Extraction of essential oils from Mentha spicata L. (Labiatae) via optimized supercritical carbon dioxide process

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Extraction of essential oils from Mentha spicata L. (Labiatae) via optimized supercritical carbon dioxide process M. Shahsavarpour a , M. Lashkarbolooki b,∗ , Mohammad Javad Eftekhari c , F. Esmaeilzadeh a a

Chemical and Petroleum Engineering Department, School of Engineering, Shiraz University, Shiraz, Iran School of Chemical Engineering, Babol Noshirvani University of Technology, Babol, Iran c Department of Petroleum Engineering, Science and Research Branch, Islamic Azad University, Iran b

a r t i c l e

i n f o

Article history: Received 1 December 2016 Received in revised form 5 February 2017 Available online xxx Keywords: Extraction process Supercritical carbon dioxide Spearmint oil Experimental data

a b s t r a c t Spearmint oil known as aromatic volatile compound of plants are widely used in food industries. In the present study, supercritical carbon dioxide (SC-CO2 ) extraction is used to extract the essential oil from spearmint leaves. In this way, a series of extraction experiments are designed to investigate the effect of different operating conditions such as extraction pressure (85–120 bar), extraction temperature (38–50 ◦ C), CO2 flow rate (0.059–0.354 g/min), mean particle size (0.177–2 mm) and dynamic extraction time (20–120 min) on efficiency of the extracted essential oils. In the last step, not only a second order polynomial equation is developed to obtain the optimal SC-CO2 extraction parameters, but also the composition of extracted essential oil at optimum operating condition is analyzed using gas chromatography-mass spectrometry (GC–MS). © 2017 Elsevier B.V. All rights reserved.

1. Introduction Spearmint oils are widely used in food, cosmetic, perfumes, beverage, pharmaceuticals and tobacco industries [1–5]. Essential oils such as spearmint oils are generally complex mixtures of monoterpenes, hydrocarbon sesquiterpenes, oxygenated monoterpenes and sesquiterpenes, and compounds derived from the secondary metabolism of plants [5,6]. One of the most common methods for extraction of spearmint oil from plant is steam distillation known as a direct leaching and rectification process [7–9]. Although steam distillation leads to a solvent-less product, it suffers two main disadvantages such as being both time and energy consuming process which also the used heat may partially hydrolyze some essential oil components and thermally degrade them. As a replacement for steam distillation, hexane-based solvent extraction is another common method leads to higher yields during oil extraction. The main advantage of this method is its mild operational conditions compared with steam distillation needs no high temperature, although solvent contamination face the safety and efficiency of this method with some extend of uncertainties. In total, not only nature of hexane extraction method is faster com-

∗ Corresponding author. E-mail address: [email protected] (M. Lashkarbolooki).

pered to steam distillation but also it is possible to manipulate the efficiency of the hexane extraction process using temperature, solvent type and the ratio of sample to solvent with more flexibility compared to steam distillation [4,10]. In the light of aforementioned shortcomings and in the light of necessity to new and efficient extraction methods, supercritical fluid-based extraction (SFE) methods are proposed during the last three decades to its unique advantages. The main characteristic of supercritical-based technologies is producing high quality solventfree extracted oil [11–15]. Regarding the advantages of supercritical carbon dioxide (SCCO2 ) [16–18], extraction of essential oils from a large number of seeds, leaves, etc. such as the peel of citrus fruits [19,20]; Teucrium polium L. [21]; corn germ [22]; cottonseed [23,24]; peanut [24]; soybean [24,25]; Juniperus communis L. [25]; caraway (Carum carvi L.) [26]; lavender [27]; sage dried leaves [28]; Kadsura oblongifolia Merr. leaves [29]; jojoba [30]; Ocimum gratissimum L. [31]; sunflower [32]; Lippia alba (Mill.) N.E. brown leaf [33]; celery [34]; palm kernels [35]; peach almond [36]; rosemary leaves [37,38]; black pepper [39,40]; red pepper [41]; oil from some Mexican spices [42]; Pimenta dioica Merrill. leaf [43]; Citrus medica cv. diamante [44]; tuberose [45]; Echium amoenum seed [3]; Trinia glauca (L.) Dumort. (Apiaceae) [46]; savory and dragonhead [47]; peppermint oil [4,9,48]; Moringa oleifera kernels [48] and spearmint leaf [49] are examined.

http://dx.doi.org/10.1016/j.supflu.2017.02.004 0896-8446/© 2017 Elsevier B.V. All rights reserved.

