Comparative Study on Rosmarinic Acid Separation by Reactive

Article pubs.acs.org/IECR

Comparative Study on Rosmarinic Acid Separation by Reactive Extraction with Amberlite LA‑2 and D2EHPA. 1. Interfacial Reaction Mechanism and Influencing Factors Lenuţa Kloetzer,† Mădălina Poştaru,† Anca-Irina Galaction,‡ Alexandra Cristina Blaga,† and Dan Caşcaval*,† †

Department of Biochemical Engineering, Faculty of Chemical Engineering and Environmental Protection, “Gheorghe Asachi” Technical University of Iasi, D. Mangeron 73, 700050 Iasi, Romania ‡ Department of Biomedical Science, Faculty of Medical Bioengineering, “Gr. T. Popa” University of Medicine and Pharmacy of Iasi, M. Kogalniceanu 9-13, 700454 Iasi, Romania ABSTRACT: The mechanism of reactive extraction of rosmarinic acid with Amberlite LA-2 (secondary amine) and D2EHPA (organophosphoric acid) was analyzed for three solvents with different polarities (n-heptane, n-butyl acetate, dichloromethane), with or without 1-octanol. Thus, in the absence of 1-octanol, the stoichiometric ratio between the rosmarinic acid and Amberlite LA-2 was 1:1 for dichloromethane, 1:2 for butyl acetate, and 1:4 for n-heptane. In the presence of 1-octanol, the formation of aminic adducts is restricted, and the structures of the interfacial complexes reflect the following stoichiometries for the interfacial reaction between the solute and Amberlite LA-2: 1:1 for dichloromethane and butyl acetate and 1:2 for n-heptane. However, regardless of 1-octanol addition to the organic phase, the extraction of rosmarinic acid with D2EHPA occurred only through the formation of an interfacial compound containing one acid molecule and two molecules of extractant for all considered solvents. In the case of reactive extraction with Amberlite LA-2, the highest extraction constant was reached for low-polarity solvents, which promotes the formation of aminic adducts. In contrast, an increase of the organic-phase polarity positively influenced the value of the extraction constant for extraction with D2EHPA.

1. INTRODUCTION Rosmarinic acid is a member of the class of phenolic compounds containing one carboxyl group, being an ester of caffeic acid and 3-(3,4-dihydroxyphenyl)lactic acid (Figure 1).1

names: rosemary, lemon balm, thyme, mint, sage, oregano, lavender, and clover).1,6,7 Therefore, even though it requires large amounts of solvents and offers rather low productivity, extraction from raw vegetable materials currently represents the direct method of rosmarinic acid production. Rosmarinic acid could also be obtained by chemical synthesis, but with high materials and energy consumption and a low yield because of the formation of a racemic mixture.7−9 In these circumstances, the production of rosmarinic acid by biosynthesis is very attractive. The biosynthesis methods use cultures of Coleus blumei, Salvia miltiorrhiza, Anchusa off icinalis, or Lavandula vera cells on glucose.3,10−15 The biosynthesis productivity was enhanced by the simultaneous cultivation of some microorganism, namely, Pseudomonas, Agrobacterium rhizogenes, or Phytium aphanidermatum.1,4,13 Moreover, the growth of plant cells, their viability, and the rosmarinic acid productivity can be improved by addition of elicitors in the culture medium (proline and analogues, dimethylsulfoxide, yeast extract, pantothenic acid). In this case, depending on the type of cultivated plant cells, the rosmarinic acid yield can be increased by up to a factor of 6 and reach between 2% and 21% based on the dry cell weight.13,16−18 Another attractive method for rosmarinic acid production is enzymatic synthesis. In this case , rosmarinic acid synthase is

Figure 1. Chemical structures of (a) rosmarinic acid, (b) Amberlite LA-2, and (c) D2EHPA.

The interest in rosmarinic acid is justified by its biological activity, as this acid exhibits antioxidant, astringent, antiinflammatory, antimutagenic, anticancer, antiallergic, antibacterial, and antiviral (anti-HIV) effects.1−4 This compound was isolated in 1958 by Scarpati and Oriente from rosemary (Rosmarinus off icinalis).5 Other plant sources have been subsequently identified, such as monocotyledonous (Cannaceae, Zosteraceae, Potamogetonaceae), dicotyledonous (Laminaceae, Boraginaceae, Curcubitaceae, Rubiaceae, etc.), as well as Blechnaceae, Anthocerotaceae, and so on (common © 2013 American Chemical Society

Received: June 7, 2013 Accepted: September 2, 2013 Published: September 2, 2013 13785

dx.doi.org/10.1021/ie4023513 | Ind. Eng. Chem. Res. 2013, 52, 13785−13794

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LA-2 and 0.015−0.24 M for D2EHPA). The volumetric ratio of the aqueous and organic phases was 1 (20 mL of each phase). The pH value of the initial aqueous solution was varied between 1 and 7. The pH adjustment was made with a solution of 3% sulfuric acid or 3% sodium hydroxide, depending on the prescribed pH value. The pH values were determined using a Consort C836 type digital pH meter and were recorded throughout each experiment. Any pH change was recorded during the extraction experiments. The extraction process was analyzed in terms of the extraction efficiency and the distribution coefficient. To calculate these parameters, the rosmarinic acid concentrations in the initial aqueous solution and in the raffinate were measured, and the mass balance was used for the entire extraction system. The acid concentration was determined by high-performance liquid chromatography (HPLC) as described in the literature.20 Each experiment was repeated two or three times under identical conditions, with the average value of the considered parameters being used. The maximum experimental error was ±5.24%.

