Journal of Chromatography A, 1406 (2015) 118–128
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Steady state preparative multiple dual mode counter-current chromatography: Productivity and selectivity. Theory and experimental verification Artak E. Kostanyan ∗ , Andrey A. Erastov Kurnakov Institute of General & Inorganic Chemistry, Russian Academy of Sciences, Leninsky Prospekt 31, Moscow 119991, Russia
a r t i c l e
i n f o
Article history: Received 9 April 2015 Received in revised form 28 May 2015 Accepted 30 May 2015 Available online 9 June 2015 Keywords: Multiple dual mode counter-current chromatography Periodic sample loading Peak equations Steady state operation
a b s t r a c t In the steady state (SS) multiple dual mode (MDM) counter-current chromatography (CCC), at the beginning of the first step of every cycle the sample dissolved in one of the phases is continuously fed into a CCC device over a constant time, not exceeding the run time of the first step. After a certain number of cycles, the steady state regime is achieved, where concentrations vary over time during each cycle, however, the concentration profiles of solutes eluted with both phases remain constant in all subsequent cycles. The objective of this work was to develop analytical expressions to describe the SS MDM CCC separation processes, which can be helpful to simulate and design these processes and select a suitable compromise between the productivity and the selectivity in the preparative and production CCC separations. Experiments carried out using model mixtures of compounds from the GUESSmix with solvent system hexane/ethyl acetate/methanol/water demonstrated a reasonable agreement between the predictions of the theory and the experimental results. © 2015 Elsevier B.V. All rights reserved.
1. Introduction A general and distinctive feature of counter-current chromatography (CCC) separation processes is the mobility of both phases [1–8], which allowed to develop and implement various dual-mode (DM) operating schemes, where both phases are countercurrently moving through a chromatographic column [9–24]. As is well known, the separation power of the countercurrent processes is much higher than that of conventional chromatography processes, as they require a much smaller number of theoretical plates to solve separation problems. In cyclic DM CCC processes (intermittent counter-current extraction (ICCE), intermittent CCC (ICCC) and multiple dual-mode CCC (MDM CCC)), the separation consists of a succession of two isocratic counter-current steps carried out in series alternating between normal phase and reversed phase operation. In ICCE, the sample is continuously introduced at the middle of the column or between two columns [10,12,13,16,17]; after several cycles, the concentration profiles of solutes eluted with both phases become constant. In ICCC, in contrast to ICCE, the sample is injected into the column during a short time, and the separation is non-steady state [10,22]. The advantage of the ICCE over
∗ Corresponding author. Tel.: +7 4959554834; fax: +7 4959554834. E-mail address: [email protected] (A.E. Kostanyan). http://dx.doi.org/10.1016/j.chroma.2015.05.074 0021-9673/© 2015 Elsevier B.V. All rights reserved.
the ICCC is high productivity, as the sample is continuously fed into the column. The benefit of the ICCC over the ICCE is that the non-steady state operation allows separating more than two components in a single stage, whereas by the ICCE only two components (or two groups of components) can be separated in one operation step. The MDM CCC differs from the ICCC in sample loading conditions: in the MDM CCC the sample is injected at the beginning of the column [9–12,18–20]. The productivity of the MDM CCC technique may be increased by the sample re-injection between each of the dual-mode steps as first proposed by Delannay et al. [20]. The theoretical approaches for the MDM method with single sample injection were proposed in Refs. [14,21]. Analytical solutions for MDM separations were presented, where the timing of the alternating phase elution steps can be adjusted [23]. Recently, basing on the cell model, a full theoretical treatment of the MDM CCC with periodic impulse injection of the sample was performed [24]. It is evident that the performance of the SS MDM CCC may be further increased by increasing the mass loading, i.e. increasing the loading time. This paper is devoted to the further development of the theory of the MDM CCC with periodic sample loading and its experimental verification. In this study, the theory was extended to develop analytical solutions for the MDM CCC with periodic non-impulse (semi-continuous) sample loading.
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First step of the j cycle: sample loading period - τs; flow period - τx SS Tank
τs Fx xs v2
XP Tank
v1
XP Pump
0
1
2
k
n
τy Fy
1
2
k
n
1−e
−at
n (at)i
τx Fx
(2)
i!
i=0
Xj(t)
t > ts
X1 (k, t) = e−a(t−ts )
Second step of the j cycle: flow period - τy 0
1 x1 (n, t) = X1 (n, t) = X1 (t) = ts x¯
(τx-τs )Fx
Yj(t)
119
k [a (t − ts )]k−i
X1 (i, ts ) ;
(k − i)!
i=0
k = 0, 1, 2, . . ., n (3)
Fy
Fig. 1. Schematic diagram for mathematical model of steady state multiple dual mode counter-current chromatography.
X1 (n, t) = X1 (t) = e−a(t−ts )
The development of analytical solutions in the present paper is based on the cell model of chromatography, which was discussed in detail in Refs. [10,22].
1 X1 (i, ts ) = X1 (k, ts ) = ts
n [a (t − ts )]n−i
(n − i)!
i=0
1−e
−ats
k (ats )i
X1 (i, ts )
;
i!
i=0
(4)
k = 0, 1, 2, . . ., n (5)
2. Theory 2.1. Mathematical model of the steady-state multiple dual mode counter-current chromatography with periodic semi-continuous sample loading As mentioned above, we consider linear chromatography and build on the equilibrium cell model. The process scheme is shown in Fig. 1. The number of the feed cell is denoted as 0, and the current number of cells as k = 0,1, 2,. . ., n. We denote the phase with which a mixture of components is fed in the system as the “x” phase, and the other phase as the “y” phase. To develop analytical solutions, we assume that: (1) MDM separation process consists of a succession of two isocratic steps: first, the “x” phase pumped as the mobile phase from the zero numbered cell to the last cell n and second, the “y” phase pumped as the mobile phase from the last cell n to the zero numbered cell. (2) The start time for every step of any cycle j is = 0. (3) The duration of the flow periods of the phases is kept constant for all the cycles: jx = x = const; jy = y = const. (4) The process starts with the “x” phase mobile. A solute dissolved in this phase, is continuously fed to the zero numbered cell in the first step of every cycle over a time s , not exceeding the run time of the first step x : s < x . The solute solution is loaded by switching the “x” phase pump (valve 1) from the fresh “x” phase tank to the solute solution tank (valve 2). After the solute loading is finished, the “x” phase pump is switched back to the fresh “x” phase tank.
