Other Methods of Fractionation

So far we have considered only the newer countercurrent methods for fractionating peptides. These will probably play a predominant role in the future, though the classical methods of fractional crystallization and precipitation should not be forgotten. They are still the most effective methods of fractionating proteins and probably larger polypeptides, such as the oxidation products of insulin (Sanger, 1949a). 

The aromatic sulfonic acids, which were developed by Bergmann for the specific precipitation of amino acids, have also been used for the separation of peptides from a partial hydrolyzate of silk (Stein et al., 1944). 

Many of the methods considered above have not been extensively used for the fractionation of peptides, so that it is impossible to know how far they may be applied and which are the most effective methods. Also it is to be expected that considerable improvements in these techniques will take place and that other new methods will be devised in the near future. 

The properties of amino acids and small peptides render them suitable to fractionation by methods employing partition chromatography. Paper chromatography is especially to be preferred for the final fractionation of a simplified peptide mixture because of the good resolutions obtained, the ease and rapidity of technique, and the possibility of using 

For the preliminary separation of a complex protein hydrolyzate into simpler peptide mixtures, ionophoretic methods are probably the most generally useful. Aromatic peptides may be separated by adsorption on charcoal and cystine peptides by oxidation. 

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Other methods of fractionation which utilize the presence of ionizable groups, either natural (such as carboxyl or amino groups), or induced (such as borate complexes), may be useful. Such methods have been discussed in reviews.248 2W Complexation (for example, with Fehling solution) was found useful in the study of a polymannose.216 An insoluble complex was formed, as with natural mannans. 

Other methods of fractionation with supercritical fluids are conceivable. One such possiblility could involve manipulation of the pressure during the destraction or upon subsequent separation of the fluid and residuum. The relative merits of such possibilities remain to be explored. Successful development of such technology will result in the ability to fractionate and characterize material currently intractable by conventional methods. 

It was pointed out initially that the various PEG grades are not perfectly uniform chemical compounds rather, they are mixtures of similar polymer species. Information on the molecular weight distribution of PEG grades is given by gel permeation chromatography or by other methods of fractionation [25—27]. 

The use of other methods of fractionation of histones (car-boxymethylcellulose, Amberlite IRC-50, DEAE-cellulose) has also... 

Electroosmotic flow in a capillary also makes it possible to analyze both cations and anions in the same sample. The only requirement is that the electroosmotic flow downstream is of a greater magnitude than electrophoresis of the oppositely charged ions upstream. Electro osmosis is the preferred method of generating flow in the capillary, because the variation in the flow profile occurs within a fraction of Kr from the wall (49). When electro osmosis is used for sample injection, differing amounts of analyte can be found between the sample in the capillary and the uninjected sample, because of different electrophoretic mobilities of analytes (50). Two other methods of generating flow are with gravity or with a pump. 

Principles and Characteristics Fractional solution procedures usually consist of consecutive extractions with solvents of increasing solvent power. These labour intensive methods benefit from a larger surface area to mass ratio. Other methods for fractionation by solubility rely on fractional precipitation through addition of a nonsolvent, lowering the temperature or solvent volatilisation (Section 3.7). 

Separation depends on the selection of a process in which the behaviour of the material is influenced to a very marked degree by some physical property. Thus, if a material is to be separated into various size fractions, a sieving method may be used because this process depends primarily on the size of the particles, though other physical properties such as the shape of the particles and their tendency to agglomerate may also be involved. Other methods of separation depend on the differences in the behaviour of the particles in a moving fluid, and in this case the size and the density of the particles are the most important factors and shape is of secondary importance. Other processes make use of differences in electrical or magnetic properties of the materials or in their surface properties. 

Optical counters allow relatively rapid measurements of the size distribution and, unlike some of the other methods of size fractionation, include volatile particles in the measurement. However, some care must be taken in interpreting the detailed shape of the size distribution spectrum because of some anomalies that have been observed for example, around the l-/xm region, interference from light that is reflected or refracted from the front and back of the particle gives a knee in many calibration curves of number of particles versus their diameter (LBL, 1979). 

In other experiments, the opposite method of fractionation by dissolution was applied. In this way G. Meissner [107] prepared a soluble fraction with a nitrogen content of 10.28%, in quantity about 4% by extracting a specimen of nitrocellulose with 12.17% N using 50 50 ether-alcohol. The insoluble part was composed of nitrocellulose of 12.32% N. 

In 1905 Hall and Smith 2 investigated all the then known methods for the removal of titanium, and tried various other processes they were unable, however, to improve on Marignac s method of fractional recrystallisation of the double potassium fluorides.3 This method has the disadvantage that in the case of the niobium salt protracted and tedious repetition is necessary before it is obtained free from titanium, and the method becomes impossible with small quantities of material.4... 

Many proliferation-associated antigens have been reported as clinically useful indicators of proliferative activity (1). Of these, the so-called proliferating cell nuclear antigen (PCNA) and Ki-67 have been identified as the most useful in both immunohistochemistry (see Chapter 27) and flow cytometry (FCM). PCNA is an auxiliary protein to DNA polymerase 8 (2,3) and is intimately associated with DNA replication, but also DNA repair (4,5). Ki-67 is a large protein associated with nuclear nonhistone proteins (6,7), and is expressed in all actively proliferating cells (8,9). Expression of these two proteins, in a cell population should equate to the growth fraction, i.e., the proportion of cells involved in an active cell cycle. However, there are apparent inconsistencies when these two proteins have been compared with one another (10) and with other methods of assessing cell proliferation (11). 

A few other methods of determining the ring content of saturated mineral oil fractions will be discussed further on in connection with the application of the viscosity in the field of graphical statistical methods. 

However, heavy crude oil and bitumen are extremely complex and very little direct information can be obtained by distillation. It is not possible to isolate and identify the constituents of the heavier feedstocks using analytical techniques that rely upon volatility. Other methods of identifying the chemical constituents must be employed. Such techniques include a myriad of fractionation procedures (Chapter 3) as well methods designed to draw inferences about the hydrocarbon skeletal structures and the nature of the heteroatomic functions. 

The lines in Fig. 7.4 are the results of theoretical calculations, using models of the respiratory tract (Yu Diu, 1982). The points are measurements with radioactive aerosols. Numerous other determinations of fractional deposition in the whole tract have been made, using non-radioactive methods to count the number of particles in the inhaled and exhaled air (Heyder et al., 1986 Schiller et al., 1988). Fractional deposition is least for particles of about 0.2 to 0.5 m diameter. Table 7.1 shows that the combined effect of sedimentation and Brownian motion is then at a minimum. 

A proper method of fractionation is indispensable in the study of the composition of coal-derived liquids (CDL). Data in Table II show large differences between solvent extraction and distillation as fractionation methods. Two fractions, ES and EI-AS, from ethanol extraction exhibited little differences from each other, while two fractions from distillation of ES revealed marked differences in molecular weight, H/C ratio, hydroxyl group content and physical appearance. 

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