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Application of ion exchange to purify acarbose from fermentation broths
Biochemical Engineering Journal 40 (2008) 130–137
Application of ion exchange to purify acarbose from fermentation broths Juan F. Rodriguez ∗ , Antonio De Lucas, Manuel Carmona, F´atima Ca˜nas Department of Chemical Engineering, University of Castilla-La Mancha, Avda. de Camilo Jos´e Cela s/n, 13004 Ciudad Real, Spain Received 11 July 2007; received in revised form 27 November 2007; accepted 28 November 2007
Abstract Acarbose is conventionally used to reduce the insuline consumption of the diabetic patients. This compound is an oligosaccharide with the general formulae C25H43NO18 and obtained from fermentation processes by certain strains of Actinoplanes Utahensis. After the fermentation process, the acarbose has to be isolated from the fermentation broth where is accompanied of a large amount of substances, such as substrates, intermediate metabolites, proteins and different salts. Four strong acid resins considering geliform and macroporous matrix types in aqueous and organic media have been tested in order to reach an easy and selective separation process. According to the experimental data, the Finex CS10GC (a gel strong cationic ion exchanger) presented the maximum acarbosa uptake and also the highest rate of ion exchange in water. The best behavior in non-aqueous media was observed with the Purolite CT151 (macroporous ion exchanger) but its maximum capacity of ion exchange was really lower than that exhibited by the Finex CS10GC resin in aqueous media. These results suggest that the acarbose removal from fermentation broths must be carried out in aqueous media to ensure the maximum usage of the resin uptake capacity. The results obtained provide a significant insight into the main equilibrium phenomena that takes place depending on the characteristics of the liquid phase. Finally, the elution of acarbose from the resin can be accomplished of a selectivity way by using a solution of 2.25N of HCl. The proposed separation method seems to be technically and economically feasible. © 2007 Elsevier B.V. All rights reserved. Keywords: Acarbose; Ion exchange; Adsorption; Kinetics; Regeneration
1. Introduction Acarbose is an oral alpha-glycosidase inhibitor used in the management of non-insulin-dependent diabetes mellitus (NIDDM). This amino sugar inhibits the final step in the digestion of complex carbohydrates to absorbable monosaccharides and consequently retards the absorption of carbohydrates from the small intestine. As a consequence, glucose coming from these carbohydrates goes to blood slowly. Thus, acarbose is usually used as a component of different commercial antidiabetic drugs to treat the type II diabetes mellitus (T2DM) [1–3]. This complex oligosaccharide is obtained by fermentation and several impurities remain in the broths at the end of the process such as sugars, different salts and metabolic products [4].
∗
Corresponding author. Tel.: +34 902204100; fax: +34 926 295318. E-mail address: [email protected] (J.F. Rodriguez).
1369-703X/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2007.11.025
Acarbose presents a solubility of 1.4 kg/l at 20 ◦ C and two pKa values 5.1 and 12.39 [5]. Due to the presence of an intramolecular nitrogen in its structure (Fig. 1), the acarbose exhibits a certain basic character, allowing its recovery by an ion exchange process with a cationic resin. The acid character of acarbose can be attributed to the dissociation of some of the hydroxyl groups of the molecule. Different methods have been applied in literature to separate the acarbose from fermentation broths and all of them are based on ion exchange using cationic resins [4,6–11]. Nevertheless, the high-molecular weight of the acarbose molecule together with the presence in the broths of a large variety of cations and metabolites (proteins, acarbose derivatives, etc.) make its high purity separation by ion exchange very difficult. The whole acarbose purification process involves several treatments in non-aqueous media. The lower solubility that the accompanying substances exhibit in alcohols or acetone allows work with less unpurified solutions. Up to now an accurate selec-
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by atomic absorption spectrometry (AAS) with a Varian 220 AS spectrophotometer. The arithmetic average particle diameter was determined using low-angle laser light scattering (LALLS) in a Mastersizer 2000, while the H+ concentration was obtained with a GLP 21 Crison pH meter. The standard uncertainty and reproducibility of measurements was found to be ±0.1%. 2.4. Equilibrium experiments Fig. 1. Acarbose structure.
tion of an ion exchanger that presents high exchange rate, large ion exchange capacity and high selectivity for the separation of acarbose from fermentation broths has not been reported in literature. According to the above mentioned, the main objective of this work was to find the best resin to carry out the acarbose separation from fermentation broths. This research presents the isotherms and kinetics of ion exchange of acarbose using four different ion-exchange resins in aqueous and non-aqueous (methanol) media. Finally, the acarbose elution curve is included to demonstrate the feasibility of this separation procedure. 2. Experimental
The experimental set consisted of nine 250 ml Pyrex containers, hermetically sealed and submerged in a temperaturecontrolled thermostatic bath. The temperature was kept constant at 25 ± 2 ◦ C. Different known masses of resin, in the H+ -form and prewetted with the solvent used, were put in contact with acarbose in two different media (organic or aqueous at its natural pH). The suspension formed by the resin and solution was vigorously agitated at 200 rpm by means of a multipoint magnetic stirrer. To ensure that the equilibrium was achieved the experiments were left under stirring overnight. At the end of this period, the mixtures were filtered to remove the ion exchange resin and the filtrate was analyzed for acarbose content by HPLC as it was described above. The resin phase concentration in equilibrium with the liquid phase was obtained by means of the following mass balance equation. (C0 − C∗ )V × 1000 WMeq
2.1. Chemicals
n∗ =
Acarbose (C25 H43 NO18 ) with a purity higher than 97.1% and d(+)-maltose monohydrate (C12 H22 O11 ·H2 O) with a purity higher than 90%, were supplied by Sigma-Aldrich. Calcium chloride, hydrogen chloride (37%, w/w), methanol (99.5%, w/w) and sodium hydroxide were supplied by Panreac. Demineralised water was conventionally treated in our laboratory (final conductivity less than 1 s/cm).
where C0 and C* are the initial and equilibrium concentrations of acarbose in the liquid phase (g/l), respectively, n* denotes the resin phase equilibrium concentration of acarbose (mequiv./g dry resin), V and W are the initial volume of acarbose solution in litres and the weight of dry ion exchange resin in grams, respectively. Meq is the molecular weight of the acarbose (645 g/equiv.).
2.2. Ion exchange resins
2.5. Batch kinetics studies
Four commercially sulfonic polystyrene-divinylbenzene strong acid cationic resins were used in this study. Their relevant properties are given in Table 1. An especial mention is need for the resin Purolite SST60, it owns a shallow shell core in which only a fraction of the particle is functionalized (being inert the particle core; only the outer shell is active). Finex resins are relatively new in the market and they are especially indicated to sugar treatments. The resins were pre-treated and converted to the H+ -form by repeated treatments in a column with 1.0N NaOH and 1.0N of HCl solutions, and thoroughly rinsed with distilled-deionised water with conductivity smaller than 1 s/cm [12–13].
Intraparticle diffusion dynamics were studied by measuring the rate of acarbose uptaking from the liquid phase by the resin in a well-mixed tank at each time. The reactor was hermetically sealed and equipped with four baffles, a standard turbine stirrer with six flat blades, and a heating system formed by a temperature-controlled thermostatic bath. The temperature was kept at 30 ± 2 ◦ C. A high agitation rate was applied in order to ensure that the film mass transfer resistance was negligible and thus, only diffusion within the resin particle was the controlling step. In a typical experiment, 10 g of the ion-exchange prewetted resin was placed in contact with 1 l of acarbose solution with a concentration of 2 g/l. The mixture was stirred for 2 h and small aliquots of 2 ml were periodically taken out, and the acarbose content was analyzed and also, this amount was not considered in the liquid phase for the following steps. On this way, the amount of acarbose taken out in each sample does not present any effect on the diffusion coefficients obtained.
