Experimental Study of the Effect of Buffer on Chromatography and

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Experimental Study of the Effect of Buffer on Chromatography and Crystallization Hybrid Process Ka Y. Fung and Ka M. Ng* Department of Chemical Engineering, Hong Kong UniVersity of Science and Technology, Clear Water Bay, Kowloon, Hong Kong

Christianto Wibowo ClearWaterBay Technology, Inc., 20311 Valley BouleVard, Suite C, Walnut, California 91789

For a chromatography-crystallization hybrid process, a buffered mobile phase that gives the best chromatographic resolution may not necessarily give a solid-liquid equilibrium phase behavior that allows the highest per-pass recovery in the crystallizer. This paper presents a conceptual and experimental study to elucidate this possible tradeoff using ibuprofen and triethylammonium acetate as the model system. Experimental results show that a buffered system with a pH of 3.5 gives the best performance with both factors considered. Introduction

Table 1. List of Common Buffers for Chromatographic Separations

Chromatography and crystallization have been used extensively in the fine chemical and pharmaceutical industry to separate and purify high value-added products. Both processes have their own advantages and disadvantages. For example, complete separation can in principle be achieved in a long chromatographic column, albeit at a high cost. In contrast, crystallization is a less expensive process, but is often hindered by the thermodynamic boundaries which limit the yield of a pure product. Therefore, to utilize the advantages of both processes, a better alternative is to partially separate the compounds by chromatography and then crystallize the enriched fractions to obtain pure products. Studies of hybrid processes have gained more attention in recent years.1-3 Specifically, a systematic procedure has been developed recently to design the chromatography-crystallization hybrid separation process.4 Design heuristics are provided to determine the basic process structure, fractionation of the chromatographic effluent, and flow sheet synthesis. Building on this previous study, this article aims at elucidating the effect of buffer on the chromatographic and crystallization hybrid process both conceptually and experimentally using ibuprofen as a model system. The Hybrid Process Effect of Buffer on the Chromatographic Process. Separation of solute molecules by chromatography is based on the various interactions between the solute molecules and the stationary phase, such as hydrogen bonding, dipole-dipole interaction, and steric hindrance. It also depends on the properties of the mobile phase such as composition, pH, and flow rate. For example, a buffer in the mobile phase alters the degree of ionization of solute molecules with weakly acidic or basic functional groups, which in turn influences solute retention and selectivity. The effect of buffer on chromatographic separation is widely recognized. For example, the use of triethylammonium acetate (TEAA) as a buffer in the separation of a racemic mixture of * To whom correspondence should be addressed. Tel.: (852) 23587238. Fax: (852) 23580054. E-mail: [email protected].

buffer

pKa

acetate borate citrate formate glycinium perchlorate phosphate trifluoroacetate tris-(hydroxymethyl)aminomethane

4.8 9.2 3.1, 4.7, 5.4 3.7 2.3, 9.8 ∼9 2.1, 7.2, 12.3 0.2 8.3

propranolol suppresses the ionization of the acidic molecule and enhances the hydrophobic interaction between the enantiomers and the chiral stationary phase. A better separation between the propranolol enantiomers was obtained when the pH of the mobile phase was increased from 4 to 6.5 Other studies also report the use of buffer to control the pH of the mobile phase to achieve a better resolution for various chiral and achiral systems, such as flavor and fragrance compounds,6 flavanones,7 beta-blocker drugs,8,9 and a racemic mixture of an antidepressant drug, fluoxetine.10 Thus, buffer selection which depends on the functional groups and pKa values of the compound to be separated is an important task. A list of commonly used buffers as well as their pKa values is presented in Table 1.11 Effect of Buffer on the Crystallization Process. Solid-liquid equilibrium (SLE) phase diagrams play an important role in the synthesis of crystallization-based processes. Let us consider a generic system consisting of a racemic mixture (AH) which forms a racemic compound (RS), a mixture of solvents (S1 and S2), and a buffer system (PQ and HQ). This system can be used to illustrate the separation of racemic propranolol using methanol and water as the solvent and TEAA and acetic acid as the buffer. With the chiral compounds and the buffer being electrolytes, the following dissociation reactions occur:

