Crystallization dialysis methods

Crystal growth can be conveniently viewed by holding the capillary horizontally under a microscope. The advantages of a dialysis method are, of course, that the capillary can be placed easily into reservoirs with different solutions. Figure kindly supplied by J. Drenth, University of Groningen and reproduced with permission. 

The development of photochemical active films of chromatophores has also involved the dialysis method. The photoactive sheet derived from Rhodopseudomonas viridis had characteristic properties of a three-dimensional thin crystal, showing linear dichroism in the absorption spectrum. That indicates a correct orientation in the arrangement of the chromatophores. 

Recently, new basal insulin formulation was designed by Jo et al. (2008). Zinc-crystallized insulin was physically loaded into hydrophobically modified glycol chitosan (HGC) nanoparticles by dialysis method (Figure 22.2). The biological activities of insulin-HGC formulations were investigated... 

Variations to the slow-equilibration method include cross-linking of crystals prior to transfer into cryobuffers (Lusty, 1999), transfer of crystals into cryobuffer by dialysis, or the introduction of the cryobuffer during crystallogenesis. 

Dialysis and recrystallisation are simple and yield enzyme of high purity. Several forms of crystals are obtained depending on the pH of the crystallising solution. The activity of lysozyme is measured by the rate of decrease of turbidity (at 570nm ) as hydrolysis of acetone-dried cell walls of the Gram-positive bacterium Micrococcus lysodeikitus (as substrate) occurs on addition of the enzyme [Hirs Methods Enzymol I 124 1968]. 

In addition to the vapor diffusion method described previously, other techniques such as the batch and micro-batch methods, bulk and micro dialysis, free interface diffusion, liquid bridge, and concentration dialysis have also been developed to produce crystals for x-ray diffraction analysis (see McPherson, 1982 and McPherson, 1999). 

Rosenkrantz (1957) has written one of the earlier articles on the utilization of fractionation procedures with infrared analysis and has listed several types of fractionation techniques chromatography, countercurrent distribution, preferential solvent extraction, sublimation, fractional crystallization, molecular distillation, dialysis, centrifugation, electrophoresis, diffusion, and freeze-drying. He has also given references to work in which these methods have been used to fractionate a large variety of biological compounds. Elvidge and Sammes (1966) have discussed many of the techniques mentioned above. 

The conventional techniques for the purification of low-molecular-weight compounds, such as distillation, sublimation, and crystallization, are not applicable to polymers. In some cases, it is possible to remove the impurities by cold or hot extraction of the finely powdered polymer with suitable solvents or by steam distillation. Separation of low-molecular-weight components from water-soluble polymers (e.g., poly(acrylic acid), poly(vinyl alcohol), poly(acryl amide)) can be accomplished by dialysis or electrodialysis. However, the most widely used method of purification is by reprecipitation in which the solution of polymer (concentration less than 5-10 wt.%) is dropped into a 4- to 10-fold excess of precipitant, with stirring. If necessary, this operation is repeated with other solvent/precipitant pairs until the impurities are no longer detectable. 

Other common strategies for inducing crystallization involve the gradual removal of solvent from a biopolymer solution, either by dialysis (Section 3.10) or vapor diffusion. In one implementation of the vapor diffusion method, a single drop of biopolymer solution hangs above an aqueous solution (the reservoir), as shown in Fig. 11.19. If the reservoir solution is more concentrated in a nonvolatile solute (for example, a salt) than is the biopolymer solution, then solvent will evaporate slowly from the drop until the vapor pressure of water in the closed container reaches a constant, equilibrium value. At the same time, the concentration of biopolymer in the drop increases gradually until crystals begin to form. 

This method is time-consuming and during the long term dialysis the protein can easily undergo irreversible changes. Moreover, only a small part of it is transformed into crystals. Preparations of pure hemerythrin from Sipunculus nudur, useful for analytical work, can be obtained by paper electrophoresis in Veronal buffer pH 8.9 (Holleman and Biserte, 1958). 

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