Crystallization mixed solvents

If the substance is found to be far too soluble in one solvent and much too insoluble in another solvent to allow of satisfactory recrystallisation, mixed solvents or solvent pairs may frequently be used with excellent results. The two solvents must, of course, be completely miscible. Recrystallisation from mixed solvents is carried out near the boiling point of the solvent. The compound is dissolved in the solvent in which it is very soluble, and the hot solvent, in which the substance is only sparingly soluble, is added cautiously until a slight turbidity is produced. The turbidity is then just cleared by the addition of a small quantity of the first solvent and the mixture is allowed to cool to room temperature crystals will separate. Pairs of liquids which may be used include alcohol and water alcohol and benzene benzene and petroleum ether acetone and petroleum ether glacial acetic acid and water. 

Coelenterazine (A) is oxidized into dehydrocoelenterazine (D) by MnC>2 in a mixed solvent of ethanol and ether (Inoue et al., 1977b). Dehydrocoelenterazine (C26H19O3N3) can be obtained as dark red crystals. It does not have the capability of chemiluminescence. The ultraviolet absorption spectrum (Fig. 5.6) shows its absorption maxima at 425 nm (e 24,400) and 536 nm (g 12,600) in ethanol. An addition of NaOH significantly increases the 536 nm peak at the expense of the 425 nm peak. Dehydrocoelenterazine can take a tautomeric structure of quinone type (not shown), in which the phenolic proton on the 2-substituent is shifted onto the N(7) of the imida-zopyrazinone ring. Dehydrocoelenterazine can be readily reduced to... 

The one-step reaction of H2prCl6] with MeC02Li under 02 in a mixed solvent of acetic acid and acetic anhydride yields the Ir11 binuclear complex [Ir2(/u-02CMe)2Cl2(C0)2].483 Crystal-structure determinations of [Ir2(/x-02CMe)2Cl2(C0)2L2], (295), where L = MeCN, DMSO, and py, are reported. The one-electron oxidation product for (295), L = py, is EPR active at 77 K the odd electron occupies the 6Ir Ir orbital. 

The crude stearone is allowed to solidify and is then broken up and dried by pressing between filter paper. The crude yield is 230-240 g. (91-95%). The product melts at 84-86° and has an acid value of zero. It is purified by dissolving in 1200 ml. of a 2 1 mixture of benzene and absolute ethanol, filtering hot, and allowing to crystallize (Note 8). After a second crystallization from the same mixed solvents, the stearone is obtained as glistening white flakes, melting clear at 89-89.5° (shrinks at 87-88°). The yield is 204-220 g. (81-87%) (Note 9). 

From this, you should get the idea that it would be good to use a mixed solvent to recrystallize compounds that are soluble in alcohol yet insoluble in water. You see, each solvent alone cannot be used. If the material is soluble in the alcohol, not many crystals come back from alcohol alone. If the material is insoluble in water, you cannot even begin to dissolve it. So, you have a mixed solvent, with the best properties of both solvents. To actually perform a mixed-solvent recrystallization you... 

Every silver lining has a cloud. More often than not, compounds recrystallized from a mixed-solvent system don t form crystals. Your compound may form an oil instead. 

All these results are encouraging for investigators planning to use X-ray diffraction in mixed solvents at subzero temperatures and the rest of the present article will be devoted to a discussion of methods and preliminary results in this field. The methodology for cryoprotection of protein crystals, its physical-chemical basis, and the specific problems raised by the crystalline state, as well as the devices used to collect data at subzero temperatures, will be described. Limitations and perspectives of the procedure will be discussed critically. First attempts to determine the structure of productive enzyme-substrate intermediates through stop-action pictures will be described, as well as investigations showing that X-ray diffraction at selected normal and subzero temperatures can reveal protein structural dynamics. 

Since use of increasing amounts of cosolvents as antifreeze could perturb the conformation and thus the activity of lysozyme, a number of experiments were carried out to try to determine conditions for investigating lysozyme reactions and lysozyme-substrate intermediates in cooled mixed solvents as a preliminary to similar investigation by X-ray diffraction on crystals. Work began with an estimate of the solubility of... 

These results suggest that the crystallographic determination of the structure of a productive enzyme-substrate complex is feasible for lysozyme and oligosaccharide substrates. They also provide the information of pH, temperature, and solvent effects on activity which are necessary to choose the best conditions for crystal structure work. The system of choice for human lysozyme is mixed aqueous-organic solvents at -25°C, pH 4.7. Data gathered on the dielectric constant, viscosity, and pH behavior of mixed solvents (Douzou, 1974) enable these conditions to be achieved with precision. 

Finally, we note that all transfers to alcohol-water mixtures or additions of alcohol to crystal mother liquor involve changes in the proton activity of the solution. Care must be taken to ensure that the pH does not change too much, or the crystal may be disrupted. Worse still, the enzymatic activity may be abolished. Control of proton activity in mixed solvents is discussed in Section III,D. If dielectric effects are controlled and pH is properly adjusted, the microenvironment of a crystalline protein will correspond closely to that of aqueous solution at room temperature. Such correspondence is essential for temporal resolution of individual steps in a catalytic reaction. 

We have seen that crystals can be safely transferred to mixed solvents and that the percentage of organic solvent may often be increased to any desired level provided that its gradual addition is coupled with a gradual reduction in temperature so as to keep the dielectric constant of the medium as near as possible to the value for the normal mother liquor. Such a result deserves explanation and comment about the behavior of the dielectric constant in mixed solvents as a function of temperature. 

While sequential transfer of crystals in cooled mixed solvents is a prerequisite to maintain crystal integrity, it has been found that changes in pH must also be taken into account and that buffers must be selected whose pH values show little change with temperature. Precise control of... 

Tables VIII-XI show examples of pon variations of several buffers. With such tables, it is easy to adjust any desired pan value in mixed solvents at any selected temperature or in a given range of temperatures. We will see in Section III,E how these values are essential to investigate safely both crystal structure and productive enzyme-substrate complexes in the crystalline state.

In recent years, it has been shown (Douzou and Maurel, 1976) that some proteins can behave as polyanions or polycations, and the stability of their solid state might be endangered at lower salt concentration due to repulsive forces between protein molecules. Much more important is the problem of enzyme activity in crystals suspended in cooled mixed solvents as a consequence of cosolvent- and temperature-induced changes in salt concentration and therefore in electrostatic potentials. 

The reaction flask is removed from the oil bath, and the solution is poured into a 250-ml. beaker to crystallize 10 ml. of benzene is used to rinse out the flask. When crystallization sets in, 50 ml. of petroleum ether (b.p. 35-55°) is added to the warm mixture with manual stirring. After the mixture has been chilled in an ice bath, the product is filtered by suction and washed with 100 ml. of a 1 1 petroleum ether-benzene mixture. It is then removed from the funnel, stirred into a slurry with 100 ml. of the mixed solvent, filtered, and again washed with 50 ml. of the solvent. These washings remove the bulk of the color, and a powdery white product remains. After standing overnight in a warm place, the product weighs 35.5-36.5 g. (74-76%) and melts at 104-105°. It may be purified further by recrystallization from benzene (3.7 ml. per g.). The yield of colorless benzoylacetanilide melting at 106-106.5° cor. is 32-34 g. 

Maleic anhydride was crystallized from sodium dried, thiophene-free benzene. The bisisobutyronitrile was crystallized from a mixed solvent containing equal volumes of benzene and toluene. The liquid monomers were purified by distillation under reduced pressure. Freshly distilled solvents were used. 

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