Cftri Thesis Final PDF
Cftri Thesis Final PDF
Cftri Thesis Final PDF
DISSERTATION BY
Harsh Sharma
Gaurav Kumar
SUBMITTED TO
TAMIL NADU
Dr. B. MANOHAR
MYSORE-570020
April 2019
1
CERTIFICATE
This is to certify that the Project Work entitled “Optimization of supercritical CO2 extraction of
Guggul and Byadgi chilly and their characterization” submitted by Harsh Sharma, Gaurav Kumar ,
Ayush Vyas Kukillaya to Vellore Institute of Technology, Vellore for the award of the Degree
of bachelor of technology in chemical engineering is a record of Bonafide work carried out by
them under my guidance in the Department of Food Engineering, CFTRI, Mysore – 570 020
during the period of Dec 2018 to April 2019.
To the best of my knowledge, the present work is the result of his original investigation
and study. No part of the dissertation has ever been submitted for any other degree at any
University. The dissertation is fit for submission.
Date: 01-04-2019
CSIR-CFTRI
2
DECLARATION
This report does not, to the best of our knowledge, contain part of our work which has been
submitted for the award of any degree either of this institute or any other university without
proper citation.
Date:01-04-2019
Gaurav Kumar
3
ACKNOWLEDGEMENT
We sincerely thank Dr. KSMS Raghava Rao, Director, CSIR- CFTRI and Dr. Navin K.
Rastogi, Head of Department, Food Engineering for providing us the opportunity and facilities
to undertake this work.
It is also with great pleasure that we wish to record our deep sense of gratitude and indebtness
to our External Guide, Dr. B. Manohar, Chief Scientist and Head of Food Engineering
Department, CFTRI, Mysore for his valuable guidance and critical worthy suggestions during
the course of our project work.
We also extend special acknowledgement to Mr. Girish K. Ghiwari, Technical officer, DTS
, Dr. Joseph G.S. ,Technical officer, FED , Mr. Ramesh T. , Technical officer, Electrical and
Mechanical Maintenance Department , CIFS and HRD and Lab-1, FED for providing their
more than valuable assistance in ensuring the progress of our Dissertation work.
We would like to thank our internal Project Guide, Dr. Aruna Singh(VIT) for her valuable
support and assistance in the proceedings of our project as well as the Department of Chemical
Engineering for their invaluable support in ensuring the success of this dissertation work.
4
LIST OF TABLES
Table 10: Variation of Color value and Pigment concentration according to BBD parameters
LIST OF FIGURES
Figure 7.a: Surface and contour plots showing effect of Pressure and Temperature vs. Yield
of guggul
Figure 7.b: Surface and contour plots showing effect of Pressure and Flowrate vs. Yield of
guggul
Figure 7.c: Surface and Contour plots showing effect of Temperature and Flowrate vs. Yield
of guggul
5
Figure 8: Plot of ln C* vs. Time
Fig 10.a: Surface and contour plots showing effect of Temperature and Flowrate vs. Yield of
chilli
Fig 10.b: Surface and contour plots showing effect of Pressure and temperature vs. Yield of
chilli
Fig 10.c: Surface and contour plots showing effect of Pressure and Flowrate vs. Yield of
chilli
6
CONTENTS
ABSTRACT
1. INTRODUCTION
1.1 GENERAL
1.2 GUGGUL
1.3 BYADGI CHILLI
2. REVIEW OF LITERATURE
2.1 GUGGUL
2.2 CARETENOID EXTRACTION FROM PEPPER
2.3 SUPERCRITICAL FLUID: CO2
2.4 SUPERCRITICAL EXTRACTION
2.5 SCFE EQUIPMENT LAYOUT AND PROCESS FLOW
2.6 BOX-BEHNKEN DESIGN
3. MATERIALS AND METHOD
3.1 RAW MATERIALS
3.1.1 GUGGUL FEED
3.1.2 BYADGI CHILLI FEED
3.2 CHEMICALS AND REAGENTS
3.3 SOLVENT EXTRACTION
3.3.1 GUGGUL EXTRACTION
3.3.2 CHILLI EXTRACTION
3.4 SUPERCRITICAL EXTRACTION OF GUGGUL AND
BYADGI CHILLI
3.5 HPLC ANALYSIS OF EXTRACT
3.6 COLOR VALUE ANALYSIS OF CHILI OLEORESIN
3.7 PIGMENT CONCENTRATION MEASUREMENT OF CHILLI
OLEORESIN
4. RESULTS AND DISCUSSION
4.1 SCFE GUGGUL EXTRACTS AND ANALYSIS
4.1.1 YIELD ANALYSIS OF GUGGUL
4.1.2 STATISTICAL ANALYSIS OF
DESIGN
4.1.3 CONTOUR PLOT AND RESPONSE
SURFACE ANALYSIS
7
4.1.4 DIFFUSIVITY STUDY FOR
EXTRACTION
4.1.5 HPLC ANALYSIS OF EXTRACT
4.2 SCFE CHILLI OLEORESIN EXTRACT
ANALYSIS
4.2.1 YIELD ANALYSIS OF CHILLI
OLEORESIN
4.2.2 STASTISTICAL ANALYSIS OF
DESIGN
4.2.3 CONTOUR PLOT AND RESPONSE
SURFACE ANALYSIS
4.2.4 DIFFUSIVITY STUDY FOR
EXTRACTION
4.2.5 COLOR VALUE AND PIGMENT
CONCENTRATION ANALYSIS
5. SUMMARY
5.1 GUGGUL
5.2 BYADGI CHILLI
6. REFERENCE
8
ABSTRACT
GUGGUL
The resin of Commiphora mukkul (Indian Bedellium) were extracted and optimized
using Box Behnken design by varying the Pressure, Temperature, and Flowrate at three
different levels .The highest yield was obtained at pressure of 400 bar, temperature of 550C and
flowrate of 35g/min to give a yield of 14.41%.The modelled quadratic equation showed an
adjusted R2 value of 97.14% and a P-Value of 0.00019(<005).Using Fick’s law,the diffusivity
at highest yield conditions was found out to be 3.23*10-9 m2/s. HPLC analysis of the extracts
showed a retention time of 20.413 min for E-guggulsterone and a highest E-guggulsterone
concentration of 3.84% at pressure of 300bar, Temperature of 450C and flowrate of 35g/min.In
general, the SCFE extracts were found to have higher E-guggulsterone concentration as
compared to solvent extracts.