2.3. Analytical methods Acarbose and maltose concentration were measured by HPLC with a detector of refraction index and using a nucleosil amino column. The calcium concentration was measured
(1)
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Table 1 Resin properties Resin
Structure Supplied form Active group Matrix type Description Capacity (equiv./l) Technology
Finex CS9GC
Finex CS10GC
Geliform
Geliform
Purolite SST60
Purolite CT151
Geliform
Macroporous
Styrene
Styrene-DVB
1.6 Shallow shell resin
1.7 Catalytic resin
Na+ –SO3 H Styrene-DVB (4.5%) 1.5
Styrene-DVB (5%) Strong acid type 1.5
3. Results and discussion
media as a pH function:
3.1. Equilibrium
R − NH+ ←→ R N + H+
3.1.1. Equilibrium in aqueous phase and its theoretical treatment Fig. 2 shows the isotherms obtained for acarbose removal in aqueous media. As can be seen, the acarbose equilibrium obtained for the four resins studied is quite favourable. The great differences between the Finex to Purolite resins are related with the resin properties since the greater flexibility of the less crosslinked resins (geliform) favours the accessibility of large ions into the swelling resin beads. The Purolite SST60 exhibits the lowest capacity. This behavior can be attributed to the non-functionalized core which is non-polar and thus the resin swelling is really poor avoiding the mobility of the acarbose. In this way, the greater rigidness of the macroporous matrix (Purolite CT151) limits the swelling of the resin and the accessibility of resin gel phase thus the capture of this large molecule takes place on the active sites located on the macropore surface. When water is used as solvent, the acarbose dissociation takes place according to its pKa values (5.1 and 12.39). The following dissociation reactions of acarbose can be proposed in aqueous
R N
Fig. 2. Isotherms of ion exchange of acarbose on four different ion exchangers in aqueous media. T = 25 ◦ C, V = 0.1 l, W = 0.5 g and C0 (acarbose) = 1–9 g/l.
pKaI =5.1
pKaII =12.39
←→
R N− + H +
(2) (3)
where [R N] is the undissociated acarbose, [R –NH+ ] the cationic form of acarbose and [R N− ] is the anionic form of acarbose concentrations, respectively. When the acarbose content is analyzed, the value measured includes the three forms of acarbose, i.e.: [R Nmeasured ] = [R N] + [R –NH+ ] + [R N− ]
(4)
where [R Nmeasured ] is the total amount of acarbose that is in the solution in any of the three forms. The concentrations of the different acarbose forms in solution as a function of the total concentration of acarbose and the pH of the liquid solution can be obtained combining the last three equations. The change with pH of the relative concentrations of three species, undissociated acarbose, cationic acarbose and anionic acarbose, with respect to the total concentration of acarbose in solution is presented in Fig. 3. The distribution curves show that the anionic acarbose form is only present at pH value greater than 10. Thus, it would be expected that at the experimental conditions when a strong acid exchanger is involved the undissociated and the cationic acarbose forms would be the main species in solution and the concentration of the anionic form of acarbose is almost negligi-
Fig. 3. Theorethical concentration distribution of acarbose species in the liquid phase at 25 ◦ C.
J.F. Rodriguez et al. / Biochemical Engineering Journal 40 (2008) 130–137
ble. According to the above comments, the following reaction scheme can be proposed to explain the equilibrium between the solid and liquid acarbose contents: The cationic form of acarbose should be loaded into the resin by a conventional ion exchange mechanism: R –NH+ + R–SO3 − H+ ⇔ R–SO3 HN–R + H+
(5)
Taking into account that the acarbose presents a group –NH with a pair of odd electrons, it is also possible that the chemical adsorption takes place also on the active acid sites of the strong acid resins used like an acid/base neutralization reaction [14]: R N + R–SO3 − H+ ⇔ R–SO3 HNR
(6)
In this way, R –NH+ and R N can be removed by the resin occupying one active centre, releasing one or zero hydrogen ions from the resin active sites, respectively. Thus chemical adsorption and ion exchange phenomena could occur in a similar way to that of nicotine and other alkalis with amine group removed by strong acid resins as it has been described in literature [14–18]. If the ideal mass action law it is used to represent the above solid–liquid equilibriums, we can obtain: For ion exchange: KIE =
IE [H+ ] qRSO 3 HNR
IE qRSO [R − NH+ ] 3H
(7)
IE where qRSO is the equilibrium concentration of acarbose in 3 HNR IE the solid phase for ion exchange, qRSO the available sites of 3H + the resin for ion exchange, [H ] the protons concentration in the liquid phase and KIE the ion exchange equilibrium constant. On the other hand, for chemical adsorption (Eq. (6)):
KAD =
AD qRSO 3 HNR
AD qRSO [R N] 3H
(8)
133
On the other hand, the following mass balance can be drawn: IE AD + qRSO = ([R NInitial ] − [R Nmeasured ]) × qRSO 3 HNR 3 HNR
V W (12)
where [R NInitial ] is the initial acarbose concentration, V is volume of the solution and W is the weight of dry resin. The Eqs. (11) and (12) indicate the global amount of acarbose removed from the liquid phase. Equaling Eqs. (11) and (12), one obtains: KIE [R − NH+ ]/[H+ ] + KAD [RN] W Q 1 + KIE [R − NH+ ]/H+ + KAD [RN] V = [R NInitial ] − [R Nmeasured ]
This equation describes the removal of acarbose by the solid in terms of three unknown parameters (KIE , KAD and Q) related to ion exchange and chemical adsorption. Experimental data were fitted to the Eq. (13) by a non-linear least-squares regression procedure (Fig. 4). It was confirmed that the measured and the calculated values of pH at the equilibrium were quite similar. The concentrations of the both acarbose forms in liquid phase at each point were obtained by using the Eqs. (2)–(4) and the pH measured. The values of the parameters obtained by fitting the experimental data to this model are shown in Table 2. According to these fitted parameters, it is possible to conclude that only the ion exchange phenomena takes place since KAD values for all the resin tested are zero. The reason for this behavior is that the neutral acarbose represents only the 15% of the total acarbose content at pH < 4.5. Besides, this is the maximum pH value obtained at the experimental conditions. Taking into account the Eq. (2) as higher is the acarbose uptake by the resin greater is the amount of H+ released to the solution decreasing the pH of it. Experimental results con-
AD is the equilibrium concentration of acarbose where qRSO 3 HNR AD the available sites of in the solid phase for adsorption, qRSO 3H the resin for adsorption and KAD the adsorption equilibrium constant. If it is considered that all the active centres could be used indistinctly for adsorption or ion exchange, it can be written: IE AD qRSO = qRSO = qRSO3 H 3H 3H
(9)
According to the above comments, the total capacity of the different resins for acarbose uptake could be expressed as IE AD + qRSO + qRSO3 H Q = qRSO 3 HNR 3 HNR
(10)
where Q is the total solid-phase capacity and qRSO3 H is the total available active sites for ion exchange or chemical adsorption. Introducing Eqs. (7)–(9) in the Eq. (10), one obtains the following one: IE AD qRSO + qRSO 3 HNR 3 HNR
=Q
[KIE [R − NH+ ]/[H+ ] + KAD [RN]] [1 + KIE [R − NH+ /[H+ ]] + KAD [RN]]
(13)
(11) Fig. 4. Reproducibility of the experimental data by the theoretical model.
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Table 2 Equilibrium parameters of acarbose on ion exchange resins in aqueous media.
