic fibres as cotton and

viscose.

Cellulose fibres can be dyed by sulphur dyes. Similar to the β-carotene dyestuff they are

both water insoluble dyes. The difference is the containing sulphur group in the sulphur dye.

The water insoluble sulphur dyes becoming water soluble through alkali reduction.

Vat dyes are also used for the dyeing of cellulosic fibres. Vat dyes are absolutely water

insoluble and due to reduction they are transformed to the water-soluble leuco form. After the

dyeing process they are getting water insoluble again by oxidation.

The highest similarities in the dye structure have β-carotene with disperse dyes for

polyester. Both are low water-soluble due to the lack of existing water-soluble making

groups and reactive groups in the dye molecule. The dye molecules are relatively small and

non-polar (Marx, 2011). Some disperse dyes hydrolyse under alkaline conditions and form

free carboxylic acids that are soluble in the alkaline solution (Broadbent et al., 2001).


Hydrolysis of a disperse dye for polyester under alkaline conditions:

Dye-NR -CH2CH2CO2CH3 (s) + HO-(aq)

¬ ®¾

Dye-NR -CH2CH2CO2

- (aq)+CH3OH(aq)

The synthetically form of β-carotene is absolutely water insoluble, but nature has not

bounded the natural β-carotene to lipoidal systems. The protein complexes of which the β-

carotene is surrounded in carrots and orange peels and colloidal structures allow the β-

carotene to be dispersed in primarily aqueous systems. (Bauernfeind, 1981)

Reactive dyeing is the most common used dyeing method in the industry. Reactive dyes are

quite small (comparable to β-carotene) and water soluble molecules. The dyeing medium is

usually alkali and a huge amount of sodium chloride is used as electrolyte addition. The

chemical binding between reactive dyes and the fibres are an atom binding. (Marx, 2011)

The fact that polyester requires dyeing temperatures of 130°C to open up the fibre structure

and let the dye molecules migrate into fibre, cotton was chosen. Cotton can be dyed at

temperatures around 60°C. The high temperatures of polyester dyeing would destroy the β-

carotene double bonds and so the colour impression. The only possibility the dye polyester

at lower temperatures is with the use of carriers. But they are highly environmentally

unfriendly. (Marx, 2011)


3. Problem description

Brämhults Juice AB is producing 8670 tons of orange peel and carrot waste every year from

the total waste of 9300 tons. In the state after the juice pressing the vegetable and fruit waste

is still a valuable source for different natural pigments that can be extracted and used as

dyestuff for the textile dyeing industry. Alone from the carrots and orange peel waste more

than 200 kg of β-carotene could be extracted and used as a new dyestuff source for the

textile dyeing industry. There is no previous research found which focus on the extraction of

β-carotene dyestuff from orange peel and carrots as further use for cotton dyeing.

The extracted β-carotene dyestuff has to be a process stable product for dye houses. The

dye process has to be secured and reproducible with the same colour result to be applicable

for a dye house. Therefore an, as possible, pure dye has to be extracted from the natural

source, orange peel and carrots residue.

3.1. Aim

The aim of this master thesis is a comprehensive study. It is about the extraction of β-

carotene dyestuff from orange peel and carrot residue from the juice pressing industry and

the analysis of the extract by HPLC on purity and yield of β-carotene. Further the usage for

the dyeing of cotton fabric will be investigated. The extract on yield and the purity of the

extract have to be competitive with synthetically produced β-carotene. The dyed cotton

fabrics are tested on their wash and light fastness to see if the dyestuff is competitive with

synthetic dyes. The aspect of environmentally friendly solvents for the extraction is neglected

in the study. This might be interesting for the further work. For the present report this was out

of scope.

3.2. Research question

Which organic solvent or mixture is most suitable for β-carotene extraction from

carrots and orange peel


Does the extracted β-carotene conform to synthetically produced β-carotene?

Which yield and purity can be extracted?

Which dyeing method is most suitable to dye cotton with the extracted β-carotene-

dye?

Can acceptable wash and light fastness be achieved on the dyed cotton

fabric?

Can a better result be achieved with using a mordant?


4. Materials and methods

All the materials and methods that were used in this work are explained and listed in the

following chapter. There were always 2 samples done for the HPLC analysis and for the

dyeing with its fastness and colour measurements, to value the results in the arithmetic

average of 2 samples.

4.1. Materials

The residue of orange peels and carrots were from the local juice company Brämhults Juice

AB, Borås. Orange peel Valencia from Egypt and small amounts from Mexico, Tunisia and

Spain and carrots from the Nantes family from Gotland, Sweden were used for the β-

carotene extraction. The orange peel residues were 0,5 – 2 cm and the carrot residue was

0,1– 0,2 cm big when received from the juice company. The organic solvents used for the β-

carotene extraction were: ethanol absolute (>99,8%) from VWR International (preliminary

tests), ethyl acetate (≥99.9%) and a mixture of acetone (≥99.9%) and hexane (≥97.0%), all

HPLC grade and petroleum ether, spectrophotometric grade (all chemicals if not other

mentioned were from Sigma Aldrich). A bleached and mercerized cotton weave fabric (150

g/m2) was used for the dyeing process.

