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salt aqueous two-phase system
Separation and Purification Technology 132 (2014) 396–400
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Extraction and purification of chlorogenic acid from ramie (Boehmeria nivea L. Gaud) leaf using an ethanol/salt aqueous two-phase system Zhijian Tan a,b, Chaoyun Wang a,⇑, Yongjian Yi a, Hongying Wang a, Mao Li a, Wanlai Zhou a, Shiyong Tan a, Fenfang Li b a b
Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, Changsha 410205, China College of Chemistry and Chemical Engineering, Central South University, Changsha 410083, China
a r t i c l e
i n f o
Article history: Received 17 December 2013 Received in revised form 12 May 2014 Accepted 17 May 2014 Available online 11 June 2014 Keywords: Ramie Chlorogenic acid Aqueous two-phase system Purification
a b s t r a c t In this paper, an aqueous two-phase system (ATPS) based on ethanol/NaH2PO4 was used in separation and purification of chlorogenic acid (CGA) from ramie leaf. CGA was firstly extracted from dried ramie leaf powder by ethanol solution, then was added into ATPS for further purification. During the ATPS extraction procedure, the influencing parameters of type and concentration of salt, extraction temperature and pH were investigated. The experiments results demonstrated the CGA were extracted into alcohol-rich phase with impurities being extracted into salt-rich phase, the phase-forming components were recycled to reduce the cost after extraction. Furthermore, the thermodynamics and kinetics during the extraction procedure were simultaneously studied for the first time. This much easier, greener and cheaper alcohol/salt ATPS was more feasible for large-scale production. Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction Ramie (Boehmeria nivea L. Gaud) is a perennial bast fiber crop belonged to Urticaceae. It originated in China and more than 90% of the total yields are produced in China, so a popular name of ‘‘China grass’’ is called in the world [1,2]. Ramie is widely used as fiber resource, however its usage for pharmaceutical purpose is less studied. There are many medical ingredients in the ramie leaf and root, such as chlorogenic acid, total polyphenol, flavonoid and so on [3]. Chlorogenic acid (CGA) is a major medical ingredient in ramie leaf, which is also found in other plants such as Eucommia ulmoides. Oliver, Lonicera japonica. Thunb, Toraxacum mongolicum Hand. Mazz., etc. CGA has many medical effects of anti-oxidant [4], anti-diabetic, anti-lipidemic [5], reducing liver inflammation and fibrosis [6], protecting human umbilical vein endothelial cells from the toxic damage [7], etc. ATPS was first introduced by Albertson for partitioning macromolecules and cell particles [8]. The most classical two types of ATPS are PEG/dextran and PEG/salt, however the high cost and high viscosity resulting in difficulties in separating products from polymer have restricted their implication [9,10]. In recent years, ATPS based on small molecular water-miscible organic solvent and salt is a type of liquid–liquid extraction technique, it has many
⇑ Corresponding author. Tel./fax: +86 731 88998501. E-mail address: [email protected] (C. Wang). http://dx.doi.org/10.1016/j.seppur.2014.05.048 1383-5866/Ó 2014 Elsevier B.V. All rights reserved.
advantages such as low cost and viscosity, easy recovery of phaseforming components and easy to scale-up in industrial production [11–14]. Alcohol/salt ATPS was applied to extract and purify lots of compounds such as anthraquinones derivatives from Aloe vera L. [11], isoflavonoids from Dalbergia odorifera T. Chen [15], anthocyanins from purple sweet potatoes [16], flavones and sugars from honeysuckle [17], lipase derived from Burkholderia cenocepacia ST8 [18], chloramphenicol in livestock meat samples [19], etc. In this paper, an ethanol/NaH2PO4 system was used in extraction and purification of CGA from ramie leaf. The affecting parameters of type and concentration of salt, pH and temperature were investigated in details. The whole flow chart of extraction and purification of CGA and recovery of alcohol and salt is shown in Fig. 1. 2. Materials and methods 2.1. Materials and reagents Ramie leaf, collected in Changsha Ramie Garden of Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences. The standard of chlorogenic acid (purity >98% by HPLC) was purchased from Staherb Natural Ingredient Co., Ltd. (Changsha, China). (NH4)2SO4, K3PO43H2O, NaH2PO42H2O, K2CO3, methanol and ethanol are of analytical grade was acquired from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Other reagents were all analytical grade and used without further treatment. De-ionized water was used to prepare the sample solutions.
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Z. Tan et al. / Separation and Purification Technology 132 (2014) 396–400 Evaporated and recycled Ramie leaf
CGA added
Dried, smashed Salt added
Homogenized
Centrifugated
Soaked, Concentraed
Alcohol-rich phase
CGA product
Salt-rich phase
CGA Alcohol solution
Alcohol salt ATPS
Homogeneous system
Evaporated and recycled
ATPS extraction procedure Methanol added
Ethanol molecules Salt molecules CGA molecules Methanol molecules
Precipitated and recycled
Fig. 1. Flow chart of extraction and purification of CGA using ethanol/salt ATPS.
2.2. Preparation of crude plant materials
K¼
Fresh ramie leaf was dried at 45 °C, smashed to powder by a grinder, the powder was sieved by a sieve with the particle size of 60 meshes (about 250 lm), then was soaked by 70% ethanol solution, an ultrasonic treatment was performed for 30 min. The mixture was filtrated, leaving behind the supernatant. After the supernatant being concentrated by a rotary evaporator, solid CGA was obtained and then dissolved by distilled water, set at 4 °C for further study.
