natural dyeing of cotton fabric with yellow pigments from...

Natural Dyeing of Cotton Fabric with yellow pigments from Pencillium

bilaiae, from Cultivated Soil

Swamy Dhas Shibila and Ayyakkannu Usha Raja Nanthini*

Department of Biotechnology, Mother Teresa Women’s University, Kodaikanal, Tamil Nadu-624101.

*Corresponding Author –[email protected]

ABSTRACT

The use of synthethic dyes in textile industry resulted in environmental pollution.

Therefore, the use of synthetic dye in textile industries must be replaced with organic non

toxic dyes. The present study is an attempt to explore the possibility of yellow pigment of

Pencillium bilaiae as colorant for cotton fabric. In this study, pigment producing fungus

was isolated and identified as Pencillium bilaiae. This extracted yellow pigment of

Pencillium bilaiae was tested for i ts colour fastness on cotton fabric in both pre-mordant

and unmordant conditions. The results showed that yellow pigment dye with unmordant

cotton fabrics at pH 5 and 7 exhibited excellent colour fastness. This extracted yellow

pigment of Pencillium bilaiae was also undergone cytotoxicity test (MTT assay) and the

result showed that the pigment was non toxic to vero cells and there was no significant

cytotoxic effect among the concentration extracts. In addition, the invesegator also used

the GC-MS analysis and confirmed that 3,5-Di-t-butylphenol compound present in the

coloured fractions. Therefore this study will provide useful alternative to textile industries.

Keyword: Yellow pigment, Pencillium bilaiae, Cotton fabric

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Volume 9, Issue 1, January 2020

INTRODUCTION

Synthetic dyes play an important role in textile, paper printing food, pharmaceutical,

leather, cosmetics. The use of synthetic dyes in textiles are more preferred due to ease of

production, less expensive, superior colouring properties and small amount needed which attracts

the market. Many of the synthetic compounds are known for their allergic, mutagenic,

carcinogenic and toxic which ultimately led to adverse effects on the health and environment

(Downham and Collins 2000). The use of natural pigments from plants and animals are prefered

because they are non toxic, non polluting and less health hazardous (Sivakumar et al., 2009).

Natural pigments isolated from bacteria and fungi and its application is more remarkable

in food, cosmetic, pharmaceutical and textile industry. They can exhibit better bio-degradability

and higher compatibility with the environment. The majority of the pigments produced by fungi

are quinones, flavonoids, melanins and azaphilones, which belong to the aromatic polyketide

chemical group (Pastre et al., 2007) and have been widely described for medicinal uses and

potential use of dyes. In the present investigation, attempts have been taken to assess the dyeing

capability of fungal pigments, Pencillium bilaiae on cotton fabrics and their cytotoxicity effect

on vero cells. Besides the main compound present in yellow pigment also identified by GC-MS.

MATERIALS AND METHODS

Sampling and fungal isolation

The soil samples were randomly collected from cultivated land at Attuvampatti,

Kodaikanal, Tamil Nadu, India. The soil samples were serially diluted with sterile saline buffer

and plated on a sterile Potato Dextrose Agar (PDA) plate. After 4 to 5 days of incubation, the

fungi colonies were individually picked with sterile inoculation loop and inoculated on a sterile

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PDA plate. The inoculated plates were incubated for 10 to 14 days at 25±2°C. The pigment

producing fungus was identified based on the colour change of media compare to the control

plate (Fig. 1). Further, the pigment producing fungus was identified based on the microscopic

characters by lactophenol cotton blue wet mound method (Leck, 1999).

