Homogeneous Fractions

Takayama and coworkers (60) introduced the h.p.l.c. separation technique for such amphiphilic molecules as lipid A, and in earlier experiments they applied paired-ion reverse-phase h.p.l.c. for the preparation of homogeneous fractions deriving from 4,-monophosphated lipid A of S. typhimur-ium. The purified preparations obtained were suitable for f.a.b. - m.s. analysis. However, monophosphated lipid A isolated in this way expressed a considerable heterogeneity with respect to the number and location of 0-acyl residues (60). In order to further improve the purification procedure, as well as to obtain lipid A derivatives suitable for n.m.r. spectroscopy, Qureshi et al. (174) prepared the dimethyl phosphate derivative of S. minnesota (R595) lipid A, which, after purification by reverse-phase h.p.l.c. (C18), could be analyzed by1 H-n.m.r. The n.m.r. spectrum of, for example, the heptaacyl lipid A dimethyl monophosphate fraction, unequivocally revealed 0-acyl substitution [14 0(3-OH)J at position 3 and a free hydroxyl group at position 4 of GlcN(I). 

Figure 2. Distribution of marker enzymes and DEHP-metabolizing enzymes in trout liver homogenate fractions. DEHP esterase and DEHP oxidase were each measured by 1-hr incubations of 0.010 ftmol of UC-DEHP in a total volume of 2 mL. Fraction (A), 2,000 g pellet (B), 10,000 g pellet (C), 100,000 g pellet and (D), 100,000 g supernatant. Relative Specific Activity = percent of total activity/ percent of total protein (14).

Fractionation and Analysis of the Lubricant Fraction. Because of the greater complexity of the lubricant fraction and the lack of sufficient quantity of material, the resolution of the lubricant fraction of the Ponca, Okla., petroleum under investigation was carried to the stage of homogeneous fractions, each containing molecules of the same size and type. 

The quantity of each of the final homogeneous fractions of both in water-white oil and extract oil portions was about 15 grams, representing V oooth part of the original crude petroleum from which it came, and consisted of compounds of substantially similar sizes and types. These fractions, although far from being pure compounds, appear to be nearer to pure compounds than any material (except n-paraffin hydrocarbons) hitherto separated from the lubricant fraction of any crude petroleum. 

Effect of Varying the Concentration of a Water Homogenate of Normal Adult Mouse Liver, Buffered with Acetate, During the Incubation with Substrate (0.00126 M Phenolphthalein fl-v-Glucosiduronic Acid).33 Homogenate Fractionated Before and After Incubation at 88°, and then Assayed... 

In short, cell fractionation, which is composed of three steps—homogenization, fractionation, and analysis—can be an excellent way to locate an enzyme within the cell. The reader is referred to the excellent commentary by ap Rees (1995), in which the rigorous criteria to follow so that a cell fractionation provides good, reliable information is summarized. The author concludes that work done on soybean protoplasts (Macdonald and ap Rees, 1983), wheat endosperm protoplasts (Entwistle and ap Rees, 1988), wheat endosperm (Tetlow et al., 1993), pea embryos (Denyer and Smith, 1988), and pea roots (Borchert et al., 1993) provides further support for the view that ADPGlc PPase essentially is confined to the plastid. 

Another option for the analysis of complex polymers is the separation with respect to chemical composition or functionality by means of interaction chromatography. In this case, functionally or chemically homogeneous fractions are obtained which can then be subjected to molar mass determination. 

The classical approach is based on the dependence of copolymer solubility on composition and chain length. A solvent/nonsolvent combination fractionating solely by molar mass would be appropriate for the evaluation of MMD, another one separating with respect to chemical composition would be suited for determining CCD or FTD. However, in reality, precipitation fractionation yields fractions which vary both in chemical composition and molar mass. Even high resolution fractionation would not improve the result. Narrower fractions can be obtained by cross-fractionation separating in two different directions. However, even in this case, it is almost impossible to obtain perfectly homogeneous fractions. 

Further studies by Badgley and Mark " on relatively homogeneous fractions of a secondary cellulose acetate obtained by controlled precipitation from acetone and methylcellosolve solutions (with methyl alcohol), showed that equations 9, 10 and 11, above, apply over relatively narrow ranges of DP. Above a DP. of about 380, equation 9 gave D.P. values higher than those obtained by osmotic pressure measurements. Modification of equation 11, to the form... 

Based on previous oflF-line results by other authors (11, 12, 14), we anticipated improved resolution of multicomponent analytes. We also hoped for chemically homogeneous fractions to measure MMDs of copolymers and blends correctly. 

In contrast to previous publications, we wanted to create chemically homogeneous fractions in the first separation and work on these for molar mass characterization. Therefore, HPLC (or LACCC) was the first and SEC the second separation method. The advantages of this setup are numerous. LC has more parameters (gradient, stationary phase, etc.) to adjust the separation according to the chemical nature of the... 

During self-similar growth of the model, the similarity dimension of the statistically homogeneous fraction that now arises can be estimated right at the percolation threshold from... 

Application of solubility parameter data is hindered by the multicomponent nature of humic substances, and homogeneous fractions are needed to obtain the necessary data for the macromolecules. Nevertheless, the available information shows that the best solvents for humic acids have polar,... 

The purposes of this chapter are (1) to present an overview of fractionation methods that have been successfully applied to aquatic humic substances (2) to examine chemical and physical fractionation mechanisms in the light of what is known about aquatic humic substance properties and structure (3) to postulate new fractionation approaches that hopefully will result in more homogeneous fractions and, ultimately, pure compounds that comprise aquatic humic substances. 

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