Buffer Composition-Primary

Although there are numerous types and compositions of buffers, three common primary buffers are most frequently used. The word primary implies to be preferred over other buffers of similar pH value and affording greater accuracy. These buffers are the pH 4.01 phthalate buffer, the pH 6.86 phosphate buffer, and the pH 9.18 borax buffer. 

The pH 4.01 buffer has a composition of 0.05 m potassium hydrogen phthalate (KHC8H4O4). It has a relatively low buffering capacity but is stable to dilution and change in temperature. It should be prepared with C02 free distilled water. It is subject to mold growth but is normally inhibited by fungicide. 

The pH 6.86 buffer has a composition of 0.025 m potassium dihydrogen phosphate (KH2PO4) and 0.025 m disodium hydrogen phosphate (Na2HP04). It is fairly stable to contamination and moderately affected by temperature and dilution. 

The pH 9.18 buffer has a composition of 0.01 m sodium tetraborate decahydrate (Na2B4O7-10H2O). In solution this salt hydrolyzes to form boric acid and sodium borate. It has good stability toward 

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Provide a reference or direct experimental proof that the conditions chosen do provide initial rate measurements. At a minimum, the percentage of substrate consumed during the course of an initial rate determination should be specified. One should also show that under initial rate conditions a doubling of enzyme concentration should exactly produce a doubling in the observed initial rate. Likewise, if an auxiliary enzyme assay is used to monitor the primary enzyme s activity the observed rate at low or high substrate concentration should not depend on the concentration of additional enzyme(s), substrate(s), or factors used for the coupled assay. 2. Describe all assay conditions (eg., concentrations of substrates, products, inhibitors, and/or activators enzyme concentration temperature pH and buffer composition ... 

Since fluorescent dyes are rather expensive, it is useful to test different conditions like temperature, incubation time, and buffer composition for the labeling reaction at small scale. In any circumstance for labeling of cysteine residues DTT and azide should be avoided. For the labeling of lysine residues primary amines must be avoided, and it has proved useful to polymerize actin before labeling to avoid modification of residues involved in polymerization. 

The running buffer composition is of primary importance to CE selectivity optimization. The buffer contains at least one anion and one cation, and one of these ions should have an adequate buffering capacity. If possible, the EOF should have the same direction as the migration of the analytes to shorten the analysis. The co-ion should have a similar mobility as the analyte to ensure a good peak shape. 

There are many different characteristics which determine if a particular solution composition will qualify as a buffer. The primary parameters are the buffering capacity, the effect dilution has on the buffer, and the pH change with temperature. Other characteristics which must be considered are shelf life, growth of mold, and absorption of carbon dioxide. 

Many other buffers have been developed for various purposes. Generally, these alternate compositions are less stable or accurate than the primary buffers described the the National Bureau of Standards. They are often employed when special conditions must be met or when the buffer is more compatible with the sample. Some of these buffers are described in Section 4.4. One scheme of buffer compositions which cover the pH range 1-10 has been developed by Clark and Lubs. These are described in Table A.3 and, generally, with others listed in the Handbook of Biochemistry [5]. 

Calibrating the electrode presents a third complication since a standard with an accurately known activity for H+ needs to be used. Unfortunately, it is not possible to calculate rigorously the activity of a single ion. For this reason pH electrodes are calibrated using a standard buffer whose composition is chosen such that the defined pH is as close as possible to that given by equation 11.18. Table 11.6 gives pH values for several primary standard buffer solutions accepted by the National Institute of Standards and Technology. 

Since the principal hazard of contamination of acrolein is base-catalyzed polymerization, a "buffer" solution to shortstop such a polymerization is often employed for emergency addition to a reacting tank. A typical composition of this solution is 78% acetic acid, 15% water, and 7% hydroquinone. The acetic acid is the primary active ingredient. Water is added to depress the freezing point and to increase the solubiUty of hydroquinone. Hydroquinone (HQ) prevents free-radical polymerization. Such polymerization is not expected to be a safety hazard, but there is no reason to exclude HQ from the formulation. Sodium acetate may be included as well to stop polymerization by very strong acids. There is, however, a temperature rise when it is added to acrolein due to catalysis of the acetic acid-acrolein addition reaction. 

As there can be significant variations in the purity of samples of a buffer of the same nominal chemical composition, it is essential that the primary buffer material used has been certified with values that have been measured with Cell I. The Harned cell is used... 

The transformation of micas and feldspars buffers the formation of acidic solutions by increasing the concentration of K , Fe, Mg, Al and Si in the pore waters. These pore solutions are not displaced and the mineral authigenesis is controlled by a local equilibrium. Under these conditions the primary mineral composition of the deposit controls the distribution of the cement. 

The primary components of LCM technology are (1) visualization of the cells of interest through microscopy, (2) transfer of near-infrared laser energy pulses to a thermolabile polymer with formation of a polymer-cell composite, and (3) removal of the polymer from the tissue surface, which shears the embedded cells of interest away from the heterogeneous tissue section (18,19). Extraction buffers applied to the polymer film solubilize the cells, liberating the molecules of interest. The DNA, RNA, or protein from the microdissected cells may be analyzed by any method with appropriate sensitivity (20,21,22,23,24). Protein extracted from microdissected cells may be used for mass spectrometric analysis, applied to reverse phase protein microarrays, or used for western blot analysis (25,26). 

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