Ct Cb
ð2Þ
where Ct and Cb are the CGA concentrations in the alcohol-rich phase and salt-rich phase, respectively. The extraction efficiency (E) of CGA in the alcohol-rich phase is determined from the Eq. (3):
E¼
K 100% K þ 1R
ð3Þ
2.5. Phase diagram 2.3. Preparation of ATPS To a 15 mL centrifuge tube, 5.0 mL distilled water, 2.0 mL ethanol, a given amount of salt and 0.5 mL CGA solution were added. Another tube with the same phase-forming components but without CGA was prepared as a blank to avoid interference. The mixture was stirred well to make the salt dissolve completely. Due to the incompatibility of alcohol and salt solution, in addition the low viscosity in system, the phase-separation can be achieved during a few seconds, two clear phases formed. In order to achieve complete phase-forming and extraction, usually a centrifugation procedure was performed. The top alcohol-rich phase was mainly composed of ethanol and CGA, and the bottom phase was the salt-rich solution containing the impurities such as polyphenols, flavonoids, metallic elements and proteins, etc. The volume of each phase was noted down. 2.4. Analysis of CGA The alcohol-rich phase was withdrawn to a tube using a syringe, diluted using methanol for the analysis of CGA concentration by a UV–Vis spectrophotometer (UV-2100, Unico, USA). The absorbance of CGA was measured at the wavelength of 326 nm. The calibration curve for analysis of CGA was Y = 0.0567X 0.0057 with R2 = 0.9997 (RSD = 0.046%, n = 5). Where Y is the absorbance, and X is the concentration of CGA in the range of 2.0–32 lg/mL. The CGA concentration in the salt-rich phase was determined by mass balance. The phase ratio (R) is defined as the Eq. (1):
R¼
Vt Vb
ð1Þ
where Vt and Vb are the volumes of alcohol-rich phase and salt-rich phase, respectively. The partition coefficient (K) is defined as the Eq. (2):
The phase diagrams were prepared by a turbidimetric titration method [20,21]. Firstly, ethanol of known mass was added into a centrifuge tube. Then, a salt solution of known mass fraction was added dropwise and the mixture was well mixed. The solution was clear at first, but after a certain amount of the salt solution being added, one further drop made the mixture turbid, then two phases formed. The mass fraction of each component was calculated. Finally, a few drops of water was added to make the mixture clear again, and the above procedure was repeated to obtain sufficient data to construct the phase diagrams. 3. Results and discussion 3.1. Selection of the optimal alcohol/salt ATPS In order to choose an optimal alcohol/salt ATPS, four kinds of organic alcohol (methanol, ethanol, 1-propanol and 2-propanol) and four kinds of inorganic salt (K3PO4, (NH4)2SO4, NaH2PO4, K2CO3) were considered as phase-forming component. Compared with methanol, 1-propanol and 2-propanol, ethanol has the advantages of lowest cost, almost no toxicity and moderate boiling point, which is more suitable for large-scale industrial production. So ethanol was chosen as the phase-forming alcohol. The phase diagrams of the ethanol/salt system was shown in Fig. 2, the abilities of four types of salt for phase separation were in an order of K3PO4 > K2CO3 > (NH4)2SO4 > NaH2PO4. To optimize the salt type and concentration, four ethanol/salt ATPSs were employed to extract CGA, and the results were shown in Table 1. The results showed that the acid systems formed by ethanol/NaH2PO4 and ethanol/(NH4)2SO4 have the better extraction ability for CGA. CGA is an acidic compound and is more stable in the acidic solution. Furthermore, ethanol/NaH2PO4 system has the relatively higher extraction efficiency than ethanol/(NH4)2SO4 system and was chosen for further studies. The optimal extraction
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Z. Tan et al. / Separation and Purification Technology 132 (2014) 396–400 Table 2 The transfer thermodynamic properties for CGA extraction from the salt-rich phase to the alcohol-rich phase in ATPS.
35
(NH4)2SO4 NaH2PO4
30
K2CO3
Salt (%, w/w)
25
K3PO4
20
T (k)
K
DG (kJ mol1)
TDS (kJ mol1)
DH (kJ mol1)
298.15 303.15 308.15 313.15 318.15
39.04 18.66 10.34 5.02 3.52
9.08 7.37 5.98 4.20 3.33
88.08 89.79 91.18 92.96 93.73
97.16
15 10
100 5
80 0
5
10
15
20
25
30
35
40
45
50
55
Ethanol (%, w/w)
60
Fig. 2. Phase diagrams for ethanol/salt ATPSs at room temperature (18 ± 2 °C).
40
20
3.2. Effect of temperature
0
/%
system was composed of 2.0 mL ethanol, 5 mL water and 3.8 g NaH2PO42H2O added (see Table 1).
