Article pubs.acs.org/crystal
Probing the Mechanisms Underlying Electrolyte-Assisted Nucleation Enhancement of DL-Alanine Guangjun Han,*,† Pui Shan Chow,† and Reginald B. H. Tan*,†,‡ †
Institute of Chemical and Engineering Sciences, A-STAR (Agency for Science, Technology and Research), 1, Pesek Road, Jurong Island, Singapore 627833 ‡ Department of Chemical and Biomolecular Engineering, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260 ABSTRACT: The effects of common electrolytes (including inorganic acids, bases, and salts) on DL-alanine nucleation in aqueous solutions have been systematically investigated via measurement of nucleation induction times. It is observed that all the electrolytes examined in this study considerably shorten the induction times, suggesting that they greatly enhance DLalanine nucleation. It is hypothesized that, in solution, an electrolyte assists the formation of the head-to-tail chains of DLalanine molecules. Such head-to-tail chains, structurally matching DL-alanine crystal packing, act as favorable precursors for DL-alanine nucleation and play a great role in enhancing the nucleation, highlighting the importance of prenucleation phenomena.
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INTRODUCTION
DL -Alanine is a simple amino acid (neutral form NH2CH3CHCOOH). In its aqueous solution, alanine molecules (both D- and L-alanine enantiomers) exist as a zwitterionic form (NH3+CH3CHCOO−)14 due to proton transfer. Upon crystallization from solution, the obtained DL-alanine crystal (space group Pna21), usually exhibiting a needle-like shape, is packed with zwitterionic monomers (as elementary building units) in a head-to-tail orientation along the polar c-axis of the crystal.14,15 Such a particular molecular orientation in packing leads to the fact that the carboxylate (COO−) groups are exposed at the −c end, while the amino (NH3+) groups are exposed at the +c end.15 Different crystalline structures (i.e., polymorphs) of DL-alanine have not been reported before.10 An earlier study15 showed that, in pure aqueous solution, the COO−-rich (001̅) face at the −c end grows much faster than the NH3+-rich faces at the +c end, exhibiting unidirectional growth at the associated supersaturations. The fast growth at the −c end was rationalized using the “relay” mechanism15 where the role of easing of desolvation within the “pockets” at the (001)̅ face at the −c end was particularly highlighted. Our recent studies11,14 revealed further interesting growth behavior of DL-alanine crystals in pure aqueous solutions. At normal supersaturations, DL-alanine nucleates and grows exceptionally slowly, taking a few days to develop to a size of 1 mm. Such a remarkably slow crystallization was partly attributed to the significant existence of alanine cyclic dimers10
Solution crystallization has been a key technique for separation and purification in many industries. It is of practical importance to control the polymorphs1−7 and habit8−13 of a crystalline product from solution crystallization. Such an importance of controlled crystallization can be well justified by these two facts: (1) the chemical and physical properties of a crystal, crucial to their applications, vary with its polymorphs; (2) the crystal habit exerts a large impact on the subsequent downstream processing (e.g., filtration, drying, and formulation) and characterization of the product crystals. The preferential formation of a particular polymorph is determined by the relative nucleation rates of the polymorphs, while the crystal habit of a given polymorph is determined by its relative face growth rates. Chemical additives2−6 (or solvents7−9) have found their applications in habit and polymorph control. An additive may be chosen or designed in such a way that it alters the relative face growth rates of a crystal, and hence a desired morphology modification of the crystals is made. Additives can also affect the relative nucleation rates of associated polymorphs of a given crystallization system so as to alter the polymorphic outcome. Despite great dedicated efforts, robust control of crystal habit and polymorph remains challenging, as the fundamentals of crystal growth and nucleation from solution crystallization are poorly understood.1−5 The crystallization of a simple amino acid, DL-alanine, provides a good example, which illustrates the challenges faced in understanding of crystal growth and nucleation. © 2014 American Chemical Society
Received: December 16, 2013 Revised: January 23, 2014 Published: February 5, 2014 1406
