The crystallization of lysozyme in the system of ionic liquid [BMIm][BF4]‐water

Lysozyme crystallization was conducted in the ionic liquid (IL) 1‐butyl‐3‐methylimidizolium tetrafluoroborate ([BMIm][BF₄]) with different buffer/IL proportions. It was found that the addition of [BMIm][BF₄] could promote the crystallization process, during which more lager single crystals with controllable morphologies could be obtained due to the manageable crystal growth velocity. A probable explanation was proposed based on the influence of the ionic polarization and kinetics in the lysozyme crystallization. Moreover, the transform in coordination number and the relative growth rate of different crystal faces were discussed. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Uncertainties in crystallization of hen‐egg white lysozyme: reproducibility issue

The reproducibility of biomacromolecular crystallization (tetragonal and orthorhombic lysozyme crystals) was studied by monitoring the evolution of protein concentration during the crystallization process using Mach‐Zehnder interferometer. It was found that formation of both tetragonal and orthorhombic crystals exhibited poor reproducibility. When the crystallization occurred under isothermal conditions, the protein concentration in the solution varied differently in different experiments under identical conditions (for both types of crystals). Moreover, in the case of orthorhombic lysozyme crystallization (under either isothermal or thermal gradient conditions), it is clear that the crystals could not be always readily formed. When formation of tetragonal lysozyme crystals was conducted at a temperature gradient condition, however, the evolution of concentration was reproducible. The phenomena found in this study revealed that biomacromolecular crystallization can be uncertain, which is probably caused by the process of nucleation. Such uncertainties will be harmful for the efforts of screening crystallization conditions for biomacromolecules. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Sedimentation as a tool for crystallization from protein mixtures

Crystals from apoferritin which is an iron‐free form of protein ferritin were obtained from protein mixtures lysozyme/apoferritin using sedimentation under high gravity. Solution containing apoferritin at concentration as high as 5mg/ml in the presence of 25mg/ml lysozyme and overlaid on 5%(w/v) CdSO₄ in 0,2M/L NaAC, pH=5 still favors apoferritin crystal formation under normal gravity conditions, but at apoferritin concentrations <0,5mg/ml (∼1,14µM/L) in 25mg/ml (∼1,71mM/L) lysozyme only the sedimentation in a centrifuge appears to be useful for separating the apoferritin molecules from the mixture followed by apoferritin crystallization in the same system. The very high molecule number ratio (∼1:10³) of two proteins is used to stress on the observed effect. (© 2006 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Phase transformation of guanosine 5′‐monophosphate in drowning‐out crystallization: Comparison of experimental results with mathematical modeling

The phase transformation of Guanosine 5′‐Monophousphate (GMP) in drowning‐out crystallization using a batch system was experimentally monitored and mathematically modeled. The solid (amorphous and crystalline GMP hydrate) and liquid phases of the GMP products were simultaneously monitored using a video microscope, FT‐IR, and UV/Vis spectroscopy during the phase transformation. For the modeling, the phase transformation was assumed to occur via the simultaneous dissolution of amorphous GMP and growth of crystalline GMP hydrate in the solution. Based on a comparison of the experimental results and model predictions, both the dissolution and growth of the GMP solids were found to contribute competitively to the phase transformation. When varying the crystallization conditions, in this case the agitation speed and feed concentration, the phase transformation was significantly promoted when increasing the agitation speed, yet independent of the feed concentration. The simple mathematical model used for the GMP phase transformation was quite successful in describing the experimental results. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Effects of forced solution flow on lysozyme crystal growth

In order to study quantitatively the effects of forced solution on crystal growth, we designed a new set of experimental equipment, in particular, a microchannel mixer was used as crystallization container so that the consumption of protein samples was much reduced and thus an exact syringe pump could be used for precise control of the flow rates. Since the mixer's section was designed to be rectangular, the solution velocity in its center was steady and constant, and thus repeatable experiments were facilitated. Experimental results showed that the effects of forced solution on protein crystal growth were different under different levels of supersaturation, and new results were obtained for cases of high supersaturation. When the supersaturation is σ = 2.3, with increasing flow rates the growth rates of the lysozyme crystal's (110) face hardly change when the flow rates are lower than 1300 μm/s, and decrease quickly afterwards. When the flow rate reaches 2000 μm/s, the crystal nearly ceases to grow. When the supersaturation is σ = 2.7, with increasing flow rates the (110) face growth rates increase at the beginning then reach the maximum values at 1700 μm/s – 1900 μm/s and decrease afterwards, approaching zero or so when the flow rate reaches 12000 μm/s. The higher the supersaturation, the larger the flow rate at which the crystal ceases to grow. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Crystallization of potassium sulfate by cooling and salting‐out using 1‐propanol in a calorimetric reactor

