Sevgi Polat,Perviz Sayan*

Department of Chemical Engineering,Faculty of Engineering,Marmara University,34722 ?stanbul,Turkey

Keywords:Glycine Oleic acid Crystallization Isoconversional methods Phase transformation

A B S T R A C T The polymorphic phase transformation of β-glycine to α-glycine was carried out both in the absence and presence of various concentrations of oleic acid used as additive at 25°C in a water/ethanol medium.The effects of oleic acid and its concentration on phase transformation time were determined by continuously measuring the ultrasonic velocity.The crystals obtained by the completion of the phase transformation were characterized by XRD,SEM,and TG/DTG.The XRD and SEM results indicated that oleic acid significantly impacted phase transformation time and the morphological characteristics of the crystals.In addition to SEM analysis,detailed crystal shape analysis was performed and the circularity,elongation,and convexity parameters were determined quantitatively.TG/DTG analyses were performed to investigate thermal decomposition behavior and to calculate the activation energies based on different kinetic models such as FWO,KAS,Starink,and Tang kinetic models.With the addition of oleic acid to the medium,the calculated activation energy values increased from 89.63–90.63 to 153.8–155.4 kJ·mol-1.The activation energy values showed that oleic acid was adsorbed on the crystal surface;this result was supported by FTIR,elemental,and Kjeldahl analyses.

1.Introduction

Glycine is the smallest protein-forming amino acid and is an all-purpose material,employed in different applications in various fields.The polymorphism phenomenon exhibited by glycine adds to its merits[1].Of the six polymorphs of glycine,three emerge under ambient conditions[2].At atmospheric temperature and pressure,the thermodynamic stability of these three polymorphs follows the order of γ-form＞α-form＞β-form[3].At room temperature,pure aqueous solution yields the α-glycine,whereas β-nucleation usually occurs only under appropriate conditions that either require alcohol or additives in the environment,or can be obtained by freezing the aqueous solution or by the sublimation of the stable form.γ-glycine nucleation usually occurs only if selective additives are present,which results in changes in molecular charge and hence,the inhibition of dimer formation[4].Polymorphs have varying mechanical,thermal,and physical properties that include compressibility,melting point,solubility,heat capacity,and crystal shape; these properties have considerable effects on the bioavailability,filtration,and tableting processes of pharmaceutical,food,and specialty materials.Therefore,consistent attainment of the desired polymorph is required to avoid specific problems resulting from the transformation between two forms during the production process[5].A thorough grasp of the phase transformation process is needed,both for control of polymorphism and in the production of high-quality final products with desired physical properties.Previous studies have revealed that operating conditions of crystallization,such as pH,temperature,initial concentration,and additives,can affect phase transformation and physical properties of the end-products.Therefore,using additives to control phase transformation and physical properties has received considerable attention[6].Some studies indicate that different polymorphs can be formed with different physical properties with the presence of additives in crystallization media.Hrkovac et al.explored the effects of sodium chloride and oxalic acid used as additives on glycine crystallization.They found that both additives and their amounts affected the solubility, the width of the metastable zone, crystal size distribution,and crystal morphology of glycine[7].A similar study performed by Yang et al.used sodium chloride as the additive,which had significant effects on the nucleation and polymorphic transformation of glycine.The presence of that additive caused a change in the nucleation of γ-glycine[8].Another study performed by Losev et al.examined the impacts of various carboxylic acids such as acetic,oxalic,malonic,succinic,maleic,glutaric,and L-malic acid on glycine polymorphism.Except for glutaric acid,all other acids studied affected the polymorphism of glycine[2].Oleic acid is a polyunsaturated fatty acid and was preferred in the present study as an additive because of its biodegradability,cost effectiveness,high efficiency,easy handling,non-toxicity,and availability.The lack of studies on the use of oleic acid as an additive in the transformation of β-to α-glycine,and the absence of a detailed shape analysis to determine the shape factors also contributed to this preference.Particle shape factors should be determined to obtain a high-quality product with desired properties.A detailed identification of the crystal shape by interpreting and depicting the shape parameters will contribute to different industrial uses of glycine.In the present study,given these goals,transformation of β-glycine to α-glycine was investigated both in a pure medium and in the presence of oleic acid.Particle shape parameters were quantitatively determined.The study aimed to investigate the effects of oleic acid on thermal decomposition behavior and to calculate the activation energies of the crystals obtained in both pure and oleic acid media using various kinetic models.

