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Effects of the mobile phase on the chromatographic separation of l -lysine and 5-aminovaleric acid
Microchemical Journal 152 (2020) 104369
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Effects of the mobile phase on the chromatographic separation of L-lysine and 5-aminovaleric acid
T
Siyeon Kima, Jung Oh Ahnb, Kyung-Min Kimc,1, , Chang-Ha Leea,1, ⁎⁎
⁎
a
Department of Chemical and Biomolecular Engineering, Yonsei University, Seoul, Korea Biotechnology Process Engineering Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea c Climate Change and Environmental Laboratory, KEPCO Research Institute, Daejeon, Korea b
ARTICLE INFO
ABSTRACT
Keywords: 5-aminovaleric acid L-lysine Derivatization method Mobile phase Simulated moving bed
5-Aminovaleric acid (5AVA), an attractive bio-based carbon-5 building block for polymer synthesis, is produced from L-lysine. However, their structural similarity makes separating 5AVA from L-lysine challenging. The simulated moving bed (SMB), continuous chromatographic separation process provides a solution with high productivity and low eluent consumption. Here, the effects of the mobile phase on the separation of 5AVA and Llysine on a C18 column are reported. Pre-column derivatization using diethylethoxymethylene malonate enhanced the detection sensitivity and separation performance, which was strongly affected by the phosphate buffer pH. A 70/30 (v/v) mobile phase of 20 mM phosphate buffer at pH 6.8 and acetonitrile showed the best separation performance in isocratic mode. The selectivity at adsorption equilibrium was determined from pulse experiments, and the optimal operating conditions of the SMB process were determined from the equilibrium constants of both components. The feasibility of separating 5AVA and L-lysine by the SMB process was confirmed with purity and recovery of greater than 99%. The SMB process using pre-column derivatization can provide the competiveness of the bio-based chemical production because separation processes typically contribute to more than 70% of the total production cost.
1. Introduction There is growing interest in the sustainable production of chemicals and materials from renewable resources to replace petroleum-based chemicals [1,2]. Requirement of environmentally friendly products, concerns about the effects on the environment and health of fossil fuel consumption, or increasingly limited fossil fuel resources facilitate the production of the bio-based chemicals. Considering the rapidly growing size of the bioplastic market [3,4], the production of bio-based platform chemicals [5] and monomers [6,7] by microbial host strains is one of the most promising approaches [8]. Serving as building blocks, biobased platform chemicals can be converted to various materials such as bioplastics [9,10]. The growth of the market for bio-based chemicals has been possible because of the emergence of new environment-friendly production methods that can replace petroleum-based chemicals [11]. 5-Aminovaleric acid (5AVA) has attracted much attention because it can be produced from L-lysine through the 5-aminovalerate pathway in
bacteria. In addition, as a platform biochemical, 5AVA can be used for the synthesis of polymer-like nylon-5 [12]. Moreover, 5AVA can be employed as a building block for carbon-5 resources, such as glutaric acid, which is used in the production of polyesters and polyamides [13,14]. To utilize 5AVA as a bio-based feedstock, a separation process is required to produce high-purity 5AVA. However, because of the structural similarity between 5AVA and L-lysine, as shown in Fig. 1, the separation of 5AVA from unreacted L-lysine after the conversion reaction in an environment-friendly and efficient manner is very challenging. The advantages and drawbacks of chromatography, pervaporation, crystallization, and membrane technologies for bio-based chemicals were well reviewed in literature [15]. Among them, the chromatographic separation using an ion exchange column is known as a common method [16]. The product liquid is sequentially treated by several steps for being acidified, adsorbed on an ion-exchange resin, eluted by the injection of an ammonia solution, and concentrated. The
Corresponding author. Corresponding author. E-mail addresses: [email protected] (K.-M. Kim), [email protected], [email protected] (C.-H. Lee). 1 Both authors are equally contributed to the study. ⁎
⁎⁎
https://doi.org/10.1016/j.microc.2019.104369 Received 17 June 2019; Received in revised form 14 October 2019; Accepted 23 October 2019 Available online 24 October 2019 0026-265X/ © 2019 Elsevier B.V. All rights reserved.
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phase [28,29]. Therefore, the selection of the optimal mobile phase should be carried out before designing the SMB process [30,31]. In this study, the effects of mobile phases on the chromatographic separation of a 5AVA and L-lysine mixture were experimentally conducted in isocratic mode. The purpose of this study was to present the possibility of bulk and continuous production of 5AVA with an SMB process. A pre-column derivatization method was applied to overcome the separation difficulty originating from the structural similarity and to increase the sensitivity for UV detection [14,32,33]. Then, the HPLC experiments were performed to evaluate the effects of the pH of the mobile phase on the separation of 5AVA and L-lysine. In addition, the parameters for adsorption equilibrium in a C18 HPLC column were obtained using the pulse input method. Using the selected mobile phase and obtained process parameters, the applicability of the SMB process for the mixture of 5AVA and L-lysine was tested by a simulation study.
Fig. 1. Molecular structures of L-lysine and 5-aminovaleric acid.
chromatographic separation can provide usually high product qualities, but lower concentration and increased waste streams need to be overcome. Although the reaction itself has been investigated in detail [11], few researchers have reported the development of efficient separation methods for the 5AVA and L-lysine mixture. The simulated moving bed (SMB) process, a multicolumn continuous chromatographic process, has many advantages, such as low solvent consumption and high productivity because downstream processes typically contribute to >70% of the overall product cost [17]. Since the SMB process inherently entails no phase change, it contributes to reducing the energy consumption in the separation step. Moreover, because various SMB operating strategies are feasible, even for the separation of fine chemicals and enantiomers, the SMB process meets the requirements of various industrial fields including pharmaceuticals and biotechnology, especially regarding purity, recovery, and productivity [18,19]. The SMB process for binary mixtures can continuously produce pure substances by using the countercurrent movement of the solid and liquid phases (Fig. 2). Since the inlet and outlet positions are periodically switched with a suitable interval in the direction of liquid flow [20], the countercurrent movement in a SMB process maximizes the mass transfer driving force and the separation performance is enhanced compared to that of a batch chromatographic method [21–23]. As a single mobile phase was used, the eluent obtained from the concentration step can be recirculated to reduce the waste stream after separating a product from the eluent. Furthermore, it was reported that an SMB process can achieve approximately four times higher productivity than that in a batch chromatography process. Thus, the dilution factor is about 2–5 times less [24]. One of the most important prerequisites for the implementation of this powerful process is the selection of an adequate mobile phase for a fixed stationary phase [25,26]. The importance of finding the correct mobile phase in high-performance liquid chromatography (HPLC) is widely recognized because the mobile phase affects factors such as the peak shape and selectivity [27]. Furthermore, because the retention time of each component varies significantly depending on the mobile phase during chromatographic separation, the separation performance of the SMB process is also highly influenced by the selected mobile
2. Mathematical models 2.1. Determination of adsorption isotherms A dynamic method (that is, the moment method) was used for the evaluation of the column characteristics using HPLC [34]. The method uses the retention time obtained from the injection of a small amount of adsorbate into a packed bed column [35]. The nth moment of the peak profile at a column length of z == L is obtained using Eq. (1).
C (t , z = L) t ndt
Mn =
(1)
0
The n
µn =
absolute moment is given by Eq. (2).
Mn = M0
The n
µn =
th
0
th
C (t , X = L) t ndt
0 0
C (t , Z = L) dt
(2)
central moment is given by Eq. (3).
µ1)ndt
C (t , z = L)(t 0
C (t , z = L) dt
(3)
By using above equations, the first absolute moment can be expressed as shown in Eqs. (4) and (5).
µ1 =
0
=
L [1 + u (1
e) p
0]
+
t 0A 2
(4)
p KAD
1+
e
(5)
p
The first absolute for a non-retained component obtained using Eq. (5) with KAD = 0 is given in Eq. (6).
L u
(µ1)inert =
1+
(1
e) p e
+
t 0A 2
(6)
Using Eqs. (3) and (4),
µ1 = µ 1
(µ1)inert =
(1
e) e
p KAD
L u
(7)
By rearranging Eq. (7), we obtain Eq. (8).
(1
µ1 e )/
= e
p KAD
L u
(8)
Here, C is the component concentration in the mobile phase, L is the length of the column, εe and εp are the interparticle and intraparticle porosities, respectively, and u is the interstitial velocity. Here, ρp is the particle density, t0A is the time duration of the injection of the adsorbate or the non-retained component in the chromatography column, and KAD is the adsorption equilibrium constant for the adsorption isotherm. Henry's constant, H, can be obtained using Eq. (9).