The mordants that were used are potassium aluminium sulphate (potassium alum)

and ferrous sulphate from Sigma Aldrich. Auxiliaries

used for the dyeing process were sodium chloride as electrolyte addition, Zenit Levelling

agent S from Zenit AB Sweden, dispersing agent Lyocol RDN fl. from Clariant Germany,

sodium hydroxide 25% (NaOH) and acetic acid 25% for the pH regulation.

(KAl(SO4)2 ×12H2O) (FeSO4

×7H2O)


4.2. Drying of the carrot and orange peel residue

Figure 7: Desiccator silica under vacuum containing orange peel and carrot pieces

The orange peel and carrot were dried

under vacuum with a vacuum pump RV12

from Edwards of 200 V before extracted.

For drying a desiccator silica with silica gel

was used. The drying was done to remove

the water from the residue to make sure

that only the residue without any water

was used for the further extraction. The

samples were dried in a dark place to

prevent degradation of the β-carotene due

to light exposure. The drying was

continued until the dry matter weight was

not changing anymore to be sure all

moisture was removed from the samples.

To achieve a higher surface area the dried

orange peel and carrot pieces were

ground in a porcelain mortar after the

drying.

Figure 8: Grinding dried orange peel and carrots in a mortar


4.3. Extraction method

Figure 9: Extraction from carrots in petroleum ether

Organic solvent extraction was used to

extract the β-carotene dyestuff from

orange peels and carrots residue. The

organic solvent extraction was carried out

with the solvents: hexane and acetone

mixture (1:1 v/v), petroleum ether and

ethyl acetate in a sample/solvent ratio of

1:15. The amounts of dried residue and

solvent used for the HPLC samples and

the dyestuff extraction were listed in the

table below. The dried ground samples

were mixed together with the solvent. The

mixture was kept at room temperature

under magnetic stirring for 30 min. After

filtration (Munktell filter paper, grade 3) the

solvent was evaporated using rotary

evaporator Büchi Rotavapor R-114 with

Büchi Waterbath B-480 and an Edwards

vacuum pump R12 of 200 V. First the

HPLC samples were done and after their

analysis the dyestuff extraction for the

dyeing was continued. From each sample

2 duplicates were done.

Table 3: Amounts of the dried residues (g) and solvents (ml) that were used for the HPLC samples and the dyestuff extraction for the cotton dyeing.

Solvents HPLC samples Dyestuff extraction

Ethyl acetate 5 g dried orange peel or carrot

75 ml solvent

Ethyl acetate was not used for the dyestuff

extraction due to the lowest yield of β-carotene

determined by HPLC.

Petroleum ether 5 g dried orange peel or carrot

75 ml solvent

27 g dried carrot

400 ml solvent

Hexane/acetone

mixture

5 g dried orange peel or carrot

75 ml solvent

27 g dried orange peel

400 ml solvent


4.4. Analysis of the dyestuff, purity and yield

The spectrum for β-carotene was determined at 450 nm. For its standard curve 4 different

concentrations of β-carotene (β-carotene standard, synthetic ≥95%, HPLC grade, crystalline

from Sigma Aldrich) in hexane/ acetone (1:1 v/v) were made spectrophotometrically in a

Biochrom Libra from Nordic Biolabs. The concentrations for the standard curve were 0,033,

0,0056, 0,00093 and 0,00015 mg/ml of β-carotene.

Reverse Phase High Performance Liquid Chromatography (RP-HPLC) was used to

analyse β-carotene in the extracts and to determine the purity and the yield. 2 x 6 samples

were analysed. β-carotene analysis was realized by a Waters Alliance Seperation Module

2695, with a UV detector, Refractive Index Detector 2414 at 450 nm. The column was a C18

Sunfire reverse phase column from Waters, 4,6 mm × 250 mm; 5,0 µm particle size and pore

size 100 Å. The mobile phase was a methanol and acetonitrile (9:1 v/v) mixture. The flow

rate was 1,0 ml/min. β-carotene standard, synthetic ≥95%, HPLC grade, crystalline from

Sigma Aldrich was used. The standard for the HPLC was prepared in the concentrations

0,033, 0,025 and 0,0167 mg/ml. With help of the standard curve the concentration of β-

carotene in the extracts was calculated to draw conclusions from how much β-carotene could

be extracted per grams of carrots or orange peel and how much β-carotene was used for the

dyeing of cotton fabric. The 12 samples for the RP-HPLC analysis were diluted with 6 ml of

the used solvents for each extraction.

4.5. Cotton dyeing

A reactive dyeing method in a dye bath ratio of 1:50 (sample/dye bath g/ml) was applied. The

sample size was 10 cm x 5,5 cm. The size was big enough to cut a sample for the washing

test of the size 10 cm x 4 cm and a sample for the light fastness test of the size 7 cm x 1,5

cm. The rest of the sample was used as a reference sample to compare the colour changes

after the fastness property tests.

The dye: The dye used for the cotton fabric dyeing is named β-carotene dyestuff of carrot or

orange peel residue in the following work. The extracts achieved by organic solvent

extraction were used without any modifications. The two highest yields of the β-carotene

extraction of orange peel and carrot residue were used for the dyeing. The petroleum ether


extract from carrots and the hexane/acetone mixture extract from orange peel. The dyes

consists of the extracted β-carotene in known yield (determined by the RP-HPLC analysis)

and the by literature determined compounds α-carotene and zeaxanthin. Besides this

allocated compounds, the β-carotene dyestuff of orange peel residue contains flavonoids,

phenolic acids, pectin and waxes.

