Supersaturation dependence of glycine polymorphism using laser-induced nucleation, sonocrystallization and nucleation by mechanical shock.

The nucleation of glycine from aqueous supersaturated solution has been studied using non-photochemical laser-induced nucleation (NPLIN), ultrasound (sonocrystallization), and mechanical shock of sample vials. It was found that at higher supersaturation, samples were more susceptible to nucleation and produced more of the γ-glycine polymorph. The results are described in terms of a mechanism common to all three nucleation methods, involving the induction of cavitation events and pressure shockwaves. The switch in preference from α- to γ-glycine was observed to occur over a narrower range of supersaturation values for NPLIN. We attribute this to induction of cavitation events with higher energies, which result in higher localized pressures and supersaturations. Experiments on NPLIN using circularly versus linearly polarized light showed no evidence for binary polarization switching control of glycine polymorphism.

commonly crystallized from aqueous solution; however, in order of increasing thermodynamic stability,  >  > . 6 The  crystal structure is centrosymmetric (space group P2 1 /n), whereas the  crystal structure (space group P2 1 ) and  crystal structure (space group P3 1 or P3 2 ) are both noncentrosymmetric. 7 Glycine exists as a zwitterion ( + NH 3 CH 2 COO -) in aqueous solution at pH ~ 6. 8 The preference for crystallization of -glycine has been explained in terms of formation of cyclic dimers in solution, since these may be considered as the building blocks of the  form. Recent experiments on freezing point depression, however, suggest that glycine exists primarily as monomers in solution. 9 Molecular dynamics studies indicate that pair interactions tend to favour open dimers rather than cyclic dimers. 10,11 Modification of solution conditions such as pH, 8,12 or additives such as salts, 13,14 have been used to bias the probability of crystallization of a specific polymorph of glycine. The mechanism of action of these additives has been ascribed to ion speciation in solution, and inhibition of growth at specific crystal faces that favour one polymorph over another. 8,[15][16][17] Studies have shown that transformation from the  to  forms can take place within about one day in aqueous solution, and that this can be accelerated by additives such as D 2 O and NaCl. 13,18 Therefore, care is required to identify and isolate products quickly after nucleation. 19 External fields have been shown to bias nucleation of glycine. Aber et al. observed that strong, static external electric fields favoured nucleation of -glycine in aqueous solutions at high supersaturation. 20 Using non-photochemical laser-induced nucleation (NPLIN) Zaccaro et al. observed that nanosecond pulses of linearly polarised light nucleated -glycine, whereas spontaneously nucleated control samples all produced -glycine. 21 In a remarkable study, Garetz et al. demonstrated that linearly polarised (LP) light nucleated -glycine, and circularly polarised (CP) light nucleated -glycine: an effect that was named polarization switching. 22 It was considered that the intense polarized electric field of the laser pulse interacts with the polarizability of prenucleating clusters, causing alignment of solute molecules, i.e., the optical Kerr effect (OKE). The putative pre-nucleating clusters have different polarizability anisotropies that mirror the symmetry of each polymorph:  has rod-shaped anisotropy and  has disk-shaped anisotropy. A subsequent detailed study showed that binary control of nucleation of versus -glycine using polarized laser light only operated in a narrow window of temperature and supersaturation. 23 Masuhara and co-workers used intense continuous-wave (CW) laser radiation to control glycine polymorphism using a laser trapping technique in D 2 O solutions. 24,25 Over a period of minutes a dense liquid became visible by eye, caused by hindered diffusion and trapping of solute. 26 Remarkably, nucleation was observed in undersaturated and saturated solutions as well as supersaturated solutions. 27 The effects of polarization were considered to be similar to those observed by Garetz et al., with CP light acting preferentially on native clusters that favour glycine. 28 At high laser powers, the trapping increased, and more -glycine was nucleated. At very high powers, heating competed with trapping, and -glycine became less favourable.
Uwada et al. focussed a CW laser beam onto a gold surface submersed in supersaturated glycine solution. 29 The absorption of the beam by the film caused formation of a persistent vapour bubble. After a short time, a dense liquid was observed near the contact point between bubble and surface, followed by formation of a crystal. The authors explained these observations in terms of thermal convection and Marangoni flow, which creates a localised, high supersaturation at the surface contact point of the bubble. They also considered that the vapour-liquid interface could promote alignment of the solute, and thereby enhance the probability of nucleation. No measurements of polymorphism were made in this study.
To discuss possible mechanisms, it is important to distinguish between different methods of laser-induced nucleation. In the case of NPLIN using pulsed lasers, the light is delivered to the sample over a very short time (< 100 ns). 30,31 Nucleation can be effected with a single laser pulse. 32,33 Typically the beam is not focussed tightly, so as to avoid optical breakdown or photochemical effects. 34 In experiments involving trapping, a CW laser beam is focussed tightly into a solution or at a solution interface. This laser trapping method is observed to change the structure of the solution over a longer period of time (minutes). 35 The trapping can also be achieved using trains of laser pulses at high repetition-rates over similar periods. 36 Different mechanisms apply to laserinduced nucleation by pulsed NPLIN or by laser trapping: although both may be described as nonphotochemical laser-induced nucleation. To avoid confusion, for the remainder of this article we deal only with pulsed NPLIN experiments and the corresponding mechanism.
The control of polymorphism using polarised laser light has significant potential uses.
However, there are uncertainties about the OKE mechanism for NPLIN. Theoretical considerations suggest that thermal fluctuations should overwhelm molecular alignment by the optical electric field. 37,38 Recent work on NPLIN of urea shows no correlation between the direction of polarization of the light and the direction of the initial crystallite needles, 39 contrary to the original report. 40 Polarization switching of glycine polymorphs by NPLIN has so-far not been reproduced. 41 Filtering of solutions has been shown to supress NPLIN. 42,43 An alternative mechanism to explain NPLIN has been described, based on formation of transient vapour cavities due to heating of impurity particles by the laser light. [43][44][45] The objective of the present work was to explore the supersaturation dependence of polymorphism in glycine solutions using different methods for inducing nucleation. The methods used were pulsed laser light (NPLIN), ultrasound and mechanical shock. We have also re-examined evidence for the effect of polarization switching.

