Héctor Uriel

Rodríguez Vera

Fabien Baillon

Fabienne Espitalier

Philippe Accart

Olivier Louisnard

Crystallization of α-glycine by anti-solvent assisted by ultrasound

Keywords:

Crystallization of α-glycine by addition of an anti-solvent (ethanol) assisted by ultrasound is studied. The experiments of crystallization are conducted at 303.15 K in a solution of 150 ml with continuous agitation by a magnetic rod. Ultrasound is then applied at powers ranging from 8 to 41 W thanks to an ultrasonic horn at 20 kHz. The supersaturation ratio (S) is followed throughout all the experiment. At the end of the experiment, the suspension is filtered, the solid is washed with ethanol and dried at 333.15 K. The resulting crystals are characterized by their final size distributions measured by laser granulometry, their morphologies observed by scanning electronic microscope (SEM) and their crystalline structures by differential scanning calorimetry (DSC). The influence of ultrasonic power (continuous 13, 28 and 40 W or pulsed modes), measured by calorimetry method, is studied for different addition rates (0.05 to 0.36 g of ethanol/min). Ultrasound permits to reduce the metastable zone width and to decrease the size of crystals due to an increase of the nucleation rate. The rate of de-supersaturation is higher in presence of ultrasound, inducing a higher nucleation rate, a higher growth rate or both. At 40 W, the decrease of supersaturation is faster, and the crystallization is finished in 40 min instead of 80 min (at 13 and 28 W) or 120 min without ultrasound. The use of pulsed ultrasound (50 on/50 off) is interesting from an economic point of view because similar results are obtained: comparable size distributions and resembling concentration profiles.

Introduction

The crystallization is an important step in the formulation of a solid as it permits to control the solid properties like the size distribution, the morphology and the crystalline structure. However, in a batch crystallization process, it is difficult to control the phenomena involved in the vessel (nucleation, growth and agglomeration) without the use of seeding [START_REF] Mersmann | Crystallization Technology Handbook[END_REF]. Ultrasound can be an interesting alternative to the use of seeding.

The effect of ultrasound on crystal size is observed and reported in many studies involving varied pharmaceutical drugs such as non-steroidal anti-inflammatory drugs: ibuprofen [START_REF] Manish | Melt sonocrystallization of ibuprofen: effect on crystal properties[END_REF], mefenamic acid [START_REF] Iyer | Ultrasound assisted crystallization of mefenamic acid: effect of operating parameters and comparison with conventional approach[END_REF], ketoprofen [START_REF] Belkacem | Effect of ultrasound on the physicochemical properties of poorly soluble drugs: antisolvent sonocrystallization of ketoprofen[END_REF], phenylbutazone [START_REF] Park | Liquid antisolvent recrystallization of phenylbutazone and the effect of process parameters[END_REF]; antibiotics or antifungals: itraconazole [START_REF] Dalvi | Analysis of nucleation kinetics of poorly water-soluble drugs in presence of ultrasound and hydroxypropyl methyl cellulose during antisolvent precipitation[END_REF], sulfamethoxazole [START_REF] Dalvi | Analysis of nucleation kinetics of poorly water-soluble drugs in presence of ultrasound and hydroxypropyl methyl cellulose during antisolvent precipitation[END_REF], sulfathiazole [START_REF] Su | Recrystallization of phenacetin and sulfathiazole using the sonocrystallization process[END_REF] and cloxacillin [START_REF] Li | Effects of sonocrystallization on the crystal size distribution of cloxacillin benzathine crystals[END_REF]; analgesics: piroxicam [START_REF] Pereira | Ultrasound influence on the solubility of solid dispersions prepared for a poorly soluble drug[END_REF], salicylic acid [START_REF] Hatkar | Process intensification of anti-solvent crystallization of salicylic acid using ultrasonic irradiations[END_REF], phenacetin [START_REF] Su | Recrystallization of phenacetin and sulfathiazole using the sonocrystallization process[END_REF]; antiepileptics: carbamazepine [START_REF] Park | Antisolvent crystallization of carbamazepine from organic solutions[END_REF]; and antilipemics: gemfibrozil [START_REF] Ambrus | Effect of sonocrystallization on the habit and structure of Gemfibrozil crystals[END_REF]. In all these studies, the particles sizes of active drug obtained are finer than those obtained with conventional crystallization methods and the size distributions become narrower in the presence of ultrasound. This decrease of size permits for instance to increase the drug dissolution rate and so, to improve the bioavailability of the drug. In addition to reducing the crystals size, ultrasound allows to control the crystalline structure. For example, in the case of piroxicam, the stable polymorphic form I is created predominantly. However, the formation of amorphous piroxicam and the metastable form of benzoic acid is also noted in the presence of ultrasound [START_REF] Gracin | Influence of ultrasound on the nucleation of polymorphs of p-aminobenzoic acid[END_REF]. The morphology of crystals can be changed by the application of ultrasound: needles shaped crystals are obtained without ultrasound for ketoprofen or mefenamic versus plate crystals with ultrasound. Finally, ultrasound causes a decrease of the induction time of metastable zone width at equivalent supersaturation [START_REF] Baillon | 28 -Crystallization and freezing processes assisted by power ultrasound[END_REF].