a=
N ; 1 − S + SKD
(6)
S=
Vy Vy + Vx
(7)
Eqs. (1) and (3) describe the profiles of the solute in the column during and after the sample loading period of the first step of the first cycle, respectively. Eqs. (2) and (4) describe the elution profiles during and after the sample loading period of the first step of the first cycle. In Eqs. (1–5) x1 (k,t) and x1 (n,t) are the solute concentration in the “x” phase; X1 (k,t) and X1 (n,t) are the normalized solute concentration in the “x” phase; x¯ = xs Fx s /Vc is the average concentration in the column; xs is the solute concentration in the feed solution; t = Fx /Vc is the normalized time; ts = s Fx /Vc is the normalized sample loading time; Fx is the volumetric flow rate of “x” phase; KD = y/x is the ratio of equilibrium phase concentrations; N = n + 1 is the total number of equilibrium cells in the column; Vx and Vy are the volumes of “x” and “y” phases in the column, respectively; Vc = Vx + Vy is the column volume; 2.1.2. j Cycle The first step (j > 1): 0 ≤ t ≤ ts
1 Xj (k, ts ) = ts
1−e
+ e−ats
Under these assumptions, the solution of the model equations is obtained as follows:
−ats
k (ats )i
i!
0 k (ats )k−i 0
(k − i)!
1 Xj (t) = Xj (n, t) = ts
0 ≤ t ≤ ts
X1 (k, t) =
1 x1 (k, ) = ts x¯
1 − e−at
k (at)i i=0
i!
1−e
k = 0, 1, 2, . . ., n (1)
Xj−1 i, ty = Xj−1 k, ty =
n (at)i
n (at)n−i 0
;
−at
0
+ e−at
Xj−1 i, ty ;
2.1.1. First cycle The first step (“x” phase flow period):
(n − i)!
(8)
i!
Xj−1 i, ty
Yj−1 k, ty KD
k = 0, 1, 2, . . ., n
(9)
(10)
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Fig. 2. Simulation of the steady-state MDM separation of the binary mixture containing compounds with partition coefficients KD1 = 0.5 and KD2 = 1. Process conditions: the duration of the sample loading period ts = 0.01; the duration of the first steps tx = 0.7; the duration of the second steps ty = 0.9. N = 100; S = 0.7. The steady-state regime is achieved after four cycles.
Xj k, ty = e
t > ts
Xj (k, tx ) = e−a(tx −ts )
−KD aty
i−k n KD aty i=k
k [a (tx − ts )]k−i 0
(k − i)!
Xj (i, ts ) ;
k = 0, 1, 2, . . ., n (11)
Xj (t) = Xj (n, t) = e−a(t−ts )
n [a (t − ts )]n−i 0
Xj (i, ts ) = Xj (k, ts )
(n − i)!
Xj (i, ts )
(12)
Yj (t) = Yj (0, t) =
(i − k)!
yj (0, t)
Xj (i, tx ) = Xj (k, tx )
x¯
Xj (i, tx ) ;
k = 0, 1, 2, . . ., n (14)
= KD e−KD at
n (KD at)i i=0
i!
Xj (i, tx )
(15)
(16)
(13)
Eqs. (8) and (11) describe the profiles of the solute in the column at the end of the sample loading period and after the first step of the current cycle j > 1, respectively; Eqs. (9) and (12) describe the elution profiles during and after the sample loading period of the first step of any cycle j > 1, respectively. In Eq. (11) tx = x Fx /Vc is the normalized run time of the first step (“x” phase flow period). The second step (“y” phase flow period) of any j cycle (j = 1,2,3,. . .):
Eq. (14) describes the distribution of the solute in the column after the second step of the current cycle j. Eq. (15) describes the elution profile in the “y” phase for the current cycle j. In Eqs. (15) yj is the solute concentration in the “y” phase; Yj is the normalized solute concentration in the “y” phase; t = Fy /Vc is the normalized time for the second step; ty = y Fy /Vc is the normalized run time of the second step (“y” phase flow period). The eluted portions (q) of the solute are determined by the following equations: With the “x” phase:
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121
A
B
Fig. 3. Simulation of the steady-state MDM separation of the binary mixture KD1 = 0.5 and KD2 = 1. Process conditions: N = 100; S = 0.7: (A) ts = 0.1; tx = 0.7; ty = 0.9. The steady-state regime is achieved after three cycles. (B) ts = 0.3; tx = 0.8; ty = 0.85. The steady-state regime is achieved after eight cycles.
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Fig. 4. Simulation of the steady-state MDM separation of the binary mixture KD1 = 0.5 and KD2 = 1. Process conditions: ts = 0.2; N = 100; S = 0.7: (A) tx = 0.7; ty = 0.9. The steady-state regime is achieved after three cycles.: (B) tx = 0.8; ty = 0.9. The steady-state regime is achieved after seven cycles.
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123
In the first cycle: Q1x = q1x = Q
tx
ts X1 (t) dt =
tx X1 (t) dt +
0
X1 (t) dt ts
0
1 X1 (k, tx ) a n
= 1−
(17)
k=0
In the j cycle (j > 1) Qjx Q
tx = qjx =
ts Xj (t) dt =
0
tx Xj (t) dt + ts
0
=1+
Xj (t) dt
1 1 Xj−1 k, ty − Xj (k, tx ) a a n
n
k=0
k=0
(18)
With the “y” phase in any j cycle Qjy Q
ty = qjy = 0
1 Yj (t) dt = a
n
Xj (k, tx ) −
k=0
n
Xj i, ty
(19)
k=0
2.2. Analysis of the mathematical model The equations presented in Section 2.1 are sufficient to simulate the profiles of solutes in the column after both steps for any cycle and the chromatograms eluted with both phases during each step and cycle for the given process parameters: KD , n, S, ts , tx and ty . When the solutes are separated and completely eluted with the phase flows within one cycle, all the cycles, including the initial ones are identical, and the process represents the steady state operation of the two-dimensional separation [25–27] performed using the same column. When the solutes cannot be separated for one cycle, they come out only by parts in the initial cycles and the steady-state regime is achieved after a certain number of cycles. In Figs. 2–4, examples of the simulation of the steady-state MDM separation of the binary mixture containing compounds with partition coefficients KD1 = 0.5 and KD2 = 1, are shown for different sample loading conditions (periods: ts = 0.01; ts = 0.1; ts = 0.2; ts = 0.3). The elution profiles in the “x” phase are calculated by Eqs. (2), (4), (9) and (12), in the “y” phase—by Eq. (15). The eluted portions of the solutes qjx and qjy are determined by Eqs. (17)–(19). After reaching steady state, the values of qjx and qjy become constant. Moreover, under steady state conditions the amount of a solute fed to the column in a single cycle must be equal to the amount of this solute, eluted with both phases during one cycle, that is: qjx + qjy = 1. In the steady state regime, concentrations vary over time during each cycle, however, the elution profiles remain the same (are repeated) in all subsequent cycles. Depending on the operating conditions the steady-state regime is achieved after three (Figs. 3A and 4A), four (Fig. 2), seven (Fig. 4B) or eight (Fig. 3B) cycles. Increasing the duration of the sample loading period from ts = 0.01 to ts = 0.1, ts = 0.2 and ts = 0.3, leads to the increase in the productivity of 10, 20 and 30 times, respectively. When ts = 0.01, the purity of the separated products is practically equal to 100% (Fig. 2). When ts = 0.1 (Fig. 3A), the purity of the first solute (KD1 = 0.5) eluted with the “x” phase is 100%, the recovery—99.0%, and the purity of the second solute (KD2 = 1) eluted with the “y” phase is 99.0%, the recovery—100%. For ts = 0.2: when tx = 0.7, ty = 0.9 (Fig. 4A), the purity of the first solute is 100%, the recovery—93.8%, and the purity of the second
Fig. 5. Simplified diagram of the experimental CPLC set up.
