Microwave Assisted Extraction of Pigments for better yield and quality.pptx

Department of Postharvest Technology 1

University of Horticultural Sciences, Bagalkot
College of Horticulture, Bengaluru
Seminar - I
Extraction of pigments through microwave assisted extraction (MAE) for
quality and yield
Major Advisor
Dr. Suresh G. J.
Assistant Professor
Dept. of Postharvest Technology
Rosemary M. Xavier
UHS19PGD379
II Ph. D.
Dept. of Postharvest Technology

Topic division
• Introduction
• Conventional methods of extraction
• Advances in extraction techniques
• Microwave assisted extraction
• Plant based pigments
• Case studies
• Advantages and disadvantages of MAE
• Future line of work
• Summary
• Conclusion
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• Extraction is the process in which one or more components are get separated selectively
from a liquid or a solid mixture by means of a immiscible solvent
• Solvent used for the extraction is known as MENSTRUM
• The inert fibrous and other insoluble materials remaining after extraction is called as
MARC
• Its major application is in isolation of phytoconstituents and nutraceuticals
INTRODUCTION
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Conventional methods of extraction
Infusion
• Pouring water to the drug
• Keep in contact with water for a stated period of time
• Filtering off the liquid
Decoction
• Crude drug is boiled with specific amount of water for a defined time
• Cooled and strained or filtered
• Suitable for water soluble heat stable compounds
Maceration
• Solid ingredients are soaked in a stoppered container
• Whole solvent allowed to stand for period of at least 3 days with agitation
• The mixture is strained and the marc is pressed
Soxhlet extraction
• Sample is placed in a thimble and fresh condensed solvent is circulated from a distillation flask
• Siphoning takes place to avoid the over flow
• The process repeated till the complete extraction achieve
Digestion • Form of maceration with slight warming
• The most used temperature 35° C – 40° C
• Used for tougher plant parts with poor solubility
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DRAWBACKS
Time consuming
Rate of mass transfer goes on decreasing
Large amount of solvent required
Difficulty in solvent recovery
Not suitable for thermolabile compounds (digestion, decoction)
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Advances in extraction techniques
Microwave assisted extraction
Ultrasound assisted extraction
Supercritical fluid extraction
Accelerated solvent extraction
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Microwave theory
• Non-ionizing electromagnetic waves of
frequency between 300MHz-300GHz
• Position – between the radio and infrared ray
Microwaves are made up of two oscillating
perpendicular fields i.e. electric field and
magnetic field
• The principle of heating is based on two
phenomenon
 Ionic conduction
 Dipole rotation
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Fig. 1- Microwave (diagrammatic representation)

Ionic conduction
• Electrophoretic migration of ions under the
influence of the changing electric field
• The resistance offered by the solution generate
friction and heat up the medium
• One of the mechanism of current
• Materials exhibiting this property used in
batteries- e.g. β- alumina
Fig. 2- Diagrammatic representation of
electrophoretic migration
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Dipole rotation
• Dipole rotation means the realignment of dipoles of the molecules with the
rapidly changing electric field
• At 2450 MHz the dipole align and randomize 4.9 x 109 times per second
Eskilsson and Bijorklund, 2000
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Fig. 3 – Diagrammatic representation of dipole rotation

27-04-2022 08:16 Department of Postharvest Technology 11
The microwave assisted extraction (MAE) system
Fig. 4 – The microwave assisted extraction (MAE) system

Micro irradiation
Moisture get heat up
Moisture evaporate
Generation of tremendous pressure on cell wall
Rupture of the cell
Leaching out phyto constituents
Swelling of the plant cell
Extraction process
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Fig. 5 – Conventional extraction Vs. Microwave extraction

Components of MAE
MAE
Magnatron
Wave guide
Applicator
Circulator
Generate microwave
Sample holder and sample is
placed here
Regulate the movement of
microwave
Direct the waves from
source to cavity
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Method development
Pre-treatment • Air drying/ freeze drying followed by grounding and /or sieving
Parameters influence on
extraction process
• Extraction time/ irradiation time
• Microwave power
• Sample to solvent ratio
Additional clean-up • Filteration, centrifugation
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Factors affecting MAE
1. Solvent
a). Solubility of analyte b). Ability to absorb microwave energy and pass it in the
form of heat depends on dissipation factor
2. Microwave power
Optimized to minimize the extraction time and prevent thermal deterioration due to
excessive heating
3. Extraction time
The amount of the analyte extracted can be improved with increasing extraction time
with the associated risk of degradation of thermolabile components
4. Temperature
Increase solubility and cause cellular pressure build up and leading to cell rupture
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Table-1. Solvents commonly used in MAE
Solvent Dielectric
constanta (ἐ)
Dipole moment b Dissipation factor,
tan ẟ (X 104)
Boiling point c
(°C)
Closed vessel
temperature
(°C)
Acetone 20.7 56 164
Acetonitrile 37.5 82 194
Ethanol 24.3 1.96 2500 78 164
Hexane 1.89 69 -°
Methanol 32.6 2.87 6400 65 151
2-propanol 19.9 1.66 6700 82 145
Water 78.3 2.3 1570 100
Hexane- Acetone (1:1) 52 156
a Determined at 20° C
b Determined at 25° C
c Determined at 101.4 kPa
d Determined at 1207 kPa
-° Indicate no microwave heating
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Applications
of MAE
Pharmaceuticals and
natural products
Phenols
Metals
Polymers
Persistent organic
pollutants
Pesticides
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Sample Microwave supported extraction
Experimental design Spectrophotometric analysis
Modelling and optimisation
storage
Chromatographic analysis
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Fig. 6- Flow chart of MAE process and analysis

