Optimization of Extraction Parameters for Natural Dye from Pterocarpus santalinus by using Response Surface Methodology

Hemanthraj.K.P.M et al. Int. Journal of Engineering Research and Applications www.ijera.com
ISSN : 2248-9622, Vol. 4, Issue 9( Version 3), September 2014, pp.100-108
www.ijera.com 100 | P a g e
Optimization of Extraction Parameters for Natural Dye from Pterocarpus santalinus by using Response Surface Methodology Hemanthraj.K.P.M1, Sudhanva.M.Desai1, Surendra Singh Bisht2. 1Department of Chemical Engineering, Dayananda Sagar College of Engineering, Bangalore, India. 2Chemistry of Forest Products Division, Institute of Wood Science and Technology, Malleshwaram, Bangalore, India. Abstract: - Pterocarpus species has been admired for centuries for its dye, beautiful color, hardness and durability. The present study deals with the extraction of natural dye from Pterocarpus wood materials. Response surface methodology was used to study the optimal conditions for the extraction of dye. Factors such as extraction temperature, extraction time, and solid to liquid ratio were identified to be significantly affecting natural dye extraction efficiency. By using three-level three-factor Box-Behnken design, the optimized conditions for dye extraction by different techniques such as Solvent, Ultrasonic and Microwave extraction method. Microwave assisted extraction method showed the highest natural dye yield percentage which is 50.0 for ethyl acetate solvent and 50.2 for methanol solvent.
Keywords: Pterocarpus species, Natural dye, Response surface methodology.
I. INTRODUCTION:
A natural dye is a substance derived from natural sources used to add a color to or change the color of something and considered as sustainable and eco- friendly [1-4]. Pterocarpus species wood is renowned for its characteristic timber of exquisite color, beauty and superlative technical qualities yielding a natural dye santalin belong to the molecular class of condensed bioflavonoid. Extraction of bioactive compounds is influenced by various process parameters such as solvent composition, pH, temperature, extraction time and solid to liquid ratio [5, 6].
Solvent extraction is a common form of chemical extraction using organic solvents e.g. hexane, ethyl acetate, ether, chloroform, benzene, ethanol, methanol etc. It is commonly used in combination with other technologies such as solidification/stabilization, precipitation and electro winning. Another typical method called Ultrasonic- assisted extraction (UAE) is a process of high extraction yields of good quality in shorter periods of time using lower amounts of solvent than traditional processes. Among the new extraction techniques, UAE is the most economical and the one with less instrumental requirements. Different plant extracts and bioactive metabolites have been obtained with this technique. Microwave assisted extraction (MAE) is a relatively latest extraction techniques which utilizes microwave energy to heat the solvent and the sample to increase the mass transfer rate of the solutes from the sample matrix into the solvent. Microwave extraction offers better selectivity less extractant use and lower energy input efficiently. MAE offers a rapid delivery of energy to a total volume of solvent and solid target matrix with subsequent heating of the solvent and solid matrix, efficiently and homogeneously. Response surface methodology (RSM) used to provide superb statistical tools for design and analysis of experiments aimed at process optimization [7]. Design of Experiments (DOE) deals many RSM designs with options depend on the number of design variables or factors, which can range from one to ten offering Box-Behnken designs (BBD) for three to seven factors require only three levels, coded as −1, 0, and +1 creating designs with desirable statistical properties. The objective of the present study is to provide an overview of existing research studies on extraction of natural dye from Pterocarpus spp. with aid of Response surface methodology.
II. MATERIALS AND METHODS
2.1 Materials: Sawdust sample were produced from Pterocarpus santalinus wood collected from Chemistry of Forest Products and Wood Properties and Engineered Wood at Institute of Wood Science and Technology, Bangalore. The HPLC grade solvents (Ethyl acetate and Methanol) used for extracting metabolites were purchased from Merck Specialties Pvt. Ltd. and HiMediaLaboratories Pvt. Ltd. Mumbai. The instrument Buchi Rota Vapor used for evaporation.