2.Experimental Method

Analytical grade α-glycine,ethanol,and oleic acid used in this study were provided by the Merck company,and triple-distilled water was used in all experiments.The experiments were carried out at 25°C in a specially designed 2 L-capacity double-coated glass crystallizer in the absence and the presence of an additive.All experiments were performed at least in triplicate.The temperature of the inner crystallizer was precisely controlled by a thermostat.Ultrasonic velocity measurements were taken by a probe placed at the base of the crystallizer.A special paddle-type blade connected to the mechanical stirrer was placed on top of the crystallizer to prevent crystal accumulation between the receiver and the transmitter under the operating conditions of the ultrasonic probe.In the experiments,500 ml of saturated α-glycine solution was placed in the crystallizer,and it was allowed to reach thermal equilibrium at 25°C.750 ml of ethanol used as antisolvent,previously held at 5°C,was rapidly added to the crystallizer and mixed with aqueous glycine solution.The β-form appeared instantly and then transformed into the α-form.After the antisolvent was added into crystallizer,this transformation process was continuously followed by measurement of ultrasonic velocity change.Ultrasonic velocity measurement was carried out with an ultrasonic sensor(Liqui Sonic 30,Senso Tech GmbH,Germany)with anofaccuracy±0.01m·s-1.At the same time,samples taken at specific time intervals were filtered rapidly,and dried.X-ray diffraction(XRD)analyses done by a Bruker D2 Phaser Table-top Diffractometer were performed to observe the transformation process.The sample was scanned in range of 10°–50°with a step size 0.01 and Cu Kαradiation from an X-ray tube at 30 kV.Oleic acid was used as an additive.The experiments were carried out at three different additive concentrations of 50,100,and 250 μl·L-1.Oleic acid was added to the media with ethanol solution.Scanning electron microscopy(SEM),morphology,thermogravimetric analysis(TGA),differential scanning calorimetry(DSC),and Fourier Transform Infrared Spectroscopy(FTIR)analyses were performed to characterize the end products obtained by completion of transformation in pure media and in the presence of oleic acid.SEM analysis was carried out in a Zeiss EVO LS 10 device to investigate the morphological changes in crystals.To collect more information about the size and shape characteristics of the crystals obtained with and without additive,the crystals were characterized by the Malvern Morphologi G3 device.This instrument allows analysis of the shape and size parameters by scanning and recording the image of all measured crystals.The 10X lens was utilized for all analyses.In addition,thermogravimetric analyses were performed on a Setaram LABSYS Evo Thermal Analyzer from 30 °C to 800 °C in an N2atmosphere at a flow rate of 20ml·min-1.Three different heating rates(5,10,20 °C·min-1)were used,and the data obtained were processed to determine the kinetic parameters using different kinetic models.DSC analysis was performed in aluminum pan with(5±0.5)mg of sample at 10 °C·min-1heating rate in N2atmosphere.Finally,FTIR analyses were carried out by a Perkin Elmer Spectrum 100 device in the range 4000 to 650 cm-1to identify the functional groups of the samples and to determine the possible adsorption properties of the additive on the surface of the glycine crystals.Elemental analyses of the crystals were performed,both to determine the elemental compositions and to show the influence of oleic acid on the C,H,N,and O composition of the crystals via a LECO-CHN-628 analyzer.

3.Results and Discussion

3.1.Ultrasonic velocity measurement

Ultrasonic velocity values measured in the experiments where the transformation process of β-glycine to α-glycine was studied in pure media and in the presence of different oleic acid concentrations are given in the Fig.1.

