Successive precipitation fractionation

Figure 8.6 Effect of successive fractionations weight fraction n-mer versus n for seven successive precipitates and final residue (calculated for R = 10 ). [Adapted from G. V. Schulz, Z. Phys. Chem. B46 137 (1940) B47.155 (1940).]...

Fig. 128.—Calculated distribution for each of eight fractions separated from the initial distribution shown by the uppermost curve. Dotted lines represent distribution of polymer remaining in the dilute phase after each successive precipitation with =10 in each case. The distribution for each fraction, obtained as the differences between successive dotted curves, is shown by a solid curve. (Schulz. )...

Fig. 13. Schematic representation of successive precipitation fractionation (SPF) and successive solution fractionation (SSF)38)...

As another example, imagine that an S-100 fraction has been prepared, ammonium sulfate has been added, and the fraction that precipitates between 30 and 50% has been obtained. This sample is subjected to affinity and ion-exchange chromatography. Have these procedures been successful in removing extraneous proteins While it is possible to answer this question by a determination of total protein content, it may be more informative to analyze each of the samples for its constituent proteins. 

Beef liver (or lung) was minced and then autolyzed for twenty-four hours before extraction with an alkaline solution saturated with ammonium sulfate. Protein was precipitated by warming the extract, and the heparin-protein complex was precipitated from the supernatant liquor on acidification. Extraction of the complex with ethanol removed fatty material, and tryptic digestion removed most of the protein. The heparin was precipitated with ethanol, redissolved in warm alkaline solution to destroy trypsin, and reprecipitated with acetone. This material, crude heparin, was isolated in a yield of 15-50 g. per 100 lb. of animal tissue. In a later paper," the purification of crude heparin by fractionation successively with Lloyd s reagent, cadmium chloride, and acetone, was described. The purified heparin w-as 100 times as active as the crude material. Scott and Charles" reported the presence of nitrogen... 

A scheme devised by Thurman and Malcolm (1981) uses XAD-8 to concentrate and isolate aquatic humic substances. According to this scheme, the sample is first filtered through 0.45 /am silver-membrane filters and acidified. After concentration on XAD-8 resin, the humic acid fraction is precipitated at pH 1. Both humic acid and fulvic acid fractions are hydrogen saturated by passing the sample through a cation-exchange resin in the H-form. These fractions are then freeze-dried to yield low-ash samples of aquatic humic and fulvic acids. This extraction scheme is outlined in Table 7. These authors successfully isolated humic substances from a number of surface and groundwaters. Even samples with DOC values of 0.7 mg C/L could be proc-... 

Fractional Precipitation Separation of the individual components from a solution of the given mixture through successive precipitations which can be made to materialize by employing techniques like salting out etc. 

Fractional precipitation as exemplified gives an initial separation of the constituents of a mixture but the product of each successive precipitation is far from being a pure compound, because precipitates carry a lot of other solutes along with them from the solution, even though the solubility of such salts may not have been exceeded. Thus, whereas fractional crystallisation is an adequate separatory technique in itself and can be used independently to obtain individual constituents of a given mixture in the pure state, fractional precipitation has to be combined with other purification processes e.g. electrophoresis to get pure compounds from the fractions obtained by successive precipitations. 

Kamide,K., Sugamiya,K. Role of concentration dependence of polymer/ solvent interaction parameter in the polymer fractionation by successive precipitational method. Makromol. Chem. 139,197 (1970). 

Galactosylceramide can be conveniently isolated from bovine brain tissue. If this organ is extracted successively with acetone, diethyl ether and then hot aqueous ethanol, sphingolipids are obtained in the final fraction. These precipitate on cooling and the residue is washed with acetone and dissolved in hot acetic acid. On cooling they can again be precipi-... 

There are two main candidate models which describe the effect of the solute clusters on hardness. The modulus hardening model by Russell and Brown" has been successfully employed for many years to link microstructural and mechanical properties. It predicts very little effect of particle size on hardening at a given volume fraction of precipitates, when particle diameters are in the range 2-4 nm. A more modern model based... 

Despite the idealized and restrictive set of assumptions, the LSW theory provides a remarkably successful description of many coarsening observations. However, for diffusion-controlled coarsening, it is often found that real systems exhibit a broader and more symmetric steady-state distribution. Furthermore, while the average precipitate size is usually observed to increase qualitatively with the predicted dependence, the rate constant is generally found to be different from that predicted by the LSW theory. For purely reaction-controlled coarsening where the mechanism is strictly a surface phenomenon, the process should be independent of the volume fraction of precipitates as long as the precipitates do not touch. On the other hand, for diffusion control, the interaction of the diffusion fields of the precipitates must be taken into account, and there have been several attempts to incorporate volume fraction effects into the LSW theory (7). 

Pig. 3. Successive precipitation (SPF) and solution fractionation (SSF) (coacervate extraction) , mother solution n, polymer rich phase and polymer-lean phase. Numbers denote fraction numbers (148). Courtesy of Society of Chemical Industry. 