Fig. 2. Scheme of the four-zone simulated moving bed process for the separation of two solutes in a fluid mixture. 2
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KAD = H
1 p (1
four performance parameters (purity, recovery, productivity, and eluent consumption), which are defined in Eqs. (19)–(23) [37,38]. Major product concentration [g/L]:
t e)
(9)
(Extract) C¯A, E ; (Raffinate) C¯B, R
2.2. Column and configuration models for SMB process
Purity [%]:
The equilibrium dispersive model was used to predict the separation performance and internal concentration profiles of the SMB process [36]. In this model, all influences from non-equilibrium conditions are contained in the apparent axial dispersion coefficient. The differential mass balance equation of the equilibrium-dispersive model can be written as Eq. (10).
Ci, j t
+ uj
Ci, j
1
+
z
qi*, j
t
t
t
2C i, j , z2
= Da, i
Da, i
uL = 2N
(Extract)
at z = 0, Ci, j =
Ciin ,j
(Extract)
(10)
for
Ci, j =
(Extract)
(Extract)
2
(13)
+ QD = Q I
2 QIV 2
1
= Ciin , I 1 QI
QE = QII
2
in Ciout , I 2 = Ci, II
1
2 + QF out Ci, II 2 QII 2
= QIII
1
+ Ci, F QF = Ciin , III 1 QIII
1
(16)
Raffinate node:
QIII
QR = QIV
2
Ciout , III
2
= Ciin , IV
1
1
= Qa
Ciout , a 1 Qa
1
(17)
(23)
The pre-column derivatization method has been widely applied in the analysis of amino acids in HPLC analysis with UV and mass spectrometric detection [14,31–33]. It has been reported that the analysis sensitivity is highly improved by using this method. In this study, diethylethoxymethylene malonate (DEEMM) was used as an organic derivatization reagent [42,43]. The diamine derivative samples for each component were obtained by the reaction of 300 μL of borate buffer (0.05 M, pH 9), 100 μL of methanol, 47 μL of water, 50 μL of organic acid sample (0.01 M/L), and 3 μL of DEEMM. After reaction, each sample was heated at 70 °C for 2 h to allow the complete degradation of excess DEEMM and other byproducts. The blank samples without organic acids were also treated in the same way.
2
= Ciin ,a
QD + QF Q + QF ; (Raffinate) D QE C¯A, E QR C¯B, R
3.2. Derivatization
1
= Ci, R
Intermediate node in zone a:
Qa
QR C¯B, R NColumn (1 t ) VColumn
L-Lysine
(15)
Feed node:
QII
(Raffinate)
HPLC grade acetonitrile and water were purchased from J.T. Baker. (purity ≥ 98%), 5-aminovaleric acid (purity ≥ 97%), thiourea (purity ≥ 99%), diethylethoxymethylene malonate, orthophosphoric acid (85%), acetic acid (100%), sodium phosphate dibasic (≥ 99%), sodium phosphate monobasic (≥ 99%), and sodium acetate (≥ 99%) were purchased from Sigma–Aldrich. All products were used as obtained without any further purification.
1
= Ci, E
QE C¯A, E ; NColumn (1 t ) VColumn
3.1. Chemicals
Extraction node:
QI
(21)
3. Materials and methods
(14)
1
QR C¯B, R × 100 QF CB, F
Here, Ncolumn is the number of columns in the SMB systems, and Vcolumn is the volume of the column. For SMB operation (Fig. 2), five operating parameters, the flow rates of each zone and the switching period, should be determined. Triangle theory provides criteria for achieving the complete separation of a binary mixture by applying equilibrium theory [39]. Because four constraints of the flow rate ratio in each zone (mj values) out of five degrees of freedom can be determined from the complete separation region in the triangle theory, one more constraint is needed: the flow rate of the feed, the production amount, the maximum allowable pressure drop related to the capacity of the pumps, which may limit the sum of the flow rates of each column, or the maximum available flow rate for a column [40,41]. In this study, the flow rate of zone I was set to the applicable marginal flow rate for the column (< 6.5 mL/min).
The node models in Eqs. (14)–(18) were combined with the abovementioned column model to simulate the SMB process with a 2–2–2–2 configuration. Desorbent node:
Ciout , IV
(Raffinate)
Eluent Consumption [L/g]:
Here, Ci,j is the concentration of the component in the liquid phase of component i (i == A, B) in column j, qi,j* is the solid phase concentration of component i in equilibrium with the fluid phase, u is the interstitial velocity, εt is the total porosity, Da,i is the apparent axial dispersion coefficient of component i, and N is the number of theoretical plates in the column. The superscripts “i” and “j” represents the component and the column number (j == I-1, I-2, II-1, II-2, III-1, III-2, IV-1, and IV-2), respectively, and “in” refers to the inlet stream. In this study, N was fixed at 100 for 5AVA and 80 for L-lysine to match the elution profiles from experiment and simulation. The solid phase concentration of component i in equilibrium with liquid phase, qi*, is given by a linear adsorption isotherm (Eq. (13)).
QIV
QE C¯A, E × 100; QF CA, F
(22)
(12)
qi* = Hi Ci
(20)
Productivity [g/(L•h)]:
(11)
Ciin ,j
C¯A, E C¯B, R × 100; (Raffinate) × 100 C¯A, E + C¯B, E C¯A, R + C¯B, R
Recovery [%]:
The initial and boundary conditions of each column are given in Eqs. (11) and (12):
at t = 0, Ci, j = 0 for 0 < z < L
(19)
(18)
2 Qa 2
Ci,jin
Ci,jout
In these equations, and are the inlet and outlet concentrations of component i in column j, respectively, Qj is the flow rate of column j, and Ci,F is the feed concentration of component i. The subscripts D, E, F, and R refer to the desorbent inlet, extract outlet, feed inlet, and raffinate outlet nodes, respectively, a refers to zone number (I, II, III, and IV). The separation performance of the SMB operation was expressed by 3
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Table 1 Composition of the mobile phases for preliminary HPLC experiments. No.
Buffer solution
Mobile phase component ratio (buffer/ acetonitrile)
1 2
H3PO4/NaH2PO4 (pH 2.8) CH3COOH/CH3COONa (pH 4.8) NaH2PO4/Na2HPO4 (pH 6.8) NaH2PO4/Na2HPO4 (pH 6.8) NaH2PO4/Na2HPO4 (pH 6.8)
70/30 (v/v) 70/30 (v/v)
3 4 5
Table 2 System and operating parameters for SMB operation. System parameters Column properties Column diameter, D [cm] Column length, L [cm] Interparticle porosity, εe [-] Total porosity, εt [-] External porosity, εe [-] Particle porosity, εp [-] The number of dispersion units, N [-] L-Lysine 5-Aminovaleric acid Linear isotherm coefficient [-] HLys (strong adsorbate) HAVA (weak adsorbate)
50/50 (v/v) 60/40 (v/v) 70/30 (v/v)
3.3. Pulse experiments The HPLC system used for pulse experiments consisted of a degasser (YL9101 Vacuum Degasser), a quaternary pump (YL9110 Quaternary Pump), an autosampler (YL9150 Plus), a column thermostat, and a UV–visible detector (YL9120). In all pulse experiments, samples prepared by the derivatization method were filtered through a cellulose acetate syringe filter (0.20 nm), and 20 μL of each sample was injected to the HPLC system at room temperature (25 °C). To screen the mobile phase, a HPLC column (250 mm × 4.6 mm, 5 μm, Waters Symmetry C18) was used at a flow rate of 1 mL/min. Five different mobile phases were prepared, as shown in Table 1. To study the effect of the pH of the buffer solution in the mobile phase on separation, 0.02 M H3PO4/NaH2PO4 buffer at pH 2.8, 0.02 M CH3COOH/ CH3COONa buffer at pH 4.8, and 0.02 M NaH2PO4/Na2HPO4 buffer at pH 6.8 were mixed with acetonitrile at a buffer-to-acetonitrile ratio of 70/30 (v/v) [44]. The mobile phases are listed as No. 1, 2, and 5 in Table 1. Then, to study the effect of the amount of buffer solution in the mobile phase on separation, at a fixed pH of the buffer solution (pH 6.8), two additional mobile phases (No. 3 and 4) using 0.02 M NaH2PO4/Na2HPO4 buffer were prepared at buffer/acetonitrile ratios of 50/50 (v/v) and 60/40 (v/v). The UV wavelength was set to 284 nm in all experiments. In addition, the HPLC experiments for pure water (blank sample for raw organic acid samples) with the mobile phase No. 5, and the blank samples with all the mobile phases were also conducted to confirm the origin of some unexpected peaks. To determine the parameters for the SMB process, pulse experiments were performed with five different flow rates (1 to 5 mL/min) and a UV wavelength of 284 nm. A HECTOR-M C18 column (250 mm × 7.8 mm, 20/45 μm) was used for the preparative chromatographic separation. The prepared derivatives samples were mixed with a mobile phase of 500 μL. The proposed method was validated in terms of linearity, the limit of detection (LOD), the limit of quantitation (LOQ), accuracy, precision, and reproducibility. Stock solutions of L-lysine and 5AVA were separately prepared by dissolving an accurately weighed amount of each organic acid in water. The stock solutions were diluted by water. Calibrated solutions at five different concentrations in the range of 1–10 mmol L − 1 and quality-control samples at three different concentrations of 1, 5, and 10 mmol L − 1 were prepared. The derivatization of each sample was conducted with the method described in Section 3.2. All the experiments for quality assurance/quality control (QA/QC) were conducted using the HECTOR-M C18 column with the mobile phase No. 5.