solute is 94.2%, the recovery—100%; when tx = 0.8, ty = 0.9 (Fig. 4B), the purity of the first solute is 98.6%, the recovery—99.5%, and the purity of the second solute is 99.5%, the recovery—98.6%. When ts = 0.3 (Fig. 3B), the purity of the first solute is 97.3%, the recovery—97.8%; the purity of the second solute is 97.8%, the recovery—97.3%. Thus, proper selection of the process operating parameters (ts , tx and ty ) for a given mixture can allow to find a suitable compromise between productivity and selectivity. To simulate the separation of complex mixtures by MDM CCC, first the values of N for the components of the mixture should be experimentally determined in conventional isocratic flow mode at operating parameters of a CCC system (S, Fx , Fy ). Then by numerical studies via the above equations the process operating parameters (ts , tx and ty ) are to be selected to ensure the required separation of the components. 3. Experimental 3.1. Apparatus To compare theory with practice, the controlled-cycle pulsed liquid–liquid chromatography (CPLC) system consisting of a series of multistage columns connected in the form of a coil, and a
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Fig. 6. SS MDM separation of aspirin (KDa = 0.5) and caffeine (KDc = 0.13). Simulated (A) and experimental (B) chromatograms of caffeine. Process conditions: ts = 0.005; tx = 0.45; ty = 1.1; N = 70; S = 0.5. In (A) concentrations X are normalized using the average concentration in the column; in (B) concentrations x are proportional to the actual units of the detector.
pulsation-cycling flow system was employed [28]. In the CPLC device, the pulsed-mixing technique is used to force the mobile phase through the columns and disperse it in the stationary phase in each stage of the columns. The operation of the CPLC system is illustrated in Supplementary data. The experimental CPLC set up (Fig. 5) consisted of four multistage columns of 6.4 mm internal diameter FEP tubing (Cole-Parmer, USA), and the purpose-built piston pump with PC regulated frequency and piston velocity for the discrete supply of the mobile phase as repetitive pulses to the apparatus. In all experiments the mobile phase portions vm = 0.55 ml were forced into the apparatus with an interval of 15 s. Each column was divided into 26 stages by at 35 mm intervals (stage volume vs = 1.1 ml) spaced horizontal perforated plates with 13 0.25 mm diameter holes, fabricated from 3 mm thick PTFE sheet. The peristaltic pump Heidolph PD 5101 (Germany) was used to fill the apparatus with the stationary phase. Akvilon UVV 101.4 M (Russia) spectrophotometers with preparative flow cells operating at 270 nm and 300 nm were used to monitor the eluents on each end outlet of the CPLC system.
3.2. Two-phase solvent system and sample solutions The solvent system consisting of hexane, ethyl acetate, methanol and water with volume ratio of 1:1:1:1 (HEMWat system 17) was used. To evaluate the performance of the MDM CCC with periodic sample loading, aspirin and caffeine from the GUESSmix [29] were used. 3.3. Experimental procedures In all the experiments, the process started in reverse phase mode with the lower phase (“x” phase) as the mobile phase and upper phase (“y” phase) as the stationary phase: the empty columns were initially filled with upper phase. It must be noted that the retained volume of the stationary phase in the CPLC system is determined by the volume of the mobile phase portions vm fed to the apparatus [28]. The volume of each portion vm is equal to the mobile phase volume in each stage of the columns. Hydrodynamic equilibration was established to achieve approximately 50%/50% upper to lower
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Fig. 7. SS MDM separation of aspirin and caffeine. Simulated (A) and experimental (B) chromatograms of aspirin. Process conditions: ts = 0.005; tx = 0.45; ty = 1.1; N = 70; S = 0.5. In (A) concentrations Y are normalized using the average concentration in the column; in (B) concentrations y are proportional to the actual units of the detector.
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Fig. 8. SS MDM separation of aspirin and caffeine. Simulated (A) and experimental (B) chromatograms of caffeine. Process conditions: ts = 0.05; tx = 0.45; ty = 1.1; N = 70; S = 0.5.
phase ratio in the columns by setting vm = 0.5vs . The sample was loaded with the “x” phase at the beginning of each cycle. The mobile phase was fed into the apparatus in separate portions; the volume of each portion was equal to the 0.5 volume of a column stage. All experiments were performed at room temperature. 4. Results and discussion Steady state MDM separation of aspirin and caffeine was performed under two different sample loading conditions: (1) with short sample loading time: the sample was fed into the apparatus in one portion of the lower phase, which corresponds to the duration of the sample loading period ts = 0.005. (2) With long sample loading time: the sample was fed with ten portions of the lower phase (the duration of the sample loading period ts = 0.05). The separation processes were at first simulated by using the theoretical relationships presented in Section 2 and then verified experimentally. For the simulation, the values of the partition coefficients (defined as the concentration in the upper phase divided by the concentration in the lower phase), and separation
efficiency of the CPLC system (measured by the theoretical plate number N) for each compound determined earlier in the single isocratic run operations in normal and reversed phase modes [23] were used. The results of the numerical simulation and experimental studies are presented in Figs. 6–9. In these figures, the chromatograms of the solutes eluted during each step of the first four cycles are shown. The figures do not show the steps in which there was no elution of the solutes with the phases. In Figs. 6 and 7, the calculated and experimental chromatograms obtained under the short sample loading time (ts = 0.005) operating conditions are compared. In Figs. 8 and 9, the calculated and experimental chromatograms for long sample loading time (ts = 0.05) are compared. In the calculations, experimentally determined values of the partition coefficients for aspirin KDa = 0.5 and caffeine KDc = 0.13 and the average value of N = 70 for both components from [23] were used. According to the results of the numerical studies, the following run conditions were selected: the duration of the first steps tx = 0.45; the duration of the second steps ty = 1.1. As shown in Figs. 7 and 9, aspirin is eluting already in the first cycle (with the upper phase at the second step), while caffeine starts
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Fig. 9. SS MDM separation of aspirin and caffeine. Simulated (A) and experimental (B) chromatograms of aspirin. Process conditions: ts = 0.05; tx = 0.45; ty = 1.1; N = 70; S = 0.5.