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which are hard to fit into the lattices of the monomer-based DLalanine crystal. Supporting this, an earlier solution thermodynamic study16 showed that alanine molecules are prone to selfassociation in supersaturated solutions; a recent quantummechanics computation17 suggested that doubly hydrogen bonded alanine cyclic dimers are more stable than alanine monomers. All of these indicate that cyclic dimers are significant species in solution. However, whether cyclic dimers are the highly dominant species in alanine aqueous solution has yet to be further investigated, given that different computational techniques17,18 can project contradicting relative stabilities of the species (e.g., glycine monomers and cyclic dimers) in solution. Furthermore, a large dead supersaturation zone exists within which the polar c-axis virtually does not grow. The existence of the dead zone, difficult to be interpreted by the “relay” mechanism,15 was explained based on strong solvent water adsorption11,14 at the faces at both the NH3+-rich +c end and the COO−-rich −c end. The inhibiting effect of solvents on face growth of other crystals4,11,19 has been documented. Effects of electrolytes on DL-alanine crystallization10,20 were also investigated. Cölfen and co-workers20 reported that sodium salts accelerate the crystallization of DL-alanine aqueous solutions. More recently, we investigated the effect of various inorganic salts10 on growth of DL-alanine crystals. Surprisingly, it was found that both the polar −c end +c ends of DL-alanine caxis underwent a revival of solvent-inhibited dead growth in the presence of every of the typical salts, with the b-axis growth affected insignificantly. Such a salt-induced c-axis growth acceleration is in contrast to the usual understanding that the strong Coulombic interaction between salt ions and DL-alanine polar c-ends could inhibit rather than promote DL-alanine c-axis growth. This interesting phenomenon was explained on a basis of surface roughening.10 In addition, significant promoting effects of strong inorganic acids and bases on DL-alanine c-axis growth10 were also revealed. In summary, inorganic electrolytes, irrespective of their types (acids, bases, and salts), generally exert a great promoting effect on DL-alanine c-axis growth. However, whether this growth promotion leads to nucleation enhancement has been an open question. In this work, we systematically investigate the effects of typical inorganic electrolytes, which include common acids, bases, and salts, on DL-alanine nucleation. We found that all these electrolytes examined in this study substantially enhance DL-alanine nucleation. The results were explained at the molecular level. The role of prenucleation phenomena21,22 in solution nucleation was highlighted. In particular, a consistent mechanism was proposed to interpret the electrolyte-assisted DL-alanine nucleation enhancement, regardless of the nature of the electrolytes.
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reported in our previous work.10 These solubility data were used to calculate DL-alanine supersaturations (σ = C/Csat, where C and Csat are the actual concentration and the solubility respectively, with salting effects taken into account) in preparing supersaturated solutions for measurement of induction times in this study. The induction times of DL-alanine nucleation from solutions in the absence and in the presence of a given additive were measured at a supersaturation level of 1.4 at 23 °C. Each experiment for induction time, typically repeated five times, was carried out in a 25 mL glass jacketed beaker at 23 °C controlled using a Julabo water circulator. In each run, a 10 mL DL-alanine solution was prepared in a properly capped glass bottle at an elevated temperature of 65 °C, with a concentration corresponding to supersaturation level of 1.4 at 23 °C with the given additive. This solution was kept agitated at 65 °C for 35 min to make sure all DL-alanine solids were fully dissolved. It was then quickly transferred to a 25 mL jacketed beaker controlled at 23 °C, agitated with a magnetic stirrer at 60 rpm. The solution was monitored using visual observation to determine the cloud point of the solution. The induction time was defined as the period from the moment at which the solution was transferred to the 25 mL jacketed beaker to the moment at which the cloud point was detected. Average induction times were reported, with a typical standard deviation of 25%. At the end of each experiment, the obtained DL-alanine crystals were harvested, and their structure (polymorphic form) was confirmed using powder X-ray diffraction (PXRD) (Bruker D8 Advance diffractometer14), despite the fact that DL-alanine is a nonpolymorphic crystallization system10,14 under usual conditions.
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RESULTS AND DISCUSSION Solubilities of DL-Alanine in Electrolyte Solutions. The solubilities of DL-alanine in the presence of acid and base electrolytes at 23 °C, measured in this study, are presented in Figure 1, with a typical standard deviation of merely 0.04 g/100
Figure 1. Solubilities of DL-alanine in acidic and basic solutions at 23 °C, showing a salting-in effect of acids and bases on DL-alanine.