E
The partitioning of CGA in ethanol/NaH2PO4 system at the temperature range of 25–45 °C was investigated with pH being not adjusted. The results were shown in Fig. 3 that when the temperature was 25 °C the extraction efficiency was maximum of 93.66%. Ethanol with low boiling easily volatilize at relative high temperature, so the extraction experiment could be just done at room temperature. The large scale production is more feasible by cutting down the cost of equipment and energy. 3.3. Effect of pH B–R (Britton–Robinson) buffer was used to adjust the pH of the aqueous solution of the range of 2.09–8.36. The pH was adjusted by mixing different solution of 0.04 M phosphoric acid, 0.04 M acetic acid, 0.04 M boric acid, and 0.20 M sodium hydroxide. The results were shown in Fig. 4 that the extraction efficiency varied little at pH 2.0–6.0 and the maximal extraction efficiency was obtained
45
40
K 35
Temperature/
30
25
Fig. 3. Extraction of CGA at different temperature.
at aqueous solution pH of 3.29. The optimal ethanol/NaH2PO4 system (2.0 mL ethanol, 5 mL water and 3.8 g NaH2PO42H2O) has a proper pH value of 3.90, the pH of ATPS extraction is unnecessary to be adjusted in the following experiments. 3.4. Thermodynamics and kinetics in extraction procedure To investigate the thermodynamics, extraction of CGA can be regarded as a transfer process of the CGA from the salt-rich phase to the alcohol-rich phase [22,23]. The Gibbs energy change (DG) is
Table 1 Extraction of CGA using different ethanol/salt ATPSs (each system contains 2.0 mL ethanol, 5.0 mL water, a given of salt and 0.5 mL crude CGA extract solution). Alcohol/salt system
Salt added (g)
Phase ratio (R)
Partition coefficient (K)
Extraction efficiency (E, %)
Ethanol/(NH4)2SO4
2.0 2.2 2.4 2.6 2.8
0.09 0.29 0.29 0.29 0.27
2.56 5.88 11.53 12.55 32.66
18.86 63.21 77.12 78.58 89.91
Ethanol/K2CO3
3.0 3.4 3.8 4.2 4.6
0.46 0.43 0.42 0.33 0.32
1.85 1.75 1.71 1.76 1.88
46.24 43.11 57.83 37.38 38.17
Ethanol/NaH2PO4
3.2 3.4 3.6 3.8 4.0
0.52 0.52 0.51 0.51 0.50
3.39 7.11 11.45 38.72 38.49
64.02 78.60 85.33 93.58 93.44
Ethanol/K3PO4
2.4 2.8 3.2 3.6 4.0
0.70 0.66 0.57 0.53 0.47
1.59 2.23 1.61 1.27 1.29
52.78 59.50 48.04 40.11 37.69
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Z. Tan et al. / Separation and Purification Technology 132 (2014) 396–400
80
syringe at different time after phase forming. The concentration of CGA in bottom phase was determined. Firstly we supposed that it is a first-order kinetics procedure. The kinetics equation Eq. (6) is as follow:
70
ln C A ¼ kt þ ln C o
60
where Co is the original concentration of CGA, CA is the concentration after the extraction time of t seconds in bottom phase, k is extraction rate constant. The CGA concentration was determined during the extraction procedure at different temperature and time. The extraction time of t is in the range of 20, 30, 40, 50 and 60 s. A straight line were obtained by plotting ln CA versus t, k values was the slope in Eq. (6), the results were shown in Table 3. The satisfactory correlation coefficient demonstrated extraction of CGA is a first-order kinetics.
100 90
50 40 30 20
/%
10 0
E
8
7
6
5
4
3
K 2
3.5. Comparison of different ATPSs in extraction of CGA
pH Fig. 4. Extraction of CGA at different pH and 298.15 K.
related to the partition coefficient (K) can be calculated by Eq. (4). The enthalpy change (DH) and entropy change (DS) were obtained from the slope and intercept of the linear Eq. (5) which plots ln K versus 1/T through Van’t Hoff approach. The obtained linear equation was ln K = 11686.29 1/T 35.58 with correlation coefficient R2 = 0.9895.
DG ¼ RT ln K ln K ¼
ð6Þ
ð4Þ
DH DS þ RT R
Extraction of CGA using PEG/salt, PEG/dextran and alcohol/salt ATPSs were studied. The phase-forming time of ATPS was recorded by visual observation. When the clear phase interface can be observed, we considered it as the phase-forming time. The viscosity of each ATPS was determined by a rotary viscosimeter (NDJ-5S, Shanghai Hengping). The results of phase-forming time, extraction efficiency and viscosity values in top and bottom phase were shown in Table 4. It can be seen that alcohol/salt ATPS has relative high extraction efficiency. Furthermore, compared with the PEG-based ATPSs, alcohol/salt system has shorter phase-forming time, smaller viscosity and lower cost, which is more potential for scale-up production.