to elute with the lower phase in the second cycle (Figs. 6 and 8). After the second cycle, the elution profiles remain approximately constant. As can be seen from Figs. 6–9, an increase in productivity of ten times had no noticeable effect on the selectivity of the separation. The results presented in these figures demonstrate in general a fair agreement between the theory and the experiment.
The reasons for the discrepancy between theory and experiment can be the displacement of the previous mobile phase in the flow line back into the device with each switching cycle and the uneven distribution of two phases through the columns. In addition, the theoretical model does not account for differences in the value of N for the first and second steps of the process.
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5. Conclusion The steady state MDM CCC is a cyclic separation process consisting of a succession of two isocratic counter-current steps and is carried out in series alternating between “x” phase and “y” phase flow periods. The duration of the flow periods of the phases is kept constant. The sample dissolved in the “x” phase is continuously fed into a CCC column at the beginning of the first step of every cycle over a constant time, not exceeding the run time of the first step. The SS MDM CCC separation is described on the basis of the equilibrium cell model. Equations are developed allowing the simulation of the chromatograms eluted from the column with the phases during each step of the cycles. There was a good agreement between the predicted and experimental separations. The SS MDM CCC separation technique may be especially useful for large scale preparative and industrial scale chromatography, since it offers a means to reduce eluent consumption and increase productivity and provides both high productivity and high resolution. Acknowledgements This work was supported by the Russian Foundation for Basic Research (project no. 15-03-02940). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chroma.2015.05. 074 References [1] M. Neves Vieiraa, F.N. Costa, G.G. Leitão, I. Garrard, P. Hewitson, S. Ignatova, P. Winterhalter, G. Jerz, Schinus terebinthifolius scale-up countercurrent chromatography (Part I): High performance countercurrent chromatography fractionation of triterpene acids with off-line detection using atmospheric pressure chemical ionization mass spectrometry, J. Chromatogr. A 1389 (2015) 39–48. [2] J.B. Friesen, S. Ahmed, G.F. Pauli, Qualitative and quantitative evaluation of solvent systems for countercurrent separation, J. Chromatogr. A 1377 (2015) 55–63. [3] W.D. Conway, Counter-current chromatography: simple process and confusing terminology, J. Chromatogr. A 1218 (2011) 6015–6023. [4] Y. Ito, Golden rules and pitfalls in selecting optimum conditions for high-speed counter-current chromatography, J. Chromatogr. A 1065 (2005) 145–168. [5] A. Berthod, T. Maryutina, B. Spivarov, O. Shpigun, I.A. Sutherland, Countercurrent chromatography in analytical chemistry (IUPAC technical report), Pure Appl. Chem. 81 (2009) 355–387. [6] W.D. Conway, Countercurrent Chromatography: Apparatus, Theory and Applications, first ed., VCH, New York, NY, 1990. [7] I. Sutherland, P. Hewitson, S. Ignatova, Scale-up of counter-current chromatography: demonstration of predictable isocratic and quasi-continuous operating modes from the test tube to pilot/process scale, J. Chromatogr. A 1216 (2009) 8787–8792. [8] I. Sutherland, D. Hawes, S. Ignatova, L. Janaway, P. Wood, Review of progress toward the industrial scale-up of CCC, J. Liq. Chromatogr. Related Technol. 28 (2005) 1877–1891.
[9] M. Englert, S. Hammann, W. Vetter, Isolation of -carotene, ␣-carotene and lutein from carrots by countercurrent chromatography with the solvent system modifier benzotrifluoride, J. Chromatogr. A 1388 (2015) 119–125. [10] P. Hewitson, I.A. Sutherland, A.E. Kostanyan, A.A. Voshkin, S. Ignatova, Intermittent counter-current extraction–equilibrium cell model, scaling and an improved bobbin design, J. Chromatogr. A 1303 (2013) 18–27. [11] E. Kitazume, S. Koikawa, L. Hui, S. Sannohe, Y. Yang, Y. Maki, Y. Ito, Sequential determination of anionic-type detergents by complexation with methylene blue using dual high speed counter-current chromatography, J. Chromatogr. A 1236 (2012) 148–151. [12] S. Ignatova, P. Hewitson, B. Mathews, I. Sutherland, Evaluation of dual flow counter-current chromatography and intermittent counter-current extraction, J. Chromatogr. A 1218 (2011) 6102–6106. [13] P. Hewitson, S. Ignatova, I.A. Sutherland, Intermittent counter-current extraction—effect of the key operating parameters on selectivity and throughput, J. Chromatogr. A 1218 (2011) 6072–6078. [14] N. Mekaoui, A. Berthod, Using the liquid nature of the stationary phase. VI. Theoretical study of multi-dual mode countercurrent chromatography, J. Chromatogr. A 1218 (2011) 6061–6071. [15] E. Hopmann, M. Minceva, Separation of a binary mixture by sequential centrifugal partition chromatography, J. Chromatogr. A 1229 (2012) 140–147. [16] A. Peng, H. Ye, J. Shi, S. He, S. Zhong, S. Li, L. Chen, Separation of honokiol and magnolol by intermittent counter-current extraction, J. Chromatogr. A 1217 (2010) 5935–5939. [17] P. Hewitson, S. Ignatova, H. Ye, L. Chen, I.A. Sutherland, Intermittent countercurrent extraction as an alternative approach to purification of Chinese herbal medicine, J. Chromatogr. A 1216 (2009) 4187–4192. [18] N. Rubio, S. Ignatova, C. Minguillon, I.A. Sutherland, Multiple dual-mode countercurrent chromatography applied to chiral separations using a (S)naproxen derivative as chiral selector, J. Chromatogr. A 1216 (2009) 8505– 8511. [19] A. Berthod (Ed.), Countercurrent Chromatography, The Support-Free Liquid. [20] E. Delannay, A. Toribio, L. Boudesocque, J.M. Nuzillard, M. Zeches-Hanrot, E. Dardennes, G. Le Dour, J. Sapi, J.