Table 4: Composition of the used β-carotene dyestuff of carrot and orange peel residue that was used for the dyeing of 0,8 g cotton fabric.

Dye name Containing β-

carotene amount

determined by RP-

HPLC analysis

Containing

compounds

determined by

literature

Other compounds

β-carotene dyestuff

of carrot residue

10,5 mg α-carotene and

zeaxanthin

-

β-carotene dyestuff

of orange peel

residue

0,1 mg α-carotene and

zeaxanthin

Flavonoids, phenolic

acids, pectin and

waxes

After the evaporation of the solvent the dye was left in the rotary evaporator beakers. The

amount for both extracts (carrot and orange peel) was so less that it was impossible to

remove the dye by a spoon. The chosen way to remove the dye from the glass beaker walls,

was to solve it in the rotary evaporator beakers. Half of the water amount used for the dye

bath (20 ml) was added to the beakers together with the 8 ml sodium hydroxide. The sodium

hydroxide showed in pre tests that it helps the extract to solve in the water. It could be

observed that the β-carotene dyestuff of orange peel residue solved readily whereas β-

carotene dyestuff of carrot residue needed to be warmed in a water bath together with the

glass beaker. Additionally the extract was scratch from the glass walls with a spattle. The

dyeing pistons were each prepared with the additives: 1,2 g sodium chloride, 4 ml levelling

agent, 0,2 ml dispersing agent and 0,8 g cotton fabric. Then the solved dye was added to the

piston. The rest of 20 ml water was to rinse the rotary evaporator bakers once more, to be

sure, that all dye was removed form the beaker. Acetic acid 25 % was added to the piston

until the pH between 4-5 was reached.


Table 5: Final dye receipt. Amount per litre water and the calculated amount that was used for the dyeing of 0,8 g cotton fabric is listed.

Chemical/Additives Amount per litre

water

Calculated amount used for the

dyeing of 0,8 g cotton fabric

Cotton - 0,8 g

Water (tap water) - 40 ml

β-carotene dyestuff of carrot

residue

Defined in table 4

β-carotene dyestuff of

orange peel residue

Defined in table 4

Salt 30 g/l 1,2 g

Levelling agent 10% 20 ml/l 4 ml

Dispersing agents, lyocol

RDN 10%

5 ml/l 0,2 ml

Sodium hydroxide 25 % 200 ml/l 8 ml

The samples were dyed in a dyeing machine, Pyro Tec MB 2 from Roaches International Ltd.

at 60°C for 60 min. with a rate of 2°C/min. After dyeing the samples were rinsed in cold water

and dried in a dryer.

4.6. Spectrophotometric measurements

Spectrophotometric measurements of the dyed samples were done with a Datacolor CHECK

Pro and evaluated by the Datacolor TOOLS software from Datacolor. The measured values

were the average of 5 measurements. As standard illuminate D65/10 (daylight) was used

and a tolerance of CMC 2:1. The values measured were K/S Kubelka Munk, which can be

seen a colour strength or colour uptake, the CIE colour parameters for lightness-darkness L*,

red-green a*, blue-yellow b*, chroma C*, hue h and ΔE. Where ΔE describes the colour


difference between the uncoloured bleached and mercerized cotton weave with coloured

ones. The Datacolor TOOLS software automatically calculated all values.

4.7. Fastness properties tests

Two fastness property tests were done on the dyed cotton samples. Xenon test (ISO-Norm

B02) and wash fastness test (ISO 105 – C).

4.7.1. Xenon test (ISO-Norm B02)

To determine the light fastness of the dyed cotton fabric xenon test according to ISO-Norm

B02 was done. The prepared samples had the size 1,5 x 7 cm. The samples were rayed by

xenon arc light up to grade 4 and analysed in D65 illuminate.

4.7.2. Wash fastness test (ISO 105 – C)

The wash fastness was ascertained by a wash fastness test according to ISO 105 – C. The

sample was together with an additional adjacent fabric, a multistripe with 6 different fabric

samples (diacetate, cotton, polyamide, polyester. acryl and wool) washed for 30 min at 60°C,

with 50 ml of detergent solution (4 g detergent /l). 25 steel balls were added. A common

household detergent was used as washing liquid. The washed samples were rinsed and

dried. Afterwards the dyed cotton samples were analysed on colour change and the

multistripe adjacent fabrics were analysed on staining with the 5 grades grey-white scale in

D65 illuminate.


5. Results and discussion

The following chapter lists the results and interprets them in graphs, tables and explanations.

Some of the results are further analysed to become clearer. The results are the arithmetic

average of 2 samples done for the HPLC analysis and for the dyeing with its fastness and

colour measurements.

Preliminary test showed that ethanol and water as solvents are not suitable for β-carotene

extraction due to their polar character (see Attachment I).

5.1. Determination of β-carotene spectrum

The determination of the spectrum was done in the range of 190 – 500 nm to determine the

visible range of β-carotene. The identified wavelength was at 450 nm that is confirmed by

literature (Bauernfeind, 1981).

Figure 10: Graph of the determined wavelength of β-carotene at 450 nm.


5.2. Results of the RP-HPLC analysis

The following graph shows the HPLC-standard curve for β-carotene.

