Invention Journal of Research Technology in Engineering & Management (IJRTEM) ISSN: 2455-3689

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Fractionation of Crude Dye Extracted From Cucurbita Pepo Leaves by Cold

Extraction Method
Shradhanjali Mohapatra1 & Pragnyashree Mishra2
1
Assistant Professor, Department of Textile and Apparel designing, College of Home Science, OUAT.
2
Assistant Professor, Department of Floriculture and Landscaping, College of Horticulture, OUAT.

ABSTRACT: Natural dyes are those dyes obtained from natural sources. The majority of natural dyes are usually collected from
roots, berries, bark, leaves, wood, fungi and lichens. Usually in ancient days people have dyed their textiles by using locally available
materials. Cold extraction for crude dyes extraction from Cucurbita pepo leaves. Theextract obtained quantitatively from cold
extraction method was 6.81g and 2.27g respectively from 100g and 50g of C. pepo dry mass taken in 750ml and 500ml of ethanol
solvent.6 components/functional groups were confirmed in crude dye fractioned with n hexane, chloroform and ethyl acetate but only
4 components/functional groups were confirmed in crude dye fractioned with acetone.

INTRODUCTION
Natural dyes are those dyes obtained from natural sources. The majority of naturaldyes are usually collected from roots, berries, bark,
leaves, wood, fungi and lichens.Usually in ancient days people have dyed their textiles by using locally available materials. Woad,
indigo, saffron, and madder were raised commercially and were important trade goods in the economics of Asia and Europe.A large
number of investigations on extraction of colorants from natural sources like plants, microbes, insect and animals and their different
kinds of applications have been done till date. In functional finishing oftextiles, coloration of food and dyesensitized solar cells natural
dyehas already appliedShahid& Islam (2013). According toGrifoniet al. (2011)with naturaldye fabrics made of vegetable fibres has
role in UV protection. Textiles offered a safestprotection from (UV) radiation exposer with the use of natural dyesincrease the UV
protection of fabrics madeof vegetable fibre.A pumpkin is a gourd-like squash of thegenus Cucurbita belongs to the
familyCucurbitaceaeCucurbita pepo or pumpkins are widely grown for commercial use and having good values in both food and
recreation. This family consists of 125 generaand 960 species, mainly in tropicaland subtropical regions. Since long time efforts have
been made by variousscientists to develop an efficient extractiontechnique for natural dye extraction.Baliarsinghet al.(2012) has
isolated principal colour compounds such as flavonoids and tannin moietiesfrom two plant species (SaracaasocaandAlbizialebbeck),
using solvent extraction method and standardised dyeing techniques, identified colourcomponents and checked their
potentialantimicrobial properties against commonhuman pathogens. Hee-OckBooaet al (2012). extracted and characterized
somenatural plant pigments and their use for cosmetics and determined antioxidantactivities, total polyphenols and flavonoids, and
antimicrobial effects of some plant pigments.Natural pigment from the petals of the Flame of forest(Buteamonosperma) flower under
differentoperating conditions such as extraction time,temperature and mass of the petals conventional extraction technique were
extracted by by Sinha et al (2012). In the present study crude dyes are extracted from C. pepo leaves by cold extraction method. The
fractionation of the dye was done with various solvents andanalysed by simple chromatographic technique.

MATERIAL AND METHODS

This experiment was conducted at OUAT, Bhubaneswar All commercially available solvents such as were carried out. The curves
were visualized under ultra violet at 254 nm in TLC.

2.1. Collection of C. pepo leaves

The leaves of Cucurbita pepo was collected from nearby areas of OUAT in November 2016.

2.2. Preparation of sample

The leaves of C. pepo were washed with tap water followed by distilled water toremove impurities/dust from the surface. The leaves
were then dried in shade and powdered by blender.