-H. Renault, Multiple dual-mode centrifugal partition chromatography, a semi-continuous development mode for routine laboratory-scale purifications, J. Chromatogr. A 1127 (2006) 45–51. [21] Y. Yang, H.A. Aisa, Y. Ito, Mathematical model of computer-programmed intermittent dual countercurrent chromatography applied to hydrostatic and hydrodynamic equilibrium systems, J. Chromatogr. A 1216 (2009) 6310–6318. [22] A.E. Kostanyan, S. Ignatova, I.A. Sutherland, P. Hewitson, Y.A. Zakhodjaeva, A.A. Erastov, Steady-state and non-steady state operation of counter-current chromatography devices, J. Chromatogr. A 1314 (2013) 94–105. [23] A.E. Kostanyan, A.A. Erastov, O.N. Shishilov, Multiple dual mode countercurrent chromatography with variable duration of alternating phase elution steps, J. Chromatogr. A 1347 (2014) 87–95. [24] A.E. Kostanyan, Multiple dual mode counter-current chromatography with periodic sample injection: steady-state and non-steady-state operation, J. Chromatogr. A 973 (1373) (2014) 81–89. [25] J. Meng, Z. Yang, J. Liang, H. Zhou, S. Wu, Comprehensive multi-channel multi-dimensional counter-current chromatography for separation of tanshinones from Salvia miltiorrhiza Bunge, J. Chromatogr. A 1323 (2014) 73–81. [26] J. Liang, Z. Yang, X. Cao, B. Wu, S. Wu, Preparative isolation of novel antioxidant flavonoids of alfalfa by stop-and-go counter-current chromatography and following on-line liquid chromatography desalination, J. Chromatogr. A 1218 (2011) 6191–6199. [27] R. Hu, X. Dai, X. Xu, C. Sun, Y. Pan, Two-dimensional counter-current chromatography: 1st traditional counter-current chromatography, 2nd acid–base elution counter-current chromatography, J. Chromatogr. A 1218 (2011) 6085–6091. [28] A.E. Kostanyan, A.A. Voshkin, N.V. Kodin, Controlled-cycle pulsed liquid–liquid chromatography. A modified version of Craig’s counter-current distribution, J. Chromatogr. A 1218 (2011) 6135–6143. [29] J.B. Friesen, G.F. Pauli, G.U.E.S.S.—a generally useful dstimate of solvent systems for CCC, J. Liq. Chromatogr. Related Technol. 28 (2005) 2777–2806.
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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Steady state preparative multiple dual mode counter-current chromatography: Productivity and selectivity. Theory and experimental verification Artak E. Kostanyan ∗ , Andrey A. Erastov Kurnakov Institute of General & Inorganic Chemistry, Russian Academy of Sciences, Leninsky Prospekt 31, Moscow 119991, Russia
a r t i c l e
i n f o
Article history: Received 9 April 2015 Received in revised form 28 May 2015 Accepted 30 May 2015 Available online 9 June 2015 Keywords: Multiple dual mode counter-current chromatography Periodic sample loading Peak equations Steady state operation
a b s t r a c t In the steady state (SS) multiple dual mode (MDM) counter-current chromatography (CCC), at the beginning of the first step of every cycle the sample dissolved in one of the phases is continuously fed into a CCC device over a constant time, not exceeding the run time of the first step. After a certain number of cycles, the steady state regime is achieved, where concentrations vary over time during each cycle, however, the concentration profiles of solutes eluted with both phases remain constant in all subsequent cycles. The objective of this work was to develop analytical expressions to describe the SS MDM CCC separation processes, which can be helpful to simulate and design these processes and select a suitable compromise between the productivity and the selectivity in the preparative and production CCC separations. Experiments carried out using model mixtures of compounds from the GUESSmix with solvent system hexane/ethyl acetate/methanol/water demonstrated a reasonable agreement between the predictions of the theory and the experimental results. © 2015 Elsevier B.V. All rights reserved.
1. Introduction A general and distinctive feature of counter-current chromatography (CCC) separation processes is the mobility of both phases [1–8], which allowed to develop and implement various dual-mode (DM) operating schemes, where both phases are countercurrently moving through a chromatographic column [9–24]. As is well known, the separation power of the countercurrent processes is much higher than that of conventional chromatography processes, as they require a much smaller number of theoretical plates to solve separation problems. In cyclic DM CCC processes (intermittent counter-current extraction (ICCE), intermittent CCC (ICCC) and multiple dual-mode CCC (MDM CCC)), the separation consists of a succession of two isocratic counter-current steps carried out in series alternating between normal phase and reversed phase operation. In ICCE, the sample is continuously introduced at the middle of the column or between two columns [10,12,13,16,17]; after several cycles, the concentration profiles of solutes eluted with both phases become constant. In ICCC, in contrast to ICCE, the sample is injected into the column during a short time, and the separation is non-steady state [10,22]. The advantage of the ICCE over
∗ Corresponding author. Tel.: +7 4959554834; fax: +7 4959554834. E-mail address: [email protected] (A.E. Kostanyan). http://dx.doi.org/10.1016/j.chroma.2015.05.074 0021-9673/© 2015 Elsevier B.V. All rights reserved.
the ICCC is high productivity, as the sample is continuously fed into the column. The benefit of the ICCC over the ICCE is that the non-steady state operation allows separating more than two components in a single stage, whereas by the ICCE only two components (or two groups of components) can be separated in one operation step. The MDM CCC differs from the ICCC in sample loading conditions: in the MDM CCC the sample is injected at the beginning of the column [9–12,18–20]. The productivity of the MDM CCC technique may be increased by the sample re-injection between each of the dual-mode steps as first proposed by Delannay et al. [20]. The theoretical approaches for the MDM method with single sample injection were proposed in Refs. [14,21]. Analytical solutions for MDM separations were presented, where the timing of the alternating phase elution steps can be adjusted [23]. Recently, basing on the cell model, a full theoretical treatment of the MDM CCC with periodic impulse injection of the sample was performed [24]. It is evident that the performance of the SS MDM CCC may be further increased by increasing the mass loading, i.e. increasing the loading time. This paper is devoted to the further development of the theory of the MDM CCC with periodic sample loading and its experimental verification. In this study, the theory was extended to develop analytical solutions for the MDM CCC with periodic non-impulse (semi-continuous) sample loading.