Table -2. Comparison of traditional Soxhlet extraction and MAE
MAE Soxhlet extraction
Brief description Sample is immersed in a microwave
absorbing solvent in a closed vessel
and irradiated with microwave energy
Sample is placed in a glass fibre thimble
and by using soxhlet extractor the sample
is repeatedly percolated with the
condensed vapours of the solvent
Extraction time 3-30 min. 3-48 h
Sample size 1-10 g 1-30g
Solvent usage 10-40 mL 100-500 mL
Investment Moderate Low
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• Natural colorants – Ubiquitous in nature
• High nutraceutical value due to their recognized biologically active properties
(Delgado and Lopez, 2002)
• Eg. of natural colorants- Chlorophylls, carotenoids, curcuminoids,
anthocyanins, betalains, bixin etc. (Cai et al., 2003)
• Consumers prefer pigments extracted from plants since they avoid the health
hazards posed by synthetic chemical colorants (Lila, 2004)
Plant based pigments
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• High temperature processing results in pigment
degradation and associated colour loss
• The addition of colorants, either synthetic or natural, to
enhance , improve or restore colour (Azeredo, 2009)
• The association of synthetic dyes and development of
ADHD (Attention Deficit Hyperactivity Disorder)
• According to European Food Safety Authority(2008) the
product should be labelled “may have an adverse effect
on activity and attention in children ” (Kanarek, 2011)
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Table-3. Naturally occurring pigments in biology
Group Alternative name Major examples Predominant colour
Tetrapyrrole
Porphyins and porphyrin
derivaties
Chlorophylls Green
Haems (Hemes) Red
Bilins (Bile pigments) Blue- green
Yellow- Red
Tetraterpenoids Carotenoids
Carotene Yellow-Red
Xanthophylls Yellow
O- Heterocyclic compound Flavonoids
Anthocyanins Blue - Red
Flavonols Yellow –White
Flavones White - Cream
Anthochlores Yellow
Quinones Phenolic compounds
Naphthaquinones Red- Blue- Green
Antraquinones Red- Purple
Allomelanins Yellow- Brown
Tannins Brown- Red
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Group Alternative name Major examples Predominant colour
N- Heterocyclic compound
Indigoids and Indole
derivatives
Betalaines Yellow- Red
Eumelains Black- Brown
Phaeomelanins Brown
Indigo Blue- pink
Substituted pyrimidines
Pterins White - Yellow
Purins Opaque white
Flavins Yellow
Phenoxazines Yellow-Red
Phenazones Yellow- purple
Metalloproteins
Cu- proteins Blue - Green
Haemerythrin Red
Haemovanadins Green
Adenochroma Purple-Red
Miscellaneous Lipofuscins Brown- grey
Fungal pigments Various but commonly yellow
Hendry and Hougton-1996
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Contd….

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Case study -1
Objectives
To evaluate the effect of microwave technology on the extraction yield of carotenoid from carrot juice
processing waste using flax seed oil as solvent and optimise the extraction condition (microwave power,
extraction time and oil to waste ratio) to achieve optimum recovery of carotenoids)
NASS Score – 9.71
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Table-4. Summary of the microwave assisted extraction parameters
Process parameters Units Levels with codes
-2 -1 0 +1 +2
Microwave power W 50 80 125 170 200
Extraction time Min. 1 3.14 6.30 9.46 12
Oil to waste ratio g/g 5:1 8:1 12.5:1 17:1 20:1
• Response surface methodology (RSM) was employed to determine the optimal conditions.
• Microwave power (X1), extraction time (X2) and oil to waste ratio (X3) were independent variables of
the system and % recovery of carotenoid (Y1) was the response of the system.
• The extraction parameters were optimized through a central composite rotatable design (CCRD).
• The experimental design consists of 20 test
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Department of Postharvest Technology
27
Fig. 7.C. Oil to waste ratio (g/g) versus
microwave power (W).
Fig 7. A. Extraction time (min)
versus microwave power (W)
Fig. 7. B. Extraction time (min)
versus oil to waste ratio (g/g)
%
Recovery
%
Recovery
%
Recovery
Extraction time (min) Extraction time (min)
Microwave power (W)
Microwave power (W)
Oil to waste ratio (g/g)
Oil to waste ratio (g/g)
7.A
7.C
7.B
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X
X
X
Y
Y
Y
Z
Z
Z