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In the shadow of the aforementioned facts, the main objective of this investigation is aimed to study and enlighten the effects of various parameters including extraction pressures (85–120 bar), extraction temperature (38–50 ◦ C), CO2 flow rates (0.059–0.354 g/min), mean particle sizes (0.177–2 mm) and dynamic extraction times (20–120 min) on the extraction of essential oils from spearmint leaves. The spearmint leaves are selected to extract its essential oil due to its unique application in different areas. The uses of spearmint oil extend beyond the daily applications and the medicine shelf including aromatherapy, food ingredient, fragrance, ingredient in pharmaceutical products, bath oil, massage oil and insecticide. In more details, for example, because of its menthol content, spearmint oil is often used in aromatherapy to help alleviate fatigue, headaches, migraines, nervousness, and even digestive problems. In addition, spearmint with its antispasmodic properties, spearmint oil can help relieve muscle pain and even abdominal pain due to menstruation [50,51].

2. Experimental section 2.1. Material The spearmint, Mentha Spicata L. widely grown in Shiraz, Iran, is an abundantly growing Labiatae species in wet places. It is a morphologically very variable kind can easily distinguish from other members of the genus Mentha by its characteristic inflorescence. The plant from the mint-family is perennial aromatic herbs and their leaves are used as the main part for extraction of essential oils. In this regard, the leaves of M. Spicata L. with moisture content of 13% grown in Shiraz-Iran were used as the model herb in the current investigation. In addition, the used CO2 was supplied from Oxi Gas Company, Shiraz-Iran (purity > 99%).

2.2. Experimental apparatus The schematic of the used apparatus is shown in Fig. 1. In brief, CO2 is inserted (A) into liquefaction section (C). Then, an air driven oil-free reciprocating pump (E) (Haskel pump) pressurized the liquefied CO2 and push it into the surge tank (G) where the pressure fluctuations is dampen. To prevent entrance of any particles into the manual pump a 5 ␮m filter is just installed before the manual pump. The pressurized carbon dioxide is then flown into a ¼¨stainless steel coil which emerged in a warm bath (F). At this time the SC-CO2 with desired pressure and temperature is passed through the bed (contained 30 g of dried leaves) which packed with packings to avoid channeling phenomenon inside the column of the dried leaves (J). Besides, the entrance and exit ports of the extraction vessel was covered with glass wool to prevent any entrainment of dried leaves into extracted essential oil. The temperature of the extraction column was controlled using a circulating hot water bath wrapped the extraction column. The measurements revealed that the temperature inside the water bath regulated within ±0.5 ◦ C using an immerged heating element with a PID controller coupled with a PT-100 thermocouple. The pressure of each vessel was monitored and controlled by a pressure-gauge (I) (Indumart, EN 837-1, Canada) with division of 1 bar in the range of (0–250) bar, and a pressure transmitter (P) (Wika, S-10, Germany) with an accuracy of 0.1 bar mounted on the top of the surge tank. The pressure of the system was kept constant at the desired pressure within ± 0.2 bar during each experiment. For all of the experiments, after 45 min static time which the system was kept under desired temperature and pressure, a heated fine needle valve (K) was opened and the SC-CO2 dissolved the desired essential oils was allowed to expand to atmospheric pressure. During the extraction process, after giving the static time to the system, a very accurate amount of SC-CO2 flow rate was passed

Fig 1. Schematic diagram of the extraction of essential oil from peppermint with supercritical carbon dioxide: A, CO2 cylinder; B, filter; C, condenser; D, check valve; E, high-pressure pump; F, warm baths; G, surge tank; H, relief valve; I, pressure gauge; J, extraction columns; K, fine needle valve; L, ice bath; M, U-tube separator; N, wet test meter; O, bubble flow meter; P, pressure transmitter; Q, water saturator; and (mv1 to mv14) stop valves.