When time-dependent ultrasonic velocity changes of crystals produced in pure media were examined,it was observed that the transformation process of β-glycine to α-glycine occurred in three stages.The first stage was the dissolution of the β-glycine,where the ultrasonic sound velocity showed a rapid increase.The second stage was the plateau region where the formation of nuclei from dissolved glycine was observed.The third region was the growth stage of α-glycine crystals where the level of the ultrasonic velocity started rising again from the plateau level.This behavior observed in the transformation process of β-glycine to α-glycine was also observed in all experiments carried out in the presence of oleic acid.Whereas the transformation process was completed in 45 min in pure medium, the duration was determined to be 50,55,and 59 min at50,100,and 250 μl·L-1oleic acid concentrations,respectively.The transformation rate was slowed by the addition of oleic acid.These results showed that there was a relationship between transformation time and oleic acid concentration.At 50 μl·L-1oleic acid concentration,the additive had almost no effect on transformation time,whereas at higher concentrations,the additive tended to prolong the transformation time.The increase in the transformation time caused the morphology of the crystals and the particle shape parameters to change.

3.2.XRD analysis

The XRD measurements were performed both to follow the transformation process of β-glycine to α-glycine and to identify the crystal structures.The results of XRD analyses on samples taken at different times from the transformation process for the pure media are given in theFig.2(a).TheXRD peaks were compared with the peaks in the Cambridge Structural Database.As Fig.2 shows,the crystals obtained at the time t=0 min were completely in the β-form and the peaks were compatible with those shown in the literature[9,10].

Fig.1.Variation of ultrasonic velocity versus time for the crystals obtained in:a)pure media,and in the presence of b)50 μl·L-1oleic acid c)100 μl·L-1oleic acid d)250 μl·L-1oleic acid.

Fig.2.XRD results of the crystals obtained(a)in pure media and(b)in the presence of different oleic acid concentrations.

XRD analysis of the sample taken at the 30th minute of the transformation showed α characteristic peaks as well as β characteristic peaks.Compared to the results for crystals obtained at the time t=0 min,the characteristic β-glycine peak intensity decreased and the new peaks at 20°and 29°,which are the characteristic peaks of α-glycine,appeared.During the subsequent stages of the transformation process,the β peaks disappeared and the structure became fully α-form.This process was complete at t=45 min for glycine obtained in pure media.The follow-up of the transformation process performed by XRD analysis was in one-to-one concurrence with the follow-up with ultrasonic velocity measurements.Taking that into consideration,the completion of the transformation time for the crystals obtained at different oleic acid concentrations was determined from velocity measurement curves;the XRD results of the samples taken during these times are shown in the Fig.2(b).

The XRD profiles of all crystals obtained in the presence of oleic acid present the typical peaks of α-glycine;however,their peak intensities changed.The characteristic peak intensities of α-glycine increased as the concentration of oleic acid increased.This increase in intensity could result from the incorporation of additive molecules within the crystal lattice.In this circumstance,internal strains and structural imperfections occur in the individual lattice symmetry.Hereby,shifts and changes take place when the additive is present compared to those in the pure media[11].

3.3.SEM analysis

To determine the effects of oleic acid on glycine morphology,the SEM images of the glycine crystals with and without oleic acid contained are shown in the Fig.3.In agreement with the existing literature reports,the crystals obtained in pure media were prismatic with a regular rod shape and a smooth surface[8,12].

The SEM images show that both the length and the width of the crystals containing additive changed compared to those in pure media.The crystals obtained in the presence of 50 μl·L-1oleic acid shortened lengthwise,and the crystals gained volume due to increases in their width.The crystals grew on each other,leading to formation of crystal aggregates.The same situation was also observed for crystals in the presence of 100 μl·L-1oleic acid,and shorter crystals were obtained.As the crystals grew on each other,more dense crystal aggregates were formed compared with those in 50 μl·L-1additive media.As the crystal aggregates were broken by the hydrodynamic conditions of the media,partial deformations were observed on the crystal surface.Crystals retained their rod form despite shortening and increase in volume.The crystals obtained in the presence of 250 μl·L-1oleic acid completely lost the rod form.The crystals obtained were similar in appearance and were uniformly stable.The aggregates formed during the phase transformation had weaker bonding compared with those at 50 and 100 μl·L-1and were easily dispersed by the hydrodynamic conditions of the media.The morphology of the glycine crystals,which was clearly visible in the SEM images,varied depending upon the increase in oleic acid concentration.This change occurred ascrystals acquired a different form depending on the change in the length to width ratio of the crystals.