Different ultrasonic systems may be used: horn, transducer or bath; with different frequencies (20 kHz-20 MHz) and powers (few Watts to hundreds of Watts).

Glycine is the smallest and simplest of the amino acids. It allows the body to synthesize many proteins, especially in the skin and muscles. It is one of the molecules found in more than 100 specialties like cataract treatment, subcutaneous rehydration and parenteral nutrition). It presents three polymorphs: α-glycine, β-glycine and γ-glycine. At room temperature, the γ-glycine is the most stable [START_REF] Yang | Polymorphism in the crystallization of glycine[END_REF][START_REF] Bazi | Antisolvent crystallization: effect of ethanol on batch crystallization of α glycine[END_REF]. It is soluble in water and insoluble in ethanol. The crystallization of glycine assisted by ultrasound has been the subject of few studies. Previous works on cooling crystallization of glycine with ultrasound in aqueous phase showed that a purer crystalline phase is formed with more regular faces with moderate ultrasonic [START_REF] Louhikultanen | Crystallization of glycine with ultrasound[END_REF] and a decrease of the induction time when the ultrasonic power increases [START_REF] Baillon | Crystallization and freezing processes assisted by power ultrasound[END_REF]. The effect of high frequency ultrasound (between 1 and 10 MHz) on the nucleation and growth of glycine has been explored by Lyczko et al. [START_REF] Devi | Ultrasound assisted nucleation and growth characteristics of glycine polymorphs -a combined experimental and analytical approach[END_REF]. They showed that ultrasound only promotes α-nucleation and completely inhibits β-and γnucleation in the system glycine-water.

In our work, the crystallization of α-glycine by addition of an antisolvent (ethanol) assisted by ultrasound is studied. Effects of ultrasonic power, addition rate and pulsed cycle application on the particle size distribution and crystallization have been studied.

Materials and methods

Pure α-glycine (GPR RECTAPUR, 99.9% purity), ethanol (NORM-APUR) for the crystallization experiments and ethanol (TechniSolv 96%) to wash crystals were purchased from VWR CHEMICALS.

Crystallization experiments

The setup used for batch experiments is shown in Fig. 1. It is similar to the one used in a previous work [START_REF] Lyczko | Effect of ultrasound on the induction time and the metastable zone widths of potassium sulphate[END_REF]. It consists of a jacketed cylindrical vessel with a diameter of 0.056 m, stirred with a magnetic bar. The height of liquid at the end of experiment is 0.06 cm. The maximal volume corresponding to this height is 150 ml. A thermocouple connected to a temperature control system (connected to a LAUDA RC 20 CP) permits to maintain the temperature constant throughout all the experiment. The temperature control system is provided by a computercontrolled LAUDA RC 20 CP thermostat bath. For the sonocrystallization, an ultrasonic horn of 1 cm tip diameter controlled by VibraCell Bioblock Scientific generator is always positioned at the same height (0.02 m from the bottom of the reactor). The frequency of the generator is 20 kHz with a power range of 13-43 W.

A solution of concentration 0.27 kg α-glycine/kg water is prepared at 308.15 K (5 K above the temperature of saturation). This solution is then cooled at 303.15 K.

Once the temperature is stabilized at 303.15 K, ethanol is added at a constant flow rate by means of a syringe pump (PHD 2000 infusion) and a 20 ml disposable syringe (TERUMO®). The injection of ethanol is carried out close to the ultrasound probe tip. The addition rate varies between 0.05 and 0.36 g of ethanol/min.

During the course of the experiment, samples are taken and filtered with syringe filters (0.2 µm). Some experiments are performed three times in order to verify repeatability. The refractive index of the filtrate is measured. The measurement of refractive index of solution permits to follow the concentration of glycine (C glycine ) all along the experiment. A calibration curve is determined for a mass ratio R and a concentration of α-glycine ranged respectively between 0 and 0.05 g ethanol/g water and 0 and 0.191 g αglycine/ g solution at 303.15 K.