0.78 25 0.03 0.53 0.05 0.52 80 100 2.140 0.174
Operating parameters Flow rate [mL/min] QF QD QII QIV Switching period, t* [s]
3.538 3.898 2.495 2.495 180
SMB process were predicted from the dynamic simulation Eqs. (10)–((18)) using gPROMS Processbuilder 1.2.0 (PSE Ltd., UK). The operating conditions of the SMB process were determined using triangle theory. 4. Results and discussions 4.1. Effect of pre-column derivatization Fig. 3 shows a comparison of the chromatograms of the raw samples and the derivatized samples. Without the pre-column derivatization step, the peaks of 5AVA and L-lysine are overlapped and the detection sensitivity is very weak (Fig. 3a). Organic compounds with conjugated system such as double bonds, hydroxyl groups, or aromatic structures tend to be adsorbed strongly. As shown in Fig. 4, the derivatives of 5AVA and L-lysine have a number of double bonds after the pre-column derivatization step. As a result, the intensities of organic acids after the derivatization step (Fig. 3b) were considerably enhanced. Furthermore, when the amino group of each component was replaced by DEEMM through the reaction step shown in Fig. 4, the retention time of each derivative was also affected in the reverse-phase chromatography. Therefore, the conversion from organic acids to stable aminoenone derivatives through the pre-column derivatization step enabled more accurate analysis because of the increased detection sensitivity, as well as facilitating enhanced separation by controlling the retention time, as shown in Fig. 3b. It was serendipitous that the pre-column derivatization step enhances the separation performance and the sensitivity of UV detection [42]. 4.2. Effect of pH and ratio of mobile phase The pKa value, is one factor indicating the strength of an acid. The buffering capacity of a species or its ability to maintain the pH of a solution is highest when the pKa and pH are close. Considering the pKa values of amino acids and the stable range of the C18 adsorbent, buffer solutions with pH between 2 and 8 were selected. Because each component has a unique pKa (4.27 for 5AVA and pKa1 of 2.20, pKa2 of 8.90 and pKa3 of 10.29 for L-lysine), ionization occurs to different degrees depending on the pH of the mobile phase. Each ionized component in the mobile phase has a different affinity for the solid phase. Therefore, it is necessary to prevent ionization that causes multiple peaks [43]. To suppress ionization, it has been reported that
3.4. System characterization for the simulation of the SMB process To design the SMB process, the adsorption isotherm parameters were determined using the moment method Eqs. (1)–((7)) using the HPLC results (HECTOR-M C18 Column). The total porosity (εt) and external porosity (εt) were calculated from the retention time of thiourea, a non-retained component. More detailed properties of the column are listed in Table 2. The separation performance and internal concentration profiles of 4
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Fig. 3. Chromatograms of L-lysine, 5-aminovaleric acid, L-lysine derivatives, and 5-aminovaleric acid derivatives in a C18 column: (a) without derivatization using phosphate buffer at pH 6.8/acetonitrile, 70/30 (v/v) and (b) with derivatization using phosphate buffer at pH 6.8/acetonitrile, 70/30 (v/v). Flow rate of 1 mL/min and UV detection at 284 nm.
the pH should differ by at least two units from the pKa of the component [45]. With respect to pH, at a pH 2.0 units higher or lower than the pKa of the desired component, the proportion of conjugate base or acid could be almost 99% [46]. In addition, for analytical purposes, HPLC in gradient mode with a pH 4.8 buffer solution is typically operated using a buffer/acetonitrile ratio from 80/20 to 40/60 (v/v) [47]. In this study, three buffer solutions with different pH values (2.8, 4.8, and 6.8)
were selected to investigate the effect of pH on separation. The separation experiments were conducted in isocratic mode after the buffer solution had been mixed with acetonitrile in a 70/30 (v/v) ratio. Then, the effect of the ratio of the mobile phase on the separation was studied using buffer/acetonitrile ratios of 50/50, 60/40, and 70/30 (v/v) at a fixed pH (6.8). The mobile phases prepared in this study are listed in Table 1.
Fig. 4. Structure of aminoenone derivatives: (a) Derivatization reaction. (b) L-Lysine derivative. (c) 5-Aminovaleric acid derivative. 5
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Fig. 5. Effect of buffer pH on the chromatograms of L-lysine and 5-aminovaleric acid derivatives in a C18 column: (a) Phosphate buffer (H3PO4/NaH2PO4) at pH 2.8/ acetonitrile, 70/30 (v/v), (b) acetic acid buffer (CH3COOH/CH3COONa) at pH 4.8/acetonitrile, 70/30 (v/v), and (c) phosphate buffer (NaH2PO4/Na2HPO4) at pH 6.8/acetonitrile, 70/30 (v/v) (from Fig. 3b). Flow rate of 1 mL/min and UV detection at 284 nm.
mobile phase is more preferable. In addition, considering ionization and separation, it is reasonable to set the pH of mobile phase to between 4.8 and 8.0, as shown in Fig. 5c, for the analysis in an isocratic mode. The effect of the ratio of buffer solution to acetonitrile in the mobile phase is presented in Fig. 6. The ratio of phosphate buffer at pH 6.8 to acetonitrile was tested at 50/50, 60/40, and 70/30 (v/v), which correspond to mobile phases No. 3–5 in Table 1. The small peaks at 3–4 min in Fig. 6b and at 2–2.5 min in Fig. 6c were expected to originate from other components for derivative reaction or retained DEEMM because the chromatograms of the blank samples also showed the peaks at the similar retention time (Figs. S2 and S3 in Supplementary information). The ionization of the compounds was suppressed by the stable aminoenone derivatives generated from the pre-column derivatization step, as well as the appropriate pH of the buffer solution. The
Fig. 5 shows the chromatograms obtained at various buffer pH values. Regardless of the pH conditions, the peak intensity and separation were much greater than those of the underivatized case (Fig. 5a). When the sample was analyzed at the highest acidic condition (mobile phase No. 1; phosphate buffer at pH 2.8/acetonitrile 70/30 (v/v)), the peaks of L-lysine and 5AVA derivatives were clearly observed in Fig. 5a. The peak at 2 min might result from a non-organic acid component because the peak at the same retention time was observed in the chromatogram of the blank sample (Fig. S1 in Supplementary Information). Two peaks were observed in mobile phase No. 2 in Table 1 (acetic acid buffer at pH 4.8/acetonitrile 70/30 (v/v)), as shown in Fig. 5b. Because of the small gap between the pH 4.8 and the pKa value of 5AVA (4.27), two peaks derived from the ionization of 5AVA were observed. Due to the stability of derivatives and operation durability, less acidic condition for a 6
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Fig. 6. Effect of the mobile phase composition on the chromatograms of L-lysine and 5-aminovaleric acid derivatives in a C18 column: (a) Phosphate buffer (NaH2PO4/Na2HPO4) at pH 6.8/acetonitrile, 50/50 (v/v), (b) phosphate buffer at pH 6.8/acetonitrile, 60/40 (v/v), and (c) phosphate buffer at pH 6.8/acetonitrile, 70/30 (v/v) (from Fig. 3b). Flow rate of 1 mL/min and UV detection at 284 nm.
