Some Important Buffers

A different procedure to avoid pH changes and also loss of potential over the baffles is to add fresh buffer continuously to the electrode vessel, which has an overflow. Thus the electrodes lie far nearer the paper without an interposed baffle system. Grabar introduced this method for agar gel electrophoresis (G9). We ourselves use a pump to circulate the buffer continuously over the two electrodes, catching the overflow in one common reservoir, where it mixes and then returns to circulation. This system proved particularly useful in continuous two-dimensional electrophoresis, as will be seen in Part II. But it will also help in the building of easily standardized electrophoretic chambers, necessary for standardization of clinical work on electrophoresis of proteins. 

The Electrical Field. At this point it is important to emphasize the difference between the potential applied at the electrodes and the potential applied to the strip itself. It is also important to remember that because of evaporation the concentration and therefore conductivity of the buffer increase and that because of the field itself ionophoresis also modifies conductivity. As a result the potential differences per centimeter are not identical everywhere on the strip but vary according to the local changes in conductivity. 

A supplementary change in conductivity is introduced by the application of the sample which often contains quantities of ions, large enough to modify seriously the original conductivity of the buffer. 

It can be seen (Fig. 21) that there is a collapse of field strength where the sample is applied if it contains too many conducting ions. This is of course responsible for bad separation, as mobility decreases with increased ionic strength and velocity is impaired by the low field, until the ions are moved on by ionophoresis. If evaporation is rapid the whole sample will be pushed forward without any separation, as it is paralyzed by its own high conductivity and high ionic strength. Only after iono- 

There is another important factor, namely the buffer flow inside the buffered substrate, which influences migration. This flow has three main sources evaporation partially caused by heat production from electrical energy the hydrodynamic result of the level of the buffer vessels with respect to one another and to the level of the paper and the electro-endosmotic effect, the interaction between the electrical forces and the buffered substrate. 

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This difference in behavior for acetic acid in pure water versus water buffered at pH = 7 0 has some important practical consequences Biochemists usually do not talk about acetic acid (or lactic acid or salicylic acid etc) They talk about acetate (and lac tate and salicylate) Why Its because biochemists are concerned with carboxylic acids as they exist in di lute aqueous solution at what is called biological pH Biological fluids are naturally buffered The pH of blood for example is maintained at 7 2 and at this pH carboxylic acids are almost entirely converted to their carboxylate anions... 

These same dependencies will, in general, apply to the heat of ionization of the buffer acid, AH. Thermodynamic quantities, namely, AH°, have been reported for some buffer substances, and it is found that A//° is temperature dependent. Bates and Hetzer studied the temperature dependence of for the important buffer tris(hydroxymethyl)aminomethane (TRIS), finding... 

This chapter starts with a simplified analysis of biological processes using the basic tools of physics, chemistry, and thermodynamics. It provides a brief description of mitochondria and energy transduction in the mitochondrion. The study of proper pathways and multi-inflection points in bioenergetics are summarized. We also summarize the concept of thermodynamic buffering caused by soluble enzymes and some important processes of bioenergetics using the linear nonequilibrium thermodynamics formulation. 

Boughtflower, R. J., Underwood, T., and Paterson, C. J. (1996) Capillary electrochromatography—some important considerations in the preparation of packed capillaries and the choice of mobile phase buffers. Chromatographia 40, 329-335. 

The interlocking nature of these reactions on a global scale has some important consequences for the composition of natural waters. For example, the concentrations of the major dissolved constituents (Ca , Mg, Na", Cl", SO/ , HCOa", SiOsfa, ) vary over a rather narrow range because of the buffering action of these reactions. (See Fig. 1-2.) For the same reason, the composition of the ocean is fairly constant with respect to these major dissolved components. 

The choice of a buffer is critical to obtaining successful CE separation of the analytes. Once the optimal wavelength for detection has been established, a buffer must be selected that does not interfere with the ability to detect the analytes of interest, maintains solubility of the analytes, maintain buffering capacity through the analysis, and produces the desired separation. These topics are covered in more detail in the following sections, with examples highlighting the importance of each parameter. Refer to Appendix 3 for some exemplary buffer systems and associated conditions for separation of various analytes. 

In an earlier study, we pointed out some important aspects of aminolysis of thioester in our enzyme model. First, the fastest rate for aminolysis of thioester was obtained in the presence of equimolar amounts of acid and base catalysts. Second, the reaction proceeded in aprotic nonpolar solvents such as benzene, ethyl acetate, dichloromethane, and so on [7]. Thus, the peptide syntheses by the enzyme model have been performed in benzene buffered with equimolar amounts of pivalic acid and triethylamine as acid and base catalysts, respectively. Third, the superiority of intramolecular aminolysis over an intermolecular one was clearly demonstrated, despite the large membered cyclic intermediate expected for the intramolecular reaction. The host 10 could achieve the synthesis of the tetrapep-tide derivative (11) by formal turnover of the intra-complex thiolysis and the intramolecular aminolysis, but its efficiency as an enzyme model has remained to be improved [3]. 

How Important buffers are for normal functioning of a body can be understood from the fact that the pH of blood is maintained strictly within the range 7.3 to 7.5. Death is more or less certain below a pH of 7.0 and above a pH of 7.9. In the laboratory, buffers are used for two main purposes (i) as reference standards for pH determination, and (ii) to maintain optimum acid-base reaction of a medium such as bacteria or tissue culture or an enzymatic reaction mixture. We will discuss more about some important biological buffers in a later section. 

Flatbed gel electrophoresis (GE) is used extensively in the analysis and characterization of proteins with a variety of protocols such as isoelectric focusing (lEF) and polyacrylamide gel electrophoresis (PAGE), but has not been specifically developed for the separation of metal-containing proteins. Therefore, before using the method for speciation of biological samples, some important parameters have to be checked. It has to be investigated in detail if speciation data are hampered by contamination or alteration of native species due to the influence of the electric field and/or denaturing buffer systems. ... 

The production of H+ ions, if left unchecked, would lower the pH of the blood and cause acidosis . This may disrupt some body functions and eventually lead to coma. The equilibrium between carbon dioxide and hydrogencarbonate is the most important buffering system in the blood. 

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