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Table 1 Decoded and coded independent variables used in optimization. Symbol

Independent variable (decoded value)

Factor levels X1 X2 X3 X4 X5

Temperature (◦ C) Pressure (bar) Solvent flow rate (g/min) Particle diameter (mm) Time (min)

coded values (38, 45, 50) (85, 100, 120) (0.059, 0.177, 0.354) (0.177, 0.5, 2) (20, 40, 60, 90, 120)

through the packed bed at the constant pressure using a metering vale and nitrogen capsule which inertly kept the pressure inside the extraction vessel constant. Finally, the extracted essential oil was precipitated and collected in a U-tube separator (M) submerged in an ice-bath (L). The point must be mentioned is that to avoid the Joule-Thomson effect and consequent clogging of the fine needle value due to sudden expansion of the SC-CO2 , The valve was heated using an element coupled with a K-type thermocouple. Finally, the extracted essential oil was weighed by an analytical balance (Sartorius, BA110S, Germany) with an accuracy of 10−4 g. Finally, the liberated gas was passed through a water saturator (Q) and wet test meter (N) (supplied from MFD, Precision Scientific Co., USA) with accuracy of 28.32 cm3 to measure the total volume of solvent used in each run. The last point must be considered is that, to clearly demonstrate the efficiency of each experiment, a parameter named yield was used. The yields can evaluate gravimetrically using the following equation:

-1 -1 -1 -1 -1

-0.6

0.1667 -0.1428 -0.2 -0.6456 -0.2

0.4

+1 +1 +1 +1 +1

Fig. 2. Extraction rate with effect of static time on yield at 40 ◦ C, 100 bar, 0.5 mm particle size and 0.177 g/min CO2 flow rate.

3. Results and discussion Yield = (g essential oil/g feed) × 100

(1)

2.3. Optimization method The design of experiments of this study was performed with the assist of the genetic algorithm method of the MATLAB toolbox to obtain the optimum values of temperature, pressure, solvent flow rate, particle diameter and dynamic extraction time to increase the yield of extracted essential oil. The coded and decoded independent variables used in genetic algorithm are shown in Table 1 [52]. For convenience of notation and solving for the coefficients in the matrix, actual Xi variables were coded as −1 to +1 (Table 1).

2.3.1. Statistical analysis In addition, multiple regressions to fit the second order equation to all dependent variables was used to statistically analyze the results of 405 experimental data points obtained during the extraction process [53].

3.1. Optimum static time In the first series of the experiments, the effect of static time on the extraction efficiency of essential oil was investigated by changing the static time between 15 and 60 min while the other operational conditions, including extraction temperature (40 ◦ C), extraction pressure (100 bar), particle size (0.5 mm) and CO2 flow rate (0.177 g/min) were held constant (see Fig. 2). By a glance in Fig. 2, it can be concluded that despite an increase in the static time from 15 min to 45 min leads to an increase in the efficiency of extraction while further increase in the static time from 45 min to 60 min introduce no considerable change in the efficiency of the extraction process. Regarding these findings, 45 min was selected as the optimum static time for extraction process in the remained experiments. This observed trend can be related to this hypothesis that higher contact time between SC-CO2 and grinded leaves leads to higher extraction efficiency due to better penetration of SC-CO2 into the leaves matrix and extraction of essential oils. 3.2. Effect of different parameters

