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Úvodní stránka podle aplikace Laboratorní vážení Buffer Preparation – Solutions, Calculation & Solving Common Problems

Buffer Preparation – Solutions, Calculation & Solving Common Problems

Why Accurate Weighing is Vital for Quality Buffer Solutions

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Buffer Preparation – solutions, calculation & solving common problems

Buffer preparation is a common process in chemistry and biochemistry laboratories. A buffer solution is a mixture of a weak acid and its conjugate base or a weak base and its conjugate acid. Buffer solutions are used to help maintain a stable pH value of another solution that is mixed with the buffer. The buffer resists changes in the pH value of the whole solution when a small amount of a different acid or alkali is introduced into the solution either through addition or via any chemical reaction within the solution. Buffer solutions are therefore very useful in a wide variety of applications in which a relatively stable pH is required. A buffer may also be called a pH buffer, hydrogen ion buffer, or buffer solution.

For example, blood contains natural buffers to maintain a stable pH of between 7.35 and 7.45 so that our enzymes work correctly. As enzyme activity varies with pH, maintaining a constant pH is essential in biochemical assays to ensure the correct level of activity is observed. In commercial applications, buffers can be found in shampoos to prevent skin irritation, in baby lotions to inhibit the growth of bacteria, and in contact lens solutions to ensure the pH level of the fluid remains compatible with that of the eye.

Preparing buffers consists of several steps: Weighing-in the components, dissolving the components, adjusting the pH and replenishing to the final volume. As the ratio of the acid to base in a buffer is directly related to the final pH, it is vital to weigh the components with a high degree of accuracy. So, it is important that the equipment used (balance, pipettes and pH meter) are properly calibrated and have sufficient accuracy. 

Buffer Preparation

Watch the Video: Buffer Solution Preparation – Simple, Convenient and Accurate

Buffer solution preparation takes time and must be done with care to ensure the buffer performs as desired. When the quality of your products or your biochemical analyses depend on the performance of your buffer solutions, you want to be sure you get them right first time.

Watch the video and see how you can save time and effort preparing accurate buffer solutions with METTLER TOLEDO precision balances and pH meters.


Jump to one of the following section to explore and learn more:

  1. How to prepare buffer solutions? Typical procedure.
  2. Buffer solution know-how
    1. How do buffer solutions work?
    2. Types of buffer solutions
    3. What to consider when preparing a buffer solution
    4. Benefits of universal buffers
    5. Additional tips for preparing and using buffers
  3. Request free support for your buffer preparation process
  4. The challenges of preparing a buffer solution
  5. METTLER TOLEDO featured solution for convenient and accurate buffer preparation
  6. Weighing guide: Efficient weighing workflows
  7. Buffer preparation – an optimized workflow with comprehensive data management
  8. How to ensure your pH meter is calibrated correctly
  9. FAQ – frequently asked questions on buffer solution preparation

Buffer Preparation - formulation workflow

How to Prepare Buffer Solutions? Typical Procedure.

Preparing buffers consists of several steps: Calculate the components (concentrations and amounts) according to the required use and target volume, weigh-in the components, dissolve the components, adjust the pH, fill-up to the final volume, label, document results and use directly or store for later usage.

  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.

Buffer Preparation Formula

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:

  • pH adjustment
    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: 
    • Buffer Preparation - second formula

    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.

Buffer Preparation Expert

Do You Need Support?

Buffer solutions are vitally important in a wide range of applications. However, buffer preparation is time-consuming and labor-intensive and must be done with care to avoid errors. If you need support with buffer preparation or want advice on which balance meets your accuracy requirements, our team of experts is here for you. Do not hesitate to contact us for help or advice.

The Challenges of Preparing a Buffer Solution

The Challenges of Preparing a Buffer Solution

Whilst the simplest buffer solution may consist just of an acid, a base and water, laboratory technicians often have to prepare several buffer solutions per day  – typically consisting of 2-5 components, but could be as many as 20. A typical laboratory may have more than 20 buffer solution recipes that are intended to produce 1 liter of buffer.

Buffer solution calculations

With a database of 20+ recipes, the laboratory technician must first be sure that the correct buffer solution recipe is selected. For buffer solution volumes other than 1 liter, it is necessary to recalculate the quantities of all the components accordingly and log the new amounts. Any errors in the calculations or in recording the new values may lead to incorrect pH values in the buffer solution. There is a higher risk of errors in a manual data recording system.

Weighing and logging weight results

When weighing in all the different buffer solution components, care must be taken to use the right amount of the right component. The actual weight of each component must be logged, and, if recording results by hand or entering the values into a computer, care must be taken to avoid transcription errors.

