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Mass transfer process and separation mechanism of four 5′-ribonucleotides on a strong acid cation exchange resin
Journal of Chromatography A 1634 (2020) 461681
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Mass transfer process and separation mechanism of four 5 -ribonucleotides on a strong acid cation exchange resin Kun Dai a, Xiaoqiang Peng a, Wei Zhuang a, Pengpeng Yang a, Pengfei Jiao b,∗∗∗, Jinglan Wu a,∗∗, Hanjie Ying a,c,∗ a
College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, National Engineering Technique Research Center for Biotechnology and Jiangsu National Synergetic Innovation Center for Advanced Materials, Nanjing, China School of Life Science and Technology, Nanyang Normal University, Nanyang, China c State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing, China b
a r t i c l e
i n f o
Article history: Received 6 July 2020 Revised 13 October 2020 Accepted 30 October 2020 Available online 11 November 2020 Keywords: 5 -ribonucleotides Ion exchange chromatography Mass transfer process Homogeneous surface diffusion model Separation mechanism Physical adsorption
a b s t r a c t 5 -ribonucleotides including adenosine 5 -monophosphate (AMP), cytidine 5 -monophsphate (CMP), guanosine 5 -monophosphate (GMP) and uridine 5 -monophosphate (UMP) have been widely used in the food and pharmaceutical industries. This work focused on the assessment of mass transfer process and separation mechanism of four 5 -ribonucleotides and counter-ion Na+ on the strong cation exchange resin NH-1. The intraparticle diffusion was determined as the rate-limiting step for the mass transfer of AMP, CMP, GMP, and Na+ on the resin NH-1 through the Boyd model. Meanwhile, a homogeneous surface diffusion model (HSDM) combing ion exchange and physical adsorption was proposed and tested against adsorption kinetic data in the batch adsorption systems. The fixed-bed film-surface diffusion model based on the HSDM was then developed and successfully predicted the concentration profiles of 5 -ribonucleotides and the change of pH at the outlet of the fixed-bed in the dynamic adsorption and separation process. Finally, the separation mechanism of 5 -ribonucleotides was presented combining model prediction and experimental results. The separation of UMP, GMP and CMP were mainly based on their differences in isoelectric points, while that of AMP and CMP were lied with the discrepancy of their physical adsorption binding capacity with the resin NH-1. © 2020 Elsevier B.V. All rights reserved.
1. Introduction Ion exchange chromatography is one of the historical and versatile procedures for the separation and purification of biochemical, such as citric acid, amino acid, bacteriocins, heparin, monoclonal antibodies and nucleic acids [1-6], since its introduction by Small et al. in 1975 [7]. Ion exchange chromatography offers many advantages over other methods for the sample purification, particularly applied for the separation of multicomponent with heterogeneous isoelectric points. Primary separation mechanism of ion exchange chromatography is based upon the discrepancy of electrostatic interactions between different adsorbates and ion exchang-
∗
Corresponding author. Corresponding author. Postal address: College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Xin Mofan Road 5, Nanjing 210 0 09, China. Fax: +86-25-58139389; Tel: +86-25-86990 0 01 ∗∗∗ Corresponding author. E-mail addresses: [email protected] (P. Jiao), [email protected] (J. Wu), [email protected] (H. Ying). ∗∗
https://doi.org/10.1016/j.chroma.2020.461681 0021-9673/© 2020 Elsevier B.V. All rights reserved.
ers, as well as the size of different adsorbate molecules due to that number of water layers shielding the surface affects the ion exchange mechanism. Besides the above mechanisms, ion exchange chromatography may be also based on physical adsorption interaction between non-ionic moieties of adsorbate molecules and the ion exchanger polymeric backbone [8,9]. Hirano et al. [10] found that the binding affinity of negatively charged resins to the uncharged aromatic solutes could be enhanced in case of existing sodium ions and arginine. They assured that the enhancement was attributed to the contributions from the cation-π interactions. Zhu et al. [11] reported the retention mechanism of lysozyme and monoclonal antibodies on the cation exchange resins were related to the collective effect of electrostatic interaction and hydrophobicity. These studies demonstrated the importance of physical adsorption (including hydrophobic interaction and van der Waals force) between the adsorbate and the adsorbent in the ion exchange process. Moreira et al. [12,13] studied the adsorption behaviors of phenylalanine and tyrosine on a strong-base anionic resin, and found the significance of physical adsorption. The contribution of physical adsorption was described as change of ion exchange
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Journal of Chromatography A 1634 (2020) 461681
selectivity coefficient. However, the joint adsorption of zwitterion and anion or zwitterion and cation in the ion exchange systems was seldom considered because of its complex separation mechanism. Actually, Jiao et al. [14] found that adsorption of zwitterions on the cation exchange resin played a significant role, which could not be ignored. 5 -ribonucleotides, including adenosine 5 -monophosphate (AMP), cytidine 5 -monophsphate (CMP), guanosine 5 monophosphate (GMP), and uridine 5 -monophosphate (UMP), are usually derived from the enzymatic hydrolysis of RNA in the bio-production processes [15]. A strong acid cation exchange resin NH-1 have been used to separate these four components successfully by our group previously [14]. The adsorption equilibrium performance on resin NH-1 was studied. A modified Helfferich equilibrium model combing ion exchange and physical adsorption was proposed. Fixed-bed column adsorption was a necessary approach to separate plenty of biochemical products from milligram to ton scales in the ion exchange chromatography [16]. The study on the mass transfer process in the batch and fixed-bed column could provide an effective route to ascertain the separation mechanism of four 5 -ribonucleotides, which was conducive to the design and optimization of subsequent continuous separation process. As we all know, an accurate chromatographic column model is pivotal in designing and optimization of the ion exchange systems especially for poly-component separation. Therefore, the mass transfer process and the separation mechanism of four 5 -ribonucleotides were illuminated by both experimental data and a appropriate mathematical model in this work. There are a lot of fixed-bed models available in literature for systematic investigation of mass transfer process in ion exchange systems. Among these models, the general rate model is commonly believed to be the most comprehensive mass transfer model for liquid column chromatography and several expressions derived from it have been put forward, such as pore diffusion model (PDM) [17], pore and surface diffusion model (PSDM) [18] and homogeneous surface diffusion model (HSDM) [19]. However, most of these models merely consider ion exchange process, taking no account of physical adsorption process, especially that of zwitterions. As a continuation of our previous research [14], this paper focused on the mass transfer process of four 5 -ribonucleotides on a strong cation exchange resin NH-1. The Boyd model was used to determine the rate-limiting step for the mass transfer of 5 ribonucleotides from bulk solution onto the resin particles. The HSDM based on the modified Helfferich equilibrium was proposed and tested against the adsorption kinetics data in different closed batch systems. Subsequently, the fixed-bed film-surface diffusion model considering the dispersion in the axial direction as well as the mass transfer process (including the external liquid-film mass transfer from bulk phase to external surface of adsorbent and the diffusion transport in the adsorbent particles) was established to describe the relevant breakthrough curves and elution concentration profiles. The dynamic column model was also employed to predict the pH profiles. The separation mechanism was explored based on these fixed bed experiments and model predictions. Moreover, as one of the components in enzymatic hydrolysate of RNA, Na+ played a vital role in the separation process of 5’-ribonucleotides and its adsorption behavior was also investigated in this work.
