pH Adjustment Ionic strength differences can arise from the buffer preparation procedure. For example, when preparing a 0. 1 M acetate buffer of pH 4.2, was 0.1 mole of sodium acetate added to 900ml of water, and then titrated to pH 4.2 with acetic acid before bringing to 1L volume? If so, the acetate concentration will be significantly higher than 0. 1M. Or, was the pH overshot, necessitating the addition of dilute Naoh to bring the ph back to target, increas- ing the ionic strength due to excess sodium? The 0.1M acetate buffer might have been prepared by dissolving 0. 1 mole sodium acetate in 1 liter of water, and the ph adjusted to 4.2 with acetic acid. Under these circumstances the final acetate concentration is anyone's guess but it will be different from the first example above The best way to avoid altering the ionic concentration of a buffer is to prepare the buffer by blending the acid and conjugate base in molar proportions based on Henderson-Hasselbalch cal culations such that the pH will be very near the target pH. This solution will then require only minimal ph adjustment. Dilute to within 5% to 10% of final volume, make any final ph adjustment hen bring to volume. Generally, select a strong acid containing a counter-ion already present in the system(e.g, Cl, PO4, and OAc) to adjust a basic buffer. The strength(concentration) of the acid should be chosen so that a minimum(but easily and reproducibly delivered) volume is used to accomplish the ph adjustment. If overshooting the ph target is a problem, reduce the concentration of the acid being used. Likewise. choose a base that contains the cations already present or known to be innocuous in the assay(Na*, K, etc. Solutions of strong acids and bases used for final pH adjustment usually are stable for long periods of time, but not forever. Was the Naoh used for pH adjustment prepared during the last ice age? Was it stored properly to exclude atmospheric CO2, whose presence can slowly neutralize the base, producing sodium bicar bonate(NaHCO3) which further alters the buffer properties and ionic strength of the solution? Buffers from Stock Solutions Stock solutions can be a quick and accurate way to store " buffer precursors:” Preparing10×to100× concentrated buffer salts can simplify buffer preparation, and these concentrated solutions can also retard or prevent bacterial growth, extending almost indefi nitely the shelf stability of the solutions PfannkochpH Adjustment Ionic strength differences can arise from the buffer preparation procedure. For example, when preparing a 0.1M acetate buffer of pH 4.2, was 0.1 mole of sodium acetate added to 900 ml of water, and then titrated to pH 4.2 with acetic acid before bringing to 1L volume? If so, the acetate concentration will be significantly higher than 0.1M. Or, was the pH overshot, necessitating the addition of dilute NaOH to bring the pH back to target, increas￾ing the ionic strength due to excess sodium? The 0.1 M acetate buffer might have been prepared by dissolving 0.1 mole sodium acetate in 1 liter of water, and the pH adjusted to 4.2 with acetic acid. Under these circumstances the final acetate concentration is anyone’s guess but it will be different from the first example above. The best way to avoid altering the ionic concentration of a buffer is to prepare the buffer by blending the acid and conjugate base in molar proportions based on Henderson-Hasselbalch cal￾culations such that the pH will be very near the target pH. This solution will then require only minimal pH adjustment. Dilute to within 5% to 10% of final volume, make any final pH adjustment, then bring to volume. Generally, select a strong acid containing a counter-ion already present in the system (e.g., Cl- , PO4 3+ , and OAc- ) to adjust a basic buffer. The strength (concentration) of the acid should be chosen so that a minimum (but easily and reproducibly delivered) volume is used to accomplish the pH adjustment. If overshooting the pH target is a problem, reduce the concentration of the acid being used. Likewise, choose a base that contains the cations already present or known to be innocuous in the assay (Na+ , K+ , etc.) Solutions of strong acids and bases used for final pH adjustment usually are stable for long periods of time, but not forever. Was the NaOH used for pH adjustment prepared during the last ice age? Was it stored properly to exclude atmospheric CO2, whose presence can slowly neutralize the base, producing sodium bicar￾bonate (NaHCO3) which further alters the buffer properties and ionic strength of the solution? Buffers from Stock Solutions Stock solutions can be a quick and accurate way to store “buffer precursors.” Preparing 10¥ to 100¥ concentrated buffer salts can simplify buffer preparation, and these concentrated solutions can also retard or prevent bacterial growth, extending almost indefi- nitely the shelf stability of the solutions. 36 Pfannkoch