= + C R I R g g RI R ( , ) 7
where RI is the refractive index measured at reference temperature of 293.15 K. The supersaturation ratio (S) is calculated by the ratio of C α- glycine (RI, R) and C α-glycine , eq (R,T). C α-glycine , eq is the equilibrium concentration of α-glycine measured for this study as function of mass ratio of ethanol at 303.15 K. At the term of the crystallization experiments, the resulting suspension is filtered and the solid is washed with ethanol, then dried in a stove (Hereaus) at 333.15 K. The crystals are characterized by their final size distributions measured by laser granulometry and their morphologies are observed by scanning electronic microscope (Hitachi TM3030 SEM). In order to verify the crystalline structure, an analysis by differential scanning calorimetry (DSC, Q200 TA Instrument) with a heating rate of 5 K/min between 293.15 and 473.15 K are conducted in a crimped aluminum pan [START_REF] Yang | Polymorphism in the crystallization of glycine[END_REF].

The influence of ultrasonic power (continuous or pulsed modes), measured by calorimetry method, is studied for different addition rates of ethanol. The limit supersaturation ratio S limit and the beginning of crystallization (time between S = 1 and S limit ) are followed and observed.

Solubility determination

Different solubility data of α-glycine in water or alcohol aqueous mixtures as a function of temperature and solvent composition are given in the literature [START_REF] Bouchard | Solubility of glycine polymorphs and recrystallization of β-glycine[END_REF]. In order to calculate the supersaturation ratio in crystallization operating conditions, the equilibrium concentration of α-glycine in water, C glycine eq -, , is measured at 303.15 K for three mass ratio of ethanol 0, 0.025 and 0.05 g ethanol/g water in the crystallization experimental set up. An excess of α-glycine solid is introduced in water at 303.15 K. The suspension is stirred at 900 rpm during 24 h. Samples are taken and filtered with syringe filters. The refractive index of filtrate is measured as a function of time.

Dissipated power

Temperature measurements are performed during the application of ultrasound in the crystallization vessel. The vessel is filled with 150 g of water and the jacket is empty in order to thermally insulate the system.

An energy balance leads to:

= + + T T Q Q hS hS e ( )

Results

Solubility

The Fig. 2 presents the evolution of α-glycine concentration as function of time during the solubility measurement at 303.15 K for a mass ratio of 0.05 g ethanol/g water. The liquid-solid equilibrium is reached very rapidly because the concentration very slightly changes over time between 0 and 1440 min (as each sampling has to be taken into account in the mass balance). In this operating condition, the equilibrium concentration is equal to 0.186 ± 0.001 g glycine/g solution.

The solubilities of α-glycine measured at 303.15 K in three ethanol water mixtures are reported in Table 1. The glycine concentrations calculated from the refractive index are compared to those measured by dry extract. A good agreement is observed between these two methods. According to the literature, the solubility of α-glycine decreases with the mass ratio ethanol-water [START_REF] Bouchard | Solubility of glycine polymorphs and recrystallization of β-glycine[END_REF][START_REF] Ferreira | Solubility of amino acids and diglycine in aqueous-alkanol solutions[END_REF]. Moreover, these α-glycine solubility data are in agreement with those available in the literature data (298 and 313 K, and 0.053 g ethanol/g water) [START_REF] Ferreira | Solubility of amino acids and diglycine in aqueous-alkanol solutions[END_REF].

The concentrations given in the rest of document are estimated from the measurement of the refractive index.

Dissipated power

The measured dissipated powers by ultrasound are reported in Table 2 in W and in W/kg solution as function of the level of graduation. The measured dissipated powers are ranged between 8 and 41 W (or 55 and 271 W/kg solution). During an operation of crystallization, it is important to use moderate power in order to control the temperature, and by that way, the supersaturation. Pulsed ultrasound is used in order to reduce the energy consumption. A thermal transfer coefficient h of 10.5 W m K 2 is found. Since the transfer coefficient h is small, it can be assumed that the system is adiabatic. The power dissipated by agitation (0.07 W), is negligible compared to the power dissipated by ultrasound.

Crystallization experiments

Operating conditions of the experiments are reported in Table 3. The initial concentration of glycine in water (0.27 g glycine/g eau), the temperature (303.15 K) and the final mass ratio of ethanol in water (0.05 g ethanol/g water) are constant for all experiments. The influence of four addition rates of ethanol, three dissipated powers and two pulsed at one graduation on the particle size distributions and supersaturation ration profiles have been studied.