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First step of the j cycle: sample loading period - τs; flow period - τx SS Tank
τs Fx xs v2
XP Tank
v1
XP Pump
0
1
2
k
n
τy Fy
1
2
k
n
1−e
−at
n (at)i
τx Fx
(2)
i!
i=0
Xj(t)
t > ts
X1 (k, t) = e−a(t−ts )
Second step of the j cycle: flow period - τy 0
1 x1 (n, t) = X1 (n, t) = X1 (t) = ts x¯
(τx-τs )Fx
Yj(t)
119
k [a (t − ts )]k−i
X1 (i, ts ) ;
(k − i)!
i=0
k = 0, 1, 2, . . ., n (3)
Fy
Fig. 1. Schematic diagram for mathematical model of steady state multiple dual mode counter-current chromatography.
X1 (n, t) = X1 (t) = e−a(t−ts )
The development of analytical solutions in the present paper is based on the cell model of chromatography, which was discussed in detail in Refs. [10,22].
1 X1 (i, ts ) = X1 (k, ts ) = ts
n [a (t − ts )]n−i
(n − i)!
i=0
1−e
−ats
k (ats )i
X1 (i, ts )
;
i!
i=0
(4)
k = 0, 1, 2, . . ., n (5)
2. Theory 2.1. Mathematical model of the steady-state multiple dual mode counter-current chromatography with periodic semi-continuous sample loading As mentioned above, we consider linear chromatography and build on the equilibrium cell model. The process scheme is shown in Fig. 1. The number of the feed cell is denoted as 0, and the current number of cells as k = 0,1, 2,. . ., n. We denote the phase with which a mixture of components is fed in the system as the “x” phase, and the other phase as the “y” phase. To develop analytical solutions, we assume that: (1) MDM separation process consists of a succession of two isocratic steps: first, the “x” phase pumped as the mobile phase from the zero numbered cell to the last cell n and second, the “y” phase pumped as the mobile phase from the last cell n to the zero numbered cell. (2) The start time for every step of any cycle j is = 0. (3) The duration of the flow periods of the phases is kept constant for all the cycles: jx = x = const; jy = y = const. (4) The process starts with the “x” phase mobile. A solute dissolved in this phase, is continuously fed to the zero numbered cell in the first step of every cycle over a time s , not exceeding the run time of the first step x : s < x . The solute solution is loaded by switching the “x” phase pump (valve 1) from the fresh “x” phase tank to the solute solution tank (valve 2). After the solute loading is finished, the “x” phase pump is switched back to the fresh “x” phase tank.
a=
N ; 1 − S + SKD
(6)
S=
Vy Vy + Vx
(7)
Eqs. (1) and (3) describe the profiles of the solute in the column during and after the sample loading period of the first step of the first cycle, respectively. Eqs. (2) and (4) describe the elution profiles during and after the sample loading period of the first step of the first cycle. In Eqs. (1–5) x1 (k,t) and x1 (n,t) are the solute concentration in the “x” phase; X1 (k,t) and X1 (n,t) are the normalized solute concentration in the “x” phase; x¯ = xs Fx s /Vc is the average concentration in the column; xs is the solute concentration in the feed solution; t = Fx /Vc is the normalized time; ts = s Fx /Vc is the normalized sample loading time; Fx is the volumetric flow rate of “x” phase; KD = y/x is the ratio of equilibrium phase concentrations; N = n + 1 is the total number of equilibrium cells in the column; Vx and Vy are the volumes of “x” and “y” phases in the column, respectively; Vc = Vx + Vy is the column volume; 2.1.2. j Cycle The first step (j > 1): 0 ≤ t ≤ ts
1 Xj (k, ts ) = ts
1−e
+ e−ats
Under these assumptions, the solution of the model equations is obtained as follows:
−ats
k (ats )i
i!
0 k (ats )k−i 0
(k − i)!
1 Xj (t) = Xj (n, t) = ts
0 ≤ t ≤ ts
X1 (k, t) =
1 x1 (k, ) = ts x¯
1 − e−at
k (at)i i=0
i!
1−e
k = 0, 1, 2, . . ., n (1)
Xj−1 i, ty = Xj−1 k, ty =
n (at)i
n (at)n−i 0
;
−at
0
+ e−at
Xj−1 i, ty ;
2.1.1. First cycle The first step (“x” phase flow period):
(n − i)!
(8)
i!
Xj−1 i, ty
Yj−1 k, ty KD
k = 0, 1, 2, . . ., n
(9)
(10)
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Fig. 2. Simulation of the steady-state MDM separation of the binary mixture containing compounds with partition coefficients KD1 = 0.5 and KD2 = 1. Process conditions: the duration of the sample loading period ts = 0.01; the duration of the first steps tx = 0.7; the duration of the second steps ty = 0.9. N = 100; S = 0.7. The steady-state regime is achieved after four cycles.
Xj k, ty = e
t > ts
Xj (k, tx ) = e−a(tx −ts )
−KD aty
i−k n KD aty i=k
k [a (tx − ts )]k−i 0
(k − i)!
Xj (i, ts ) ;
k = 0, 1, 2, . . ., n (11)
Xj (t) = Xj (n, t) = e−a(t−ts )
n [a (t − ts )]n−i 0
Xj (i, ts ) = Xj (k, ts )
(n − i)!
Xj (i, ts )
(12)
Yj (t) = Yj (0, t) =
(i − k)!
yj (0, t)
Xj (i, tx ) = Xj (k, tx )
x¯
Xj (i, tx ) ;
k = 0, 1, 2, . . ., n (14)
= KD e−KD at
n (KD at)i i=0
i!
Xj (i, tx )
(15)
(16)
(13)
Eqs. (8) and (11) describe the profiles of the solute in the column at the end of the sample loading period and after the first step of the current cycle j > 1, respectively; Eqs. (9) and (12) describe the elution profiles during and after the sample loading period of the first step of any cycle j > 1, respectively. In Eq. (11) tx = x Fx /Vc is the normalized run time of the first step (“x” phase flow period). The second step (“y” phase flow period) of any j cycle (j = 1,2,3,. . .):
Eq. (14) describes the distribution of the solute in the column after the second step of the current cycle j. Eq. (15) describes the elution profile in the “y” phase for the current cycle j. In Eqs. (15) yj is the solute concentration in the “y” phase; Yj is the normalized solute concentration in the “y” phase; t = Fy /Vc is the normalized time for the second step; ty = y Fy /Vc is the normalized run time of the second step (“y” phase flow period). The eluted portions (q) of the solute are determined by the following equations: With the “x” phase:
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121
A
B
Fig. 3. Simulation of the steady-state MDM separation of the binary mixture KD1 = 0.5 and KD2 = 1. Process conditions: N = 100; S = 0.7: (A) ts = 0.1; tx = 0.7; ty = 0.9. The steady-state regime is achieved after three cycles. (B) ts = 0.3; tx = 0.8; ty = 0.85. The steady-state regime is achieved after eight cycles.
