Nucleation and seeding

As described in Chapter 2, new crystals may be formed by primary nucleation (homogeneous and heterogeneous), secondary nucle- 

Nucleation. There is relatively little information available on nucleation rates of biochemicals from solution, yet the control of nucleation rates controls the number of particles produced and has a direct bearing on crystal size for a given yield of crystallizing product. Uncontrolled nucleation either heterogeneously from contaminants or as secondary particles produced from existing crystals can greatly affect the desired size distributions. The general principles of nucleation theory as discussed in Chapter 2 apply to biochemical systems. 

Very little work has been done on nucleation rate expressions under high supersaturations, on the nature of the solids formed during rapid nucleation (which may be nonequilibrium and/or amorphous phases), or on the relative nucleation rates of polymorphs or optical isomers. Devices to achieve high supersaturations such as jet mixers or supercritical fluid precipitators must be designed properly to achieve rapid mixing and uniform crystallization conditions. Research in these areas will facilitate production of fine particles of desired crystallinity and morphology. 

Seeding. Seeding is a common practice in many batch crystallizations in an attempt to control particle size uniformity. However, it is limited by the ability to produce seeds of the proper crystal structure having an appropriately narrow size distribution 

If seeds with a particular size distribution (number distribution) are introduced, at time zero, into a batch crystallizer (without crystals initially present), then the PSD at later times is described using the population balance equation (Randolph and Larsen 1988, or Chapter 4 of this book) where it has been assumed no breakage or agglomeration occurs. 

In some circumstances the rate of formation of nuclei is enhanced by the preliminary formation of an amorphous precipitate, which is usually more soluble than the crystal. With enolase, a significant portion of dissolved protein was in equilibrium with the precipitate, and crystals grew from this mixture [29]. However, frequently the outcome is not so happy. There are no general rules which favour crystal growth except that crystals grow best if supersaturation is approached slowly and there are no heterogeneous nucleation sites such as dust particles, impurities, etc. 

Controlled nucleation may be achieved once seed crystals are available. Seeding is best carried out using a very few very small crystals. For example, with phosphorylase b, a large (0.4 x 0.4 x 1 mm) crystal was crushed in a small volume (1 ml) of solution. The solution was left to settle for 10 minutes and the supernatant serially diluted by lO. Additions to the crystallisation trial were made so that the final dilution of seed solution was 10 to 10.  

With pig aspartate aminotransferase, crystals were obtained by seeding with chicken enzyme crystals [42]. 

Alber et al. [43], have observed an interesting phenomenon with yeast triosephos-phate isomerase when crystallised from poly(ethylene glycol), which they termed oiling-out . At high concentrations of PEG ( 20%) the protein formed droplets which coalesced. On dilution most of these dissolved, leaving a few droplets which subsequently converted to crystals. The authors comment that these observations are not limited to triosephosphate isomerase nor to poly(ethylene glycol). 

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Zeolite beta characteristically replaces ZSM-20 at long age times in the standard synthesis (JL) - represented by experiment 1 in Table 1. In most of these syntheses zeolite beta seems to be an unavoidable low level impurity. The experiments detailed in Table 2 examine the effects of various nucleation and seeding methods on ZSM-20... 

The overall i-th moments are defined as summation of nucleated and seeded crystallization moments. 

Moment equations (combining the nucleated and seeded crystals together) ... 

The concept of this widely used technique consists in the decoupling of (i) nucleation and seed formation at high supersaturation, and (ii) growth of the seed crystals to a continuous layer at low supersaturation. Usually, in the crystal growth step a new nucleation can be avoided and only the seeds grow to the molecular sieve layer. Therefore, the layers obtained by secondary growth are less polycrystalline. If the support surface was covered by a homogeneous and dense seed layer, relative thin zeolite and MOF layers can be obtained. 

Kortan A R, Hull R and Opila R L 1990 Nucleation and growth of CdSe on ZnS quantum orystallite seeds and vise versa in inverse mioelle media J. Am. Chem. Soc. 112 1327... 

Several features of secondary nucleation make it more important than primary nucleation in industrial crystallizers. First, continuous crystallizers and seeded batch crystallizers have crystals in the magma that can participate in secondary nucleation mechanisms. Second, the requirements for the mechanisms of secondary nucleation to be operative are fulfilled easily in most industrial crystallizers. Finally, low supersaturation can support secondary nucleation but not primary nucleation, and most crystallizers are operated in a low supersaturation regime that improves yield and enhances product purity and crystal morphology. 

The seeding-growth procedure is a popular technique that has been used for a century to synthesize metal particles in solution. Recent studies have successfully led to control the dimensionality of the particles where the sizes can be manipulated by varying the ratio of seed to metal salt [23-25]. The step-by-step particle enlargement is more effective than a one-step seeding method to avoid secondary nucleation [26,27]. This mechanism involves a two-step process, i.e. nucleation and then successive growth of the particles as illustrated in Scheme 1. 

Whenever the solubility curve is crossed for the less stable Form II there is a risk that it will nucleate and contaminate the product. This situation is very probable when the solubility curves of the two polymorphs lie close together, as shown in Figure 21 of the Cimetidine case study. The addition of seed crystals of Form I, close to its solubility curve, and minimization of the supersaturation during the growth process is a good method of control in this instance. Solvent selection to extend the width of the Form II metastable zone would also be desired, as discussed in section 2.4.4. 

Carefully selected seed crystals are sometimes added to a crystalliser to control the final product crystal size. The rapid cooling of an unseeded solution is shown in Figure 15.20a in which the solution cools at constant concentration until the limit of the metastable zone is reached, where nucleation occurs. The temperature increases slightly due to the release of latent heat of crystallisation, but on cooling more nucleation occurs. The temperature and concentration subsequently fall and, in such a process, nucleation and growth cannot... 

Crystallization is generally preceded by two types of nucleation. The primary nucleation occurs with the formation of clusters of molecules at the submicron level. When the concentration exceeds saturation to afford supersaturation, the clusters become nuclei. The secondary nucleation is caused by particles due to primary nucleation or seeds. There are many strategies to achieve supersaturation to initiate crystallization such as cooling, evaporation, and antisolvent addition. 

Although the self-assembly of polymeric structures can involve nucleation and elongation steps (See Actin Assembly Kinetics Microtubule Assembly Kinetics), one can simplify the assembly process through what is known as seeded assembly. At an initial monomer concentration [M], seeded assembly is induced by the addition of pre-assembled polymeric structures consequently the polymer number concentration must remain constant. The rate of monomer incorporation into indefinite length polymers can be written as follows ... 

After the bath attained its equilibrium temperature, the crystallizer was charged with about 400 ml of liquid and was inserted into the bath. After about 30 minutes the system attained a constant temperature and a subcooling (difference of equilibrium temperature and constant temperature before initiation of crystaUization) was established. Introduction of the seed crystals ( ter being allowed to warm for a period of a few seconds) on the specially prepared stirrer initiated crystallization (secondary nucleation) and resulted in a change in the temperature of the crystallizer (figure 2). The temperature of the crystallizer attained an uilibrium value of a few minutes after nucleation occurred. The concentration of the sucrose solutions was measured using a refractometer (.1% accuracy). 

The most commonly used technique for separation of nucleation and growth is seeding (Stura, 1999 Bergfors, 2003 and references therein). Although very successful, seeding often involves handling of... 

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