BYADGI CHILLI
Box Behnken design was carried out to optimize the supercritical carbon dioxide
oleoresin yield of Byadgi chilli by varying Pressure, Temperature and Flowrate at three
different levels .The highest oleoresin yield was obtained as 7.126% at pressure of 400 bar,
temperature of 550C and flowrate of 35g/min. The highest colour value and pigment
concentration were obtained as 114985 and 89.81g/Kg respectively at pressure of 400 bar.
Temperature of 350C and flowrate of 35 g/min. The developed predictive equation showed an
adjusted R2 value of 98.51%, F-value of 103.62 and a p-value of 0.00032.The diffusivity of
capsanthin and capsorubin in supercritical carbon dioxide was calculated using Fick’s law to
be 1.221*10-10 m2/s. The oleoresin yield , color value and pigment concenteration of the extract
obtained by SCFE technique was lower by about 20-30% as compared to extract obtained by
solvent extraction technique.
9
CHAPTER 1
INTRODUCTION
1.1 GENERAL
During the last two decades the emphasis on extraction of essential compounds from natural
sources have gained severe emphasis for usage in various applications that include with major
applications being in the field of nutraceuticals and pharmaceuticals. The traditional extraction
techniques of these compounds include solvent extraction, steam distillation which are
currently used for commercial applications. Although these methods are convenient to use,
they have their fair share of downsides such as requirement additional separation process, usage
of harsh chemicals, and release of toxic chemicals into the environment. The conventional
processes are also highly non-selective in nature and require heat input at one or the other stage
thereby leading to the thermal degradation of heat sensitive molecules [1].Furthermore some
of these techniques like solvent extraction tend to leave residual solvent in the extract which is
not suitable for medicinal usage. Soxhlet extraction being one of the most common method
used for solid-liquid extraction , comes with major drawbacks like long extraction time
requirement, large amount of solvent wastage and incapability to provide constant agitation
during the extraction process[2].
These conventional methods are not suitable for extraction of medicinal compounds which
require high degree of purity and nil solvent residue in the extract. In such cases, supercritical
fluid extraction (SCFE) is regarded as the best method for extraction. The main advantages of
supercritical extraction are residue free extracts, low to moderate temperature of processing,
high rates of mass transfer and easy product separation amongst others.
1.2 GUGGUL
Guggul is regarded as one of the most important herbal compounds for medicinal use and is
of high commercial importance to the pharmaceutical industry. The major players in
manufacture of Guggul are: Sami Labs Pvt. Ltd. (Bangalore, India), Maypro Industries
(U.S.A), Himalaya Wellness (India) amongst many others. Although there are various
manufacturers of Guggul in India and across the globe using SCFE, there is no research article
available regarding the optimized extraction condition for extraction of the same. Hence it is
10
of high importance that such data is made available so that further research can be carried out
and to assist the commercialisation of the product.
India has a domination over the spice market by being largest producer and exporter of
chilli pepper and also having the largest area under cultivation, with most popular being
Sannam, Byadgi and Guntur varieties of chillies [3]. Peppers are considered to be excellent
food colorant having antioxidant compounds present in them [4].The compounds present in
peppers can be broadly categorised as Capsaicinoids and carotenoids. The capsaicinoids are
responsible for the pungency in the pepper fruits with the most common capsaicinoid being
capsaicin (trans-8 methyl-N-vanillyl-6-nonenamide) and dihydrocapsaicin (8 methyl-
Nvanillylnonanamide)[5].Carotenoids are the compounds responsible for imparting colour to
the chilli peppers and are commercially very important. The red color in chilli pepper is due to
mainly two carotenoids: Capsanthin and capsorbium [6].
Bydagi chilli is a variety of chilli indigenous to the state of Karnataka (India) and is
renowned for its high carotenoid content and low capsaicinoid content, thereby making it very
suitable for use as food and which has led to tremendous increase in oleoresin demand for the
same.
11
CHAPTER 2
REVIEW OF LITERATURE
2.1 GUGGUL
‘Guggul’ is the extract obtained from the oleo-gum resin of Commiphora mukul( Indian
Bedillium).The resin comes in light yellow to dark brown colour with an aromatic odour[7].
