Standard buffers

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. 

The measurement of pH using the operational ceU assumes that no residual Hquid-junction potential is present when a standard buffer is compared to a solution of unknown pH. Although this may never be stricdy tme, especially for complex matrices, the residual Hquid-junction potential can be minimised by the appropriate choice of a salt-bridge solution and caHbration buffer solutions. 

Prepare the solutions and measure the pH at one temperature of the kinetic study. Of course, the pH meter and electrodes must be properly calibrated against standard buffers, all solutions being thermostated at the single temperature of measurement. Carry out the rate constant determinations at three or more tempertures do not measure the pH or change the solution composition at the additional temperatures. Determine from an Arrhenius plot of log against l/T. Then calculate Eqh using Eq. (6-37) or (6-39) and the appropriate values of AH and AH as discussed above. 

Details for the preparation of the solutions referred to in the table are as follows (note that concentrations are expressed in molalities) all reagents must be of the highest purity. Freshly distilled water protected from carbon dioxide during cooling, having a pH of 6.7-7.3, should be used, and is essential for basic standards. De-ionised water is also suitable. Standard buffer solutions may be stored in well-closed Pyrex or polythene bottles. If the formation of mould or sediment is visible the solution must be discarded. 

For most purposes it is not necessary to follow the procedures given above for the preparation of standard buffer solutions the buffer tablets which are available from laboratory suppliers, when dissolved in the specified volume of distilled (de-ionised) water, produce buffer solutions suitable for the calibration of pH meters. 

Before the pH electrode is used, it should be calibrated using two (or more) buffers of known pH. Many standard buffers are commercially available, with an accuracy of 0.01 pH unit. Calibration must be performed at the same temperature at which the measurement will be made care must be taken to match the temperature of samples and standards. The exact procedure depends on the model of pH meter used. Modem pH meters, such as the one shown in Figure 5-8, are microcomputer controlled, and allow double-point calibration, slope calculation, temperature adjustment, and accuracy to 0.001 pH unit, all with few basic steps. The electrode must... 

In sugar refinery control operations, pH electrodes should not be, as unfortunately they sometimes are, calibrated with standard buffer solutions, then placed on stream in sugar liquors, and assumed to read equivalent pH. Implicit in this operation is the equivalence of pH electrode response in dilute aqueous buffers at 24°C and in high Brix sugar solutions at elevated temperatures. Such equivalence does not exist. 

Isolated chromaffin cells were maintained in suspension culture and loaded with the fluorescent calcium indicator Fura 2 as previously described (28). 2 x 10 cells/ml were added into a cuvette containing standard buffer without (dotted line) or with (full line) 2 mM calcium. At the arrow, 10" M pardaxin was added. A rise in was... 

Pectolytic activity was also studied in batch reactors, following the reaction progress in thermostated quartz cuvettes. The reaction medium (3 cm ) was prepared with 1.5 g/L pectin in the standard buffer and 0.063 mg of enzyme. The absorbance of the reaction mixture against the substrate blank was continuously recorded at the spectrophotometer (Perkin Elmer Lambda 2, USA). Typical reaction time was 15 minutes, but initial reaction rates were estimated considering only the absorbances recorded during the first 200 seconds, range of totally linear response. 

The important question arises of the actual precision of pH measurement in analytical control. In this connection, it has become common practice to standardize pH determinations, on standard buffer solutions with pH regions where the pH of the solution under test is to be expected. As currently commercially available pH meters, pH electrodes and buffer solutions are of outstanding quality, the reliability of the pH measurement becomes shifted to the performance of the measuring electrochemical cell here as first principle the same cell should be used for the test solution and the standard solution, so that according to the Bates-Guggenheim convention... 

Solubility measurement at a single pH [37-39] under equilibrium conditions is largely a labor-intensive procedure, requiring long equilibration times (12h-7 days). It s a simple procedure. The drug is added to a standard buffer solution (in a flask) until saturation occurs, indicated by undissolved excess dmg. The thermostated saturated solution is shaken as equilibration between the two phases is established. After microfiltration or centrifugation, the concentration of the substance in the supernatant solution is then determined using HPLC, usually with UV detection. If a solubility-pH profile is required, then the measurement needs to be performed in parallel in several different pH buffers. 

