Optimal design of crystallization processes for the recovery of a slow

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Optimal design of crystallization processes for the recovery of a slow nucleating sugar with a complex chemical equilibrium in aqueous solution: the case of lactose Elena Simone, Arwen Tyler, Daniel Kuah, Xiaofan Bao, Michael Ries, and Daniel Baker Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.8b00323 • Publication Date (Web): 09 Jan 2019 Downloaded from http://pubs.acs.org on January 9, 2019

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Organic Process Research & Development

Optimal design of crystallization processes for the recovery of a slow nucleating sugar with a complex chemical equilibrium in aqueous solution: the case of lactose

Elena Simone1*, Arwen I.I. Tyler1, Daniel Kuah1, Xiaofan Bao1, Michael E. Ries2, Daniel Baker2 1

Food Colloids and Processing Group, School of Food Science and Nutrition, University of Leeds, LS2 9JT, Leeds, UK 2

School of Physics and Astronomy, University of Leeds, LS2 9JT, Leeds, UK

*Corresponding author. Email: [email protected]. Telephone number: +44 (0) 113 343 1424.

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Table of Content


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Abstract Lactose is the major carbohydrate in milk and, similarly to other sugars, it can exist as two anomers in solution, the α and β forms, with a ratio depending on factors including temperature and pH (mutarotation equilibrium). Lactose is extracted from whey mostly to prevent environmental pollution. In fact, the presence of this sugar can contribute to a dramatic increase in the biological oxygen demand (BOD) of whey, making its direct disposal potentially dangerous for the environment. However, preserving our ecosystem is not the only reason why lactose is recovered. Purified lactose is, in fact, a high value product, commonly used as an excipient in pharmaceutical formulations and as a carrier in dry powder inhalers. Despite the increasing interest that lactose crystallization has recently received, a full understanding of this process is still missing, particularly the link between the process parameters of the crystallization step and the properties of the final product in terms of crystalline structure, purity and particle size and shape distribution. This work is, to the authors’ knowledge, the most comprehensive study of lactose crystallization, exploring cooling and anti-solvent operations, for the determination of the effect of several operative conditions on: (i) the kinetics of nucleation, growth and agglomeration; (ii) the yield of lactose recovery from solution; (iii) the final crystal size and shape distributions; and (iv) the purity of the obtained crystals.

Keywords: Lactose, crystallization, anti-solvent, anomeric purity, polymorphs



1. Introduction Lactose is the major carbohydrate in milk and it is derived from whey, the liquid remaining after milk has been curdled and strained during the production of cheese and yogurt. 1-5 This sugar is widely used as a food supplement or pharmaceutical excipient.6 In order to obtain purified lactose the whey is first concentrated by water evaporation and then lactose is precipitated via cooling crystallization. Many factors can affect the latter operation including, in particular, the complex equilibrium of lactose molecules in solution, the existence of multiple crystal structures (polymorphs) for this sugar and the slow kinetics of primary nucleation and growth for some of the lactose polymorphs. The molecules of lactose can exist in solution in the form of two anomers, the α and β. The change from one anomer to the other is called mutarotation and the equilibrium point for this conversion depends on the pH, temperature, presence of impurities as well as solvent composition.7-10 Both anomers can precipitate from solution in different crystalline structures as shown in Figure 1. 11,12

Figure 1: Mutarotation equilibrium and possible crystal structures of lactose.


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The lactose crystals normally precipitated from water at ambient temperature are α-lactose monohydrate. Because of its stability, this form is the most commonly used as pharmaceutical excipient or food supplement. β-lactose crystals can be obtained in water at a temperature above 93 ⁰C. This crystallization behaviour is due to the different solubility of β-lactose and α-lactose monohydrate in water. In fact, the β-lactose is more soluble at ambient temperature but its solubility is not as much affected by temperature as the α-lactose monohydrate; therefore, at high temperature it becomes the less soluble form, which is easier to nucleate.13,14 In terms of morphology the α-lactose monohydrate crystals are characterized by a typical tomahawk shape, while β-lactose crystals are normally needle-like with high aspect ratio.15-17 Lactose can exist also as anhydrous α-lactose crystals obtained via dehydration of the α-lactose monohydrate form18 or in amorphous form.19 . Because of the mutarotation equilibrium both types of anomers are present during the nucleation and growth of a specific crystal structure. This can affect the purity of the precipitated crystals;20,21 in fact, even the commercially available α-lactose monohydrate normally contains 1-5% w/w of β-lactose incorporated in the crystal structure as a solid solution. Furthermore, the α-lactose monohydrate form is characterized by slow kinetics of growth and nucleation. 1,3,5 For these reasons it is very difficult and time-consuming for dairy industries to achieve a high yield of recovery and to obtain large crystals with a narrow size distribution and consistent purity and crystal structure. Different crystallization conditions (e.g., temperature, supersaturation, crystallizer type) were found to greatly influence nucleation rate, growth and properties of lactose crystals.7,8,10,17,19,2326