Fig. Supersaturation ratio as a function of time for different addition rates

and a mass ratio of ethanol in water of 0.05 g ethanol/g water in silent condition (a) and with a dissipated power of 28.3 W (b). The full lines represent the theoretical supersaturation ratio in absence of crystallization. The dotted lines are drawn to show the trends. a-in silent condition (empty square marker 0.36 g ethanol/min, empty diamond marker 0.19 g ethanol/min, empty circle marker 0.09 g ethanol/min, empty triangle marker 0.046 g ethanol/min). bwith dissipated power of 28.3 W (filled square marker 0.36 g ethanol/min, filled diamond marker 0.19 g ethanol/min, filled circle marker 0.09 g ethanol/ min, filled triangle marker 0.046 g ethanol/min).

To identify the form of glycine crystallized (γ or α), DSC analyses have been made. The presence of an endothermic peak around 441 K corresponds to the phase transformation of γ-glycine to α-glycine [START_REF] Bazi | Cristallisation discontinue et semi-continue de la glycine. Etude de l'influence d'un anti-solvant[END_REF]. The Fig. 3 presents the thermograms obtained for three solids (raw product, glycine crystallized with and without ultrasound). The absence of an endotherm means that the pure metastable α-form is obtained whatever the operating conditions. Ultrasound doesn't modify in this case the crystalline structure.

Influence of flowrate

Four addition rates of ethanol have been tested in silent condition and with a dissipated power of 28 W. Profiles of supersaturation ratio, morphologies and size distributions of final crystals and limit supersaturation ratio S limit in silent condition and with a dissipated power of 28 W are reported in Fig. 4 to Fig. 5. For each experiment, three phases are observed. In the first phase, logically, the supersaturation ratio increases linearly with time due to the addition of ethanol. During this phase, no crystallization occurs. A decrease of this supersaturation ratio for S = S limit is then observed in a second phase due to the crystallization (nucleation and growth) of α-glycine. A plateau is attained in the last phase: S tends toward 1. The solution is considered saturated. The limit supersaturation ratio (1.13 without ultrasound and 1.08-1.10 with ultrasound) does not depend of the addition rate of ethanol ranged between 0.09 and 0.36 g ethanol/min (Fig. 4). This maximal supersaturation ratio is lower (1.09 without ultrasound and 1.04 with ultrasound) at low addition rate 0.05 g ethanol/min. In term of metastable zone width, it increases when the addition rate increases in silent conditions and with ultrasound. With the same flowrates, this width decreases when ultrasound is applied. Some information on nucleation can be deduced from the metastable zone width as function of the addition rate of ethanol by plotting the logarithm of the total amount of ethanol per water (m ethanol in kg ethanol/kg water) added as function of addition rate m ethanol (kg ethanol/kg water/s) according to the Nyvlt model [START_REF] Karpinski | Metastable zone width in salting-out crystallization[END_REF][START_REF] Zhang | Metastable zone width, crystal nucleation and growth kinetics measurement in anti-solvent crystallization of βartemether in the mixture of ethanol and water[END_REF] ) or complex functions m and K of both nucleation and growth kinetics parameters if the first crystals are not detected immediately. Values of n (or m) and k N (or K) are respectively equal to 2.4 and 12.5 kg ethanol/kg water/s in silent conditions and 2.7 and 366.2 kg ethanol/kg water/s with ultrasound (28 W). The nucleation order values are in agreement with values ranged between 2.47 and 2.65 found by Bazi et al. for the Dolder® glycine obtained by antisolvent crystallization [START_REF] Bazi | Antisolvent crystallization: effect of ethanol on batch crystallization of α glycine[END_REF]. By considering that the crystals are detected immediately, the nucleation rate increases in presence of ultrasound.

The resulting crystals are smaller, and the crystal size distributions are narrower when ultrasound is used (Fig. 6). The images obtained by SEM are in agreement with the particle size distribution (Fig. 5). The size distributions of crystals obtained in silent condition are very large but few effects on mean volume diameter d 4,3 are observed as function of the addition rate. In presence of ultrasound, an increase of d 4,3 is observed with the addition rate (between 0.04 and 0.09 g/min), which is in agreement with the works of Vishwakarma et al. [START_REF] Vishwakarma | Intensified oxalic acid crystallization using ultrasonic reactors: understanding effect of operating parameters and type of ultrasonic reactor[END_REF] and is due, in our case, to agglomeration and a decrease of this diameter with addition rate (between 0.09 and 0.36 g/min). No agglomerates were observed at 0.19 and 0.36 g/min. At low addition rate, ultrasound seems to favor agglomeration.