L − 1 with a regression coefficient of 0.9951 for 5AVA and 0.9998 for Llysine. The limit of detection (LOD) and limit of quantitation (LOQ) were calculated with the formula: LOD = 3σ/S, LOQ = 10σ/S, where S is the slope of the calibration curve and σ is the standard deviation of intercept. The LOD values of 13.6 μmol L − 1 for 5AVA and 13.5 μmol L − 1 for L-lysine, and the LOQ values of 45.3 μmol L-1 for 5AVA and 45.1 μmol L − 1 for L-lysine were obtained, indicating the high sensitivity of the method. The accuracy, precision, and reproducibility of retention time were evaluated from the chromatogram of the quality control samples at three concentration levels with three replicates. The accuracy expressed as recovery (%) ranged from 86.9 to 110.4% while the precision as RSD (%) were less than 8.8%. The reproducibility of retention time expressed as RSD (%) was less than 0.5%. As a result, the method used in
increased pH of the buffer solution resulted in an enhancement in the difference in the retention time between the two peaks. With respect to the sharpness of peaks, the use of a small amount of buffer solution is recommended, as shown in Fig. 6a. However, considering the selectivity of the L-lysine derivative to the 5AVA derivative, a larger portion of buffer solution in the mobile phase (70%) was better (Fig. 6c). 4.3. Quality assurance / quality control The results of method validation are summarized in Table 3. A daily standard calibration curves (n == 5) of amino acid derivatives were generated to evaluate the linearity of the method using the mobile phase No. 5 in Table 1, phosphate buffer at pH 6.8/acetonitrile, 70/30 (v/v). Linear relationships were obtained in the range of 1–10 mmol 7
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Table 3 Statistical parameters using proposed method for linearity, limit of detection (LOD), limit of quantitation (LOQ), accuracy, precision, and reproducibility. Analytes Concentration range (mmol L Linear equation a Regression coefficient (R) LOD (μmol L − 1) LOQ (μmol L − 1) Recovery (n == 9,%)
−1
)
RSD (%, peak area, n == 9) Retention time (min) RSD (%, retention time, n == 9) a
Low Medium High Low Medium High
5AVA
L-lys
1–10 Y == 1.4861E6X - 736.1 0.9951 13.6 45.3 110.4 99.9 102.7 2.23 2.63 2.80 3.054 0.5
1–10 Y == 1.2358E6X + 135.5 0.9998 13.5 45.1 107.1 86.9 101.3 8.80 1.42 1.51 6.933 0.1
X: concentration of organic acid (mmol L − 1); Y: peak area of organic acid.
this study showed good accuracy, precision, and reproducibility. 4.4. Determination of isotherm and separation performance of SMB To determine the optimal operating conditions and to simulate the SMB process, the column porosity and adsorption isotherm parameters are required from preliminary experiments and information. Since the pulse samples were dilute, the moment method was applied to the chromatographic peaks with the assumption of a linear isotherm. For a linear isotherm system, accurate adsorption equilibrium constants can be provided by the first moment method. The adsorption equilibrium constant (KAD) of each organic acid was determined by using Eq. (8), and the Henry's constants (Hi) were calculated using Eq. (9) [48]. The experimental data were well fitted by linear regression, having high coefficient of determination values (R2 > 0.99), as shown in Fig. 7. The Henry's constants of the L-lysine and 5AVA derivatives were 2.140 and 0.174, respectively, and the adsorption parameters obtained from the first moment could be used reasonably for the SMB process. From the adsorption isotherm parameters, the complete separation region in triangle theory was determined at the flow rate ratio of each zone (mI, mII, mIII, and mIV) in Fig. 8. In this study, the flow-rate ratio of separation zones mII and mIII in Fig. 2 was determined at the triangle vertex shown by a circle in Fig. 8, which was the largest difference between mII and mIII for the maximized flow rate of the feed. For zones I and IV, the mI and mIV values were arbitrarily chosen with a margin from the lower and upper limits, respectively (Fig. 8). The switching
Fig. 8. Complete separation region and operating points.
period was set to 180 s for the maximum flow rate at zone I, and the detailed operating conditions are presented in Table 2. For the simulation, it was assumed that (1) the SMB consists of eight identical HECTOR-M C18 columns and (2) 20 mM phosphate buffer at pH 6.8/acetonitrile 70/30 (v/v) was used as the mobile phase. The total concentration was fixed at 0.2 g/L, but the ratio of L-lysine to 5AVA in the feed was tested at 1:1, 1:2, and 2:1, as presented in Table 4. As expected from the chromatogram results, a purity and recovery of each component higher than 99% could be achieved simultaneously because the peaks were well separated by the developed mobile phase. The increase of a component in the feed led to a slight reduction in the purity of the corresponding component, whereas the recovery remained constant. Important factors to consider were the variation in eluent consumption and productivity when a specific component was present in small quantities. Compared with the variation in purity, the changes in eluent consumption and productivity were much higher for both components. Fig. 9 shows the internal concentration profiles at the beginning and the end of the switching period when the SMB process reached a cyclic steady state. The transportation of 5AVA propagated along with the mobile phase in zones II and III to the raffinate node, whereas the Llysine migrated in the opposite direction from zones III to I along with the column. Thus, a high concentration profile of each main component at each outlet port was formed, and this enabled both products to be obtained with high purity and recovery. The internal concentration of each component increased as its concentration in the feed increased.
Fig. 7. Dependence of the first moment of the chromatographic response curve on L/u values of L-lysine and 5-aminovaleric acid in a HPLC column with 20 mM phosphate buffer at pH 6.8/acetonitrile (70/30 (v/v)) as the mobile phase. 8
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Table 4 Separation performance of SMB. Run
Feed conc. [g/L] L-Lys 5AVA
Purity [%] L-Lys
5AVA
Recovery [%] L-Lys 5AVA
Eluent consumption [L g − 1] L-Lys 5AVA
Productivity [g L − 1 h − 1] L-Lys 5AVA
1 2 3
0.1 0.067 0.133
99.50 99.75 99.00
99.54 99.01 99.77
99.54 99.54 99.54
0.49 0.65 0.33
21.08 15.85 31.47
0.1 0.133 0.067
99.49 99.49 99.49
The impurity of each product resulted from the propagation of the other component, which was observed at the beginning of the switching period in the extract product node and at the end of the switching period in the raffinate product node. Various operating strategies for reducing the impurity concentration or treating the contaminated part of the product have been reported because this is common in conventional SMB processes [20]. For example, the contaminated part of each product is simply discarded in the partial-discard operation to improve the purity [49]. Alternatively, the discarded part can be recycled to the feed in recycling partial-discard operation, enabling the recovery of further product [50]. Another example is Backfill SMB, in which the product is recycled to the feed or intermediate node in zones II and III [51]. Table 4 and Fig. 9 indicate that, if the content of one component is too less in the feed, the other component can be produced efficiently with respect to all the performance parameters. If both components are important regardless of concentration in the feed, further study of the operating strategies will be required to improve eluent consumption
0.49 0.33 0.65
21.09 31.48 15.86
and productivity. 5. Conclusion The effects of pH and amount of buffer in the mobile phase on the separation of L-lysine and 5-aminovaleric acid using a simulated moving bed process were studied. A pre-column derivatization step was essential for chromatographic separation because the adsorption selectivity and UV detection sensitivity were enhanced by the generated aminoenone group. Among the mobile phases used, 20 mM phosphate buffer at pH 6.8/ acetonitrile 70/30 (v/v) was selected as the optimal mobile phase. Since the organic acids could be ionized at low pH values, the selection of the optimal pH condition to suppress the ionization was the most important factor considering the pKa values of the components. However, considering the subsequent isolation of separated compounds, acetic acid buffer (pH 4.8) was recommended when L-lysine is the only desired product. The difference in the retention times of the L-
Fig. 9. Internal concentration profiles of the SMB at cyclic steady state. 9
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lysine and 5AVA derivatives could be controlled by changing the amount of buffer solution in the mobile phase. In this study, a sufficiently high ratio of the buffer solution in the mobile phase resulted in improved selectivity with good intensity. The conventional SMB process with 2–2–2–2 configuration using C18 columns was simulated based on the isotherm parameters obtained from the moment method. Thus, the separation feasibility confirmed that 5AVA and L-lysine could be successfully produced from their mixture at purity and recovery of greater than 99% by using a four-zone SMB process. The eluent consumption exhibited 0.33–0.65 L/g, depending on feed concentration. The result indicated high reduction of eluent in SMB separation compared to about 7 ml of eluent consumption to separate 2 μg of each diamine derivative sample in a batch separation (350 L/g). Furthermore, the waste stream of a mobile phase would be significantly reduced by recirculating the eluent obtained from the subsequent step. From the results of this study, the SMB process will be a reliable technique for the separation of L-lysine, 5AVA and, possibly, other organic acids. Furthermore, because a specific organic acid with high concentration is the target product in organic acid mixtures, the SMB process could be a powerful separation method. However, if the concentration of the target organic acid is relatively low, the development and selection of efficient operating strategies will be important with respect to eluent consumption and productivity. Furthermore, the mobile phase composition is important in practical processes because the products from the SMB process should be isolated from the mobile phase. Therefore, since a volatile buffer has a greater potential to reduce the cost and energy for the subsequent isolation step, the study for a volatile buffer between pH 4.8 and 6.8 is needed in future to protect ionization, retain high separation efficiency and lower the separation cost.