Most of the work conducted on lactose consists of seeded cooling crystallization experiments

while in situ nucleation is rarely investigated because of the very slow kinetics of primary nucleation. Furthermore, in some cases the induction time is not measured in situ23 but via offline sampling and optical measurements;8,9 such sampling can be a source of error in the 5 ACS Paragon Plus Environment


measurement of induction time. The effect of the kinetic of mutarotation equilibrium on the rates of nucleation and growth of lactose polymorphs was also investigated.8 It was found that despite having a slow kinetic the mutarotation equilibrium is not a rate-limiting factor for the growth and nucleation of lactose crystals. However, the β-lactose molecules present in solution can act as both nucleation and growth inhibitors for the α-lactose monohydrate crystals.21,22 This can explain the very slow kinetics of primary and secondary nucleation as well as growth for this specific crystal structure, although the exact mechanism is still not clear. Recently the use of ultrasound (sono-crystallization) has been found to increase the primary nucleation rate and reduce the induction time for lactose crystallization in both pure water and mixtures of water and an anti-solvent, with higher recovery yields achieved (above 70% in pure water).27-32 However, sono-crystallization was found to induce the precipitation and growth of needle like crystals instead of the characteristic tomahawk shaped α-lactose monohydrate ones.29,31 Furthermore, this technique can be difficult to control because of the impracticality of obtaining a uniform ultrasound field throughout a large industrial scale crystallizer.6 Anti-solvent crystallization is another viable option to increase the recovery yield of lactose.13,30,31,33 This technique is rarely used in the dairy industry because of the need of an organic solvent that has to be subsequently separated and recovered from the water solution. However, anti-solvent crystallization presents several advantages over both cooling and evaporation crystallization processes.34,35 In fact, it can be conducted at ambient temperature, with minimal heat induced degradation of the solution and it is less energy-intensive than evaporative crystallization.13,14 In this work, both seeded cooling and anti-solvent experiments were conducted to determine the effect of process parameters on the characteristic properties of lactose crystals, including: morphology, size distribution, level of agglomeration, crystal structure and purity.


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Furthermore, the effect of such conditions on the overall recovery yield were evaluated. To the authors’ knowledge, this work is the most comprehensive study conducted on lactose crystallization, looking at all the crystal parameters that can affect product quality and using two different, highly controlled crystallization techniques. The outcomes of the experiments shown in the paper will allow a more efficient design of the crystallization step for lactose or other compounds with a similar complex solution equilibrium and polymorphism.

2. Materials and Methods 2.1. Crystallization set-up and experiments Acetone from VWR International UK Ltd., extra pure α-lactose monohydrate from Fisher Scientific UK Ltd. and distilled water were used for the experiments carried out in this work. The α-lactose monohydrate crystals were used without any further purification both to prepare the lactose solutions for all the performed experiments and as seed crystals for the seeded experiments. Lactose crystals were produced in a 500 mL jacketed vessel equipped with overhead stirring (PTFE pitch blade impeller) as shown in Figure 2. The temperature inside the vessel was controlled using a PT-100 temperature probe connected to a Huber Ministat 230 thermoregulator (Huber UK & Ireland Ltd., Derby, UK). An Optek C4000 turbidity probe (Optek-Danulat GmbH, Essen, Germany) was used to monitor the evolution of all the crystallization experiments and a Watson-Marlow 101U peristaltic pump (Watson-Marlow Ltd., Falmouth, UK) was used for the anti-solvent addition experiments. Lactose crystals were produced by both anti-solvent addition and seeded cooling crystallization. Preliminary experiments with in situ nucleation were carried out but the kinetics of primary nucleation were found to be very slow: solutions saturated at 50⁰C and left at 5⁰C 7 ACS Paragon Plus Environment


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did not show any sign of crystals after 2-3 days of continuous stirring. Therefore, this crystallization strategy was not further explored.

Figure 2: Schematic of the rig used for the crystallization experiments.

For the seeded crystallization experiments α-lactose monohydrate was dissolved in 500 mL distilled water in the jacketed reactor at a temperature that was 10⁰C higher than the saturation temperature. Two saturation temperatures, 50 and 55 °C (corresponding to about 45 and 55 g of lactose in 100 g of water) were used for this set of experiment. The solubility of α-lactose monohydrate was calculated using the following equation:8,14 𝑔

𝑆𝑙𝑎𝑐𝑡(100𝑔 𝑠𝑜𝑙𝑣𝑒𝑛𝑡) = 0.0151𝑇2 ― 0.1256𝑇 + 14.081 ,

(1)

where 𝑇 is the temperature expressed in C. After complete dissolution of the solid material, the hot clear solution was filtered using a vacuum filtration system in order to remove any undissolved impurities or dust.


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Figure 3: (a) Example of a temperature profile used for seeded cooling crystallization experiments; (b) Typical temperature profile used for the anti-solvent crystallization experiments.

After that, the solution was cooled down to a temperature 4.5 C below the saturation temperature and α-lactose monohydrate seed crystals were added to the solution. The temperature was then decreased linearly down to 6 C (see Figure 3a). The equilibrium ratio between β and α molecules at such temperature is around 1.62, corresponding to around 62%



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of β-lactose molecules in solution. The ratio between β and α-lactose molecules in water as a function of temperature can be expressed with the following equation:8 𝛽 ― 𝑙𝑎𝑐𝑡𝑜𝑠𝑒 𝛼 ― 𝑙𝑎𝑐𝑡𝑜𝑠𝑒

(2)

= ―0.0024𝑇 + 1.6353

where 𝑇 is the temperature expressed in C. It is worth noticing that, according to equation 2, β molecules are prevalent in water solution and during the cooling profile for seeded experiments their amount only changed from around 60.4-60.6% to 61.8% of the total dissolved lactose. After seeding crystals were left in suspension for 23 hrs, with overhead stirring at 400 rpm. After that they were filtered, washed and dried at room temperature. The recovery yield of each experiment of different conditions was calculated from the mass of dry lactose crystals recovered. Recovery yields of different operating conditions were compared. The total lactose crystal yield (Y) was calculated using equation (3): 𝑌 (%) =

𝑀𝑎𝑠𝑠 𝑜𝑓 𝑙𝑎𝑐𝑡𝑜𝑠𝑒 𝑟𝑒𝑐𝑜𝑣𝑒𝑟𝑒𝑑 (𝑔) 𝑀𝑎𝑠𝑠 𝑜𝑓 𝑡𝑜𝑡𝑎𝑙 𝑙𝑎𝑐𝑡𝑜𝑠𝑒 𝑖𝑛 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 (𝑔)