Figure 11: HPLC-standard curve of β-carotene

The standard or calibration curve was done by three different concentrations of β-carotene in

mg per ml solvent. Those are listed in the table below together with the area. The standard

curve proceeds linearly.

Table 6: Three different concentrations used for the HPLC-standard curve

Sample Area Concentration (mg/mL)

Standard 1 1389337 0,034

Standard 2 1019366 0,025

Standard 3 704341 0,0167

y = 41,479,150.34x R² = 1.00

0

200000

400000

600000

800000

1000000

1200000

1400000

1600000

0 0.01 0.02 0.03 0.04

Are

a

Concentration (mg/ml)


The RP-HPLC analysis identified clearly β-carotene in the extracts of carrot and orange

peel at a retention time around 55 min (Figure 12-13) and a purity of more than 50 % in the

carrot extract. The differences in the orange peel and carrot extract were the amount of other

compounds that were shown by the HPLC. An identification of the other main compounds

was done with help of literature.

RP-HPLC analysis in this work was based on the article by Ben-Amotz and Fishier (1998).

Due to their work other peaks besides β-carotene were allocated. They identified three main

peaks of carrots: β-, α-carotene and zeaxanthin. According to them β-carotene showed the

highest area and zeaxanthin the lowest. Another compound with a very low area was lutein.

With the determined retention times in the HPLC graphs of the carrot extract compounds,

these three compounds could be identified in the orange peel samples as well (Figure 12,

13). The purity of carotenes in the extract is with an area of β- and α-carotene of more than

90 % and less other compounds very high. Besides the allocated compounds (α-, β-carotene

and zeaxanthin) orange peel contains flavonoids, phenolic acids, pectin and waxes (Wang et

al., 2008).

Figure 12: HPLC graph at 450 nm showing β-carotene, α-carotene and zeaxanthin extracted from carrot residue

Figure 13: HPLC graph at 450 nm showing β-carotene, α-carotene and zeaxanthin extracted from orange peel residue


There was no significant difference in the purity and amount between the different solvents.

The lowest amount of β-carotene was extracted by ethyl acetate with 0,26 mg/g of carrots

and 0,0027 mg/g of orange peel. The highest amount of β-carotene from carrots was

extracted by petroleum ether with 0,39 mg/g and from orange peel respectively with 0,0036

mg/g from petroleum ether and hexane/acetone mixture, (Table 4).

Table 7: β-carotene content in carrots and orange peel (mg/g of carrots or orange peel)

Organic solvent Carrots Orange peel

Ethyl acetate 0,26 0,0027

Petroleum ether 0,39 0,0036

Hexane/acetone (1:1 v/v) 0,30 0,0036

The extracted amount varies only minimal between the different solvents. The highest

extraction yield for both carrot and orange peel residue, achieved petroleum ether. Whereas

the difference to the lowest extraction yield with ethyl acetate is only minimal for carrot and

orange peel extracted β-carotene. The amount of extracted β-carotene increased with

decreasing polarity of the solvents.

Neither water as a polar solvent nor ethanol as an organic solvent is suitable for extracting

the non-polar β-carotene. No relevant peaks were allocated at a wavelength of 450 nm with

the HPLC of these extracts. Hence nor or too less β-carotene or other carotenes were

extracted. The same result was obtained by Dumbravă et al. (2010) the extracted amount

with ethanol from orange peel was to low for determination in a visible range. Ethanol has a

quite low relative solubility of ethanol for β-carotene with 30 mg/L (Craft and Soares, 1992)

compared with the used solvents (ethyl acetate, petroleum ether and hexane/acetone) were

acetone had the lowest relative solubility with 200 mg/L and hexane the highest with 600

mg/L. From the article by Yi and Yoo (2010) can be assumed that mostly flavonoids are

extracted by ethanol.

The selection of the solvents was done under the criteria of the polarity of the solvent and the

boiling point. For the evaporation of the solvent in the rotary evaporator it is important that


the solvents boiling temperature is as low as possible. Degradation of heat sensitive β-

carotene is therefore avoided.

To solve the non-polar β-carotene, solvents of the same character were used with the

exception of the hexane and acetone mixture. A mixture of the non-polar solvent hexane and

the polar solvent acetone helped to extract the highest amount of natural β-carotene.

According to Ghazi (1999) “…naturally occurring carotenoids are found inside cells and

surrounded by an aqueous protoplasmic medium, so acetone can bind water and force

carotene to be out of solution.“

5.3. Mordanting and Dyeing

Preliminary test showed that mordanting with ferrous sulphate adulterated the light shades of

β-carotene dye on cotton. The differentiation between ferrous sulphates own colour in

orange-red shades and orange shades of β-carotene dyestuff of carrot and orange peel

residue is difficult. This state of investigation is only based on the original colour of β-

carotene dyestuff of carrot and orange peel residue on cotton. Therefore the research on

ferrous sulphate as mordant was not investigated further.

The results for the vat dyeing method (see appendix II) of cotton with β-carotene dyestuff of

carrot and orange peel residue extracted by the organic solvents ethyl acetate and

hexane/acetone did not show any colour uptake of the cotton. The vat dyeing bath was at

room temperature around 20°C. That had the consequence that the cotton fabric was barley

coloured because the colour pigments didn’t diffuse into the fibre. When changing the

method vat dyeing in reactive dyeing where the cotton fabric was dyed in the same

concentration and the ratio 1:40 at 80°C for 90 min, the fabric didn´t show any colour uptake.

