A buffer is a chemical system that stabilizes the acidity or alkalinity of a solution, resisting significant changes in pH when a small amount of acid or base is added. This resistance is achieved by maintaining a balance between a weak acid and its conjugate base. The sodium phosphate buffer system is widely employed in biological and chemical laboratories because its effective range is close to the neutral pH found in living organisms. In experiments such as enzyme assays, cell culture, and chromatography, slight shifts in pH can dramatically alter results. The phosphate system is useful in the range of pH 5.8 to 8.0, making it a versatile tool for mimicking physiological environments.
Essential Ingredients and Stock Solutions
The preparation of a sodium phosphate buffer requires two specific sodium salts of phosphoric acid: Monobasic Sodium Phosphate ($\text{NaH}_2\text{PO}_4$), which acts as the weak acid, and Dibasic Sodium Phosphate ($\text{Na}_2\text{HPO}_4$), which serves as the conjugate base. These components neutralize added hydrogen or hydroxide ions, maintaining a steady pH.
Concentrated stock solutions of each salt are typically prepared individually, usually at 1.0 M, to allow for easy dilution later. For example, preparing a 1 M stock requires dissolving approximately 142.0 grams of anhydrous $\text{Na}_2\text{HPO}_4$ or 120.0 grams of anhydrous $\text{NaH}_2\text{PO}_4$ in deionized water and bringing the volume to one liter. Note that if hydrated forms are used, the required weighed mass must be adjusted.
High-purity deionized water must be used for all dissolutions to avoid introducing contaminants that could affect the final pH. The salts should be dissolved completely in a volume of water less than the final desired volume, usually about 800 milliliters, before the solution is topped up to the one-liter mark. The dibasic salt, in particular, is hygroscopic and readily absorbs moisture from the air, which necessitates careful and quick weighing to ensure accuracy.
Standard Protocol for Mixing the Buffer
The final desired concentration and the target pH determine the precise volumes of the monobasic and dibasic stock solutions to be combined. The ratio of the conjugate base ($\text{Na}_2\text{HPO}_4$) to the weak acid ($\text{NaH}_2\text{PO}_4$) dictates the resulting pH of the buffer solution. A buffer with a pH lower than the phosphate system’s second dissociation constant (approximately 7.21) will require a greater proportion of the monobasic salt, while a higher pH requires more of the dibasic salt.
To make a buffer of a specific molarity, such as a 0.1 M solution, one must first decide on the total final volume. The total moles of phosphate needed is calculated by multiplying the target molarity by the volume. Using published tables or the Henderson-Hasselbalch equation, the ratio of the two components needed to achieve the target pH is determined, which then translates into the specific volumes of the 1 M stock solutions to be mixed.
For example, a common biological buffer at pH 7.2 requires mixing the two stock solutions at a ratio that favors the dibasic salt slightly. If preparing one liter of 1 M buffer at pH 7.2, one would mix 280 milliliters of the monobasic stock with 720 milliliters of the dibasic stock. To create a working solution of 0.1 M, these volumes are scaled down and then diluted with deionized water to the final volume. After the calculated volumes of the two stocks are combined, the solution must be thoroughly mixed before moving to the next stage of fine-tuning.
Fine-Tuning the Buffer pH
While mixing the calculated volumes provides a strong approximation of the target pH, the value must be confirmed and adjusted using a calibrated pH meter. For a precise and reproducible experiment, the pH must be verified and corrected to the exact specification.
The pH meter should be calibrated with at least two standard buffer solutions that bracket the target pH before any measurements are taken, ensuring the sensor provides an accurate reading in the desired range. The electrode of the calibrated meter is then submerged in the prepared phosphate solution, and the reading is allowed to stabilize.
If the measured pH is lower than the target, a small amount of a strong base, such as a concentrated sodium hydroxide ($\text{NaOH}$) solution, is added dropwise while stirring to raise the pH. Conversely, if the measured pH is higher than the target value, a strong acid, typically hydrochloric acid ($\text{HCl}$), is added dropwise to lower the pH. This process requires patience and small, dropwise additions, as the buffer is designed to resist pH changes, preventing large additions from being effective and potentially leading to overshooting the target. This adjustment is always performed before the solution is brought to its final volume with deionized water, as adding the remaining water after adjustment will not alter the established concentration ratio of the acid and base components.
Storage and Stability Considerations
Proper storage is necessary to maintain the integrity and functionality of the buffer over time. Once the pH has been precisely adjusted, the solution should be transferred to a clean, sealed container, ideally one that has been sterilized. The container must be clearly labeled with the buffer’s name, its concentration, the exact pH, the date of preparation, and the preparer’s initials.
Phosphate buffers are susceptible to microbial contamination, especially when stored at near-neutral pH values, which can be indicated by the solution appearing cloudy. To mitigate this risk, the buffer is often stored in a refrigerator at 4 °C, which slows down the growth of microorganisms and generally extends the shelf life to about one to two weeks for most applications. If the buffer is prepared under sterile conditions, such as through autoclaving, the shelf life can be extended significantly, sometimes up to a year, provided the container remains sealed and no cloudiness develops. Storing the buffer at room temperature is possible, but it should be used quickly, typically within 48 hours, as stability and sterility cannot be guaranteed for longer periods.