(3)

with the mass of total lactose in solution including both the mass of lactose added initially and the lactose seed crystals. For the seeded cooling crystallization experiments the different operating parameters tested were: (i) initial lactose solution concentration; (ii) amount of seed crystals; (iii) cooling rate; and (iv) stirring rate. For the anti-solvent crystallization experiments, an initial clear aqueous solution of lactose was prepared with the same methodology described for the seeded cooling crystallizations. The initial volume of such aqueous solution was calculated based on a total final volume (solvent plus anti-solvent) of 500 mL. The clear solution was then cooled down to the set final


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temperature and kept for 2 hrs to allow the solution to reach the mutarotation equilibrium between α and β lactose molecules (see Figure 3b).13 According to equation 2 the amount of β molecules in solution at equilibrium for the three chosen temperatures of 15, 25 and 35°C corresponded to respectively 61.5, 61.2 and 60.8% of the total dissolved lactose. After the equilibration time, the anti-solvent (acetone) was added. Two addition procedures were used: (1) sudden addition of all the anti-solvent; (2) linear addition using a peristaltic pump. The suspension was kept stirring for 2 hrs and the crystals were filtered, washed and dried at room temperature. The operating parameters tested for this set of experiments were: (1) Initial solute concentration of lactose in water; (2) w/w ratio solvent/antisolvent; (3) stirring rate; (4) temperature; and (5) flow rate addition. Furthermore, two seeded anti-solvent experiments were conducted. Seeds were added (a) at 4.5 C supersaturation (b) just before the anti-solvent addition. The suspensions were stirred for 2 hrs and then crystals were filtered, washed and dried at room temperature. During each experiments few drops of slurry were collected every 30 minutes and crystals and analysed by optical microscopy to check nucleation, growth and agglomeration. 2.2. Measurement of crystal size distributions All crystals obtained in solution were vacuum filtered, dried and studied with an optical microscope (Nikon Optiphot 200, Nikon UK Limited, Kingston Upon Thames, UK ) and a Malvern Mastersizer 3000 (Malvern Panalytical Ltd, Malvern, UK). Using a combination of both these techniques allowed a better qualitative characterization of the crystal size distribution, particularly for needle-shaped crystals or agglomerates, and a clearer understanding of the differences among analysed samples. For the analysis with the Malvern Mastersizer, samples were dispersed in a saturated solution of α-lactose monohydrate at room temperature for the measurement of crystal size distribution 11 ACS Paragon Plus Environment


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(CSD) with the Mastersizer. Each sample was analysed with the Mastersizer in triplicate and three repeats were performed for each measurement and averaged. Data analysis was carried out using the Mastersizer 3000 software (version v3.4) and Microsoft Excel 2016. The results were reported on a volume basis, so the crystal size was expressed by the volume mean diameter (VMD), which corresponds to the D [4, 3] statistic calculated by the Mastersizer 3000 software: 𝑛

𝐷[4,3] =

∑1𝐷4𝑖 ∙ 𝑉𝑖 𝑛

∑1𝐷3𝑖 ∙ 𝑉𝑖

,

(4)

where: 𝐷𝑖: size classes from Mastersizer, μm 𝑛: the total number of size classes 𝑉𝑖: the volume density of each size class, % 2.3. Attenuated total reflection (ATR) Fourier Transform Infrared (FTIR) spectroscopy for crystal structure determination The crystal structure of each sample was determined by attenuated total reflectance (ATR) Fourier Transform Infrared (FTIR) spectroscopy. The ATR-FTIR instrument used was a Nicolet iS 50 Spectrometer (Thermo Scientific) equipped with OMNIC software (version 7.0). Dry solid samples were analysed directly, without any preliminary preparation. A principal component analysis of the ATR-FTIR data within selected spectral regions (between 850-960, 1600-1700 and 3100-3600 cm-1), which include the most evident differences between lactose crystal structures, 11,12 was performed. Calculation were carried out using the pca function in Matlab R2015b. Data were mean centered and variance scaled before being processed. The Matlab pca function uses the singular value decomposition (SVD) algorithm and the principal


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component scores are in order of variance explained (the first score explains most variance, followed by the second score etc.). 2.4. X-ray scattering for crystal structure determination X-ray experiments for further structural analysis were carried out in-house and at Diamond Light Source (DLS, Didcot, UK). In-house, the small and wide angle X-ray scattering (SWAXS) camera setup (SAXSpace, Anton Paar, Austria) uses a line-shaped beam of Cu Kα radiation with a wavelength λ = 0.154 nm. All studied solid samples were filled into vacuumtight, 4 mm glass capillaries and measured at 25 °C. A Mythen X-ray detector (Dectris Ltd., Baden, Switzerland) system was used to record the 1D scattering patterns and the SAXStreat and SAXSQuant software (Anton Paar, Graz, Austria) were used to pre and post process the data. X-ray experiments at DLS were carried out on beamline I22. The synchrotron X-ray beam was tuned to a wavelength of 0.069 nm. The distance between the sample and detector was set at 2.2 m and the 2D powder diffraction patterns were recorded on a Pilatus 2M (Dectris Ltd) and Pilatus P3-2M-DLS-L (Silicon hybrid pixel detector, Dectris Ltd) detectors for the SAXS and WAXS patterns respectively. Diffraction images were analysed using the DAWN software.36 Only the WAXS area was used in this work; the obtained two dimensional WAXS patterns were radially integrated to give one-dimensional scattering intensity profiles. Such profiles were then compared to the x-ray powder diffraction patterns calculated with the Mercury software (version 3.9) using data from the Cambridge Crystallographic Data Centre (references LACTOS01 for α-monohydrate lactose, EYOCUQ01 for the stable α-lactose anhydrous and BLACTO02 for β-lactose).37,38 2.5. Measurement of crystal purity Crystal purity in terms of the ratio between α and β molecules within each crystalline sample was quantified using proton nuclear magnetic resonance.20,39 A Bruker Ultra-shield-plus 400 13 ACS Paragon Plus Environment


