Ionic strength protocols

It is not possible to prescribe specific pretreatment procedures here because these can only be decided upon when the system and the purpose of the experiments has been properly defined. However, a wealth of information exist in various biochemical reference books on how to isolate various biological compounds. The recommended techniques and methods could be used as part of the trace element speciation protocol often after slight modification, taking into consideration the following points First, the trace element blank levels have to be low, less than 10% of the total concentration in the sample. Second, the regents used should not interfere with subsequent analytical determinations. Third, the experimental conditions should not deviate markedly from those found in vivo, especially the pH and ionic strength of the medium. 

Trimesic acid, on the other hand, appeared to be so strongly adsorbed to the anion-exchange resin that it was incompletely recovered in the eluate. As discussed earlier, glycine also appeared to bind tightly and to elute incompletely by the standard protocol. Both of these compounds probably could be eluted with aqueous eluents of high ionic strength, but this process would create the problem of recovering the solute from the eluate. Volatile buffers may offer a solution to this problem. 

Craane-van Hinsberg, W.H.M., et al. 1994. Iontophoresis of a model peptide across human skin in vitro Effects of iontophoresis protocol, pH and ionic strength on peptide flux and skin impedance. Pharm Res 11 1296. 

In addition to the preceding complexities, the P450 enzymes have some unique characteristics that complicate the design of experimental protocols. Because of the broad substrate selectivities for these enzymes, the enzymes are not optimized for the metabolism of a particular substrate. Therefore, the reaction conditions (i.e., pH, ionic strength, temperature) that result in optimum velocities for a given reaction are dependent on both the enzyme and the substrate. To further complicate matters, the velocities for these enzymes tend to vary greatly with changes in these reaction conditions. This variation may well be due to the dependence of the reaction velocity on several pathways in the catalytic cycle. 

Appropriate extraction protocols and sample preparation procedures must be well-documented by the developer and presented in a manner clearly understood by someone unfamiliar with the method. The effects of extraction and sample preparation on antibody performance must be determined. Specific areas such as pH, organic solvents, and ionic strength should also be addressed. 

It is possible to refine the above analysis, because the (literature values of the) pKa s of amino acids mostly lie in the range from 1.9 to 2.4, while those of lactic and acetic acid are 3.86 and 4.76 respectively. These numbers are sufficiently far apart to allow their separate numerical analysis. Because of activity effects on both the equilibrium constants and the pH measurements, the precise values of the pKa s depend on the ionic strength of the sample, and it is therefore best to treat the pKa s as adjustable parameters, that are allowed to vary within rather narrow pH ranges. Here, then, is the analysis protocol we will follow initially. We will consider five separate ranges, one each for the amino acids (pH 1.9 to 2.7), lactic acid (3.4 to 4.0) and acetic acid (4.4 to 5.0), plus one for total nitrogen (8.8 to 10.8) and one... 

While this method is commonly used (De Nobih and Fornasier (1994), Kiichler et al. (1994), Mazid (1988), BeUn et al. (1993), Shaw et al (1994), Burba et al (1998), Buffle et al (1978), Crum et al (1996), Aiken (1984), Amy et al (1987), Reinhard (1984), Amy et al (1992), Hepplewhite (1995)), most authors have used different filtration protocols for their fractionation experiments. Cells can be operated in series (cascade) or in parallel, volumes and concentrations are varied, and some authors refill the cell with pure water to keep the cell concentration constant. All these factors influence the results obtained, and solution chemistry, pH, and ionic strength may also influence results. Generally reported size results are above the expected sizes of FA and FIA. UF MWCOs used are usually 0.5-1, 3, 5, 10, 30 kDa. 

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