A study was made on a isothermal process for the crystallization of potassium sulfate as an alternative to the cooling process. The process employs addition of 1‐propanol to aqueous salt solutions to achieve the “saltingout” of the K₂SO₄. This work was carried out using an automated Mettler Toledo model RC1 reactorcrystallizer with 800 ml capacity, and controlled isothermally at 25 °C to test the crystallization of K₂SO₄ by addition of the alcohol, and from 50 to 10 °C for the cooling crystallization. In both systems, the line of nucleation points was shown to be approximately parallel to the saturation curve, with an average width of 13°C or 3 % mass for crystallization by cooling, compared with 0.2 to 1 % by salting‐out. In experiments on crystallization by cooling, the K₂SO₄ crystals were 0.27 mm in mean size, showed 7 % agglomeration, and contained 8.5 % moisture. Crystals obtained by salting‐out had a mean size of 0.79 mm, 28 % agglomeration, and 9‐10 % moisture content. A crystal shape factor of approximately of 0.7 was obtained in both systems, apart from the agglomeration.     

Layered crystals of apo‐ and holoferritin grown by alternating crystallization

We report on the use of alternating crystallization for deposition of layers of different (though closely related) proteins in a single crystal. Investigations were carried out with the unique protein couple consisting of two forms of ferritin, apoferritin and holoferritin from horse spleen, which, despite being of quite different molecular masses, still possess identical organic shells. Crystals of both proteins were used as substrates for subsequent contiguous growth of the partner protein in perfect alignment. We observed continuous growth of combined (onion‐like) single crystals; artificial structures of biological macromolecules can be designed in this way. The homoepitaxial layered growth shows in an unambiguous way that protein crystallization depends only on the surface protein conformation and amino‐acid composition, but not on the internal molecule structure. The limitations of protein crystal growth for designing layered structures of biological macromolecules were revealed by growing of heterogeneous protein crystals onto pre‐existing protein crystalline substrates. Tetragonal crystals of hen egg‐white lysozyme were grown onto cubic apoferritin crystals used as substrates. It was observed that the lysozyme crystals were not lattice‐matched to the ‘host’ apoferritin crystals; this led to mere aggregates of different crystals. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

In situ monitoring and control of lysozyme concentration during crystallization in a hanging drop

Fiber optic Raman spectroscopy combined with a partial least-squares regression model was demonstrated as a monitor of lysozyme concentration during crystallization in a hanging drop experiment in real time. Raman spectral features of the buffer and protein were employed to build the regression model. The use of fiber optic technology coupled with Raman spectroscopy, which is ideal for use with aqueous solutions, results in a powerful noninvasive probe of the changing environment within the solution. Lysozyme concentrations were monitored in experiments at a constant reservoir ionic strength. Data from these uncontrolled experiments were used to determine rates of supersaturation, induction times, and the number and size of the resultant lysozyme crystals. Control experiments were performed by introducing step changes in the reservoir ionic strength. The step changes were initiated by comparing in situ rates of supersaturation with the rates of supersaturation calculated from the uncontrolled data. Monitoring the concentration changes of the lysozyme within the hanging drop permits a measurement of the level of supersaturation of the system and enhances the possibility of dynamic control of the crystallization process.     

Study of lactose crystallization in water‐acetone solutions

This paper describes a study on the process of lactose crystallization using a water‐acetone solution. The selection of lactose was based on its significance for the pharmaceutical and food industries and on the fact that the crystallization of this organic compound has been little studied and is, unlike inorganic compounds, complex. The objective was to achieve lactose batch crystallization of solutions by analyzing the crystal growth under different operating conditions. To determine solubility curves, the experiments were carried out based on gravimetric methods. All the crystallization experiments were performed according to the methodology proposed by Nývlt in 1985, who uses the temperature at which the first crystals appear (nucleation) to establish the width of the metastable zone and the induction time. The results showed that crystals with different average diameters, shape factors, and recovered mass were obtained for different water‐acetone compositions. (© 2007 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Spontaneous crystallization of sodium chloride from aqueous‐ethanol solutions; Part 3: Examination of a model of the crystallization process