MHz NMR equipped with Bruker Topspin 1.5 software was used for the experiments. Dimethyl sulfoxide (DMSO)-d6 (d, 99.9%) with 0.05% v/v Trimethylsilane (TMS) purchased from Goss Scientific Instruments Ltd was used as the solvent to dissolve the lactose samples. This solvent was chosen because lactose presents a very slow mutarotation equilibrium in it. This allows a reliable measurement of the ratio between α/β molecules in the solid crystals, even after complete dissolution. Furthermore, the TMS present in the solvent can be used as reference when comparing the chemical shifts. Around 3-4 mg of solid sample was dissolved in 1 mL of solvent and measured within the following hour. The temperature was kept constant at 20⁰C and 1H spectra were recorded by taking 16-32 scans using a 90⁰ proton “zg” pulse sequence with a pulse length of 6.5 µs. The sweep width was set to 12.45 ppm with an acquisition time of 0.5 s. A Fourier transform was applied to the raw data to calculate the 1H spectrum in the Top Spin software (version 3.5). Spectra were manually phase corrected and a baseline correction was applied before peak integration. The ratio between the α and β anomers was calculated by dividing the areas of the two doublets corresponding to each molecule type: 6.3 and 6.65 ppm for α and β molecules respectively.20

3. Results and Discussion 3.1. Seeded cooling crystallization experiments The results and operating conditions for all the seeded cooling crystallization experiments are shown in Table 1 and Figure 4. Figure 4 also shows the initial size distribution of the seed crystals. Despite the long duration of each experiment (around 23 hrs) the recovery yield for all the runs was below 60%, ranging from 24 to 55%. This is likely due to the slow growth and secondary nucleation rates of α-lactose monohydrate in water. The literature shows that the kinetics of mutarotation is faster than the growth rate of α-lactose monohydrate, hence this 14 ACS Paragon Plus Environment

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equilibrium is not directly a limiting factor for crystal growth. However, mutarotation affects the concentration of molecules of β-lactose in solution (around 60% of all the molecules for the whole cooling profile) and this anomer has been found to inhibit the already slow growth of α-lactose monohydrate.21,22 Because of such slow growth kinetics, the cooling rate after seeding did not have a significant effect on either the VMD or the yield, as shown by comparing runs 2 and 4 (it should be noted that the final crystal size distributions, shown in Figure 4b, overlap). Table 1: Experimental conditions and recovery yield after 23 hrs for the seeded cooling crystallization experiments.

Run

Initial lactose concentration in solution (g lactose/100g water)

Stirring rate (rpm)

Seed (% w/w)

Cooling rate (⁰C/min)

Recovery yield

VMD (μm)

Seed crystals

/

/

/

/

/

71

1

45

400

0.1

-0.25

28%

130

2

45

400

1

-0.25

44%

83

3

45

400

2

-0.25

43%

87

4

45

400

1

-0.15

42%

82

5

55

400

1

-0.25

55%

78

6

45

500

0.1

-0.25

30%

149

7

45

600

0.1

-0.25

24%

110

The amount of seed crystals used had a more significant effect, which is evident from comparison of runs 1, 2 and 3. Increasing the amount of seeds from 0.1% to 1% w/w of the total lactose initially dissolved in solution generated an increase in yield from 28% to 44%. However, such improvement is mainly due to an increase in the rate of secondary nucleation rather than crystal growth as demonstrated by the reduction of VMD from 130 to 83 μm. Figure 4c also shows clearly the more significant crystal growth recorded for run 1, in comparison with runs 2 and 3. Nevertheless, a further increase in the amount of seeds up to 2% w/w of the total lactose initially dissolved in solution did not impact significantly on either recovery yield or VMD. Similarly to the cooling rate, increasing the speed of the overhead stirrer from 400 to 15 ACS Paragon Plus Environment


600rpm did not affect the process. The recovery yield remained between 24 and 30% while the final VMD did not change significantly, as it is also shown in Figure 4d where the final crystal size distributions for runs 1, 6 and 7 overlap. A more significant increase in the recovery yield was observed when increasing the initial lactose concentration of the clear solution from 45 to 55 g/100 g water (run 2 compared to run 5).

Figure 4: Crystal size distributions from Malvern Mastersizer for lactose samples obtained by seeding cooling crystallization at (a) different initial lactose concentration in water; (b) different cooling rates; (c) different amount of seeds (as a weight fraction of the dissolved lactose) and (d) different stirring rates.

The amount of recovered lactose increased from 44 to 55% but the final crystal size distributions did not significantly differ, as shown in Figure 4a. This is due to preferential 16 ACS Paragon Plus Environment

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secondary nucleation instead of growth as the initial dissolved lactose concentration increases (similarly to the effect of increasing amount of seed crystals). Crystals of α-lactose monohydrate with characteristic tomahawk morphology were obtained in all experiments. In fact, despite the presence of both anomers in solution, only the α-lactose monohydrate is saturated enough in water to nucleate and grow.