(R)-AH T (R)-A- + H+

(1)

(S)-AH T (S)-A- + H+

(2)

+

PQ T P + Q

-

HQ T H+ + Q-

(3) (4)

Therefore, there are four electrolytes ((R)-AH, (S)-AH, PQ, and HQ), two cations (H+ and P+), three anions ((R)-A-, (S)-A-,

10.1021/ie0602052 CCC: $33.50 © 2006 American Chemical Society Published on Web 06/10/2006

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and Q-), and two nondissociating compounds (S1 and S2), giving a total of 11 components. The system has to obey eqs 1-4 and the condition of electroneutrality. As the phase diagram is plotted under isobaric and isothermal conditions, the number of additional constraints for the system equals 7. Since at least one phase has to be present in the system, there is a maximum of 5 degrees of freedom for the phase diagram. Using the general expressions for ionic coordinates developed by Wibowo and Ng,12 we get

R(P+ ) ) R(Q-) )

[P+] [P+] + [H+] [Q-]

[Q-] + [(R)-A-] + [(S)-A-]

R((S) - A-) )

[(S) - A-] [Q-] + [(R)-A-] + [(S)-A-]

R(S1) ) R(S2) )

[S1] [P+] + [H+] [S2] +

[P ] + [H+]

(5)

(6)

(7)

(8)

(9)

As the dimensionality is larger than 3, it is impossible to plot the phase behavior on a piece of paper. Proper projections or cuts need to be chosen to view the subsystems of the phase diagram, which in aggregate form a mental picture of the complete system.12 As the mobile phase has a fixed ratio of S1 to S2 (S1:S2 ) r1), it is convenient to take a cut at constant solvent composition. In such a four-dimensional cut, the two solvent coordinates are lumped into one:

R(S1/S2) )

[S1] + [S2] [P+] + [H+]

(10)

As the desired pH of the mobile phase for chromatographic separation can be achieved by mixing PQ and HQ in a certain proportion, another cut at a fixed ratio of PQ to HQ (PQ:HQ ) r2) can be taken to further reduce the dimensionality. The structure of the resulting three-dimensional phase diagram is shown in Figure 1, along with its vertical projection along the solvent coordinate. The projection features three crystallization regions or compartments, one for (R)-AH, another for (S)-AH, and a third one for RS. In a hybrid separation process, the buffer used in the chromatographic process is usually carried forward to the crystallization process, affecting the solubility of the crystallizing components (Figure 2). Figure 2a shows a three-dimensional prism which is obtained by taking a Janecke projection, with S1/S2 as vertex, of a four-dimensional cut at a constant S1 to S2 ratio. Figures 2b and 2c show two cuts taken at different values of r2, each corresponding to a different pH of the mobile phase. These cuts intersect the saturation boundaries in the phase diagram at different locations, resulting in an apparent shift of those boundaries as shown in the cuts. Note that a cut with the (R)-AH to (S)-AH edge is the most convenient for representing the crystallization of (R)-AH, (S)-AH, or RS. Flow Sheet Synthesis and Tradeoffs. Process flow sheet for the resolution of a racemic mixture of (R)-AH and (S)-AH (i.e., the feed F) can be synthesized using the procedure developed by Fung et al.4 Let us assume that (R)-AH and (S)-AH exit

Figure 1. Three-dimensional phase diagram for the generic system with cuts at S1:S2 ) r1 and PQ:HQ ) r2, along with the vertical projection along the solvent coordinate.

