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 Pfannkoch
pH 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, increasing 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 calculations 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 bicarbonate (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
The pH of the stock solutions should not be adjusted prior to dilution; the pH is the negative log of the H* ion concentration so dilution by definition will result in a pH change. Always adjust the ph at the final buffer concentrations unless the procedure explicitly indicates that the diluted buffer is at an acceptable p nd ionic concentation, as in the case with some hybridization and electrophoresis buffers(Gallagher, 1999) Filtration n many applications a buffer salt solution is filtered prior to mixing with the other buffer components. An inappropriate filter can alter your solution if it binds with high affinity to one of the solution components. This is usually not as problematic with polar buffer salts as it can be with cofactors, vitamins, and the like. This effect is very clearly demonstrated when a solution is prepared with low levels of riboflavin. After filtering through a PTFE filter the filter becomes bright yellow and the riboflavin disappears from the solution Incomplete Procedural Information If you ask one hundred chemists to write down how to adjust the pH of a buffer, you'll probably receive one hundred answers, d only two that you can reproduce. It is simply tedious to describe in detail exactly how buffer solutions are prepared. When ading procedures, read them with an eye for detail: Are all details of the procedure spelled out, or are important aspects left out? The poor soul who tries to follow in the footsteps of those who have gone before too often finds the footsteps lead to a cliff Recognizing the cliff before one plunges headlong over it is a learned art. A few prototypical signposts that can alert you of an impending large first step follow Which salts were used to prepare the"pH 4 acetate buffer"? Sodium or potassium? What was the final concentration? Was pH adjustment done before or after the solution was brought to final volume? If the solution was filtered, what type of filter was used? What grade of water was used? What was the ph of the starting water source What Is the Storage Lifetime of a Buffer? a stable buffer has the desired ph and buffer capacity intended when it was made. The most common causes of buffer failure are The Preparation of Buffers and Other Solutions
The pH of the stock solutions should not be adjusted prior to dilution; the pH is the negative log of the H+ ion concentration, so dilution by definition will result in a pH change. Always adjust the pH at the final buffer concentrations unless the procedure explicitly indicates that the diluted buffer is at an acceptable pH and ionic concentation, as in the case with some hybridization and electrophoresis buffers (Gallagher, 1999). Filtration In many applications a buffer salt solution is filtered prior to mixing with the other buffer components. An inappropriate filter can alter your solution if it binds with high affinity to one of the solution components. This is usually not as problematic with polar buffer salts as it can be with cofactors, vitamins, and the like. This effect is very clearly demonstrated when a solution is prepared with low levels of riboflavin. After filtering through a PTFE filter, the filter becomes bright yellow and the riboflavin disappears from the solution. Incomplete Procedural Information If you ask one hundred chemists to write down how to adjust the pH of a buffer, you’ll probably receive one hundred answers, and only two that you can reproduce. It is simply tedious to describe in detail exactly how buffer solutions are prepared.When reading procedures, read them with an eye for detail: Are all details of the procedure spelled out, or are important aspects left out? The poor soul who tries to follow in the footsteps of those who have gone before too often finds the footsteps lead to a cliff. Recognizing the cliff before one plunges headlong over it is a learned art. A few prototypical signposts that can alert you of an impending large first step follow: • Which salts were used to prepare the “pH 4 acetate buffer”? Sodium or potassium? What was the final concentration? • Was pH adjustment done before or after the solution was brought to final volume? • If the solution was filtered, what type of filter was used? • What grade of water was used? What was the pH of the starting water source? What Is the Storage Lifetime of a Buffer? A stable buffer has the desired pH and buffer capacity intended when it was made. The most common causes of buffer failure are The Preparation of Buffers and Other Solutions 37