Figure 5: Absorbance signal from the turbidity probe during seeded cooling crystallization experiments at (a) different initial solution concentration but same seed amount (0.1% w/w of the total dissolved lactose) and cooling rate (-0.25 ⁰C/min); (b) different initial solution concetration but same amount of seeds (1% w/w of the total dissolved lactose) and cooling rate (-0.25 ⁰C/min); (c) different amounts of seed crystals at the same initial solution concentration (45 g/100 g water) and cooling rate (-0.25 ⁰C/min); (d) different



cooling rates but same amount of seeds (1% w/w of the total dissolved lactose) and initial solution concentration (45g/100 g water).

Some agglomerates were identified at the end of runs 2 to 5 where higher recovery yields were achieved. This is due to the large amount of crystals generated from secondary nucleation, which increases the chances of crystal-to-crystal collision and particle aggregation. A turbidity probe was used to monitor the amount of crystals in suspension during the seeded crystallization experiments and to qualitatively estimate differences in growth and secondary nucleation kinetics. Figure 5 shows the trends for the absorbance recorded by the turbidity probe during each experiment: the gradient of each curve is related to both secondary nucleation and growth kinetics. The four curves of Figure 5a and 5b show the effect of the initial lactose concentration; as expected, an increase in initial lactose concentration determines a significant increase in secondary nucleation and growth rates. Figure 5c shows the absorbance trends for different amounts of seed crystals added. An increase from 0.1% to 1% w/w determines a significant increase in the gradient of the absorbance curves, due to an increase of the secondary nucleation rate. However, increasing the seed crystals amount from 1% to 2% w/w of the total solid initially dissolved in solution did not affect the absorbance curve significantly. This is in accordance with the crystal size distributions (CSDs) measured by laser diffraction as well as the values of the final recovery yield. It is worth noticing that all the parameters tested for seeded experiments have a direct effect on the rates of growth and secondary nucleation. Nevertheless, only the cooling rate could also affect the rate of mutarotation equilibrium. The experimental results show that increasing the cooling rate from -0.15 to -0.25 C/min did not significantly affect either the CSD or the final recovery yield. Most likely the growth and secondary nucleation rates of α-lactose monohydrate are slower than both the used cooling rates and the kinetics of mutarotation. Hence, no significant effect could be observed by changing these two parameters. 18 ACS Paragon Plus Environment

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3.2. Anti-solvent crystallization experiments Table 2 shows the experimental conditions used for the anti-solvent experiments performed. Acetone was used as the solvent and the effect of the following parameters was tested: (1) initial concentration of lactose in water; (2) ratio acetone/water at the end of the experiments; (3) flow rate of acetone in the acqueous solution of lactose; (3) stirring rate; (4) temperature of the aqueous solution; (5) seeded versus unseeded experiments. The main difference observed in the anti-solvent experiments in comparison with the seeded ones is the formation of crystals with a high aspect ratio needle-like morphology, with a large tendency towards agglomeration rather than the tomahawk morphology typical of α-lactose monohydrate crystals.


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Table 2: Experimental conditions and recovery yield after 2 hrs for the anti-solvent experiments.

Run

8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

Initial lactose concentration in solution (g lactose/100 g water) 15 20 25 30 15 15 15 15 15 15 15 15 25 25 25 25 25 25 25 25

Mass ratio of acetone in the final solvent (w/w)

Flow rate of acetone (ml/min)

Stirring rate (rpm)

Temperature (⁰C)

Seed (% w/w)

Seeding temperature (⁰C)

Recovery yield

VMD (μm)

Crystal structure precipitated

60% 60% 60% 60% 50% 70% 80% 60% 60% 60% 60% 60% 60% 60% 50% 50% 60% 60% 60% 60%

/ / / / / / / / / / / / 5 10 5 10 5 10 5 5

400 400 400 400 400 400 400 500 600 700 400 400 400 400 400 400 400 400 400 400

15 15 15 15 15 15 15 15 15 15 25 35 6 6 6 6 15 15 6 6

/ / / / / / / / / / / / / / / / / / 1 1

/ / / / / / / / / / / / / / / / / / 27 6

59% 76% 82% 85% 2% 82% 81% 55% 68% 24% 48% 33% 90% 83% 75% 76% 81% 82% 84% 80%

232 248 233 322 n.a. 126 37 117 101 39 141 182 102 170 223 216 333 256 135 130

β-lactose + α-lactose monohydrate β-lactose + α-lactose monohydrate β-lactose + α-lactose monohydrate β-lactose + α-lactose monohydrate α-lactose monohydrate β-lactose + α-lactose monohydrate β-lactose + α-lactose monohydrate β-lactose + α-lactose monohydrate β-lactose + α-lactose monohydrate α-lactose monohydrate α-lactose monohydrate α-lactose monohydrate β-lactose + α-lactose monohydrate β-lactose + α-lactose monohydrate β-lactose + α-lactose monohydrate β-lactose + α-lactose monohydrate β-lactose + α-lactose monohydrate β-lactose + α-lactose monohydrate β-lactose + α-lactose monohydrate β-lactose + α-lactose monohydrate


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Figure 6 shows microscopic images of lactose crystals obtained from different experiments. The needle-like crystals obtained are not pure α-lactose monohydrate but mixtures of α-lactose monohydrate and β-lactose crystals in different amounts, as shown in more in detail by the ATR-FTIR and WAXS data of the next section. The precipitation of β-lactose in addition to the expected α-lactose monohydrate is due to the low solubility of this structure in acetone. After adding the antisolvent into the aqueous lactose solution, a high level of supersaturation for both anomers is generated, which allows the primary nucleation of both β-lactose and αlactose monohydrate.

(a)

(b)

(d)

(c)

Figure 6: (a) Crystals of β-lactose (agglomerates) obtained via anti-solvent addition with final concentration of acetone in the solvent of 60% w/w; (b) Crystals of β-lactose (agglomerates) obtained via anti-solvent addition at stirring rate of 600 rpm; (c) Crystals of α-lactose obtained via anti-solvent addition with final concentration of acetone in the solvent of 50% w/w; (d) Crystals of α-lactose obtained via seeded cooling crystallization of a solution at 45% w/w initial concentration of lactose.