The main active ingredient of Guggul which account for the medicinal activity are E -
gugulsterone (E-4,17(20)-trans-pregnadiene-3, 16-dione) and Z-guggulsterone (Z-4,17(20)-
cis-pregnadiene-3,16-dione) stereoisomers[8]. Guggulsterones have been found to possess
medicinal properties such as anti‑inflammatory [9,10],muscle relaxing,[11]
hypocholesterolemic and anti‑obesity[12‑13] and many more.The conventional extraction of
guggul is done using ethyl acetate [14] and the extract is standardised to 2.5%-7.5%
guggulsterones for medicinal usage by use of techniques like HPLC [15]. The genuine samples
of guggul contain 1%of volatile oil [16] and between 1.0% and 1.5% of guggulsterones (𝑍 and
𝐸) [17].
12
oleoresin has also been carried out and resulted in highest red pigment concentration of
66.7g/kg when carried out at two stages of 2000 psi followed by 6000-7000 psi [20].
13
Density(g/cm3) 10-3 0.3 1
Diffusivity(cm2/s) 0.2 10-3 10-5
Viscocity(g/cm s) 10-4 10-4 10-2
14
Figure 2:SCFE equipment
For extraction process, CO2 was supplied from the cylinder into the condenser(2 phase
heat exchanger) which condensed the CO2 into liquid state.Then the Liquid CO2 was sent into
the preheater where it was heated before going into the reciprocating type CO2 pump which
created the flowrate and built up the pressure head as required.Next the SCF entered the
extractor from bottom where it dissolved the solute from the solid into itself. The extractor was
equipped with a heating element so as to heat the SCF to the required temperature. From the
extractor ,the SCF containing the solute entered into a vaporizer which heated the SCF further
to assist proper separation. Finally the SCF entered into the separator which was operated at
low pressure ( 700-900 psig) , where it changed its phase into gaseous state, leaving behind the
solute. The gaseous CO2 was recycled back to the condenser and the cycle was repeated. An
ABPR was used to control the pressure of the extraction line.The PFD of the equipment is
shown in figure 3 and the specifications are shown in table 2
15
Figure3: Schematic diagram of SCFE equipment: TI-Temperature Indicator, PI-Pressure
Indicator, T01-CO2 cylinder, HE01-Condenser, HE02-Pre-heater, HE03-Vaporizer, HE04-
Chiller, E01-500ml extractor, E02-100ml extractor, P01-CO2 pump, P02- Solvent pump
COMPONENT SECIFICATION
CO2 PUMP Type: reciprocating (dual head)
Cooling: water coled
Flowrate: 25gm/min -50gm/min
Operating pressure: 60-600 bar
EXTRACTION VESSEL Volume: 100 ml
Heating: Jacketed
MoC: Stainless steel
EXTRACTION LINE MoC: Stainless steel
Maximum Temperature: 800C
CONDENSER Type: Shell and Tube
Heating Type :Electrical
Working Temp: <1100C
Table 2: Technical specifications of the SCFE equipment
16
2.6 BOX-BEHNKEN DESIGN
Response surface method(RSM) is a design scheme in which changes in the
independent variables are made and the response of the dependent variable is analysed to
understand the relationship between them. Box-Behnken Design(BBD) is a multivariate RSM
in which simultaneous changes are made in the levels of all the variables. It is a second order
multivariate technique that involves three level incomplete factorial design and is utilized for
the analysis of critical experiment condition that include the extreme values. The number of
experiments (N) required for the development of BBD is defined as N=2k(k-1)+Co, where k is
the number of variables and Co is the number of central points. A typical three variable system
is analyzed at three levels of -1,0,1 amounting to 15 experiments according to BBD.
17
CHAPTER 3
MATERIALS AND METHOD
High quality Guggul resin was purchased from online seller “Attar
Ayurved (Rajasthan India) and stored under airtight conditions.The resin was grinded to reduce
the particle size to 410µm.
Export grade Byadgi chilli was purchased from local market at Mysore ( Karnataka
,India).The seeds were separated from the pericarp using a hammer mill.The pericarp was
further reduced to particle size of 713 µm by fine grinding.The chilli feed was stored under
airtight conditions.
18
3.2 CHEMICALS AND REAGENTS
Analytical grade Ethyl Acetate , Ethanol, and acetone were procured from
“Sisco Research Laboratories”, (New Mumbai, India).HPLC grade Methanol and Acetonitrile
were also purchased from “Sisco Research Laboratories”, (Mumbai, India). E -Guggulsterone
standard was purchased from “Sigma Aldrich” and stored at -20oC until further use. Food grade
CO2 was sipplied by “Penya Industrial Gases”,(Mysore,India).
20 gms of the powdered gum resin was extracted with 250 ml of Ethyl acetate
with constant stirring for 2 hrs.The residue was further extracted for 2 hrs using fresh ethyl
acetate (250ml) under mid reflux conditions.The extracts were collected and were treated with
activated charcoal for 2hrs. Finally the solvent removal was carried out using Rotary
Evaporator. The extract was weighed and the yield was found to be 33% w/w of initial
powdered gum. The sample was further analyzed using HPLC for guggulsterone content.
20 grams of chilli powder was fed into the soxhlet apparatus and extraction was carried
out using 250 ml ( 1:12 ratio) of Acetone at 600C for three hours.The solvent saperation from
the extract was carried out using a rotary evaporation.The extracted oleoresin was subjected to
further analysis.
For the extraction 30 gms of feed (Wo) was loaded into the extraction vessel.The
extraction time was kept constant at 3hrs for every sample. The pressure , temperature and flow
rate of CO2 was varied at three levels according to BBD method as shown in Table 3.The
separator temperature was maintained at 35oC and separator pressure was at 900 psig. The
extracts were collected under separator pressure , weighed (W1) and stored in an airtight flask
for further analysis. After every extraction run, the SCFE system was purged with CO 2 for
19
removal of any residual extract remaining in the system.The yield percentage of extract was
W1
calculated as: { Wo*100}.