The reason for this might be that at the temperature of 80°C the β-carotene degraded or

oxidized and the colour was destructed.

Regarding the results from the HPLC analysis the dyeing was done by the highest

concentration of β-carotene achieved. That was for carrots with petroleum ether as solvent

and for orange peel with hexane and acetone mixture. The extracts achieved by the organic

solvent extraction were not further modified. The composition of the dye is listed in table 4.

According to Ali et al. (2009) the best colour and fastness results for a mordanted cotton


fabric in colour coordinates L*, a*, b*, C*, h*, wash and light fastness was achieved by post

mordanting with 10% of alum owf of cotton.

Fair dyeing results were achieved with the dyeing at 60°C for 60min. The dye results on

cotton fabric with β-carotene dyestuff of carrot and orange peel residue are shown in figure

14-15. The same adjustments gave good results in the dyeing of cotton with unripe Citrus

grandis Osbeck extract done by Yi and Yoo (2010).

Figure 14: β-carotene dyestuff of carrot residue, dyed on cotton, mordanted and unmordanted

Figure 15: β-carotene dyestuff of orange peel residue, dyed on cotton, mordanted and unmordanted

The pH of the dye solution was adjusted to 4-5 were the β-carotene is according to Qian et

al. (2012) stable. The goods – dye bath ratio was 1:50. The dyed cotton fabric was for both

β-carotene dyes fairly even dyed. Even though the β-carotene concentration of the dyed

cotton with β-carotene dyestuff of orange peel residue is 100 times lower (0,1 mg β-carotene

is contained in the dyestuff) than the concentration in the β-carotene dyestuff of carrot

residue dyed samples (10,5 mg) their colour uptake ΔE is higher (Table 8-9). This might be

due to the different compounds that the β-carotene dyestuff of orange peel residue contains.


The different compounds might have more functional groups and a better absorbency on the

cotton fibres than the β-carotene itself has. The β-carotene that is mostly present in the β-

carotene dyestuff of carrot residue has a lack of functional groups that makes it difficult to

absorb on the cotton fibres, which resulted in a lower colour uptake indicated by ΔE.

5.4. Spectrophotometric colour measurements

The β-carotene dyestuff of carrot residue, dyed cotton fabric was dyed in a yellow-reddish

shade. The cotton fabric was evenly dyed. Compared to the lower concentrations of levelling

agent in some preliminary tests the evenness of the colour was good with 100ml/l for both β-

carotene dyestuffs. The levelling agent lowered the rate of absorption of the β-carotene

dyestuff of carrot and orange peel residue on the cotton fabric, which resulted in a more

evenly dyed substrate.

The results for the colorimetric measurements of cotton weave fabric, dyed with β-carotene

dyestuff of carrot residue are given in table 5. The measurements were done by the CIE

colour system. The K/S values were for both unmordanted and mordanted samples the

same. The post mordanted samples were little bit lighter in colour due to the slightly higher L*

value. However the L* values for both unmordanted and mordanted samples were high

compared to the low values of C*. Considering both values L* and C* together the shades of

β-carotene dyestuff of carrot residue dyed cotton weave fabric were light and weak in colour

saturation. The a* and b* values indicate the shades of reddish yellow. The mordanted

samples were compared to the unmordanted samples slightly more colour saturated (C*) and

the mordant bind more dyestuff on the cotton fabric means more colour absorbed on the

cotton fabric (ΔE). The shade is more yellowish and less reddish.

Table 8: Colour coordination of the dyed cotton fabrics with carrot extract

Carrot

K/S L* a* b* C* h* ΔE

Unmordant 0,15 85,65 8,37 12,13 14,76 55,50 25,99

Alum post mordant 0,15 88,25 6,31 14,78 16,07 66,90 26,92


The ΔE shows that the colouration of β-carotene dyestuff of orange peel residue dyed on

cotton is higher than the colouration with β-carotene dyestuff of carrot residue. This might be

due to the higher amount of different compounds extracted from the orange peel. The

different compounds have different molecule sizes and functional groups and therefore a

different affinity to absorb on cotton fibres. Whereas in the β-carotene dyestuff of carrot

residue most compounds are - and β-carotene, which have due to the lack of functional

groups a lower affinity to cotton fibres.

Table 9: Colour coordination of the dyed cotton fabrics with orange peel extract

Orang-peel


Unmordant 0,31 90,32 -1,68 27,86 27,91 93,40 39,20

Alum post mordant 0,40 88,96 -3,16 27,49 27,67 96,54 39,25

The results for the colorimetric measurements of cotton weave fabric dyed with β-carotene

dyestuff of orange peel residue are given in table 6. The measurements were as well done

by the CIE colour system. The shade of orange peel extract dyed on cotton fabric was

greenish yellow. The cotton fabric was evenly dyed. The colour uptake (ΔE) was for the

mordanted samples again slightly higher than for the unmordanted samples. The mordant

alum increased the absorption of the dyestuff molecules on the cotton fabric. The colour

strength K/S is higher for the mordanted samples. The unmordanted samples were

compared with the post mordanted samples with alum slightly lighter due to their higher L*

value. The C* values for colour saturation were for the post mordanted samples little lower,

which means the colour saturation decreases with mordanting. Considering both values L*

and C* the shades of both unmordanted and mordanted cotton fabric dyed with orange peel

extract are light and weak in shade. The slight negative values of a* and the more positive b*

values indicate shades of greenish yellow and slightly more greenish and less yellowish for

the mordanted samples.