In the case of antisolvent addition, the nucleation rates for both type of crystals are faster than the mutarotation equilibrium, which is even slower in mixtures of water and acetone compared 21 ACS Paragon Plus Environment


to pure water.40 Therefore, such equilibrium cannot sustain the conversion of β-lactose into αlactose molecules and, therefore, both β and α-lactose monohydrate crystals can precipitate. However, in runs 12, 17, 18 and 19 only α-lactose monohydrate crystals were obtained and in runs 13, 14 and 15 mixtures with a larger amount of α-lactose monohydrate than the β-lactose crystals were produced. In all cases the recovery yield was considerably lower compared to the runs in which β-lactose crystals were precipitated, suggesting that this latter form has faster kinetics of nucleation and growth compared to the α-lactose monohydrate in the solvents tested. Run 12 had the lowest percentage of acetone and initial dissolved lactose concentration used (50% w/w of the final solvent mass). In this case, therefore, a lower supersaturation was generated at the addition of the anti-solvent, which was not enough to allow the precipitation of β-lactose. In run 18 and 19 the anti-solvent was added at a higher temperatures (25 and 35 ⁰C), determining both lower supersaturation and a slightly higher initial concentration of α molecules in solution that might have favoured the nucleation and growth of α-lactose monohydrate. The relationship between stirring speed and the nucleated structure is not clear. Mixtures of β-lactose and α-lactose monohydrate were obtained at 400 and 600 rpm, whilst only α-lactose monohydrate was nucleated at 700 rpm and a mixture with mostly α-lactose monohydrate crystals was produced at 500 rpm. The poor quality of mixing at high stirring rate might have had a negative effect on the consistency of this set of experiments. In terms of lactose recovery, anti-solvent crystallization generated higher yields than seeded cooling experiments, with the exception of the few runs in which only α-lactose monohydrate was nucleated. Furthermore, experiments were considerably faster and a reasonable lactose recovery could be achieved in just 2 hrs. The data from laser diffraction also shows larger VMD values of the CSD (see penultimate column of Table 2) in comparison with the ones obtained with seeded cooling experiments. 22 ACS Paragon Plus Environment

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Figure 7: Crystal size distributions from Malvern Mastersizer for lactose samples obtained via anti-solvent addition at (a) different initial lactose concentration in water; (b) different amount of acetone in the final solution (w/w); (c) different operating temperatures and (d) different stirring rates.

However, this is probably due to the formation of large agglomerates of smaller needle-like crystals rather than on faster crystal growth, as shown in Figures 6a and 6b. Considering the higher recovery yield and the presence of a large number of small needle-like crystals at the end of each experiment it can be concluded that anti-solvent crystallization determines higher primary and secondary nucleation rates for β-lactose crystals compared to the seeded experiments.



Similarly to that observed in the seeded experiments, an increase in the initial lactose concentration resulted in a higher recovery yield as shown by comparing runs 9 and11. Larger crystals are also observed, as shown in in the CSD from laser diffraction of Figure 7a. Increasing the ratio of anti-solvent to lactose solution also improves the recovery yield and generates larger crystals. However, as shown in Figure 7b, 80% w/w acetone/lactose solution generated a bimodal CSD with a large amount of fine crystals precipitated because of the high supersaturation during the addition of acetone. Besides, large agglomerates formed during the experiment. Figure 7d shows the effect of stirring rate on the CSD of lactose crystals: higher stirring speeds generated smaller crystals but with a narrower distribution. In the case of run 16 (600 rpm) this is probably due to reduced crystal agglomeration generated by the high shear stress present in the system. In runs 15 and 17 a higher amount of α-lactose monohydrate crystals were nucleated, compared to the other runs. As previously observed, this crystal structure has a different morphology and slower kinetics of nucleation and growth compared to β-lactose. For these reasons the CSD for these two runs are narrower and have a lower VMD than run 14, in which 400 rpm was used as stirring rate. Finally, the effect of the solution temperature can be observed in Figure 7c. Increasing the temperature of the aqueous solution during anti-solvent addition determined nucleation of pure α-lactose monohydrate with narrow size distribution. The recovery yield after two hours was comparable with the much longer seeded experiments and crystals obtained in runs 18 and 19 were larger than the ones generated in runs 1-7. The higher temperature and the addition of an organic solvent had a positive effect on the growth rate of α-lactose monohydrate. This is due to a reduced solubility of lactose in acetone/water mixtures compared to pure water. It is also possible that the slightly lower initial concentration of molecules of the growth inhibitor β-


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lactose (61.2 and 60.8% of the total molecules in solution instead of 61.5% at 15°C) might have favoured the nucleation of the α-lactose monohydrate form. In runs 20 to 27 the anti-solvent was pumped at a constant rate into the lactose solution instead of being added instantaneously. Comparing runs 24-25 to run 10, which had the same initial lactose concentration, acetone/solution ratio and stirring rate, the recovery yield does not significantly change with the anti-solvent addition rates used. Figure 8a shows the final CSD obtained at the end of runs 20 to 23, for two different anti-solvent addition rates of 5 and 10 mL/min. It is not clear the effect of the anti-solvent addition rate on the final VMD of the obtained crystals. In runs 22-23 a final ratio acetone/solution of 50% and a temperature of 6 C were used. A lower recovery yield but higher VMD was achieved compared to runs 24-25, as shown in Figure 8b. This is due to lower supersaturation and therefore, lower nucleation rate for both β-lactose and α-lactose monohydrate, as also suggested by the formation of a lower number of fine crystals at the end of the runs. In runs 20-21 a lower temperature (6 C instead of 15 C) was used compared to runs 24-25. An increase in yield and a lower VMD was observed as a result of a higher supersaturation at nucleation and, therefore, higher nucleation rate. Figure 8c shows a comparison between the CSD obtained at the end of runs 20 and 24. The final VMD for 15 ⁰C is clearly higher than the one obtained at 6 ⁰C. An increase from 102 to 170 μm of the VMD was observed by increasing the flow rate from 5 to 10 mL/min at 60% acetone and 6 ⁰C (runs 20 and 21). However, when comparing runs 22 and 23 the VMD of the final CSD decreases when increasing the acetone flowrate. Additionally, only a small change in the VMD (around -3.2%) was observed between runs 22 and 23. The high level of agglomeration for these samples, which can affect the laser diffraction