The Guggul samples were further analyzed by using HPLC for the estimation of E-
guggulsterone content and the chilli samples were analyzed using UV-visible
spectrophotometry for color value and pigment concenteration.
HPLC Analysis was carried out using Waters 515 HPLC system coupled with
a SHIMADZU (CTO-10AS) column oven and a Waters 2489 UV/Visible Detector. Analysis
was carried out in a Waters RP-C18 Analytical column (4.6 x 250 mm, particle size 5.0µm).
The software used for analysis was ‘Empower pro’.The sample was dissolved in 1ml of
methanol and passed through a 0.22µm filter. Mobile phase consisting of Acetonitrile(A) and
0.1% acetic acid in water (B) was used at a flowrate of 1ml/min with gradation as shown in
figure 6. The amount of E-guggulsterone was estimated by comparing the area under the curve
to the reference E-guggulsterone sample.
20
3.6 COLOR VALUE ANALYSIS OF CHILLI OLEORESIN
0.01% of oleoresin sample in acetone was prepared and the absorbance was taken at 458 nm
using (SHIMADZU UV-1201) UV spectrophotometer.
CV=A458*61000 eqn.1
Where A458 is the absorbance value at 458 nm and 61000 is the constant.
𝑎 10000
C = 2100x eqn.2
𝑊
Where ‘a’ is the absorbance of sample , 2100 is the A1%1cm for capsanthin/capsorubin in acetone
at 462 nm, W is the weight of oleoresin sample (1gm) and 10000 is the dilution factor to obtain
0.01% concenteration.
21
CHAPTER 4
The Box Behnken Design (BBD) under Response surface methodology was carried out to
optimize the Yield(Y) based on three variables: Temperature (T), Pressure (P) and flow rate
(F) at three different levels: -1, 0, +1 as shown in table (3) .The experiment plan is showed in
table (4) and table (5) for Guggul and Byadgi chilli respectively and comprised of 13 different
experiments and 2 repetition which accounted for overall 15 experiments in total. A second
order multivariate polynomial was used to approximate the relationship between the yield and
the independent variable as shown in equation (eqn.1)
Y=𝛽0 + ∑ 𝛽𝑖 𝑋𝑖 +∑ 𝛽𝑖𝑖 𝑋𝑖2 +∑ ∑ 𝛽𝑖𝑖 𝑋𝑖 𝑋𝑗 eqn 1
22
4.1 SCFE GUGGUL EXTRACTS AND ANALYSIS:
The BBD design showed highest yield at conditions of (P=400 bar, T=55oC, F=35gm/min) and
the lowest yield at conditions of (P=200, T=45 oC, F=45gm/min).
The quadratic equation fit for predicted yield was obtained as:
% Yp = 16.42 - 0.0853 P - 1.364 T + 1.716 F - 0.000028 P*P + 0.01141 T*T - 0.02821 F*F
+ 0.001495 P*T + 0.001626 P*F - 0.00196 T*F eqn.2
The R2 value of the model was found to be 98.98% and the adjusted R2 was found to be 97.14%
which suggested that 97.14% variation in the yield was due to the independent variable and
only 2.86% variation could not be explained by the model.The overall relative error in the
measured (Yo) and the predicted (Yp) percentage yield for the experimental setup was
calculated to be 3.042%, thereby indicating the preciseness and efficiency of the developed
model.
23
4.1.2 STATISTICAL ANALYSIS OF DESIGN
The statistical analysis of the BBD experiment was carried out MINITAB18 software. The high
F value (53.76) ,low P value (0.00019) for the model and high P value (0.0197) for the lack of
fit indicated that regression model was entirely significant in nature as shown in table (5) .The
analysis of variance showed that the linear parameters (P,T,F) were highly significant
(p<0.05).The quadratic terms of Temperature (T) and Flow rate (F) were also found to be
significant (p<0.05) but the quadratic term of pressure was found to have a p-value of 0.313
which demonstrated its insignificance. There was significant interaction between Pressure-
Temperature (0.001<p<0.05) and Pressure-Flow rate (0.001<p<0.05) for the extract yield but
the interaction between Temperature-Flow rate was found to be insignificant due to high p-
value (p>0.4518).
Analysis of Variance
Source DF Adj SS Adj MS F-Value P-Value
Model 9 112.192 12.4658 53.76 0.00019
Linear 3 31.378 10.4592 45.11 0.00048
P 1 4.045 4.0448 17.44 0.00868
T 1 6.796 6.7956 29.31 0.00291
F 1 14.225 14.2252 61.35 0.00054
Square 3 36.324 12.1079 52.22 0.00034
P*P 1 0.291 0.2911 1.26 0.31341
T*T 1 4.808 4.8083 20.74 0.00609
F*F 1 29.376 29.3762 126.69 0.0001
2-Way Interaction 3 19.669 6.5564 28.28 0.00146
P*T 1 8.943 8.9428 38.57 0.00158
P*F 1 10.572 10.5723 45.6 0.00108
T*F 1 0.154 0.1542 0.67 0.45182
Error 5 1.159 0.2319
Lack-of-Fit 3 1.001 0.3338 4.23 0.19724
Pure Error 2 0.158 0.079
Total 14 113.352
Table 5: Statistical analysis of model
24
4.1.3 CONTOUR PLOT AND RESPONSE SURFACE ANALYSIS:
2-D Contour plot and 3-D Response surface are very helpful in indicating the
relationship between independent variable and the response. The relationship between 2
variables and the yield was plotted keeping the third variable fixed at ‘0’ level. Fig 7.a shows
the effect of temperature and pressure on the yield keeping the flow rate constant at 35gm/min.