Table 3 Equilibrium parameters of acarbose on ion exchange resins in organic media
Resins
Chemical adsorption
Equilibrium parameters
Finex CS9GC Finex CS10GC Purolite SST 60 Purolite CT 151 Av. dev. (%) = mental data.
Q (mol/kg)
KIE
3.58 3.92 1.44 2.03
0.0456 0.0830 0.0965 0.0335
m i=1
ABS
exp
theor qObs −qObs i exp qObs i
i
Av. dev. (%) KID
0.0
(dm3 /mol) 11.2 10.6 8.4 7.0
Resin
KAD (dm3 /mol)
Q (mol/kg)
Finex CS9GC Finex CS10GC Purolite SST 60 Purolite CT 151
1.767 0.392 0.338 0.735
0.424 0.921 0.365 1.270
Swelling (%) (water/methanol) 17.5 18.9 −7.2 8.3
× 100/m; m: total number of experi-
firm that the acid pH favours the acarbose removal by this type of resins. On the other hand, Q values obtained indicate that the Finex resins should be used in an aqueous medium and mainly the Finex CS10GC with a value of 3.92 mol kg−1 and an equilibrium constant greater than the Finex CS9GC resin presents. 3.1.2. Equilibrium in organic phase and its theoretical treatment Fig. 5 shows the isotherms obtained for acarbose removal in methanol. As can be seen, all the equilibriums seem to be favourable, indicating a high affinity of the resins for this compound. Although the methanol (amphoteric solvent) is partially ionized into anions and cations its ionic product is lower than that of water [19]. Thus, it can be expected that the non-dissociated form of acarbose will be the main specie in the organic phase. According to the above comments, the chemical adsorption would be the main mechanism of acarbose removal by these types of resins. The experimental data were fitted to the Eq. (13) doing KIE values equals to zero. Adsorption parameters are shown in Table 3
and also the swelling ratio of the particles in water with respect to methanol media. As can be seen, the macroporous resin exhibits the maximum capacity although its swelling is lower than that of the geliform resins. The analogous results obtained in both media for the maximum capacity of Purolite CT 151 indicate that the removal of large molecules takes place on the active sites located on the macropores as it was discussed above. On the other hand, the bead size of the geliform resins shrinks in methanol media except the non-functionalized core of Purolite SST60. The decrease of the exchange capacity in methanol confirms that the available capacity decreases as the polarity of the pure solvent decreases, as expected [20,21]. With the aim of comparing the available ion exchange capacity of each resin for the uptake of acarbose the maximum ion exchange capacity in aqueous media were obtained by titration the resin active centres with a NaOH solution. The results are showed in Table 4. As can be seen in Table 4 not all the fixed charges of the resins are available for acarbose uptake indicating that ion exclusion inside the resin takes place due to the big size of acarbose molecule [21]. The decrease of the available capacity for acarbose uptake is larger in methanol than water, confirming that the available capacity decreases as the polarity of the pure solvent decreases as it was discussed above. In this case the macroporous resin exhibits the lower reduction in capacity. This can be explained taking into account that the acarbose removal is happening mainly in the surface of macroporous and so that its greater crosslinking make more rigid the matrix and is not affected by changing the solvent. The maximum load capacities obtained in aqueous media were several times higher than the values obtained in methanol as it was expected. In methanol media the available resin capacity Table 4 Saturation capacity with acarbose (Q) and the total capacity obtained with NaOH (n∞ ) of resins
Fig. 5. Isotherms of acarbose removal on four different ion exchangers in methanol media. T = 25 ◦ C. V = 0.1 l, W = 0.5 g and C0 (acarbose) = 1–9 g/l. Solid lines: theoretical model.
MEDIA
Resin
Q (mol/kg)
n∞ (mol/kg)
Total capacity used (%)
H2 O
Finex CS9GC Finex CS10GC Purolite SST 60 Purolite CT 151
3.58 3.92 1.44 2.03
3.680 4.304 2.613 3.058
97.8 91.1 55.1 66.4
MeOH
Finex CS9GC Finex CS10GC Purolite SST 60 Purolite CT 151
0.424 0.921 0.365 1.270
1.780 4.228 2.359 2.804
23.8 21.8 15.5 45.3
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to acarbose decreases greatly with respect to aqueous media because of the large size of acarbose and the moderate shrinkage of resin beads in methanol limits the accessibility of a large part of the resin active sites. According to maximum capacity values, Finex resins should be used in an aqueous medium and mainly the Finex CS10GC with a value of 3.92 mol kg−1 . Nevertheless, the macroporous resin Purolite CT151 could be a good choice in non-aqueous media. These results also allow to conclude that the worse resin to carry out this process is the Purolite SST60. 3.2. Kinetic A homogeneous model which assumes that a quasihomogeneous phase exists inside the solid particle was employed in previous works to obtain the effective diffusion coefficients with satisfactory results [22]. It is an useful way to obtain effective diffusion coefficients even in the case of macroporous resin in order to compare their exchange ability with other kinds of resins [23–24]. Intraparticle transport in ion exchange resins is generally controlled by the interdiffusion of counterions. The most general solution which supposes intraparticle controlled diffusion from a well-stirred solution of limited volume was given by Crank [25]: F (t) =
Mt C 0 − Ct = M∞ C0 − C ∗
= 1−
∞ 6α(α + 1) exp (−p2 Deff t/R2 ) n
n=1
9 + 9α + 9p2n α2
P
3pn 3 + αp2n
(15)
and α=
VC0 Wn∞
Table 5 Intraparticle effective diffusion coefficients of acarbose into the resins studied MEDIA
Resin
D (m)
Deff (×10−8 ) (cm2 /s)
H2 O
Finex CS9GC Finex CS10GC Purolite SST 60 Purolite CT 151
369.229 369.366 594.914 795.307
27.8 93.1 34.0 58.1
MeOH
Finex CS9GC Finex CS10GC Purolite SST 60 Purolite CT 151
349.856 348.661 610.01 774.279
1.35 4.17 6.65 15.4
(14)
where Mt and M∞ are the amounts of acarbose in the particles at time t and at the infinite, respectively, and pn are non-zero roots and defined by the following equations: tan (pn ) =
Fig. 6. Kinetics of acarbose uptake in aqueous media. V = 1 l, W = 10 g, T = 30 ◦ C, speed = 500 rpm and C0 (acarbose) = 2 g/l. Solid lines: predicted curves.
takes place in this phenomenon favours the ionic mobility of the ions respect the neutral species. The model proposed was able to fit properly the experimental data and the results are shown in Table 5. According to the results, the diffusion coefficients of acarbose in an aqueous medium are at least one order of magnitude higher than in methanol for the FINEX resins [27]. This is in good agreement with its greater swelling and accessibility. The greater swelling of unfunctionalized core Purolite SST60 in methanol
(16)
Intraparticle effective diffusivity values (Deff (cm2 /s)) were determined by fitting the experimental data to Eq. (14) using a non-linear regression method based on the Marquardt’s algorithm [26]. The average particle diameter of the resin in each kind of mixture was measured by Masterziser 2000. In order to test which of the solvents (H2 O, CH3 OH) had the strongest effect on the elimination rate a set of experiments of acarbose removal were carried out in both media. Figs. 6 and 7 show the kinetic for all resins in the both mediums. While in aqueous media the equilibrium is attached in 20 min in methanol media are necessary almost 2 h. As it was discussed above as more polar the solvent is the resin bead experiment a grater swelling and thus easier is the access of the acarbose to active centres. Besides, in aqueous media the main transport mechanism is the ion exchange and the electrical potential that
Fig. 7. Kinetics of acarbose uptake in methanol media. V = 1 l, W = 10 g, T = 30 ◦ C, speed = 500 rpm and C0 (acarbose) = 2 g/l. Solid lines: predicted curves.