RESEARCH ARTICLE OPEN ACCESS

2.2 Methods 2.2.1 Batch Solvent Extraction method (BSEM): 1g of the wood powder was mixed with various volumes of ethyl acetate solvent and methanol respectively to give a solid to liquid ratio ranging from 1:50 to 1:300(g/mL).The flask containing sample powder along with solvent was incubated in thermostatic water bath at various temperatures (30–60∘C) and various time intervals (15–300min). Observe the change in color within solvent and filtering it into filtrate and residue. Extract containing colored filtrate is subjected to evaporation in rotavapor at 60°C of water bath temperature. Metabolite extract is remained in the RB is weighed and transferred to ependoffs for the analysis. 2.2.2 Ultrasonic Assisted Extraction (UAE): 1g of the wood powder was mixed with various volumes of ethyl acetate and methanol solvent respectively to give a solid to liquid ratio ranging from 1:50 to 1:250(g/mL) and allowed for gentle mixing. The beaker was then placed into the ultrasonic bath aided with grill containing distilled water for extraction process. Parameter optimized was ultrasonic temperature for the range of 30-80oC and extraction time for 5-30 minutes. All extracts were filtered and evaporated in rotavapour at 60°C of water bath temperature and is dried to get solid sample, the weight was measured and transferred to ependoffs for the analysis and interpretation procedures. 2.2.3 Microwave Assisted Extraction (MAE): 1g of sawdust samples were sieved and was mixed with various volumes of ethyl acetate and methanol solvent respectively to give a solid to liquid ratio ranging from 1:50 to 1:250 (g/mL) and allowed for gentle mixing in container and placed in micro treatment chamber (oven) with variable extraction time for range 5-30 mins at ranging temperature of 30-60°C respectively and microwave power of 800W was used for the extraction work. Further it is filtered, filtrate was evaporated in rotary evaporator at 60°C of water bath temperature and is dried to get solid sample, and the weight was measured and transferred to ependoffs for the analysis and interpretation procedures. 2.2.4. Statistical screening and optimization design of experiments:
A Box-Behnken model for three factors or variables was adopted in this study as the experimental design model using Design Expert software (version 6.0.8.Stat Ease Inc., Minneapolis, MN, USA). This method is preferred as design model since relatively few combinations of the variables are adequate to estimate potentially complex response function. In total 17 experiments are needed in each sample of individual solvent to calculate its 10 coefficients of the second order polynomial equation which was, fitted on the experimental data. Percentage recovery of dye was taken as response of the system while the three process parameter i.e., temperature, extraction time and solid to liquid ratio were taken as input independent variables with respect to Solvent, Microwave and Ultrasonic extraction methods. The system was stated by the following equation:
푌 = 푏0+ 푏1A+ 푏2B+ 푏3C+ 푏11A2+ 푏22B2+ 푏33C2+ 푏12AB+ 푏13AC+ 푏23BC Where 푏0 is the intercept; 푏1, 푏2, and 푏3 are linear coefficients; 푏11, 푏22, and 푏33 are squared coefficients; 푏12, 푏13, and 푏23 are interaction coefficients and the experimental variables are temperature (A), Extraction time (B) and Solid to liquid ratio (C).The model adequacies were checked in terms of the values of 푅2 and Analysis of variance (ANOVA) was engaged to determine the significance of the models [9].
III. RESULTS AND DISCUSSION
3.1. Box-Behnken Analysis: In this study, BBD was used for three process variables (extraction temperature, extraction time, and solid to liquid ratio) at three levels. The design points fall within a safe operational limit, within the nominal high and low levels, as BBD does not contain any points at the vertices of the cubic region. Design arrangements and responses of Experimental and Predicted values of Ethyl acetate (EtOAc) and Methanol (MeOH) solvents for Solvent Extraction method, Ultrasonic assisted extraction and Microwave assisted extraction method were generated are given in Table No.1. 3.2. Statistical Analysis: Multiple regression analysis of the data yielded, the following equation for the recovery of natural dye using Ethyl acetate and Methanol recovery of Batch Solvent Extraction Method in terms of coded factors:
Y1 = +25.72- 0.46* A+ 0.17* B+ 2.01* C+ 0.65* A2+ 0.23* B2+ 0.15* C2- 0.35* A * B- 0.22* A * C- 0.70* B * C Y2 = +34.46- 0.76* A+ 0.55* B+ 2.41* C+ 0.82* A2+ 0.70* B2+ 0.57*C2- 0.17* A * B- 0.95* A * C- 0.13* B * C