tained from a water purifier (EPED 30 TH, Nanjing, China). NH4 H2 PO4 (purity: ≥ 99%) supplied by Xilong Chemical Co., Ltd (Shantou, China) and methanol (purity: ≥ 99.9%) supplied by Tedia Company, Inc. (Fairfield, OH, USA) were utilized to prepare mobile phase for HPLC assay without further purification. Other chemicals, including NaCl, HCl, and NaOH, were of analytical grade from local sources. The strong acid cation exchange resin NH-1 in hydrogen form was supplied by National Engineering Technique Research Center and employed as the adsorbent, which had an average diameter of 0.4 mm. Physicochemical properties of the resin NH-1 were summarized in Table S1. The pretreatment of the adsorbent involved successively being washed with 1mol/L NaOH, deionized water, 1mol/L HCl and deionized water, ensuring a consistent condition of resin for all the experiments. 2.2. Analytical methods The concentrations of 5’-ribonucleotides were determined by Agilent 1200 HPLC system (Agilent Technologies, CA, USA) equipped with a UV detector (254 nm) and a Zorbax SB-Aq C18 column (Agilent, CA, USA, 250 × 4.6 mm, 5 μm) by means of gradient elution. The column was maintained at room temperature, and the mobile phase flow rate was set to 1 mL/min. The mobile phase consisted of solvent A (2.3 g/L NH4 H2 PO4 and 3.5% methanol aqueous mixed solution) and solvent B (pure methanol). Gradient elution profiles (15 min) were listed in Table S2. The pH value of solution was determined by pH meter (Polilyte Plus ARC 120, Hamilton Bonaduz AG, Switzerland) and the concentration of Na+ was measured by Na+ densimeter (DWS-51, Shanghai Instrument Electric Science Instrument Limited by Share Ltd, Shanghai, China). 2.3. Experimental methods 2.3.1. Batch kinetic adsorption experiments Batch kinetic adsorption experiments were performed to investigate the single/binary/ternary-component adsorption kinetics of 5’-ribonucleotides (including AMP, CMP and GMP) and Na+ onto the strong acid cation exchange resin NH-1 at the temperature of 293 ± 1 K. In a typical experiment, 10.0 g of wet resin were added into 500 mL Erlenmeyer flask containing 250 mL single/binary/ternary-component systems of different initial concentrations, closed with glass stoppers on a magnetic stirrer shaking at 250 rpm. After a given period of time interval, the supernatant was sampled with syringes and then filtered through 0.45 μm membranes to determine concentrations of 5’-ribonucleotides and Na+ until equilibrium reached. The total volume of the samples taken from the flask was around 10 mL, which was merely amounted to 4% of the overall volume of the solution in the flask. Hence, the volume of the solution could be supposed to be unchanged throughout the experiments. The pH of solution was influenced by the concentration of common ion (Cl− ). In order to evaluate the impact of pH on the adsorption kinetics in single component adsorption process, the concentration of Cl− in initial solution was adjusted (the pH of solution was changed accordingly) by adding HCl aqueous solution. Other operation procedures were consistent with the experiments mentioned above. 2.3.2. Dynamic column adsorption experiments Dynamic column adsorption experiments were carried out to evaluate column adsorption performance of 5’-ribonucleotides and Na+ in the single component adsorption system onto the strong acid cation exchange resin NH-1 at 293 ± 1 K. A glass column (diameter of 1.46 cm and length of 13.7 cm) connected with a thermostatic water bath (Ningbo Xinzhi Biotechnology Co. Ltd.,
2. Materials and methods 2.1. Materials 5’-ribonucleotides (purity: ≥ 99%) were supplied by Nanjing Biotogether Co., Ltd (Nanjing, China). Deionized water was ob2
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Journal of Chromatography A 1634 (2020) 461681
DC-0530, China) using a water jacket to keep constant temperature, packed with about 18 g NH-1 resin, was exploited to conduct column adsorption experiments. The specific operating conditions were presented in Table S3. The feed stream with different initial concentrations was pumped into the column in a down-flow mode through a Longer constant-flow peristaltic pump (BT100-1L, Shanghai Mersey Scientific Instrument Co. Ltd., China). The effluent at the column outlet was collected by automatic sampling instrument (BSZ-100, Shanghai Huxi Analysis Instrument Factory Co. Ltd., China) and the concentrations of outlet were determined at predetermined time intervals until the concentrations approached to that of feed solutions. The pH values of column outlet were simultaneously monitored to evaluate the dynamic adsorption process.
where Cb,i represeted the bulk liquid phase concentration of the component i (mol/L), V indicated the volume of the bulk solution (mL), m was the mass of the resin (g) and qav,i showed the average adsorption amount of the component i (mmol/g). The mass conservation equations of CMP+ between the bulk liquid phase and the solid phase were expressed as Eq. (4) and Eq. (5):
2.3.3. Dynamic column elution experiments Dynamic column elution experiments were conducted in a glass column with inner diameter of 1.93 cm and length of 18.9 cm, filled with about 39 g resin NH-1. The volumetric flow rates of injection and elution were both 0.16 mL/min and the flow was constant in a downward direction. The real enzymatic hydrolysate of 37 mL (preparation method was detailed in Supplementary Materials) was loaded as a feed solution and deionized water was used as desorbent. Other operation conditions and monitoring methods were same as described in section 2.3.2.
The mass conservation equations of AMP+ and GMP+ between the bulk liquid phase and the solid phase were given as Eq. (6) and Eq. (7):
dCb,CMPt dqav,CMP+ V = −m dt dt qav,CMP+
•
•
•
3 r 3p
r p qA/GMP+/± r 2 dr
(7)
0
(8)
r = r p , qi = f (Cb,i pH )
(9)
t = 0, qi = 0
(10)
The boundary condition at r = rp exhibited the adsorption equilibrium relationship of the adsorbate on the surface of the resin particles, which was described by the modified Helfferich equilibrium model containing ion exchange and physical adsorption (the electric neutral conditions in the liquid phase, 5’-ribonucleotides dissociation equations, and the equations for the adsorption equilibrium relationship of 5’-ribonucleotides on the solid surface could be referred to the literature [14]). During the solution process, the surface diffusion coefficient was regarded as an optimization variable. The surface diffusion coefficient Ds,i was obtained by adjusting the optimization variable to minimize the following objective function shown as Eq. (11):
(1)
Minimum =
N i=1
Cb,exp − Ccal Cb,exp
2
(11)
where N was the total numbers of experimental points, while Cb,exp and Ccal demonstrated the experimental and calculated result, respectively. The Boyd model, fixed-bed film-surface diffusion model, and numerical solution, as well as the correlations of model parameters were all discussed specially in Supplementary Materials. 3. Results and discussion 3.1. Mass transfer of 5’-ribonucleotides and Na+ in resin NH-1 particles
(2)
The adsorption kinetic curves of AMP, CMP, GMP and Na+ on the resin NH-1 in the single-component adsorption systems were shown in Fig. 1 (a) and the relationships between Bt and t were
r p qi r 2 dr
(6)
∂ qi =0 ∂r
r = 0,
where qi was the concentration of the component i (mmol/g) in the solid phase (at time t), Ds,i meant the surface diffusion coefficient (cm2 /min) of the component i, and r signified the radial coordinate in the particle (cm). As reported in reference [14], except cationic forms (AMP+ /CMP+ /GMP+ ) of 5’-ribonucleotides, AMP± and GMP± could also be adsorbed by NH-1, while adsorption of CMP± on NH-1 was negligible. Hence, the mass conservation equations of 5’-ribonucleotides in the bulk liquid phase were different from that of Na+ . The mass conservation equations of Na+ between the bulk liquid phase and the solid phase were figured out as Eq. (2) and Eq. (3) [12]:
qav,i =
3 r 3p
where AMPt , CMPt and GMPt represented the total 5’ribonucleotides of AMP, CMP and GMP, respectively. Initial and boundary conditions were provided as Eq. (8-10) [21]:
The surface diffusion equation of 5’-ribonucleotides cationic, zwitterionic forms or Na+ in resin particles was represented as Eq. (1) [20]:
dCb,i dqav,i V = −m dt dt
(5)
0
qav,A/GMP+/± =
The resin particles were spherical with uniform in size and internal structure. Only occurring along the radial direction of the resins particles diffusion was considered. The internal surface diffusion of particles was the rate-limiting step of the entire ion exchange process. Common ions (including Cl− , OH− and 5’-ribonucleotides negative ions) could not enter into the resin particles. The adsorption equilibrium achieved instantly on the external surface of resin particles.
2 ∂ qi ∂ qi 2 ∂ qi = Ds,i + ∂t r ∂r ∂ r2
qCMP+ r 2 dr
The homogeneous surface diffusion mode (HSDM) developed in this work contained the physical adsorption and the ion exchange. This model was mainly based on the following assumptions:
•
r p
3 = 3 rp
dCb,A/GMPt dqav,A/GMP+ dqav,A/GMP± V = −m + dt dt dt
2.4. Models and theory
•
(4)
(3)
0
3
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Journal of Chromatography A 1634 (2020) 461681
Fig. 1. (a) Adsorption kinetic curves and (b) Boyd plots of AMP, CMP, GMP and Na+ in the single-component adsorption systems. Table 1 Ion exchange selectivity, free and surface diffusion coefficients of 5 -ribonucleotide cations, zwitters, and Na+ .
a
KH Dm (cm2 /min) Ds (cm2 /min)
Na+
AMP+
AMP±
CMP+
GMP+
GMP±
1.1 7.8E-4 5.0E-5
1.95 4.39E-4 6.0E-6
2.5 4.39E-4 1.0E-5
0.81 4.38E-4 1.5E-5
0.9 4.44E-4 1.5E-5
2.0 4.44E-4 2.0E-5
KH a : Ion exchange selectivity coefficients for 5’-ribonucleotides cations and Na+ relative to H+ and distribution coefficients of 5’-ribonucleotides zwitter ions (All the KH a values were from the reference [14]).