Molecular identification of fungi

The genomic DNA was extracted from the fungal mycelium as described by Doyle and

Doyle (1987). The universal primers described by White et al., (1990) were (Forward primer

ITS1-5'-TCCGTAGGTGAACCTGCGG-3' and reverse primer ITS4-5'-TCCTCCG-

CTTATTGATATGC-3') used to amplify specific region ITS1, the ITS gene amplification

reaction mixture (30µl) consisted of 2X master mix (Amplicon, Denmark) with 10 ng of

template DNA and 10 pmol of each primer. PCR amplification was carried out in Agilent

SureCycler 8800® gradient PCR machine. The following cyclic conditions were executed:

initial denaturation for 5 min at 95oC followed by 35 cycles of denaturation for 45 sec at 94

oC;

annealing for 1 min at 59oC; extension for 1 min at 72

oC followed by a final extension at 72

oC

for 5 min. The amplified PCR products were electrophoresed along with 1 kb Ready- to -Use

DNA marker on 2% gel. The amplified PCR product was visualized and photographed using gel

documentation system (Gelstan, Mediccare Scientific, India).

rDNA-ITS Sequencing

The PCR amplified ITS product was purified and sequenced using DNA sequencing

services (Eurofins Scientific, Bangalore). The obtained sequences were subjected for noise

editing using Bioedit software v 7.0.9 (Hall, 1999). The edited sequences were blast with

GenBank Nucleotide Database (http://www.ncbi.nlm.nih.gov/BLAST/) using the algorithm Blast

N (Altschul et al., 1990) to identify matches with existing reference sequences. The best hit was

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defined as the sequence with highest maximum identity to the query sequence. The gene

sequences of the fungal isolates was deposited in GenBank and accession numbers was obtained.

Screening of Dye

The cotton fabrics (105 GSM, 40 count, 17 diameter and 34 inches) used in these studies

was purchased from Sakthi Knit, Tirupur, Tamil Nadu, India. The cotton fabric was sliced into

pieces of 30 cm × 30 cm dimension. The cotton fabrics were pre mordant with 5% of alum

(ammonium potassium sulphate) and 10% ferrous sulphate on weight of fabric (o.w.f). Initially,

the cotton fabrics were boiled in alum with sodium carbonate (0.03 g) at 70°C for 1 hour and left

in the bath at room temperature for overnight. For iron mordanting, the cotton fabrics were

boiled with ferrous sulfate solution at 70 ºC for 10 min and left to cool in the bath. After pre-

mordanting, the cotton fabrics were squeezed and air dried at room temperature (Moeyes, 1993)

The yellow coloured culture filtrate from fungus was used to dye cotton fabrics. The pH

of culture filtrate was measured and adjusted to pH to 5, 7 and 9 with vinegar and ammonium

solution. The pre-mordant and unmordant fabrics were immersed in the fungal filtrate and was

heated in a water bath at 70 to 80 ºC for 1 hour. Then, the dyed cotton fabrics were washed with

cold water to remove the unfixed pigment and dried at room temperature overnight.

Colour Fastness

All the dyed cotton fabrics were assessed for colour fastness to washing, perspiration and

rubbing according to standard methods, ISO 105: C 06-1997 (washing), ISO 105: E04-1996

(perspiration) and ISO 105: X12-2002 (Rubbing) described by The Regional Laboratory, Textile

Committee, Chennai, India.

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Purification of fungal pigment

The fungus culture filtrate was mixed with ethyl acetate 1:3 ration and agitated 24 hours

at 100 rpm. After, the solvent was separated from culture filtrate by separation funnel. The

pigment extract was concentrated using rotary vacuum evaporator. About 25 cm of glass column

(34 × 2.5 cm dimensions) was filled with Silica gel (60-120 mesh size, Merck, India). The crude

pigment extract was loaded on to the top of the column. The following solvents were used as

elution solvent at increasing polarity ratio such as, hexane, chloroform, ethyl acetate and

methanol. The coloured fractions were collected individually and further used for pigment

identification.

GC-MS Analysis of coloured fraction

The eluted coloured fractions were subjected to analyse in gas chromatography connected

with mass spectrometry (GC-MS) using Agilent Technologies GC systems (Model GC-

7890A/MS-5975C, Agilent Technologies, Santa Clara, CA, USA). Relative quantity of the

chemical compounds present in each of the elution mixtures were expressed as percentage based

on peak area produced in the chromatogram.