2.3. Cold Extraction

100g of sample wassoaked in 500ml of ethanol solvent for twoweeks. 250 ml of ethanol was added to the soaked sample material in
two batches for one week after removal of previous batch ethanol. Then all the layerswere separated and then mixed and filtered.In
Rotary evaporator at reduced pressure, the ethanol filtrate wasevaporated to get dark green residue. Then Residue wasplaced in the
water bath at the temperature 70oC for 4 hours. By this method ethanol content got evaporated and the ethanol extract which was
collected in a vialand stored for fractionation.
2.4. Concentration of C. pepo leaves coldextract with rotary evaporator

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 Fractionation of Crude Dye Extracted From Cucurbita Pepo Leaves by Cold Extraction Method
C. pepo (leaves) extract was kept in a cleaned flask. Temperature of rotary evaporatorwas set at 78 oC for removal of solvent. The
concentrated extract was stored for Fractionation and other analyticaltest.
2.5. Fractionation of C. pepo (leaves) coldextract
For fractionation the concentrated coldextract was taken. The solvent used were n-hexane,chloroform, ethyl acetate and acetone. All
the solvents were added to the conc. cold extract of C. pepo leaves till the extract were dissolved completely in the solvent.Fractions
were stored in vials for fractionation in TLC.

RESULTS AND DISCUSSIONS

3.1. Extraction of crude dye from C. pepoleaves
Two methods of dye extraction, cold extractionand hot extraction were evaluated. Thequantitative amount of extract obtained from
coldextraction method is 6.81 g and 2.27grespectively while from hot extraction method is2.48g as in Table 1 and Table 2. From the
tables it can be concluded thaton the basis of quantitative yield of dye hot extractionmethod is better than cold extractionmethod due
tothe influenceof temperature on the extraction process.

It is noted from the literature that the extractconcentration increases due to increasing temperature. Hot extractionis better than cold
extraction because it`s arapid process of extraction, extraction isgood due to the involvement of temperature and due to the use offilter
paper in hot extraction no filtration is required. Usually a lowboiling solvent like n-hexane are used in hotextraction methods. Though
hot extraction is better than cold extraction when quantitative yield is considered but in the sense of cost efficacy and availability cold
extraction is better than hot extraction as cold extraction canbe carried out in a simple pot but for hotextraction a heating source,
condenser,round bottom flask and the most importantelectricity is required.

Table1.Quantitative production of crude extract of the leaves of C. pepo using ethanol solvent.
S.no Sample Solvent volume Weight of Weight of beaker Weight of extract
weight (g) (ml) beaker (g) +extract (g)
1 100 750 161.83 168.64 6.81
2 50 500 161.83 164.10 2.27

Table 2. Quantitative production of crude extract of the leaves of C. pepo using n-hexane solvent
. Sample weight Solvent volume Weight of beaker Weight of beaker Weight of extract
(g) (ml) (g) +extract (g)
50 500 35.18 37.66 2.48

Table 3. Fractionation with n-hexane

No. of spots Distance trveled by Distance traveled by Rf value
solvent front spots
1 3.6 cm 3.42cm Rf1 = 0.98
2 3.6 cm 3.3cm Rf2 = 0.92
3 3.6 cm 3.0cm Rf3 = 0.83
4 3.6 cm 2.3cm Rf4 = 0.63
5 3.6 cm 1.2cm Rf6 = 0.21
6 3.6 cm 0.8cm Rf5 = 0.32

Table 4.Fractionation with Chloroform

No. of spots Distance trveled by Distance traveled by Rf value
solvent front spots
1 3.3 cm 3.2cm Rf1 = 0.92
2 3.3 cm 2.6cm Rf2 = 0.79
3 3.3 cm 2.3cm Rf3 = 0.69
4 3.3 cm 1.7cm Rf4 = 0.51
5 3.3 cm 1.3cm Rf6 = 0.38
6 3.3 cm 0.9cm Rf5 = 0.26

Table 5. Fractionation with Ethyl acetate fraction

No. of spots Distance trveled by Distance traveled by Rf value
solvent front spots
1 3.9cm 3.3cm Rf1 = 0.85