The following equation for the recovery of natural dye using Ethyl acetate and Methanol recovery of Ultrasonic extraction method in terms of coded factors:
Y1= +29.50+0.038*A+2.81*B+2.15*C-0.14*A2- 3.34*B2+ 0.44*C2- 0.28*A*B- 0.55*A*C- 1.00*B* C Y2= +39.52+0.038* A+2.08 * B+1.51* C-0.22* A2- 2.40* B2+0.78* C2+0.15* A * B+0.33* A * C-1.50* B * C The following equation for the recovery of natural dye using Ethyl acetate and Methanol recovery of Microwave extraction method in terms of coded factors:
Y1=+44.70+0.36*A+1.38*B+1.29*C+1.64*A2- 0.59*B2+1.44*C2+0.050*A*B-0.68*A*C- 0.100*B*C Y2=+49.70+0.73*A+1.35*B+1.07*C+0.012*A2- 1.69*B2-0.29*C2+0.075*A*B-0.63*A*C- 0.025*B*C Where Y1 is Ethyl acetate response variable and Y2 is Methanol response variable. The student t- distribution and the corresponding p-values along with the f-values of EtOAc and MeOH responses are listed in Table No.2 respectively. In ANOVA for Response Surface Quadratic Model of Ethyl Acetate recovery of Batch Solvent Extraction method, the Model F-value of 4.36 implies the model is significant and there is only a 3.26% chance that a "Model F-Value" this large could occur due to noise. Values of "Prob > F" less than 0.0500 indicate model terms are significant. In this case Solid to Liquid feed ratio (C) are significant model terms. Values greater than 0.1000 indicate the model terms are not significant. The "Lack of Fit F-value" of 1.58 implies the Lack of Fit is not significant relative to the pure error. The values for the coefficient of determination 푅2=0.8486 and Adjusted 푅2 =0.6539 represents the proportion of variation in the yield or response in the model. A negative Pred R-Squared= -0.4215 implies that the overall mean is a better predictor of obtained response than the current model.
Whereas ANOVA for Response Surface Quadratic Model of Ethyl Acetate recovery of Ultrasonic extraction method, the Model F-value of 3.34 implies there is a 6.28% chance that a "Model F- Value" this large could occur due to noise. Values of "Prob > F" less than 0.0500 indicate model terms are significant. In this case Extraction time, Solid to Liquid (Feed) ratio and Squared Extraction time coefficients are significant model terms. The values for the coefficient of determination 푅2=0.5687 and Adjusted 푅2 =0.8113 represents the proportion of variation in the yield or response in the model. A negative Pred R-Squared=-2.0188 implies that the overall mean is a better predictor of obtained response than the current model. In case of Microwave extraction method the Model F-value of 2.87 implies there is an 8.94% chance that a "Model F-Value" this large could occur due to noise. Values of "Prob > F" less than 0.0500 indicate model terms are significant. In this case Extraction time and Solid to liquid (feed) ratio are significant model terms. The values for the coefficient of determination 푅2= 0.7866 and Adjusted 푅2 = 0.5122 represents the proportion of variation in the yield or response in the model. A negative Pred R-Squared= -2.4147 implies that the overall mean is a better predictor of obtained response than the current model. In ANOVA for Response Surface Quadratic Model of Methanol recovery of Batch Solvent Extraction method, the Model F-value of 18.21 implies the model is significant. There is only a 0.05% chance that a "Model F-Value" this large could occur due to noise. Values of "Prob>F" less than 0.0500 indicate model terms are significant. In this case Extraction temperature, Extraction time, Solid to liquid (Feed) ratio, squared temperature coefficient and Temperature-Solid to liquid interaction coefficients are significant model terms. The "Lack of Fit F-value" of 0.56 implies the Lack of Fit is not significant relative to the pure error. The values for the coefficient of determination 푅2= 0.9590 and adjusted 푅2 = 0.9064 represents the proportion of variation in the yield or response in the model. The "Pred R-Squared" of 0.7602 is in reasonable agreement with the "Adj R-Squared" of 0.9064. Whereas ANOVA for Response Surface Quadratic Model of Methanol recovery, the Model F- value of 2.98 implies there is an 8.22% chance that a " A negative Pred R-Squared= -1.3706 implies that the overall mean is a better predictor of obtained response than the current model and ANOVA for Response Surface Quadratic Model of Methanol recovery, the Model F-value of 4.94 implies the model is significant. There is only a 2.34% chance that a "Model F-Value" this large could occur due to noise. Values of "Prob > F" less than 0.0500 indicate model terms are significant. In this case Extraction time, Solid to liquid ration and squared Extraction time coefficients are significant model terms.