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Fig. 8. Concentration of maltose in the effluent stream during the loading procedure on the resin bed of H+ -form Finex CS10GC of the artificial fermentation broth. C0 (acarbose) = 2 g/l, C0 (maltose) = 2.22 g/l, C0 (calcium) = 0.22 g/l and F = 2 l/h. Bed diameter = 40 mm and bed length = 465 mm.
media does not produce an increase of diffusion coefficient. It can only be explained if it is assumed that the functionalized shell shrink in methanol making difficult the diffusion while at the same time the inner core is swelled by the solvent. Finally the macroporous resin Purolite CT151 (that usually has a high DVB content) shows the smaller change of the diffusion coefficient between water and methanol media confirming again that its more rigid matrix remain almost unaffected by the solvent [16,27–30].The effective intraparticle diffusivities obtained are in the range found in literature. Deff = 1 × 10−6 cm2 /s for inorganic ions and Deff = 1 × 10−8 cm2 /s for organic molecules with high molecular weight [14,31,32]. Comparing between them, the macroporous resin Purolite CT 151 exhibits a diffusion coefficient in the same range than the geliform resins in aqueous media. Nevertheless, the diffusion coefficient of acarbose in methanol is almost an order of magnitude greater for the macroporous resin. That difference in methanol can be explained taking into account that the large acarbose ion diffuses better in the open macroporous structure of the Purolite CT 151 resin than in the shrunk polymer network of the other geliform resins [33]. According to Table 5, the fastest mobility of the acarbose is into the Finex CS10GC in aqueous medium. Thus, based on the both, equilibrium and kinetics studies, it may be stated that Finex CS10GC could be selected as the best exchanger to carry out the acarbose purification in aqueous medium. Nevertheless, if in a previous purification step of the fermentation broths a non-aqueous solvent was employed the resin Purolite CT 151 could be the best choice. 3.3. Fixed bed column experiments The artificial fermentation broth used as feed stream to the bed resin of Finex CS10GC was constituted of acarbose (2 g/l), maltose (2.22 g/l) and a small amount of calcium (0.22 g/l). In each loading cycle no more than 2 l of the artificial broth were passed through the column being this amount not enough to reach the breakthrough point of the resin bed. Consequently, all the acarbose and calcium were retained in the bed and only maltose passed through the bed with only slight changes in con-
Fig. 9. Elution curves of acarbose and calcium from Finex CS10GC with HCl 2.25N. F = 2 l/h. Bed diameter = 40 mm and bed length = 465 mm.
centration with respect to the inlet solution as can be seen in Fig. 8. At the end of the charge step the column was washed with water to eliminate the compounds not retained by the resin. 3.3.1. Loading and elution test Two litres of an aqueous artificial fermentation broth were pumped through the fresh resin at a flow rate of 2 l/h after that, the column was washed with water to eliminate the compounds not retained by the resin. According to the analysis, the acarbose and also the Ca2+ ions fed were completely retained by the resin while the maltose was not removed from the liquid phase. The elution process was carried out with hydrochloric acid as regenerant agent. It was found that a concentration of HCl (2.25N) allows to release the both species acarbose and Ca2+ ions from the resin. Besides, according to Fig. 9 a selective elution of both species (acarbose and Ca2+ ) can be obtained. The acarbose does not appear in the effluent until the almost the complete elution of Ca2+ ions and thus, it is possible to draw that the acarbose elutes selectively after the rest of metal cations present in the resin and represented in this experiment by Ca2+ ion. The general trend of the elution process seems to be clear. These results suggest that Finex CS10GC could definitively be used to separate the acarbose from fermentation broths allowing to reach a good separation from other cations in aqueous solutions. 4. Conclusions The equilibrium for the acarbose uptake from these mixtures is favourable. A good fitting of the experimental equilibrium data was obtained by an ideal mass action law model. According to the experimental results, the acarbose purification should be carried out in aqueous medium and use for this purpose the resin Finex CS10GC. This geliform strong cation exchanger showed the biggest capacity and the highest rate for acarbose removal between the studied resins. The macroporous resin Purolite CT151 could be a good choice in non-aqueous media and the Purolite SST60 resin is not appropriated for this purification purpose. Finally, the recovery of acarbose from dilute aqueous solutions by ion exchange using Finex CS10GC as exchanger in the H+ -form appears to be a suitable technology.
J.F. Rodriguez et al. / Biochemical Engineering Journal 40 (2008) 130–137
References [1] M.J. Kim, S.B. Lee, H.S. Lee, S.Y. Lee, J.S. Baek, D. Kim, T.W. Moon, J.F. Robyt, K.H. Park, Comparative study of the inhibition of ␣glucosidase, ␣-amylase, and cyclomaltodextrin glucanosyltransferase by acarbose, isoacarbose, and acarviosine-glucose, Arch. Biochem. Biophys. 371 (1999) 277–283. [2] M. Taifo, L. Sungsook, H.G. Floss, The byosinthesis of acarbose and validamycin, Chem. Rec. 1 (2001) 300–310. [3] G. Jermendy, Can type 2 diabetes mellitus be considered presentable? Diabetes Res. Clin. Pract. 68S1 (2005) S73–S81. [4] E. Rauenbusch, Highly pure acarbose, US Patent 4,904,769, 1990. [5] Y. Daali, S. Cherkaoui, X. Cahours, E. Varesio, J.L. Veuthey, High performance liquid chromatography of acarbose and its metabolite on porous graphitic carbon column, J. Sep. Sci. 25 (2005) 280–284. [6] W. Frommer, B. Junge, U. Keup, L. M¨uller, W. Puls, D. Schmidt, Amino sugar derivatives, US Patent 4,062,950, 1977. [7] C. Hong, K. Kim, B. Choi, G. Choi, A process for preparing acarbose with high purity, WO 01/30796 A1, 2001. [8] V. Keri, L. Deak, Method for purification of acarbose, US Patent 0,111,320 A1, 2002. [9] P. Lange, E. Rauenbusch, Process for the purification of acarbose with polymers, US Patent 4,767,850, 1998. [10] C. Lawton, A process for the purification of acarbose, WO 99/07720, 1998. [11] E. Rauenbusch, D. Schmidt, Process for isolating glucopyranose compound from culture broths, US Patent 4,174,439, 1979. [12] E. Costa, A. De Lucas, M.E. Gonz´alez, Ion exchange. Determination of interdiffusion coefficients, Ind. Eng. Chem. Fundam. 23 (1984) 400–405. [13] A. De Lucas, J. Zarca, P. Ca˜nizares, Ion-exchange equilibrium of Ca2+ , Mg2+ , K+ , Na+ and H+ ions on Amberlite IR-120: experimental determination and theoretical prediction of the ternary and quaternary equilibrium data, Sep. Sci. Technol. 27 (1992) 823–841. [14] H. Yoshida, K. Shimizu, T. Kataoka, Adsorption of amine and paints on H-form resin from electrodeposition wastewater, AIChE J. 36 (1990) 1815–1820. [15] A. De Lucas, P. Ca˜nizares, M.A. Garc´ıa, J. G´omez, J.F. Rodr´ıguez, Recovery of nicotine from aqueous extracts of tobacco wastes by an H+ -form strong-acid ion exchanger, Ind. Eng. Chem. Res. 37 (1998) 4783–4791. [16] S. Melis, J. Markos, G. C˜ao, Ion-exchange equilibria of amino acid on a strong acid resin, Ind. Eng. Chem. Res. 35 (1996) 1912–1920. [17] H. Yoshida, D. Ruthven, Adsorption of gaseosous ethylamine on H-form strong-acid ion exchangers, AIChE J. 35 (1989) 1869–1875.