from the column as two symmetric, overlapping peaks. The effluent is divided into three fractions (Figure 3a). The solvent fraction is recycled to the column as stream 1. Stream 2 contains the (S)-AH peak up to the intersection of the two peaks. Stream 6 starts from the intersection and includes the rest of the (R)AH peak. Crystallization of the desired enantiomer such as (S)-AH from the chromatographic effluent is effected by solvent evaporation. The composition change can be represented as movements on the phase diagram, as illustrated in Figure 3b. As the phase diagram is a solvent-free projection, the chromatographic effluent (2) and the composition after solvent evaporation (4) lie on the same point. Note that in order to produce a pure enantiomer, the feed point of the crystallizer (4) has to be located inside the compartment of the desired product bounded by (S)AH-B-C-D. Furthermore, the mother liquor composition (5) also has to lie within the same compartment. Otherwise, the racemic compound (RS) may also precipitate out and lower the product purity. The location of the saturation surfaces in Figure 1 represents the upper limit on the per-pass yield of the product in the crystallizer. As illustrated in Figure 2, the location of saturation boundaries is different at a different pH of the mobile phase. Operating at a pH which provides the best resolution may correspond to a low recovery in the crystallizer. This leads to a large solute recycle flow rate, making the process unfavorable. Two scenarios are discussed below to illustrate the tradeoff. Again consider the generic system described by eqs 1-4. Assume that phase behaviors as shown in Figures 4a and 4b are obtained for buffers at two pH values (pH1 and pH2). Clearly, the buffer at pH1 gives a larger compartment for the pure product, when compared to the buffer at pH2. However, the

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Figure 2. (a) Three-dimensional phase diagram for the generic system with a cut at S1:S2 ) r1 and taking Janecke projection with S1/S2 as vertex; (b) and (c) are cuts at two different PQ:HQ ) r2, each corresponding to a different pH of the mobile phase.

Figure 3. (a) Synthesized flow sheet for the generic system; (b) corresponding process paths on the phase diagram.

buffer at pH2 gives a crystallizer feed richer in (S)-AH due to a better resolution. As mentioned, the mother liquor composition has to lie in the same compartment as the product to prevent cocrystallization of other components. Comparing the two figures reveals that the maximum per-pass recovery is higher for the buffered system at pH1, which means that the tradeoff between resolution and recovery has to be considered to determine the pH that would give the best performance. In an extreme case such as that in Figure 4c, the phase behavior is so undesirable that the crystallizer feed lies in the compartment of a racemic compound, resulting in an infeasible operation. The solute recycle ratio (the solute flow rate in the recycle stream and the solute flow rate in the feed) which is related to the recovery of a component in the crystallizer can be used for comparing the performance of a flow sheet operating with the same buffer but at different pH values. A larger solute recycle flow rate requires more mobile phase to completely dissolve the solute at the feed point of the chromatographic column, and thus a longer batch time is needed if the column dimensions and mobile phase flow rate are fixed. This increases the operating cost and decreases the productivity of the process. Experimental Study A model system consisting of ibuprofen (Ibu) as the compound to be separated and triethylammonium acetate (TEAA) as the buffer was experimentally studied to investigate the effects of buffer on the hybrid process, particularly chromatographic resolution and SLE behavior.

Figure 4. Phase behaviors at (a) pH1, (b) pH2, and (c) pH3.

Materials. Racemic ibuprofen and its S-enantiomer (enantiopurity >99%) were purchased from Tokyo Chemical Industry

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Figure 6. (a) Experimental chromatogram and (b) resolution of racemic ibuprofen at different pH.

Figure 5. Phase behavior by obtaining a cut at (S1 + S2):(PQ + HQ) ) r3 of the three-dimensional phase diagram. Table 2. r2 and r3 for Obtaining Different Buffer pH Values pH

r2

r3

3.5 4.02 5.02 5.6 6.36

0.186 0.452 1.369 1.626 1.734

38.58 76.70 142.30 152.47 156.18

and Fluka, respectively. HPLC-grade methanol from Merck with 99.9% purity, glacial acetic acid (purity >99.7%) from Fisher, and triethylamine (purity >99%) from RDH were used as the solvent. All the purchased chemicals were used without further purification. The water used in the experiment was doubledeionized and had a resistivity of 18.2 MΩ-cm at room temperature. Chromatographic Process. Chromatographic separations were performed using an Agilent 1100 series HPLC system with an (R,R)-Whelk-O1 chiral column from Regis Technologies, Inc. The mobile phase consisted of 50 vol % methanol and 50 vol % water. Triethylammonium acetate was added as buffer to the mobile phase, prepared by using 1 vol % triethylamine solution which was adjusted to the desired pH by adding glacial acetic acid. The mobile phase was filtered and degassed before