A very simple method to do so is called the Successive Precipitation Fractionation (SPF). The initial solution of the polydisperse polymer 5 in a suitable solvent A forms the feed phase F. A change of temperature results in a phase separation. The polymer-rich phase II is removed and forms fraction 1. The polymer-lean phase 7 serves directly as the feed phase for step 2. A further change of temperature results in a second phase separation (step 2). Again, the polymer-rich phase II is removed forming fraction 2 whereas the polymer-lean phase / is the feed phase of the next step. This procedure is continued. The feed phase of each step equals the polymer-lean phase I of the preceding step. At the end of this process instead of the widely distributed original polymer there are several fractions (1, 2,- ) with narrow molar-mass distributions. 

SEC Size exclusion chromatography SPF Successive precipitation fractionation... 

Figure 3 presents the schemes for successive precipitation fractionation (SPF) and successive solution fractionation (SSF). In both cases, by lowering the temperature (or adding nonsolvent) a homogeneous polymer solution (called feed phase F) splits into two coexisting phases, a polymer-lean sol phase I and a polymer-rich gel phase II, which are then separated. In SPF (Fig. 3a), the polymer is isolated from phase II as fraction FI. Phase I directly forms the feed phase for the next fractionation step, etc. 

The cross-fractionation (CF) of copolymers suggested by Rosenthal and White [64] is a combination of several successive precipitation procedures and is also... 

Fig. 19 Comparison of three different fractionation methods circles cross-fractionation, crosses successive precipitation fractionation using solvent mixture 1, and stars successive precipitation fractionation using solvent mixture 2...

Fig. 20 Comparison of three different Iractionatimi methods circles cross-ftactionation, crosses snccessive precipitation fractionation using solvent mixture 1, and stars successive precipitation fractionation using solvent mixture 2, with regard to the integral distribution of the original copolymer by (a) molecular weight, and (b) chemical composition. The lines represent the distribution of the original copolymer...

The second, by Kamm et al. (338) is based on the following procedure the crude extract is treated with ammonium sulfate and filtered. The dry precipitate is suspended in concentrated acetic acid, and the acetic solution is fractionated by precipitation with ethyl ether and petrol ether. Kamm s method was successfully used by du Vigneaud (465) in his work on the purification and analysis of the posterior pituitary hormones. Stehle and Frazer (548) published a variation of this technique using methanol and ethyl acetate as solvents, employed separately or in a mixture. 

Judged from Fig. 31, successive extraction into a large number of fractions gives the best result achievable both with the generalized Beall and with Schulz s evaluation. Successive precipitation is not a suitable method. This view is supported by Fig. 32, which shows the evaluation of successive precipitation fractionations of an exponential distribution (M = 10 5 = 2) 23,70), into 16 fractions each the overall concentrations are 0.01, 0.003 and 0.001. We see again that the results deteriorate in this order. The cumulative Schulz curves are now distinctly inferior to those found by the generalized Beall method. The insensitivity of integral curves for details in the differential distribution is once more illustrated. 

Melcher and Fraij (1980) successfully used cryo-precipitation at -20 °C to separate a zein 11 extract obtained from com meal into a methionine-rich polypeptide fraction (cryo-precipitate) and a cryo-supematant containing predominantly polypeptides with amino acid composition similar to that of the zein 1 proteins. The cryo-precipitate was readily dissolved in 70% ethanol containing mercaptoethanol and precipitated again on cooling, indicating that the cryo-precipitaion was reversible. 

In order to determine whether fraction was lodate (or some oxidation state readily exchangeable with lodate), a known amount of potassium lodate was added to a solution containing the fraction and successive precipitations of silver lodate were carried out. The fraction of total lodate precipitated was determined In each case as well as the percentage of total activity carried. These experiments showed that oxidation of carrier-free Iodide In acidic cerium (IV) solution, at 105°C, for 75 minutes, yielded a Z fraction which was lodate or some Intermediate completely exchangeable with lodate at room temperature, over one-half hour, less than 20 percent of the 1 fraction formed was lodate. 

Fit a 1500 ml. bolt-head flask with a reflux condenser and a thermometer. Place a solution of 125 g. of chloral hydrate in 225 ml. of warm water (50-60°) in the flask, add successively 77 g. of precipitated calcium carbonate, 1 ml. of amyl alcohol (to decrease the amount of frothing), and a solution of 5 g. of commercial sodium cyanide in 12 ml. of water. An exothermic reaction occurs. Heat the warm reaction mixture with a small flame so that it reaches 75° in about 10 minutes and then remove the flame. The temperature will continue to rise to 80-85° during 5-10 minutes and then falls at this point heat the mixture to boiling and reflux for 20 minutes. Cool the mixture in ice to 0-5°, acidify with 107-5 ml. of concentrated hydrochloric acid. Extract the acid with five 50 ml. portions of ether. Dry the combined ethereal extracts with 10 g. of anhydrous sodium or magnesium sulphate, remove the ether on a water bath, and distil the residue under reduced pressure using a Claiseii flask with fractionating side arm. Collect the dichloroacetic acid at 105-107°/26 mm. The yield is 85 g. 

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