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measurements, might be the reason for the lack of a clear trend for the VMD in these experiments. Finally, two seeded antisolvent experiments (runs 26-27) were carried out.

(a)

(b)

(d)

(c)

Figure 8: Crystal size distributions from Malvern Mastersizer for lactose samples obtained via anti-solvent addition at (a) different antisolvent addition rates runs 20 to 23; (b) different amount of acetone in the final solution (w/w), runs 20, 22; (c) different operating temperatures at antisolvent addition (runs 20 and 24) and (d) different seeding temperatures, runs 26 and 27.

Crystals of α-lactose monohydrate were used as seeds in order to promote growth of this structure. However, β-lactose crystals were nucleated during both experiments and only needle-like crystals were observed at the end of each run. This shows that primary nucleation of the β-lactose structure is possibly faster than both growth and secondary nucleation of the 26 ACS Paragon Plus Environment

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α-lactose monohydrate form. Modifying the temperature at which seed crystals were added from 6 to 27⁰C did not significantly affect either the recovery yield or the final VMD measured with laser diffraction, as shown in Figure 8d. 3.3. Crystal structure determination The structure of lactose samples obtained from both the seeded cooling and anti-solvent experiments was determined via ATR-FTIR spectroscopy and WAXS. Figure 9 shows the three ATR-FTIR regions used for the determination of the crystal structure of each lactose sample: (a) 850 – 960 cm-1; (b) 3100 – 3600 cm-1 and (c) 1600 – 1700 cm-1. These regions were chosen based on literature11,12 as well as a preliminary assessment with known samples. The characteristic peak of α-lactose anhydrous at 855 cm-1 was not identified in any of these samples and therefore all the crystals obtained were either α-lactose monohydrate, β-lactose or mixtures of the two forms. The β-lactose structure has a distinctive peak at 948 cm-1 which is not present in any of the other possible lactose structures.11,12 Furthermore, β-lactose crystals can be distinguished from the α-lactose monohydrate ones by the presence of a peaks at 892 cm-1 and 3447 cm-1. Characteristic peaks for the monohydrate α-lactose structure are at 916, 1654 and 3520 cm-1. Figures 9a to c show the ATR-FTIR spectra of selected samples in the chosen region. It is clear that the formed mixtures of α-lactose monohydrate and β-lactose crystals have different ratios of these two structures. In order to qualitatively compare all samples, principal component analysis was carried out on the data. Figure 9d shows the scores of the first principal component (PC1) plotted against the second principal component (PC2). A cluster with all the pure α-lactose monohydrate samples can be identified at values of the PC1 score < 30 and PC2 scores < 5. With increasing amount of β-lactose crystals in the samples both PC1 and PC2 scores increase. Samples with a high amount of this structure have positive PC1 scores as shown in Figure 9d. 27 ACS Paragon Plus Environment


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Run 8 Run 16 Run17 (α-lactose monohydrate) Run 13

(b)

(a) Run 8 Run 16 Run 17 (α-lactose monohydrate) Run 13

β

(c)

(d)

Figure 9: ATR-FTIR spectra of α and β lactose in the regions between (a) 850-960 cm-1; (b) 1600-1700 cm1

and (c) 3100-3600 cm-1. Scores of the first and second principal components for each analyzed sample are

shown in (d).

Data from X-ray scattering of selected samples are shown in Figure 10a, while Figure 10b shows the x-ray powder diffraction pattern calculated with the Mercury software, based on data from the literature.36-39 28 ACS Paragon Plus Environment

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All scattering plot has been normalized, with the maximum intensity of the scattered signal equal to 1.

β – lactose peak α-lactose monohydrate peak

(a)

β-lactose α-lactose anhydrous α-lactose monohydrate

(b)

Figure 10: (a) Wide angle x-ray scattering data for selected lactose samples obtained via anti-solvent addition crystallization (b) Predicted XRD powder patterns calculated with the Mercury software, references LACTOS01 for α-monohydrate lactose, EYOCUQ01 for the stable anhydrous α-lactose and BLACTO02 for β-lactose.



Such expedient is applied in order to take into account differences in peak intensity that might exist among samples, and that are due to different crystal packing within the capillaries (e.g., large agglomerates will pack differently inside the capillary compared to single crystals). Mixtures of both α-lactose monohydrate and β-lactose crystals shows the characteristic peaks of the β-lactose structure at 10.52⁰ and 20.97⁰ as well as the α-monohydrate peaks at 12.26, 16.42 and 19.86⁰. It is worth noting that none of the analysed samples presented any of the characteristic peak associated with the α-lactose anhydrate form, particularly the high intensity one at 19.44⁰. Slight irregular shifts and peak broadening (variable peak full width at half maximum, FWHM) can be noticed for most samples. This is most likely due to a distorsion of the crystalline structure generated by inclusions of β-lactose molecules in the α-lactose monohydrate structure and vice versa. The NMR results also confirm this, as shown in the next section. 3.4. Purity measurement The purity of each solid sample was evaluated using proton NMR. Dry solid samples were dissolved in DMSO, transferred into NMR tubes and analysed within an hour from their preparation. In DMSO, the mutarotation equilibrium is very slow and thus allows the quantification of the ratio between α and β-lactose molecules in the solid crystals produced.20 Figure 11 shows the typical 1H-NMR spectra obtained for lactose in DMSO; the region between 6 and 7ppm can be used for the identification and quantification of α and β molecules.