use and then was pumped into the column at a flow rate of 1 mL/min under isocratic mode. UV absorbance of ibuprofen was set at a wavelength of 264 nm. Separation of racemic ibuprofen with mobile phases of different pH was carried out to observe its effect on chromatographic separation. To determine the required amount of solvent (containing buffer) to completely dissolve the solute at the inlet of the chromatographic column, SLE phase behavior, particularly those cuts at a fixed (S1 + S2) to (PQ + HQ) ratio (r3) as shown in Figure 5, is needed. To avoid solid formation anywhere in the process except the crystallizers, the feed and outlet streams of the column should be sufficiently dilute to lie in the unsaturated region of the phase diagram. Crystallization Process. The system is similar to the generic system discussed above with AH being ibuprofen, S1 being methanol, S2 being water, PQ being TEAA, and AQ being AcOH. The ratio of methanol to water is assumed to be fixed throughout the process, which corresponds to r1 ) 0.792. The values of r2 and r3 that are required to obtain the different pH with this buffer system are shown in Table 2. The following experimental procedure was used to measure the SLE phase behavior as a function of pH. Mixtures of various compositions of racemic ibuprofen and (S)-ibuprofen were prepared. These mixtures were partially dissolved in a solvent with the same composition as the mobile phase used in the chromatographic separation (containing methanol, water, and buffer in different proportions). A temperature-controlled sonication bath set at 25 °C was used to provide the required mixing. All mixtures were left overnight to allow equilibrium to be reached. The liquid phase was then extracted using a syringe and passed through a filter to remove any undissolved solids.

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Figure 7. Phase diagram of ibuprofen in solvent (methanol and water) and buffer (TEAA and AcOH) at 25 °C, corresponding to the generic phase behavior illustrated in Figure 5.

The sample was then diluted with solvent in order to prevent any solid precipitation during analysis. The sample was analyzed using HPLC to obtain the solubility of ibuprofen. As the phase diagram of an enantiomeric system is symmetric, SLE measurements were carried out to measure the saturation curves rich in (S)-ibuprofen on the right-hand side of the lower phase diagram in Figure 5. From the HPLC results, a simple mass balance was performed to obtain the amounts of R and S in the undissolved solids. If the solids contained only S or an

equal proportion of R and S, the mother liquor represented points on the saturation curve. If the solids contained more S than R, then the mother liquor represented the double saturation point, such as point C in Figure 3b. A similar procedure was also used to locate the double saturation point such as point B for a system without buffer, which corresponds to the intersection of the double saturation troughs with the (R)-AH to (S)-AH edge. Thus, the double saturation trough such as line BC can be located.

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Figure 9. Solute recycle ratio of the flow sheet at different pH of mobile phase and crystallization solvent.

Figure 8. Phase diagram of ibuprofen in solvent (methanol and water) and buffer (TEAA and AcOH) at 25 °C, corresponding to the generic phase behavior illustrated in Figure 3b.

Results and Discussion Chromatographic separations of racemic ibuprofen were carried out at different pH values of the mobile phase: 3.5, 4.02, 5.02, 5.6, and 6.36. Among the five pH values, the best separation was obtained at pH ) 5.6. The corresponding experimental chromatogram is shown in Figure 6a. As discussed in the systematic procedure of Fung et al.,4 the experimental chromatogram has to be resolved using models provided by Pap and Papai13 into two component peaks for flow sheet synthesis. The separation performance can be quantified by resolution R, defined as

tr,i+1 - tr,i wi+1 + wi

Ri,i+1 ) 2

(11)