able to catalyze the reaction between 3-(3,4-dihydroxyphenyl)lactic acid and caffeoyl coenzyme A.19 Currently, the separation and purification of rosmarinic acid from plant extracts, cells cultures, or enzyme media are achieved by ion exchange, electrodialysis, electrophoresis, physical extraction, supercritical fluid extraction, and nonfacilitated pertraction.6,12,15,20,21 In many separation technologies, physical extraction constitutes a viable solution, because of its technical accessibility and high efficiency. The use of physical extraction for rosmarinic acid separation is possible because this acid is soluble in water-immiscible solvents, such as esters, ethers, paraffins, chloroform, and long-chain alcohols.5,20 However, its distribution coefficient between aqueous and organic phases is relatively low (values greater than unity are obtained only for ethyl acetate and diisopropyl ether20). Moreover, its reextraction from the organic phase requires the use of an aqueous alkaline solution, and the chemical stability of rosmarinic acid is strongly affected for pH values higher than 8.22 For these reasons, in this work, we investigated the separation of rosmarinic acid by an alternative technique, namely, reactive extraction. Because the chemical structure of rosmarinic acid contains both acidic and basic groups, reactive extraction was carried out using two types of extractants: an extractant from among high-molecular-weight amines, namely, lauryl trialkylmethylamine (Amberlite LA-2), and an organophosphoric derivative, namely, di-(2-ethylhexyl) phosphoric acid (D2EHPA) (Figure 1). In these studies, the effects of the extraction system characteristics (extractant type, solvent polarity, 1-octanol addition) and operating conditions (pH value of the aqueous phase, extractant concentration, mixing intensity) on the interfacial reaction mechanism and efficiency of reactive extraction were analyzed.

3. RESULTS AND DISCUSSION Rosmarinic acid has a complex structure, containing multiple groups that could react with the considered extractants. Because the acidic or basic characters of a group, as well as its size, control the separation efficiency by reactive extraction, the possible active groups were initially identified. Except for the pKa value associated with the carboxylic group (pKa1 = 2.826), no data are available in the literature regarding the acidic or basic character of the ionizable groups of rosmarinic acid. For this reason, using the software ACD/ Laboratories,27 we identified five ionizable groups, namely, one carboxylic group and four phenolic groups, and calculated the pKa values for the phenolic groups (Table 1 corresponding to Figure 1).

2. MATERIALS AND METHODS The experiments were carried out using an extraction column with vibratory mixing, which offers a high interfacial area and the possibility of rapidly reaching the equilibrium state. The laboratory equipment was described in detail in previous articles23,24 and consisted of a glass column with an internal diameter of 36 mm and a height of 250 mm, provided with a thermostatic jacket where the thermal agent (water and ethylene glycol mixture) was circulated at 25 °C. The phases were mixed by means of a perforated disk with a 45-mm diameter and 20% free section. The vibrations had a frequency of 50 s−1 and an amplitude of 5 mm. The perforated disk position was maintained at the initial contact interface between the aqueous and organic phases. The extraction time was 1 min. The resulting emulsion was broken in a centrifugal separator at 6000 rpm. The initial concentration of rosmarinic acid in aqueous solution was 10 g·L−1 (0.027 M). The reactive extraction was carried out with three solvents having different dielectric constants, namely, n-heptane (dielectric constant of 1.90 at 25 °C25), n-butyl acetate (dielectric constant of 5.01 at 25 °C25), and dichloromethane (dielectric constant of 9.08 at 25 °C25). 1-Octanol (≥98%, Merck) (dielectric constant of 10.3 at 25 °C25) was dissolved in the above-mentioned solvents at a volumetric fraction of 0.10 or 0.20. The extractants, Amberlite LA-2 and D2EHPA, were separately dispersed in the organic phase. The extractant concentrations in the organic phase varied in the range of 5−80 g·L−1 (0.013−0.21 M for Amberlite

Table 1. Rosmarinic Acid pKa Values Calculated by ACD/ Labs Software at 25 °Ca

a

ionizable group

pKa

−COOH −OH (7) −OH (8) −OH (24) −OH (25)

2.78 ± 0.10 9.33 ± 0.10 12.33 ± 0.31 9.77 ± 0.10 12.65 ± 0.20

Functional group numbering is indicated in Figure 2.