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[18] H. Yoshida, K. Shimizu, T. Kataoka, Recovery of amine and paints from electrodeposition wastewater by an H-form ion exchanger: desorption process, Ind. Eng. Chem. Res. 31 (1992) 934–941. [19] C. Reichardt, Solvent and Solvents Effects in Organic Chemistry, VCH, Marburg, 1988. [20] A. De Lucas, P. Ca˜nizares, J.F. Rodr´ıguez, I. Gracia, Potassium removal from water–methanol–polyol mixtures by ion exchange on amberlite 252, Chem. Eng. J. 66 (1997) 137–147. [21] A. De Lucas, J.L. Valverde, M.C. Romero, J. G´omez, J.F. Rodr´ıguez, The ion exchange equilibrium of Na+ /K+ in nonaqueous and mixed solvents on a strong acid cation exchanger, Chem. Eng. Sci. 57 (2002) 1943– 1954. [22] A. De Lucas, P. Ca˜nizares, J.F. Rodr´ıguez, Ion exchange kinetics for the removal of potassium from crude polyols on strong acid resins, Sep. Sci. Technol. 32 (1997) 1805–1820. [23] F. Helfferich, Ion Exchange, McGraw Hill Book Co. Inc., New York, 1962. [24] T. Huang, K. Li, Ion-exchange kinetics for calcium radiotracer in batch system, Ind. Eng. Chem. Fundam. 12 (1973) 50–55. [25] J. Crank, The Mathematics of Diffusion, Clarendon Press, Oxford, 1975. [26] D.W. Marquard, An algorithm for least-squares estimation of nonlinear parameters, J. Soc. Ind. Appl. Math. 11 (1963) 471–481. [27] G. Marcelin, D.C. Cronauer, R.F. Vogel, M.E. Prudich, J. Solash, Shale oil denitrogenation with ion exchange. 2. Evaluation of ion-exchange adsorbents and resin treatment procedures, Ind. Eng. Chem. Proc. Des. Dev. 25 (1986) 747–756. [28] T.Y. Yan, P. Shu, Regeneration of ion-exchange resin in nonaqueous media, Ind. Eng. Chem. Res. 26 (1987) 753–755. [29] J.F. Rodr´ıguez, A. De Lucas, J.R. Leal, J.L. Valverde, Determination of intraparticle diffusivities of Na+ /K+ in water and water/alcohol mixed solvents on a strong acid cation exchanger, Ind. Eng. Chem. Res. 41 (2002) 3019–3027. [30] M. Per´ez-Collado, C. Molero, F. Larrondo, P. Sanchez, L. Rodriguez, J.F. Rodr´ıguez, Removal of caesium catalyst from polyols by ion exchange on amberlite 252, React. Funct. Polym. 64 (2005) 139–150. [31] M.J. Slater, The Principles of Ion Exchange Technology, Butterworth–Heinemann, Oxford, 1991. [32] H. Yoshida, M. Yoshikawa, T. Kataoka, Parallel transport of BSA by surface and pore diffusion in strongly basic chitosan, AIChE J. 40 (1994) 2034–2044. [33] A. De Lucas, P. Ca˜nizares, J.F. Rodr´ıguez, Removal of alkaline catalysts from polyols by ion exchange: selection of an ion exchange resin, Sep. Sci. Technol. 30 (1995) 125–140.
Application of ion exchange to purify acarbose from fermentation broths Juan F. Rodriguez ∗ , Antonio De Lucas, Manuel Carmona, F´atima Ca˜nas Department of Chemical Engineering, University of Castilla-La Mancha, Avda. de Camilo Jos´e Cela s/n, 13004 Ciudad Real, Spain Received 11 July 2007; received in revised form 27 November 2007; accepted 28 November 2007
Abstract Acarbose is conventionally used to reduce the insuline consumption of the diabetic patients. This compound is an oligosaccharide with the general formulae C25H43NO18 and obtained from fermentation processes by certain strains of Actinoplanes Utahensis. After the fermentation process, the acarbose has to be isolated from the fermentation broth where is accompanied of a large amount of substances, such as substrates, intermediate metabolites, proteins and different salts. Four strong acid resins considering geliform and macroporous matrix types in aqueous and organic media have been tested in order to reach an easy and selective separation process. According to the experimental data, the Finex CS10GC (a gel strong cationic ion exchanger) presented the maximum acarbosa uptake and also the highest rate of ion exchange in water. The best behavior in non-aqueous media was observed with the Purolite CT151 (macroporous ion exchanger) but its maximum capacity of ion exchange was really lower than that exhibited by the Finex CS10GC resin in aqueous media. These results suggest that the acarbose removal from fermentation broths must be carried out in aqueous media to ensure the maximum usage of the resin uptake capacity. The results obtained provide a significant insight into the main equilibrium phenomena that takes place depending on the characteristics of the liquid phase. Finally, the elution of acarbose from the resin can be accomplished of a selectivity way by using a solution of 2.25N of HCl. The proposed separation method seems to be technically and economically feasible. © 2007 Elsevier B.V. All rights reserved. Keywords: Acarbose; Ion exchange; Adsorption; Kinetics; Regeneration
1. Introduction Acarbose is an oral alpha-glycosidase inhibitor used in the management of non-insulin-dependent diabetes mellitus (NIDDM). This amino sugar inhibits the final step in the digestion of complex carbohydrates to absorbable monosaccharides and consequently retards the absorption of carbohydrates from the small intestine. As a consequence, glucose coming from these carbohydrates goes to blood slowly. Thus, acarbose is usually used as a component of different commercial antidiabetic drugs to treat the type II diabetes mellitus (T2DM) [1–3]. This complex oligosaccharide is obtained by fermentation and several impurities remain in the broths at the end of the process such as sugars, different salts and metabolic products [4].
∗
Corresponding author. Tel.: +34 902204100; fax: +34 926 295318. E-mail address: [email protected] (J.F. Rodriguez).
1369-703X/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2007.11.025
Acarbose presents a solubility of 1.4 kg/l at 20 ◦ C and two pKa values 5.1 and 12.39 [5]. Due to the presence of an intramolecular nitrogen in its structure (Fig. 1), the acarbose exhibits a certain basic character, allowing its recovery by an ion exchange process with a cationic resin. The acid character of acarbose can be attributed to the dissociation of some of the hydroxyl groups of the molecule. Different methods have been applied in literature to separate the acarbose from fermentation broths and all of them are based on ion exchange using cationic resins [4,6–11]. Nevertheless, the high-molecular weight of the acarbose molecule together with the presence in the broths of a large variety of cations and metabolites (proteins, acarbose derivatives, etc.) make its high purity separation by ion exchange very difficult. The whole acarbose purification process involves several treatments in non-aqueous media. The lower solubility that the accompanying substances exhibit in alcohols or acetone allows work with less unpurified solutions. Up to now an accurate selec-
J.F. Rodriguez et al. / Biochemical Engineering Journal 40 (2008) 130–137
131
by atomic absorption spectrometry (AAS) with a Varian 220 AS spectrophotometer. The arithmetic average particle diameter was determined using low-angle laser light scattering (LALLS) in a Mastersizer 2000, while the H+ concentration was obtained with a GLP 21 Crison pH meter. The standard uncertainty and reproducibility of measurements was found to be ±0.1%. 2.4. Equilibrium experiments Fig. 1. Acarbose structure.