Experimental validation of optimized condition
• The optimum conditions were determined by a quadratic model as 165 W of microwave
power , 9.39 min of extraction time, 8.06:1 g/g of oil to waste ratio to obtain optimum
value of recovery (77.87%) of carotenoid
• The carotenoid extraction (three times) were performed at optimum condition to validate
the suggested model.
• The experimental results gave 77.48±0.16% recovery of carotenoids which was close to
the predicted value

• It was found that microwave power and extraction time are significantly effective in extracting
carotenoids from carrot waste to flaxseed oil
• A 77.48% recovery of carotenoids was achieved successfully at optimum conditions (165 W of
microwave power, 9.39 min of extraction time and 8.06:1 g/g of oil to waste ratio).
• The extraction yield for conventional method was about 50% in the first 30 minutes of
extraction
• The carotenoid extraction using oil under microwave irradiation is a promising and efficient
process for both waste uses and enrichment of oil.
• Alternative method based on innovative and fast technology
Inference
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NASS score – 7.42
Case study II
Objective
To study the effect of microwave power, irradiation time and ethanol solvent concentration in MAE on the
extraction efficiency of anthocyanins.
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Process parameters Units
Levels with code
-1 0 +1
Microwave power Watt 350 425 500
Irradiation time s 30 60 90
Ethanol solvent
concentration
%, v/v 20 50 80
• Analysis was based on 0.1 g sour cherry peel samples
• Microwave power (X1), irradiation time (X2) and EtOH solvent concentration (X3) were the independent
variables, set down according to groundwork experiments.
• Experiment was composed of 20 runs and 6 central points
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3D surface plot for total anthocyanin of the sour cherry peels extract as a function of
Fig 8. A- EtOH solvent concentration to irradiation time(Microwave power kept constant as 500W)
Fig. 8.B –Ethanol solvent concentration to microwave power (irradiation time kept constant as 90 s)
Fig. 8. C- Irradiation time to microwave power (EtOH solvent concentration kept constant as 80 %).
TA
(mg-cyn-3-glu/g
FM)
TA
(mg-cyn-3-glu/g
FM)
TA
(mg-cyn-3-glu/g
FM)
Microwave power
(Watt)
Irradiation
time (S)
EtOH solvent
concentration (%)
8.A
EtOH solvent
concentration (%)
Irradiation
time (S)
8.B 8.C
Microwave
power (Watt)
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Inference
• Sour cherry peels were extracted by an alternative green method.
• Samples were found to be rich in phenolic and anthocyanin contents
• The optimization study has suggested that 500W of microwave power, 90 s of
irradiation time and 80 per cent ethanol are optimum conditions to obtain maximum
total anthocyanin (12.47 mg-cyanidin-3-glucoside/ g- FM) from sour cherry peels.
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Case study III
NASS score – 12.31
Objective
To optimize the MAE method to obtain maximum curcumin and
antioxidant content in extracts for five natural deep elutectic solvents
(Binary combinations of choline chloride, lactic acid, fructose and
sucrose) Chemical structure of curcumin
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-1 0 +1
Microwave temperature °C 45 60 75
Extraction time Min. 5 17.5 30
Solvent to solid ratio mL/0.2g 10 15 20
Solvent code Constituents Molar ratio
NADES 1 Fructose: choline chloride: water 2:5:5
NADES 2 Sucrose: choline chloride : water 1:4:4
NADES 3 Fructose: lactic acid: water 1:5:5
NADES 4 Sucrose: lactic acid: water 1:5:7
NADES 5 Lactic acid: choline chloride: water 1:1;2
Table -7. Composition and solvent code for studied NADESs
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Fig. 9 -Contour plots for the effects
of temperature and time on
curcumin content of turmeric at a
constant optimal solvent-to-solid
ratio related to corresponding
NADES
NADES-1 NADES-3
NADES-2
NADES-4 NADES-5

Fig -10. Contour plots for the effects
of temperature and time on CC of
turmeric at an optimal constant
time related to corresponding
NADES
NADES-1 NADES-2
NADES-4
NADES-3
NADES-5