Regardless of the extractant type, the reactive extraction of rosmarinic acid is based on the formation of a strong hydrophobic compound at the interface between the aqueous and organic phases. The mechanism and kinetics of the interfacial reaction and the optimum operating conditions depend on the extractant type and polarity of the organic phase. 3.1. Reactive Extraction without 1-Octanol in the Organic Phase. Similarly to the extraction of other carboxylic acids, the carboxylic group of the solute is involved in the reactive extraction with Amberlite LA-2 (E), and the process occurs by means of the interfacial interactions between these two compounds. The interfacial interactions could be of the hydrogen-bonding type if the rosmarinic acid is undissociated in the aqueous phase or of the ionic type if the acid is partially dissociated 13786



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Figure 2. Influence of the pH value of the aqueous phase on the efficiency of the reactive extraction of rosmarinic acid with (a) Amberlite LA-2 and (b) D2EHPA without 1-octanol (extractant concentration = 40 g·L−1): (■) dichloromethane, (□) butyl acetate, (▲) n-heptane.

rosmarinic acid with Amberlite LA-2 is reached at a pH of 2 in the aqueous solution (Figure 2a). Owing to the superior ability of solvents with higher polarity to solubilize dissociated molecules, the maximum extraction degree of rosmarinic acid was recorded for dichloromethane, being 1.07 times higher than that for butyl acetate and 1.27 times higher than that for n-heptane. Reactive extraction with D2EHPA occurs if the phenolic groups of rosmarinic acid exist in the protonated form in the aqueous phase and thus in the highly acidic domain. However, the strongly acidic domain also induces the protonation of the extractant, which hinders its reaction with rosmarinic acid.29 In this case, because of these contrary effects of pH, the maximum extraction efficiency is reached for pH 3 (Figure 2b). The maximum extraction degree of rosmarinic acid with D2EHPA is less evident than that with Amberlite LA-2, because, in the strongly acidic domain, the carboxylic group is not ionized, which allows for possible physical coextraction with a positive effect on solute transfer into the organic phase. The differences in extraction efficiency related to the three solvents are maintained in the case of reactive extraction with D2EHPA. For the optimum pH value, the extraction efficiency recorded for dichloromethane was 1.05 times greater than that for butyl acetate and 1.22 times greater than that for n-heptane. The extraction efficiencies of the two extractants are comparable, regardless of the solvent used. However, for the same concentration of extractants in the organic phase and at the specific optimum pH value, the decrease of solvent polarity leads to a maximum difference of 4−7% in favor of D2EHPA. These differences increase significantly for pH variations between 3 and 5, becoming 30−40%, but this pH interval is not feasible for the reactive extraction of rosmarinic acid. To analyze the extraction mechanism of rosmarinic acid, HA, in both cases, it was assumed that n molecules of extractant, E, and one acid molecule participate in the formation of the interfacial compound by means of hydrogen bonding, as mentioned before. Therefore, the reactive extraction of rosmarinic acid with Amberlite LA-2 can be described by the interfacial equilibrium

RA−COOH(aq) + n E(org) ⇄ RA−COOH·En(org)

Moreover, depending on the structures of the system components and the solvent polarity, acidic or aminic adducts could be formed at the interface. However, because of the steric hindrance induced by the voluminous molecule of rosmarinic acid and the fact that the initial concentration of acid is lower than that of aminic extractant, the formation of acidic adducts at the interface is not possible. Therefore, the interfacial compound could be of ammonium salt type, resulting from neutralization of the solute carboxylic group with one extractant molecule, or of aminic adduct type for n ≥ 2.28 The formation of these molecular associations, a phenomenon that is more pronounced in solvents with low dielectric constants, improves the hydrophobicity of the interfacial compound.28 Because rosmarinic acid contains phenolic groups with strong basic character, it can react also with acidic extractants, including D2EHPA and HP. In this case, the reactive extraction occurs by means of an interfacial reaction of the ion-exchange type, which requires the solute to be in protonated form in the aqueous phase RA(O+H 2)m (aq) + pHP(org) ⇄ RA(O+H 2)m P−m ·(p − m)HP(org) + mH+(aq)

where m = 1−4. The formation of adducts including many solute molecules is hindered in these extraction systems as well, because of the voluminous molecules of rosmarinic acid. In both cases, the pH of the aqueous phase controls the efficiency of reactive extraction. Regardless of the extractant type or solvent polarity, from Figure 2, it can be seen that the efficiency of reactive extraction increases initially with an increase of the pH value of the aqueous solution, reaches a maximum level, and then decreases. In the case of reactive extraction with Amberlite LA-2, the maximum of extraction degree is the result of phenolic group ionization in the strongly acidic pH domain, and this phenomenon limits the extraction efficiency. However, the increase of the pH value leads to the dissociation of the carboxylic group, which then becomes unable to react with the extractant. Thus, regardless of the experimental solvent phase, because of these two contrary effects induced by the increase of pH, the highest efficiency in the reactive extraction of

HA(aq) + n E(org) ⇄ HAEn(org)

In this circumstance, the distribution coefficient, D, is calculated with the expression 13787


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solvent polarity on the structure of the interfacial compound. The experimental results are presented in Figure 3.

