Buffers calibration

The difference in the conductivity of the calibration buffers and sample can cause a very large error on the sample measurement, due to junction potentials in different environments. Solid samples should be dissolved in purified water. It is necessary that the water be carbon dioxide-free. The presence of dissolved carbon dioxide will cause significant bias in the measurement of samples with low buffering capacity. For pH measurements with an accuracy of 0.01 to 0.1 pH unit, the limiting factor is often the electrochemical system (i.e., the characteristics of the electrodes and the solution in which they are immersed). 

The pH measurement system is calibrated against primary standard buffers admitted either manually or automatically into the sample chamber. The buffers are phosphate solutions that should meet National Institutes of Standards and Technology (NIST) specifications. Calibration buffers meeting NIST specifications are available from the manufacturer of an instrument, usually in containers of appropriate size and shape for mounting as a reservoir on the instrument. The pH values of the low and high calibrator buffers axe set by the manufacturer but always lie close to 6.8 and 7.4 at 37 °C. The tolerance of the specified values should be less than or equal to an SD of 0.003 to achieve SDs of 0.005 to 0.01 in measuring blood pH. Unopened containers should be stored at room temperature. When visual observation or an instrument display warns of low concentrations in the reservoir, recommended practice is to replace the reservoir with a newly opened container rather than to replenish fluid in the current one. Pooling several almost empty containers is not recommended. 

The pH adjustment step of MPA might also pose potential problems. The pH calibration buffers often contain a preservative that might contaminate MPA if the pH electrode is dipped during pH adjustment (see Figure 5.14). This problem can be eliminated by pouring out small aliquots into separate containers to check pH. 

Prepare calibration solutions with defined free [Ca ] by adding aqueous CaCl to the calibration buffer and adjusting the pH to 7.0. Use the association constant 4.831 x lO " M for Ca-EGTA at an ionic strength of 100 mM see Note 13). 

It is critical that pH electrodes are calibrated often as the slope of their response curve will change with temperature, as defined by the Nernst equation [2], Calibration should be performed with at least two certified pH calibration buffers that bracket the expected pH of the sample. Calibration is performed as described in the manufacturer s instructions provided with the pH meter. Calibration should be checked by measuring the pH of the first calibration buffer. The result should be no more than 0.02 pH units different from the certified pH of the buffer after adjusted for temperature. pH measurements are best performed when calibration buffers and samples are held at a constant temperature. 

The pH of solid dosage form and API is measured directly in administered solutions or after material is dissolved. The pH of an API is measured after the compound is dissolved completely in a portion of water, and the sample is allowed to come to the same temperature as that of the calibration buffers. If API does not dissolve in water, the compound cannot react with the water molecules creating a new equilibrium of water and hydronium ions. Even small amounts of polar organic solvents alter the true pH measurement. 

The lack of need for a reference electrode and some type of easily fouled liquid junction. Therefore, there is no signal drift. It is calibration free. No calibration buffers required. pH response stored permanently. 

The naming of the total and seawater pH scales is far from consistent in the literature, so that it is advisable to check carefully >Vhich scale is actually used, e.g., from the definition of or from the calibration buffers used. 

The substrates used for lipase assay were tributyrin (C4 0), tricaproin (C6 0), tricaprylin (C8 0), tricaprin (C10 0), tri-laurin (C12 0) from Sigma. Sodium caseinate (Alanate 185) was supplied by the N.Z. Dairy Board, L-a-ledthin was from Sigma, color key pH calibration buffers were from BDH Chemicals and Q-sepharose was from Pharmacia LKB. 

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