ð5Þ 3.6. Recycle of phase-forming components
The DG, DH and DS values were calculated by Eqs. (4) and (5) are shown in Table 2. All the DG values are negative, reflecting that the extraction of CGA is spontaneous and preferential partitioning in the alcohol-rich phase. Partitioning of CGA in ATPS is marked by negative values for DH and TDS with the DH being greater in value than the DS. Thus it can be concluded that extraction of CGA form salt-rich phase to alcohol-rich phase is an exothermic, spontaneous process [24,25]. The extraction of CGA by alcoholbased ATPS is highly dependent on temperature. There are also some other researchers reported that temperature can greatly influence the partition of target materials in different types of ATPS [26–28]. In order to measure the kinetics in the extraction procedure, the tube was placed in the thermostat water bath at different temperature, the sample in bottom phase was quickly withdrawn by a
In order to reduce the cost in extraction of CGA using ethanol/ salt ATPS, we have tried to recycle the phase-forming components of ethanol and salt. When the extraction is finished, alcohol-rich phase and salt-rich phase were separated away from each other. Ethanol solution in top phase was recycled by evaporation and distillation, then processed by calcium oxide to obtain high pure ethanol. The salt in bottom phase can be recycled by dilution crystallization. After a certain amount of methanol being added into the salt solution with full stirring, NaH2PO4 can be precipitated out. Methanol has lowest boiling point and was easily recycled than other organic alcohols, which cannot easily form azeotrope with water and is to be evaporated out at relatively lower temperature [29]. To optimize the amount of methanol, 0.5–5 times volume of
Table 3 The kinetic equations of ATPS extraction of CGA at different temperature. Temperature (K) 298.15 308.15 318.15
Kinetic equation ln C = 0.0224t + 4.850 ln C = 0.0283t + 4.862 ln C = 0.0313t + 4.840
Reaction rate constant (k, s1) 2
2.24 10 2.83 102 3.13 102
Correlation coefficient (R2) 0.9792 0.9942 0.9914
Table 4 Comparison of phase-forming time, viscosity values and extraction efficiencies of different ATPSs. Type of ATPS (concentration %, w/w)
Phase-forming time (s)
Viscosity in top/bottom phase (mpa s)
Extraction efficiency (%)
PEG2000 (15%)/NaH2PO4 (25%) PEG6000 (15%)/NaH2PO4 (25%) PEG10000 (15%)/NaH2PO4 (25%) PEG6000 (15%)/Dextran 40,000 (6%) Ethanol (15.2%)/NaH2PO4 (28.1%)
120 ± 5 140 ± 5 170 ± 5 320 ± 5 18 ± 3
24.1 ± 0.3 (PEG-rich phase)/3.2 ± 0.3 (salt-rich phase) 35.7 ± 0.3 (PEG-rich phase)/4.5 ± 0.2 (salt-rich phase) 89.6 ± 0.5 (PEG-rich phase)/4.8 ± 0.3 (salt-rich phase) 26.5 ± 0.2 (PEG-rich phase)/124.5 ± 0.5 (Dextran-rich phase) 2.3 ± 0.2 (alcohol-rich phase)/1.5 ± 0.2 (salt-rich phase)
85.5 ± 0.3 87.4 ± 0.3 84.3 ± 0.2 75.9 ± 0.2 94.2 ± 0.3
(PEG-rich phase) (PEG-rich phase) (PEG-rich phase) (PEG-rich phase) (alcohol-rich phase)
400
Z. Tan et al. / Separation and Purification Technology 132 (2014) 396–400
100 90
Salt recovery ratio/ %
80 70 60 50 40 30 20 10 0 0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
Vmethanol added/ VSalt-rich phase Fig. 5. Recovery ratio of NaH2PO4 by dilution crystallization using methanol.
methanol was added into the salt solution. The results in Fig. 5 showed that when 1.5 times volume of methanol was added, the recovery ratio of NaH2PO4 reached to about 80%. To overall consider the cost, we think 1.5 times volume of methanol was enough for recovery of NaH2PO4. When CGA was extracted using the recycled salt, the extraction efficiency hardly decreased after recycling of 3 times. That is because the impurities dissolved in the methanol solution with the crystalline salt being not polluted. 4. Conclusion In this paper, an alcohol/salt ATPS was used to extract and purify CGA from ramie leaf by a single-step procedure. The optimized extraction conditions were as follow: ethanol/NaH2PO4 was chosen as the extraction system, the maximum extraction of 95.76% was obtained at 25 °C and aqueous solution pH of 3.29. In addition, the thermodynamics and kinetics was explored simultaneously in the extraction procedure for the first time, which can well interpret the extraction mechanism of alcohol/salt ATPS. Lastly, the phase-forming components were recycled, which can reduce the cost making this system more possible for large-scale production of CGA. Acknowledgements This work was financially supported by the earmarked fund for the China Agriculture Research System (Project No. CARS-19-08B). References [1] L.J. Liu, C.Y. Lao, N. Zhang, H.Q. Chen, G. Deng, C. Zhu, D.X. Peng, The effect of new continuous harvest technology of ramie (Boehmeria nivea L. Gaud.) on fiber yield and quality, Ind. Crop Prod. 44 (2013) 677–683. [2] L. Wang, T. Zhang, H. Yan, M. Peng, Z. Fang, Modification of ramie fabric with a metal-ion-doped flame-retardant coating, J. Appl. Polym. Sci. 129 (2013) 2986–2997. [3] S. Sancheti, S. Sancheti, M. Bafna, H.R. Kim, Y.H. You, S.Y. Seo, Evaluation of antidiabetic, antihyperlipidemic and antioxidant effects of Boehmeria nivea root extract in streptozotocin-induced diabetic rats, Revista Brasileira De Farmacognosia, Braz. J. Pharmacogn. 21 (2011) 146–154. [4] J.G. Xu, Q.P. Hu, Y. Liu, Antioxidant and DNA-protective activities of chlorogenic acid isomers, J. Agric. Food Chem. 60 (2012) 11625–11630.