[HAEn(org)] [HA(aq)]

(1)

where [HA(aq)] is the overall acid concentration in the aqueous phase and [HAEn(org)] represents the overall concentration of extracted compound at the equilibrium state in the solvent phase. According to the interfacial equilibrium, the extraction constant, KE, can be calculated as KE =

[HAEn(org)] [HA(aq)][E(org)]n

(2)

Thus, the concentration of extracted compound is given by [HAEn(org)] = KE[HA(aq)][E(org)]n

(3)

Because this study on the interfacial mechanism was carried out at the optimum pH value of 2, the concentration of rosmarinic acid molecules with undissociated −COOH groups in the aqueous phase, [HA(aq)], was calculated by means of the overall acid concentration in the aqueous phase and the concentration of dissociated acid, [A−(aq)] [HA(aq)] = [HA(aq)] − [A−(aq)]

Figure 3. Influence of the Amberlite LA-2 concentration on the reactive extraction efficiency of rosmarinic acid (pH 2): (■) dichloromethane, (□) butyl acetate, (▲) n-heptane.

Using the data from Figure 3 and plotting eq 8 , the straight lines in Figure 4 were obtained for the three considered

(4)

The dissociation constant, Ka1, corresponding to −COOH groups, is defined according to the equilibrium HA(aq) ⇄ A−(aq) + H+(aq) K a1 =

[A−(aq)][H+] [HA(aq)]

(5)

Considering eqs 4 and 5, the expression for the concentration of undissociated rosmarinic acid from the aqueous phase becomes [HA(aq)] =

[HA(aq)] 1+

K a1 [H+]

(6)

By combining the eqs 1, 3, and 6, the following relationship for the distribution coefficient is obtained D=

Figure 4. Graphical representation of the straight lines given by eq 8 for solvents without 1-octanol (pH 2): (■) dichloromethane, (□) butyl acetate, (▲) n-heptane.

KE[E(org)]n 1+

K a1 [H+]

(7)

solvents. The values of the slopes of these straight lines depend on solvent polarity, as given in Table 2. These results indicate the modification of the chemical structure of the interfacial compound as a function of the polarity of the organic phase. Thus, reactive extraction with Amberlite LA-2 in a low-polarity solvent (n-heptane) occurs with the formation of an interfacial aminic adduct with four extractant molecules. In the extraction systems with n-butyl acetate and dichloromethane, solvents with higher dielectric constants, two and one molecules of aminic extractant, respectively, participate in the formation of the interfacial compound. The above results are confirmed by the variation of extraction efficiency plotted in Figure 3. Thus, it can be observed that, for n-heptane, the extraction efficiency increased significantly with increasing extractant concentration to 40 g· L−1 (0.11 M), a limit that corresponds to a 1:4 molar ratio between rosmarinic acid and extractant. On the other hand, for n-butyl acetate, the extraction efficiency increased strongly with increasing extractant concentration only for Amberlite LA-2

In logarithmic form, the correlation in eq 7 represents the equation of a straight line ⎛ K a1 ⎞ ln D + ln⎜1 + ⎟ = ln KE + n ln[E(org)] ⎝ [H+] ⎠

(8)

The initial concentration of Amberlite LA-2 was higher than the initial concentration of rosmarinic acid; therefore, [E(org)] was assumed to be the initial concentration of extractant in the solvent phase. By means of the above algorithm, from the slope of the straight line given by eq 8, it is possible to determine the number of aminic extractant molecules that participate in the interfacial reaction with the solute, and the value of the extraction constant can be established from the intercept of the straight line. The influence of the Amberlite LA-2 concentration on the extraction efficiency, for all three considered solvents, was studied to establish the number of aminic extractant molecules that react with rosmarinic acid, as well as the influence of 13788



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Table 2. Number of Extractant Molecules Included in the Interfacial Compound Structure, n, for Solvents without and with 1-Octanol solvent

KE =

number of extractant molecules (n)

=

Amberlite LA-2 Extractant n-heptane n-heptane + 1-octanol n-butyl acetate n-butyl acetate + 1-octanol dichloromethane dichloromethane + 1-octanol D2EHPA Extractant n-heptane n-heptane + 1-octanol n-butyl acetate n-butyl acetate + 1-octanol dichloromethane dichloromethane + 1-octanol

3.94 2.06 1.93 0.94 1.08 1.03

⇒[Hm + 1A (aq)] =

D=

2.08 2.07 1.95 1.92 2.10 2.11

(11)

KE[E(org)]n [H+(aq)]m

(12)

The linearized form of eq 12 is obtained by applying the logarithm ln D + m ln[H+] = ln KE + n ln[E(org)]

(13)

Similarly to the extraction with Amberlite LA-2, from the slope and intercept of the straight line given by eq 13, it is possible to determine the number of organophosphoric extractant molecules that participate in the interfacial reaction with the solute and the value of the extraction constant, respectively, considering [E(org)] as the initial concentration of D2EHPA. The dependence between the extraction efficiency and the D2EHPA concentration is plotted in Figure 5 for all studied

Figure 5. Influence of the D2EHPA concentration on the reactive extraction efficiency of rosmarinic acid (pH 3): (■) dichloromethane, (□) butyl acetate, (▲) n-heptane.