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Author contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. *These authors contributed equally. (match statement to author names with a symbol) Funding sources We gratefully acknowledge the financial support from the DAPA/ ADD of Korea (Converged Energy Materials Research Center). Declaration of Competing Interest The authors declare no competing financial interest Supplementary materials Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.microc.2019.104369. References [1] C.M. Rochman, M.A. Browne, B.S. Halpern, B.T. Hentschel, E. Hoh, H.K. Karapanagioti, L.M. Rios-Mendoza, H. Takada, S. Teh, R.C. Thompson, Classify plastic waste as hazardous, Nature 494 (2013) 169–171 https://doi.org/10.1038/ 494169a. [2] A.K. Mohanty, M. Misra, L.T. Drzal, Sustainable bio-composites from renewable resources: opportunities and challenges in the green materials world, J. Polym. Environ. 10 (2002) 19–26 https://doi.org/10.1023/A:1021013921916. [3] J.J. Bozell, G.R. Petersen, Technology development for the production of biobased products from biorefinery carbohydrates-the US department of energy's "Top 10" revisited, Green Chem. 12 (2010) 539–554 https://doi.org/10.1039/B922014C. [4] J.V. Bomtempo, F.C. Alves, F.D. Oroski, Developing new platform chemicals: what is required for a new bio-based molecule to become a platform chemical in the bioeconomy? Faraday Discuss 202 (2017) 213–225 https://doi.org/10.1039/ c7fd00052a.
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Effects of the mobile phase on the chromatographic separation of L-lysine and 5-aminovaleric acid
T
Siyeon Kima, Jung Oh Ahnb, Kyung-Min Kimc,1, , Chang-Ha Leea,1, ⁎⁎
⁎
a
Department of Chemical and Biomolecular Engineering, Yonsei University, Seoul, Korea Biotechnology Process Engineering Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea c Climate Change and Environmental Laboratory, KEPCO Research Institute, Daejeon, Korea b
ARTICLE INFO
ABSTRACT
Keywords: 5-aminovaleric acid L-lysine Derivatization method Mobile phase Simulated moving bed
5-Aminovaleric acid (5AVA), an attractive bio-based carbon-5 building block for polymer synthesis, is produced from L-lysine. However, their structural similarity makes separating 5AVA from L-lysine challenging. The simulated moving bed (SMB), continuous chromatographic separation process provides a solution with high productivity and low eluent consumption. Here, the effects of the mobile phase on the separation of 5AVA and Llysine on a C18 column are reported. Pre-column derivatization using diethylethoxymethylene malonate enhanced the detection sensitivity and separation performance, which was strongly affected by the phosphate buffer pH. A 70/30 (v/v) mobile phase of 20 mM phosphate buffer at pH 6.8 and acetonitrile showed the best separation performance in isocratic mode. The selectivity at adsorption equilibrium was determined from pulse experiments, and the optimal operating conditions of the SMB process were determined from the equilibrium constants of both components. The feasibility of separating 5AVA and L-lysine by the SMB process was confirmed with purity and recovery of greater than 99%. The SMB process using pre-column derivatization can provide the competiveness of the bio-based chemical production because separation processes typically contribute to more than 70% of the total production cost.
1. Introduction There is growing interest in the sustainable production of chemicals and materials from renewable resources to replace petroleum-based chemicals [1,2]. Requirement of environmentally friendly products, concerns about the effects on the environment and health of fossil fuel consumption, or increasingly limited fossil fuel resources facilitate the production of the bio-based chemicals. Considering the rapidly growing size of the bioplastic market [3,4], the production of bio-based platform chemicals [5] and monomers [6,7] by microbial host strains is one of the most promising approaches [8]. Serving as building blocks, biobased platform chemicals can be converted to various materials such as bioplastics [9,10]. The growth of the market for bio-based chemicals has been possible because of the emergence of new environment-friendly production methods that can replace petroleum-based chemicals [11]. 5-Aminovaleric acid (5AVA) has attracted much attention because it can be produced from L-lysine through the 5-aminovalerate pathway in
bacteria. In addition, as a platform biochemical, 5AVA can be used for the synthesis of polymer-like nylon-5 [12]. Moreover, 5AVA can be employed as a building block for carbon-5 resources, such as glutaric acid, which is used in the production of polyesters and polyamides [13,14]. To utilize 5AVA as a bio-based feedstock, a separation process is required to produce high-purity 5AVA. However, because of the structural similarity between 5AVA and L-lysine, as shown in Fig. 1, the separation of 5AVA from unreacted L-lysine after the conversion reaction in an environment-friendly and efficient manner is very challenging. The advantages and drawbacks of chromatography, pervaporation, crystallization, and membrane technologies for bio-based chemicals were well reviewed in literature [15]. Among them, the chromatographic separation using an ion exchange column is known as a common method [16]. The product liquid is sequentially treated by several steps for being acidified, adsorbed on an ion-exchange resin, eluted by the injection of an ammonia solution, and concentrated. The
Corresponding author. Corresponding author. E-mail addresses: [email protected] (K.-M. Kim), [email protected], [email protected] (C.-H. Lee). 1 Both authors are equally contributed to the study. ⁎
⁎⁎
https://doi.org/10.1016/j.microc.2019.104369 Received 17 June 2019; Received in revised form 14 October 2019; Accepted 23 October 2019 Available online 24 October 2019 0026-265X/ © 2019 Elsevier B.V. All rights reserved.
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phase [28,29]. Therefore, the selection of the optimal mobile phase should be carried out before designing the SMB process [30,31]. In this study, the effects of mobile phases on the chromatographic separation of a 5AVA and L-lysine mixture were experimentally conducted in isocratic mode. The purpose of this study was to present the possibility of bulk and continuous production of 5AVA with an SMB process. A pre-column derivatization method was applied to overcome the separation difficulty originating from the structural similarity and to increase the sensitivity for UV detection [14,32,33]. Then, the HPLC experiments were performed to evaluate the effects of the pH of the mobile phase on the separation of 5AVA and L-lysine. In addition, the parameters for adsorption equilibrium in a C18 HPLC column were obtained using the pulse input method. Using the selected mobile phase and obtained process parameters, the applicability of the SMB process for the mixture of 5AVA and L-lysine was tested by a simulation study.
Fig. 1. Molecular structures of L-lysine and 5-aminovaleric acid.
chromatographic separation can provide usually high product qualities, but lower concentration and increased waste streams need to be overcome. Although the reaction itself has been investigated in detail [11], few researchers have reported the development of efficient separation methods for the 5AVA and L-lysine mixture. The simulated moving bed (SMB) process, a multicolumn continuous chromatographic process, has many advantages, such as low solvent consumption and high productivity because downstream processes typically contribute to >70% of the overall product cost [17]. Since the SMB process inherently entails no phase change, it contributes to reducing the energy consumption in the separation step. Moreover, because various SMB operating strategies are feasible, even for the separation of fine chemicals and enantiomers, the SMB process meets the requirements of various industrial fields including pharmaceuticals and biotechnology, especially regarding purity, recovery, and productivity [18,19]. The SMB process for binary mixtures can continuously produce pure substances by using the countercurrent movement of the solid and liquid phases (Fig. 2). Since the inlet and outlet positions are periodically switched with a suitable interval in the direction of liquid flow [20], the countercurrent movement in a SMB process maximizes the mass transfer driving force and the separation performance is enhanced compared to that of a batch chromatographic method [21–23]. As a single mobile phase was used, the eluent obtained from the concentration step can be recirculated to reduce the waste stream after separating a product from the eluent. Furthermore, it was reported that an SMB process can achieve approximately four times higher productivity than that in a batch chromatography process. Thus, the dilution factor is about 2–5 times less [24]. One of the most important prerequisites for the implementation of this powerful process is the selection of an adequate mobile phase for a fixed stationary phase [25,26]. The importance of finding the correct mobile phase in high-performance liquid chromatography (HPLC) is widely recognized because the mobile phase affects factors such as the peak shape and selectivity [27]. Furthermore, because the retention time of each component varies significantly depending on the mobile phase during chromatographic separation, the separation performance of the SMB process is also highly influenced by the selected mobile
2. Mathematical models 2.1. Determination of adsorption isotherms A dynamic method (that is, the moment method) was used for the evaluation of the column characteristics using HPLC [34]. The method uses the retention time obtained from the injection of a small amount of adsorbate into a packed bed column [35]. The nth moment of the peak profile at a column length of z == L is obtained using Eq. (1).
C (t , z = L) t ndt
Mn =
(1)
0
The n
µn =
absolute moment is given by Eq. (2).
Mn = M0
The n
µn =
th
0
th
C (t , X = L) t ndt
0 0
C (t , Z = L) dt
(2)
central moment is given by Eq. (3).
µ1)ndt
C (t , z = L)(t 0
C (t , z = L) dt
(3)
By using above equations, the first absolute moment can be expressed as shown in Eqs. (4) and (5).
µ1 =
0
=
L [1 + u (1
e) p
0]
+
t 0A 2
(4)
p KAD
1+
e
(5)
p
The first absolute for a non-retained component obtained using Eq. (5) with KAD = 0 is given in Eq. (6).
L u
(µ1)inert =
1+
(1
e) p e
+
t 0A 2
(6)
Using Eqs. (3) and (4),
µ1 = µ 1
(µ1)inert =
(1
e) e
p KAD
L u
(7)
By rearranging Eq. (7), we obtain Eq. (8).