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Separation and Purification Technology journal homepage: www.elsevier.com/locate/seppur
Extraction and purification of chlorogenic acid from ramie (Boehmeria nivea L. Gaud) leaf using an ethanol/salt aqueous two-phase system Zhijian Tan a,b, Chaoyun Wang a,⇑, Yongjian Yi a, Hongying Wang a, Mao Li a, Wanlai Zhou a, Shiyong Tan a, Fenfang Li b a b
Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, Changsha 410205, China College of Chemistry and Chemical Engineering, Central South University, Changsha 410083, China
a r t i c l e
i n f o
Article history: Received 17 December 2013 Received in revised form 12 May 2014 Accepted 17 May 2014 Available online 11 June 2014 Keywords: Ramie Chlorogenic acid Aqueous two-phase system Purification
a b s t r a c t In this paper, an aqueous two-phase system (ATPS) based on ethanol/NaH2PO4 was used in separation and purification of chlorogenic acid (CGA) from ramie leaf. CGA was firstly extracted from dried ramie leaf powder by ethanol solution, then was added into ATPS for further purification. During the ATPS extraction procedure, the influencing parameters of type and concentration of salt, extraction temperature and pH were investigated. The experiments results demonstrated the CGA were extracted into alcohol-rich phase with impurities being extracted into salt-rich phase, the phase-forming components were recycled to reduce the cost after extraction. Furthermore, the thermodynamics and kinetics during the extraction procedure were simultaneously studied for the first time. This much easier, greener and cheaper alcohol/salt ATPS was more feasible for large-scale production. Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction Ramie (Boehmeria nivea L. Gaud) is a perennial bast fiber crop belonged to Urticaceae. It originated in China and more than 90% of the total yields are produced in China, so a popular name of ‘‘China grass’’ is called in the world [1,2]. Ramie is widely used as fiber resource, however its usage for pharmaceutical purpose is less studied. There are many medical ingredients in the ramie leaf and root, such as chlorogenic acid, total polyphenol, flavonoid and so on [3]. Chlorogenic acid (CGA) is a major medical ingredient in ramie leaf, which is also found in other plants such as Eucommia ulmoides. Oliver, Lonicera japonica. Thunb, Toraxacum mongolicum Hand. Mazz., etc. CGA has many medical effects of anti-oxidant [4], anti-diabetic, anti-lipidemic [5], reducing liver inflammation and fibrosis [6], protecting human umbilical vein endothelial cells from the toxic damage [7], etc. ATPS was first introduced by Albertson for partitioning macromolecules and cell particles [8]. The most classical two types of ATPS are PEG/dextran and PEG/salt, however the high cost and high viscosity resulting in difficulties in separating products from polymer have restricted their implication [9,10]. In recent years, ATPS based on small molecular water-miscible organic solvent and salt is a type of liquid–liquid extraction technique, it has many
⇑ Corresponding author. Tel./fax: +86 731 88998501. E-mail address: [email protected] (C. Wang). http://dx.doi.org/10.1016/j.seppur.2014.05.048 1383-5866/Ó 2014 Elsevier B.V. All rights reserved.
advantages such as low cost and viscosity, easy recovery of phaseforming components and easy to scale-up in industrial production [11–14]. Alcohol/salt ATPS was applied to extract and purify lots of compounds such as anthraquinones derivatives from Aloe vera L. [11], isoflavonoids from Dalbergia odorifera T. Chen [15], anthocyanins from purple sweet potatoes [16], flavones and sugars from honeysuckle [17], lipase derived from Burkholderia cenocepacia ST8 [18], chloramphenicol in livestock meat samples [19], etc. In this paper, an ethanol/NaH2PO4 system was used in extraction and purification of CGA from ramie leaf. The affecting parameters of type and concentration of salt, pH and temperature were investigated in details. The whole flow chart of extraction and purification of CGA and recovery of alcohol and salt is shown in Fig. 1. 2. Materials and methods 2.1. Materials and reagents Ramie leaf, collected in Changsha Ramie Garden of Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences. The standard of chlorogenic acid (purity >98% by HPLC) was purchased from Staherb Natural Ingredient Co., Ltd. (Changsha, China). (NH4)2SO4, K3PO43H2O, NaH2PO42H2O, K2CO3, methanol and ethanol are of analytical grade was acquired from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Other reagents were all analytical grade and used without further treatment. De-ionized water was used to prepare the sample solutions.
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Z. Tan et al. / Separation and Purification Technology 132 (2014) 396–400 Evaporated and recycled Ramie leaf
CGA added
Dried, smashed Salt added
Homogenized
Centrifugated
Soaked, Concentraed
Alcohol-rich phase
CGA product
Salt-rich phase
CGA Alcohol solution
Alcohol salt ATPS
Homogeneous system
Evaporated and recycled
ATPS extraction procedure Methanol added
Ethanol molecules Salt molecules CGA molecules Methanol molecules
Precipitated and recycled
Fig. 1. Flow chart of extraction and purification of CGA using ethanol/salt ATPS.
2.2. Preparation of crude plant materials
K¼
Fresh ramie leaf was dried at 45 °C, smashed to powder by a grinder, the powder was sieved by a sieve with the particle size of 60 meshes (about 250 lm), then was soaked by 70% ethanol solution, an ultrasonic treatment was performed for 30 min. The mixture was filtrated, leaving behind the supernatant. After the supernatant being concentrated by a rotary evaporator, solid CGA was obtained and then dissolved by distilled water, set at 4 °C for further study.