5.5. Results of the wash fastness test (ISO 105 – C)

The results for the wash fastness test according to ISO 105-C are given in the table below.

Grade range from 1-5, where 1 is very bad and 5 very good.

Table 10: Results of the wash fastness test of unmordanted and post mordanted (alum) dyed cotton fabrics of orange peel and carrot extract.

The results for the colour change of unmordanted and mordanted samples dyed with β-

carotene dyestuff of orange peel residue were of grade 1-2. Most of the colour was washed

away by the washing test. The samples were partially coloured after the test in much lighter

and weaker colour saturation. The results for the colour change of β-carotene dyestuff of

carrot residue dyed cotton fabrics were only a grade 1. Almost no colour was left on the

cotton samples after washing at 60°C for 30 min. Whereas the colour change gave poor

results for both β-carotene dyestuff of carrot and orange peel residue dyed on cotton fabric

the staining results were with the grades 4-5 for all samples very good. The poor results for

colour change of the washing test for both β-carotene dyestuffs of carrot and orange peel

residue dyed on cotton weave fabric is interpreted by the lack of functional groups and the

relative small molecule sizes of the β-carotene dye and their consequential low affinity

towards the textile fibres (Marx, 2011). Yi and Yoo (2010) achieved same results for orange

peel dyestuff. Their poorest washing grade was for a dye concentration of 100 % owf that

was increased to a grade 4 for a dye concentration of 800 % owf. A possibility to improve the

wash fastness properties could be the increase of dye concentration as done by Yi and Yoo

(2010).

Orange peel Carrots

Colour change Staining Colour change Staining

Unmordant 1-2 4-5 1 4-5

Alum post mordant 1-2 4-5 1 4-5


5.6. Results of the xenon test (ISO-Norm B02)

The xenon test samples are graded between the ranges from 1-8, where 1 is very bad and 8

is very good. The results are listed in the table below.

Table 11: Results of the xenon light fastness test of unmordanted and post mordanted (alum) dyed cotton fabrics of orange peel and carrot extract.

The light fastness properties of β-carotene dyestuff of carrot residue dyed on cotton (2 for

unmordanted and 3 for mordanted samples) were poorer compared to the β-carotene

dyestuff of orange peel residue dyed samples. The results for β-carotene dyestuff of orange

peel residue were reasonable, with the grade 3 for unmordanted and 4 for mordanted

samples. The light fastness was improved by post mordanting the samples. The mordanted

samples of both β-carotene dyestuffs of carrot and orange peel residue were compared with

their unmordanted counterparts each with 1 grade better. The poorer results of the light

fastness for β-carotene dyestuff of carrot residue could be interpreted due to the high

sensitivity of β-carotene in light exposure and its higher and purer amount in this dye

compared with the β-carotene dyestuff of orange peel residue (Liaaen-Jensen, 1989). In its

natural form β-carotene is higher concentrated for example in carrots and it is surrounded by

proteins that can keep it more stable (Bauernfeind, 1981). Another reason is the small

molecule size and the resulting lower affinity towards the cotton fibres (Marx, 2011).

The research by Saleh et al. (2013) (explained in the literature on p. 10 ) showed great

promise that dyeing on cotton fabric with the dyestuff containing; chlorophyll a, and b and β-

carotene extracted from banana leaves by acetone is possible. They concluded that the

dyeing resulted in high colour strength, and high fastness properties. Comparing their results

of unmordanted samples with the results in this report (also unmordanted samples) in colour

Orange peel Carrots

Unmordant 3 2

Alum post mordant 4 3


strength and fastness properties, a conflict arises. The K/S values were with 0,52 higher

compared to the results of β-carotene dyestuff of carrot residue with 0,15 and β-carotene

dyestuff of orange peel residue with 0,31. The lightness L* is lower with a value of 77,01

compared to 90,32, (β-carotene dyestuff of orange peel residue) and 85,65 (β-carotene

dyestuff of carrot residue). They concluded a light fastness of grade 4 whereas this report

graded 3 for β-carotene dyestuff of orange peel residue and grade 2 for β-carotene dyestuff

of carrot residue. The wash fastness test at 40°C gave a grade of 4 and in this report 1-2 (β-

carotene dyestuff of orange peel residue) and 1 (β-carotene dyestuff of carrot residue) was

achieved. Neither high colour strength nor high fastness properties could be concluded in

this report and made the initial evidence that the dyeing on cotton with β-carotene dyestuff

could work invalid.


6. Conclusions

The work showed that the organic solvent extraction of β-carotene from orange peel and

carrots residue from the juice pressing industry is possible. The three different organic

solvents; ethyl acetate, petroleum ether and hexane/acetone mixture were chosen for the

extraction of β-carotene. Amongst them petroleum ether was slightly better for the β-

carotene extraction from both orange peel and carrot residue due to its strong non-polar

character.

The dyeing with β-carotene dyestuff of carrot and orange peel residue is only reasonable

possible and might not be an alternative for the dyeing industry, due to its poor fastness

properties and the unstable character of the β-carotene molecule to light and heat exposure.