At higher pressure, increase in temperature resulted in increased yield. On the contrary, at
pressure below 250 bar, increasing the temperature up to 450C led to decrease in the yield and
again started increasing as temperature was increased from 450C to 550C .The relationship
between pressure and flow rate can be seen in fig 7.b, keeping the temperature constant at
450C.At any flowrate, increase in pressure resulted in increase in yield. At any constant
pressure the yield increased with increase in flowrate up to 35gm/min and then started
decreasing with increase in flow rate. This can be explained by the fact that at low flow rate
the ratio of weight of feed (W0) to CO2 flow per min (W0/F) is < 1, which resulted in incomplete
extraction due to less fluid availability for extraction. As the flow gradually increased to
35gm/min the ratio became > 1 which resulted in sufficient fluid for extraction and yield
reached its maxima. Subsequent increase in flowrate to 45gm/min the ratio became even higher
(>1.5) which resulted in increased superficial velocity and less residence time of the fluid inside
the extractor which led to decrease in interaction time between the feed and fluid, ultimately
leading to decrease in the yield. The effect of variation in temperature and flowrate on the yield,
keeping the pressure constant at 300 bar is demonstrated in fig 7.c. At any temperature, the
yield increased up to flow rate of 40gm/min and then started decreasing. At constant flow , the
increase in temperature led to increase in the yield, but changeover to decreasing phenomena
can be seen at 450C .
25
Surface Plot of response vs P, T
15
%YIELD 10
400
5
300
0 P
40
48 200
T 56
300
P
250
200
35 40 45 50 55
T
Figure 7.a: Surface and contour plots showing effect of Pressure and Temperature vs. Yield
26
Contour Plot of response vs P, F
400
response
< 6
6 – 8
8 – 10
10 – 12
350 12 – 14
> 14
300
P
250
200
25 30 35 40 45
F
Figure 7.b: Surface and contour plots showing effect of Pressure and Flowrate vs. Yield
27
Surface Plot of response vs T, F
15
%YIE LD 10
56
5 4
48
T
24 40
30
36
F 42
45
T
40
35
25 30 35 40 45
F
Figure 7.c: Surface and Contour plots showing effect of Temperature and Flowrate vs. Yield
28
4.1.4 DIFFUSIVITY STUDY FOR EXTRACTION
The diffusivity at the point of highest yield was modelled using Fick’s diffusion model
which is a gradient based model stating that flux is proportional to the concentration gradient
with diffusion taking in the direction of decreasing concentration.
The generalized form of the diffusion equation is given as follows [26]:
𝜕2 𝐶 𝜕𝐶
= 𝜕𝑡 eqn 3
𝜕𝑟 2
By applying the initial and boundary conditions, the solution of the above equation is given as:
[27]
6 𝐷𝑒 𝑡
C*=∑∞
𝑛=1 (𝑛𝜋)2 𝑒𝑥𝑝 {−(𝑛𝜋)
2
} eqn.4
𝑟2
𝐶−𝐶0
Where C*= with 𝐶 , 𝐶0 and 𝐶∞ being the average solute concentration in the solvent,
𝐶0 −𝐶∞
the total solute present in the feed and the solute concentration in the solvent at infinite time
respectively.
𝐷 𝑡
For Fourier number (F0= 𝑟𝑒2 ) greater than 0.1, all the higher terms of the equation can be ignored
6 𝐷𝑒 𝑡
C*=∑∞
𝑛=1 𝑒𝑥𝑝 {−(𝜋)2 } eqn.5
𝜋2 𝑟2
For our experiment, the value of diffusivity (De) was found out at the condition giving the
highest yield (P=400 bar, T=550C and F= 35gm/min). The experiment was conducted for 6 hrs
and sample was collected after every hour. The total % yield at end of 6 hrs. (𝐶∞ ) was found
to be 17.85%.The initial amount of solute in the sample (𝐶0 ) was taken as the total extract
obtained by solvent extraction, which in this case was 33.785%. By plotting graph of ln(C*) vs
Time (fig.8) the value of diffusivity was found to be 3.23*10-9 m2/s.
29
3.5
2.5
ln(C*{π2/6})
1.5
0.5
0
0 2000 4000 6000 8000 10000 12000 14000 16000 18000 20000
Time (s)
y = -8E-05x + 3.018
R² = 0.9179
Diffusivity of Guggul in supercritical CO2 was found to be very high as compared to some
other compounds , as shown in table 8.This demonstrates the high efficiency of supercritical
carbon dioxide in extraction of Guggul from its resin.