+

where m = 1−4 and n ≥ m. The elucidation of the mechanism of reactive extraction with D2EHPA is more difficult, because the number of phenolic groups participating in the interfacial reaction is not known exactly. According to the interfacial equilibrium, the distribution coefficient can be calculated by the relationship

solvents. In the absence of any previous data regarding the number of phenolic groups participating in the interfacial reaction, m, using the results given in Figure 5, eq 13 was plotted for all four possible values of m. Therefore, regardless of the solvent polarity and the number of phenolic groups of rosmarinic acid participating in the interfacial reaction with the extractant, from Figure 6, it can be observed that all slopes have an approximate value of 2 (Table 2). According to the initial assumption about the interfacial reaction mechanism, namely, n ≥ m in all cases, possible real behavior is indicated only by the straight lines corresponding to the values m = 1 and m = 2. The combination m = 1 and n = 2 suggests that one active −OH group reacts with two D2EHPA molecules, indicating the solvation of rosmarinic acid. However,

1

[H+(aq)] [HAEn(org)] m = [Hm + 1Am +(aq)] [Hm + 1Am +(aq)]

1 [H+(aq)]m + 1 m KE[E(org)]n

(10)

Thus, the relationship for calculating the distribution coefficient, D, becomes

Hm + 1A (aq) + n E(org) ⇄ HAEn(org) + mH (aq)

D=

1 [H+(aq)]m + 1 m [Hm + 1Am +(aq)][E(org)]n m+

concentrations below 20 g·L−1 (0.53 M), a value related to a solute/extractant molar ratio of 1:2, with the increase becoming slower for higher concentrations of amine. The least extended domain of extractant concentration corresponding to the important influence of this parameter on extraction efficiency was recorded for dichloromethane (up to 10 g·L−1 Amberlite LA-2, or 0.027 M). This result suggests that each reactant molecule participates with one molecule in the formation of the interfacial compound. Thus, the decrease of solvent polarity facilitates the formation of aminic adducts by hydrogen-bonding interactions and increases the number of extractant molecules included in these adducts. Thus, also for rosmarinic acid extraction, it was confirmed that the behavior of Amberlite LA-2 is similar to that previously reported in the literature for aminic extractants.28 The experiments regarding the mechanism of reactive extraction of rosmarinic acid with D2EHPA were also carried out at the optimum pH value of the aqueous phase, pH 3. In this case, the phenolic groups are the active ones and the efficiency of extraction is influenced by the ability of these groups to react with the extractant. The interfacial reaction can be described by the equilibrium m+

[HAEn(org)][H+(aq)]m [Hm + 1Am +(aq)][E(org)]n

(9)

where [Hm+1Am+(aq)] and [HAEn(org)] indicate the overall concentrations of acid and extracted compound, respectively, in the two phases at the equilibrium state. Based on the interfacial equilibrium, the extraction constant, KE, is given by 13789



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Figure 6. Graphical representation of the straight lines given by eq 13 for solvents without 1-octanol (pH 3): m = (■) 1, (□) 2, (▲) 3, (▼) 4.

Table 3. Expressions and Values of Extraction Constants for the Studied Extraction Systems without 1-Octanol solvent

valuea

extraction constant Amberlite LA-2 Extractant

n-heptane n-butyl acetate dichloromethane

KE =

KE = KE =

2.89 × 104 L4·mol−4

[HAE4(org)] 4

[HA(aq)][E(org)]

6.62 × 102 L2·mol−2

[HAE 2(org)] 2

[HA(aq)][E(org)] [HAE(org)] [HA(aq)][E(org)]

1.04 × 102 L·mol−1

D2EHPA Extractant n-heptane

KE = n-butyl acetate

KE = dichloromethane

KE = a

[HAE 2(org)][H+(aq)]2

2.01 × 10−4

[H3A2 +(aq)][E(org)]2 [HAE 2(org)][H+(aq)]2

5.62 × 10−4

[H3A2 +(aq)][E(org)]2 [HAE 2(org)][H+(aq)]2

8.38 × 10−4

[H3A2 +(aq)][E(org)]2

Including units.

ionizable groups.30,31 For this reason, contrary to the extraction with Amberlite LA-2, the number of D2EHPA molecules that react with the solute is not modified by changing the solvent from n-heptane to dichloromethane, that is, by increasing the solvent polarity. This result demonstrates that solvated

the solvent polarity represents an important factor controlling the efficiency and mechanism of the extraction of ionizable solutes. The dielectric constant is considered to be a characteristic of solvent−solute interactions by limiting solute solvation by the solvent or extractant, mainly for solutes with 13790



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Figure 7. Influence of the pH value of the aqueous phase on the efficiency of the reactive extraction of rosmarinic acid with (a) Amberlite LA-2 and (b) D2EHPA with 1-octanol (extractant concentration = 40 g·L−1, 1-octanol volumetric fraction = 0.2): (■) dichloromethane, (□) butyl acetate, (▲) n-heptane.