2.4. Analytical analysis In the current investigation, a gas chromatography-mass spectrometry (Varian Saturn Model 3400, Sun Fernando, USA), connected to an Ion Trap Detector, operated at an electron energy of 70 eV. A DB-1 fused-silica column (60 m, 0.25 mm, 0.25 ␮m film thickness) was used to detect the components of the extracted essential oil. The injected sample volume was 1 ␮m. Oven temperature was programmed at 50 ◦ C to 270 ◦ C at 4 ◦ C/min. The column was used with carrier gas, helium (purity >99.99 mol%) and flow rate of 4 ml/min with injection temperature of 280 ◦ C. Mass spectra were recorded from 40 to 420 mass units with a scan time of 5 ␮s. The sample components were identified by a mass spectra matching with Wiley5 and NIST90 Registry Mass Spectra Data Bases, when possible the identification was verified by comparison of retention index and mass spectrum with a reference compound.

In the next series of the experiments, the effects of other parameters including extraction pressure and temperature, particle size, and SC-CO2 flow rate were examined while the static time was kept constant at 45 min. Regarding this purpose, the effect of extraction pressures of 85, 100 and 120 bar; CO2 flow rates of 0.059, 0.177, 0.354 g/min; extraction temperatures of 38, 45 and 50 ◦ C and the mean particle sizes of 0.177, 0.5 and 2 mm were investigated using a total number of 405 different extraction experiments (see Table 2)The obtained results revealed that among the examined operational parameters, extraction temperature and pressure introduced the most significant effects on the extraction yield. Typically, Fig. 3 shows the concomitant effect of dynamic time and pressure on the yield% for the case of 0.354 g/min carbon dioxide flow rate and mean particle size of 2 mm. The obtained results revealed that the dynamic time and extraction pressure has a direct relation. In more details, for all of the studied isobars, an increases

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Table 2 The yield% of essential oil extracted of from Iranian spearmint leaves. Condition

Time (min)

Q = 0.059 g/min

Q = 0.177 g/min

Q = 0.354 g/min

Particle diameter (mm)

Particle diameter (mm)

Particle diameter (mm)

