Buffer strength standard

Standardized assay conditions across all CYP enzymes are recommended because buffer strength, composition, and pH can affect enzyme kinetics. 

Adequate buffer strength was important for peak shape and sensitivity. Initially, a buffer of 0.01 M phosphate was used. This buffer gave a reasonable peak shape for a small volume of standard compound, but distorted peaks, and hence, poor limits of detection were observed for tobacco samples. A possible source for this peak distortion could be the presence of a high concentration of NaCl (e.g., approximately 5 M) produced from the acid-base neutralization, which caused slower solute diffusion in the injected solution than in bulk mobile phase. This was further confirmed by obtaining a similarly distorted peak from a standard solution of MH which was 6 M in NaCl. The peak shape of MH in tobacco samples was improved by using a stronger buffer (0.04 M phosphate). This also greatly reduced other possible causes of peak distortion, one of which may be that the pH of the neutralized tobacco solution may not be neutral. 

Variability in the liquid junction potential (Section 15-2) was hypothesized to lead to the variability in the pH measurements. Standard buffers used for pH meter calibration typically have ion concentrations of -0.05 M, whereas rainwater samples have ion concentrations two or more orders of magnitude lower. To test the hypothesis that junction potential caused systematic errors, a pure HCl solution with a concentration near 2 X lO"" M was used as a pH calibration standard in place of high ionic strength buffers. The data in panel b were obtained, with good results from all but the first lab. The standard deviation of all 17 measurements was reduced from 0.077 pH unit with the standard buffer to 0.029 pH unit with the HCl standard. It was concluded that junction potential is the cause of most of the variability between labs and that a low ionic strength standard is appropriate for rainwater pH measurements. 

Another approach to matrix matching, which does not rely on knowing the exact composition of the sample s matrix, is to add a high concentration of inert electrolyte to all samples and standards. If the concentration of added electrolyte is sufficient, any difference between the sample s matrix and that of the standards becomes trivial, and the activity coefficient remains essentially constant. The solution of inert electrolyte added to the sample and standards is called a total ionic strength adjustment buffer (TISAB). 

Accuracy and Interpretation of Measured pH Values. The acidity function which is the experimental basis for the assignment of pH, is reproducible within about 0.003 pH unit from 10 to 40°C. If the ionic strength is known, the assignment of numerical values to the activity coefficient of chloride ion does not add to the uncertainty. However, errors in the standard potential of the cell, in the composition of the buffer materials, and ia the preparatioa of the solutioas may raise the uacertaiaty to 0.005 pH unit. 

Direct analysis with the fluoride lon-selective electrode requires addition of total ionic strength adjustor buffer solution (TISAB) to the standard and to unknown samples Some advantages of this addition are that it provides a constant background ion strength, ties up interfenng cations such as aluminum or iron, which form a complex with fluoride ions, and maintains the pH between 5 0 and 5 5 According to the manufacturer s claim, reproducibility of direct electrode measurement IS 2 0%, and the accuracy for fluonde ion measurement is 0 2% [27]... 

A 10 mM ionic strength universal buffer mixture, consisting of Good zwitterio-nic buffers, [174] and other components (but free of phosphate and boric acid), is used in the pION apparatus [116,556], The 5-pKa mixture produces a linear response to the addition of base titrant in the pH 3-10 interval, as indicated in Fig. 7.53. The robotic system uses the universal buffer solution for all applications, automatically adjusting the pH with the addition of a standardized KOH solution. The robotic system uses a built-in titrator to standardize the pH mapping operation. 

It is often more convenient to relate the potentiometer reading directly to concentration by adjusting the ionic strength and hence the activity of both the standards and samples to the same value with a large excess of an electrolyte solution which is inert as far as the electrode in use is concerned. Under these conditions the electrode potential is proportional to the concentration of the test ions. The use of such solutions, which are known as TISABs (total ionic strength adjustment buffers), also allows the control of pH and their composition has to be designed for each particular assay and the proportion of buffer to sample must be constant. 

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