where tr,i and wi are the retention time and the peak width for component i, respectively. Here, i + 1 refers to the component with a longer retention time. Figure 6b shows the resolution of the system at different pH of the mobile phase. In the range of pH covered in this study, complete separation was never achieved. This is not a problem since complete separation may not be the most economical option in a commercial hybrid process anyway. Experimental results for the constant r3-cuts illustrated in Figure 5 for the ibuprofen system are shown in Figure 7. As the solubility of ibuprofen in the solvent is very low, the saturation boundaries are close to the solvent vertex. To allow a clearer view of the saturation boundaries, the region close to the solvent vertex is enlarged. The saturation curves for R-Ibu, S-Ibu, and RS for pH ) 4.02 is omitted as they are too close to those for pH ) 3.5. It can be observed that the solubility of ibuprofen is higher at a higher pH. In other words, a larger unsaturated region exists for a system with a buffer composition corresponding to a higher pH. The results of the SLE phase behavior used for crystallizer design are summarized in Figure 8. Only results for pH at 3.5, 5.02, and 6.36 are shown because those for pH at 4.02 and 5.6 are too close to the others. The double saturation points for the system without buffer, marked as squares, occur at mass fractions 0.23 and 0.77 of (S)-ibuprofen. Here, the square on the right corresponds to point B in Figure 3b. These two double

saturation points are connected by straight lines to the double saturation points at different pH values to obtain an approximate location of the compound crystallization boundaries (for example, line BC in Figure 3b). The experimental data indicate that the double saturation troughs are more tilted for buffers at lower pH, giving a relatively larger compartment for the product. With the chromatogram and phase diagram, the flow sheet for the hybrid separation process, which turned out to be similar to the flow sheet described in Figure 3a, can be synthesized. Material balance calculations for the overall process were performed for buffer at different pH to evaluate its effect on the plant performance. Three issues need particular attention in performing material balance calculations. First, the chromatography feed should be sufficiently dilute such that the feed and the outlet streams of the column are located in the unsaturated region of a phase diagram. Second, the location of boundaries and saturation surfaces limit the per-pass recovery in a crystallizer, which leads to tradeoffs between resolution and recovery as discussed above. With use of Figure 7, the amount of solvent required to completely dissolve the chemicals can be identified. As the saturation boundaries are closer to the solvent vertex at lower pH, more solvent is required for processes operating at a buffer of lower pH. To allow fair comparisons between resolution and recovery, the amount of solvent added to the column is kept the same for all cases. The least soluble condition (pH ) 3.5) is used as the reference for fixing the amount of solvent added to the system. Third, the solubility of (R)-AH and (S)-AH in streams 5 and 9 (Figure 3a), respectively, are required in the material balance calculations. These data might not have been included in Figure 7 and were obtained as follows. A mixture of (R)-AH, (S)-AH, and buffer with a composition such as stream 6 was prepared using the isothermal soliddisappearance method.14 Solvent was added to the mixture until the solids completely dissolved while keeping the temperature of the system constant at 25 °C. The resulting composition corresponds to a point on the saturation surface of the phase diagram. Solute recycle ratio is used for performance comparison. The solute recycle ratio for processes operating at different pH is shown in Figure 9, which shows a maximum for the buffered system at a pH of 5.02. For the pH range studied, the lowest solute recycle ratio was obtained at a pH of 3.5, which is recommended as the operating pH of the buffer for the ibuprofen system despite the fact that the chromatographic separation performance at this pH is the worst. Conclusions In chromatographic separations, buffers are commonly added to the mobile phase for enhancing the resolution. The same

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buffer, however, is usually carried forward to the crystallizer in a hybrid process, where it affects the solubility of the components. Therefore, a pH that provides the best separation in the chromatographic column may not give the best recovery in the crystallizer. To elucidate the effect of buffer on a hybrid process, experiments were conducted for separating racemic ibuprofen with methanol and water as the mobile phase and TEAA as the buffer. Chromatographic results show that the best separation is achieved for a buffered system at a pH of 5.6, but crystallization solvent at this pH gives a relatively higher solubility. This in turn leads to a lower per-pass recovery in a crystallizer and a larger solute recycle ratio. Material balance shows that the lowest solute recycle ratio occurs at a pH of 3.5, a pH that gives the worst chromatographic separation. This study is not merely a demonstration of the tradeoff, nor is it limited to ibuprofen. More importantly, it shows a generic procedure which, by combining an understanding of SLE phase behavior and experimental methods, can assist a research engineer or chemist to conceptualize a chromatographycrystallization hybrid process. This procedure can be extended by considering simulated moving beds, supercritical chromatography, solid solutions, etc. Also, we can consider the case where the pH in each process is optimized separately. Of course, adjusting the pH before chromatography and before crystallization will increase process complexity and cost. Further work in these directions is underway. Acknowledgment Research support of the Research Grants Council (Grant HKUST602704) is gratefully acknowledged. Notation R ) resolution, dimensionless tr ) retention time, s w ) peak width, s Subscript i ) component index