Ct Cb
ð2Þ
where Ct and Cb are the CGA concentrations in the alcohol-rich phase and salt-rich phase, respectively. The extraction efficiency (E) of CGA in the alcohol-rich phase is determined from the Eq. (3):
E¼
K 100% K þ 1R
ð3Þ
2.5. Phase diagram 2.3. Preparation of ATPS To a 15 mL centrifuge tube, 5.0 mL distilled water, 2.0 mL ethanol, a given amount of salt and 0.5 mL CGA solution were added. Another tube with the same phase-forming components but without CGA was prepared as a blank to avoid interference. The mixture was stirred well to make the salt dissolve completely. Due to the incompatibility of alcohol and salt solution, in addition the low viscosity in system, the phase-separation can be achieved during a few seconds, two clear phases formed. In order to achieve complete phase-forming and extraction, usually a centrifugation procedure was performed. The top alcohol-rich phase was mainly composed of ethanol and CGA, and the bottom phase was the salt-rich solution containing the impurities such as polyphenols, flavonoids, metallic elements and proteins, etc. The volume of each phase was noted down. 2.4. Analysis of CGA The alcohol-rich phase was withdrawn to a tube using a syringe, diluted using methanol for the analysis of CGA concentration by a UV–Vis spectrophotometer (UV-2100, Unico, USA). The absorbance of CGA was measured at the wavelength of 326 nm. The calibration curve for analysis of CGA was Y = 0.0567X 0.0057 with R2 = 0.9997 (RSD = 0.046%, n = 5). Where Y is the absorbance, and X is the concentration of CGA in the range of 2.0–32 lg/mL. The CGA concentration in the salt-rich phase was determined by mass balance. The phase ratio (R) is defined as the Eq. (1):
R¼
Vt Vb
ð1Þ
where Vt and Vb are the volumes of alcohol-rich phase and salt-rich phase, respectively. The partition coefficient (K) is defined as the Eq. (2):
The phase diagrams were prepared by a turbidimetric titration method [20,21]. Firstly, ethanol of known mass was added into a centrifuge tube. Then, a salt solution of known mass fraction was added dropwise and the mixture was well mixed. The solution was clear at first, but after a certain amount of the salt solution being added, one further drop made the mixture turbid, then two phases formed. The mass fraction of each component was calculated. Finally, a few drops of water was added to make the mixture clear again, and the above procedure was repeated to obtain sufficient data to construct the phase diagrams. 3. Results and discussion 3.1. Selection of the optimal alcohol/salt ATPS In order to choose an optimal alcohol/salt ATPS, four kinds of organic alcohol (methanol, ethanol, 1-propanol and 2-propanol) and four kinds of inorganic salt (K3PO4, (NH4)2SO4, NaH2PO4, K2CO3) were considered as phase-forming component. Compared with methanol, 1-propanol and 2-propanol, ethanol has the advantages of lowest cost, almost no toxicity and moderate boiling point, which is more suitable for large-scale industrial production. So ethanol was chosen as the phase-forming alcohol. The phase diagrams of the ethanol/salt system was shown in Fig. 2, the abilities of four types of salt for phase separation were in an order of K3PO4 > K2CO3 > (NH4)2SO4 > NaH2PO4. To optimize the salt type and concentration, four ethanol/salt ATPSs were employed to extract CGA, and the results were shown in Table 1. The results showed that the acid systems formed by ethanol/NaH2PO4 and ethanol/(NH4)2SO4 have the better extraction ability for CGA. CGA is an acidic compound and is more stable in the acidic solution. Furthermore, ethanol/NaH2PO4 system has the relatively higher extraction efficiency than ethanol/(NH4)2SO4 system and was chosen for further studies. The optimal extraction
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Z. Tan et al. / Separation and Purification Technology 132 (2014) 396–400 Table 2 The transfer thermodynamic properties for CGA extraction from the salt-rich phase to the alcohol-rich phase in ATPS.
35
(NH4)2SO4 NaH2PO4
30
K2CO3
Salt (%, w/w)
25
K3PO4
20
T (k)
K
DG (kJ mol1)
TDS (kJ mol1)
DH (kJ mol1)
298.15 303.15 308.15 313.15 318.15
39.04 18.66 10.34 5.02 3.52
9.08 7.37 5.98 4.20 3.33
88.08 89.79 91.18 92.96 93.73
97.16
15 10
100 5
80 0
5
10
15
20
25
30
35
40
45
50
55
Ethanol (%, w/w)
60
Fig. 2. Phase diagrams for ethanol/salt ATPSs at room temperature (18 ± 2 °C).
40
20
3.2. Effect of temperature
0
/%
system was composed of 2.0 mL ethanol, 5 mL water and 3.8 g NaH2PO42H2O added (see Table 1).