3.3. Plot Response Surface Design
To visualize the relationship between
response and experimental levels of the independent
variables for the natural dye extraction, three
dimensional (3D) surface plots were constructed
according to the quadratic polynomial model
equation.
The variation of Ethyl acetate recovery and
Methanol recovery with Extraction time and
Temperature are graphically presented in above Fig
No.1. As the Extraction time of both solvents
increases and Temperature were decreased, the
Natural dye recovery increased significantly with
curvature contour lines.
DESIGN-EXPERT Plot
Ethyl acetate recovery
X = A: TEMPERATURE
Y = B: EXTRACTION TIME
Actual Factor
C: FEED RATIO = 150.00
25.6346
26.1228
26.611
27.0993
27.5875
30.00
37.50
45.00
52.50
60.00
15.00
86.25
157.50
228.75
300.00
A: TEMPERATURE
B: EXTRACTION TIME
DESIGN-EXPERT Plot
Methanol recovery
X = A: TEMPERATURE
Actual Factor
34.2027
35.0177
35.8326
36.6476
37.4625
Methanol recovery
30.00
37.50
45.00
52.50
60.00
15.00
86.25
157.50
228.75
300.00
A: TEMPERATURE
B: EXTRACTION TIME
Fig No.1: Extraction time Vs Temperature of
Batch Solvent Extraction Method for Sample
.
DESIGN-EXPERT Plot
X = A: TEMPERATURE
Actual Factor
22.9
24.701
26.5021
28.3031
30.1042
30.00
42.50
55.00
67.50
80.00
5.00
11.25
17.50
23.75
30.00
A: TEMPERATURE
B: EXTRACTION TIME
DESIGN-EXPERT Plot
Methanol recovery
X = A: TEMPERATURE
Actual Factor
34.7125
36.0296
37.3466
38.6637
39.9808
Methanol recovery
30.00
42.50
55.00
67.50
80.00
5.00
11.25
17.50
23.75
30.00
A: TEMPERATURE
B: EXTRACTION TIME
Ultrasonic Assisted Extraction Method for Sample
temperature are graphically presented in above Fig
increases and temperature were decreased, the
DESIGN-EXPERT Plot
X = A: TEMPERATURE
Actual Factor
42.7226
43.9263
45.1301
46.3338
47.5375
30.00
37.50
45.00
52.50
60.00
5.00
11.25
17.50
23.75
30.00
A: TEMPERATURE
B: EXTRACTION TIME
DESIGN-EXPERT Plot
Methanol recovery
X = A: TEMPERATURE
Actual Factor
46.025
47.2033
48.3816
49.5598
50.7381
Methanol recovery
30.00
37.50
45.00
52.50
60.00
5.00
11.25
17.50
23.75
30.00
A: TEMPERATURE
B: EXTRACTION TIME
Microwave Assisted Extraction Method for
Sample

Table No.1: Box-Behnken Design arrangement and Responses for Batch Solvent, Ultrasonic Assisted and Microwave extraction method Exp EtOAc: Experimental Ethyl acetate, Pred EtOAc: Predicted Ethyl acetate, Exp MeOH: Experimental Methanol, Pred MeOH: Predicted Methanol. Table No. 2: ANOVA for Response Surface Quadratic Model of Ethyl Acetate and Methanol Recovery

.
