# Buffer Preparation – Solutions, Calculation & Solving Common Problems

## Why Accurate Weighing is Vital for Quality Buffer Solutions

### Buffer Preparation

1. Select recipe from database
2. Recalculate recipe quantities according to the required buffer volume
3. Weigh compounds into the vessel
4. Dissolve the compounds in a suitable solvent (typically water)
5. Check and adjust the pH value by using a pH meter
6. Top up the solution to the required volume
7. Transfer to a storage bottle and label
8. Document results

### How Do Buffer Solutions Work?

Buffers are aqueous systems that resist changes in pH when small amounts of acid or base are added and are composed of a weak acid and its conjugate base. A buffer keeps the pH of a solution constant by absorbing protons that are released during reactions or by releasing protons when they are consumed by reactions. The discovery that partially neutralized solutions of weak acids or bases are resistant to changes in pH, when small amounts of strong acids or bases are added, led to the concept of the ‘buffer’.

A conjugate base is an acid that has lost a proton.
HA  ↔  H+ + A-
Acid  ↔  proton + conjugate base

A conjugate acid is a base that has gained a proton.
A + H+  ↔  H+A
Base + proton  ↔  conjugate acid

An equilibrium between the dissociated and undissociated form is established.
For example, weak acetic acid partially dissociates in water producing an acetate ion:
CH3COOH  ↔  H+ + CH3COO-
The undissociated acetic acid, the hydrogen ions and the dissociated ion exist in equilibrium in solution.

Sodium acetate also dissociates in water to produce the same acetate ion:
CH3COONa  ↔  Na+ + CH3COO-
The undissociated sodium acetate and the ions of sodium and acetate exist in equilibrium in solution.

An aqueous solution of a mixture of acetic acid and sodium acetate can therefore absorb H+ ions from the addition of an acid through the combining of the hydrogen ions with the acetate base to produce acetic acid. Furthermore, when OH- ions are introduced into the solution through the addition of an alkali, they combine with acid molecules (H+) to produce water. In this way, as the system attempts to restore the equilibrium, the solution resists changes to the pH value. This is how a buffer solution works.

What happens after adding acid to the buffer?

When some strong acid (more H+) is added to an equilibrium mixture of the weak acid and its conjugate base, the equilibrium is shifted to the left, in accordance with Le Chatelier’s principle.

What happens after adding a base to the buffer?

Similarly, if a strong base is added to the mixture, the hydrogen ion concentration decreases by less than the amount expected for the quantity of base added. This is because the reaction shifts to the right to accommodate for the loss of H+ in the reaction with the base.

Types of Buffer Solutions

Buffer solutions consisting of a weak acid and its conjugate base are known as an acidic buffers and have a pH < 7. A buffer made with acetic acid (weak acid) and sodium acetate (conjugate base) is an acidic buffer and has a pH of around 4.75.
Buffer solutions consisting of a weak base and its conjugate acid are known as alkaline buffers and have a pH > 7. An example of an alkaline buffer is an aqueous solution of ammonium hydroxide (weak base) and ammonium chloride (conjugate acid) which has a pH of 9.25.

What to Consider When Preparing a Buffer Solution

Buffer solutions work best when their pH is similar to that of the system or solution being investigated. In the study of enzymes in human biology, a system is required that matches the pH of blood, i.e. 7.35 to 7.45 otherwise the enzymes will not function correctly. If the buffer system has a pH outside of the desired range, this will also negatively impact analyses.

It is therefore necessary to be able to prepare buffer solutions of a specific pH and this can be done in different ways:

The crystalline acid or base is first dissolved in a volume of water equating to around 60-70% of the final required volume of buffer solution. The pH is tested and then adjusted. If a crystalline acid is used, the pH is adjusted using a base that will not generate ions that may interfere with the system under investigation. If a crystalline base is used, the pH is adjusted using a suitable acid. Once the desired pH is achieved, the volume of the buffer solution can be topped up with water to obtain the required volume.

• Mixing acid and base solutions
In this method, a solution of the acid or base is mixed with a solution of the associated salt. The concentrations of the source solutions must be the same as the required buffer solution. The solutions can be mixed in varying ratios to achieve differing pH values in the final buffer solution. Alternatively, the pH can be monitored while adding one solution to the other.