30
Fig 9.a: E-Guggulsterone standard at 1mg/ml
31
Fig 9.c: E-Guggulsterone standard at 0.25mg/ml
32
Fig 9.e: Guggul solvent extract
33
Fig 9.g: HPLC of SCFE of guggul at (200 bar, 550C, 35g/min)
34
Fig 9.i: HPLC of SCFE of guggul at (400 bar, 550C, 35g/min)
35
Fig 9.j: HPLC of SCFE of guggul at (200 bar, 450C, 45g/min)
36
Fig 9.m: HPLC of SCFE of guggul at (400 bar, 450C, 45g/min)
37
Fig 9.o: HPLC of SCFE of guggul at (300 bar, 350C, 45g/min)
38
Fig 9.q: HPLC of SCFE of guggul at (300 bar, 550C, 45g/min)
39
Pressure(P) Temperature(T) Flowrate (F) % E-guggulsterone
200 35 35 2.267
200 55 35 1.7148
400 35 35 2.2593
400 55 35 2.80
200 45 25 1.305
200 45 45 2.383
400 45 25 3.12
400 45 45 1.91
300 35 25 2.63
300 35 45 2.602
300 55 25 2.093
300 55 45 2.77
300 45 35 3.84
Table 6:Variation of % E-guggulsterone in extract with change in extraction conditions
40
200 45 25 1.53 1.37
According to BBD experiments, the highest yield of 7.126% was obtained at conditions of
(P= 400 bar, T= 550C and F=35 g/min) which corresponds to the level of (1, 1,0)while the
lowest yield of 1.067% was obtained at conditions of (P=200 bar, T=350C and F=35 g/min)
corresponding to the level of (-1,-1,0)
%Yp= -2.02 - 0.00703 P + 0.009 T + 0.070 F + 0.000025 P*P - 0.00050 T*T - 0.00277 F*F
+ 0.000097 P*T + 0.000286 P*F + 0.00185 T*F eqn 6
The R2 value of the model was found to be 99.47% thereby indicating the highly precise
predictive function of the developed model. The adjusted R2 value of the model was found to
be 98.51% which illustrates the fact that 98.51% of the variation in the yield was due to the
independent variables and only 1.49% of the variation was due to other factors which could
not be explained by the model.
The overall relative error between the observed yield (Yo) and predicted yield (Yp) was found
to be 4.39 % thereby indicating efficiency and preciseness of the response model that was
carried out.
41
4.2.2 STATISTICAL ANALYSIS OF DESIGN
The statistical analysis of the BBD experiment was carried out MINITAB18 software. The
model depicted high F-value (103.62), a low p-value (0.00032) which signify that the
developed quadratic model was highly significant in nature .The high p-value of lack of fit of
the model (0.3875<p<0.05) illustrated the highly precise fit for the model and high proximity
between observed and predicted value. All the linear terms in the model showed a high F-value
and a very low p-value (<0.05) which translates into high significance of the linear terms in the
developed model. The quadratic terms of Pressure, Temperature and Flow rate were found to
have high F-value and a low p-value (<0.05) which illustrates the fact that the quadratic terms
of the independent variables also had a highly significant contribution towards the developed
model. There was a significant interaction between pressure-temperature (0.00012<p<0.05)
and pressure-flowrate (0.0058<p<0.05) towards the yield while the interaction between
flowrate-temperature had an insignificant effect towards the yield which is demonstrated in its
high p-value (0.05<p<0.156).The analysis is clearly illustrated in table 8
Analysis of Variance
42
Error 5 0.2438 0.04876
Total 14 143.6528
Table 8: Analysis of variance of the developed fit
The analysis of contour and surface plots are often employed to understand the trend of
interaction between the dependent and independent variable. All the interactions between two
independent variables and the response was plotted by keeping the third variable fixed at ‘0
level. The effect of temperature and flowrate on the yield is illustrated in (Fig 9.a).The yield
was found to increase with increase in either flowrate or temperature or with increase in both
temperature and flowrate. The increase in yield with increasing temperature is attributed to
increasing fluid density for extraction at fixed pressure .The increase in yield due to increase
in flowrate is due to increase in fluid availability for extraction which results in improved solute
dissolution into the supercritical fluid. Temperature range between 450C-550C and flowrate
between 35g/min -45g/min was found to be most optimal for carrying out the extraction
Figure (Fig 9.b) illustrates the effect of pressure and flowrate on the yield. The increase in
pressure and flowrate resulted in increased yield. The increase in yield due to increase in
pressure is attributed to higher penetrating effect of the solvent into the solid matrix at higher
pressure. Pressure above 350 bar and flowrate above 35g/min was found to have highest yields.
The effect of temperature and pressure is illustrated in (fig 9.c).Increase in pressure or
temperature or both led to an increase in the yield. Keeping the temperature constant, increase
in pressure from 200 bar (‘-1 level) to 400 bar (‘+1 level) resulted in an increase in yield by
almost three times, as mentioned in literature[27].The increase in yield due to increase in
pressure and temperature is due to increase in density of the extracting fluid which allows
higher solubility of solute into the extracting media.
43
Fig 10.a: Surface and contour plots showing effect of Temperature and Flowrate vs. Yield of chilli
44
Fig 10.b: Surface and contour plots showing effect of Pressure and temperature vs. Yield of chilli
45
Fig 10.c: Surface and contour plots showing effect of Pressure and Flowrate vs. Yield of chilli
46
4.2.4 DIFFUSIVITY STUDY FOR EXTRACTION
The diffusivity at the parameters resulting in highest yield (P=400 bar, T=550C and F=35g/min)
yield was modelled using Fick’s diffusion model to find the diffusivity of chilly extract into
the supercritical fluid. Fick’s law states that the flux is directly proportional to the concentration
gradient with diffusion taking place in the direction of decreasing concentration.