tion of an ion exchanger that presents high exchange rate, large ion exchange capacity and high selectivity for the separation of acarbose from fermentation broths has not been reported in literature. According to the above mentioned, the main objective of this work was to find the best resin to carry out the acarbose separation from fermentation broths. This research presents the isotherms and kinetics of ion exchange of acarbose using four different ion-exchange resins in aqueous and non-aqueous (methanol) media. Finally, the acarbose elution curve is included to demonstrate the feasibility of this separation procedure. 2. Experimental
The experimental set consisted of nine 250 ml Pyrex containers, hermetically sealed and submerged in a temperaturecontrolled thermostatic bath. The temperature was kept constant at 25 ± 2 ◦ C. Different known masses of resin, in the H+ -form and prewetted with the solvent used, were put in contact with acarbose in two different media (organic or aqueous at its natural pH). The suspension formed by the resin and solution was vigorously agitated at 200 rpm by means of a multipoint magnetic stirrer. To ensure that the equilibrium was achieved the experiments were left under stirring overnight. At the end of this period, the mixtures were filtered to remove the ion exchange resin and the filtrate was analyzed for acarbose content by HPLC as it was described above. The resin phase concentration in equilibrium with the liquid phase was obtained by means of the following mass balance equation. (C0 − C∗ )V × 1000 WMeq
2.1. Chemicals
n∗ =
Acarbose (C25 H43 NO18 ) with a purity higher than 97.1% and d(+)-maltose monohydrate (C12 H22 O11 ·H2 O) with a purity higher than 90%, were supplied by Sigma-Aldrich. Calcium chloride, hydrogen chloride (37%, w/w), methanol (99.5%, w/w) and sodium hydroxide were supplied by Panreac. Demineralised water was conventionally treated in our laboratory (final conductivity less than 1 s/cm).
where C0 and C* are the initial and equilibrium concentrations of acarbose in the liquid phase (g/l), respectively, n* denotes the resin phase equilibrium concentration of acarbose (mequiv./g dry resin), V and W are the initial volume of acarbose solution in litres and the weight of dry ion exchange resin in grams, respectively. Meq is the molecular weight of the acarbose (645 g/equiv.).
2.2. Ion exchange resins
2.5. Batch kinetics studies
Four commercially sulfonic polystyrene-divinylbenzene strong acid cationic resins were used in this study. Their relevant properties are given in Table 1. An especial mention is need for the resin Purolite SST60, it owns a shallow shell core in which only a fraction of the particle is functionalized (being inert the particle core; only the outer shell is active). Finex resins are relatively new in the market and they are especially indicated to sugar treatments. The resins were pre-treated and converted to the H+ -form by repeated treatments in a column with 1.0N NaOH and 1.0N of HCl solutions, and thoroughly rinsed with distilled-deionised water with conductivity smaller than 1 s/cm [12–13].
Intraparticle diffusion dynamics were studied by measuring the rate of acarbose uptaking from the liquid phase by the resin in a well-mixed tank at each time. The reactor was hermetically sealed and equipped with four baffles, a standard turbine stirrer with six flat blades, and a heating system formed by a temperature-controlled thermostatic bath. The temperature was kept at 30 ± 2 ◦ C. A high agitation rate was applied in order to ensure that the film mass transfer resistance was negligible and thus, only diffusion within the resin particle was the controlling step. In a typical experiment, 10 g of the ion-exchange prewetted resin was placed in contact with 1 l of acarbose solution with a concentration of 2 g/l. The mixture was stirred for 2 h and small aliquots of 2 ml were periodically taken out, and the acarbose content was analyzed and also, this amount was not considered in the liquid phase for the following steps. On this way, the amount of acarbose taken out in each sample does not present any effect on the diffusion coefficients obtained.
2.3. Analytical methods Acarbose and maltose concentration were measured by HPLC with a detector of refraction index and using a nucleosil amino column. The calcium concentration was measured
(1)
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Table 1 Resin properties Resin
Structure Supplied form Active group Matrix type Description Capacity (equiv./l) Technology
Finex CS9GC
Finex CS10GC
Geliform
Geliform
Purolite SST60
Purolite CT151
Geliform
Macroporous
Styrene
Styrene-DVB
1.6 Shallow shell resin
1.7 Catalytic resin
Na+ –SO3 H Styrene-DVB (4.5%) 1.5
Styrene-DVB (5%) Strong acid type 1.5
3. Results and discussion
media as a pH function:
3.1. Equilibrium
R − NH+ ←→ R N + H+
3.1.1. Equilibrium in aqueous phase and its theoretical treatment Fig. 2 shows the isotherms obtained for acarbose removal in aqueous media. As can be seen, the acarbose equilibrium obtained for the four resins studied is quite favourable. The great differences between the Finex to Purolite resins are related with the resin properties since the greater flexibility of the less crosslinked resins (geliform) favours the accessibility of large ions into the swelling resin beads. The Purolite SST60 exhibits the lowest capacity. This behavior can be attributed to the non-functionalized core which is non-polar and thus the resin swelling is really poor avoiding the mobility of the acarbose. In this way, the greater rigidness of the macroporous matrix (Purolite CT151) limits the swelling of the resin and the accessibility of resin gel phase thus the capture of this large molecule takes place on the active sites located on the macropore surface. When water is used as solvent, the acarbose dissociation takes place according to its pKa values (5.1 and 12.39). The following dissociation reactions of acarbose can be proposed in aqueous
R N
Fig. 2. Isotherms of ion exchange of acarbose on four different ion exchangers in aqueous media. T = 25 ◦ C, V = 0.1 l, W = 0.5 g and C0 (acarbose) = 1–9 g/l.
pKaI =5.1
pKaII =12.39
←→
R N− + H +
(2) (3)
where [R N] is the undissociated acarbose, [R –NH+ ] the cationic form of acarbose and [R N− ] is the anionic form of acarbose concentrations, respectively. When the acarbose content is analyzed, the value measured includes the three forms of acarbose, i.e.: [R Nmeasured ] = [R N] + [R –NH+ ] + [R N− ]
(4)
where [R Nmeasured ] is the total amount of acarbose that is in the solution in any of the three forms. The concentrations of the different acarbose forms in solution as a function of the total concentration of acarbose and the pH of the liquid solution can be obtained combining the last three equations. The change with pH of the relative concentrations of three species, undissociated acarbose, cationic acarbose and anionic acarbose, with respect to the total concentration of acarbose in solution is presented in Fig. 3. The distribution curves show that the anionic acarbose form is only present at pH value greater than 10. Thus, it would be expected that at the experimental conditions when a strong acid exchanger is involved the undissociated and the cationic acarbose forms would be the main species in solution and the concentration of the anionic form of acarbose is almost negligi-
Fig. 3. Theorethical concentration distribution of acarbose species in the liquid phase at 25 ◦ C.
