Fractionation methods with dialysis

Ribonucleic acid can be eluted electrophoretically directly from the gel without the intervening process of searching for zones. For cylindrical gels, the simplest method is to cover the end of the tube with dialysis tubing, so as to leave a dead space, from which RNA can be recovered with a hypodermic syringe. This is, however, too primitive for most purposes, for one can only select one s fractions by guesswork. 

Most problems with this procedure have involved tracer impurities and the separation of bound and free labeled fractions. Several separation techniques have been used, including equilibrium dialysis, membrane ultrafiltration, and steady-state gel filtration. Their deficiencies include a requirement for a large sample volume, the need for complicated correction of sample volume changes that occur during the separation, and difficulties of collecting and measuring radioactivity in numerous fractions of each sample. Equilibrium dialysis has been used most often in the past, but serious errors often arise from the sample dilution required by this method. Symmetrical dialysis of undiluted samples is reported to be less susceptible to tracer contamination and dilution effects. Ultrafiltration also appears to overcome these problems and to obviate errors caused by dilution. 

In the second method, ultrafiltration, centrifugal force is applied for a few minutes to a sample of the compound in plasma, pushing the unbound fraction through a permeability selective membrane. LC/MS/MS analysis of the ultrafiltrate and retentate is then done. This has the advantage of being faster than dialysis. Methods like 96-well plate have been developed here too. As in HTS, edge effects have been noted with these, and one study comparing such a method with results from individually run samples for a dozen research compounds showed an value of 0.60. ... 

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. 

Water soluble protein with a relative molecular mass of ca. 32600, which particularly contains copper and zinc bound like chelate (ca. 4 gram atoms) and has superoxide-dismutase-activity. It is isolated from bovine liver or from hemolyzed, plasma free erythrocytes obtained from bovine blood. Purification by manyfold fractionated precipitation and solvolyse methods and definitive separation of the residual foreign proteins by denaturizing heating of the orgotein concentrate in buffer solution to ca. 65-70 C and gel filtration and/or dialysis. 

Gel filtration with Sephadex was used by Ghassemi and Christman [426] to make separations by molecular size on water samples concentrated by vacuum evaporation. Fluorescence was also used as one method for following the fractionation. Molecular size was also used by Gjessing [427] but with pressure dialysis as the method of separation. A similar method of concentration and separation was used by Brown [428] to follow the dispersion of these materials as fresh and salt water mixed in the Baltic Sea. 

The first fractionation of urinary ampholytes in this way was carried out by Boulanger et al. (BIO) in 1952 with the use of ion-exchange resins. They had designed this procedure previously for the fractionation of ampholytes in blood serum (B8). According to this method, deproteinized urine was subjected to a double initial procedure aiming at the separation of low-molecular weight substances from macro-molecular ones. One of the methods consisted of the fractionation of urinary constituents by means of dialysis, the second was based on the selective precipitation of urinary ampholytes with cadmium hydroxide, which, as had previously been demonstrated, permits separation of the bulk of amino acids from polypeptides precipitated under these circumstances. Three fractions, i.e., the undialyzable part of urine, the dialyzed fraction, and the so-called cadmium precipitate were analyzed subsequently. 

A constant observation when the MRP were separated by various methods was that antioxidative effect was found in many different fractions. Both the dialysates and the retentates from dialysis were antioxidative to some extent. All the electrophoresis fractions exhibited some antioxidative effect. Attempts to separate the MRP by column chromatography on Sephadex G-50 have resulted in several fractions with some antioxidative effect, and so on. This indicates that several antioxidative products are formed by the Maillard reaction, possibly differing in molecular size and chemical structure, but perhaps with one single antioxidative functional group in common, such as a free radical function. However, it can not be excluded that the MRP contain a few entirely different antioxidants with different modes of action. Various mechanisms have also been suggested. Eichner (6) claimed MRP to inactivate the hydroperoxides formed by the lipid oxidation. There are also reports on the complex binding of metals by MRP (18, 19). 

Techniques can be classified into two main categories those that detect total metal concentrations and those that detect some operationally defined fraction of the total. Methods which detect total concentrations such as inductively coupled plasma spectrometry, neutron activation analysis, atomic absorption spectrometry and atomic emission spectrometry have no inherent speciation capabilities and must be combined with some other separation technique(s) to allow different species to be detected (approach A in Fig. 8.2). Such separation methods normally fractionate a sample on the basis of size, e.g. filtration/ultrafiltration, gel filtration, or a combination of size and charge, e.g. dialysis, ion exchange and solvent extraction (De Vitre et al., 1987 Badey, 1989b Berggren, 1989 1990 Buffle et al., 1992). In all instances the complexes studied must be relatively inert so that their concentrations are not appreciably modified during the fractionation procedure. 

The comparison of a number of dialytic extracts with the parent coals is given in Table 1L These results indicate that the elemental composition of the dialytic extract closely mirrors that of the organic fraction of the coal. Similar conclusions were reached when coal liquids were separated via the dialytic method. The conclusion that dialysis does not concentrate any particular compound type deserves further investigation, since obtaining a representative sample is crucial to the utility of the method. In Table H, it is particularly interesting to note that in each case the "organic sulfur 1 from the classical coal analysis is almost identical to the sulfur content directly determined on the dialytic extract. 

At the close of the dialysis the plasma was centrifuged to remove small amounts of precipitate and the clear plasma resulting was subjected to starch-block preparative zone electrophoresis by the technique of Kunkel and Slater (15). At the close of the electrophoresis 1-cm. segments of the starch block were cut and transferred to sintered glass funnels, and the proteins were quantitatively eluted with five successive 2-ml. aliquots of 0.9% sodium chloride. The filtrates containing the protein fractions were then made up to a constant volume and mixed, and small aliquots were analyzed for total protein content by the method of Lowry et al. (18). 

Florisil columns are used for the separation of lipids and the target compounds [1, 4, 7]. Zook et al. [9] used a dialysis technique with a polyethylene film for the removal of lipids, followed by gel permeation chromatography (GPC), using S-X3 Bio beads with dichloromethane/n-hexane (50 50, v/v), carbon column chromatography, and florisil columns. De Boer et al. [8] have tried to avoid the use of florisil, because of the extensive pre-treatment and its relative instability. They used GPC, Bio beads S-X3 with dichloromethane/ m-hexane (50 50, v/v) for the separation of lipids and TCPM and TCPMe. The GPC elution was carried out twice and was followed by a silica gel column fractionation to separate TCPM and TCPMe from the PCBs. The recovery of a TCPM spike in a seal blubber extract in this method was 90%. 

DON At present, there are three basic approaches used to isolate DON for tracer experiments wet chemical isolation, ion retardation or dialysis, described above. The suitability of a method is judged by its ability to remove aU of the DIN forms (NH4+, NOs", and N02 ) and to isolate the DON pool with high efSciency. Ultrafiltration is not a suitable isolation method for use in determining DON release rates because it only isolates the higher MW fraction and thus allows the LMW moieties, which are likely important short-term release products, to be lost. 

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