Inference
• The extraction temperature was found to be the most significant operational factor on
the extraction of curcumin
• In order to obtain the highest curcumin yields, optimal extraction temperature,
extraction time and solvent-to-solid ratio as independent variables were determined
related to NADES type in the ranges of 64.7–71.8°C, 15.4–21.6 min and 14.5–16.5
mL/0.2 g, respectively.
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Case study IV
NASS score – 8.60
Objective
To evaluate the extractability of betalains via microwave assisted extraction and thermal degradation of
resulted extracts on heating at specific times and temperature.
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Table -8. Summary of the microwave assisted extraction parameters
-1 0 +1
Microwave power W 100 450 800
Extraction time s 30 90 150
Solvent to solid ratio w/v 0.1 0.15 0.2
Table- 9. Composition and solvent code
Solvent code Constituents composition
PW Pure water -
AW Acidified water 0.5% ascorbic acid
EW Ethanol – water 15% ethanol
AEW Acidified ethanol water 15% ethanol+ 0.5% ascorbic acid +
water
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Fig. 11. Yield % of each sample extract with different solvents
at varied conditions of MAE
A- Pure water
B – Acidified water
C – Ethanol water
D- Acidified ethanol water
Table -Process variables for MAE of betalain color compounds
from the peel of beetroot

Inference
• Pure water is a very good solvent for the extraction of betalains
• The optimum condition for the extraction of betalains (115.89 mg 100g-1 FW)were found
as 800 W microwave power, 150 sec of irradiation time and 0.2 (w/v) as solvent to
sample ratio
• Beetroot peels can be effectively utilized as a source of betalain on commercial scale and
minimize the wastage from beetroot processing industries
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Case study V
NASS score – 10.24
Objective
To evaluate the microwave assisted extraction (MAE) of saffron major compounds- picrocrocin, safranal and
crocin through response surface methodology (RSM)
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Table-10. Summary of the microwave assisted extraction
parameters
-2 -1 0 +1 +2
Microwave temperature °C 45 65 85 105 125
Extraction time Min. 10 15 20 25 30
Ethanol concentration (%) 0 25 50 75 100
20 Runs were conducted in triplicate, including six replications for the central point
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12. A Crocin content when the ethanol
concentration is kept constant at 50 %
12. B. Crocin content when the
time is kept constant at 20 min
12. C. Crocin content when the
temperature is kept constant at 80°C

Inference
• The effects of MAE temperature and ethanol concentration were found to be more
effective on all the responses than that of MAE time.
• According to the optimization algorithm of RSM, the temperature of 95.91 °C, time of
30 min and ethanol concentration of 59.59 % were determined to be the optimum
circumstances of the process (optimum recovery in molecular extinction coefficient,
picrocrocin- 256.59, safranal- 57.21 and crocin-106.54)
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Table -11. Conclusion of case studies
Compound extracted
Optimum process parameters
References
Microwave
power (W)
Temperature
(°C)
Solvent details Extraction
time
Carotenoid – carrot
(77.48%)
165 - Solvent to sample ratio-
8.06:1 gg-1
Flax seed oil as solvent
9.39 min. Aysel et al., 2020
Anthocyanin – sour cherry
peel
(12.47 mg-cyn-3-gluc g-1
FM)
500 - 80% Ethanol 90 sec. Ebru et al., 2021
Curcumin – turmeric
(13.94- 31.99 mg/g DM)
- 64.7- 71.8 14.5- 16.5 mL/.2g
NADES
15.4-21.6 min. Khadija et al.,
2021
Betalaines – beetroot peel
(115.89 mg 100g-1 FW)
800 - Solvent to sample ratio-
0.2 (w/v)
150 sec. Zin and
Banvolgyi, 2021
Crocin – saffron
(106.54 molecular extinction
coefficient)
- 95.91 59.59% Ethanol 30 min. Sarfarazi et al.,
2019

Advantages of MAE
• Reduction in extraction time
• MAE allows for a significant reduction in the organic solvent consumption
• Possibility of running multiple samples
Disadvantages of MAE
• The extraction solvent must be able to absorb microwaves
• Clean up step is needed
• Waiting time for the vessel to cool down
Disadvantages of MAE
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Future line of work
• More research is needed to improve the understanding of extraction
mechanism, remove technical barriers, improve the design and scale up of
the novel extraction system for their better industrial application
• Development of green solvent to minimize the use of chemical solvents
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Summary
• Extraction is the crucial step in the analysis of any compound
• Traditional extraction methods are replaced by advanced extraction methods
• Microwave assisted extraction method less time and solvent consuming
• Optimization of process variables are very important for each compound
• For extraction of pigments MAE is highly recommendable
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Conclusion
• The MAE method provides advantages such as shorter extraction time,
automation convenience, multiple sample processing and low solvent
requirement compared to other extraction techniques.
• The MAE method has the potential to be an efficient and sustainable
procedure in the pharmaceutical, natural dye and food industries for the
extraction of bioactive compounds.
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