J.F. Rodriguez et al. / Biochemical Engineering Journal 40 (2008) 130–137
ble. According to the above comments, the following reaction scheme can be proposed to explain the equilibrium between the solid and liquid acarbose contents: The cationic form of acarbose should be loaded into the resin by a conventional ion exchange mechanism: R –NH+ + R–SO3 − H+ ⇔ R–SO3 HN–R + H+
(5)
Taking into account that the acarbose presents a group –NH with a pair of odd electrons, it is also possible that the chemical adsorption takes place also on the active acid sites of the strong acid resins used like an acid/base neutralization reaction [14]: R N + R–SO3 − H+ ⇔ R–SO3 HNR
(6)
In this way, R –NH+ and R N can be removed by the resin occupying one active centre, releasing one or zero hydrogen ions from the resin active sites, respectively. Thus chemical adsorption and ion exchange phenomena could occur in a similar way to that of nicotine and other alkalis with amine group removed by strong acid resins as it has been described in literature [14–18]. If the ideal mass action law it is used to represent the above solid–liquid equilibriums, we can obtain: For ion exchange: KIE =
IE [H+ ] qRSO 3 HNR
IE qRSO [R − NH+ ] 3H
(7)
IE where qRSO is the equilibrium concentration of acarbose in 3 HNR IE the solid phase for ion exchange, qRSO the available sites of 3H + the resin for ion exchange, [H ] the protons concentration in the liquid phase and KIE the ion exchange equilibrium constant. On the other hand, for chemical adsorption (Eq. (6)):
KAD =
AD qRSO 3 HNR
AD qRSO [R N] 3H
(8)
133
On the other hand, the following mass balance can be drawn: IE AD + qRSO = ([R NInitial ] − [R Nmeasured ]) × qRSO 3 HNR 3 HNR
V W (12)
where [R NInitial ] is the initial acarbose concentration, V is volume of the solution and W is the weight of dry resin. The Eqs. (11) and (12) indicate the global amount of acarbose removed from the liquid phase. Equaling Eqs. (11) and (12), one obtains: KIE [R − NH+ ]/[H+ ] + KAD [RN] W Q 1 + KIE [R − NH+ ]/H+ + KAD [RN] V = [R NInitial ] − [R Nmeasured ]
This equation describes the removal of acarbose by the solid in terms of three unknown parameters (KIE , KAD and Q) related to ion exchange and chemical adsorption. Experimental data were fitted to the Eq. (13) by a non-linear least-squares regression procedure (Fig. 4). It was confirmed that the measured and the calculated values of pH at the equilibrium were quite similar. The concentrations of the both acarbose forms in liquid phase at each point were obtained by using the Eqs. (2)–(4) and the pH measured. The values of the parameters obtained by fitting the experimental data to this model are shown in Table 2. According to these fitted parameters, it is possible to conclude that only the ion exchange phenomena takes place since KAD values for all the resin tested are zero. The reason for this behavior is that the neutral acarbose represents only the 15% of the total acarbose content at pH < 4.5. Besides, this is the maximum pH value obtained at the experimental conditions. Taking into account the Eq. (2) as higher is the acarbose uptake by the resin greater is the amount of H+ released to the solution decreasing the pH of it. Experimental results con-
AD is the equilibrium concentration of acarbose where qRSO 3 HNR AD the available sites of in the solid phase for adsorption, qRSO 3H the resin for adsorption and KAD the adsorption equilibrium constant. If it is considered that all the active centres could be used indistinctly for adsorption or ion exchange, it can be written: IE AD qRSO = qRSO = qRSO3 H 3H 3H
(9)
According to the above comments, the total capacity of the different resins for acarbose uptake could be expressed as IE AD + qRSO + qRSO3 H Q = qRSO 3 HNR 3 HNR
(10)
where Q is the total solid-phase capacity and qRSO3 H is the total available active sites for ion exchange or chemical adsorption. Introducing Eqs. (7)–(9) in the Eq. (10), one obtains the following one: IE AD qRSO + qRSO 3 HNR 3 HNR
=Q
[KIE [R − NH+ ]/[H+ ] + KAD [RN]] [1 + KIE [R − NH+ /[H+ ]] + KAD [RN]]
(13)
(11) Fig. 4. Reproducibility of the experimental data by the theoretical model.
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Table 2 Equilibrium parameters of acarbose on ion exchange resins in aqueous media.
Table 3 Equilibrium parameters of acarbose on ion exchange resins in organic media
Resins
Chemical adsorption
Equilibrium parameters
Finex CS9GC Finex CS10GC Purolite SST 60 Purolite CT 151 Av. dev. (%) = mental data.
Q (mol/kg)
KIE
3.58 3.92 1.44 2.03
0.0456 0.0830 0.0965 0.0335
m i=1
ABS
exp
theor qObs −qObs i exp qObs i
i
Av. dev. (%) KID
0.0
(dm3 /mol) 11.2 10.6 8.4 7.0
Resin
KAD (dm3 /mol)
Q (mol/kg)
Finex CS9GC Finex CS10GC Purolite SST 60 Purolite CT 151
1.767 0.392 0.338 0.735
0.424 0.921 0.365 1.270
Swelling (%) (water/methanol) 17.5 18.9 −7.2 8.3
× 100/m; m: total number of experi-
firm that the acid pH favours the acarbose removal by this type of resins. On the other hand, Q values obtained indicate that the Finex resins should be used in an aqueous medium and mainly the Finex CS10GC with a value of 3.92 mol kg−1 and an equilibrium constant greater than the Finex CS9GC resin presents. 3.1.2. Equilibrium in organic phase and its theoretical treatment Fig. 5 shows the isotherms obtained for acarbose removal in methanol. As can be seen, all the equilibriums seem to be favourable, indicating a high affinity of the resins for this compound. Although the methanol (amphoteric solvent) is partially ionized into anions and cations its ionic product is lower than that of water [19]. Thus, it can be expected that the non-dissociated form of acarbose will be the main specie in the organic phase. According to the above comments, the chemical adsorption would be the main mechanism of acarbose removal by these types of resins. The experimental data were fitted to the Eq. (13) doing KIE values equals to zero. Adsorption parameters are shown in Table 3
and also the swelling ratio of the particles in water with respect to methanol media. As can be seen, the macroporous resin exhibits the maximum capacity although its swelling is lower than that of the geliform resins. The analogous results obtained in both media for the maximum capacity of Purolite CT 151 indicate that the removal of large molecules takes place on the active sites located on the macropores as it was discussed above. On the other hand, the bead size of the geliform resins shrinks in methanol media except the non-functionalized core of Purolite SST60. The decrease of the exchange capacity in methanol confirms that the available capacity decreases as the polarity of the pure solvent decreases, as expected [20,21]. With the aim of comparing the available ion exchange capacity of each resin for the uptake of acarbose the maximum ion exchange capacity in aqueous media were obtained by titration the resin active centres with a NaOH solution. The results are showed in Table 4. As can be seen in Table 4 not all the fixed charges of the resins are available for acarbose uptake indicating that ion exclusion inside the resin takes place due to the big size of acarbose molecule [21]. The decrease of the available capacity for acarbose uptake is larger in methanol than water, confirming that the available capacity decreases as the polarity of the pure solvent decreases as it was discussed above. In this case the macroporous resin exhibits the lower reduction in capacity. This can be explained taking into account that the acarbose removal is happening mainly in the surface of macroporous and so that its greater crosslinking make more rigid the matrix and is not affected by changing the solvent. The maximum load capacities obtained in aqueous media were several times higher than the values obtained in methanol as it was expected. In methanol media the available resin capacity Table 4 Saturation capacity with acarbose (Q) and the total capacity obtained with NaOH (n∞ ) of resins
Fig. 5. Isotherms of acarbose removal on four different ion exchangers in methanol media. T = 25 ◦ C. V = 0.1 l, W = 0.5 g and C0 (acarbose) = 1–9 g/l. Solid lines: theoretical model.