cient of Na+ was higher than other three 5’-ribonucleotides cations (AMP+ , CMP+ and GMP+ ), which was assigned to the contributions from the higher molecular diffusion coefficient of Na+ and similar adsorption equilibrium constants [14]. The adsorption equilibrium constant of AMP+ was higher than that of CMP+ and GMP+ , while the molecular diffusion coefficients of these three 5’ribonucleotides were analogous. The surface diffusion coefficient of AMP+ was lower than that of CMP+ and GMP+ . HSDM were employed to predict the adsorption kinetic curves of 5’-ribonucleotides and Na+ under different initial concentrations. As plotted in Fig. 3., HSDM with single surface diffusion coefficient predicted the kinetic curves with a good fitness under different initial concentrations, which indicated that the surface diffusion coefficients of 5’-ribonucleotides and Na+ were hardly affected within the tested concentration range. The adsorption kinetic experiments of 5’-ribonucleotides and Na+ on the resin NH-1 in two-component and three-component adsorption systems were also conducted to investigate the competitive adsorption. UMP was hardly adsorbed on the resin NH1 and eluted faster than the other three 5’-ribonucleotides. Na+ was tightly bound to the resin NH-1 and eluted slower than 5’ribonucleotides. Therefore, the adsorption rate of UMP and Na+ by resin NH-1 were insusceptible by other three 5’-ribonucleotides during mass transfer process. As exhibited in Fig. 4., Adsorption kinetic data and predicted curves were drawn under HSDM of twocomponent and three-component of 5’-ribonucleotides. It could be seen clearly that the surface diffusion coefficients obtained by fitting the experimental data of 5’-ribonucleotides adsorption kinetics in a single-component adsorption system could also be used to predict the multi-component adsorption kinetic curves satisfactorily. The results indicated that the surface diffusion coefficients of 5’-ribonucleotides were not significantly affected by other components. The potential reason was that the concentrations of
displayed in Fig. 1 (b). The linear plots passing through origin of coordinates indicated that intraparticle diffusion was the ratelimiting step for the mass transfer of AMP, CMP, GMP and Na+ into the resin NH-1 particles. Generally, the effect of the external film mass transfer resistance was usually negligible compared to that of the intraparticle mass transfer resistance of the adsorbent particles [22-24], which may be due to high agitation rate and thin external film. Therefore, the external film mass transfer resistance in the batch experiments was not considered in this work. The adsorption kinetic curves of 5’-ribonucleotides under different concentrations of co-ion (Cl− ) and the adsorption kinetic curves of counter-ion Na+ were viewed in Fig. 2. and Fig. S1. As a kind of gel type adsorbent, the resin NH-1 possessed small particle pore diameter which was generally less than 2 nm even under swelling condition. Consequently, the HSDM, regardless of the pore diffusion and external film mass transfer resistance, was used to simulate the mass transfer kinetics of 5’-ribonucleotides and Na+ . As curved in Fig. 2., the HSDM fitted the adsorption kinetic data of 5’-ribonucleotides and Na+ well. Moreover, the surface diffusion coefficients and ion exchange selectivity coefficients of various forms of 5’-ribonucleotides were demonstrated in Table 1. According to Miyabe et al. [25], the surface diffusion coefficient of strong reserved component was lower than that of weak reserved component due to difference of their affinity with the stationary phase. The interaction between 5’-ribonucleotides cations and NH1 was more remarkable than that between zwitterions and NH-1. Consequently, the surface diffusion coefficients of AMP+ and CMP+ were lower than that of AMP± and CMP± , respectively, which was consistent with the results obtained in Table 1. The surface diffusion coefficient in the stationary phase was proportional to the molecular diffusion coefficient in the bulk liquid phase and inverse to the adsorption equilibrium constant between the solid and liquid phases [26]. In this work, the surface diffusion coeffi4
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Journal of Chromatography A 1634 (2020) 461681
Fig. 2. Adsorption kinetic curves of (a) AMP, (b) CMP, (c) GMP under different co-ions concentrations and (d) adsorption kinetic curves of Na+ .
5’-ribonucleotides were low and no obvious competitive adsorption occurred. Moreover, the satisfactory degree of fitting between the experimental and predicted results indicated that the proposed HSDM was suitable to describe the adsorption process of 5’-ribonucleotides and Na+ , which further proved that both physical adsorption and ion exchange were involved during the mass transfer process of 5’-ribonucleotides on the resin NH-1.
in Fig. 5 (b), during the adsorption process of AMP, the pH at the column outlet decreased rapidly at the initial stage followed by a slower decrease until the pH value approached that of the feed solution. The pH decrease was attributed to the ions exchange between AMP+ and hydrogen ions on NH-1. And then, the hydrogen ions exchanged from the resin flowed out from the fixed-bed column along with the mobile phase. Neutral deionized water was original resident in the column of the solution. The pH value of the AMP feed solution was about 3.0. Therefore, the deceleration rate of pH slowed down and finally approached 3.0 after penetration arrived. Similar trend for CMP and GMP could be also observed in Fig. S2. As surveyed in Fig. 5 (c), the breakthrough curves of Na+ under different initial concentration were recorded. It was generally believed that the uptake of Na+ and the release of H+ from the strong acid cation exchange resin NH-1 were performed at a stoichiometry of 1:1. Therefore, the total adsorption amount of Na+ in the fixed-bed column was almost constant owing to the fixed mass of resin NH-1 in the column. The saturation of NH-1 resin under a higher concentration of Na+ was achieved by adding a small amount of NaCl solution, which meant a shorter stoichiometric time at the same flow rate. As charted in Fig. 5(d), the pH at the outlet of the column plunged initially and then remained constant during the adsorption of Na+ , followed by a skyrocket increase until approaching the pH of feed solutions. The reason for the sharp decline was the replacement of hydrogen ions by Na+ on the resin
3.2. Mass transfer of 5’-ribonucleotides and Na+ in the column packed with NH-1 As discovered in Fig. 5., the uptake breakthrough curves of AMP and Na+ at different concentrations of feed solutions as well as pH at the column outlet were surveyed. The uptake breakthrough curves of GMP and CMP were presented in Fig. S2. The satisfactory agreement was attained between the predicted and experimental data including adsorbate concentration and pH at the column outlet, which proved the applicability of the fixed-bed filmsurface diffusion model. The stoichiometric time (penetrated 50% of the concentration of feed solution) of three 5’-ribonucleotides at different concentrations nearly kept constant. The pH values of 5’-ribonucleotides solutions were almost the same (as seen in Table S3) within the tested concentration range. In addition, the adsorption isotherms of the three 5’-ribonucleotides were all linear. As pictured in Fig. 5 (a), the stoichiometric time of AMP was hardly affected by the different initial concentration. As depicted 5
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Journal of Chromatography A 1634 (2020) 461681
Fig. 3. Adsorption kinetic curves of (a) AMP, (b) CMP, (c) GMP, and (d) Na+ on NH-1 under different initial concentrations.
NH-1. When the hydrogen ions were completely replaced, the pH at the column outlet increased drastically and tended to be stable at the pH of feed solution. The fixed-bed film-surface diffusion model was used to predict the uptake breakthrough curves of 5’-ribonucleotides and Na+ under different concentrations (as shown in Fig. 5. and Fig. S2.). The model parameters used in these predictions and the average relative deviations between the predicted and experimental values were summarized in Table S3. It can be seen that the average relative deviations (ARD) were all less than 11%, which indicated that the fixed-bed film-surface diffusion model was suitable to predict the fixed-bed mass transfer process of 5’-ribonucleotides and Na+ .
ble of predicting chromatographic profiles of UMP and CMP. These slight deviations could be generated by the different predicted and actual values under adsorption equilibrium model parameters and mass transfer model parameters. Additionally, non-uniformities of radial fixed-bed column packed may also lead to that according to Astrath et al. [27]. However, the predicted elution and end time of GMP and AMP were in good agreement with the experimental data, further indicating the model capable of illustrating the separation process of 5’-ribonucleotides. As described in Fig. 6 (b), the pH variations at the column outlet could be generally simulated by the fixed-bed film-surface diffusion model. These results demonstrated the accuracy of the fixed-bed column model and interpreted that adsorption process of 5’-ribonucleotides onto NH-1 resin was dominated by ion exchange and physical adsorption. As documented in Fig. 6 (a), UMP run out first without being adsorbed by the resin NH-1. And then, GMP and CMP flowed out the column in succession accompanied with two significant peaks of pH profile at the outlet. The anionic form of GMP drained out earlier than that of CMP owing to a lower isoelectric point compared to CMP. The sequential outflow of GMP and CMP resulted in a pH of the solution at the column outlet reduction with occurence of two significant peaks of pH profile. AMP flowed out finally on account of tighter attachment (physical adsorption) with resin. Besides a stronger hydrophobic force of AMP [14], the ion exchange equilibrium constants of AMP+ and AMP± were higher than that of CMP+ , which caused a longer retention time of AMP. Due to
3.3. Separation of 5’-ribonucleotides in the fixed-bed packed with resin NH-1 The results of breakthrough experiments confirmed that fixedbed film-surface diffusion model could well predict the change of effluent concentration and pH during the adsorption of 5’ribonucleotides and Na+ . This model was used to predict the separation process of four 5’-ribonucleotides. The experimental separation results of the 5’-ribonucleotides and predicted concentration profiles at the column outlet were simulated by the fixed-bed film-surface diffusion model in Fig. 6 (a). In spite of the slight deviations between the experimental and predicted values of GMP and AMP, the fixed-bed film-surface diffusion mode could be capa6
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Journal of Chromatography A 1634 (2020) 461681
Fig. 4. Adsorption kinetics curves of (a) AMP and CMP, (b) AMP and GMP, (c) GMP and CMP, (d) CMP, GMP and AMP on NH-1 in multicomponent systems.