3.4.Morphology analysis

The morphological analysis was performed detailed using a Morphologi G3 instrument to better demonstrate the effect of oleic acid on glycine morphology,and to describe the particle shape parameters quantitatively. Firstly, particle size distributions determined by considering the circle equivalent(CE)diameter,the diameter of a circle with the same area as the particle,of α-glycine obtained by the completion of the phase transformation in pure media and in the presence of 250 μl·L-1oleic acid are shown in Fig.4.Whereas the D(0.5)value of α-glycine crystals obtained in pure media was 131.6 μm,this value was 68.68 μm in the presence of 250 μl·L-1oleic acid.The particle distributions for the crystals obtained in both media showed a normal distribution;no bimodal distribution was observed.The addition of oleic acid to media resulted in a narrow distribution.

To obtain more information about the shape of the crystals and to quantify the shape of the crystals,the particle shape factors such as circularity,elongation,and convexity were determined.Circularity is the ratio of the perimeter of a circle with the same area as the particle divided by the perimeter to the actual particle image, and elongation is determined as [1-width/length]. Both circularity and elongation values range from0 to 1: the circularity of a perfect circle is 1,whereas this value for a narrow rod is closer to 0.The elongation value of a circle is 0 whereas a rod has a high elongation.The other shape factor,convexity,is calculated by dividing the convex hull perimeter by the actual particle perimeter.The convexity of a smooth shape is 1,whereas the convexity of a very irregular object is closer to 0[13–15].Circularity,elongation,and convexity values of the crystals are given in Fig.4.

Fig.3.The SEM images of the glycine crystals obtained in:(a)in pure media,and in the presence of(b)50 μl·L-1(c)100 μl·L-1(d)250 μl·L-1oleic acid concentrations.

Fig.4.The particle size distribution of the glycine crystals obtained (a) and variation of the crystal shape factor values of the crystals obtained (b) in puremedia and in the presence of 250 μl·L-1additive.

The high sensitivity (HS) circularity value of the crystals produced in pure media was 0.552, but this value was 0.678 in the presence of oleic acid. This was an indication that the shape of the crystals transformed to a more rounded form. The decrease of the elongation value with the additive supported this change. The convexity value was determined to be about 0.960 for both media.Aspect ratios obtained by dividing the width of the crystal by its length were also calculated to determine the agglomeration tendency of the crystals.The aspect ratio mean value was 0.483 in pure media increased to 0.649 with the addition of oleic acid to the media,indicating that agglomeration of the crystals increased in terms of shape analysis.The images obtained from SEM analysis support this view.

3.5.TG/DTG and DSC analyses

TG and thermogravimetric derivative(DTG)curves showing the thermal decomposition patterns of α-glycine crystals obtained in pure media and in the presence of 250 μl·L-1oleic acid at different heating rates are shown in Fig.5.

Examining the TG and DTG curves for the crystals obtained in pure media,it was observed that thermal decomposition of α-glycine crystals can usually be divided into two steps.These steps were related to the decomposition of the amino group and carboxyl group in the glycine.In the first step,the mass losses for 5,10,and 20 °C·min-1heating rates were the same,and almost 50% were in the temperature range 245 to 300 °C,253 to 326 °C,and 280 to 340 °C,respectively.After amino group decomposition,the decomposition of carboxyl group for 5,10,and 20 °C·min-1heating rates started to occur between 316 and 460°C,326 and 520 °C,and 359 and 535 °C,respectively.The mass losses were almost 30%.The results obtained were consistent with previously published studies[16].The characteristic decomposition temperatures such as initial temperature,Ti,maximum peak temperature,Tmax,and final temperature,Tf,for the crystals obtained in pure and impure media are given in Table 1.

Fig.5.(a)TGA and(b)DTG curves of glycine obtained in pure media;(c)TGA and(b)DTG curves of glycine obtained in 250 μl·L-1oleic acid.