MEDIA
Resin
Q (mol/kg)
n∞ (mol/kg)
Total capacity used (%)
H2 O
Finex CS9GC Finex CS10GC Purolite SST 60 Purolite CT 151
3.58 3.92 1.44 2.03
3.680 4.304 2.613 3.058
97.8 91.1 55.1 66.4
MeOH
Finex CS9GC Finex CS10GC Purolite SST 60 Purolite CT 151
0.424 0.921 0.365 1.270
1.780 4.228 2.359 2.804
23.8 21.8 15.5 45.3
J.F. Rodriguez et al. / Biochemical Engineering Journal 40 (2008) 130–137
135
to acarbose decreases greatly with respect to aqueous media because of the large size of acarbose and the moderate shrinkage of resin beads in methanol limits the accessibility of a large part of the resin active sites. According to maximum capacity values, Finex resins should be used in an aqueous medium and mainly the Finex CS10GC with a value of 3.92 mol kg−1 . Nevertheless, the macroporous resin Purolite CT151 could be a good choice in non-aqueous media. These results also allow to conclude that the worse resin to carry out this process is the Purolite SST60. 3.2. Kinetic A homogeneous model which assumes that a quasihomogeneous phase exists inside the solid particle was employed in previous works to obtain the effective diffusion coefficients with satisfactory results [22]. It is an useful way to obtain effective diffusion coefficients even in the case of macroporous resin in order to compare their exchange ability with other kinds of resins [23–24]. Intraparticle transport in ion exchange resins is generally controlled by the interdiffusion of counterions. The most general solution which supposes intraparticle controlled diffusion from a well-stirred solution of limited volume was given by Crank [25]: F (t) =
Mt C 0 − Ct = M∞ C0 − C ∗
= 1−
∞ 6α(α + 1) exp (−p2 Deff t/R2 ) n
n=1
9 + 9α + 9p2n α2
P
3pn 3 + αp2n
(15)
and α=
VC0 Wn∞
Table 5 Intraparticle effective diffusion coefficients of acarbose into the resins studied MEDIA
Resin
D (m)
Deff (×10−8 ) (cm2 /s)
H2 O
Finex CS9GC Finex CS10GC Purolite SST 60 Purolite CT 151
369.229 369.366 594.914 795.307
27.8 93.1 34.0 58.1
MeOH
Finex CS9GC Finex CS10GC Purolite SST 60 Purolite CT 151
349.856 348.661 610.01 774.279
1.35 4.17 6.65 15.4
(14)
where Mt and M∞ are the amounts of acarbose in the particles at time t and at the infinite, respectively, and pn are non-zero roots and defined by the following equations: tan (pn ) =
Fig. 6. Kinetics of acarbose uptake in aqueous media. V = 1 l, W = 10 g, T = 30 ◦ C, speed = 500 rpm and C0 (acarbose) = 2 g/l. Solid lines: predicted curves.
takes place in this phenomenon favours the ionic mobility of the ions respect the neutral species. The model proposed was able to fit properly the experimental data and the results are shown in Table 5. According to the results, the diffusion coefficients of acarbose in an aqueous medium are at least one order of magnitude higher than in methanol for the FINEX resins [27]. This is in good agreement with its greater swelling and accessibility. The greater swelling of unfunctionalized core Purolite SST60 in methanol
(16)
Intraparticle effective diffusivity values (Deff (cm2 /s)) were determined by fitting the experimental data to Eq. (14) using a non-linear regression method based on the Marquardt’s algorithm [26]. The average particle diameter of the resin in each kind of mixture was measured by Masterziser 2000. In order to test which of the solvents (H2 O, CH3 OH) had the strongest effect on the elimination rate a set of experiments of acarbose removal were carried out in both media. Figs. 6 and 7 show the kinetic for all resins in the both mediums. While in aqueous media the equilibrium is attached in 20 min in methanol media are necessary almost 2 h. As it was discussed above as more polar the solvent is the resin bead experiment a grater swelling and thus easier is the access of the acarbose to active centres. Besides, in aqueous media the main transport mechanism is the ion exchange and the electrical potential that
Fig. 7. Kinetics of acarbose uptake in methanol media. V = 1 l, W = 10 g, T = 30 ◦ C, speed = 500 rpm and C0 (acarbose) = 2 g/l. Solid lines: predicted curves.
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Fig. 8. Concentration of maltose in the effluent stream during the loading procedure on the resin bed of H+ -form Finex CS10GC of the artificial fermentation broth. C0 (acarbose) = 2 g/l, C0 (maltose) = 2.22 g/l, C0 (calcium) = 0.22 g/l and F = 2 l/h. Bed diameter = 40 mm and bed length = 465 mm.
media does not produce an increase of diffusion coefficient. It can only be explained if it is assumed that the functionalized shell shrink in methanol making difficult the diffusion while at the same time the inner core is swelled by the solvent. Finally the macroporous resin Purolite CT151 (that usually has a high DVB content) shows the smaller change of the diffusion coefficient between water and methanol media confirming again that its more rigid matrix remain almost unaffected by the solvent [16,27–30].The effective intraparticle diffusivities obtained are in the range found in literature. Deff = 1 × 10−6 cm2 /s for inorganic ions and Deff = 1 × 10−8 cm2 /s for organic molecules with high molecular weight [14,31,32]. Comparing between them, the macroporous resin Purolite CT 151 exhibits a diffusion coefficient in the same range than the geliform resins in aqueous media. Nevertheless, the diffusion coefficient of acarbose in methanol is almost an order of magnitude greater for the macroporous resin. That difference in methanol can be explained taking into account that the large acarbose ion diffuses better in the open macroporous structure of the Purolite CT 151 resin than in the shrunk polymer network of the other geliform resins [33]. According to Table 5, the fastest mobility of the acarbose is into the Finex CS10GC in aqueous medium. Thus, based on the both, equilibrium and kinetics studies, it may be stated that Finex CS10GC could be selected as the best exchanger to carry out the acarbose purification in aqueous medium. Nevertheless, if in a previous purification step of the fermentation broths a non-aqueous solvent was employed the resin Purolite CT 151 could be the best choice. 3.3. Fixed bed column experiments The artificial fermentation broth used as feed stream to the bed resin of Finex CS10GC was constituted of acarbose (2 g/l), maltose (2.22 g/l) and a small amount of calcium (0.22 g/l). In each loading cycle no more than 2 l of the artificial broth were passed through the column being this amount not enough to reach the breakthrough point of the resin bed. Consequently, all the acarbose and calcium were retained in the bed and only maltose passed through the bed with only slight changes in con-
Fig. 9. Elution curves of acarbose and calcium from Finex CS10GC with HCl 2.25N. F = 2 l/h. Bed diameter = 40 mm and bed length = 465 mm.
centration with respect to the inlet solution as can be seen in Fig. 8. At the end of the charge step the column was washed with water to eliminate the compounds not retained by the resin. 3.3.1. Loading and elution test Two litres of an aqueous artificial fermentation broth were pumped through the fresh resin at a flow rate of 2 l/h after that, the column was washed with water to eliminate the compounds not retained by the resin. According to the analysis, the acarbose and also the Ca2+ ions fed were completely retained by the resin while the maltose was not removed from the liquid phase. The elution process was carried out with hydrochloric acid as regenerant agent. It was found that a concentration of HCl (2.25N) allows to release the both species acarbose and Ca2+ ions from the resin. Besides, according to Fig. 9 a selective elution of both species (acarbose and Ca2+ ) can be obtained. The acarbose does not appear in the effluent until the almost the complete elution of Ca2+ ions and thus, it is possible to draw that the acarbose elutes selectively after the rest of metal cations present in the resin and represented in this experiment by Ca2+ ion. The general trend of the elution process seems to be clear. These results suggest that Finex CS10GC could definitively be used to separate the acarbose from fermentation broths allowing to reach a good separation from other cations in aqueous solutions. 4. Conclusions The equilibrium for the acarbose uptake from these mixtures is favourable. A good fitting of the experimental equilibrium data was obtained by an ideal mass action law model. According to the experimental results, the acarbose purification should be carried out in aqueous medium and use for this purpose the resin Finex CS10GC. This geliform strong cation exchanger showed the biggest capacity and the highest rate for acarbose removal between the studied resins. The macroporous resin Purolite CT151 could be a good choice in non-aqueous media and the Purolite SST60 resin is not appropriated for this purification purpose. Finally, the recovery of acarbose from dilute aqueous solutions by ion exchange using Finex CS10GC as exchanger in the H+ -form appears to be a suitable technology.
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