Figure 8. Influence of the pH value of the aqueous phase on the amplification factor for the reactive extraction of rosmarinic acid with (a) Amberlite LA-2 and (b) D2EHPA (extractant concentration = 40 g·L−1): (■) dichloromethane + 10 vol % 1-octanol, (□) dichloromethane + 20 vol % 1octanol, (●) butyl acetate + 10 vol % 1-octanol, (○) butyl acetate + 20 vol % 1-octanol, (▲) n-heptane + 10 vol % 1-octanol, (Δ) n-heptane + 20 vol % 1-octanol.

taking into consideration the data presented in Figures 4 and 6 (Table 3). Thus, for the reactive extraction with Amberlite LA-2, the formation of aminic adducts leads to an increase of the extraction constant by a factor of about 280 from dichloromethane to n-heptane, a variation that indicates a significant shifting of the interfacial equilibrium to the right. However, if the mechanism of the interfacial reaction remains the same for all solvents, as in the case of extraction with D2EHPA, the organic-phase polarity exhibits a direct favorable effect on the extraction equilibrium. Thus, in varying the dielectric constant from that of n-heptane to that of dichloromethane, the value of the extraction constant was increased by a factor of 4.17. 3.2. Reactive Extraction with 1-Octanol in the Organic Phase. To improve the efficiency of extraction, a second solvent immiscible with the aqueous phase can be added to the organic phase to increase the organic-phase polarity and, implicitly, facilitate the solubilization of polar molecules. For this reason, 1-octanol was used in the experiments. Independent of the solvent used, the addition of 1-octanol in the organic phase does not change the shapes of the dependences between the reactive extraction efficiency and pH of the aqueous phase. However, its addition improves the extraction degree, an effect that is more pronounced for solvents with lower polarity (Figure 7).

compounds are not formed in this case and, consequently, that the combination m = 1 and n = 2 does not describe the real behavior. In these circumstances, the only possible mechanism of reactive extraction of rosmarinic acid with D2EHPA is based on the interfacial reaction of one molecule of solute with two molecules of extractant, that is, m = n = 2. Because of their high basicity and steric hindrance, the phenolic groups from positions 8 and 25 will each react with one extractant molecule (Figure 1). This conclusion on the mechanism of reactive extraction of rosmarinic acid with D2EHPA is also confirmed by the variation of the degree of reactive extraction with extractant concentration, plotted in Figure 5 for all three solvents. Thus, independent of the solvent polarity, a significant increase in extraction efficiency with increasing extractant concentration in the organic phase occurs up to 20 g·L−1 D2EHPA (0.06 M). Above this concentration of D2EHPA, its influence on extraction efficiency becomes less important. This value of D2EHPA concentration corresponds to the formation of a hydrophobic interfacial compound from one molecule of solute and two molecules of extractant. The solvent polarity and the formation of interfacial adducts have a strong influence on the extraction constant, mainly through the mechanism and extraction efficiency. The extraction constants for the two extractants were calculated 13791



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compounds due to the addition of 1-octanol in the organic phase, the straight lines described by eqs 8 and 13 were plotted and analyzed for the three solvents containing 20 vol % alcohol (Figures 9 and 10).

Compared to the extraction without 1-octanol, at the optimum pH values corresponding to the reactive extraction with Amberlite LA-2 and D2EHPA, the reduction of the solvent polarity from dichloromethane to n-heptane was found to lead to an increase of the extraction degree of 1−14% for extraction with Amberlite LA-2 and 1−12% for extraction with D2EHPA. These differences were amplified at higher pH values of the aqueous solution. The increase in solvent ability to solubilize the polar/ dissociated molecules of rosmarinic acid in the presence of 1octanol diminished the differences in the values of extraction yield, thus flattening the shape of the recorded curves. At the same time, the extraction degrees for all three solvents became closer. To emphasize the positive influence of 1-octanol on the reactive extraction efficiency, in Figure 8, the dependences between the amplification factor and the pH value of the aqueous phase are plotted for the studied extraction systems. (The amplification factor is defined as the ratio between the rosmarinic acid extraction degrees for the solvent with and without 1-octanol, respectively.) These variations suggest that the effect of 1-octanol addition is more important for lowpolarity solvents. Thus, compared to the extraction without 1octanol, for a 1-octanol volumetric fraction of 0.20, the maximum increase in the extraction degree of rosmarinic acid with Amberlite LA-2 was a factor of 1.4 for dichloromethane, 1.64 for butyl acetate, and 2.48 for n-heptane (Figure 8a). Similarly, the efficiency of reactive extraction with D2EHPA increased by a factor of 1.37 for dichloromethane, 1.87 for butyl acetate, and 2.19 for n-heptane (Figure 8b). However, depending on the extractant type, differences in the shapes describing the dependencies between the amplification factors and pH can be observed from Figure 8. In the case of reactive extraction with Amberlite LA-2, regardless of the solvent polarity and 1-octanol concentration, an increase in pH value induces a sinusoidal variation of the amplification factor, its minimum level being reached at the optimum pH for extraction (pH 2) and its maximum at pH 6. This variation is a consequence of additional solubilization of ionized rosmarinic acid molecules in the presence of 1-octanol at pH values lower or higher than the optimum value. The pronounced dissociation of carboxyl groups at neutral or alkaline pH also reduces the solubility in polar solvents and leads to a decrease of the amplification factor for pH > 6. This variation in the amplification factor becomes more evident with increasing solvent dielectric constant and 1-octanol volumetric fraction. For reactive extraction with the organophosphoric extractant, the variation of the amplification factor is modified. This parameter decreases slowly up to the pH value corresponding to the maximum extraction efficiency and increases continuously for higher pH values of the aqueous phase. Because the carboxylic group is undissociated in the pH domain below the optimum value (pH 3) and this group does not react with D2EHPA, the effect of 1-octanol addition is less important, especially for dichloromethane and butyl acetate. Dissociation of the −COOH group at higher pH values facilitates the increase of the magnitude of the positive influence of 1-octanol, leading to the enhancement of the amplification factor. As in the previous case, the variation of the amplification factor is less obvious for low-polarity solvents, namely, n-heptane. To establish the interfacial reaction mechanisms and identify the possible changes in the structure of the interfacial