EXPERIMENTAL SECTION
g of H2O. The solubility increases significantly with the increase in acid or base concentration, showing that these acids and bases have a great salting-in effect on DL-alanine. This salting-in effect may be largely attributed to the formation of alanine ions which are similar to glycine ions23 formed in acidic and basic solutions. The solubilities of DL-alanine crystals in aqueous solutions in the presence of typical inorganic salts at 23 °C have been reported before.10 For easier reference, the associated solubilities used in this study are shown in Figure 2. It should be noted that the acetate salts10 (e.g., sodium acetate, NaAc)
DL-Alanine
(99%) was from Sigma-Aldrich and used as received. Three types of electrolytes, namely, acids, bases, and salts, were used as additives in DL-alanine crystallization. The purity of the two bases, NaOH and KOH (pellets), was 99.9%, while the three acids (H2SO4, HCl, HNO3) and six typical inorganic salts (NaCl, NaAc, KNO3, Ca(NO3)2, MgSO4, (NH4)2SO4) were of analytical grade. Ultrapure water (Millipore, resistivity 18.2 MΩ cm and filtered with pore size 0.22 μm) was used for solution preparation. Precise solubilities of DL-alanine in basic and acidic solutions at 23 °C were measured in this study using an isothermal method.10,23 The solubilities of DL-alanine in various salt solutions at 23 °C had been 1407
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from disintegration2,24 into liquid, thus facilitating their stabilization and hence favoring the nucleation process. The growth of structured nuclei, difficult to be directly studied due to its small size and time scale, may be reasonably reflected by the growth kinetics of mature crystals having the same structure of the nuclei, assuming that their growth behaviors are similar. In other words, the fast growth rate of mature crystals is an indication of quick development of nuclei. A previous screening study10 has revealed that the DL-alanine c-axis grew faster in acidic and basic solutions than in pure (additive free) solution, an outcome of both pH-induced proton transfer (hence easing of desolvation)4,23 at one DL-alanine polar c end and surface roughening10,15,25 at the other polar c end. For a better examination of the impact of common acids and bases on DLalanine growth kinetics, quantitative growth rates were systematically measured (Figure 4) in this study using the Figure 2. DL-Alanine solubilities in various salt solutions at 23 °C, showing that all the salts, except sodium acetate (NaAc), have a salting-in effect on DL-alanine.
exert a significant salting-out effect on DL-alanine solubility, while other salts exert either virtually no effect or salting-in effect on DL-alanine. The significances of the different salting effects will be elaborated in Results and Discussion. Promoting Effects of Acid and Base Electrolytes on DLAlanine Nucleation. The experiments for induction times were performed to examine the effects of these typical acids and bases on DL-alanine nucleation. The measured average induction times (Figure 3) show that, in pure solution, it Figure 4. Impact of acids and bases on DL-alanine c-axis growth at supersaturation σ = 1.4 at 23 °C, showing that acids and bases tremendously promote the growth along DL-alanine c-axis. Standard deviation (based on three runs): 20%.
method described in our previous studies.10,14 It was found that, although DL-alanine b-axis growth remains slow and is largely unaffected, the c-axis growth is significantly promoted by all these acid and bases, and it highly dominates the overall growth rate of DL-alanine crystal. Therefore, it is reasonable to suggest that the development of the unstable DL-alanine nuclei from subcritical size to critical size is faster in acidic and basic solutions than in pure solution, contributing to the observed DL-alanine nucleation enhancement to a certain extent. Prenucleation phenomena (e.g., aggregation and ordering),21,22 happening before the formation of the well-structured nuclei in solution, play a great role in affecting nucleation process. Additive-associated prenucleation phenomena can promote the nucleation5 of a given crystal (or polymorph), as was demonstrated in our recent study,5 which revealed that strong acids and bases accelerate γ-glycine nucleation. This γglycine nucleation acceleration was primarily attributed to the charge-directed molecular organization, hence the formation of glycine head-to-tail chains which match γ-glycine structure and are considered as favorable nucleation precursors of γ-glycine. Since DL-alanine is highly akin to γ-glycine in both molecular structure (glycine NH3+CH2COO− and alanine NH3+CH3CHCOO−) and crystal packing (Figure 5), nucleation of DL-alanine can also be promoted by acids and bases in a similar way. An acid is taken as an example to briefly show how
Figure 3. Impact of acids and bases on induction times of DL-alanine nucleation at supersaturation σ = 1.4 at 23 °C, showing that acids and bases tremendously accelerate DL-alanine nucleation. Standard deviation (based on three runs): 25%.
takes more than two days for the cloud point of a DL-alanine solution to be detectable, suggesting that DL-alanine nucleation is very slow; acids and bases drastically shorten the induction times of DL-alanine nucleation from 2900 min to as short as merely 2 min, which corresponds to a nucleation enhancement factor of up to approximately 1500; acids are more effective than bases in shortening induction time of DL-alanine nucleation. This great shortening in induction time, a good indication of nucleation enhancement, may be attributed to growth kinetics of the unstable (subcritical-sized) structured nuclei and prenucleation phenomena21,22 of DL-alanine in acidic and basic solutions. It may be understandable that fast growth of the unstable nuclei from subcritical size to critical size helps prevent them 1408
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nucleation in various salt solutions (with pHs ranging from 5.5 to 6.7), measured in this study, are presented in Figure 6. It
Figure 5. Structure comparison between (a) DL-alanine packed with NH3+CH3CHCOO− and (b) γ-glycine packed with NH3+CH2COO−, showing the similar head-to-tail orientation along their polar c-axes, with the −c end exposing the carboxylate (COO−) groups and the +c end exposing the amino (NH3+) groups. Built in Materials Studio. Color scale of atoms in packing: C (dark gray), N (blue), O (red), and H (white).