Experimental methods
Molarity is defined as moles of solute per litre of the final solution (not per litre of solvent), whereas molality is defined as moles of solute per kg of solvent. Sun et al. specified glycine concentrations in molarity; 23 but the supersaturations quoted appear to be more consistent (to within 5%) with ratios defined by molality. In the present work, we report supersaturation as S = C/C sat , with concentrations in molality, and C sat is the saturation concentration (solubility). In the present work we reference all supersaturations relative to the -glycine polymorph. The solubility of -glycine used was 3.01 mol kg -1 at 20 °C and 3.34 mol kg -1 at 25 °C, as determined from a polynomial fit to published data. 46 We note that the supersaturations reported by Clair et al. 41 were based on the solubility data of Yang et al., 47  Glycine was obtained from Sigma Aldrich (33226, puriss p. a., 99.7-101%) and used without further purification. Analysis indicated the as-purchased solid was a mixture of and -glycine. The basic optical setup has been described elsewhere. Laser pulses (5.6 ns) were obtained from a Q-switched Nd 3+ : YAG laser (Quantel Brilliant) with wavelength of 1064 nm. The beam was passed through a Glan-laser polarizer to control the transmitted power and to ensure purity of the linear polarization. The mean power of the beam was 0.62 W, and its diameter (2.5 mm) was obtained by Galilean telescope. The sample tube acts as a cylindrical lens, loosely focusing the beam in the horizontal plane. The refractive index of the solution was estimated to be n = 1.4. We calculate the energy densities as 1.3 J cm -2 (input) and 3.0 J cm -2 (exit), corresponding to peak power densities of 0.21 GW cm -2 (input) and 0.5 GW cm -2 (exit). The irradiated volume was 0.045 cm 3 . The exit peak power density was similar to the high-intensity value (0.46 GW cm -2 ) used by Sun et al. Each sample was exposed to pulses at 10 Hz for 60 s. The temperature was not controlled during exposure. In total 283 samples were exposed (see Supporting Information for tables of data). Sonocrystallization was used as an alternative method of nucleation for comparison against NPLIN. Samples were prepared as described above, but contained in glass vials (Gilson C4000-1, diameter 12 mm, sample volume 2 cm 3 ). Samples were aged for up to 7 days at 25 °C and then exposed by placing in a standard laboratory ultrasonic bath (Elmasonic S30H, 37 kHz, effective power 80 W) for up to 120 s. In total 213 samples were exposed to ultrasound. Another method of nucleation tested was mechanical shock. Samples were prepared in glass vials (Murray & Co. T102/V1, diameter 20 mm, sample volume 4 cm 3 ), aged for 3 days at 25 °C and then exposed to shock by placing them harshly and abruptly onto a flat surface. In total 48 samples were exposed to mechanical shock.
Following exposure, samples were stored in a temperature-controlled oven and checked for the presence of crystals after 1-7 hours. Crystals were extracted, washed briefly with water, dried using compressed air, and ground to a powder using a mortar and pestle. According to Boldyreva et al. the ,  and  polymorphs are stable with respect to this procedure. 19 The powder was analysed using powder X-ray diffraction (pXRD, Bruker D2 PHASER) or attenuated totalreflectance Fourier-transform infrared spectroscopy (ATR-FTIR, Perkin Elmer UATR Two) to determine the polymorph obtained (see Supporting Information for details). 13 Samples were counted as follows: N was the number of solutions exposed, n was the total number of samples nucleated, and n g was the number of samples giving only -glycine. The fraction of samples that nucleated was calculated as f = n / N, and the fraction of samples that nucleated -glycine only was calculated as f g = n g / n.