long stagnation in the column and a large number of eluent, pH at the outlet did not show significant change compared with three others. When AMP adsorbed on the resin completely escaped from fixed-bed column, the pH value of column outlet would gradually increase owing to the introduction of eluent of deionized water. The fixed-bed film-surface diffusion model proposed in this paper was also used to predict the changes of pH and uptake amount of Na+ inside the column. As recorded in Fig. 7., the uptake quantity of Na+ (in Fig. 7 (a)) and pH at the axial position (from 0 to column length) of the fixed-bed column during the loading of 5 -ribonucleotides solution (0~233.3 min) (in Fig. 7 (b)) as well as the elution process (>233.3 min) (Fig. 7 (c)) were systematically investigated. In Fig. 7 (a), the saturated area of Na+ along with the axial position increased with the increase of loading time. The fixed-bed column was filled with neutral deionized water before the loading of 5 -ribonucleotides solution, so the pH profile was around 7.0 and described as the curve at 0 min in Fig. 7 (b). As the injection proceeds, the pH of saturated zone approached that of 5 -ribonucleotides solution while un-adsorbed zone kept neutral, which was accordant with the curves of 10 min and 20 min in Fig. 7 (b). It was worth noting that hydrogen ions would be eluted along the axis position of the column due to the presence of Na+ , so a horizontal line with a lower pH value (about 1.0) appeared. When the sample size reached up to a certain amount, the replaced hydrogen ions penetrated and flowed out from the col-
umn. At this time, the pH inside the column remained unchanged about 1.0 except the saturated zone, which could interpret the curves tendency of 40min, 80min, 140min, 180min and 233.3min in Fig. 7 (b). During elution of 5 -ribonucleotides, the pH of saturated zone increased while that of elution zone became lower. In addition, hydrogen ions were also eluted in the fixed-bed column due to ion exchange. Thus, the pH profiles in Fig. 7 (c) were obtained from the elution process of 5 -ribonucleotides and hydrogen ions. Comparing the three different curves in Fig. 7 (c), the pH values corresponding to inflection points increased and the curves were broaden, which indicated that 5 -ribonucleotides and hydrogen ions moved along the axial position of the column. 5 ribonucleotides at the column inlet had been completely eluted, and partial Na+ in the resin might exchange with hydrogen ions in deionized water, causing the solution to be weakly alkaline (pH>7). Before the axial position of the fixed-bed column at 2.5 cm, the lower pH could be attributed to the desorption of GMP in this area. Some GMP molecules adsorbed on the resin were eluted and entered into the mobile phase. During elution process at 380 min, hydrogen ions exchanged from the resin almost completely flowed out the column. And the heterogeneity of pH inside the column was generated by the different elution speeds of four different kinds of 5 -ribonucleotides. The isoelectric point of GMP was lower than that of AMP and CMP. Therefore, GMP was transformed 7
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Journal of Chromatography A 1634 (2020) 461681
Fig. 5. Uptake breakthrough curves of (a) AMP, (c) Na+ as well as pH at the column outlet of (b) AMP, (d) Na+ in fixed-bed columns packed with NH-1.
Fig. 6. (a) Concentration profiles of 5 -ribonucleotides and (b) pH at the outlet of column during separation of 5 -ribonucleotides.
8
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Fig. 7. (a) Uptake amount of Na+ and (b) pH at the axial position of the fixed-bed column during the loading of 5 -ribonucleotides solution; (c) pH at the axial position of the fixed-bed column during the elution.
into anionic form and then flowed out the column earlier. As can be seen from Fig. 6 (a), the retention time of CMP was shorter than AMP during desorption, which were assigned to contributions from the tighter affinity between AMP and the resin [14]. Affinity between 5 -ribonucleotides and the resin NH-1 was associated with the hydrophobicity of bases in 5 -ribonucleotide molecules. In summary, presence of Na+ caused a rapid pH decrease inside the column. Separability of UMP, GMP and CMP was mainly based on their isoelectric differences, while that of AMP and CMP depended on their affinity with the resin NH-1.
CRediT authorship contribution statement Kun Dai: Methodology, Investigation, Writing - original draft. Xiaoqiang Peng: Validation, Formal analysis, Visualization. Wei Zhuang: Validation, Formal analysis, Visualization. Pengpeng Yang: Resources, Writing - review & editing, Supervision. Pengfei Jiao: Resources, Writing - review & editing, Supervision. Jinglan Wu: Writing - review & editing. Hanjie Ying: Writing - review & editing. Acknowledgement This project was supported in part by the National Key R&D Program of China (2017YFD040040X). We would also like to acknowledge the financial support provided by 21878153, BK20151452 and XTD1819.
4. Conclusion In this work, the mass transfer process of four different kinds of 5 -ribonucleotides was studied by Boyd model and the result demonstrated that intraparticle diffusion was the rate-limiting step. HSDM combining ion exchange and physical adsorption was also employed to match the adsorption kinetics data of 5 ribonucleotides in both single and multiple component systems with good degree of fitting. Additionally, the surface diffusion coefficients of four different 5 -ribonucleotides were hardly affected by initial concentration and the collective existence of multicomponent. Subsequently, the fixed-bed film-surface diffusion model was established on account of axial diffusion, convective mass transfer, liquid film diffusion, surface diffusion, and adsorption equilibrium of 5 -ribonucleotides. This model predicted the breakthrough curves and elution profiles of 5 -ribonucleotides well. Moreover, excellent agreement of the predicted and experimental pH variations of solution at the outlet further manifested the feasibility of fixed-bed film-surface diffusion model. Separation mechanism of 5 -ribonucleotides was illustrated by combining model prediction and experimental results. Separability of UMP, GMP and CMP was based on the their isoelectric points differences, while that of AMP and CMP was lied with the discrepancy of their affinity with resin NH-1. In view of of scabrous separation of four different kinds of 5 -ribonucleotides, this work would also shed light on the design and optimization of nucleotiodes and other multi-component systems through continuous ion exchange chromatography separation process in the future.
Supplementary materials Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.chroma.2020.461681. References [1] J. Wang, X. Wen, D. Zhou, Production of citric acid from molasses integrated with in-situ product separation by ion-exchange resin adsorption, Bioresource Technol 75 (20 0 0) 231–234. [2] N. Tomiya, E. Ailor, S.M. Lawrence, M.J. Betenbaugh, Y.C. Lee, Determination of Nucleotides and Sugar Nucleotides Involved in Protein Glycosylation by High-Performance Anion-Exchange Chromatography: Sugar Nucleotide Contents in Cultured Insect Cells and Mammalian Cells, Anal. Biochem 293 (2001) 129–137. [3] C. Saint-Hubert, A. Durieux, E. Bodo, J.-P. Simon, Large scale purification protocol for carnocin KZ 213 from Carnobacterium piscicola, Biotechnol. Lett 31 (2009) 519–523. [4] J. Kammerer, D.R. Kammerer, R. Carle, Impact of saccharides and amino acids on the interaction of apple polyphenols with ion exchange and adsorbent resins, J, Food Eng 98 (2010) 230–239. [5] J.M. Fasciano, N.D. Danielson, Ion chromatography for the separation of heparin and structurally related glycoaminoglycans: A review, J, Sep. Sci. 39 (2016) 1118–1129. [6] A.M. Ramos-de-la-Peña, J. González-Valdez, O. Aguilar, Protein A chromatography: Challenges and progress in the purification of monoclonal antibodies, J, Sep. Sci. 42 (2019) 1816–1827. [7] H. Small, T.S. Stevens, W.C. Bauman, Novel ion exchange chromatographic method using conductimetric detection, Anal. Chem. 47 (1975) 1801–1809. [8] J.S. Fritz, Factors affecting selectivity in ion chromatography, J, Chromatogr. A 1085 (2005) 8–17. [9] S. Rahmani, M. Mohseni, The role of hydrophobic properties in ion exchange removal of organic compounds from water, Can. J. Chem. Eng 95 (2017) 1449–1455. [10] A. Hirano, K. Iwashita, T. Ura, S. Sakuraba, K. Shiraki, T. Arakawa, T. Kameda, The binding affinity of uncharged aromatic solutes for negatively charged resins is enhanced by cations via cation-π interactions: The case of sodium ion and arginine, J, Chromatogr. A 1595 (2019) 97–107. [11] M. Zhu, G. Carta, Protein adsorption equilibrium and kinetics in multimodal cation exchange resins, Adsorption 22 (2016) 165–179.