(1
µ1 e )/
= e
p KAD
L u
(8)
Here, C is the component concentration in the mobile phase, L is the length of the column, εe and εp are the interparticle and intraparticle porosities, respectively, and u is the interstitial velocity. Here, ρp is the particle density, t0A is the time duration of the injection of the adsorbate or the non-retained component in the chromatography column, and KAD is the adsorption equilibrium constant for the adsorption isotherm. Henry's constant, H, can be obtained using Eq. (9).
Fig. 2. Scheme of the four-zone simulated moving bed process for the separation of two solutes in a fluid mixture. 2
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KAD = H
1 p (1
four performance parameters (purity, recovery, productivity, and eluent consumption), which are defined in Eqs. (19)–(23) [37,38]. Major product concentration [g/L]:
t e)
(9)
(Extract) C¯A, E ; (Raffinate) C¯B, R
2.2. Column and configuration models for SMB process
Purity [%]:
The equilibrium dispersive model was used to predict the separation performance and internal concentration profiles of the SMB process [36]. In this model, all influences from non-equilibrium conditions are contained in the apparent axial dispersion coefficient. The differential mass balance equation of the equilibrium-dispersive model can be written as Eq. (10).
Ci, j t
+ uj
Ci, j
1
+
z
qi*, j
t
t
t
2C i, j , z2
= Da, i
Da, i
uL = 2N
(Extract)
at z = 0, Ci, j =
Ciin ,j
(Extract)
(10)
for
Ci, j =
(Extract)
(Extract)
2
(13)
+ QD = Q I
2 QIV 2
1
= Ciin , I 1 QI
QE = QII
2
in Ciout , I 2 = Ci, II
1
2 + QF out Ci, II 2 QII 2
= QIII
1
+ Ci, F QF = Ciin , III 1 QIII
1
(16)
Raffinate node:
QIII
QR = QIV
2
Ciout , III
2
= Ciin , IV
1
1
= Qa
Ciout , a 1 Qa
1
(17)
(23)
The pre-column derivatization method has been widely applied in the analysis of amino acids in HPLC analysis with UV and mass spectrometric detection [14,31–33]. It has been reported that the analysis sensitivity is highly improved by using this method. In this study, diethylethoxymethylene malonate (DEEMM) was used as an organic derivatization reagent [42,43]. The diamine derivative samples for each component were obtained by the reaction of 300 μL of borate buffer (0.05 M, pH 9), 100 μL of methanol, 47 μL of water, 50 μL of organic acid sample (0.01 M/L), and 3 μL of DEEMM. After reaction, each sample was heated at 70 °C for 2 h to allow the complete degradation of excess DEEMM and other byproducts. The blank samples without organic acids were also treated in the same way.
2
= Ciin ,a
QD + QF Q + QF ; (Raffinate) D QE C¯A, E QR C¯B, R
3.2. Derivatization
1
= Ci, R
Intermediate node in zone a:
Qa
QR C¯B, R NColumn (1 t ) VColumn
L-Lysine
(15)
Feed node:
QII
(Raffinate)
HPLC grade acetonitrile and water were purchased from J.T. Baker. (purity ≥ 98%), 5-aminovaleric acid (purity ≥ 97%), thiourea (purity ≥ 99%), diethylethoxymethylene malonate, orthophosphoric acid (85%), acetic acid (100%), sodium phosphate dibasic (≥ 99%), sodium phosphate monobasic (≥ 99%), and sodium acetate (≥ 99%) were purchased from Sigma–Aldrich. All products were used as obtained without any further purification.
1
= Ci, E
QE C¯A, E ; NColumn (1 t ) VColumn
3.1. Chemicals
Extraction node:
QI
(21)
3. Materials and methods
(14)
1
QR C¯B, R × 100 QF CB, F
Here, Ncolumn is the number of columns in the SMB systems, and Vcolumn is the volume of the column. For SMB operation (Fig. 2), five operating parameters, the flow rates of each zone and the switching period, should be determined. Triangle theory provides criteria for achieving the complete separation of a binary mixture by applying equilibrium theory [39]. Because four constraints of the flow rate ratio in each zone (mj values) out of five degrees of freedom can be determined from the complete separation region in the triangle theory, one more constraint is needed: the flow rate of the feed, the production amount, the maximum allowable pressure drop related to the capacity of the pumps, which may limit the sum of the flow rates of each column, or the maximum available flow rate for a column [40,41]. In this study, the flow rate of zone I was set to the applicable marginal flow rate for the column (< 6.5 mL/min).
The node models in Eqs. (14)–(18) were combined with the abovementioned column model to simulate the SMB process with a 2–2–2–2 configuration. Desorbent node:
Ciout , IV
(Raffinate)
Eluent Consumption [L/g]:
Here, Ci,j is the concentration of the component in the liquid phase of component i (i == A, B) in column j, qi,j* is the solid phase concentration of component i in equilibrium with the fluid phase, u is the interstitial velocity, εt is the total porosity, Da,i is the apparent axial dispersion coefficient of component i, and N is the number of theoretical plates in the column. The superscripts “i” and “j” represents the component and the column number (j == I-1, I-2, II-1, II-2, III-1, III-2, IV-1, and IV-2), respectively, and “in” refers to the inlet stream. In this study, N was fixed at 100 for 5AVA and 80 for L-lysine to match the elution profiles from experiment and simulation. The solid phase concentration of component i in equilibrium with liquid phase, qi*, is given by a linear adsorption isotherm (Eq. (13)).
QIV
QE C¯A, E × 100; QF CA, F
(22)
(12)
qi* = Hi Ci
(20)
Productivity [g/(L•h)]:
(11)
Ciin ,j
C¯A, E C¯B, R × 100; (Raffinate) × 100 C¯A, E + C¯B, E C¯A, R + C¯B, R
Recovery [%]:
The initial and boundary conditions of each column are given in Eqs. (11) and (12):
at t = 0, Ci, j = 0 for 0 < z < L
(19)
(18)
2 Qa 2
Ci,jin
Ci,jout
In these equations, and are the inlet and outlet concentrations of component i in column j, respectively, Qj is the flow rate of column j, and Ci,F is the feed concentration of component i. The subscripts D, E, F, and R refer to the desorbent inlet, extract outlet, feed inlet, and raffinate outlet nodes, respectively, a refers to zone number (I, II, III, and IV). The separation performance of the SMB operation was expressed by 3
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Table 1 Composition of the mobile phases for preliminary HPLC experiments. No.
Buffer solution
Mobile phase component ratio (buffer/ acetonitrile)
1 2
H3PO4/NaH2PO4 (pH 2.8) CH3COOH/CH3COONa (pH 4.8) NaH2PO4/Na2HPO4 (pH 6.8) NaH2PO4/Na2HPO4 (pH 6.8) NaH2PO4/Na2HPO4 (pH 6.8)
70/30 (v/v) 70/30 (v/v)
3 4 5
Table 2 System and operating parameters for SMB operation. System parameters Column properties Column diameter, D [cm] Column length, L [cm] Interparticle porosity, εe [-] Total porosity, εt [-] External porosity, εe [-] Particle porosity, εp [-] The number of dispersion units, N [-] L-Lysine 5-Aminovaleric acid Linear isotherm coefficient [-] HLys (strong adsorbate) HAVA (weak adsorbate)
50/50 (v/v) 60/40 (v/v) 70/30 (v/v)
3.3. Pulse experiments The HPLC system used for pulse experiments consisted of a degasser (YL9101 Vacuum Degasser), a quaternary pump (YL9110 Quaternary Pump), an autosampler (YL9150 Plus), a column thermostat, and a UV–visible detector (YL9120). In all pulse experiments, samples prepared by the derivatization method were filtered through a cellulose acetate syringe filter (0.20 nm), and 20 μL of each sample was injected to the HPLC system at room temperature (25 °C). To screen the mobile phase, a HPLC column (250 mm × 4.6 mm, 5 μm, Waters Symmetry C18) was used at a flow rate of 1 mL/min. Five different mobile phases were prepared, as shown in Table 1. To study the effect of the pH of the buffer solution in the mobile phase on separation, 0.02 M H3PO4/NaH2PO4 buffer at pH 2.8, 0.02 M CH3COOH/ CH3COONa buffer at pH 4.8, and 0.02 M NaH2PO4/Na2HPO4 buffer at pH 6.8 were mixed with acetonitrile at a buffer-to-acetonitrile ratio of 70/30 (v/v) [44]. The mobile phases are listed as No. 1, 2, and 5 in Table 1. Then, to study the effect of the amount of buffer solution in the mobile phase on separation, at a fixed pH of the buffer solution (pH 6.8), two additional mobile phases (No. 3 and 4) using 0.02 M NaH2PO4/Na2HPO4 buffer were prepared at buffer/acetonitrile ratios of 50/50 (v/v) and 60/40 (v/v). The UV wavelength was set to 284 nm in all experiments. In addition, the HPLC experiments for pure water (blank sample for raw organic acid samples) with the mobile phase No. 5, and the blank samples with all the mobile phases were also conducted to confirm the origin of some unexpected peaks. To determine the parameters for the SMB process, pulse experiments were performed with five different flow rates (1 to 5 mL/min) and a UV wavelength of 284 nm. A HECTOR-M C18 column (250 mm × 7.8 mm, 20/45 μm) was used for the preparative chromatographic separation. The prepared derivatives samples were mixed with a mobile phase of 500 μL. The proposed method was validated in terms of linearity, the limit of detection (LOD), the limit of quantitation (LOQ), accuracy, precision, and reproducibility. Stock solutions of L-lysine and 5AVA were separately prepared by dissolving an accurately weighed amount of each organic acid in water. The stock solutions were diluted by water. Calibrated solutions at five different concentrations in the range of 1–10 mmol L − 1 and quality-control samples at three different concentrations of 1, 5, and 10 mmol L − 1 were prepared. The derivatization of each sample was conducted with the method described in Section 3.2. All the experiments for quality assurance/quality control (QA/QC) were conducted using the HECTOR-M C18 column with the mobile phase No. 5.