Results
A plot of the fraction of samples nucleated using LP laser pulses is shown in Figure 1. As expected, the nucleation fraction generally increases with supersaturation. 23, 32 Samples exposed to sonocrystallization and mechanical shock show similar nucleation fractions, and these methods were more effective than NPLIN at S = 1.4.
The nucleation fractions (f) for NPLIN observed in the present work are close to previous results of Sun et al. 23 and Javid et al., 42  Plots showing the fraction of nucleated samples that produced -glycine are presented in Figure 2. The fractions of -glycine samples obtained by NPLIN with linearly polarised light shown in Fig. 2(b) are higher than those reported by Javid et al. 41,42 This may be due to differences in the experimental conditions employed, which can be amplified by the steep slope for the switch from to -glycine for NPLIN. In the work of Javid et al. the experiments were conducted over a longer timeframe (4 days) following exposure to laser light. 42 Their inductiontime data suggested that nucleation at longer times was spontaneous, yielding almost exclusively -glycine, which would reduce the fraction of -glycine reported.
The sonocrystallization and mechanical shock results appear to show a continuous increase in preference for -glycine with increasing S. Assuming extreme values of 0 and 1 for f g at these limits, the data were modelled using a logistic function The parameter k models the rate of increase of f g with S, and S c is the mid-point value (where f g = 0.5). Least-squares fitting of the data yielded the parameters k = 9.4 ± 1.1 and S c = 1.53 ± 0.01 for sonocrystallization; k = 15.1 ± 1.5 and S c = 1.47 ± 0.01 for mechanical shock nucleation; and k = 80.5 ± 7.1 and S c = 1.42 ± 0.01 for the NPLIN data.
In comparison to externally induced nucleation, we found that fewer samples nucleated spontaneously within the timeframe of our experiments. These samples were found to be mostly (but not exclusively) the -glycine polymorph, the form usually obtained from aqueous solution. 8,51 At S = 1.5, by spontaneous nucleation 7/8 samples were found to be -glycine and 1/8 was glycine.  The vertical dashed lines in Figure 2  Our results (Fig. 2a) are not consistent with this window at S = 1.4 and 1.45.
The results of tests for polarization switching at 20 °C (S = 1.5) are shown in Table 1. The total fraction of samples nucleated at 20 °C (0.11) was much smaller than at 25 °C (0.68). We did not see binary switching of polymorph with polarization. We note that the fraction of -glycine samples nucleated using LP light is lower than the value plotted in Fig. 2

Discussion
In the following discussion, we focus on two observations that stand out from the results: (1) the results for sonocrystallization, nucleation by mechanical shock, and NPLIN show similar trends in fractions of samples nucleated as a function of supersaturation, although they are not identical; (2) the results do not reproduce the polarization switching effect for glycine as reported by Garetz and co-workers.