Influence of dissipated power

The Fig. 7 presents the supersaturation ratio for a mass ratio of ethanol/water of 0.05 as function of time in silent conditions and with three dissipated power: 13, 28 and 40 W; for an addition flowrate of 0.19 g/min, S limit decreases in presence of ultrasound that is equivalent to a narrower metastable zone width. However, no difference is observed between experiments conducted with an ultrasonic power of 13 and 28 W. At 40 W, the decrease of supersaturation is faster, and the crystallization is finished at 40 min instead of 80 min (at 13 and 28 W) or 120 min without ultrasound. These observations suggest that an increase of power linked with cavitation effects induces an increase of nucleation and growth rates in agreement with previous results [START_REF] Vishwakarma | Intensified oxalic acid crystallization using ultrasonic reactors: understanding effect of operating parameters and type of ultrasonic reactor[END_REF]. The shape of crystals obtained at 13 W is less regular than the shape of those obtained without ultrasound or at 28 W (Fig. 8). Lots of agglomerates are observed at 13 W.

Crystallization by anti-solvent effect is very sensitive to the mixing between the solvent and the solution, and the effect of mixing intensity on the properties of the crystals is well-known even in standard crystallization conditions: a good mixing allows to avoid high-supersaturation local spots in the vessel, and for example, the position of ethanol addition itself is known to influence the crystal size distribution [START_REF] Mullin | Industrial Crystallization[END_REF]. In the present experiments under silent conditions, a rather poor mixing is expected, resulting in large local supersaturation gradients, which yield in turn very dispersed crystal sizes. Cavitation is known to enhance mixing either by acoustic (macroscopic) streaming, acting as if additional mechanical stirring were present [START_REF] Gondrexon | Experimental study of the hydrodynamic behaviour of a high frequency ultrasonic reactor[END_REF][START_REF] Kumar | Experimental quantification of chemical elects of hydrodynamic cavitation[END_REF][START_REF] Tsochatzidis | Determination of velocity, size and concentration of ultrasonic cavitation bubbles by the phase-Doppler technique[END_REF][START_REF] Dahlem | The radially vibrating horn: a scaling-up possibility for sonochemical reactions[END_REF][START_REF] Chouvellon | Velocity study in an ultrasonic reactor[END_REF][START_REF] Mandroyan | Modification of the ultrasound induced activity by the presence of an electrode in a sonoreactor working at two low frequencies (20 and 40 kHz). Part I: active zone visualization by laser tomography[END_REF][START_REF] Hihn | Respective contribution of cavitation and convective flow to local stirring in sonoreactors[END_REF][START_REF] Louisnard | A viable method to predict acoustic streaming in presence of cavitation[END_REF], or by micro- streaming caused by bubble spherical and/or non-spherical oscillations [START_REF] Kornfeld | On the destructive action of cavitation[END_REF][START_REF] Birkin | Investigation of noninertial cavitation produced by an ultrasonic horn[END_REF][START_REF] Birkin | Efficient mass transfer from an acoustically oscillated gas bubble[END_REF], and bubble jetting [START_REF] Blake | Acoustic cavitation: the fluid dynamics of non-spherical bubbles[END_REF][START_REF] Crum | Surface oscillations and jet development in pulsating bubbles, C8-285-C8-288[END_REF][START_REF] Prabowo | Surface oscillation and jetting from surface attached acoustic driven bubbles[END_REF]. Thus, under ultrasound, the intense cavitation near the sonotrode tip may enhance mixing and smooth out spatially the supersaturation, producing a narrower crystal size distribution as evidenced in Fig. 9. It should be noted that the latter figure also shows that crystals obtained at 13 W are slightly smaller than at 28 W, with a comparable dispersity. This trend is opposed to what is usually observed in sono-crystallization by cooling [START_REF] Baillon | 28 -Crystallization and freezing processes assisted by power ultrasound[END_REF], and suggests that this result might also be attributed specifically to mixing effects. Whether the latter is connected with nucleation, growth or agglomeration remains to be explained.

From these experiments, we can conclude that ultrasound has an influence on the crystallization rate (nucleation and growth rates) due to the fact that for the same profile of de-supersaturation, two different size distributions are obtained. For a dissipated power ranged of 13 W (minimal power), a mean volume diameter d 4,3 of 90 µm is observed and for 28 W a value of 126 µm is obtained. The rate of de-supersaturation is higher in presence of ultrasound inducing a higher nucleation rate or a higher growth rate or both. From experimental results, modeling of crystallization will permit to conclude on crystallization rates.