2

0.5

0.177

2

0.5

0.177

2

0.5

0.177

T = 50 ◦ C P = 120 bar

20 40 60 90 120

0.036 0.091 0.130 0.163 0.188

0.043 0.103 0.156 0.194 0.226

0.047 0.118 0.180 0.224 0.256

0.071 0.186 0.260 0.294 0.325

0.094 0.197 0.255 0.310 0.348

0.100 0.209 0.284 0.355 0.379

0.110 0.182 0.234 0.273 0.294

0.114 0.192 0.217 0.277 0.312

0.130 0.232 0.286 0.317 0.340

T = 50 ◦ C P = 100 bar

20 40 60 90 120

0.035 0.078 0.115 0.142 0.166

0.047 0.096 0.132 0.156 0.172

0.043 0.100 0.140 0.161 0.179

0.061 0.127 0.185 0.225 0.248

0.103 0.224 0.282 0.328 0.348

0.101 0.228 0.281 0.322 0.340

0.062 0.112 0.136 0.160 0.170

0.044 0.100 0.140 0.179 0.195

0.078 0.144 0.195 0.225 0.251

T = 50 ◦ C P = 85 bar

20 40 60 90 120

0.032 0.097 0.121 0.144 0.156

0.017 0.040 0.057 0.069 0.078

0.016 0.034 0.048 0.059 0.068

0.054 0.130 0.158 0.189 0.205

0.038 0.083 0.117 0.149 0.173

0.034 0.080 0.102 0.117 0.130

0.057 0.096 0.128 0.152 0.158

0.036 0.097 0.114 0.126 0.129

0.031 0.054 0.071 0.083 0.087

T = 45 ◦ C P = 120 bar

20 40 60 90 120

0.053 0.142 0.206 0.257 0.296

0.096 0.205 0.281 0.346 0.382

0.091 0.214 0.295 0.366 0.407

0.131 0.287 0.356 0.399 0.436

0.098 0.288 0.403 0.492 0.521

0.128 0.293 0.413 0.499 0.536

0.108 0.204 0.283 0.348 0.393

0.149 0.270 0.359 0.420 0.448

0.164 0.302 0.395 0.432 0.461

T = 45 ◦ C P = 100 bar

20 40 60 90 120

0.068 0.141 0.199 0.248 0.281

0.044 0.143 0.214 0.279 0.341

0.075 0.188 0.265 0.328 0.361

0.079 0.217 0.321 0.397 0.429

0.109 0.253 0.344 0.423 0.468

0.115 0.259 0.360 0.429 0.471

0.142 0.332 0.385 0.389 0.391

0.241 0.302 0.359 0.398 0.434

0.144 0.252 0.330 0.393 0.424

T = 45 ◦ C P = 85 bar

20 40 60 90 120

0.049 0.101 0.144 0.168 0.187

0.031 0.062 0.087 0.104 0.118

0.021 0.044 0.065 0.081 0.093

0.058 0.151 0.224 0.279 0.312

0.059 0.133 0.187 0.223 0.241

0.036 0.081 0.108 0.129 0.144

0.077 0.142 0.189 0.194 0.196

0.049 0.087 0.118 0.128 0.131

0.046 0.076 0.093 0.099 0.101

T = 38 ◦ C P = 120 bar

20 40 60 90 120

0.062 0.143 0.203 0.258 0.283

0.056 0.147 0.201 0.255 0.292

0.067 0.140 0.204 0.259 0.299

0.098 0.206 0.302 0.373 0.413

0.069 0.263 0.382 0.455 0.483

0.114 0.257 0.377 0.469 0.499

0.106 0.174 0.233 0.279 0.303

0.115 0.191 0.249 0.291 0.316

0.116 0.213 0.263 0.299 0.328

T = 38 ◦ C P = 100 bar

20 40 60 90 120

0.073 0.147 0.196 0.244 0.266

0.056 0.126 0.179 0.229 0.277

0.071 0.146 0.196 0.239 0.279

0.096 0.209 0.312 0.367 0.390

0.069 0.247 0.337 0.402 0.435

0.074 0.252 0.332 0.404 0.441

0.097 0.166 0.221 0.265 0.269

0.091 0.161 0.219 0.258 0.283

0.103 0.178 0.232 0.258 0.278

T = 38 ◦ C P = 85 bar

20 40 60 90 120

0.046 0.098 0.143 0.183 0.207

0.039 0.083 0.114 0.141 0.159

0.021 0.057 0.082 0.098 0.106

0.077 0.200 0.259 0.313 0.339

0.057 0.137 0.206 0.255 0.289

0.087 0.175 0.225 0.259 0.285

0.091 0.152 0.197 0.227 0.231

0.057 0.097 0.133 0.156 0.174

0.025 0.065 0.085 0.099 0.105

in the dynamic time lads to a considerable enhancement in the extraction efficiency (see Fig. 3). In addition, the effect of pressure on the extraction yield% for 0.177 g/min carbon dioxide flow rate and 0.5 mm mean particle size at extraction time of 120 min and three studied temperature are shown in Fig. 4. A similar trend was reported by Toribio et al. [54] for the extraction of lipids using supercritical carbon dioxide. They have reported that the amount of lipids extracted increased with the dynamic extraction time. Their analysis revealed that the amount of the extract obtained in each fraction of 30 min is about 70% of the lipids were extracted, and after 150 min of extraction, the percentage of remaining lipids were lower than 1.0%. In the next stage of this investigation, the effect of extraction temperature on the extraction efficiency of the essential oil was investigated. A close examination in Figs. 4 and 5 revealed that the extraction of essential oil of spearmint from leaves in SC-CO2 shows a complex relation between the extraction time, tempera-

