The shape of a crystalline organic solid has a major impact on its downstream processing and on its end-product quality, issues that are becoming increasingly important in the specialty and fine chemical, as well as the pharmaceutical and life science, industries. Though it is widely known that improved crystal shapes can be achieved by varying the conditions of crystallization (such as solvent type and impurity levels), there is far less understanding of how to effect such a change. Until recently, most methods for predicting crystal shapes were based exclusively on the internal crystal structure, and hence could not account for solvent or impurity effects. New approaches, however, offer the possibility of accurately predicting the effects of solvents. Models for predicting crystal shape are reviewed, as well as their utility for process and product design.

Modeling crystal shapes of organic materials grown from solution

As a supercooled melt at 150 °C, the chiral compound 1,1‘-binaphthyl racemizes rapidly. The melt solidifies as a conglomerate of crystals, each consisting exclusively of either R-(−)- or S-(+)-enantiomer. We find that crystallization performed with a 2.00 g sample with constant stirring produces a large enantiomeric excess (mean 77%) in almost every crystallization. The predominance of R-(−) or S-(+) was random. Unstirred 2.00 g samples of binaphthyl produce a much lower enantiomeric excess (mean 20%) with optical activity centered around zero similar to an earlier report.1 Thus, chiral symmetry breaking can be realized in crystallization from a melt by the mere act of stirring, as it can be in crystallization from a solution.

Chiral Symmetry Breaking in Stirred Crystallization of 1,1‘-Binaphthyl Melt

Crystallization is used industrially for the recovery and purification of many inorganic and organic materials. However, very little is reported on the application of bulk crystallization for proteins. In this work, ovalbumin was selected as a model protein to investigate the feasibility of using bulk crystallization for the recovery and purification of proteins. A stirred 1-L seeded batch crystallizer was used to obtain the crystal growth kinetics of ovalbumin in ammonium sulfate solutions at 30 degrees C. The width of the metastable region, in which crystal growth can occur without any nucleation, is equivalent to a relative supersaturation of about 20. The bulk crystallizations were undertaken within this range (using initial relative supersaturations less than 10) and nucleation was not observed. The ovalbumin concentration in solution was measured by UV absorbance and checked by crystal content measurement. Crystal size distributions were measured both by using a Malvern Mastersizer and by counting crystals through a microscope. The crystal growth rate was found to have a second-order dependence upon the ovalbumin supersaturation. While there is no discernible effect of ammonium sulfate concentration at pH 4.90, there is a slight effect at higher pH values. Overall the effect of ammonium sulfate concentration is small compared to the effect of pH, for which there is a 10-fold increase in the growth rate constant, k(Gsigma) over the range pH 4.6-5.4. To demonstrate the degree of purification which can be achieved by bulk crystallization, ovalbumin was crystallized from a solution containing conalbumin (80,000 Da) and lysozyme (14, 600 Da). After one crystallization and a crystal wash, ovalbumin crystals were produced with a protein purity greater than 99%. No contamination by the other proteins was observed when using overloaded sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) stained with Coomassie blue stain and only trace amounts of lysozyme were observed using a silver stain. The presence of these other proteins in solution did not effect the crystal growth rate constant, k(Gsigma). The study demonstrates the feasibility of using bulk crystallization for the recovery and purification of ovalbumin. It should be readily applicable to other protein systems.

Protein purification by bulk crystallization: the recovery of ovalbumin.

Modelling and optimization of seeded batch crystallizers

While bulk crystallization from impure solutions is used industrially as a purification step for a wide variety of materials, it is a technique that has rarely been used for proteins. Proteins have a reputation for being difficult to crystallize and high purity of the initial crystallization solution is considered paramount for success in the crystallization. Although little is written on the purifying capability of protein crystallization or of the effect of impurities on the various aspects of the crystallization process, recent published reports show that crystallization shows promise and feasibility as a purification technique for proteins. In order to further examine the issue of purity in macromolecule crystallization this study investigates the effect of the protein impurities, avidin, ovalbumin and conalbumin, at concentrations up to 50%, on the solubility, crystal face growth rates and crystal purity, of the protein lysozyme. Solubility was measured in batch experiments while a computer controlled video microscope system was used to measure the f {101} and {101} lysozyme crystal face growth rates. While little effect was observed on solubility and high crystal purity was obtained (>99.99%), the effect of the impurities on the face growth rates varied from no effect to a significant face specific effect leading to growth cessation, a phenomenon that is frequently observed in protein crystal growth. The results shed interesting light on the effect of protein impurities on protein crystal growth and strengthen the feasibility of using crystallization as a unit operation for protein purification.

The effect of protein impurities on lysozyme crystal growth.

Crystallization was studied as a separation process in various applications. Two applications of interest are flue-gas desulfurization and phosphate removal from waste water.

Ken Toyokura

Papers

Crystallization as a separations process