Cytotoxicity of crude fungal pigment

The normal cell line (Vero) was obtained from National Centre for Cell Science (NCCS),

Pune and grown in Eagles Minimum Essential Medium containing 10% fetal bovine serum

(FBS). One hundred microlitres of cell suspension (10,000 cells) was seeded per well of 96-well

plates and incubated at 37 °C, 5% CO2, 95% air and 100% relative humidity to allow for cell

attachment. After 24 h the cells were treated with serial concentrations of the yellow crude

pigments. The plates were incubated for an additional 48 hours at 37 °C, 5% CO2, 95% air and

100% relative humidity. The medium containing without samples were served as control and

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triplicate was maintained for all concentrations. After 48 hours of incubation, 15 µl of MTT (5

mg/ml) in phosphate buffered saline (PBS) was added to each well and incubated at 37 °C for 4

hours. The medium with MTT was then flicked off and the formed formazan crystals were

solubilized in 100 µl of DMSO and then measured the absorbance at 570 nm using micro plate

reader.

The percentage of cell viability was calculated with respect to control as follows

% Cell viability = [A] Test / [A]control × 100

RESULTS

The microscopic characteristics of yellow pigment producing fungus showed distinctive

morphological characteristics of Pencillium bilaiae such as presence of spore bearing Penicilli,

conidiophores terminated by cluster of flask shaped phialides and spores (conidia) were

produced in dry chains from the tips of the phialides.

Molecular identification of pigment fungi

The amplified PCR product was resolved in gel at molecular weight of ~600 bp. The

sequence was subjected to NCBI blast which showed that 100% similarity with previously

submitted NCBI nucleotide sequence of Pencillium bilaiae and constructed phylogentic tree

(Fig. 1) . The yellow pigment producing fungi rDNA –ITS sequences was deposited in NCBI

and accession numbers KY611856 was obtained.

Screening of Dye

The effect of washing and rubbing on yellow pigment dyed cotton fabrics was shown in Table 1.

and Fig.2,3 &4.

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Colour Fastness to Washing

After washing, the cotton fabrics, dyed with yellow fungal pigment showed colour

changes of fastness which were rated from negligible to considerable colour changes. The cotton

fabrics which were unmordant and dyed with yellow fungal pigment at pH 5 and 7 showed

negligible colour change compared to other dyed cotton fabrics. The unmordant cotton fabrics,

dyed with yellow pigment at pH 5 and 7 noticed negligible colour staining on white fabric

whereas, unmordant cotton fabrics dyed with yellow pigment at pH 9 observed negligible to

slight colour staining on white fabric.

Colour Fastness to Perspiration

In acid perspiration, negligible colour change was observed in unmordant cotton fabrics

which were dyed with yellow pigment at pH 5 and pH 7. Similarly, the unmordant cotton fabric

dyed with yellow pigment at pH-5 and pH-7 exhibited negligible colour staining on test white

fabrics.

In alkali perspiration, the unmordant cotton fabrics, dyed with yellow pigment at pH 5

and 7 showed negligible colour change. The unmordant cotton fabrics, dyed with yellow pigment

at pH 5 and 7 exhibited negligible colour staining on tested white cotton fabrics.

Colour Fastness to Rubbing

After dry rubbing, the unmordant cotton fabrics, dyed with yellow pigment at pH 5 and 7

exhibited negligible colour change. Negligible colour staining was noticed in unmordant cotton

fabrics which dyed at pH-5 and pH-7.

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In wet rubbing, the colour fastness of cotton fabrics without pre-mordant dyed with

yellow fungal pigment at pH-5 and pH-7 showed negligible colour change. Negligible colour

staining was noticed in unmordant cotton fabrics, dyed with yellow pigment at pH-5 and pH-

7.

Purification of yellow pigment

The yellow fungal pigments were purified by silica gel column chromatography and

obtained 9 different coloured fractions. The fraction containing colour pigment was analysed by

GC-MS analysis, based on their polarity order. About, 14 different compounds were present in

the 9 fractions. The retention time, molecular formula, molecular weight, and the fragmentation

of these compounds were presented in Table 2. Based on the abundance, 3,5-Di-t-butylphenol

(Fig. 6) is the major compounds present in most of the elution mixtures.

