Precipitation with Organic Solvents

Attributable to its fast performance and high convenience this procedure is usually employed for routine isolations. Unfortunately, the use of organic solvents bears some disadvantages because they are noxious. Moreover, by treatment of haemolysate with chloroform/ethanol a considerable coprecipitation and/or denaturation of superoxide dismutase is seen. 

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Grace and Co.) is added to the mixture which is refluxed for 5-10 min or until the gas evolution is complete. The product, often an aziridine, is obtained by pouring the reaction mixture into water and either by filtering or by extracting the precipitate with organic solvent followed by a water wash and drying. 

Exudate collection in trap solutions usually requires subsequent concentration steps (vacuum evaporation, lyophilization) due to the low concentration of exudate compounds. Depending on the composition of the trap solution, the reduction of sample volume can lead to high salt concentrations, which may interfere with subsequent analysis or may even cause irreversible precipitation of certain exudate compounds (e.g., Ca-citrate, Ca-oxalate, proteins). Therefore, if possible, removal of interfering salts by use of ion exchange resins prior to sample concentration is recommended. Alternatively, solid-phase extraction techniques may be employed for enrichment of exudate compounds from the diluted trap solution (11,22). High-molecular-weight compounds may be concentrated by precipitation with organic solvents [methanol, ethanol, acetone 80% (v/v) for polysaccharides and proteins] or acidification [trichloroacetic acid 10% (w/v), per-... 

Elimination of Cellulases from Xylanases. Classical methods of protein fractionation can be used for to separate cellulases and xylanases on a large scale only when they differ considerably in molecular weight or isoelectric point. The Tricho-derma harzianum enzymes were separated by ultrafiltration because the xylanase was smaller and passed through the membrane into the ultrafiltrate 18). Fractional precipitation with organic solvents is another possibility (7). 

ADP-ribosylation of tfie samples is carried out in glass tubes (approximate volume 5 ml) in case the reaction has to be followed by precipitation with organic solvents. Therefore, the markings on the tubes should withstand solvents. Each tube contains 10 (xl of preactivated toxin (100 ng PT/tube) and 20 [il of membrane suspension. The reaction is readily started by addition of 30 1 of the P-NAD" -containing buffer, resulting in a total volume of 60 (il per tube. ADP-ribosylation of the thoroughly vortexed sample is conducted for 30 min at 30°C or tor 20 min at 37°C while the tubes are agitated con-tinously. 

The study carried out on this process showed the possibility of decreasing significantly the production costs,and time of operation, increasing product quality and process safety by using ultrafiltration in place of purification by precipitation with organic solvents (e.g. methanol). 

In HPLC, alcohols and especially acetonitrile are often added to the biological samples to remove (serum) proteins. In CE, in addition to the removal of proteins, the presence of acetonitrile and small-chain alcohols in the sample leads to stacking, as discussed earlier. Protein removal can also be accomplished by alcohols such as ethanol and by acids such as trichloroacetic acid. In CE, precipitation with acids is less desirable than the precipitation with organic solvents, as it increases the salt load. Following precipitation, proteins can also be dissolved in the appropriate buffers and assayed by CE. 

The data are then plotted as log [>>]M versus Vg, as shown In Figure 2. When the test sample is examined in the same column, the product [ i]M can be read directly from the calibration curve. For calculation of the molecular weight, either of two approaches me(y be followed. If the project is of limited scope, one can determine the intrinsic viscosity of the sample, and calculate M from the product [ ]H obtained directly from the universal calibration curve. For projects with a broader scope, it would be desirable to fractionate a preparation with a broad distribution of molecular weights into fractions with narrow distributions by either preparative SEC or established chemical methods (e.g. selective precipitation with organic solvents). The molecular weights and intrinsic viscosities of these samples would then be determined experimentally, followed by the calculation of the Mark-Houwink constants. Finally, the molecular weight of any sample can be calculated from the [>i]M value read from the curve, by the equation [ i]M =... 

Commercial bromelain is currently prepared by centrifugation, ultrafiltration and freeze-drying of cooled pineapple juice, which produces a yellowish powder. Stem bromelain has been generally prepared from the juice of pineapple wastes (mainly stems) by precipitation with organic solvents (e.g., acetone and methanol) or by ultrafiltration. ... 

One of the most important methods is salting out with a neutral salt such as ammonium sulfate, sodium sulfate, or magnesium sulfate, since highly concentrated solutions of these salts can be prepared. The salt content of a protein solution is increased stepwise, and after each addition the precipitated protein is centrifuged off. The pH may be varied to allow multiple fractionation (around their isoelectric points proteins become least soluble). Another method is precipitation with organic solvents (alcohol, acetone) according to E. Cohn. This must be carried out under refrigeration (cf. Chapt. IV-9). 

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