DESIGN-EXPERT Plot
X = A: TEMPERATURE
Actual Factor
22.9
24.701
26.5021
28.3031
30.1042
30.00
42.50
55.00
67.50
80.00
5.00
11.25
17.50
23.75
30.00
A: TEMPERATURE
B: EXTRACTION TIME
DESIGN-EXPERT Plot
Methanol recovery
X = A: TEMPERATURE
Actual Factor
34.7125
36.0296
37.3466
38.6637
39.9808
Methanol recovery
30.00
42.50
55.00
67.50
80.00
5.00
11.25
17.50
23.75
30.00
A: TEMPERATURE
B: EXTRACTION TIME
Ultrasonic Assisted Extraction Method for Sample
DESIGN-EXPERT Plot
X = A: TEMPERATURE
Actual Factor
42.7226
43.9263
45.1301
46.3338
47.5375
30.00
37.50
45.00
52.50
60.00
5.00
11.25
17.50
23.75
30.00
A: TEMPERATURE
B: EXTRACTION TIME
DESIGN-EXPERT Plot
Methanol recovery
X = A: TEMPERATURE
Actual Factor
46.025
47.2033
48.3816
49.5598
50.7381
Methanol recovery
30.00
37.50
45.00
52.50
60.00
5.00
11.25
17.50
23.75
30.00
A: TEMPERATURE
B: EXTRACTION TIME
Microwave Assisted Extraction Method for
Sample
curvature contour lines
DESIGN-EXPERT Plot
X = A: TEMPERATURE
Y = C: FEED RATIO
Actual Factor
B: EXTRACTION TIME = 157.50
23.8385
25.1852
26.5318
27.8784
29.225
30.00
37.50
45.00
52.50
60.00
50.00
100.00
150.00
200.00
250.00
A: TEMPERATURE
C: FEED RATIO
DESIGN-EXPERT Plot
Methanol recovery
X = A: TEMPERATURE
Y = C: FEED RATIO
Actual Factor
32.607
34.449
36.291
38.133
39.975
Methanol recovery
30.00
37.50
45.00
52.50
60.00
50.00
100.00
150.00
200.00
250.00
A: TEMPERATURE
C: FEED RATIO
Fig No.4: Feed ratio Vs Temperature of Batch
Solvent Extraction Method for Sample
Methanol recovery with Solid to Liquid Feed ratio

and Temperature are graphically presented in above
Fig No.4. As the Solid to Liquid Feed ratio of both
solvents increases and Temperature were decreased,
the Natural dye recovery increased significantly with
and temperature are graphically presented in above
solvents increases and temperature were decreased,
the Natural dye recovery increased non-significantly
with linear contour lines.
DESIGN-EXPERT Plot
X = A: TEMPERATURE
Y = C: FEED RATIO
Actual Factor
27.0625
28.4125
29.7625
31.1125
32.4625
30.00
42.50
55.00
67.50
80.00
50.00
100.00
150.00
200.00
250.00
A: TEMPERATURE
C: FEED RATIO
DESIGN-EXPERT Plot
Methanol recovery
X = A: TEMPERATURE
Y = C: FEED RATIO
Actual Factor
38.275
39.1956
40.1163
41.0369
41.9576
Methanol recovery
30.00
42.50
55.00
67.50
80.00
50.00
100.00
150.00
200.00
250.00
A: TEMPERATURE
C: FEED RATIO
Fig No.5: Feed ratio Vs Temperature of
Ultrasonic assisted Extraction Method for Sample
DESIGN-EXPERT Plot
X = A: TEMPERATURE
Y = C: FEED RATIO
Actual Factor
44.3411
45.5996
46.8581
48.1165
49.375
30.00
37.50
45.00
52.50
60.00
50.00
100.00
150.00
200.00
250.00
A: TEMPERATURE
C: FEED RATIO
DESIGN-EXPERT Plot
Methanol recovery
X = A: TEMPERATURE
Y = C: FEED RATIO
Actual Factor
47
47.9034
48.8068
49.7101
50.6135
Methanol recovery
30.00
37.50
45.00
52.50
60.00
50.00
100.00
150.00
200.00
250.00
A: TEMPERATURE
C: FEED RATIO
Fig No.6: Feed ratio Vs Temperature of
Microwave assisted Extraction Method for
Sample
and temperature are graphically presented in above
solvents increases and temperature were increased,
the Natural dye recovery increased significantly with
DESIGN-EXPERT Plot
X = B: EXTRACTION TIME
Y = C: FEED RATIO
Actual Factor
A: TEMPERATURE = 45.00
23.2125
24.5688
25.925
27.2813
28.6375
15.00
86.25
157.50
228.75
300.00
50.00
100.00
150.00
200.00
250.00
B: EXTRACTION TIME
C: FEED RATIO
DESIGN-EXPERT Plot
Methanol recovery
Y = C: FEED RATIO
Actual Factor
32.4538
33.9809
35.5081
37.0353
38.5625
Methanol recovery
15.00
86.25
157.50
228.75
300.00
50.00
100.00
150.00
200.00
250.00
B: EXTRACTION TIME
C: FEED RATIO
Fig No.7: Feed ratio Vs Extraction time of Batch
Solvent Extraction Method for Sample

and Extraction Time are graphically presented in
above Fig No.7. As the Solid to Liquid Feed ratio of
both solvents increases and Extraction Time were
decreased, the Natural dye recovery increased
significantly with curvature contour lines.