Influence of pulsation

Experiments with pulsed ultrasound are conducted with a mass ratio of 0.05 g ethanol/g water and an addition flowrate of 0.19 g ethanol/ min: 50 on/50 off and 25 on/70 off equivalent to a dissipated power of 13.4 and 8 W. Results on morphology an in size distribution of crystals are compared to results obtained with continuous application of ultrasound (Figs. 10 and11). The size distributions and the profiles of supersaturation ratio are identical (not shown) whatever the operating conditions. The pulsed ultrasound condition (50 on/50 off) seems to be interesting as similar results are obtained: comparable size distributions and reassembling concentration profiles. However fine crystals (needles) are observed in the case of pulsed ultrasound at low power like without ultrasound (Fig. 5).

no ultrasound = 13 W Fig. 8. SEM images of the resulting crystals obtained with an addition rate of 0.19 g ethanol/min and a mass ratio of ethanol of 0.05 g ethanol/g water in silent condition and with ultrasound (13 and 28 W).

Conclusion

In this work, the feasibility of a crystallization of glycine by antisolvent effect assisted by ultrasound is shown. At the operating conditions tested, the metastable form of glycine (α-form) is generated. Ethanol addition rates and ultrasound can modify the starting point of the crystallization, the maximal supersaturation ratio (lower supersaturation) and the crystal size distributions (obtained crystals are smaller which may mean a higher nucleation rate). An identification of nucleation kinetic parameters confirms that the nucleation rate increases in presence of ultrasound.

Fig. 1 .

 1 Fig. 1. Crystallization experimental setup.

Fig. 2 .

 2 Fig. 2.Example of the evolution of a-glycine concentration at 303.15 K for a mass ratio of 0.05 g ethanol/g water during the measurement of solubility.

2 , 8 Fig. 3 .

 283 Fig. 3. DSC analysis of three samples: raw material (right y-axis), glycine crystallized without and with ultrasound (left y-axis) for a mass ratio of 0.05.

  and the Y-intercept of the straight line obtained give the nucleation parameters n and k n (

Fig. 5 .

 5 Fig. 5. SEM images of resulting crystals obtained with different addition rates and a mass ratio of ethanol of 0.05 g ethanol/g water.

Fig. 6 .

 6 Fig. 6. Size distributions of resulting crystals for the experiment conducted in silent condition (a) and with a dissipated power of 28 W (b). a-in silent condition (empty square marker 0.36 g ethanol/min, empty diamond marker 0.19 g ethanol/min, empty circle marker 0.09 g ethanol/min, empty triangle marker 0.046 g ethanol/min). b-with dissipated power of 28.3 W (filled square marker 0.36 g ethanol/min, filled diamond marker 0.19 g ethanol/min, filled circle marker 0.09 g ethanol/min, filled triangle marker 0.046 g ethanol/min).

Fig. 7 .

 7 Fig. 7. Supersaturation ratio as a function of time for the experiment conducted with an addition rate of 0.19 g ethanol/min in silent condition and with ultrasound (13, 28 and 40 W). Filled square marker 40 W, filled diamond marker 28 W, filled triangle marker 13 W and empty diamond marker 0 W.

Fig. 9 .

 9 Fig. 9. Size distributions of resulting crystals for the experiment conducted with an addition rate of 0.19 g ethanol/min in silent condition and with ultrasound (13 and 28 W).

Fig. 10 .

 10 Fig. 10. SEM images of resulting crystals obtained with pulsed and continuous ultrasound application with an addition rate of 0.19 g/min.

Fig. 11 .

 11 Fig. 11. Size distributions of resulting crystals for the experiment conducted with continuous ultrasound (28 W) and pulsed ultrasound of 13 W (on 50/off 50) and 8 W (on 25/off 75).

Table 1

 1 Solubility of α-glycine in an ethanol-water mixture at 303.15 K.

		1	10	100	1000
	T (K)	R (g ethanol/g water)	C (RI,R) (g glycine/g solution)	C (dry extract) (g glycine/g solution)
	333.15 333.15 333.15	0.000 0.025 0.050	0.219 0.202 0.186		0.212 0.197 0.182
	Table 2 Dissipated power.
	Level/Pulsations	(W)		(W/kg)
	3 (100%) 5 (25%) 5 (50%) 5 (75%) 5 (100%) 7 (100%)		13 8 11 13 29 41		89 55 75 89 190 271

Time (min) C (g glycine/g of solution)

	0.200
	0.195
	0.190
	0.185
	0.180
	0.175
	0.170

Table 3

 3 Operating conditions of the crystallization experiments performed at 303.15 K with a mass ratio of 0.05 g ethanol/g water, the initial concentration of glycine was 0.27 kg α-glycine/kg water. The coefficient h and the stirring power Q stirring are determined with experiments respectively without stirring and without ultrasound and with stirring and without ultrasound.