Figure 9. Graphical representation of the straight lines given by eq 8 for solvents with 20 vol % 1-octanol (pH 2): (■) dichloromethane, (□) butyl acetate, (▲) n-heptane.

Figure 10. Graphical representation of the straight lines given by eq 13 for solvents with 20 vol % 1-octanol (pH 2): (■) dichloromethane, (□) butyl acetate, (▲) n-heptane.

For the extraction system containing Amberlite LA-2 and 1octanol, the values of the slopes of the straight lines corresponding to eq 8 were obtained from Figure 9 and are presented in Table 2. Compared to the results recorded for the reactive extraction of rosmarinic acid with Amberlite LA-2 without 1-octanol, in the presence of alcohol, the number of aminic extractant molecules included in the chemical structure of the interfacial compound is halved for n-heptane and butyl acetate. This phenomenon is due to the hindrance of the formation of aminic adducts with increasing polarity of the organic phase. Therefore, the structure of the compounds formed at the interface between the aqueous phase and nheptane becomes HAE2, whereas for extraction in butyl acetate, it is HAE. The addition of 1-octanol to dichloromethane, which has the highest dielectric constant among the investigated solvents, does not influence the extraction mechanism of rosmarinic acid with Amberlite LA-2, as the structure of the extracted compound remains the same as that corresponding to the extraction without 1-octanol. In the case of reactive extraction with D2EHPA, for m = 2 in eq 13, the straight lines obtained for a 20 vol % concentration 13792



Article

Table 4. Expressions and Values of Extraction Constants for the Studied Extraction Systems without 1-Octanola) solvent

valueb

extraction constant Amberlite LA-2 Extractant

n-heptane

KE =

n-butyl acetate dichloromethane

4.39 × 102 L2·mol−2

[HAE 2(org)] 2

[HA(aq)][E(org)]

KE =

[HAE(org)] [HA(aq)][E(org)]

KE =

[HAE(org)] [HA(aq)][E(org)]

88.23 L·mol−1 1.64 × 102 L·mol−1

D2EHPA Extractant n-heptane

KE = n-butyl acetate

KE = dichloromethane

KE = a

8.25 × 10−4

[HAE 2(org)][H+(aq)]2 2+

2

[H3A (aq)][E(org)]

1.46 × 10−3

[HAE 2(org)][H+(aq)]2 2+

2

[H3A (aq)][E(org)]

2.08 × 10−3

[HAE 2(org)][H+(aq)]2 2+

2

[H3A (aq)][E(org)]

b

Alcohol volumetric fraction = 0.2. Including units.

different dielectric constants (n-heptane, n-butyl acetate, and dichloromethane), with and without 1-octanol, indicated that the extraction process occurs by means of an interfacial reaction between the solute and the extractant. The mechanism of the interfacial reaction is controlled by the extractant type and organic-phase polarity. Regardless of the used solvent, the maximum efficiency for extraction with Amberlite LA-2 was reached at pH 2, whereas for extraction with D2EHPA, it occurred at pH 3. Considering the optimum pH values for the two extractants, the efficiency of extraction with D2EHPA was 4−7% higher. However, the mechanism of interfacial reaction depends on the type of extractant and solvent polarity. Thus, in the case of extraction with Amberlite LA-2 in the absence of 1-octanol, the reduction of the dielectric constant of the organic phase promotes the formation of amine adducts and the modification of the interfacial equilibrium expression. For this reason, the structure of the interfacial hydrophobic compounds formed by rosmarinic acid and aminic extractant was changed from HAE for dichloromethane to HAE2 for butyl acetate and HAE4 for nheptane. When 1-octanol was added to the extraction system, because of the increase in the organic-phase polarity, the formation of aminic adducts was hindered, and the structure of the interfacial complexes became HAE for dichloromethane and butyl acetate and HAE2 for n-heptane. Independent of the solvent polarity or the presence 1-octanol in the organic phase, the extraction of rosmarinic acid with D2EHPA occurs by means of the interfacial formation of a hydrophobic compound of the type HAE2. The value of the extraction constant is influenced by extraction mechanism. For extraction with Amberlite LA-2, the highest values of extraction constant were obtained when aminic adducts formed, at lower polarity of the organic phase. In contrast, for extraction with D2EHPA, an increase of the organic-phase polarity positively influenced the value of the extraction constant.