For each heating rate,TG and DTG curves had the same shapes,but the increase in heating rate from 5 to 20°C·min-1caused the decomposition temperature to shift higher. The maximum peak temperature corresponding to maximum mass loss rate for 5,10,and 20 °C·min-1heating rates was determined as281,286,and 305°C,respectively.This shifting was related to thermal lag.In one published study,the decomposition of the samples was delayed by the decrease of heat transfer efficiency at higher temperature[17].In TG and DTG curves for the crystals obtained in 250 μl·L-1oleic acid,the mass loss increased slightly.For instance, the total solid residue at 800°C was20.8%and 19.6%for crystals in the absence and presence of oleic acid,respectively.The higher total mass loss in additive media could be related to the oleic acid content of the samples.Comparing the DTG curves of the crystals obtained in the absence of additive, the maximum peak temperature increased in the presence of additive for each heating rate.Whereas the peak temperature was 286 °C for pure media,this value was 294 °C due to the oleic acid adsorbed on the crystal surface.

In addition to the TG/DTG analyses,the DSC analysis was performed for the crystals obtained in the absence and the presence of 250 μl·L-1oleic acid and the results were given in Fig.6.

Table 1Characteristic decomposition temperatures(°C)of the crystals obtained in pure and impure media for each heating rate

According to the DSC curve for the crystals obtained in pure media,the first peak observed at 251.2 was responsible for the presence of trace amount of β polymorph in the crystal.The second peak,around 255 °C was due to the melting point of the α-polymorph.Compared to the pure media,the presence of oleic acid in crystallization media led to a slight shift in DSC peaks toward higher temperature.

Fig.6.The DSC curves of the crystals obtained in pure and impure media.

Table 2The general kinetic model equations for FWO,KAS,Starink,and Tang models

3.6.Kinetic analysis

To investigate the effects of oleic acid on thermal decomposition behavior and kinetics of α-glycine crystals,the activation energy,an important kinetic parameter,was calculated using different kinetic models for the crystals.The fundamental kinetic equation for solid state thermal decomposition is generally expressed as in Eq.(1):

where t,k(T)and f(x)represent the reaction time,the reaction rate constant,and the reaction model,respectively.The rate constant,k(T)can be defined by the Arrhenius equation as in Eq.(2).

where A is the pre-exponential or frequency factor(min-1),E is the activation energy(kJ·mol-1),T is the absolute temperature(K),and R is the ideal gas constant(8.314 J·mol-1·K-1).

The conversion of the decomposed sample or mass loss fraction x is shown in Eq.(3):

where W0andWfare the initial and final sample mass,respectively,and Wtis the sample mass at time t.For a constant heating rate,β,(°C·min-1)can be defined as

For non-isothermal analysis,by substituting Eq.(4)into Eq.(1),the equation is given by Eq.(5).

By integrating the Eqs.(5),(6)is obtained:

where p(u)and g(x)represent the temperature integral and the integrated reaction model,respectively.The solution of this equation can be obtained using various kinetic methods that include different approaches.In the present study,the isoconversional method was used to calculate the activation energy because of the practical solution,not requiring the assumption of specific reaction models and enabling determination of the effect of activation energy on conversion degree.Certain kinetic models based on isoconversional method are proposed to calculate the activation energy.Evaluations were done based on the Flynn-Wall-Ozawa (FWO), Kissinger-Akahira-Sunose (KAS), Starink, and Tang models [18–21]. The general model equations and the plotting methods are shown in Table 2.

The activation energy can be determined from the slope of plotted lines for each model.In the present study,the kinetic parameter for the crystals obtained in pure and impure media was determined for the main decomposition region in the temperature range 245 to 340°C and 260 to 353 °C,respectively.The calculated values of activation energy and correlation coefficient(R2)in the conversion range between 0.1 and 0.9 with an interval 0.1 are given in Table 3.The calculated activation energy values as a function of conversion for the crystals obtained in pure media were very close each other,with high R2values.This result showed that decomposition of the crystals obtained in pure media had similar kinetic behavior for all conversion values,did not include complex reactions,and were attributable only to amino group decomposition.The average activation energy of the crystals obtained inpure media was90.6,89.6,90.5,and90.6kJ·mol-1using the FWO,KAS,Starink,and Tang models,respectively.

Table 3The activation energy(E,kJ·mol-1)and R2values of the crystals obtained with and without oleic acid using the KAS,FWO,Starink,and Tang models

Fig.7.Activation energy distribution based on conversion for crystals obtained in pure media in the presence of 250 μl·L-1additive.