Nucleation mechanisms
Figures 1(b) and 2(b) show that sonocrystallization and mechanical shock nucleation produce very similar results, in terms of both the fractions of samples nucleated and -glycine produced as a function of supersaturation. All the nucleation methods show an increasing propensity to form -glycine at higher supersaturations (Fig. 2).
Louhi-Kultanen et al. studied crystallization of glycine with ultrasound at 20 kHz. 52 They found that nucleation by cooling with ultrasound produced larger crystals than cooling alone. The fraction of -glycine obtained at 20-30 °C (estimated S = 1.22) was f g ~ 0.05-0.13, in good agreement with our results for sonocrystallization shown in Fig 2(b). Renuka Devi et al. also studied the effect of ultrasound at much higher frequencies (1-10 MHz) but observed nucleation of -glycine only: 53 this may be due to weaker cavitation effects (see below) at frequencies > 1 MHz. 54 The fraction of -glycine in Fig. 2(b) appears to increase smoothly from S = 1.3 to 1.7.
Modelling with Eq. 1 shows that the mid-point supersaturations (S c = 1. 47  The mechanisms for nucleation by ultrasound or by shock are not fully understood. 55 It has been known for a long time that mechanical shock can induce nucleation. 56,57 Ultrasound has been used to nucleate crystals in a variety of systems, and it is known that ultrasound causes cavitation. 58 For both of these methods it is generally accepted that nucleation results from induction of localized zones of high pressure, which increase local supersaturation. However, at the present time it is not clear whether transient high-pressure waves are sufficient alone, whether bubble interfaces are responsible, or the extent to which foreign particles are involved. 55 As outlined in Section 1, the OKE mechanism for NPLIN, based on laser-induced molecule alignment, has been cast into doubt. 37,[42][43][44][45] An alternative mechanism has been put forward, involving transient heating of particles, leading to formation of cavities and pressure shockwaves in the solution. 43,45 This alternative mechanism for NPLIN is very similar to those believed to operate for ultrasound and mechanical shock, and would account for the similarities in the behaviour shown in Figs 1 and 2. We speculate that the reason for the steeper transition from  to  for NPLIN is due to the cavitation events proceeding at higher energies compared to mechanical shock or sonocrystallization. These more-energetic cavitation events would result in higher localized pressures and supersaturations, which favour -glycine. 28 An unexplored source of differences between results of different groups lies with the identity of the particles that are heated by the laser. We believe these are impurity nanoparticles at very low concentrations, since on average only a few nuclei per cm 3 are produced. 32 As we have noted elsewhere, these particles could vary depending on the materials, vessels and procedures employed. 43 Particles that are smaller, or that absorb less energy from the laser, may produce lower-energy cavitation events. Further experiments are underway to investigate these issues.

Polarization switching
We do not see the binary polarization switching for NPLIN of glycine, reported by Garetz and co-workers, 21 28 The fact that crystallization occurs after minutes, and that crystallization takes place even in undersaturated (S = 0.68) solutions, demonstrates the sustained influence of the laser on the solution. As noted in the Introduction, however, we limit our ongoing discussion to polarization switching in the (nanosecond) pulsed NPLIN experiments used here.
Why has the binary polarization switching observed by Garetz and co-workers not been reproduced? One pitfall, from a statistical viewpoint, has been the low number of samples tested.
In Fig. 1  The sample size in Table 1 62 These studies do suggest that the polarization of light can influence nucleation.
This may be due to the polarization dependence of absorption by impurity nanoparticles, rather than influence on putative solute clusters with different polarizabilities. In future studies it would be useful to conduct statistical analyses, e.g., analysis of variance (ANOVA) on product polymorph distributions, provided sufficient sample sizes can be obtained.

Conclusions
In summary, we have conducted a study of the effect of supersaturation on crystallization of aqueous glycine using three different methods: non-photochemical laser-induced nucleation, nucleation by ultrasound (sonocrystallization) and nucleation by mechanical shock. The results show that the fraction of samples nucleated, and the fraction of samples that form -glycine, both increase with increasing supersaturation. The results are consistent with changes to the solution structure, possibly formation of dimers and clusters at higher supersaturations. We have proposed a mechanism that is common to all three methods of nucleation, where induced cavitation and pressure shockwaves cause localized increases in pressure and supersaturation. The switch in preference from -glycine to -glycine happened over a smaller range of supersaturation for NPLIN compared to the other two nucleation methods. We attribute this to the production of cavitation events with higher energies, causing higher localized supersaturations that favour glycine. Using circular and linear polarizations of laser light, we were unable to reproduce the binary polarization switching effect for glycine reported by Garetz and co-workers. Further experimental and modelling work is needed towards understanding the role of solute structures in glycine crystallization.