122
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Fig. 4. Simulation of the steady-state MDM separation of the binary mixture KD1 = 0.5 and KD2 = 1. Process conditions: ts = 0.2; N = 100; S = 0.7: (A) tx = 0.7; ty = 0.9. The steady-state regime is achieved after three cycles.: (B) tx = 0.8; ty = 0.9. The steady-state regime is achieved after seven cycles.
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123
In the first cycle: Q1x = q1x = Q
tx
ts X1 (t) dt =
tx X1 (t) dt +
0
X1 (t) dt ts
0
1 X1 (k, tx ) a n
= 1−
(17)
k=0
In the j cycle (j > 1) Qjx Q
tx = qjx =
ts Xj (t) dt =
0
tx Xj (t) dt + ts
0
=1+
Xj (t) dt
1 1 Xj−1 k, ty − Xj (k, tx ) a a n
n
k=0
k=0
(18)
With the “y” phase in any j cycle Qjy Q
ty = qjy = 0
1 Yj (t) dt = a
n
Xj (k, tx ) −
k=0
n
Xj i, ty
(19)
k=0
2.2. Analysis of the mathematical model The equations presented in Section 2.1 are sufficient to simulate the profiles of solutes in the column after both steps for any cycle and the chromatograms eluted with both phases during each step and cycle for the given process parameters: KD , n, S, ts , tx and ty . When the solutes are separated and completely eluted with the phase flows within one cycle, all the cycles, including the initial ones are identical, and the process represents the steady state operation of the two-dimensional separation [25–27] performed using the same column. When the solutes cannot be separated for one cycle, they come out only by parts in the initial cycles and the steady-state regime is achieved after a certain number of cycles. In Figs. 2–4, examples of the simulation of the steady-state MDM separation of the binary mixture containing compounds with partition coefficients KD1 = 0.5 and KD2 = 1, are shown for different sample loading conditions (periods: ts = 0.01; ts = 0.1; ts = 0.2; ts = 0.3). The elution profiles in the “x” phase are calculated by Eqs. (2), (4), (9) and (12), in the “y” phase—by Eq. (15). The eluted portions of the solutes qjx and qjy are determined by Eqs. (17)–(19). After reaching steady state, the values of qjx and qjy become constant. Moreover, under steady state conditions the amount of a solute fed to the column in a single cycle must be equal to the amount of this solute, eluted with both phases during one cycle, that is: qjx + qjy = 1. In the steady state regime, concentrations vary over time during each cycle, however, the elution profiles remain the same (are repeated) in all subsequent cycles. Depending on the operating conditions the steady-state regime is achieved after three (Figs. 3A and 4A), four (Fig. 2), seven (Fig. 4B) or eight (Fig. 3B) cycles. Increasing the duration of the sample loading period from ts = 0.01 to ts = 0.1, ts = 0.2 and ts = 0.3, leads to the increase in the productivity of 10, 20 and 30 times, respectively. When ts = 0.01, the purity of the separated products is practically equal to 100% (Fig. 2). When ts = 0.1 (Fig. 3A), the purity of the first solute (KD1 = 0.5) eluted with the “x” phase is 100%, the recovery—99.0%, and the purity of the second solute (KD2 = 1) eluted with the “y” phase is 99.0%, the recovery—100%. For ts = 0.2: when tx = 0.7, ty = 0.9 (Fig. 4A), the purity of the first solute is 100%, the recovery—93.8%, and the purity of the second
Fig. 5. Simplified diagram of the experimental CPLC set up.
solute is 94.2%, the recovery—100%; when tx = 0.8, ty = 0.9 (Fig. 4B), the purity of the first solute is 98.6%, the recovery—99.5%, and the purity of the second solute is 99.5%, the recovery—98.6%. When ts = 0.3 (Fig. 3B), the purity of the first solute is 97.3%, the recovery—97.8%; the purity of the second solute is 97.8%, the recovery—97.3%. Thus, proper selection of the process operating parameters (ts , tx and ty ) for a given mixture can allow to find a suitable compromise between productivity and selectivity. To simulate the separation of complex mixtures by MDM CCC, first the values of N for the components of the mixture should be experimentally determined in conventional isocratic flow mode at operating parameters of a CCC system (S, Fx , Fy ). Then by numerical studies via the above equations the process operating parameters (ts , tx and ty ) are to be selected to ensure the required separation of the components. 3. Experimental 3.1. Apparatus To compare theory with practice, the controlled-cycle pulsed liquid–liquid chromatography (CPLC) system consisting of a series of multistage columns connected in the form of a coil, and a
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Fig. 6. SS MDM separation of aspirin (KDa = 0.5) and caffeine (KDc = 0.13). Simulated (A) and experimental (B) chromatograms of caffeine. Process conditions: ts = 0.005; tx = 0.45; ty = 1.1; N = 70; S = 0.5. In (A) concentrations X are normalized using the average concentration in the column; in (B) concentrations x are proportional to the actual units of the detector.
pulsation-cycling flow system was employed [28]. In the CPLC device, the pulsed-mixing technique is used to force the mobile phase through the columns and disperse it in the stationary phase in each stage of the columns. The operation of the CPLC system is illustrated in Supplementary data. The experimental CPLC set up (Fig. 5) consisted of four multistage columns of 6.4 mm internal diameter FEP tubing (Cole-Parmer, USA), and the purpose-built piston pump with PC regulated frequency and piston velocity for the discrete supply of the mobile phase as repetitive pulses to the apparatus. In all experiments the mobile phase portions vm = 0.55 ml were forced into the apparatus with an interval of 15 s. Each column was divided into 26 stages by at 35 mm intervals (stage volume vs = 1.1 ml) spaced horizontal perforated plates with 13 0.25 mm diameter holes, fabricated from 3 mm thick PTFE sheet. The peristaltic pump Heidolph PD 5101 (Germany) was used to fill the apparatus with the stationary phase. Akvilon UVV 101.4 M (Russia) spectrophotometers with preparative flow cells operating at 270 nm and 300 nm were used to monitor the eluents on each end outlet of the CPLC system.