E
The partitioning of CGA in ethanol/NaH2PO4 system at the temperature range of 25–45 °C was investigated with pH being not adjusted. The results were shown in Fig. 3 that when the temperature was 25 °C the extraction efficiency was maximum of 93.66%. Ethanol with low boiling easily volatilize at relative high temperature, so the extraction experiment could be just done at room temperature. The large scale production is more feasible by cutting down the cost of equipment and energy. 3.3. Effect of pH B–R (Britton–Robinson) buffer was used to adjust the pH of the aqueous solution of the range of 2.09–8.36. The pH was adjusted by mixing different solution of 0.04 M phosphoric acid, 0.04 M acetic acid, 0.04 M boric acid, and 0.20 M sodium hydroxide. The results were shown in Fig. 4 that the extraction efficiency varied little at pH 2.0–6.0 and the maximal extraction efficiency was obtained
45
40
K 35
Temperature/
30
25
Fig. 3. Extraction of CGA at different temperature.
at aqueous solution pH of 3.29. The optimal ethanol/NaH2PO4 system (2.0 mL ethanol, 5 mL water and 3.8 g NaH2PO42H2O) has a proper pH value of 3.90, the pH of ATPS extraction is unnecessary to be adjusted in the following experiments. 3.4. Thermodynamics and kinetics in extraction procedure To investigate the thermodynamics, extraction of CGA can be regarded as a transfer process of the CGA from the salt-rich phase to the alcohol-rich phase [22,23]. The Gibbs energy change (DG) is
Table 1 Extraction of CGA using different ethanol/salt ATPSs (each system contains 2.0 mL ethanol, 5.0 mL water, a given of salt and 0.5 mL crude CGA extract solution). Alcohol/salt system
Salt added (g)
Phase ratio (R)
Partition coefficient (K)
Extraction efficiency (E, %)
Ethanol/(NH4)2SO4
2.0 2.2 2.4 2.6 2.8
0.09 0.29 0.29 0.29 0.27
2.56 5.88 11.53 12.55 32.66
18.86 63.21 77.12 78.58 89.91
Ethanol/K2CO3
3.0 3.4 3.8 4.2 4.6
0.46 0.43 0.42 0.33 0.32
1.85 1.75 1.71 1.76 1.88
46.24 43.11 57.83 37.38 38.17
Ethanol/NaH2PO4
3.2 3.4 3.6 3.8 4.0
0.52 0.52 0.51 0.51 0.50
3.39 7.11 11.45 38.72 38.49
64.02 78.60 85.33 93.58 93.44
Ethanol/K3PO4
2.4 2.8 3.2 3.6 4.0
0.70 0.66 0.57 0.53 0.47
1.59 2.23 1.61 1.27 1.29
52.78 59.50 48.04 40.11 37.69
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Z. Tan et al. / Separation and Purification Technology 132 (2014) 396–400
80
syringe at different time after phase forming. The concentration of CGA in bottom phase was determined. Firstly we supposed that it is a first-order kinetics procedure. The kinetics equation Eq. (6) is as follow:
70
ln C A ¼ kt þ ln C o
60
where Co is the original concentration of CGA, CA is the concentration after the extraction time of t seconds in bottom phase, k is extraction rate constant. The CGA concentration was determined during the extraction procedure at different temperature and time. The extraction time of t is in the range of 20, 30, 40, 50 and 60 s. A straight line were obtained by plotting ln CA versus t, k values was the slope in Eq. (6), the results were shown in Table 3. The satisfactory correlation coefficient demonstrated extraction of CGA is a first-order kinetics.
100 90
50 40 30 20
/%
10 0
E
8
7
6
5
4
3
K 2
3.5. Comparison of different ATPSs in extraction of CGA
pH Fig. 4. Extraction of CGA at different pH and 298.15 K.
related to the partition coefficient (K) can be calculated by Eq. (4). The enthalpy change (DH) and entropy change (DS) were obtained from the slope and intercept of the linear Eq. (5) which plots ln K versus 1/T through Van’t Hoff approach. The obtained linear equation was ln K = 11686.29 1/T 35.58 with correlation coefficient R2 = 0.9895.
DG ¼ RT ln K ln K ¼
ð6Þ
ð4Þ
DH DS þ RT R
Extraction of CGA using PEG/salt, PEG/dextran and alcohol/salt ATPSs were studied. The phase-forming time of ATPS was recorded by visual observation. When the clear phase interface can be observed, we considered it as the phase-forming time. The viscosity of each ATPS was determined by a rotary viscosimeter (NDJ-5S, Shanghai Hengping). The results of phase-forming time, extraction efficiency and viscosity values in top and bottom phase were shown in Table 4. It can be seen that alcohol/salt ATPS has relative high extraction efficiency. Furthermore, compared with the PEG-based ATPSs, alcohol/salt system has shorter phase-forming time, smaller viscosity and lower cost, which is more potential for scale-up production.