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Figure 11: 1H NMR spectrum of run 8 sample. The dried powder was fully dissolved in DMSO before being analyzed.

The protons of the hydroxyl group at carbon C1 have different chemical shifts for the two anomers and two doublets in such regions are associated with the α anomer (6.3 ppm) and the β anomer (6.65 ppm) as shown in the magnified box of Figure 11. Table 3 shows the results obtained from analysis of the NMR spectra. A higher amount of α-lactose molecules were identified in the samples obtained from seeded experiments (runs 1-7) and in runs 17-19 and 12. Such results are in accordance with the ATR-FTIR spectroscopy and WAXS data which identified only the α-lactose monohydrate crystal structure in these samples. It is worth noting that ATR-FTIR and WAXS could identify and quantify differences in the molecular arrangement within the solid phase but do not provide a direct quantification of the amount of the two different lactose molecules. As β-lactose molecules are present in solution during the formation of α-lactose monohydrate crystals, this anomer can be incorporated in the main crystal structure for up to 3-5% w/w. In fact, even for the pure α-lactose monohydrate crystals (runs 1-7) small amounts of β-molecules could be identified in the solid samples. 31 ACS Paragon Plus Environment


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Most samples obtained from anti-solvent crystallization experiments (runs 8-28) contained a higher amount of β-lactose molecules, most likely confined in the β-lactose crystals identified with ATR-FTIR spectroscopy as well as WAXS. The higher percentage of β-lactose molecules measured is 56% w/w, corresponding to run 1, while the total amount of this type of molecule for all the anti-solvent experiments ranges between 4 and 56%, without any particular trend related to the operating conditions. Table 3: Percentages of α and β molecules in the solid samples, quantified with proton NMR. Run Seeds 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

% α molecules (w/w)

% β molecules (w/w)

98% 97% 98% 100% 98% 100% 99% 99% 67% 47% 54% 57% 94% 88% 44% 84% 57% 96% 94% 96% 62% 56% 53% 62% 64% 63% 58% 53% 53%

2% 3% 2% 0% 2% 0% 1% 1% 33% 53% 46% 43% 6% 12% 56% 16% 43% 4% 6% 4% 38% 44% 47% 38% 36% 37% 42% 47% 47%

This indicates a general difficulty in controlling the outcome of anti-solvent crystallization experiments, probably because lactose crystals are produced via primary nucleation which is notoriously difficult to predict for polymorphic systems in particular.41 It is worth noticing that 32 ACS Paragon Plus Environment

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β-lactose crystals were always simultaneously nucleated in a physical mixture with α-lactose monohydrate particles, as identified by WAXS and ATR-FTIR. However, these solid samples presenting both β and α-monohydrate crystals were fully dissolved in DMSO before the NMR analysis. After dissolution it is not possible to distinguish between β molecules contained in the β-lactose solid crystals and the ones incorporated as impurity in the α-lactose monohydrate structure and vice versa. However, since β molecules were found to be incorporated in the pure monohydrate crystals during the seeded experiments, it is reasonable to assume that both solid structures can incorporate molecules of the other anomers as impurity. Such a hypothesis would also explain the presence of shifts and broadening in the x-ray scattering peaks, as a result of a distortion of the crystalline lattice caused by the presence of impurities.

4. Conclusions A systematic study of lactose crystallization under different operating conditions, taking into account the most important process parameters, has been carried out in this work. Different off-line and in-situ characterization techniques were used to analyse the produced crystals. Anti-solvent and seeded experiments were performed and the crystals were checked in terms of size, morphology and recovery yield, as well as crystal structure and anomeric purity. Seeded experiments allowed better control over the structure nucleated and the overall purity: only the α-lactose monohydrate crystal was precipitated and grown with a purity above 97% (only up to 3% w/w of β-lactose molecules). Furthermore, unimodal size distributions could be obtained with low levels of crystal agglomeration using seeded experiments. However, because of the slow kinetics of nucleation and growth of the α-lactose monohydrate crystals, the final recovery yield after 23 hrs of experiment was always below 50%. Anti-solvent crystallization processes allowed higher recovery yield (over 90%) with lower total batch time but with poor control



over the crystal structure nucleated and the final anomeric purity. In fact, both the α-lactose monohydrate and β-lactose crystals were precipitated. The initial solution concentration seems to be the most important factor affecting both seeded and anti-solvent crystallization. In fact, increasing the solution concentration generates a higher recovery yield of the lactose crystals, in particular the α-lactose monohydrate form for the seeded experiments and both α-lactose monohydrate and β-lactose crystals for the anti-solvent experiments. The stirring rate did not seem to significantly affect the recovery yield and final crystal size distribution of either seeded or anti-solvent experiments. It was found that the final ratio of solvent to anti-solvent have the strongest effect on the final recovery yield. Increasing the amount of acetone determines a higher yield of recovery but also lower crystal purity, with β-lactose crystals co-precipitating with the α-lactose monohydrate form.

Acknowledgements Professor Zoltan K. Nagy (Loughborough University) is acknowledged for the use of the ATRFTIR facilities in the Chemical Engineering Department at Loughborough University.

Declarations of interest: none

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