In the present work the examination of the proposed earlier model of spontaneous crystallization process was done. The model describes kinetics of the crystallization process after the end of the induction period. To test the model the published data on crystallization and aggregation kinetics of sodium chloride at its spontaneous crystallization from supersaturated aqueous‐ethanol solutions were used. It was found an excellent coincidence of the experimental and theoretical data on concentration of the salt and the total number of crystals in solution at crystallization. The model allows predicting with satisfactory accuracy kinetics of crystallization using such general parameters of sodium chloride as the specific surface energy and the height of the nucleus‐bridges between crystals at coalescence. (© 2007 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Rate of sodium chloride attrition fragment generation in a mixed‐suspension crystallizer

Attrition of crystals in industrial crystallization is the major source of secondary nucleation and has strong effects on product quality. This work describes attrition in industrial crystallizers using an empirical engineering model based on dimensionless groups describing crystal properties, suspension properties, and crystallizer geometry and operating conditions. Here the attrition rate of sodium chloride crystals in a small scale mixed‐suspension crystallizer is studied, varying the following parameters: impeller speed, parent crystal size, suspension density, draft‐tube impeller clearance, off‐bottom impeller clearance, impeller type, and impeller material. It was found that the attrition rate depends on most of the variables investigated. The direction of the dependence is predictable based on intuitive modeling. An empirical power law model based on dimensionless groups predicted by Buckingham‐Pi theory (using variables mentioned above) gives a good fit to the data.     

Melt growth and post‐grown annealing of semiinsulating (CdZn)Te by vertical gradient freeze method

We present results of development of CdZnTe semi‐insulating crystals prepared by Vertical Gradient Freeze method in a 4‐zone furnace. We applied the way of growth of the crystal from the top when the first crystallization seed is created on the surface of the melt. The typical height of the crystals is 5 cm. Resistivity and photoconductivity profiles measured along the growth axis by contactless method are compared and their mutual correlation is explained based on a model of relative shift of the Fermi level and the midgap level present in the material. The influence of the Fermi level on electron trapping and recombination is summarized. We present here results of a two‐step annealing method aimed at reduction of Te inclusions while keeping the resistivity high. We employed CdTe:Cl VGF grown samples to eliminate Te inclusions observed in as grown crystals by two‐step post grown annealing in Cd and Te atmosphere and present a model of the processes leading to high resistivity material after annealing.     

Spontaneous crystallization of potassium chloride from aqueous and aqueous‐ethanol solutions; Part 3: Model of the crystallization process

A model of spontaneous crystallization process was proposed. The model describes kinetics of the crystallization process after the end of the induction period. To test the model the published earlier data on crystallization and aggregation kinetics of potassium chloride at its spontaneous crystallization from supersaturated aqueous and aqueous‐ethanol solutions were used. It was found excellent coincidence of the experimental and theoretical data on concentration of the salt and the total number of crystals in solution at crystallization. Somewhat change for the worse was at the theoretical calculations of crystal size distribution at the end of the crystallization process. It indicated that the ways of calculation of size of crystals and their weight fraction in deposit were very approximate. The model allows predicting with satisfactory accuracy kinetics of crystallization using such general parameters of potassium chloride as the specific surface energy and the height of the nucleus‐bridges between crystals at coalescence. It needs further test of the model for other salts. (© 2006 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Determination of reactive crystallization kinetics of 5‐(difluromethoxy)‐2‐mercapto‐1H‐benzimidazole in a batch crystallizer

5‐(difluromethoxy)‐2‐mercapto‐1H‐benzimidazole (DMB) was crystallized via the reaction between sodium salt of DMB and acetic acid. In this work, we firstly measured the solubility data of DMB in binary ethanol‐water mixture at different temperatures. Then, the reactive crystallization kinetics of DMB was determined in the batch crystallizer. Based on the population balance and mass balance, the kinetics model of reactive crystallization process of DMB was established. And the nucleation rate, size‐independent crystal growth and agglomeration kernel were determined by method of classes. Finally, based on the population balance, numeric simulation was done using the above crystallization kinetics in order to verify its reliability.     