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 Fractionation of Crude Dye Extracted From Cucurbita Pepo Leaves by Cold Extraction Method

2 3.9 cm 3.0cm Rf2 = 0.77

3 3.9 cm 2.2cm Rf3 = 0.56
4 3.9 cm 1.6cm Rf4 = 0.40
5 3.9 cm 1.1cm Rf6 = 0.27
6 3.9 cm 0.7cm Rf5 = 0.16

Table 6.Fractionation with Acetone

No. of spots Distance travelled by Distance travelled by Rf value
solvent front spots

1 3.5 cm 2.0 cm Rf1 = 0.56

2 3.5 cm 1.5 cm Rf1 = 0.42
3 3.5 cm 1.0 cm Rf1 = 0.26
4 3.5 cm 0.7 cm Rf1 = 0.18

Table 7.Percent yield/ Percent recovery of cold extract of C. pepo

Mass of plant leaves: 100g
S. No Fractionation solvent Percent Yield (w/w)
1 Ethanolic extract 6.81%
2 . n-hexane 3.42%
3 Chloroform 0.77%
4 Acetone 2.48%

TLC and UV-Vis study of variousfractions of C. pepo cold extract

At ambienttemperature and pressure in 30% of mobile phase (n-hexane 7ml + ethyl acetate 3ml) system a good TLC was developed
for various fraction of C. pepo cold extract.The solvents used for fractionation were nhexane, chloroform and acetone. The extracted
dye consist of six components while the TLC was developed with n hexane, chloroform and acetone fractions but the extracted dye
consist of four components while TLC was developed with acetone fraction. All components have distinct Rf values which are shown
in the Tables 4-7. Due to variation of solvent used in fractionation deviation are seen in all cases.

6components/functional groups were confirmed in crude dye fractioned with n hexane, chloroform and ethyl acetate but only 4
components/functional groups were confirmed in crude dye fractioned with acetone. Maximum Rfvalues were observed
forchloroform, like Rf1 for chloroform is 0.92 cm. For n-hexane, ethyl acetate and acetone were 0.98cm, 0.85cm and
0.56cmrespectively. All fractions except acetone, 6spots were observed on developed TLCwhile acetone has 5 spots on
developedTLC. Similar results have been observed by Shah et al. 2013.

Conclusion
From the experiment it can be concluded that the cold extraction method is more suitable while cost and availability is concerned but
hot water extraction method is more rapid. Among all samples while 100 gm sample is dissolved in 750 ml the crude extract will be
more.The crude dye fractionation with different solvents in TLC showedinmajor thedye has six distinct components.

REFERENCES
[1] Shahid, M.&Ul-Islam, S. (2013). Recent advancements in natural dye applications. Journal ofCleaner Production, 53: 310–331.
[2] Grifoni D.,Bacci, L.,Zipoli, G., Albanese, L. and Sabatini, F.(2011). The role of natural dyes inthe UV protection of fabrics made of vegetable
fibers. Dyes and Pigments, 91: 279–285.
[3] Baliarsingh, S., Panda, A. K., Jena, J., Das, T. & Das, N. B. (2012). Exploring sustainable techniqueon natural dye extraction from native
plants for textile: identification of colourants, colourimetric analysis of dyed yarns and their antimicrobial evaluation. Journal of Cleaner
Production, 37: 257–264.
[4] Booa H.O., Hwangb, S. J.,Baec, C. S., Parkc, S. H. ,Buk-GuHeod&Gorinsteine, S. (2012).Extraction and characterization of some natural
plant pigments. Industrial Crops and Products, 40: 129–135.
[5] Sinha K., SahaaP.D. ,Datta B. S. (2012). Extraction of natural dye from petals of Flame of forest(Buteamonosperma) flower: Process
optimization using response surface methodology(RSM). Dyes and Pigments, 94: 212–216.
[6] Shah, N., Farman, S., Hussain,Z., Arain,M. B. &Sulaiman Sham, S. (2013). Pakhtunkhwa J. Life Sci. 1 (03):130-144

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