Cytotoxicity of crude fungal pigment

The cytotoxicity effect of crude yellow pigment against normal vero cell line was carried

out by MTT assay Fig 6. Result of percent viability vero cell line treated with yellow pigment

shown in Graph 1. The cell viability of vero cells was slightly decreased with increasing

concentration of yellow pigment. The cell viability were 98.8, 97.17, 95.80,93.14, 90,48

percentage with yellow pigment concentrations 12.5,25, 50, 100, 200 mg respectively. The result

showed there was no significant cytotoxic effect of different concentrations of yellow pigment

on the normal vero cell line.

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DISCUSSION

In the present investigation, the yellow pigment producing fungus was isolated from soil.

Similarly, many authors previously isolated pigment producing fungi from soil. In 2009,

Velmurugan et al. isolated five fungi namely, Monascus purpureus, Isaria farinosa, Emericella

nidulans,Fusarium verticillioides and Penicillium purpurogenum from soils collected from

Western Ghats, Nilgris. They have used the purified fungi pigments to dye cotton fabric and

leather samples. Poorniammal et al., (2013) isolated pigment producing fungus, Thermomyces

sp. from soil. The pigments were extracted and tested their dyeing capacity on cotton, silk and

wool fabrics. In 2013, Gupta et al, isolated Trichoderma spp. from soil, collected from the

premises of the Institute of Home Economics, University of Delhi, and screened their dying

ability on silk and wool fabrics. Devi (2014) isolated two fungi species, Trichoderma sp. and

Aspergillus sp. from soil samples randomly collected from Womens Christian college Chennai.

The pigments were extracted and tested their dying property to silk, cotton and silk cotton

fabrics. Ketut et al. (2016) isolated red pigment producing fungus, Penicillium purpurogenosum

from goat milk contaminated soil at Sepang village, Buleleng Bali, Indonesia and the pigments

were extracted and tested their ability to dye on silk and cotton fabrics.

Molecular identification of pigment producing fungi

The conventional fungal identification mainly based on morphological characters such as

spore-producing structures formed as a result of asexual (mitosis) or sexual (meiosis)

reproduction (Hyde et al., 2010). The understanding the evolution of morphological characters

very important to identification of fungal species. However, this approach helped to

classification of fungi at the ordinal or familial level (Wang et al., 2016), may not always

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perform well for species level (Lutzoni et al., 2004). Also, the morphological characters which

misleading due to the hybridization (Olson and Stenlid, 2002; Hughes et al., 2013), cryptic

speciation (Harrington and Rizzo, 1999; Giraud et al., 2008; Foltz et al , ; Kohn, ;

cking et al , and convergent evolution Brun and ilar, Thus, the identification

of fungi based on morphology alone can be challenging, especially when nonexperts are dealing

with cultures of fungi.

In 1990, White et al. developed the unique fungal DNA barcode by specific primers. The

primers are used to amplify the range of 450-750 base pairs (bp) which is divided into three

groups of the variable spacers ITS1 and ITS2, and the 5.8S gene. The ITS sequences showed

high interspecies diversity among fungi. According to many previous different studies, the ITS

region is a suitable target region for detecting and identifying fungi species, including Alternaria,

Aspergillus, Candida, Cryptococcus, Fusarium, Talaromyces australis, Penicillium murcianum

and Zygomycetes by rDNA –ITS sequencing (Hernández et al., 2018; Behzadi and Behzadi,

2011, Sessitsch et al., 2006, Blaalid et al., 2013, Nilsson et al., 2008).

In the present study, the isolated yellow pigment producing fungus was identified as

Pencillium bilaiae by rDNA – ITS sequencing. Similarly, many authors previously used to

identify pigment producing fungus. In 2017, Chadni et al. isolated pigment producing fungi from

spoiled mango and tested against cotton fabrics. They have used rDNA-ITS sequencing

techniques to identify the pigment producing fungus, Talaromyces verruculosus. Hernández et

al., (2018) isolated two pigments producing fungi isolated from rotten wood samples and

identified as Talaromyces australis (red) and Penicillium murcianum (yellow) by rDNA–ITS

sequencing.