These calculated values were nearly the same for all models studied;this was an indication of the accuracy and reliability of the results.With the addition of the oleic acid to the media,the calculated activation energy values increased significantly.In other words,the minimum energy requirement of the crystals obtained in 250 μl·L-1oleic acid for starting degradation was augmented.The average activation energy of the crystals obtained in additive media was 155.4,153.8,154.9,and 154.0kJ·mol-1in the FWO, KAS, Starink, and Tang models, respectively.As shown in Fig. 7,while the calculated activation energy for pure media was almost same for whole conversion degree,this trend changed with different conversion value for additive media.

The fluctuation observed in activation energy was attributable to the sample content and it showed that the reaction mechanism was not same.Compared to the pure media,the decomposition of the glycine crystals containing oleic acid was more difficult, and this process included a more complex interaction. This indicated that oleic acid affected the decomposition of the crystals and that oleic acid was adsorbed on the crystal surface.Furthermore,the results obtained by kinetic analysis were in good agreement with DTG results.The presence of oleic acid in the media resulted in a shift to higher temperature in the both maximum peak temperatures,and increased values of the calculated activation energy.

3.7.FTIR and Elemental analyses

The functional groups of the crystals obtained in pure media and in the presence of 250 μl·L-1oleic acid as determined by means of FTIR analysis;the spectra are shown in Fig.8.The obtained spectrum for pure media was in good agreement with published results[5,22,23].

The absorbance peak between 2200 and 3300 cm-1and the peak seen at 1499 cm-1were indicative of N–H stretching vibrations of NH3+group.The absorbance peaks at 1599 and 1406 cm-1were responsible for COO-group asymmetric and symmetric stretching,respectively.The peak that appeared at 1444 cm-1can be attributed to the bending vibration of the C--H bonds of the CH2group.Moreover,the other peaks at 891 and 693 cm-1were attributable to C--C symmetric stretching and O--C=O bending vibrations,respectively.Compared to FTIR spectra obtained in the pure media,the slight shifting behavior and an increase in intensity was observed.In addition,the specific peak at 1718 cm-1peak indicating oleic acid was observed in the FTIR spectrum obtained in the additive media.The peak at 1285 cm-1was also related to the C--O stretching of carboxylic group in oleic acid.Moreover,the C--H stretching vibration band in the double bond(C=C--H)at 3009 cm-1indicated the presence of oleic acid[23].These peaks indicated the presence of oleic acid adsorbed on the crystal surface.The crystals obtained were further characterized by elemental analysis.The elemental composition of the crystals obtained in pure media was 31.99%C,6.69%H,and 18.55%N.When oleic acid was added to the media,this composition changed slightly.The crystals obtained in 250 μl·L-1oleic acid consisted of 32.38%C,6.73%H,and 18.47%N.The presence of the additive caused an increase in carbon and hydrogen content but a decrease in nitrogen content.In addition to the elemental analysis,the nitrogen quantification of the crystal end products were determined by Kjeldahl analysis using Gerhardt VAPODEST?20.The nitrogen contents of crystals obtained in pure media and in the presence of additive media were 19.6%and 19.4%,respectively.This result was consistent with both elemental and FTIR analyses,confirming that oleic acid had adsorbed on the glycine crystal surface.

Fig.8.FTIR spectra of the crystals obtained in pure media(a)and impure media(b).

4.Conclusions

The present study revealed that oleic acid used as an additive had significant effects on the polymorphic phase transformation of glycine from the β to the α form.The presence of oleic acid in crystallization media decreased the transformation rate,and a higher additive concentration tended to extend the transformation time.Compared to pure media,the existence of oleic acid affected the crystal morphology in terms of both shapes and sizes of the crystals.Changes to the length and width of the crystals occurred in the presence of oleic acid:this additive enhanced the narrow crystal-size distribution.From the morphological point of view,the crystals obtained in pure media had a more elongated shape.In comparison,the circularity value of the crystals with the presence of oleic acid increased from 0.552 to 0.678 as the additive concentration increased.When the crystals were characterized by TG/DTG analysis,the value of the activation energy calculated using KAS,FWO,Starink,and Tang kinetic models for crystals obtained in pure media was lower than that for crystals obtained in oleic acid media.The increase in activation energy was attributed to the adsorption of additive on the crystal surface.The existence of oleic acid on the crystal was proven by FTIR,elemental,and Kjeldahl analyses.