Measurement of temperature and concentration dependences of refractive index of hen‐egg‐white lysozyme solution

This paper reports the measurement of temperature and concentration dependences of the refractive index of hen‐egg‐white lysozyme (HEWL) solution, and the temperature dependence of refractive index of the quartz container, which are indispensable for the efforts to measure HEWL concentration of the solution during the processes like crystal growth and dissolution using interferometer. As all of these dependences might be different under different observation wavelengths, temperatures and solution compositions, it is necessary to measure them for specific conditions. In this paper, the desired dependences were measured without knowing the actual value of the refractive index. The measurement method was introduced and the measurement experiments were undertaken at wavelength 780 nm. The dependences obtained will be also useful for those studies employing the same wavelength and solution conditions. (© 2003 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Spontaneous crystallization of potassium chloride from aqueous and aqueous‐ethanol solutions Part 1: Kinetics and mechanism of the crystallization process

Kinetics of spontaneous crystallization of potassium chloride from aqueous and aqueous‐ethanol solutions were studied. During the crystallization of the salt the electrical conductance and optical transmission of the supersaturated solutions were measured automatically. For monitoring of the total surface of growing potassium chloride crystals at the crystallization the turbidimetric method was used. The growth rate and activation energy of the crystals were determined. The crystal growth rate was proportional to supersaturation. When the volume fraction of ethanol in the solution increased from 0 to 25.76%, the activation energy of the growth process did not change and was about 60 kJ·mol^‐1. Aggregation of the crystals was found. The aggregation kinetics of the crystals may be described approximately by the famous Smoluchowski equation for coagulation of colloidal particles. (© 2004 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Correlating dynamic amino acid properties with success rate of crystallization of proteins from Bacteroides vulgatus

To enhance the success rate of protein crystallization, many studies were conducted to determine the relationship between amino acid properties and the success rate of protein crystallization. Although those were successful, new efforts should be made to search for the new factors, which affect protein crystallization. In this study, two dynamic amino acid properties were used to correlate with the success rate of crystallization of proteins from Bacteroides vulgatus, because the amino acid properties used in previous studies were steady. As previously done, logistic regression and neural network were used to model that relationship, and the results were compared against those obtained from each of 532 amino acid properties, which severed as benchmark. The results demonstrated that dynamic amino acid properties should be taken into consideration of protein crystallization. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

A modeling approach for the non‐isothermal antisolvent crystallization of a solute with weak temperature dependent solubility

Antisolvent crystallization is a crystallization technique, which is normally operated isothermally. However, non‐isothermal operation is on occasions performed for solutes that have temperature dependent solubility. This paper shows that it is beneficial to operate antisolvent crystallizers non‐isothermally even for solutes whose solubility is weakly dependent on temperature. In this context, it demonstrates the joint control of particle mean size and size distribution coefficient of variation. A non‐isothermal crystallization model‐based framework is developed for the sodium chloride‐ethanol‐water system and validated for both isothermal and non‐isothermal operations. This framework was used to systematically determine both the optimal antisolvent feed rate and temperature profiles that minimize the coefficient of variation while producing a specified mean crystal size. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Heterogeneous Nucleation of Hen‐Egg‐White Lysozyme — Molecular Approach

The heterogeneous nucleation of hen‐egg‐white lysozyme (HEWL) crystals has been repeatedly investigated using a double‐(thermal)‐pulse technique, thus detaching nucleation from growth stage. n(t) dependencies of the nucleus number n, on templates of poly‐L‐lysine, vs time, t were plotted and the steady‐state nucleation rates I were determined. They were compared with the results obtained earlier for surfaces rendered hydrophobic (by means of hexamethyl‐disilazane) as well as for bare glass surfaces. In the present paper we determine the number of HEWL molecules in the (heterogeneously formed) critical nucleus. It turned out that it is build of 3 (to 4) HEWL molecules on glass substrate and 8 molecules for both hexamethyl‐disilazane and poly‐L‐lysine templates. The energy Ak required for heterogeneous formation of a critical nucleus on poly‐L‐lysine has been calculated, on the basis of the steady‐state nucleation rates I. Intermolecular binding energy in the HEWL crystal lattice has been estimated again (approximately 10‐9 erg/molecule). This time the basis was the adhesion of HEWL crystals to poly‐L‐lysine substrate.     

Crystallization of silicalite‐1 from clear synthesis solutions: Effect of template concentration on crystallization rate and crystal size

Synthesis of silicalite‐1 powders and membranes from initially clear solutions with different tetrapropylammonium hydroxide or bromide concentrations was studied. While tetrapropylammonium bromide acts only as template, tetrapropylammonium hydroxide provides both the template and hydroxyl ions to the synthesis medium. The effects of template and hydroxyl ion concentration on the product yield, crystallization rate and crystal size were investigated. Pure and highly crystalline silicalite‐1 was obtained with all compositions. The nucleation time decreases from 100 h to 20 h and the crystal size decreases from 3.5 μm to 0.35 μm as the template amount x is increased from 5 to 30 moles at a batch composition of 80SiO₂.xTPAOH.1500H₂O at 95 °C. Yield of silicalite‐1 passes through a maximum at intermediate TPA concentration. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)