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Fastness properties of Dyed cotton fabrics

The present study, the cotton fabric dyed with yellow pigment without pre mordant at

pH5 and pH7 exhibited a negligible colour change in the wash, perspiration and rubbing fastness

studies. This result corroborates with previous finding of Sharma et al. (2012) that they have

extracted pigments from three fungi namely Trichoderma virens, Alternaria alternata,

Curvularia lunata and dyed on wool and silk fabrics. The result showed that the unmordanted

dyed wool and silk fabrics exhibited more intense colours in wash and rubbing fastness studies.

Hinsch et al. (2015) extracted pigments from three fungi, Chlorociboria aeruginosa (xylindein,

green), Scytalidium cuboideum (draconin red, red) and Scytalidium ganodermophthorum

(yellow) and were used to dye multi fabric test strips. The result showed xylindein and draconin

red exhibited colour fastness to washing and xylindein good fastness to perspiration. Hernandez

et al. (2018) isolated five fungi spices, Talaromyces australis, Penicillium murcianum,

Talaromyces sp. Trichoderma spirale and Fusarium oxysporum from rotten wood samples. They

have selected two fungi species such as T. australis and P. murcianum from the out of five

due to their colour and yield of dyes. The isolated pigments were dyed on wool samples and

noticed that unmordant or without fixing agent treated wool samples which dyed with pigments

kept their colour in acceptable range after washing.

Cytotoxicity of Crude fungal pigment

Textile manufacturing industry used a wide range of chemicals, which are harmful to the

environment, workers who working in textile processing and most importantly for consumers

(Kadir et al., 2016). The level of toxicity is varied depends on the textile processing stage. The

most common diseases reported among the textile workers are allergic reactions and irritation to

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the skin and respiratory tract (Hatch et al., 1984). Some research finding, reported textile dyes

have potential for mutagenicity (Schneider et al., 2004; Mathur and Bhatnagar, 2007) and

genotoxicity (Sharma and Sobti, 2000).

In the present investigation was aimed to study the adverse effect of fungal pigments on

vero cells by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. This

is an advance significant quantitative assay over traditional techniques which have been used

commonly for proliferation and cytotoxicity assays. In this technique, the yellow compound

tetrazolium ring is reduced by mitochondrial dehydrogenases to the water insoluble blue

formazan compound, depending on the living cells (Mosmann, 1983).

Similarly, many authors previously studied the cytotoxicity effect of dyes. Klemola et al.

(2007) investigated the cytotoxicity of reactive dyes and dyed fabrics using human keratinocyte

HaCaT cells in vitro. Kadir et al. (2016) explored cytotoxicity and neurotoxicity effect of natural

dyes in the form of liquid and dyed silk fabrics.

CONCLUTION

From the results it can be concluded that 3,5-Di-t-butylphenol is a major compound

present in the yellow fungal extract. The unmordant cotton fabric dyed with yellow fungal

pigment at pH -5 and 7 showed better colour fastness in wash, perspiration and rubbing

compared to other dyed fabrics. The cytotoxicity test (MTT assay) result showed that the

pigment was non toxic to vero cells and there was no significant cytotoxic effect among the

concentration extracts or any Cytotoxicity effects against tested cell lines.

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ACKNOWLEDGEMENT

The authors acknowledged the Department of Biotechnology, Mother Teresa Women's

University for providing the necessary facilities to carried out this research.

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Fig 2. Unmordant cotton fabrics dyed with yellow fungal pigment at different pH

Fig 3. Iron pre-mordant cotton fabrics dyed with yellow fungal pigment at different pH

Fig 4. Alum pre-mordant cotton fabrics dyed with yellow fungal pigment at different

Control pH 5 pH 7 pH9



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Fig. 5. Structure of 3,5-Di-t-butylphenol

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Fig 6. The effects of the five different concentrations of crude yellow pigment

were used for the susceptibility testing in the vero cell lines.