	Addition rate of ethanol (g/min)	Ultrasound Dissipated power (graduation)	Ultrasound on (%)	Time of experiment (min)	d 4,3 (µm)	Beginning of crystallization (min)	S limit
	0.05 0.09 0.19 0.36 0.18 0.19 0.04 0.09 0.19 0.36 0.18 0.18	0 0 0 0 13 (3) 41 (7) 28 (5) 28 (5) 28 (5) 28 (5) 8 (5) 13 (5)	0 0 0 0 100 100 100 100 100 100 25 75	120 120 120 120 120 60 120 120 120 120 120 120	143 145 186 168 90 75 167 126 96 116 122	100 68 37 18 31 21 40 45 27 11 31 33	1.095 1.130 1.130 1.133 1.108 1.075 1.035 1.083 1.100 1.081 1.107 1.124


Jose Carranza-Rojas

Alexis A J Joly
email: alexis.joly@inria.fr

Pierre Bonnet

Hervé H G Goëau

Erick Mata-Montero

Jose Carranza
email: jcarranza@itcr.ac.cr

Automated Herbarium Specimen Identification using Deep Learning

Keywords: Biodiversity Informatics, Computer Vision, Deep Learning, Plant Identification, Herbaria

. Computer vision and machine learning approaches applied to herbarium sheets are promising (Wijesingha and Marikar 2012) but are still not well studied compared to automated species identification from leaf scans or pictures of plants taken in the field.

In a recent study, we evaluate the accuracy with which herbarium images can be potentially exploited for species identification with deep learning technology (Carranza-Rojas et al.

2017), particularly Convolutional Neural Networks (CNN) [START_REF] Szegedy | Going deeper with convolutions[END_REF]. This type of network allows automatic learning of the most prominent visual patterns in the images since they are trainable end-to-end (thus, differentiable), as opposed to previous approaches that use custom, hand-made feature extractors. A first challenge is to use herbarium sheet images alone to automatically identify the species of plants mounted on herbarium sheets. Secondly, we propose studying if the combination of herbarium sheet images with photos of plants in the field (Joly et al. 2015, Carranza-Rojas and[START_REF] Carranza-Rojas | Combining leaf shape and texture for Costa Rican plant species identification[END_REF]) is a viable idea to train models that provide accurate results during identification. Finally, we explore if herbarium images from one region with a specific flora can be used in transfer learning (a technique in deep learning that first allows training a model with a dataset and then once trained, uses the weighted results to train another model with that knowledge as the baseline) to another region with other species; for example, in a region under-represented in terms of collected data.

Our evaluation shows that the accuracy for species identification with deep learning technology, based on herbarium images, reaches 90.3% on a dataset of more than 1200 European plant species. This could potentially lead to the creation of a semi-, or even fully automated system to help taxonomists and experts with their annotation, classification, and revision works.

In this paper, we take a closer look at the accuracy levels achieved with respect to the first two challenges. We evaluate the accuracy levels for each species included in the dataset, which encompasses 253,733 images, 1,204 species.

Acknowledgements

Thanks to the National Museum of Costa Rica for their help with the collection, identification, and digitization of samples in the Costa Rican leaf-scan dataset. Special thanks to the Costa Rica Institute of Technology for partially sponsoring this research. We would also like to thank the large community that has actively engaged in iDigBio initiatives, for the valuable access to their herbarium data.

Hosting institution

Costa Rica Institute of Technology, Costa Rica INRIA, France CIRAD, France


S Ho-Van
email: son.ho-van@emse.fr

B Bouillot
email: bouillot@emse.fr

D Garcia

J Douzet

A Cameirao

S Maghsoodloo-Babakhani

J M Herri

Morphology of Cyclopentane Hydrates in Saline Water

Keywords: Clathrates, Cyclopentane Hydrates, Crystallization, Brine, Morphology

This effort focuses on morphology of cyclopentane hydrates formed from pure and saline water.