ture and pressure. In more details, it can be seen from Fig. 4 that at the constant pressure of 85 bar, an increase in the extraction temperature can reduce the extraction time. This observed trend can be related to this fact that as the temperature increases, the density of SC-CO2 which is directly related to its solvating power reduces which means lower solubility of essential oils in SC-CO2 . A similar trend was observed by Nemoto et al. [55] for extraction of 88 Pesticides regarding the aforementioned relation between the temperature and efficiency. But, the amazing point is that as the extraction pressure increases to the pressures higher than 85 bar, the effect of extraction temperature on the extraction efficiency becomes more complex. In more details. For extraction pressure of 100 and 120 bar, as the extraction temperature increases from 35 ◦ C to 45 ◦ C, the extraction efficiency increases while further increases in the extraction temperature from 45 ◦ C to 50 ◦ C leads to a reduction in the extraction efficiency. This observed trend can be related to the dual effect of extraction temperature. In details, an increase

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Table 3 Obtained coefficients of second order polynomial model for supercritical fluid extraction. Coefficient

Optimum Value

Coefficient

Optimum Value

Coefficient

Optimum Value

a0 a1 a2 a3 a4 a5 a11

0.3609 −0.0280 0.0619 −0.0252 −0.0252 0.1024 −0.0612

a12 a13 a14 a15 a22 a23 a24

0.0060 −0.0045 −0.0045 −0.0142 −0.0480 −0.0112 −0.0262

a25 a33 a34 a35 a44 a45 a55

0.0333 −0.0704 −0.0005 −0.0009 −0.0010 −0.0012 −0.0010

Fig. 3. Effect of pressure on the extraction yield% for carbon dioxide flow rate of 0.354 g/min, mean particle size of 2 mm and temperatures of 38 and 50 ◦ C.

Fig. 6. Effect of temperature and CO2 flow rate on the extraction yield for pressure of 120 bar, mean particle size of 0.5 mm and extraction time of 120 min.

Fig. 7. Effect of CO2 flow rate on the extraction yield% for temperature of 50 ◦ C, mean particle size of 0.5 mm and pressure of 85 and 120 bar. Fig. 4. Effect of pressure and temperature on the extraction yield for 0.177 g/min carbon dioxide flow rate, 0.5 mm mean particle size and extraction time of 120 min.

Fig. 5. Effect of temperature on the extraction yield% for carbon dioxide flow rate of 0.354 g/min, mean particle size of 0.177 mm and pressure of 120 bar.

in the extraction temperature can reduce the density of SC-CO2 consequently reduces the solavating power as aforementioned, but on the other hand the extraction temperature enhancement also may enhance the sublimation pressure of the essential oils (see Table 3). In other words, it is the net effect of these two competing factors dictates the solubility enhances or reduces. Similar trends are reported by hezave et al. [56,57] regarding the solubility of cyproheptadine and fluoxetine hydrochloride. Moreover, similar to the results obtained for vegetable oils and seed due to the retrograde crystallization phenomenon of solubility at higher pressures (100 and 120 bar) for the CO2 flow rate of 0.177 g/min and mean particle size of 0.177 mm, the extraction yield obtained at the end of the extraction period was about 1.07 fold when the temperature increased from 38 to 45 ◦ C (see Table 3) [58–60]. In the next stage, the effect of the SC-CO2 flow rate on the extraction yield was examined (see Figs. 6–8). A close examination in the obtained results revealed that increasing the CO2 flow rate from 0.059 to 0.177 g/min for all the examined mean particle sizes of 0.177, 0.5 and 2 mm leads to an increase in the extraction efficiency

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Fig. 10. Effect of mean particle size on the extraction yield% for temperature of 45 ◦ C, CO2 flow rate of 0.177 g/min and pressure of 85 and 120 bar.

Fig. 8. Effect of CO2 flow rate on the extraction yield for pressure of 120 bar, temperature of 50 ◦ C, extraction time of 120 min and the studied mean particle size.