DESIGN-EXPERT Plot
Y = C: FEED RATIO
Actual Factor
20.6375
23.5615
26.4855
29.4095
32.3335
5.00
11.25
17.50
23.75
30.00
50.00
100.00
150.00
200.00
250.00
B: EXTRACTION TIME
C: FEED RATIO
DESIGN-EXPERT Plot
Methanol recovery
Y = C: FEED RATIO
Actual Factor
32.8125
35.0704
37.3283
39.5862
41.844
Methanol recovery
5.00
11.25
17.50
23.75
30.00
50.00
100.00
150.00
200.00
250.00
B: EXTRACTION TIME
C: FEED RATIO
Fig No.8: Feed ratio Vs Extraction time of
ultrasonic Assisted Extraction Method for Sample
and Extraction Time are graphically presented in
above Fig No.8. As the Solid to Liquid Feed ratio of
both solvents increases and Extraction Time were
decreased, the Natural dye recovery increased
DESIGN-EXPERT Plot
Y = C: FEED RATIO
Actual Factor
42.4031
43.8304
45.2578
46.6851
48.1125
5.00
11.25
17.50
23.75
30.00
50.00
100.00
150.00
200.00
250.00
B: EXTRACTION TIME
C: FEED RATIO
DESIGN-EXPERT Plot
Methanol recovery
Y = C: FEED RATIO
Actual Factor
45.275
46.6431
48.0113
49.3794
50.7475
Methanol recovery
5.00
11.25
17.50
23.75
30.00
50.00
100.00
150.00
200.00
250.00
B: EXTRACTION TIME
C: FEED RATIO
Fig No.9: Feed ratio Vs Extraction time of
Microwave assisted Extraction Method for
Sample
The variation of Ethyl acetate recovery and Methanol
recovery with Solid to Liquid Feed ratio and
Extraction Time are graphically presented in above
Fig No.4.9.As the Solid to Liquid Feed ratio of both
solvents increases and Extraction Time were
increased, the Natural dye recovery increased
3.4. Verification of Optimized Condition and
Predictive Model
Optimization requires goals to be set for the
variables and response where all goals then get
combined into one desirability function. To find a
good set of conditions that will meet all the goals, the
three variables extraction temperature extraction
time, solid to liquid, were set within range while
ethyl acetate and methanol recovery was set at
maximum. For response, the “importance” was set at
5 in order to meet the objective of getting maximum
recovery. By applying the desirability function
approach, the optimum level of various parameters
was obtained as showed in Table No.3 provided with
experimental values for obtained optimal conditions.

Table No. 3: Optimum conditions and the predicted and experimental value of responses at the optimum conditions.
Extraction methods
Responses
Temperature (°C)
Extraction Time (min)
Feed Ratio (1gm/ml)
EtOAc recovery (%)
MeOH recovery (%)
Batch solvent
Predicted
31.01
57.57
249.67
29.3351
39.6676
Experimental
31
57
250
29.10
39.50
Ultrasonic Assisted
Predicted
58.69
21.01
249.41
32.2277
41.8215
Experimental
59
21
249
32.0
42.0
Microwave Assisted
Predicted
30.00
23.84
250.00
49.845
50.6
Experimental
30.0
24.0
250.00
50.0
50.2
IV. CONCLUSION
In the present study, response surface methodology was used to optimize the Solvent, Ultrasonic and Microwave assisted extraction of natural dye from Pterocarpus santalinus wood. Box- Behnken design was used to determine the optimum process parameters and the multiple regression analysis for predicting responses were obtained. Under optimum condition Microwave assisted extraction method showed the highest natural dye yield percentage which is 50.0 for ethyl acetate solvent and 50.2 for methanol solvent. Microwave assisted extraction method dictates the quality, economics and environmental impact of any processing plant. It shows a highly promising future with drastic reduction in extraction time resulting in higher sample throughput without significant losses in analyte recovery. In this study among three parameters Solid to liquid ratio for Solvent extraction method, Extraction time for Ultrasonic extraction method and extraction time and solid to liquid for microwave assisted extraction method is found to be most prominent factor affecting the efficiency of dye extraction.
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Optimization of Extraction Parameters for Natural Dye from Pterocarpus santalinus by using Response Surface Methodology

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