0.78 25 0.03 0.53 0.05 0.52 80 100 2.140 0.174
Operating parameters Flow rate [mL/min] QF QD QII QIV Switching period, t* [s]
3.538 3.898 2.495 2.495 180
SMB process were predicted from the dynamic simulation Eqs. (10)–((18)) using gPROMS Processbuilder 1.2.0 (PSE Ltd., UK). The operating conditions of the SMB process were determined using triangle theory. 4. Results and discussions 4.1. Effect of pre-column derivatization Fig. 3 shows a comparison of the chromatograms of the raw samples and the derivatized samples. Without the pre-column derivatization step, the peaks of 5AVA and L-lysine are overlapped and the detection sensitivity is very weak (Fig. 3a). Organic compounds with conjugated system such as double bonds, hydroxyl groups, or aromatic structures tend to be adsorbed strongly. As shown in Fig. 4, the derivatives of 5AVA and L-lysine have a number of double bonds after the pre-column derivatization step. As a result, the intensities of organic acids after the derivatization step (Fig. 3b) were considerably enhanced. Furthermore, when the amino group of each component was replaced by DEEMM through the reaction step shown in Fig. 4, the retention time of each derivative was also affected in the reverse-phase chromatography. Therefore, the conversion from organic acids to stable aminoenone derivatives through the pre-column derivatization step enabled more accurate analysis because of the increased detection sensitivity, as well as facilitating enhanced separation by controlling the retention time, as shown in Fig. 3b. It was serendipitous that the pre-column derivatization step enhances the separation performance and the sensitivity of UV detection [42]. 4.2. Effect of pH and ratio of mobile phase The pKa value, is one factor indicating the strength of an acid. The buffering capacity of a species or its ability to maintain the pH of a solution is highest when the pKa and pH are close. Considering the pKa values of amino acids and the stable range of the C18 adsorbent, buffer solutions with pH between 2 and 8 were selected. Because each component has a unique pKa (4.27 for 5AVA and pKa1 of 2.20, pKa2 of 8.90 and pKa3 of 10.29 for L-lysine), ionization occurs to different degrees depending on the pH of the mobile phase. Each ionized component in the mobile phase has a different affinity for the solid phase. Therefore, it is necessary to prevent ionization that causes multiple peaks [43]. To suppress ionization, it has been reported that
3.4. System characterization for the simulation of the SMB process To design the SMB process, the adsorption isotherm parameters were determined using the moment method Eqs. (1)–((7)) using the HPLC results (HECTOR-M C18 Column). The total porosity (εt) and external porosity (εt) were calculated from the retention time of thiourea, a non-retained component. More detailed properties of the column are listed in Table 2. The separation performance and internal concentration profiles of 4
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Fig. 3. Chromatograms of L-lysine, 5-aminovaleric acid, L-lysine derivatives, and 5-aminovaleric acid derivatives in a C18 column: (a) without derivatization using phosphate buffer at pH 6.8/acetonitrile, 70/30 (v/v) and (b) with derivatization using phosphate buffer at pH 6.8/acetonitrile, 70/30 (v/v). Flow rate of 1 mL/min and UV detection at 284 nm.
the pH should differ by at least two units from the pKa of the component [45]. With respect to pH, at a pH 2.0 units higher or lower than the pKa of the desired component, the proportion of conjugate base or acid could be almost 99% [46]. In addition, for analytical purposes, HPLC in gradient mode with a pH 4.8 buffer solution is typically operated using a buffer/acetonitrile ratio from 80/20 to 40/60 (v/v) [47]. In this study, three buffer solutions with different pH values (2.8, 4.8, and 6.8)
were selected to investigate the effect of pH on separation. The separation experiments were conducted in isocratic mode after the buffer solution had been mixed with acetonitrile in a 70/30 (v/v) ratio. Then, the effect of the ratio of the mobile phase on the separation was studied using buffer/acetonitrile ratios of 50/50, 60/40, and 70/30 (v/v) at a fixed pH (6.8). The mobile phases prepared in this study are listed in Table 1.
Fig. 4. Structure of aminoenone derivatives: (a) Derivatization reaction. (b) L-Lysine derivative. (c) 5-Aminovaleric acid derivative. 5
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Fig. 5. Effect of buffer pH on the chromatograms of L-lysine and 5-aminovaleric acid derivatives in a C18 column: (a) Phosphate buffer (H3PO4/NaH2PO4) at pH 2.8/ acetonitrile, 70/30 (v/v), (b) acetic acid buffer (CH3COOH/CH3COONa) at pH 4.8/acetonitrile, 70/30 (v/v), and (c) phosphate buffer (NaH2PO4/Na2HPO4) at pH 6.8/acetonitrile, 70/30 (v/v) (from Fig. 3b). Flow rate of 1 mL/min and UV detection at 284 nm.
mobile phase is more preferable. In addition, considering ionization and separation, it is reasonable to set the pH of mobile phase to between 4.8 and 8.0, as shown in Fig. 5c, for the analysis in an isocratic mode. The effect of the ratio of buffer solution to acetonitrile in the mobile phase is presented in Fig. 6. The ratio of phosphate buffer at pH 6.8 to acetonitrile was tested at 50/50, 60/40, and 70/30 (v/v), which correspond to mobile phases No. 3–5 in Table 1. The small peaks at 3–4 min in Fig. 6b and at 2–2.5 min in Fig. 6c were expected to originate from other components for derivative reaction or retained DEEMM because the chromatograms of the blank samples also showed the peaks at the similar retention time (Figs. S2 and S3 in Supplementary information). The ionization of the compounds was suppressed by the stable aminoenone derivatives generated from the pre-column derivatization step, as well as the appropriate pH of the buffer solution. The
Fig. 5 shows the chromatograms obtained at various buffer pH values. Regardless of the pH conditions, the peak intensity and separation were much greater than those of the underivatized case (Fig. 5a). When the sample was analyzed at the highest acidic condition (mobile phase No. 1; phosphate buffer at pH 2.8/acetonitrile 70/30 (v/v)), the peaks of L-lysine and 5AVA derivatives were clearly observed in Fig. 5a. The peak at 2 min might result from a non-organic acid component because the peak at the same retention time was observed in the chromatogram of the blank sample (Fig. S1 in Supplementary Information). Two peaks were observed in mobile phase No. 2 in Table 1 (acetic acid buffer at pH 4.8/acetonitrile 70/30 (v/v)), as shown in Fig. 5b. Because of the small gap between the pH 4.8 and the pKa value of 5AVA (4.27), two peaks derived from the ionization of 5AVA were observed. Due to the stability of derivatives and operation durability, less acidic condition for a 6
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Fig. 6. Effect of the mobile phase composition on the chromatograms of L-lysine and 5-aminovaleric acid derivatives in a C18 column: (a) Phosphate buffer (NaH2PO4/Na2HPO4) at pH 6.8/acetonitrile, 50/50 (v/v), (b) phosphate buffer at pH 6.8/acetonitrile, 60/40 (v/v), and (c) phosphate buffer at pH 6.8/acetonitrile, 70/30 (v/v) (from Fig. 3b). Flow rate of 1 mL/min and UV detection at 284 nm.