Fig. 9. Effect of mean particle size on the extraction yield for pressure of 100 bar, CO2 flow rate of 0.059 g/min, extraction time of 120 min and three studied temperature.

while further increase in flow rate from 0.177 to 0.354 g/min leads to a reduction in the extraction yield. The observed trend can be related to the dual behavior of the carbon dioxide flow rate. In more details, the mass transfer coefficient increases with SC-CO2 flow rate enhancement which consequently decreases the mass transfer resistance, because of an increase in convection. On the other hand, Nemoto et al. [55] has reported that a lower flow rate resulted in a lower linear velocity and usually increases the extraction efficiency as a result of an extended contact between the supercritical fluid and the analytes. Moreover, a lower flow rate increases the trapping efficiency of analytes at the analyte trap. This is the net effect of these competing factors dictates the flow rate leads to an increase or decrease of extraction efficiency. The obtained results for the effect of particle size are shown in Figs. 8–10. As can be seen, although the highest extraction yield was related to the particles with lower mean size especially for temperature of 50 ◦ C, the obtained trend was rather more complex compared with the other examined parameters. The worth mentioning point is that the impact of particle size on the yield of extraction was completely obvious after the first 60 min. In total, smaller particles leads to higher yields for extraction pressure of 100 and 120 bar. In addition, there was an inverse trend between the particle size and the extraction yield for low extraction pressure such as 85 bar which is near to the critical pressure where the

controlling parameter is solubility. In addition, the other hypothesis behind the lower extraction efficiency of larger particles can be related to this fact that for larger particles, the majority of internal part of the leaves is not efficiently contacted by the SC-CO2 and can partially participate in extraction process. So it seems that if enough static or dynamic time introduces into the system the equal extraction efficiency obtains for all the examined mean particle sizes. On the other hand, for large particles, the amount of the destructed cells on the particle surface is insignificant if compared to the intact cells while for the grinded leaves, higher damage to the herb cells would be observed. In addition, lower particle size may produce bed caking with the formation of channels along the extraction bed in which the supercritical solvent can just pass through the bed without sufficient contact between the particles and SC-CO2 therefore decreasing the extraction efficiency. The other possibility can raise from this fact that the segregation of leaves or seeds in fractions with different oil contents may be a common occurrence in supercritical extraction experiments. For example, del Valle and Uquiche [61] have reported that the integral yield of oil approached an asymptotic value that was dependent on the particle size of the substrate: 57.1 g oil/kg dry oil-free substrate (large particles), 171.0 g/kg (medium-size particles), or 391.5 g/kg (small particles). In addition, they have explained that based on gravimetric determinations and microscopic analysis, their sizeclassification process segregated seed parts having different oil contents. Particles ≥0.85 mm were mainly composed of tough, lignified testa fragments devoid of oil, whereas particles ≤0.425 mm contained mostly brittle, oil-rich germ fragments. The segregation of seed in fractions with different oil contents may be a common occurrence in supercritical extraction experiments, especially for seeds with thick and/or hard testa and small germ, whose fractions can be separated by sieving [61]. 3.3. Optimization of supercritical fluid extraction As previously mentioned, in the current study a second order polynomial model was applied to express the yield as a function of independent variables: Y = a0 + a1 × X 1 + a2 × X 2 + a3 × X 3 + a4 × X 4 + a5 × X 5 + a11 × X 1 × X 1 + a12 × X 1 × X2 + a13 × X 1 × X3 + a14 × X 1 × X4 + a15 × X 1 × X5 + a22 × X 2 × X 2 + a23 × X 2 × X3 + a24 × X 2 × X4 + a25 × X 2 × X5 + a33 × X3 × X3 + a34 × X 3 × X4 + a35 × X 3 × X5 + a44 × X 4 × X 4 + a45 × X 4 × X5 + a55 × X 5 × X 5

(2)

Where Y is the dependent variables (yield), X as the independent variables (temperature, pressure, solvent flow rate, particle diam-

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Table 4 Calculated coefficients of second order polynomial model for supercritical fluid extraction. Factor levels