Gelsema, W. J. de Ligny, C. L. Remijnse, A. G. Blijleven, H. A., pH-measurements in alcohol-water mixtures, using aqueous standard buffer solutions for calibration, Reel. Trav. Chim. Pays-Bas 85, 647-660 (1966). 

Various chromogenic reagents have been used for the spectrophotometric determination of boron in seawater. These include curcumin [108,109], nile blue [110], and more recently 3,5 di-tert butylcatechol and ethyl violet [111]. Uppstroem [108] added anhydrous acetic acid (1 ml) and propionic anhydride (3 ml) to the aqueous sample (0.5 ml) containing up to 5 mg of boron per litre as H3BO3 in a polyethylene beaker. After mixing and the dropwise addition of oxalyl chloride (0.25 ml) to catalyse the removal of water, the mixture is set aside for 15-30 minutes and cooled to room temperature. Subsequently, concentrated sulfuric-anhydrous acetic acid (1 1) (3 ml) and curcumin reagent (125 mg curcumin in 100 ml anhydrous acetic acid) (3 ml) are added, and the mixed solution is set aside for at least 30 minutes. Finally 20 ml standard buffer solution (90 ml of 96% ethanol, 180 g ammonium acetate - to destroy excess of protonated curcumin - and 135 ml anhydrous acetic acid diluted to 1 litre... 

Standardization of pH meters is essential and must be done on a regular basis. The starting point in standardization is to adjust the meter and electrodes using a standard buffer solution of pH 7.00 and adjusting the meter to this pH. The second step is to set a second point, which is in the range of pH levels expected to occur in the measurements. Thus, if the pH values are all expected to be acidic, the second point will commonly be set using a buffer of 4.01 if they are expected to be basic, then a pH 10.00 buffer will be used. If both acidic and basic pH values are likely, the meter can be standardized using all three buffers. 

Standard buffers can be purchased as prepared solutions or as powders that are dissolved in distilled or deionized water. In the latter case, the powder is typically dissolved in 100 mL of distilled or deionized water for use. Buffers may be color-coded such as green or yellow for pH 7.00, pink for pH 4.01, and blue for pH 10.00. 

For example, to prepare a pH = 9 buffer solution, one would prepare a solution of ammonium chloride (refer to Table 5.1), and then add a solution of sodium hydroxide while stirring and monitoring the pH with a pH meter. The preparation is complete when the pH reaches 9. The required conjugate acid-base pair would be NH3 - NHj. Recipes for standard buffer solutions can be useful. Table 5.2 gives specific directions for preparing some popular buffer solutions. 

TABLE 8 Standard Buffer Recipes for Initial Experiments... 

Most determinations may be performed using standard buffers without any changes. However, in some cases when analytes co-migrate, modification may be required. 

The pH values derived by the standard buffers are temperature dependent. The pH values between 0 and 60 °C are given in Table 7.5, which also indicates the change of pH at 1 1 dilution (ApHi i). 

V. Baelum, O. Fejerskov, A. Kuseler, Approximal plaque pH follow/ing topical applications of standard buffers in vivo. Caries Res. 28 (1994) 116-122. 

Since phosphate and borate buffers are well described in the literature, the power of CZE in peptide chemistry will be illustrated with two examples that emphasize less conventional conditions than those offered by the manufacturers, such as using TEAP solvent systems or standard buffers in the presence of MeCN. These were utilized for some unusual separations. 

Under these reducing conditions of hydrolysis of tryptophan peptides, cystine is reduced to cysteine and its coelution with proline using standard buffer gradients, makes quantitation difficult. Thus, cysteine and cystine are generally derivatized prior to acid hydrolysis by oxidation to cysteic acid with performic acid 21 or alkylation, upon reduction in the case of cystine, with iodoacetic acid 21 or, more appropriately, with 4-vmylpyridine)22 23 50 Conversion of cysteine into 5- 3-(4-pyridylethyl)cysteine bears the additional advantage of suppressing epimerization via the thiazoline intermediate, thus allowing for standardization of the acid-hydrolysis dependent racemization of cysteine in synthetic peptides)24 ... 

The pH of the standard buffer and sample can vary with temperature due to shifts in their chemical equilibrium. Since the actual pH of the most common... 

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