The lack of functional groups in the β-carotene molecule made it difficult or impossible for it

to absorb on the cotton fibres and to achieve fair fastness properties. The used mordant

alum could increase the light fastness of both dyes with 1 grade, but not the wash fastness.

In general the further use of residues of the juice pressing industry for textile dye extraction

might be a valuable source for textile dyestuffs. The further usage of the residues might be a

value-adding step for the waste that would otherwise only be used as fodder or biogas

production.


7. Future research

The work showed that dyeing with β-carotene dyestuff of carrot and orange peel residue

from the juice pressing industry is only reasonable possible. The lack of functional groups in

the β-carotene dyestuff molecule makes the dyeing on cotton fabric difficult. An HPLC

analysis of the dyed cotton fabric could detect, which of the extracted compounds were really

absorbed on the fabric. It would be interesting to investigate if other fibres can improve the

fastness properties. Fibres such as wool (protein fibres) that are commonly used for the

dyeing with natural extracts due to their acid and alkaline functional groups, polypropylene

that has a low Tg at 35°C and is mostly suitable for fat- and oil soluble dyestuffs or polyester

(dyed with carriers to keep a low dyeing temperature).

The extraction of other compounds from residues of the juice pressing industry, which own

more functional groups, might be possible for the further work. Compounds such as

xantophylls (lutein or zeaxanthin) that belong to the group of carotenoids with hydrophilic

character could be a better dyestuff for cotton dyeing than β-carotene containing dyestuffs

with hydrophobic character.


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Master thesis by Susan Hecker I

APPENDIX

II Master thesis by Susan Hecker

APPENDIX I

Aqueous extraction

a) Carrots

20 g carrots (ground in the Kenwood blender until the pieces were ca. 1 mm)

200 ml tap water

All together was put in an Erlenmeyer flask and heated up in a water bath to 100°C

for 1 h.

Stirring with magnetic stirring at level 7.

After 1 hours filtration (Büchner funnel)

Rotary evaporator:

Evaporation of the water in the rotary evaporator at 40°C.

Storage of the residue (oily, caramelized liquid) in small glass bottles in no light.

b) Orange peel

20 g orange peel (ground in the Kenwood blender until the pieces were ca. 1 mm)

200 ml tap water


for 1 h.



Rotary evaporator:

Evaporation of the water in the rotary evaporator at 40°C.


Master thesis by Susan Hecker III

Organic solvents extraction with ethanol

a) Carrots

20 g carrots (as got from Brämhults juice; ground state)

200 ml 80 % aqueous ethanol solution (800 ml ethanol: 200 ml H2O)


for 4 h.



Rotary evaporator:

Evaporation of the solvent (ethanol) in the rotary evaporator at 40°C.

Solve the residue with distilled water and evaporate the water again in the rotary

evaporator.


b) Orange peel

20 g orange peel (ground in the Kenwood blender until the pieces were ca. 1 mm)

200 ml 80% aqueous ethanol solution (800 ml ethanol: 200 ml H2O)


for 4 h.



Rotary evaporator:

Evaporation of the solvent (ethanol) in the rotary evaporator at 40°C.

Solve the residue with distilled water and evaporate the water again in the rotary

evaporator.


IV Master thesis by Susan Hecker

Results of the HPLC analysis

The following table and graphs show the result of the HPLC analysis of β-carotene by

aqueous and ethanol extraction. For both solvents the β-carotene content was not

extracted or too low for the detection in the visible light at a wavelength of 450 nm.

Table 12: β-carotene content in carrots and orange peel (μg/g)

Solvent Carrots Orange peel

Water 0 0

80 % aqueous ethanol solution 0 0

Figure 16: HPLC from carrot extract in aqueous solution at 450 nm

Figure 17: HPLC from orange peel extract in aqueous solution at 450 nm

Figure 18: HPLC from carrot extract in 80 % aqueous ethanol solution at 450 nm

Master thesis by Susan Hecker V

Figure 19: HPLC from orange peel in 80 % aqueous ethanol solution at 450 nm

VI Master thesis by Susan Hecker

APPENDIX II

Chemical structures of the solvents used

Chemical structure of ethyl acetate:

Chemical structure of acetone:

Chemical structure of hexane:

Master thesis by Susan Hecker VII

APPENDIX III

Vat dye

Mercerized and bleached cotton was dyed as vat dye with the following parameters. First

the stock vat was made in a warm water bath to solve the sodium hydrosulphide. Then it

was add to the dye bath together with the fabric. Under magnetic stirring it was left for 10

min, then it was oxidized in the air for around 5 min. This procedure was repeated 2-3

times. All four samples didn´t show a recognizable colouration.