Value

◦

−0.2258 0.9849 −0.2518 −0.9217 0.7987

X1 X2 X3 X4 X5 a

Independent variable Temperature ( C) Pressure (bar) Solvent flow rate (g/min) Particle diameter (mm) Time (min)

Optimized conditionsa 42.6 119.7 0.169 0.248 109.9

At the optimum conditions, the yield of essential oil would be 0.4894%.

eter, dynamic extraction time) are the constant variables of the proposed model. The proposed model was coupled with multiple regression analysis to find its constants and regression coefficients. These constants and regression coefficients are given in Table 3. Correlation coefficients (r2 ) and mean square error (MSE) are 0.9540 and 0.0016, respectively. The r2 and MSE are defined with the following equations, respectively:

2 1  exp . Yi − Yical. N N

MSE =

(3)

i N  

r2 =

exp .

Yi

i

−Y

2

N  

−

N   i

exp . Yi

exp .

Yi

−Y

− Yical.

2 (4)

2

i exp .

Where N is the number of experimental data points, Yi experimental value of the yield,

Yical. is

is the ith

the yield predicted with the

GA and Y is the average value of the experimental yield data. Table 3 The worth mentioning point is that the quantity (yield) of essential was considered as the objective function of this modeling process to find the optimum values of the operating conditions. The optimum values for the independent variables of temperature, pressure, CO2 flow rate, mean particle diameter and dynamic extraction time calculated by the genetic algorithm method are tabulated in Table 4. 3.4. Extracted compound at optimum conditions As the last examination, the extracted essential oils were analyzed by a gas chromatography-mass spectrometry (Varian Saturn Model 3400, Sun Fernando, USA). The results revealed that the extracted essential oil is significantly comprised of Carvon (45.964%), Pulegone (13.893%) and Limonene (12.807%) (see Table 5).

Table 5 Chemical composition (%) of extracted oil at optimum conditions. No

Name of Compound

Percentage

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

␣- Thujene Camphene Sabinene ␤- Pinene Myrcene Limonene 1,8- Cineole (Z)- ␤- Ocimene (E)- ␤- Ocimene Cis- Sabinene Hydrate Borneol Isopulegone ␣- Terpineol Dihydro Carvone Pulegone Carvone Piperitenone ␤- Bourbonene ␤- Caryophyllene ␣- Humulene Germacrene D Bicyclogermacrene

0.609 0.156 0.605 0.912 0.586 12.807 7.777 0.153 0.073 0.082 0.924 0.785 0.251 3.440 13.893 45.964 1.841 1.263 5.515 0.725 1.241 0.408

increase of this parameter leads to a reduction in extraction yield. In the second stage of this investigation, a second order polynomial equation was developed to mathematically obtain the optimal SCF extraction parameters. The obtained results revealed that the optimum conditions using polynomial equation were 42.6 ◦ C and 119.7 bar for extraction temperature and pressure, respectively, 0.169 g/min for CO2 flow rate and, 0.248 mm for mean particle size and 109.9 min for static time of extraction process. Finally, the composition of the extracted components of the optimum operating condition was analyzed by GC–MS which revealed that the extracted oil is comprises of mostly Carvon (45.964%), Pulegone (13.893%) and Limonene (12.807%).

References 4. Conclusion The extraction of essential oil (Mentha Spicata Leaves) using SCCO2 based extraction technology was used to find the optimum operational conditions including extraction temperature and pressure, CO2 flow rate, static time and mean particle sizes using yield as the objective function. Although obtained results revealed a direct relation between the extraction pressure and extraction yield, a more complex trend was observed for extraction temperature since this parameter can introduce dual effect on the solubility of essential oils or other compounds by manipulating the density of the supercritical solvent and the sublimation pressure of the solutes. In other words, it can be concluded that a cross over pressure of 100 bar and cross over temperature of 45 ◦ C for extraction pressure of higher than 100 bar can be reported for the investigated herb. Also, the results demonstrated that an increase in the CO2 flow rate increased the extraction yield at the first stage while the further

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