ð5Þ 3.6. Recycle of phase-forming components
The DG, DH and DS values were calculated by Eqs. (4) and (5) are shown in Table 2. All the DG values are negative, reflecting that the extraction of CGA is spontaneous and preferential partitioning in the alcohol-rich phase. Partitioning of CGA in ATPS is marked by negative values for DH and TDS with the DH being greater in value than the DS. Thus it can be concluded that extraction of CGA form salt-rich phase to alcohol-rich phase is an exothermic, spontaneous process [24,25]. The extraction of CGA by alcoholbased ATPS is highly dependent on temperature. There are also some other researchers reported that temperature can greatly influence the partition of target materials in different types of ATPS [26–28]. In order to measure the kinetics in the extraction procedure, the tube was placed in the thermostat water bath at different temperature, the sample in bottom phase was quickly withdrawn by a
In order to reduce the cost in extraction of CGA using ethanol/ salt ATPS, we have tried to recycle the phase-forming components of ethanol and salt. When the extraction is finished, alcohol-rich phase and salt-rich phase were separated away from each other. Ethanol solution in top phase was recycled by evaporation and distillation, then processed by calcium oxide to obtain high pure ethanol. The salt in bottom phase can be recycled by dilution crystallization. After a certain amount of methanol being added into the salt solution with full stirring, NaH2PO4 can be precipitated out. Methanol has lowest boiling point and was easily recycled than other organic alcohols, which cannot easily form azeotrope with water and is to be evaporated out at relatively lower temperature [29]. To optimize the amount of methanol, 0.5–5 times volume of
Table 3 The kinetic equations of ATPS extraction of CGA at different temperature. Temperature (K) 298.15 308.15 318.15
Kinetic equation ln C = 0.0224t + 4.850 ln C = 0.0283t + 4.862 ln C = 0.0313t + 4.840
Reaction rate constant (k, s1) 2
2.24 10 2.83 102 3.13 102
Correlation coefficient (R2) 0.9792 0.9942 0.9914
Table 4 Comparison of phase-forming time, viscosity values and extraction efficiencies of different ATPSs. Type of ATPS (concentration %, w/w)
Phase-forming time (s)
Viscosity in top/bottom phase (mpa s)
Extraction efficiency (%)
PEG2000 (15%)/NaH2PO4 (25%) PEG6000 (15%)/NaH2PO4 (25%) PEG10000 (15%)/NaH2PO4 (25%) PEG6000 (15%)/Dextran 40,000 (6%) Ethanol (15.2%)/NaH2PO4 (28.1%)
120 ± 5 140 ± 5 170 ± 5 320 ± 5 18 ± 3
24.1 ± 0.3 (PEG-rich phase)/3.2 ± 0.3 (salt-rich phase) 35.7 ± 0.3 (PEG-rich phase)/4.5 ± 0.2 (salt-rich phase) 89.6 ± 0.5 (PEG-rich phase)/4.8 ± 0.3 (salt-rich phase) 26.5 ± 0.2 (PEG-rich phase)/124.5 ± 0.5 (Dextran-rich phase) 2.3 ± 0.2 (alcohol-rich phase)/1.5 ± 0.2 (salt-rich phase)
85.5 ± 0.3 87.4 ± 0.3 84.3 ± 0.2 75.9 ± 0.2 94.2 ± 0.3
(PEG-rich phase) (PEG-rich phase) (PEG-rich phase) (PEG-rich phase) (alcohol-rich phase)
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Z. Tan et al. / Separation and Purification Technology 132 (2014) 396–400
100 90
Salt recovery ratio/ %
80 70 60 50 40 30 20 10 0 0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
Vmethanol added/ VSalt-rich phase Fig. 5. Recovery ratio of NaH2PO4 by dilution crystallization using methanol.
methanol was added into the salt solution. The results in Fig. 5 showed that when 1.5 times volume of methanol was added, the recovery ratio of NaH2PO4 reached to about 80%. To overall consider the cost, we think 1.5 times volume of methanol was enough for recovery of NaH2PO4. When CGA was extracted using the recycled salt, the extraction efficiency hardly decreased after recycling of 3 times. That is because the impurities dissolved in the methanol solution with the crystalline salt being not polluted. 4. Conclusion In this paper, an alcohol/salt ATPS was used to extract and purify CGA from ramie leaf by a single-step procedure. The optimized extraction conditions were as follow: ethanol/NaH2PO4 was chosen as the extraction system, the maximum extraction of 95.76% was obtained at 25 °C and aqueous solution pH of 3.29. In addition, the thermodynamics and kinetics was explored simultaneously in the extraction procedure for the first time, which can well interpret the extraction mechanism of alcohol/salt ATPS. Lastly, the phase-forming components were recycled, which can reduce the cost making this system more possible for large-scale production of CGA. Acknowledgements This work was financially supported by the earmarked fund for the China Agriculture Research System (Project No. CARS-19-08B). References [1] L.J. Liu, C.Y. Lao, N. Zhang, H.Q. Chen, G. Deng, C. Zhu, D.X. Peng, The effect of new continuous harvest technology of ramie (Boehmeria nivea L. Gaud.) on fiber yield and quality, Ind. Crop Prod. 44 (2013) 677–683. [2] L. Wang, T. Zhang, H. Yan, M. Peng, Z. Fang, Modification of ramie fabric with a metal-ion-doped flame-retardant coating, J. Appl. Polym. Sci. 129 (2013) 2986–2997. [3] S. Sancheti, S. Sancheti, M. Bafna, H.R. Kim, Y.H. You, S.Y. Seo, Evaluation of antidiabetic, antihyperlipidemic and antioxidant effects of Boehmeria nivea root extract in streptozotocin-induced diabetic rats, Revista Brasileira De Farmacognosia, Braz. J. Pharmacogn. 21 (2011) 146–154. [4] J.G. Xu, Q.P. Hu, Y. Liu, Antioxidant and DNA-protective activities of chlorogenic acid isomers, J. Agric. Food Chem. 60 (2012) 11625–11630.
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