Literature Cited (1) Lim, B. G.; Ching, C. B.; Tan, R. B. H.; Ng, S. C. Recovery of (-)-Praziquantel from Racemic Mixtures by Continuous Chromatography and Crystallization. Chem. Eng. Sci. 1995, 50, 2289. (2) Lorenz, H.; Sheehan, P.; Seidel-Morgenstern, A. Coupling of Simulated Moving Bed Chromatography and Fractional Crystallization for Efficient Enantioseparation. J. Chromatogr. A 2001, 908, 201. (3) Wibowo, C.; O’Young, L. A Hybrid Route to Chirally Pure Products. Chem. Eng. Prog. 2005, 101 (11), 22. (4) Fung, K. Y.; Ng, K. M.; Wibowo, C. Synthesis of ChromatographyCrystallization Hybrid Separation Processes. Ind. Eng. Chem. Res. 2005, 44, 910. (5) Ching, C. B.; Fu, P.; Ng, S. C.; Xu, Y. K. Effect of Mobile Phase Composition on the Separation of Propranolol Enantiomers Using a Perphenylcarbamate β-cyclodextrin Bonded Chiral Stationary Phase. J. Chromatogr. A 2000, 898, 53. (6) Ng, S. C.; Ong, T. T.; Fu, P.; Ching, C. B. Enantiomer Separation of Flavour and Fragrance Compounds by Liquid Chromatography Using Novel Urea-covalent Bonded Methylated β-cyclodextrins on Silica. J. Chromatogr. A 2002, 968, 31. (7) Lai, X. H.; Ng, S. C. Enantioseparation on Mono(6A-N-allylamino6A-deoxy)permethylated β-cyclodextrin Covalently Bonded Silica Gel. J. Chromatogr. A 2004, 1059, 53. (8) Chen, L.; Zhang, L. F.; Ching, C. B.; Ng, S. C. Synthesis and Chromatographic Properties of a Novel Chiral Stationary Phase Derived from Heptakis(6-azido-6-deoxy-2,3-di-O-phenylcarbamoylated)-β-cyclodextrin Immobilized onto Amino-functionalized Silica Gel via Multiple Urea Linkages. J. Chromatogr. A 2002, 950, 65. (9) Wang, X.; Ching, C. B. Liquid Chromatographic Retention Behavior and Enantiomeric Separation of Three Chiral Center β-blocker Drug (Nadolol) Using Heptakis (6-Azido-6-deoxy-2,3-di-O-phenylcarbamolyted) β-Cyclodextrin Bonded Chiral Stationary Phase. Chirality 2002, 14, 798. (10) Yu, H.; Ching, C. B.; Fu, P.; Ng, S. C. Enantioseparation of Fluoxetine on a New β-cyclodextrin Bonded Phase Column by HPLC. Sep. Sci. Technol. 2002, 37, 1401. (11) Meyer, V. R. Solvent Properties. Practical High-Performance Liquid Chromatography, 3rd ed.; Wiley: New York, 1988. (12) Wibowo, C.; Ng, K. M. Visualization of High-dimensional Systems via Geometric Modeling with Homogeneous Coordinates. Ind. Eng. Chem. Res. 2002, 41, 2213. (13) Kwok, K. S.; Chan, H. C.; Chan, C. K.; Ng, K. M. Experimental Determination of Solid-Liquid Equilibrium Phase Diagrams for Crystallization-Based Process Synthesis. Ind. Eng. Chem. Res. 2005, 44, 3788. (14) Pap, T. L.; Papai, Z. Application of a New Mathematical Function for Describing Chromatographic Peaks. J. Chromatogr. A 2001, 930, 53.

ReceiVed for reView February 18, 2006 ReVised manuscript receiVed April 16, 2006 Accepted April 18, 2006 IE0602052