3.2. Two-phase solvent system and sample solutions The solvent system consisting of hexane, ethyl acetate, methanol and water with volume ratio of 1:1:1:1 (HEMWat system 17) was used. To evaluate the performance of the MDM CCC with periodic sample loading, aspirin and caffeine from the GUESSmix [29] were used. 3.3. Experimental procedures In all the experiments, the process started in reverse phase mode with the lower phase (“x” phase) as the mobile phase and upper phase (“y” phase) as the stationary phase: the empty columns were initially filled with upper phase. It must be noted that the retained volume of the stationary phase in the CPLC system is determined by the volume of the mobile phase portions vm fed to the apparatus [28]. The volume of each portion vm is equal to the mobile phase volume in each stage of the columns. Hydrodynamic equilibration was established to achieve approximately 50%/50% upper to lower
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125
Fig. 7. SS MDM separation of aspirin and caffeine. Simulated (A) and experimental (B) chromatograms of aspirin. Process conditions: ts = 0.005; tx = 0.45; ty = 1.1; N = 70; S = 0.5. In (A) concentrations Y are normalized using the average concentration in the column; in (B) concentrations y are proportional to the actual units of the detector.
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Fig. 8. SS MDM separation of aspirin and caffeine. Simulated (A) and experimental (B) chromatograms of caffeine. Process conditions: ts = 0.05; tx = 0.45; ty = 1.1; N = 70; S = 0.5.
phase ratio in the columns by setting vm = 0.5vs . The sample was loaded with the “x” phase at the beginning of each cycle. The mobile phase was fed into the apparatus in separate portions; the volume of each portion was equal to the 0.5 volume of a column stage. All experiments were performed at room temperature. 4. Results and discussion Steady state MDM separation of aspirin and caffeine was performed under two different sample loading conditions: (1) with short sample loading time: the sample was fed into the apparatus in one portion of the lower phase, which corresponds to the duration of the sample loading period ts = 0.005. (2) With long sample loading time: the sample was fed with ten portions of the lower phase (the duration of the sample loading period ts = 0.05). The separation processes were at first simulated by using the theoretical relationships presented in Section 2 and then verified experimentally. For the simulation, the values of the partition coefficients (defined as the concentration in the upper phase divided by the concentration in the lower phase), and separation
efficiency of the CPLC system (measured by the theoretical plate number N) for each compound determined earlier in the single isocratic run operations in normal and reversed phase modes [23] were used. The results of the numerical simulation and experimental studies are presented in Figs. 6–9. In these figures, the chromatograms of the solutes eluted during each step of the first four cycles are shown. The figures do not show the steps in which there was no elution of the solutes with the phases. In Figs. 6 and 7, the calculated and experimental chromatograms obtained under the short sample loading time (ts = 0.005) operating conditions are compared. In Figs. 8 and 9, the calculated and experimental chromatograms for long sample loading time (ts = 0.05) are compared. In the calculations, experimentally determined values of the partition coefficients for aspirin KDa = 0.5 and caffeine KDc = 0.13 and the average value of N = 70 for both components from [23] were used. According to the results of the numerical studies, the following run conditions were selected: the duration of the first steps tx = 0.45; the duration of the second steps ty = 1.1. As shown in Figs. 7 and 9, aspirin is eluting already in the first cycle (with the upper phase at the second step), while caffeine starts
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Fig. 9. SS MDM separation of aspirin and caffeine. Simulated (A) and experimental (B) chromatograms of aspirin. Process conditions: ts = 0.05; tx = 0.45; ty = 1.1; N = 70; S = 0.5.
to elute with the lower phase in the second cycle (Figs. 6 and 8). After the second cycle, the elution profiles remain approximately constant. As can be seen from Figs. 6–9, an increase in productivity of ten times had no noticeable effect on the selectivity of the separation. The results presented in these figures demonstrate in general a fair agreement between the theory and the experiment.
The reasons for the discrepancy between theory and experiment can be the displacement of the previous mobile phase in the flow line back into the device with each switching cycle and the uneven distribution of two phases through the columns. In addition, the theoretical model does not account for differences in the value of N for the first and second steps of the process.
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5. Conclusion The steady state MDM CCC is a cyclic separation process consisting of a succession of two isocratic counter-current steps and is carried out in series alternating between “x” phase and “y” phase flow periods. The duration of the flow periods of the phases is kept constant. The sample dissolved in the “x” phase is continuously fed into a CCC column at the beginning of the first step of every cycle over a constant time, not exceeding the run time of the first step. The SS MDM CCC separation is described on the basis of the equilibrium cell model. Equations are developed allowing the simulation of the chromatograms eluted from the column with the phases during each step of the cycles. There was a good agreement between the predicted and experimental separations. The SS MDM CCC separation technique may be especially useful for large scale preparative and industrial scale chromatography, since it offers a means to reduce eluent consumption and increase productivity and provides both high productivity and high resolution. Acknowledgements This work was supported by the Russian Foundation for Basic Research (project no. 15-03-02940). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chroma.2015.05. 074 References [1] M. Neves Vieiraa, F.N. Costa, G.G. Leitão, I. Garrard, P. Hewitson, S. Ignatova, P. Winterhalter, G. Jerz, Schinus terebinthifolius scale-up countercurrent chromatography (Part I): High performance countercurrent chromatography fractionation of triterpene acids with off-line detection using atmospheric pressure chemical ionization mass spectrometry, J. Chromatogr. A 1389 (2015) 39–48. [2] J.B. Friesen, S. Ahmed, G.F. Pauli, Qualitative and quantitative evaluation of solvent systems for countercurrent separation, J. Chromatogr. A 1377 (2015) 55–63. [3] W.D. Conway, Counter-current chromatography: simple process and confusing terminology, J. Chromatogr. A 1218 (2011) 6015–6023. [4] Y. Ito, Golden rules and pitfalls in selecting optimum conditions for high-speed counter-current chromatography, J. Chromatogr. A 1065 (2005) 145–168. [5] A. Berthod, T. Maryutina, B. Spivarov, O. Shpigun, I.A. Sutherland, Countercurrent chromatography in analytical chemistry (IUPAC technical report), Pure Appl. Chem. 81 (2009) 355–387. [6] W.D. Conway, Countercurrent Chromatography: Apparatus, Theory and Applications, first ed., VCH, New York, NY, 1990. [7] I. Sutherland, P. Hewitson, S. Ignatova, Scale-up of counter-current chromatography: demonstration of predictable isocratic and quasi-continuous operating modes from the test tube to pilot/process scale, J. Chromatogr. A 1216 (2009) 8787–8792. [8] I. Sutherland, D. Hawes, S. Ignatova, L. Janaway, P. Wood, Review of progress toward the industrial scale-up of CCC, J. Liq. Chromatogr. Related Technol. 28 (2005) 1877–1891.
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