Figure 6. Impact of inorganic salts on induction times of DL-alanine nucleation at supersaturation σ = 1.4 at 23 °C, showing that inorganic salts generally accelerate DL-alanine nucleation to a great extent. Standard deviation (based on three runs): 25%.
an acidic environment favors DL-alanine nucleation through charge-directed molecular organization and chain formation. In an acidic solution, some of alanine zwitterions (NH3+CH3CHCOO−), similar to glycine zwitterions,3−5 are converted to alanine cations (NH3+CH3CHCOOH) via protonation. These alanine cations themselves are not likely to undergo self-aggregation due to repulsion among themselves, hardly favoring alanine nucleation from the point of view of solute self-aggregation. However, they interact with the rest of zwitterions more strongly because ion−dipole interaction between an cation and a zwitterion is greater than dipole− dipole interaction between alanine zwitterions, forming a positively charged and ordered head-to-tail open dimer (zwitterion)COO−···+H3N(cation). (For comparison, a negatively charged open head-to-tail dimer (anion)COO−···+H3N(zwitterion) is formed in basic solution.) Such a charged dimer can further interact with other zwitterions, developing a longer head-to-tail open alanine chain. Since these head-to-tail open alanine chains structurally match the dominant head-to-tail linear chains in DL-alanine crystal structure (Figure 5a), they can serve as effective nucleation precursors of DL-alanine crystal, hence greatly accelerating DL-alanine nucleation. This analysis, by analogy, is also applicable to the promoting effect of a base on DL-alanine nucleation. It is seen that both quick development of the unstable nuclei and favorable prenucleation phenomena play roles in DL-alanine nucleation enhancement. However, similar to acid- and baseassisted γ-glycine nucleation enhancement,5 it is reasonable to suggest that the induced head-to-tail alanine molecular ordering rather than fast growth of nuclei is the key factor that primarily governs DL-alanine nucleation. In other words, DL-alanine nucleation enhancement in acidic and basic solutions has more to do with the formation of the head-to-tail alanine chains which is more likely the rate-controlling step. Perhaps alanine cations formed in an acidic solution are more prone to inducing favorable head-to-tail alanine chains than alanine anions formed in a basic solution. That would partly explain why acids are generally more effective than bases in promoting DL-alanine nucleation. The associated inorganic cations (e.g., Na+ from NaOH) and anions (e.g., Cl− from HCl) also play a significant role in affecting DL-alanine nucleation, which will be elaborated below. Promoting Effects of Inorganic Salt Electrolytes on DLAlanine Nucleation. The induction times of DL-alanine
was found that all the salts examined here, consisting of typical inorganic cations (Na+, K+, NH4+, Ca2+, and Mg2+) and anions (Cl−, NO3−, SO42−, and Ac−), regardless of their salting effects (salting-in or salting-out), tremendously shorten the induction times of DL-alanine nucleation, with enhancement factors ranging from 50 to 500, suggesting that the salt-associated DL-alanine nucleation acceleration is a general phenomenon. In addition, the divalent salts (Ca(NO3)2, MgSO4, (NH4)2SO4) are more effective than the univalent salts (NaCl, KNO3, and NaAc), with NaAc (which exerts a salting-out effect) being the least effective in enhancing DL-alanine nucleation. Upon initial consideration, this great salt-induced nucleation enhancement of DL-alanine may primarily be due to quick development of the unstable nuclei2,24 from subcritical size to critical size, as was discussed earlier. This consideration arises from the observed salt-associated crystal growth acceleration10 which indicates a quick evolution of the ordered nuclei. However, perusal of the crystal growth rates10 and the induction times (Figure 6) reveals that evolution of nuclei alone does not fully account for this phenomenon of the great salt-induced nucleation acceleration, as quick evolution of nuclei does not generally correspond to a short induction time in the presence of salts. Instead, in many cases, very slow crystal growth rates hence slow evolution of the unstable nuclei can correspond to considerably shorter induction times, as illustrated in Figure 7. This implies that the rate at which the ordered nuclei are developed from subcritical size to critical size does not seem to primarily govern DL-alanine nucleation in the presence of a salt. This great salt-associated nucleation enhancement is not due to the change in solution pH either, as the pHs [5.5−6.7] of these alanine-salt solutions (especially NaCl-alanine and KNO3alanine solutions) are practically neutral, very close to the pH (= 6.2) of the pure alanine solution. In fact, even at pHs of 4.3 (in the presence of 0.05 m HNO3) and 8.5 (in the presence of 0.1m NaOH) which are significantly beyond the solution pH range of [5.5 − 6.7] in the presence of salts, the induction times (720 and 540 min respectively. Figure 3) were far longer than those short induction times (as short as 5 min. Figure 6) at high concentrations of salts. This indicates that the limited pH change (