Declaration of Competing Interest The authors declare no competing financial interest. The coauthors have reviewed the manuscript and approved it for submission to this journal for consideration. 9
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[12] M.J.A. Moreira, Gando-Ferreira L.M, Separation of phenylalanine and tyrosine by ion-exchange using a strong-base anionic resin. I. Breakthrough curves analysis, Biochem. Eng. J. 67 (2012) 231–240. [13] M.J.A. Moreira, L.M. Gando-Ferreira, Separation of phenylalanine and tyrosine by ion-exchange using a strong-base anionic resin. II. Cyclic adsorption/desorption studies, Biochem. Eng. J. 67 (2012) 241–250. [14] P. Jiao, J. Wu, Y. Wang, J. Zhou, W. Zhuang, Y. Chen, D. Liu, C. Zhu, H. Ying, Combined ion exchange and adsorption equilibria of 5 -ribonucleotides on the strong acid cation exchange resin NH-1, J, Chem. Technol. Biot. 92 (2017) 1678–1689. [15] M.D. Benaiges, J. López-Santin, C. Solà, Production of 5 -ribonucleotides by enzymatic hydrolysis of RNA, Enzyme Microb. Tech. 12 (1990) 86–89. [16] V. Russo, D. Masiello, M. Trifuoggi, M.D. Serio, R. Tesser, Design of an adsorption column for methylene blue abatement over silica: From batch to continuous modeling, Chem. Eng. J. 302 (2016) 287–295. [17] W.-N. Chu, Q.-C. Wu, S.-J. Yao, D.-Q. Lin, High-throughput screening and optimization of mixed-mode resins for human serum albumin separation with microtiter filter plate, Biochem. Eng. J. 131 (2018) 47–57. [18] M. Kavand, N. Asasian, M. Soleimani, T. Kaghazchi, R. Bardestani, Film– Pore-[Concentration-Dependent] Surface Diffusion model for heavy metal ions adsorption: Single and multi-component systems, Process Saf. Environ 107 (2017) 486–497. [19] A. Ma, A. Abushaikha, S.J. Allen, G. Mckay, Ion Exchange Homogeneous Surface Diffusion Modelling by Binary Site Resin for the Removal of Nickel Ions from Wastewater in Fixed Beds, Chem. Eng. J 358 (2019) 1–10.
[20] R. Leyva-Ramos, C.J. Geankoplis, Model simulation and analysis of surface diffusion of liquids in porous solids, Chem. Eng. Sci. 40 (1985) 799–807. [21] D. Roy, G.-T. Wang, D.D. Adrian, A simplified solution technique for carbon adsorption model, Water Res 27 (1993) 1033–1040. [22] G.A. Heeter, The modelling and analysis of the dynamic performance of perfusion chromatography in fixed bed and periodic countercurrent column operations and parameter estimation and model discrimination using experimental data, Radioehlektronika 17 (1997) 90–92. [23] G.A. Heeter, A.I. Liapis, Affinity adsorption of adsorbates into spherical monodisperse and bidisperse porous perfusive and purely diffusive adsorbent particles packed in a column parameter estimation in the Laplace transform domain, J, Chromatogr. A 760 (1997) 55–69. [24] W. Qian, X. Lin, X. Zhou, X. Chen, J. Xiong, J. Bai, H. Ying, Studies of equilibrium, kinetics simulation and thermodynamics of cAMP adsorption onto an anion-exchange resin, Chem. Eng. J 165 (2010) 907–915. [25] K. Miyabe, G. Guiochon, Analysis of the Surface Diffusion of Alkylbenzenes and p-Alkylphenols in Reversed-Phase Liquid Chromatography Using the Surface-Restricted Molecular Diffusion Model, Anal. Chem 73 (2001) 3096–3106. [26] K. Miyabe, G. Guiochon, Correlation between Surface Diffusion and Molecular Diffusion in Reversed-Phase Liquid Chromatography, J, Phys. Chem. B 105 (2001) 9202–9209. [27] D.U. Astrath, F. Lottes, D.T. Vu, W. Arlt, E.H. Stenby, Experimental investigation of liquid chromatography columns by means of computed tomography, Adsorption 13 (2007) 9–19.
10
Contents lists available at ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Mass transfer process and separation mechanism of four 5 -ribonucleotides on a strong acid cation exchange resin Kun Dai a, Xiaoqiang Peng a, Wei Zhuang a, Pengpeng Yang a, Pengfei Jiao b,∗∗∗, Jinglan Wu a,∗∗, Hanjie Ying a,c,∗ a
College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, National Engineering Technique Research Center for Biotechnology and Jiangsu National Synergetic Innovation Center for Advanced Materials, Nanjing, China School of Life Science and Technology, Nanyang Normal University, Nanyang, China c State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing, China b
a r t i c l e
i n f o
Article history: Received 6 July 2020 Revised 13 October 2020 Accepted 30 October 2020 Available online 11 November 2020 Keywords: 5 -ribonucleotides Ion exchange chromatography Mass transfer process Homogeneous surface diffusion model Separation mechanism Physical adsorption
a b s t r a c t 5 -ribonucleotides including adenosine 5 -monophosphate (AMP), cytidine 5 -monophsphate (CMP), guanosine 5 -monophosphate (GMP) and uridine 5 -monophosphate (UMP) have been widely used in the food and pharmaceutical industries. This work focused on the assessment of mass transfer process and separation mechanism of four 5 -ribonucleotides and counter-ion Na+ on the strong cation exchange resin NH-1. The intraparticle diffusion was determined as the rate-limiting step for the mass transfer of AMP, CMP, GMP, and Na+ on the resin NH-1 through the Boyd model. Meanwhile, a homogeneous surface diffusion model (HSDM) combing ion exchange and physical adsorption was proposed and tested against adsorption kinetic data in the batch adsorption systems. The fixed-bed film-surface diffusion model based on the HSDM was then developed and successfully predicted the concentration profiles of 5 -ribonucleotides and the change of pH at the outlet of the fixed-bed in the dynamic adsorption and separation process. Finally, the separation mechanism of 5 -ribonucleotides was presented combining model prediction and experimental results. The separation of UMP, GMP and CMP were mainly based on their differences in isoelectric points, while that of AMP and CMP were lied with the discrepancy of their physical adsorption binding capacity with the resin NH-1. © 2020 Elsevier B.V. All rights reserved.
1. Introduction Ion exchange chromatography is one of the historical and versatile procedures for the separation and purification of biochemical, such as citric acid, amino acid, bacteriocins, heparin, monoclonal antibodies and nucleic acids [1-6], since its introduction by Small et al. in 1975 [7]. Ion exchange chromatography offers many advantages over other methods for the sample purification, particularly applied for the separation of multicomponent with heterogeneous isoelectric points. Primary separation mechanism of ion exchange chromatography is based upon the discrepancy of electrostatic interactions between different adsorbates and ion exchang-
∗
Corresponding author. Corresponding author. Postal address: College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Xin Mofan Road 5, Nanjing 210 0 09, China. Fax: +86-25-58139389; Tel: +86-25-86990 0 01 ∗∗∗ Corresponding author. E-mail addresses: [email protected] (P. Jiao), [email protected] (J. Wu), [email protected] (H. Ying). ∗∗
https://doi.org/10.1016/j.chroma.2020.461681 0021-9673/© 2020 Elsevier B.V. All rights reserved.
ers, as well as the size of different adsorbate molecules due to that number of water layers shielding the surface affects the ion exchange mechanism. Besides the above mechanisms, ion exchange chromatography may be also based on physical adsorption interaction between non-ionic moieties of adsorbate molecules and the ion exchanger polymeric backbone [8,9]. Hirano et al. [10] found that the binding affinity of negatively charged resins to the uncharged aromatic solutes could be enhanced in case of existing sodium ions and arginine. They assured that the enhancement was attributed to the contributions from the cation-π interactions. Zhu et al. [11] reported the retention mechanism of lysozyme and monoclonal antibodies on the cation exchange resins were related to the collective effect of electrostatic interaction and hydrophobicity. These studies demonstrated the importance of physical adsorption (including hydrophobic interaction and van der Waals force) between the adsorbate and the adsorbent in the ion exchange process. Moreira et al. [12,13] studied the adsorption behaviors of phenylalanine and tyrosine on a strong-base anionic resin, and found the significance of physical adsorption. The contribution of physical adsorption was described as change of ion exchange
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selectivity coefficient. However, the joint adsorption of zwitterion and anion or zwitterion and cation in the ion exchange systems was seldom considered because of its complex separation mechanism. Actually, Jiao et al. [14] found that adsorption of zwitterions on the cation exchange resin played a significant role, which could not be ignored. 5 -ribonucleotides, including adenosine 5 -monophosphate (AMP), cytidine 5 -monophsphate (CMP), guanosine 5 monophosphate (GMP), and uridine 5 -monophosphate (UMP), are usually derived from the enzymatic hydrolysis of RNA in the bio-production processes [15]. A strong acid cation exchange resin NH-1 have been used to separate these four components successfully by our group previously [14]. The adsorption equilibrium performance on resin NH-1 was studied. A modified Helfferich equilibrium model combing ion exchange and physical adsorption was proposed. Fixed-bed column adsorption was a necessary approach to separate plenty of biochemical products from milligram to ton scales in the ion exchange chromatography [16]. The study on the mass transfer process in the batch and fixed-bed column could provide an effective route to ascertain the separation mechanism of four 5 -ribonucleotides, which was conducive to the design and optimization of subsequent continuous separation process. As we all know, an accurate chromatographic column model is pivotal in designing and optimization of the ion exchange systems especially for poly-component separation. Therefore, the mass transfer process and the separation mechanism of four 5 -ribonucleotides were illuminated by both experimental data and a appropriate mathematical model in this work. There are a lot of fixed-bed models available in literature for systematic investigation of mass transfer process in ion exchange systems. Among these models, the general rate model is commonly believed to be the most comprehensive mass transfer model for liquid column chromatography and several expressions derived from it have been put forward, such as pore diffusion model (PDM) [17], pore and surface diffusion model (PSDM) [18] and homogeneous surface diffusion model (HSDM) [19]. However, most of these models merely consider ion exchange process, taking no account of physical adsorption process, especially that of zwitterions. As a continuation of our previous research [14], this paper focused on the mass transfer process of four 5 -ribonucleotides on a strong cation exchange resin NH-1. The Boyd model was used to determine the rate-limiting step for the mass transfer of 5 ribonucleotides from bulk solution onto the resin particles. The HSDM based on the modified Helfferich equilibrium was proposed and tested against the adsorption kinetics data in different closed batch systems. Subsequently, the fixed-bed film-surface diffusion model considering the dispersion in the axial direction as well as the mass transfer process (including the external liquid-film mass transfer from bulk phase to external surface of adsorbent and the diffusion transport in the adsorbent particles) was established to describe the relevant breakthrough curves and elution concentration profiles. The dynamic column model was also employed to predict the pH profiles. The separation mechanism was explored based on these fixed bed experiments and model predictions. Moreover, as one of the components in enzymatic hydrolysate of RNA, Na+ played a vital role in the separation process of 5’-ribonucleotides and its adsorption behavior was also investigated in this work.