𝝏𝟐 𝑪 𝝏𝑪
= 𝝏𝒕 eqn 3
𝝏𝒓𝟐
By applying the initial and boundary conditions, the solution of the above equation is given as:
𝟔 𝑫𝒆 𝒕
C*=∑∞
𝒏=𝟏 (𝒏𝝅)𝟐 𝒆𝒙𝒑 {−(𝒏𝝅)
𝟐
} eqn 4
𝒓𝟐
𝐶−𝐶0
Where C*= with 𝐶 , 𝐶0 and 𝐶∞ being the average solute concentration in the solvent,
𝐶0 −𝐶∞
the total solute present in the feed and the solute concentration in the solvent at infinite time
respectively.
𝐷 𝑡
For Fourier number (F0= 𝑟𝑒2 ) greater than 0.1, all the higher terms of the equation can be ignored
𝟔 𝑫𝒆 𝒕
C*=∑∞
𝒏=𝟏 𝒆𝒙𝒑 {−(𝝅)𝟐 } eqn 5
𝝅𝟐 𝒓𝟐
In our experimentation at the mentioned parameters, the time run was carried out for 6 hrs and
the extract was collected after every one hour. The total % yield at end of 6 hrs. (𝐶∞ ) was found
to be 9.17%.The initial amount of solute in the sample (𝐶0 ) is generally taken as the total solute
that can be extracted using Soxhlet extraction. In our case the value was found to be 10.98%.
By plotting graph of ln(C*) vs. Time (fig.10) the value of diffusivity was found to be 1.221*10-
10
m2/s
47
Chart Title y = -0.0004x + 8.0356
R² = 0.9482
8
6
ln(C*{π2/6})
0
0 2000 4000 6000 8000 10000 12000 14000 16000 18000 20000
TIME (S)
The comparison of effective diffusivity of chilli with other compounds is shown in table 8.
SC-CO2 +
Coffee Beans 15-25 313-333 (3 - 7) ×10-12
Water
Guaraná
SC-CO2 20 328 0.46×10-14
seeds
Byadgi
SC-CO2 40 333 1.221*10-10
chilli
Table 9: Comparision of effective diffusivity of Guggul and Chilli to those in literature
48
The diffusivity of chili pigments were found to be higher than most of the compounds shown
in table which signifies the effectiveness of supercritical CO2 in extracting color pigments from
chili powder.
The color value of the extracted oleoresin and pigment concentration of capsanthin and
capsorubin were analyzed using the spectrophotometric methods mentioned earlier and the
results are shown in table 9 .The highest color value of 114985 was obtained corresponding to
P=400 bar, T=550C, F=35g/min while the lowest color value was obtained corresponding to
P=200 bar, T=350C, F=35g/min.The same results were obtained for pigment concentration with
highest pigment concentration of 89.81 g/Kg at operational level of (P=400 bar, T=550C,
F=35g/min) and the lowest pigment concentration of 42.66 g/Kg at operational level of (P=200
bar, T=350C, F=35g/min).The highest oleoresin yield was obtained at (+1,+1,0) level while the
highest color value and pigment concentration was obtained at (+1,-1,0).This demonstrates the
fact that increase in temperature increases the oleoresin yield but at the same time brings down
the color value and pigment concentration. This can be attributed to the fact that increase in
temperature leads to thermal degradation of red pigments (capsanthin and capsorubin) as
supported by [28]. On the contrary, increase in pressure and flowrate resulted in increase in the
color value and pigment concentration. An increase in pressure from 200 bar to 400 bar keeping
the temperature and flow rate fixed at 350C and 35g/min respectively, increased the color value
and pigment concentration from 53802 and 42.667 g/Kg to 114985 and 89.81 g/Kg
respectively. This demonstrates that the color value and pigment concentration almost doubled
when pressure was doubled from 200 bar to 400 bar.
49
Pigment
P(bar) T(0C) F(g/kg) concentration(g/Kg) Color Value
Table 10: Variation of Color value and Pigment concentration according to BBD parameters
50
CHAPTER 5
SUMMARY
5.1 GUGGUL
The current optimization experiment showed that the highest yield was obtained at high
temperature and pressure with medium flow rate (P=400 bar, T=550C and F= 35gm/min),The
selectivity towards extraction of E-guggulsterone was more at moderate operating
parameters(P=300 bar, T=450C and F= 35gm/min) that led to 3.84% of E-guggulsterone in
the extract. The Box Behnken design showed all the parameters except the quadratic term of
pressure and the interactive term of flow and temperature had very high significance on the
yield. The extract obtained using SCFE showed high guggulsterone content than solvent
extraction which makes SCFE as a very viable alternative for solvent extraction.
51
CHAPTER 6
REFERENCE
1) Kooi-Yeong Khaw, Marie-Odile Parat, Paul Nicholas Shaw and James Robert Falconer,
Solvent Supercritical Fluid Technologies to Extract Bioactive Compounds from Natural
Sources: A Review, Molecules 2017, 22, 1186
2) M.D. Luque de Castro, L.E. GarcõÂa-Ayuso, Soxhlet extraction of solid materials: an
outdated technique with a promising innovative future, Analytica Chimica Acta 369 (1998)
1-10.
3) Somashekhar IC, Dr. JK Raju and Dr. Hema Patil, The role of information in enhancing
the agribusiness supply chain performance: A case study of dry chilli, International Journal
of Applied Research 2016; 2(12): 586-593
4) Navarro J.M., Flores P., Garrido C., Martinez V. (2006), Changes in the contents of
antioxidant compounds in pepper fruits at different ripening stages, as affected by salinity,
Food Chemistry 96, 66–73.
5) Ravishankar G.A., Suresh B., Giridhar P., Ramachandra Rao S., Sudhakar Johnson T.