L − 1 with a regression coefficient of 0.9951 for 5AVA and 0.9998 for Llysine. The limit of detection (LOD) and limit of quantitation (LOQ) were calculated with the formula: LOD = 3σ/S, LOQ = 10σ/S, where S is the slope of the calibration curve and σ is the standard deviation of intercept. The LOD values of 13.6 μmol L − 1 for 5AVA and 13.5 μmol L − 1 for L-lysine, and the LOQ values of 45.3 μmol L-1 for 5AVA and 45.1 μmol L − 1 for L-lysine were obtained, indicating the high sensitivity of the method. The accuracy, precision, and reproducibility of retention time were evaluated from the chromatogram of the quality control samples at three concentration levels with three replicates. The accuracy expressed as recovery (%) ranged from 86.9 to 110.4% while the precision as RSD (%) were less than 8.8%. The reproducibility of retention time expressed as RSD (%) was less than 0.5%. As a result, the method used in
increased pH of the buffer solution resulted in an enhancement in the difference in the retention time between the two peaks. With respect to the sharpness of peaks, the use of a small amount of buffer solution is recommended, as shown in Fig. 6a. However, considering the selectivity of the L-lysine derivative to the 5AVA derivative, a larger portion of buffer solution in the mobile phase (70%) was better (Fig. 6c). 4.3. Quality assurance / quality control The results of method validation are summarized in Table 3. A daily standard calibration curves (n == 5) of amino acid derivatives were generated to evaluate the linearity of the method using the mobile phase No. 5 in Table 1, phosphate buffer at pH 6.8/acetonitrile, 70/30 (v/v). Linear relationships were obtained in the range of 1–10 mmol 7
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Table 3 Statistical parameters using proposed method for linearity, limit of detection (LOD), limit of quantitation (LOQ), accuracy, precision, and reproducibility. Analytes Concentration range (mmol L Linear equation a Regression coefficient (R) LOD (μmol L − 1) LOQ (μmol L − 1) Recovery (n == 9,%)
−1
)
RSD (%, peak area, n == 9) Retention time (min) RSD (%, retention time, n == 9) a
Low Medium High Low Medium High
5AVA
L-lys
1–10 Y == 1.4861E6X - 736.1 0.9951 13.6 45.3 110.4 99.9 102.7 2.23 2.63 2.80 3.054 0.5
1–10 Y == 1.2358E6X + 135.5 0.9998 13.5 45.1 107.1 86.9 101.3 8.80 1.42 1.51 6.933 0.1
X: concentration of organic acid (mmol L − 1); Y: peak area of organic acid.
this study showed good accuracy, precision, and reproducibility. 4.4. Determination of isotherm and separation performance of SMB To determine the optimal operating conditions and to simulate the SMB process, the column porosity and adsorption isotherm parameters are required from preliminary experiments and information. Since the pulse samples were dilute, the moment method was applied to the chromatographic peaks with the assumption of a linear isotherm. For a linear isotherm system, accurate adsorption equilibrium constants can be provided by the first moment method. The adsorption equilibrium constant (KAD) of each organic acid was determined by using Eq. (8), and the Henry's constants (Hi) were calculated using Eq. (9) [48]. The experimental data were well fitted by linear regression, having high coefficient of determination values (R2 > 0.99), as shown in Fig. 7. The Henry's constants of the L-lysine and 5AVA derivatives were 2.140 and 0.174, respectively, and the adsorption parameters obtained from the first moment could be used reasonably for the SMB process. From the adsorption isotherm parameters, the complete separation region in triangle theory was determined at the flow rate ratio of each zone (mI, mII, mIII, and mIV) in Fig. 8. In this study, the flow-rate ratio of separation zones mII and mIII in Fig. 2 was determined at the triangle vertex shown by a circle in Fig. 8, which was the largest difference between mII and mIII for the maximized flow rate of the feed. For zones I and IV, the mI and mIV values were arbitrarily chosen with a margin from the lower and upper limits, respectively (Fig. 8). The switching
Fig. 8. Complete separation region and operating points.
period was set to 180 s for the maximum flow rate at zone I, and the detailed operating conditions are presented in Table 2. For the simulation, it was assumed that (1) the SMB consists of eight identical HECTOR-M C18 columns and (2) 20 mM phosphate buffer at pH 6.8/acetonitrile 70/30 (v/v) was used as the mobile phase. The total concentration was fixed at 0.2 g/L, but the ratio of L-lysine to 5AVA in the feed was tested at 1:1, 1:2, and 2:1, as presented in Table 4. As expected from the chromatogram results, a purity and recovery of each component higher than 99% could be achieved simultaneously because the peaks were well separated by the developed mobile phase. The increase of a component in the feed led to a slight reduction in the purity of the corresponding component, whereas the recovery remained constant. Important factors to consider were the variation in eluent consumption and productivity when a specific component was present in small quantities. Compared with the variation in purity, the changes in eluent consumption and productivity were much higher for both components. Fig. 9 shows the internal concentration profiles at the beginning and the end of the switching period when the SMB process reached a cyclic steady state. The transportation of 5AVA propagated along with the mobile phase in zones II and III to the raffinate node, whereas the Llysine migrated in the opposite direction from zones III to I along with the column. Thus, a high concentration profile of each main component at each outlet port was formed, and this enabled both products to be obtained with high purity and recovery. The internal concentration of each component increased as its concentration in the feed increased.
Fig. 7. Dependence of the first moment of the chromatographic response curve on L/u values of L-lysine and 5-aminovaleric acid in a HPLC column with 20 mM phosphate buffer at pH 6.8/acetonitrile (70/30 (v/v)) as the mobile phase. 8
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Table 4 Separation performance of SMB. Run
Feed conc. [g/L] L-Lys 5AVA
Purity [%] L-Lys
5AVA
Recovery [%] L-Lys 5AVA
Eluent consumption [L g − 1] L-Lys 5AVA
Productivity [g L − 1 h − 1] L-Lys 5AVA
1 2 3
0.1 0.067 0.133
99.50 99.75 99.00
99.54 99.01 99.77
99.54 99.54 99.54
0.49 0.65 0.33
21.08 15.85 31.47
0.1 0.133 0.067
99.49 99.49 99.49
The impurity of each product resulted from the propagation of the other component, which was observed at the beginning of the switching period in the extract product node and at the end of the switching period in the raffinate product node. Various operating strategies for reducing the impurity concentration or treating the contaminated part of the product have been reported because this is common in conventional SMB processes [20]. For example, the contaminated part of each product is simply discarded in the partial-discard operation to improve the purity [49]. Alternatively, the discarded part can be recycled to the feed in recycling partial-discard operation, enabling the recovery of further product [50]. Another example is Backfill SMB, in which the product is recycled to the feed or intermediate node in zones II and III [51]. Table 4 and Fig. 9 indicate that, if the content of one component is too less in the feed, the other component can be produced efficiently with respect to all the performance parameters. If both components are important regardless of concentration in the feed, further study of the operating strategies will be required to improve eluent consumption
0.49 0.33 0.65
21.09 31.48 15.86
and productivity. 5. Conclusion The effects of pH and amount of buffer in the mobile phase on the separation of L-lysine and 5-aminovaleric acid using a simulated moving bed process were studied. A pre-column derivatization step was essential for chromatographic separation because the adsorption selectivity and UV detection sensitivity were enhanced by the generated aminoenone group. Among the mobile phases used, 20 mM phosphate buffer at pH 6.8/ acetonitrile 70/30 (v/v) was selected as the optimal mobile phase. Since the organic acids could be ionized at low pH values, the selection of the optimal pH condition to suppress the ionization was the most important factor considering the pKa values of the components. However, considering the subsequent isolation of separated compounds, acetic acid buffer (pH 4.8) was recommended when L-lysine is the only desired product. The difference in the retention times of the L-
Fig. 9. Internal concentration profiles of the SMB at cyclic steady state. 9
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lysine and 5AVA derivatives could be controlled by changing the amount of buffer solution in the mobile phase. In this study, a sufficiently high ratio of the buffer solution in the mobile phase resulted in improved selectivity with good intensity. The conventional SMB process with 2–2–2–2 configuration using C18 columns was simulated based on the isotherm parameters obtained from the moment method. Thus, the separation feasibility confirmed that 5AVA and L-lysine could be successfully produced from their mixture at purity and recovery of greater than 99% by using a four-zone SMB process. The eluent consumption exhibited 0.33–0.65 L/g, depending on feed concentration. The result indicated high reduction of eluent in SMB separation compared to about 7 ml of eluent consumption to separate 2 μg of each diamine derivative sample in a batch separation (350 L/g). Furthermore, the waste stream of a mobile phase would be significantly reduced by recirculating the eluent obtained from the subsequent step. From the results of this study, the SMB process will be a reliable technique for the separation of L-lysine, 5AVA and, possibly, other organic acids. Furthermore, because a specific organic acid with high concentration is the target product in organic acid mixtures, the SMB process could be a powerful separation method. However, if the concentration of the target organic acid is relatively low, the development and selection of efficient operating strategies will be important with respect to eluent consumption and productivity. Furthermore, the mobile phase composition is important in practical processes because the products from the SMB process should be isolated from the mobile phase. Therefore, since a volatile buffer has a greater potential to reduce the cost and energy for the subsequent isolation step, the study for a volatile buffer between pH 4.8 and 6.8 is needed in future to protect ionization, retain high separation efficiency and lower the separation cost.
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