tained from a water purifier (EPED 30 TH, Nanjing, China). NH4 H2 PO4 (purity: ≥ 99%) supplied by Xilong Chemical Co., Ltd (Shantou, China) and methanol (purity: ≥ 99.9%) supplied by Tedia Company, Inc. (Fairfield, OH, USA) were utilized to prepare mobile phase for HPLC assay without further purification. Other chemicals, including NaCl, HCl, and NaOH, were of analytical grade from local sources. The strong acid cation exchange resin NH-1 in hydrogen form was supplied by National Engineering Technique Research Center and employed as the adsorbent, which had an average diameter of 0.4 mm. Physicochemical properties of the resin NH-1 were summarized in Table S1. The pretreatment of the adsorbent involved successively being washed with 1mol/L NaOH, deionized water, 1mol/L HCl and deionized water, ensuring a consistent condition of resin for all the experiments. 2.2. Analytical methods The concentrations of 5’-ribonucleotides were determined by Agilent 1200 HPLC system (Agilent Technologies, CA, USA) equipped with a UV detector (254 nm) and a Zorbax SB-Aq C18 column (Agilent, CA, USA, 250 × 4.6 mm, 5 μm) by means of gradient elution. The column was maintained at room temperature, and the mobile phase flow rate was set to 1 mL/min. The mobile phase consisted of solvent A (2.3 g/L NH4 H2 PO4 and 3.5% methanol aqueous mixed solution) and solvent B (pure methanol). Gradient elution profiles (15 min) were listed in Table S2. The pH value of solution was determined by pH meter (Polilyte Plus ARC 120, Hamilton Bonaduz AG, Switzerland) and the concentration of Na+ was measured by Na+ densimeter (DWS-51, Shanghai Instrument Electric Science Instrument Limited by Share Ltd, Shanghai, China). 2.3. Experimental methods 2.3.1. Batch kinetic adsorption experiments Batch kinetic adsorption experiments were performed to investigate the single/binary/ternary-component adsorption kinetics of 5’-ribonucleotides (including AMP, CMP and GMP) and Na+ onto the strong acid cation exchange resin NH-1 at the temperature of 293 ± 1 K. In a typical experiment, 10.0 g of wet resin were added into 500 mL Erlenmeyer flask containing 250 mL single/binary/ternary-component systems of different initial concentrations, closed with glass stoppers on a magnetic stirrer shaking at 250 rpm. After a given period of time interval, the supernatant was sampled with syringes and then filtered through 0.45 μm membranes to determine concentrations of 5’-ribonucleotides and Na+ until equilibrium reached. The total volume of the samples taken from the flask was around 10 mL, which was merely amounted to 4% of the overall volume of the solution in the flask. Hence, the volume of the solution could be supposed to be unchanged throughout the experiments. The pH of solution was influenced by the concentration of common ion (Cl− ). In order to evaluate the impact of pH on the adsorption kinetics in single component adsorption process, the concentration of Cl− in initial solution was adjusted (the pH of solution was changed accordingly) by adding HCl aqueous solution. Other operation procedures were consistent with the experiments mentioned above. 2.3.2. Dynamic column adsorption experiments Dynamic column adsorption experiments were carried out to evaluate column adsorption performance of 5’-ribonucleotides and Na+ in the single component adsorption system onto the strong acid cation exchange resin NH-1 at 293 ± 1 K. A glass column (diameter of 1.46 cm and length of 13.7 cm) connected with a thermostatic water bath (Ningbo Xinzhi Biotechnology Co. Ltd.,
2. Materials and methods 2.1. Materials 5’-ribonucleotides (purity: ≥ 99%) were supplied by Nanjing Biotogether Co., Ltd (Nanjing, China). Deionized water was ob2
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Journal of Chromatography A 1634 (2020) 461681
DC-0530, China) using a water jacket to keep constant temperature, packed with about 18 g NH-1 resin, was exploited to conduct column adsorption experiments. The specific operating conditions were presented in Table S3. The feed stream with different initial concentrations was pumped into the column in a down-flow mode through a Longer constant-flow peristaltic pump (BT100-1L, Shanghai Mersey Scientific Instrument Co. Ltd., China). The effluent at the column outlet was collected by automatic sampling instrument (BSZ-100, Shanghai Huxi Analysis Instrument Factory Co. Ltd., China) and the concentrations of outlet were determined at predetermined time intervals until the concentrations approached to that of feed solutions. The pH values of column outlet were simultaneously monitored to evaluate the dynamic adsorption process.
where Cb,i represeted the bulk liquid phase concentration of the component i (mol/L), V indicated the volume of the bulk solution (mL), m was the mass of the resin (g) and qav,i showed the average adsorption amount of the component i (mmol/g). The mass conservation equations of CMP+ between the bulk liquid phase and the solid phase were expressed as Eq. (4) and Eq. (5):
2.3.3. Dynamic column elution experiments Dynamic column elution experiments were conducted in a glass column with inner diameter of 1.93 cm and length of 18.9 cm, filled with about 39 g resin NH-1. The volumetric flow rates of injection and elution were both 0.16 mL/min and the flow was constant in a downward direction. The real enzymatic hydrolysate of 37 mL (preparation method was detailed in Supplementary Materials) was loaded as a feed solution and deionized water was used as desorbent. Other operation conditions and monitoring methods were same as described in section 2.3.2.
The mass conservation equations of AMP+ and GMP+ between the bulk liquid phase and the solid phase were given as Eq. (6) and Eq. (7):
dCb,CMPt dqav,CMP+ V = −m dt dt qav,CMP+
•
•
•
3 r 3p
r p qA/GMP+/± r 2 dr
(7)
0
(8)
r = r p , qi = f (Cb,i pH )
(9)
t = 0, qi = 0
(10)
The boundary condition at r = rp exhibited the adsorption equilibrium relationship of the adsorbate on the surface of the resin particles, which was described by the modified Helfferich equilibrium model containing ion exchange and physical adsorption (the electric neutral conditions in the liquid phase, 5’-ribonucleotides dissociation equations, and the equations for the adsorption equilibrium relationship of 5’-ribonucleotides on the solid surface could be referred to the literature [14]). During the solution process, the surface diffusion coefficient was regarded as an optimization variable. The surface diffusion coefficient Ds,i was obtained by adjusting the optimization variable to minimize the following objective function shown as Eq. (11):
(1)
Minimum =
N i=1
Cb,exp − Ccal Cb,exp
2
(11)
where N was the total numbers of experimental points, while Cb,exp and Ccal demonstrated the experimental and calculated result, respectively. The Boyd model, fixed-bed film-surface diffusion model, and numerical solution, as well as the correlations of model parameters were all discussed specially in Supplementary Materials. 3. Results and discussion 3.1. Mass transfer of 5’-ribonucleotides and Na+ in resin NH-1 particles
(2)
The adsorption kinetic curves of AMP, CMP, GMP and Na+ on the resin NH-1 in the single-component adsorption systems were shown in Fig. 1 (a) and the relationships between Bt and t were
r p qi r 2 dr
(6)
∂ qi =0 ∂r
r = 0,
where qi was the concentration of the component i (mmol/g) in the solid phase (at time t), Ds,i meant the surface diffusion coefficient (cm2 /min) of the component i, and r signified the radial coordinate in the particle (cm). As reported in reference [14], except cationic forms (AMP+ /CMP+ /GMP+ ) of 5’-ribonucleotides, AMP± and GMP± could also be adsorbed by NH-1, while adsorption of CMP± on NH-1 was negligible. Hence, the mass conservation equations of 5’-ribonucleotides in the bulk liquid phase were different from that of Na+ . The mass conservation equations of Na+ between the bulk liquid phase and the solid phase were figured out as Eq. (2) and Eq. (3) [12]:
qav,i =
3 r 3p
where AMPt , CMPt and GMPt represented the total 5’ribonucleotides of AMP, CMP and GMP, respectively. Initial and boundary conditions were provided as Eq. (8-10) [21]:
The surface diffusion equation of 5’-ribonucleotides cationic, zwitterionic forms or Na+ in resin particles was represented as Eq. (1) [20]:
dCb,i dqav,i V = −m dt dt
(5)
0
qav,A/GMP+/± =
The resin particles were spherical with uniform in size and internal structure. Only occurring along the radial direction of the resins particles diffusion was considered. The internal surface diffusion of particles was the rate-limiting step of the entire ion exchange process. Common ions (including Cl− , OH− and 5’-ribonucleotides negative ions) could not enter into the resin particles. The adsorption equilibrium achieved instantly on the external surface of resin particles.