of 1-octanol in the solvents are plotted in Figure 10. Similarly to the reactive extraction of rosmarinic acid with D2EHPA in the absence of 1-octanol, two extractant molecules per solute molecule participate in the formation of the interfacial hydrophobic compound (Table 2). This result confirms that interfacial solvated complexes do not form in the reactive extraction with D2EHPA. Being directly related to the mechanism of reactive extraction, the extraction constant values for the two extractants and three solvents were modified by addition of 1-octanol to the organic phase (Table 4). As in the case of extraction without 1-octanol, for the reactive extraction with Amberlite LA-2, the values of KE suggest the importance of the solvation of the interfacial compound by the extractant molecules in moving the interfacial equilibrium toward the formation of hydrophobic rosmarinic acid−aminic extractant adducts. Therefore, compared to the extraction systems without 1-octanol, the reduction of the number of extractant molecules included in the structure of the interfacial complex in the presence of 1-octanol determines a significant decrease of the extraction constant for butyl acetate and n-heptane. Moreover, the highest values of KE obtained for the extraction systems with alcohol correspond to the formation of an interfacial adduct containing two aminic molecules, namely, for extraction in n-heptane. If the interfacial reaction mechanism is not changed in the presence of 1octanol, as in the case of extraction in dichloromethane, the presence of alcohol exhibits a favorable effect on the extraction constant. The same positive influence of 1-octanol on the extraction constant was also observed for the extraction with D2EHPA, because the extraction mechanism was not changed by addition of this alcohol to the organic phase. Thus, KE increases for all solvents compared to the extraction without 1-octanol. The most significant increase of KE was found for extraction in nheptane, the solvent with the lowest polarity: upon addition of 20 vol % 1-octanol, KE was enhanced by a factor of 4.13, compared to 2.48 for dichloromethane.

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4. CONCLUSIONS The study on reactive extraction of rosmarinic acid with Amberlite LA-2 and D2EHPA dissolved in three solvents with

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Notes

(21) Baskan, S.; Oztekin, N.; Bedia Erim, F. Determination of Carnosic Acid and Rosmarinic Acid in Sage by Capillary Electrophoresis. Food Chem. 2007, 101, 1748. (22) Shen, D. Development of Anti-Inflammatory Agents from the Aromatic Plants, Origanum spp. and Mentha spp. and Analytical Methods on the Quality Control of Bioactive Phenolic Compounds. Ph.D. Thesis, Rutgers University, New Brunswick, NJ, 2008. (23) Caşcaval, D.; Galaction, A. I.; Nicuţa,̆ N.; Blaga, A. C. Selective Separation of Gentamicins from the Biosynthetic Mixture by Reactive Extraction. Sep. Purif. Technol. 2007, 57, 264. (24) Caşcaval, D.; Poştaru, M.; Galaction, A. I.; Kloetzer, L.; Blaga, A. C. Fractionation of Carboxylic Acids Mixture Obtained by P. acidipropionici Fermentation Using Pertraction with Tri-n-Octylamine and 1-Octanol. Ind. Eng. Chem. Res. 2013, 52, 2685. (25) Weast, R. C., Ed. Handbook of Chemistry and Physiscs, 54th ed.; CRC Press: Boca Raton, FL, 1974. (26) Rein, M. J.; Heinonen, M. J. Stability and Enhancement of Berry Juice Color. Agric. Food Chem. 2004, 52, 3106. (27) ADC/I-Lab 2.0; Advanced Chemistry Development, Inc.: Toronto, ON, Canada, 2010−2013; available at https://ilab.acdlabs. com/iLab2 (accessed March 2013). (28) Schuegerl, K. Solvent Extraction in Biotechnology; SpringerVerlag: Berlin, 1994. (29) Caşcaval, D.; Galaction, A. I.; Turnea, M. Study of the Influence of Solute and Carrier Characteristics on Facilitated Pertraction Mechanism in Pseudosteady-State Conditions. J. Membr. Sci. 2009, 328, 228. (30) Prezhdo, V. V.; Jagello, M.; Melnik, I. I.; Prezhdo, M. V. Solvent Effects in Extraction of Carboxylic Acids. Sep. Sci. Technol. 2002, 3, 2875. (31) Uslu, H. Distribution of Gibberellic Acid from the Aqueous Phase to the Organic Phase. J. Chem. Eng. Data 2012, 57, 902.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Grant ID PN-II-ID-PCE-2011-30088 authorized by The National Council for Scientific Research - Executive Unit for Financing Higher Education, Research, Development, and Innovation (CNCS-UEFISCDI)

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