Depending on how much of each of the buffer solution components is required and the minimum weight of the balance, it may be necessary to use two different balances. This complicates the process and reduces efficiency.

Correct performance of pH meters

Checking the pH level of the buffer solution is of vital importance. However, if the pH meter used is not calibrated and maintained correctly, pH readings may be inaccurate. Using a buffer solution with an incorrect pH may seriously impact subsequent analyses or product quality.

Documentation

The final buffer solution must be carefully labelled with all information to avoid any mix-ups. The expiry date is important to ensure the buffer solution is still effective when it is used. All data relating to the buffer solution preparation must be logged and stored securely for future reference and traceability.

Accurate Buffer Preparation

METTLER TOLEDO Featured Solution for Convenient and Accurate Buffer Preparation

METTLER TOLEDO's XSR precision balances simplify buffer solution preparation and significantly reduce the workload for laboratory technicians.

Saved recipes

Up to 50 buffer solution recipes can be saved directly on the balance as weighing methods. By saving the most commonly used buffer solution recipes on the balance, it is quick to retrieve the method needed and start working. The balance provides step-by-step instructions so analysts can easily keep track of where they are in the buffer preparation process.

Automatic calculations and weighing-in guide

At the start of each method, the required volume of the buffer solution is entered and the component quantities are calculated automatically. As each ingredient is weighed in, the SmartTrac weighing guide displays the actual weight against the target weight graphically. The colored bar turns green as soon as the weight of the added component is within the predefined tolerance range. This enables analysts to weigh in more quickly and with greater certainty.

Efficient processes and automatic data recording

After each component has been added, the balance saves the actual weight result and then tares automatically, assisting in the weighing process and eliminating the need to manually record the results. The balance can be set to print out the weight results automatically once all the ingredients have been added.

Convenient pH measurement

METTLER TOLEDO's SevenExcellence pH meters are the perfect choice for accurate pH measurements. These easy-to-use instruments offer touchscreen operation, direct measurements at the touch of a button, and intelligent sensor management.

pH measurements are only as accurate as the calibration solutions used. METTLER TOLEDO also offers a selection of quality pH calibration solutions to match your specific requirements.

Buffer Preparation Guide

Guide: Biological Buffer Preparation

Discover how you can make your buffer preparation process easy and reliable.

Download the guide and get useful tips and hints to help improve the productivity of your preparation process, ensure quality standards and traceability for buffer solutions.

  • Step-by-step SOP user guidance and accessible buffer recipes directly on the balance screen improves workflows
  • Accurate weighing in compounds is vital for the quality of buffer solutions
  • Exact and reliable pH measurement is important for the correct preparation of buffers
  • Using and operating pipettes the correct way helps to ensure that pH buffer is properly adjusted
  • Automatic calculation, labeling and reporting ensure safe data transfer and data traceability 

Buffer Preparation – An Optimized Workflow with Comprehensive Data Management

Buffer Preparation – An Optimized Workflow with Comprehensive Data Management


METTLER TOLEDO Excellence Line precision balances (XPR and XSR) and SevenExcellence pH meters can be connected to our proprietary LabX laboratory software to create a fully optimized system that provides a range of key benefits:

  • Automatic calculations and documentation
  • All results saved in a secure database
  • Full traceability
  • Central management of tasks, users and instruments

Support with FDA 21CFR part 11 compliance

LabX provides comprehensive data management functions, practically eliminating manual data transcription – even detailed labels can be printed automatically. With a centralized database, all connected users and instruments can access the same stored SOPs for buffer preparation. This ensures that every analyst carries out buffer preparation with identical settings and parameters. All results and process metadata are saved automatically in a secure database so full traceability is assured.

Buffer Preparation –PH Meter Calibration

How to Ensure Your pH Meter is Calibrated Correctly

 

To accurately calibrate your pH meter, you need accurate calibration buffer solutions of known pH! METTLER TOLEDO offers a range of quality pH buffers and guarantees maximum precision with NIST/DIN buffers.

Each buffer is supplied with a test certificate to help you ensure compliance and traceability. Whether you just need a technical buffer or a buffer certified by an accredited body, we have the right buffers to meet your needs.

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FAQ - Buffer Solution Preparation

FAQ – Frequently Asked Questions on Buffer Solution Preparation

 

Select your Question

  1. How can I make weighing for buffer preparation more efficient?
  2. Which is the best balance for buffer solution preparation?
  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.
  4. How can I be sure of the pH reading of my buffer solution?
  5. What if I add more of one ingredient than is needed?


 

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.