2 ∂ qi ∂ qi 2 ∂ qi = Ds,i + ∂t r ∂r ∂ r2
qCMP+ r 2 dr
The homogeneous surface diffusion mode (HSDM) developed in this work contained the physical adsorption and the ion exchange. This model was mainly based on the following assumptions:
•
r p
3 = 3 rp
dCb,A/GMPt dqav,A/GMP+ dqav,A/GMP± V = −m + dt dt dt
2.4. Models and theory
•
(4)
(3)
0
3
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Journal of Chromatography A 1634 (2020) 461681
Fig. 1. (a) Adsorption kinetic curves and (b) Boyd plots of AMP, CMP, GMP and Na+ in the single-component adsorption systems. Table 1 Ion exchange selectivity, free and surface diffusion coefficients of 5 -ribonucleotide cations, zwitters, and Na+ .
a
KH Dm (cm2 /min) Ds (cm2 /min)
Na+
AMP+
AMP±
CMP+
GMP+
GMP±
1.1 7.8E-4 5.0E-5
1.95 4.39E-4 6.0E-6
2.5 4.39E-4 1.0E-5
0.81 4.38E-4 1.5E-5
0.9 4.44E-4 1.5E-5
2.0 4.44E-4 2.0E-5
KH a : Ion exchange selectivity coefficients for 5’-ribonucleotides cations and Na+ relative to H+ and distribution coefficients of 5’-ribonucleotides zwitter ions (All the KH a values were from the reference [14]).
cient of Na+ was higher than other three 5’-ribonucleotides cations (AMP+ , CMP+ and GMP+ ), which was assigned to the contributions from the higher molecular diffusion coefficient of Na+ and similar adsorption equilibrium constants [14]. The adsorption equilibrium constant of AMP+ was higher than that of CMP+ and GMP+ , while the molecular diffusion coefficients of these three 5’ribonucleotides were analogous. The surface diffusion coefficient of AMP+ was lower than that of CMP+ and GMP+ . HSDM were employed to predict the adsorption kinetic curves of 5’-ribonucleotides and Na+ under different initial concentrations. As plotted in Fig. 3., HSDM with single surface diffusion coefficient predicted the kinetic curves with a good fitness under different initial concentrations, which indicated that the surface diffusion coefficients of 5’-ribonucleotides and Na+ were hardly affected within the tested concentration range. The adsorption kinetic experiments of 5’-ribonucleotides and Na+ on the resin NH-1 in two-component and three-component adsorption systems were also conducted to investigate the competitive adsorption. UMP was hardly adsorbed on the resin NH1 and eluted faster than the other three 5’-ribonucleotides. Na+ was tightly bound to the resin NH-1 and eluted slower than 5’ribonucleotides. Therefore, the adsorption rate of UMP and Na+ by resin NH-1 were insusceptible by other three 5’-ribonucleotides during mass transfer process. As exhibited in Fig. 4., Adsorption kinetic data and predicted curves were drawn under HSDM of twocomponent and three-component of 5’-ribonucleotides. It could be seen clearly that the surface diffusion coefficients obtained by fitting the experimental data of 5’-ribonucleotides adsorption kinetics in a single-component adsorption system could also be used to predict the multi-component adsorption kinetic curves satisfactorily. The results indicated that the surface diffusion coefficients of 5’-ribonucleotides were not significantly affected by other components. The potential reason was that the concentrations of
displayed in Fig. 1 (b). The linear plots passing through origin of coordinates indicated that intraparticle diffusion was the ratelimiting step for the mass transfer of AMP, CMP, GMP and Na+ into the resin NH-1 particles. Generally, the effect of the external film mass transfer resistance was usually negligible compared to that of the intraparticle mass transfer resistance of the adsorbent particles [22-24], which may be due to high agitation rate and thin external film. Therefore, the external film mass transfer resistance in the batch experiments was not considered in this work. The adsorption kinetic curves of 5’-ribonucleotides under different concentrations of co-ion (Cl− ) and the adsorption kinetic curves of counter-ion Na+ were viewed in Fig. 2. and Fig. S1. As a kind of gel type adsorbent, the resin NH-1 possessed small particle pore diameter which was generally less than 2 nm even under swelling condition. Consequently, the HSDM, regardless of the pore diffusion and external film mass transfer resistance, was used to simulate the mass transfer kinetics of 5’-ribonucleotides and Na+ . As curved in Fig. 2., the HSDM fitted the adsorption kinetic data of 5’-ribonucleotides and Na+ well. Moreover, the surface diffusion coefficients and ion exchange selectivity coefficients of various forms of 5’-ribonucleotides were demonstrated in Table 1. According to Miyabe et al. [25], the surface diffusion coefficient of strong reserved component was lower than that of weak reserved component due to difference of their affinity with the stationary phase. The interaction between 5’-ribonucleotides cations and NH1 was more remarkable than that between zwitterions and NH-1. Consequently, the surface diffusion coefficients of AMP+ and CMP+ were lower than that of AMP± and CMP± , respectively, which was consistent with the results obtained in Table 1. The surface diffusion coefficient in the stationary phase was proportional to the molecular diffusion coefficient in the bulk liquid phase and inverse to the adsorption equilibrium constant between the solid and liquid phases [26]. In this work, the surface diffusion coeffi4
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Journal of Chromatography A 1634 (2020) 461681
Fig. 2. Adsorption kinetic curves of (a) AMP, (b) CMP, (c) GMP under different co-ions concentrations and (d) adsorption kinetic curves of Na+ .
5’-ribonucleotides were low and no obvious competitive adsorption occurred. Moreover, the satisfactory degree of fitting between the experimental and predicted results indicated that the proposed HSDM was suitable to describe the adsorption process of 5’-ribonucleotides and Na+ , which further proved that both physical adsorption and ion exchange were involved during the mass transfer process of 5’-ribonucleotides on the resin NH-1.
in Fig. 5 (b), during the adsorption process of AMP, the pH at the column outlet decreased rapidly at the initial stage followed by a slower decrease until the pH value approached that of the feed solution. The pH decrease was attributed to the ions exchange between AMP+ and hydrogen ions on NH-1. And then, the hydrogen ions exchanged from the resin flowed out from the fixed-bed column along with the mobile phase. Neutral deionized water was original resident in the column of the solution. The pH value of the AMP feed solution was about 3.0. Therefore, the deceleration rate of pH slowed down and finally approached 3.0 after penetration arrived. Similar trend for CMP and GMP could be also observed in Fig. S2. As surveyed in Fig. 5 (c), the breakthrough curves of Na+ under different initial concentration were recorded. It was generally believed that the uptake of Na+ and the release of H+ from the strong acid cation exchange resin NH-1 were performed at a stoichiometry of 1:1. Therefore, the total adsorption amount of Na+ in the fixed-bed column was almost constant owing to the fixed mass of resin NH-1 in the column. The saturation of NH-1 resin under a higher concentration of Na+ was achieved by adding a small amount of NaCl solution, which meant a shorter stoichiometric time at the same flow rate. As charted in Fig. 5(d), the pH at the outlet of the column plunged initially and then remained constant during the adsorption of Na+ , followed by a skyrocket increase until approaching the pH of feed solutions. The reason for the sharp decline was the replacement of hydrogen ions by Na+ on the resin
3.2. Mass transfer of 5’-ribonucleotides and Na+ in the column packed with NH-1 As discovered in Fig. 5., the uptake breakthrough curves of AMP and Na+ at different concentrations of feed solutions as well as pH at the column outlet were surveyed. The uptake breakthrough curves of GMP and CMP were presented in Fig. S2. The satisfactory agreement was attained between the predicted and experimental data including adsorbate concentration and pH at the column outlet, which proved the applicability of the fixed-bed filmsurface diffusion model. The stoichiometric time (penetrated 50% of the concentration of feed solution) of three 5’-ribonucleotides at different concentrations nearly kept constant. The pH values of 5’-ribonucleotides solutions were almost the same (as seen in Table S3) within the tested concentration range. In addition, the adsorption isotherms of the three 5’-ribonucleotides were all linear. As pictured in Fig. 5 (a), the stoichiometric time of AMP was hardly affected by the different initial concentration. As depicted 5
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Journal of Chromatography A 1634 (2020) 461681
Fig. 3. Adsorption kinetic curves of (a) AMP, (b) CMP, (c) GMP, and (d) Na+ on NH-1 under different initial concentrations.