(2003), Biotechnological studies on capsicum for metabolites production and plant
improvement, Capsicums: 97-122.
6) Weissenberg M., Schaeffler, I.L., Menagem, E., Barzilai, M, Levy, A. 1997, Isocratic non-
aqueous reversed high performance liquid chromatographic separation of capsanthin and
capsorubin in red peppers (capsicum annuum L.), paprika and oleoresin. J. Chromatog,
656: 89 95. www.zarc.com/english/cap-stun/ tech_ info /oc/
7) S.M. Ghoreishi, E. Heidari, Extraction of epigallocatechin gallate from green tea via
modified supercritical CO2: Experimental, modeling and optimization, J. of Supercritical
Fluids 72 (2012) 36– 45
8) Code of Federal Regulations][Title 21, Volume 3][Revised as of April 1, 2018][CITE:
21CFR184.1240]
9) Prerna Sarup, Suman Bala, and Sunil Kamboj, Pharmacology and Phytochemistry of Oleo-
Gum Resin of Commiphora wightii (Guggul), Scientifica, Volume 2015, Article ID 138039
10) Syed Ghulam Musharraf, Naveed Iqbal, Muhammad Arif Ahmed, Shumiala Mazhar, and
Muhammad Iqbal Choudhary, Screening of E and Z-Guggulsterones in the Gum-Resin
Exudates of some common plants and method validation in Raw, Extracted and
Pharmaceutical Formulations of Commiphora mukul by HPLC, Journal of Liquid
Chromatography & Related Technologies, 34:2103–2117, 2011
52
11) Gujral ML, Sareen K, Tangri KK, Amma MK, Roy AK, Antiarthritic and
anti‑inflammatory activity of gum guggul (Balsamodendron mukul), ). Indian J Physiol
Pharmacol 1960; 4:267‑73.
12) Francis JA, Raja SN, Nair MG, Bioactive terpenoids and guggulusteroids from
Commiphora mukul gum resin of potential anti‑inflammatory interest, Chem Biodivers
2004;1:1842‑53.
13) Allam AF, el‑Sayad MH, Khalil SS, Laboratory assessment of the molluscicidal activity of
Commiphora molmol (Myrrh) on Biomphalaria alexandrina, Bulinus truncatus and
Lymnaeacailliaudi, J Egypt Soc Parasitol 2001;31:683‑90.
14) Tripathi SN, Sastri VV, Satyavati GV, Experimental and clinical studies on the effects of
guggul (C. mukul) in hyperlipidemia and thrombosis, J Res Indian Med 1968; 2:10.
15) Satyavati GV, Dwarakanath C, Tripathi SN, Experimental studies on the
hypocholesterolemic effect of Commiphora mukul (Guggul), Indian J Med Res
1969;57:1950‑62.
16) Bombardelli et al, Commiphora mukul extracts and therapeutical applications thereof,
United States Patent 5,273,747, Dec. 28, 1993
17) Vladimir Badmaev, Muhammed Majeed, Barbara Pacchetti, Lakshmi Prakash,
Standardization of Commiphora mukul in Dislipidemia and Cardiovascular Disease,
MUTRA foods 2003 2(2) 45-51
18) Desikacharya Sampathu et al, Process of Extracting Chilli (Capsicum) Oleoresin, US
7,097.867 B2. Aug. 29, 2006
19) Jar_en-Gal_an, M., Nienaber, U., & Schwartz, S. J. (1999), Paprika (Capsicum annuum L.)
oleoresin extraction with supercritical carbon dioxide, Journal of Agriculture and Food
Chemistry, 47, 3558–3564.
20) Manuel Jare´n-Gala´n, Uwe Nienaber and Steven J. Schwartz, Paprika (Capsicum
annuum) Oleoresin Extraction with Supercritical Carbon Dioxide, J. Agric. Food Chem.
1999, 47, 3558, 3564
21) Ayurvedic Pharmacopoeia of India, Department of Indian Systems of Medicine and
Homeopathy, Ministry of Health and Family Welfare, Government of India, New Delhi,
India, 1st edition, 2001
22) N. L. Urizar and D. D.Moore, “Gugulipid: a natural cholesterollowering agent,” Annual
Review of Nutrition, vol. 23, pp. 303– 313, 2003.
23) FAO JECFA Monographs 5 (2008)
24) Verghese, J., Balakrishnan, K.V. & Kurian, T. (1992). Profile of quality determinants in
capsicum extract processing. Indian Spices, 29, 4–11.
53
25) Aguerre, R., Gabitto, J., Chirife, J., Utilization of Fick’s second law for the evaluation of
diffusion coefficients in food processes controlled by internal diffusion, International
Journal of Food Science & Technology, 20, 623-629 (1985).
26) Gadkari, P.V., Balaraman, M, Extraction of catechins from decaffeinated green tea for
development of nanoemulsion using palm oil and sunflower oil based lipid carrier systems,
Journal of Food Engineering, 147, 14-23(2015).
27) Jar_en-Gal_an, M., Nienaber, U., & Schwartz, S. J. (1999), Paprika (Capsicum annuum L.)
oleoresin extraction with supercritical carbon dioxide, Journal of Agriculture and Food
Chemistry, 47, 3558–3564.
28) Effat I. Seliem, Khaled F. Mahmoud, Azza A. Amin and Manal F. Salama, Extraction of
oleoresin by supercritical fluid extraction, capsulation and its physiochemical properties,
Int.J.Curr.Res.Aca.Rev.2015; 3(5):1-11
54