Seeded crystallisation

Figure 6.8 Liquid-liquid diffusion Saturated solution of biological macromolecule (e.g. protein) is placed in a sealed capillary environment in contact with precipitant solution. Slow mixing and liquid diffusion creates sufficient precipitant gradient in the macromolecule solution to "seed" crystallisation.

Seeded crystallisation entails introducing a pre[Pg.270]

The following preparation works excellently if it is carried out in a laboratory which has become inoculated by the methylglucoside from previous preparations, or alternatively if some of the glucoside is available for "seeding the solution of the crude material otherwise there may be a considerable delay before crystallisation starts. 

Boil the mixture gently on a sand-bath for 4 hours and then decant into a conical flask and cool. Seed the cold solution if necessary with a trace of a-methylglucoside. The glucoside separates as colourless crystals. When crystallisation ceases, filter the glucoside at the pump, drain, wash quickly with a small quantity of methanol, and then recrystallise from a minimum of methanol. For this purpose methanol of good quality, but not necessarily anhydrous, should be used. The a-methylglucoside is obtained as colourless crystals, m,p. 165°. Yield, 6-7 g. 

If difficulty is experienced in inducing the first crude crop of the a-glucoside to ciystallise, place a few drops of the solution on a watch glass and expose freely to the air, with occasional scratching meanwhile keep the main volume of the solution securely corked in the conical flask. After an interval of varying length (possibly several days), partial crystallisation occurs in the material on the watch-glass. Then seed the solution with this material crystallisation of the first main crop will rapidly follow. 

Then filter off the solid azoxybenzene at the pump, wash it thoroughly with water, and drain well. Recrystallise from a minimum of m ethylated spirit, allowing the hot solution to cool spontaneously (with occasional stirring) until crystallisation starts, and then cool in ice-water. If crystallisation is delayed, seed the solution with a trace of the crude product if on the other hand the azoxybenzene separates at first as an emulsion, add methylated spirit, drop by drop, with stirring until the solution is clear, and then allow the cooling to proceed as before. The... 

The separation of the solid phase does not occur readily with some liquid mixtures and supercooling is observed. Instead of an arrest in the cooling curve at /, the cooling continues along a continuation of c/ and then rises suddenly to meet the line f g which it subsequently follows (Fig. 1,13, 1, iii). The correct freezing point may be obtained by extrapolation of the two parts of the curve (as shown by the dotted line). To avoid supercooling, a few small crystals of the substance which should separate may be added (the process is called seeding ) these act as nuclei for crystallisation. 

Crystallization Method. Such methods as mechanical separation, preferential crystallisation, and substitution crystallisation procedures are included in this category. The preferential crystallisation method is the most popular. The general procedure is to inoculate a saturated solution of the racemic mixture with a seed of the desired enantiomer. Resolutions by this method have been reported for histidine (43), glutamic acid (44), DOPA (45), threonine (46), A/-acetyl phenylalanine (47), and others. In the case of glutamic acid, the method had been used for industrial manufacture (48). 

Although 16 different crystalline modifications have been identified (24,25), the a-pentahydrate is the stable form below 48°C. Solutions of sodium thiosulfate in the absence of seed crystals can be easily supercooled below their normal crystallisation temperatures. The dotted line extension of the dihydrate phase in Figure 1 is an indication that, if supercooling takes place below this line, solutions normally giving the pentahydrate may form the dihydrate [36989-90-9] s1ste2id. 

The water solubiUty of kernite is shown in Figure 5 and in Table 9. Kernite is the stable phase in contact with its solutions from 58.2°C to ca 95°C (71). Its rate of crystallisation is, however, much slower than that of the pentahydrate. Large kernite crystals can be grown slowly by seeding saturated borax solutions. 

Cevadine, C32H49O9N (crystallised veratrine), was first isolated in a crystalline condition from sabadilla seeds by G. Merek, who called it veratrine, and was subsequently obtained by Schmidt and Koppen,i and by Wright and Luff, whose name cevadine has been generally adopted, though Ahrens suggested that it should be called crystallised veratrine. The yield of total alkaloids from the seeds is said to be from 6 to 7 gm. per kilo, of which 0-8 to 0-9 gm. may be obtained as crystalline cevadine, 0-5 to 0-6 as veratridine and 0-2 to 0-3 as sabadilline. 

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