NH-1. When the hydrogen ions were completely replaced, the pH at the column outlet increased drastically and tended to be stable at the pH of feed solution. The fixed-bed film-surface diffusion model was used to predict the uptake breakthrough curves of 5’-ribonucleotides and Na+ under different concentrations (as shown in Fig. 5. and Fig. S2.). The model parameters used in these predictions and the average relative deviations between the predicted and experimental values were summarized in Table S3. It can be seen that the average relative deviations (ARD) were all less than 11%, which indicated that the fixed-bed film-surface diffusion model was suitable to predict the fixed-bed mass transfer process of 5’-ribonucleotides and Na+ .
ble of predicting chromatographic profiles of UMP and CMP. These slight deviations could be generated by the different predicted and actual values under adsorption equilibrium model parameters and mass transfer model parameters. Additionally, non-uniformities of radial fixed-bed column packed may also lead to that according to Astrath et al. [27]. However, the predicted elution and end time of GMP and AMP were in good agreement with the experimental data, further indicating the model capable of illustrating the separation process of 5’-ribonucleotides. As described in Fig. 6 (b), the pH variations at the column outlet could be generally simulated by the fixed-bed film-surface diffusion model. These results demonstrated the accuracy of the fixed-bed column model and interpreted that adsorption process of 5’-ribonucleotides onto NH-1 resin was dominated by ion exchange and physical adsorption. As documented in Fig. 6 (a), UMP run out first without being adsorbed by the resin NH-1. And then, GMP and CMP flowed out the column in succession accompanied with two significant peaks of pH profile at the outlet. The anionic form of GMP drained out earlier than that of CMP owing to a lower isoelectric point compared to CMP. The sequential outflow of GMP and CMP resulted in a pH of the solution at the column outlet reduction with occurence of two significant peaks of pH profile. AMP flowed out finally on account of tighter attachment (physical adsorption) with resin. Besides a stronger hydrophobic force of AMP [14], the ion exchange equilibrium constants of AMP+ and AMP± were higher than that of CMP+ , which caused a longer retention time of AMP. Due to
3.3. Separation of 5’-ribonucleotides in the fixed-bed packed with resin NH-1 The results of breakthrough experiments confirmed that fixedbed film-surface diffusion model could well predict the change of effluent concentration and pH during the adsorption of 5’ribonucleotides and Na+ . This model was used to predict the separation process of four 5’-ribonucleotides. The experimental separation results of the 5’-ribonucleotides and predicted concentration profiles at the column outlet were simulated by the fixed-bed film-surface diffusion model in Fig. 6 (a). In spite of the slight deviations between the experimental and predicted values of GMP and AMP, the fixed-bed film-surface diffusion mode could be capa6
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Fig. 4. Adsorption kinetics curves of (a) AMP and CMP, (b) AMP and GMP, (c) GMP and CMP, (d) CMP, GMP and AMP on NH-1 in multicomponent systems.
long stagnation in the column and a large number of eluent, pH at the outlet did not show significant change compared with three others. When AMP adsorbed on the resin completely escaped from fixed-bed column, the pH value of column outlet would gradually increase owing to the introduction of eluent of deionized water. The fixed-bed film-surface diffusion model proposed in this paper was also used to predict the changes of pH and uptake amount of Na+ inside the column. As recorded in Fig. 7., the uptake quantity of Na+ (in Fig. 7 (a)) and pH at the axial position (from 0 to column length) of the fixed-bed column during the loading of 5 -ribonucleotides solution (0~233.3 min) (in Fig. 7 (b)) as well as the elution process (>233.3 min) (Fig. 7 (c)) were systematically investigated. In Fig. 7 (a), the saturated area of Na+ along with the axial position increased with the increase of loading time. The fixed-bed column was filled with neutral deionized water before the loading of 5 -ribonucleotides solution, so the pH profile was around 7.0 and described as the curve at 0 min in Fig. 7 (b). As the injection proceeds, the pH of saturated zone approached that of 5 -ribonucleotides solution while un-adsorbed zone kept neutral, which was accordant with the curves of 10 min and 20 min in Fig. 7 (b). It was worth noting that hydrogen ions would be eluted along the axis position of the column due to the presence of Na+ , so a horizontal line with a lower pH value (about 1.0) appeared. When the sample size reached up to a certain amount, the replaced hydrogen ions penetrated and flowed out from the col-
umn. At this time, the pH inside the column remained unchanged about 1.0 except the saturated zone, which could interpret the curves tendency of 40min, 80min, 140min, 180min and 233.3min in Fig. 7 (b). During elution of 5 -ribonucleotides, the pH of saturated zone increased while that of elution zone became lower. In addition, hydrogen ions were also eluted in the fixed-bed column due to ion exchange. Thus, the pH profiles in Fig. 7 (c) were obtained from the elution process of 5 -ribonucleotides and hydrogen ions. Comparing the three different curves in Fig. 7 (c), the pH values corresponding to inflection points increased and the curves were broaden, which indicated that 5 -ribonucleotides and hydrogen ions moved along the axial position of the column. 5 ribonucleotides at the column inlet had been completely eluted, and partial Na+ in the resin might exchange with hydrogen ions in deionized water, causing the solution to be weakly alkaline (pH>7). Before the axial position of the fixed-bed column at 2.5 cm, the lower pH could be attributed to the desorption of GMP in this area. Some GMP molecules adsorbed on the resin were eluted and entered into the mobile phase. During elution process at 380 min, hydrogen ions exchanged from the resin almost completely flowed out the column. And the heterogeneity of pH inside the column was generated by the different elution speeds of four different kinds of 5 -ribonucleotides. The isoelectric point of GMP was lower than that of AMP and CMP. Therefore, GMP was transformed 7
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Fig. 5. Uptake breakthrough curves of (a) AMP, (c) Na+ as well as pH at the column outlet of (b) AMP, (d) Na+ in fixed-bed columns packed with NH-1.
Fig. 6. (a) Concentration profiles of 5 -ribonucleotides and (b) pH at the outlet of column during separation of 5 -ribonucleotides.
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Fig. 7. (a) Uptake amount of Na+ and (b) pH at the axial position of the fixed-bed column during the loading of 5 -ribonucleotides solution; (c) pH at the axial position of the fixed-bed column during the elution.
into anionic form and then flowed out the column earlier. As can be seen from Fig. 6 (a), the retention time of CMP was shorter than AMP during desorption, which were assigned to contributions from the tighter affinity between AMP and the resin [14]. Affinity between 5 -ribonucleotides and the resin NH-1 was associated with the hydrophobicity of bases in 5 -ribonucleotide molecules. In summary, presence of Na+ caused a rapid pH decrease inside the column. Separability of UMP, GMP and CMP was mainly based on their isoelectric differences, while that of AMP and CMP depended on their affinity with the resin NH-1.
CRediT authorship contribution statement Kun Dai: Methodology, Investigation, Writing - original draft. Xiaoqiang Peng: Validation, Formal analysis, Visualization. Wei Zhuang: Validation, Formal analysis, Visualization. Pengpeng Yang: Resources, Writing - review & editing, Supervision. Pengfei Jiao: Resources, Writing - review & editing, Supervision. Jinglan Wu: Writing - review & editing. Hanjie Ying: Writing - review & editing. Acknowledgement This project was supported in part by the National Key R&D Program of China (2017YFD040040X). We would also like to acknowledge the financial support provided by 21878153, BK20151452 and XTD1819.
4. Conclusion In this work, the mass transfer process of four different kinds of 5 -ribonucleotides was studied by Boyd model and the result demonstrated that intraparticle diffusion was the rate-limiting step. HSDM combining ion exchange and physical adsorption was also employed to match the adsorption kinetics data of 5 ribonucleotides in both single and multiple component systems with good degree of fitting. Additionally, the surface diffusion coefficients of four different 5 -ribonucleotides were hardly affected by initial concentration and the collective existence of multicomponent. Subsequently, the fixed-bed film-surface diffusion model was established on account of axial diffusion, convective mass transfer, liquid film diffusion, surface diffusion, and adsorption equilibrium of 5 -ribonucleotides. This model predicted the breakthrough curves and elution profiles of 5 -ribonucleotides well. Moreover, excellent agreement of the predicted and experimental pH variations of solution at the outlet further manifested the feasibility of fixed-bed film-surface diffusion model. Separation mechanism of 5 -ribonucleotides was illustrated by combining model prediction and experimental results. Separability of UMP, GMP and CMP was based on the their isoelectric points differences, while that of AMP and CMP was lied with the discrepancy of their affinity with resin NH-1. In view of of scabrous separation of four different kinds of 5 -ribonucleotides, this work would also shed light on the design and optimization of nucleotiodes and other multi-component systems through continuous ion exchange chromatography separation process in the future.
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Declaration of Competing Interest The authors declare no competing financial interest. The coauthors have reviewed the manuscript and approved it for submission to this journal for consideration. 9
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