• Using Henderson-Hasselbach equation
The Henderson-Hasselbach equation can be used to estimate the pH of a buffer solution using the dissociation constant, pKa. When a weak acid (HA) is in solution and the dissociation into hydrogen ions (H+) and its conjugate base (A-) is in equilibrium, the dissociation constant is a measure of the strength of the acid at this equilibrium point. The Henderson-Hasselbach equation is as follows:

• Where: pKa is the dissociation constant of the weak acid
[A-] is the concentration of the conjugate base at equilibrium
[HA] is the concentration of the acid at equilibrium

In the case where the concentration at equilibrium of the conjugate base and the acid are the same, the pH becomes equal to the dissociation constant. At this point, the buffer solution provides the maximum buffering capability.
The Henderson-Hasselbach equation is also used for determining the dissociation constant of weak acids via direct measurement of pH

Benefits of Universal Buffers
Universal buffer solutions consist of a combination of several acid-base pairs. This enables universal buffers to maintain the pH value of a solution across a wider range and can therefore be used in many more applications.

Additional Tips for Preparing and Using Buffers

• Create a buffer preparation SOP
It is a good idea to document the buffer preparation process and be sure that everyone follows the same procedure to ensure consistency and reproducibility. The SOP should include details of the materials used and the precise steps of when and how to add the components and measure the pH. The SOP may also include many of the points mentioned below.

• Wear protective clothing
Appropriate personal protective equipment (PPE), for example protective eyewear and clothing must be worn, especially when working with strong acids or bases.

• Check for microbial contamination (especially in biological applications)
Before using any buffer, check the container for microbial contamination. Buffer solutions with a pH close to neutral are particularly susceptible to contamination. You may see some cloudiness in the solution or contaminants that have settled on the bottom.

• Use your pH meter correctly
Your pH meter should be regularly calibrated and maintained to ensure accurate pH measurements. The electrode should be properly prepared prior to use and sufficient buffer solution should be used to ensure that the electrode junction is correctly submerged. Be sure to wait for a stable pH value before taking the reading and rinse the electrode with distilled water afterwards. Make sure to use your pH meter at the ambient temperature or use an electrode with integrated temperature probe.

• Be aware of the temperature
The amount of dissociation can vary with temperature. Your buffer solution should therefore be prepared at the same temperature at which you will perform your analyses. Also make sure that the temperature at which you calibrate the electrode is the same as the temperature at which you measure.

• Be aware of the concentration
It is common practice to dilute stock buffer solutions to the required concentration for use in analyses. However, changing the concentration can affect the amount of dissociation. As pH is a measure of hydrogen ions (H+), a change in dissociation can cause a change in pH. After dilution pH should be rechecked prior to using the buffer.

## 1. How can I make weighing for buffer preparation more efficient?

On METTLER TOLEDO XPR/XSR balances, you can save your own specific weighing methods. For buffer preparation, this is achieved by using the 'Simple formulation with templates' method on the balance to store each buffer component, with its target weight and tolerance interval, as a recipe on the balance. When the method is started, you are guided through each step of the procedure. No time is wasted checking a recipe sheet and there is no uncertainty about where you are in the procedure. Weight results are saved automatically and can be printed out at the end so you save a considerable amount of time by eliminating the need to record all the weight results by hand.

## 2. Which is the best balance for buffer solution preparation?

The best balance depends on your individual requirements. You need to know the largest and the smallest amounts you want to weigh as well as how accurately you need to weigh them (i.e. what the tolerance or allowable error is). METTLER TOLEDO offers a free service to help you choose the right balance to meet your application requirements. Simply ask your local representative for a free balance recommendation. The same service also determines if your existing balance meets your requirements.

## 3. During buffer solution preparation, there's such a lot of data to record. How can I avoid making mistakes? I need an error-free solution.

First, connect a barcode reader to your balance. This will enable you to electronically record sample IDs, lot numbers, and order numbers etc. Second, connect a printer to your balance. With the METTLER TOLEDO P-5x line of printers, the metadata and the date and time can be automatically printed out along with the results at the end of the weighing procedure. An alternative option is to connect your balance to LabX laboratory software which offers comprehensive data management including customizable reports that can be sent straight to your LIMS or ERP.

## 4. How can I be sure of the pH reading of my buffer solution?

Periodic calibration and regular checks of your pH meter are necessary in order to be sure of its accuracy. Consult the guide and learn more about METTER TOLEDO's solution for easy calibration of a pH meter. And check out this video: pH Calibration ‒ A Handy How-To Guide.

## 5. What if I add more of one ingredient than is needed?

If you accidentally weigh in too much of one ingredient, it is possible to add additional amounts of the other ingredients rather than discard the materials weighed out up to that point. If calculating the amounts manually, this can be challenging and there is a high risk of errors. You should also be aware that the additional amounts required may be very small and your balance may not be suitable for this. A second balance with a higher degree of accuracy and a lower minimum weight may be required. If your balance is connected to LabX, LabX can perform the necessary recalculations for you and keep track of your progress in the procedure.