In vitro cytotoxicity assay used for vero normal cell line with fungal extracts

The cell viability of vero cells was increased concentration of yellow pigment. The cell

viability decreased such as 98.8 (A), 97.17 (B), 95.80 (C),93.14 (D), 90,48% (E) extract

pigment concentrations 12.5,25, 50, 100, 200 mg respectively. All cells were observed after

48 hours of incubation using inverted phase contrast microscopy at 20X magnification.

C A

D A

E A

A A

B A

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Table 1. Fastness properties of dyed cotton fabrics.

CC – Change in colour; S – Staining on white fabric

1-Much change, 2-Considerable change, 3-Noticeable change, 4-Slight change and 5-

Negligible change

S.

No

Sample* Wash Fastness Perspiration Fastness Rubbing Fastness

CC S Acid Alkali Dry Wet

CC S CC S CC S CC S

1 Without Mordant @

pH5

5 5 5 5 5 5 5 5 5 5

2 Without Mordant @

pH7

5 5 5 5 5 5 5 5 5 5

3 Without Mordant @

pH9

2/3 4/5 3/4 4 4/5 4/5 4 4/5 3 4

5 Iron Mordant pH5 2 4 4 4 4 4/5 4/5 4 3 4

6 Iron Mordant pH7 3 4 4/5 4/5 4 4/5 4/5 4 3/4 4

7 Iron Mordant pH9 3/4 4 4 4/5 4 4 4/5 4 4 4

9 Alum Mordant pH5 2/3 4 3/4 4/5 4/5 4/5 4/5 4 4 4

10 Alum Mordant pH7 3 4 4 4 4/5 4/5 4/5 4 4 4

11 Alum Mordant pH9 2/3 4 3 4 4 4 4/5 4/5 3/4 4

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Table 2. Fungal yellow colour compounds of ethyl acetate extract at different elution

mixtures.

Eluent mixture Retention

Time

Molecular

weight (m/z)

Compound Name

CH50:ET50

9.49,

10.57,

10.82 and

13.14,

11.79,

12.03,

280, 242,

322, 284

9-Eicosene, Pentadecanoic acid, 9-

Tricosene, Octadecanoic acid

CH70:ET30

9.53,

10.85,

11.87,

12.76,

13.37,

13.56

No major

fragments

-

ET20:MET 80

7.28, 9.76,

10.54,

11.77

206, 280,

242, 284

3,5-Di-t-butylphenol, Methyl [n-

(salicyl)-3-amidepropanoyl,

Pentadecanoic acid, Octadecanoic

acid

ET10:MET 90 10.54 211

9,9-

Dimethoxybicyclo[3.3.1]nonane-

2,4-dione

ET50:MET 50

7.28, 8.03,

8.10, 9.50,

10.82,

12.03 and

13.15,

206, 252,

226, 280,

238, 322,

3,5-Di-t-butylphenol, 3-

Octadecane, 2,3,5,8-

Tetramethyldecane, 3-Eicosene, 9-

Eicosene, 9-Tricosene,

ET60:MET 40

7.27, 9.49

and 10.81,

12.03 and

13.14

206, 280, 322 3,5-Di-t-butylphenol, 3-Eicosene,

9-Tricosene

ET70:MET 30

9.48,

10.54,

11.77,

13.14

280, 256,

284, 364

3-Eicosene, Hexadecanoic acid,

Octadecanoic acid, 9-Hexacosene

ET80:MET 20

7.27, 8.02,

9.49,

10.55,

11.77,

12.03 and

13.14

206, 252,

280, 256,

284, 322

3,5-Di-t-butylphenol, 3-

Octadecane, 3-Eicosene,

Hexadecanoic acid, Octadecanoic

acid, 9-Tricosene

ET90:MET10

7.27, 8.10,

8.75,

10.58,

11.81,

12.04 and

13.15

206, 198,

282, 256,

284, 322

3,5-Di-t-butylphenol,

Tetradecane, Hexachlorobenzene,

Hexadecanoic acid, Octadecanoic

acid, 9-Tricosene,

CH-Chloroform; ET-Ethyl acetate; MET-Methanol.

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