Different brine solutions of Na 2 SO 4 , NaCl, NaCl-MgCl 2 , or NaCl-KCl-MgCl 2 are considered at different concentrations and subcoolings. Especially, a small close reactor cell was used under a microscope with neither agitation nor seeds. Results show that the CPH crystals morphology is approximately comparable at the same subcooling, while the size of the individual crystals drops significantly with increasing of subcooling, regardless the type of salts and their concentrations.

Introduction

Cyclopentane hydrates (CPH) morphology is crucial in CPH-based desalination, or water treatment, applications since this affect strongly the desalting process and of course the equipment design. A few studies on the CPH morphology exist in literature [1][2][3][START_REF] Martinez De Baños | Mechanisms of formation and dissociation of cyclopentane hydrates[END_REF][START_REF] Delroisse | [END_REF]. Usually, only small glass vessels (about 4ml) are used. Moreover, they present an horizontal interface, no agitation, the system only consider NaCl and is seeded [1,2]. Therefore, this new effort examines other saline systems, under different concentrations, to study CPH morphology (NaCl, NaCl-MgCl 2 , NaCl-KCl-MgCl 2 , and Na 2 SO 4 ). Each time, three different subcoolings are used. A nonmixing small reactor cell (1.2ml) is used, without agitation, nor seed. Moreover, note that the geometry of the system presents a vertical interface between liquid water and cyclopentane, making the set-up easy to observe under a microscope.

Experimental observations

Chemicals utilized (Cyclopentane, NaCl, KCl, MgCl 2 , and Na 2 SO 4 ) were all provided by Sigma-Aldrich [6,7]. The schematic of reactor system and experimental protocol was well described by Ho-van et al [8]. The microscope is employed to observe and record the CPH crystallization, mainly at the interface between water and CP liquid. Photos of the individual hydrate crystals are taken for classification and comparison.

Effects of electrolytes and initial subcooling on the CPH morphology are illustrated in Figure 1.

Three different initial subcoolings are considered in pure water and in presence of six different brine solutions (2.5°C, 3.5°C and 4.3°C). Note that, in presence of salts, the subcooling does not remain the same during the hydrate formation. Indeed, the CPH generation process excludes the salts. Hence the salinity of the brine increases along with the crystallization. Therefore, since the temperature is kept constant, only the initial subcooling is chosen for each experiment.

All pictures of individual crystals have been taken at the end of each experiment (after days), when the crystal growth has ended. In this figure, the grain boundaries are delineated in black.

First, individual crystals' size at lower subcooling (2.5°C) is systematically bigger than that at higher subcoolings (3.5°C or 4.3°C). At 4.3°C initial subcooling, the approximate size 0.02-0.3mm, while the size is 0.05-0.5mm at 3.5°C, and 0.1-0.9mm at 2.5°C. This is similar to the observations of Sakemoto et al [1] and Kishimoto et al. [2]. Moreover, lower initial subcooling leads to polygons (2.5°C), while at higher subcoolings the smaller crystals take the form of ). This expected observation can be attributed to the higher crystallization rate at high driving force, leading to a reduction in the crystals size, as described in detail elsewhere [1][2][3][START_REF] Martinez De Baños | Mechanisms of formation and dissociation of cyclopentane hydrates[END_REF]9]. However, note that, in these experiments, only 2D pictures are taken. Therefore, all photos are just planar sections of 3D crystals. Hence, sword-like shapes observed can be a slice of bigger cubic CPH crystals. Nevertheless, observations are consistent with literature [1,2], and a bettersuited system would be needed to acquire more precise data on CPH morphology.

Second, it is important to notice that the size of CPH crystals is usually the same for all systems at same initial subcooling. Therefore, the kind of salt is not a significant factor to discuss the crystals' shape in the system. Only the effect of subcooling is relevant. Again, this observation agree well with literature [1,2].

Finally, the hydrate crystals in the water phase (right) are likely to form with a bigger size compared with those in the CP phase (left), as seen in Figure 2. Indeed, there is a quick growth toward the CP phase and a slower growth rate into the water phase. Ho-Van et al. [8] observed this phenomenon, took videos, and measured the interface growth speed. In the CP phase the individual crystals are more difficult to detect and seemingly stick together as a polycrystalline layer because of their very small size. Many channels and capillaries, which are supposed to be the ways for water and CP migration, can also be seen on the hydrate structure surface.

Figure 1 .

 1 Figure 1. Morphology of individual CPH crystals under different subcoolings in brine

Figure 2 .

 2 Figure 2. Crystals shape in the CP (left) and water (right) phases at initial subcooling of 3.5°C