Table 13: Different parameters for vat dyeing on cotton fabric

Sample 1 Sample 2 Sample 3 Sample 4

Residue Carrot Orange peel Carrot Orange peel

Solvent Ethyl acetate Ethyl acetate Hexane/acetone Hexane/acetone

Cotton sample weight 2,9 g 1,7 g 0,4 g 0,5 g

Stock vat

Organic solvent (ethyl acetate or hexane/acetone (1:1 v/v) 4 ml 4 ml 4 ml 4 ml

β-carotene 1,3 mg/g 0,014 mg/g 1,5 mg/g 0,018 mg/g

Water 50°C 10 ml 10 ml 10 ml 10 ml

Sodium hydroxide 25 % 1 ml 1 ml 1 ml 1 ml

Sodium hydrosulphide 0,6 g 0,6 g 0,6 g 0,6 g

Dye bath

Water ca. 20°C 300 ml 100 ml 100 ml 50 ml

Sodium hydroxide 25% 0,1 ml 0,1 ml 0,1 ml 0,1 ml

Sodium hydrosulphide 0,1 g 0,1 g 0,1 g 0,1 g

Sodium chloride 10 g 10 g 15 g 5 g

Stock vat ca. 15 ml ca. 15 ml ca. 15 ml ca. 15 ml

Heated in water bath at ca. 60°C

VIII Master thesis by Susan Hecker

APPENDIX IV

HPLC results

The following table 20-31 show the results of the HPLC for the analysis of β-carotene. The

retention time is around 56 min. for all samples and the area is between 32.635 – 162.668

for orange peel and 8.634.783 – 16.041.328 for carrot. There are two samples of each

solvent. In the report the calculations for the concentration and the used β-carotene for

dyeing was done based on the average value of this two samples.

Table 14: Results of the determination of β-carotene by HPLC

Sample Residue Solvent Retention Time Area

Concentration (mg/mL)

1 Orange peel Ethyl acetate 55,40 32.635 0,001

7 Orange peel Ethyl acetate 56,22 152.876 0,004

3 Orange peel Hexane/acetone 56,62 162.668 0,004

9 Orange peel Hexane/acetone 56,06 86.549 0,002

2 Orange peel Petroleum ether 56,65 111.816 0,003

8 Orange peel Petroleum ether 56,10 142.022 0,003

4 Carrot Ethyl acetate 56,45 8.634.783 0,207

10 Carrot Ethyl acetate 56,07 9.135.152 0,219

6 Carrot Hexane/acetone 56,32 10.856.273 0,260

12 Carrot Hexane/acetone 56,03 10.454.314 0,247

5 Carrot Petroleum ether 56,31 10.747.621 0,258

11 Carrot Petroleum ether 56,03 16.041.328 0,393

The HPLC graphs of each sample at the wavelength 450 nm are the following:

Figure 20: HPLC graph of sample 1 β-carotene from orange peel, extracted in ethyl acetate

Master thesis by Susan Hecker IX

Figure 21: HPLC graph of sample 7 β-carotene from orange peel, extracted in ethyl acetate

Figure 22: HPLC graph of sample 3 β-carotene from orange peel, extracted in hexane/acetone

Figure 23: HPLC graph of sample 9 β-carotene from orange peel, extracted in hexane/acetone

Figure 24: HPLC graph of sample 2 β-carotene from orange peel, extracted in petroleum ether

X Master thesis by Susan Hecker

Figure 25: HPLC graph of sample 8 β-carotene from orange peel, extracted in petroleum ether

Figure 26: HPLC graph of sample 4 β-carotene from carrot, extracted in ethyl acetate

Figure 27: HPLC graph of sample 10 β-carotene from carrot, extracted in hexane/acetone


Master thesis by Susan Hecker XI


Figure 30: HPLC graph of sample 5 β-carotene from carrot, extracted in petroleum ether

Figure 31: HPLC graph of sample 11 β-carotene from carrot, extracted in petroleum ether

XII Master thesis by Susan Hecker

APPENDIX V

Spectrophotometric and fastness property results

The following tables 12-14 show the results of all samples for the spectrophotometric

measurements of colour properties, the wash fastness test after ISO 105-C and light

fastness after ISO Norm B02. The values mentioned in the report are the average value of

the two samples.

Table 15: Colour coordination of the all dyed cotton fabrics with carrot extract

Carrot


Unmordant Sample 1 0,08 89,10 9,25 11,97 15,13 52,32 24,64

Unmordant Sample 2 0,22 82,21 7,48 12,29 14,39 58,68 27,34

Alum post mordant Sample 1 0,15 87,83 6,27 14,42 15,73 66,52 26,70

Alum post mordant Sample 2 0,15 88,68 6,34 15,14 16,42 67,28 27,14

Table 16: Colour coordination of the all dyed cotton fabrics with orange peel extract

Orange peel


Unmordant, sample 1 0,33 90,72 -2,19 29,61 29,69 94,23 40,92

Unmordant, sample 2 0,29 89,91 -1,17 26,11 26,14 92,57 37,49

Alum post mordant, sample 1 0,40 89,03 -3,39 27,50 27,71 97,02 39,28

Alum post mordant, sample 2 0,41 88,89 -2,92 27,48 27,64 96,07 39,22

Master thesis by Susan Hecker XIII

Table 17: Results of the wash fastness test of all unmordanted and post mordanted (alum) dyed cotton fabrics of orange peel and carrot extract.

Table 18: Results of the light fastness test of all unmordanted and post mordanted (alum) dyed cotton fabrics of orange peel and carrot extract.

Orange peel Carrots

Colour change Staining Colour change Staining

Unmordant, sample 1 1-2 4-5 1 4-5

Unmordant, sample 2 1-2 4-5 1 4-5

Alum post mordant, sample 1 1-2 4-5 1 4-5

Alum post mordant sample 2 1-2 4-5 1 4-5

Orange peel Carrots

Unmordant, sample 1 2-3 2

Unmordant, sample 2 4 colour darkend 2

Alum post mordant, sample 1 4 3

Alum post mordant sample 2 4 3

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