Buffers and Buffering


 How to prepare a buffer

    A compound can buffer the pH of a solution only when its concentration is sufficient and when the pH of the solution is close (within about one pH unit) to its pKa.  To make a buffer you must first pick a compound whose pK  is close to the pH  you want for the solution, and then decide what the buffer concentration should be.  Typically, buffer concentrations are between 1 mM and 200 mM, depending on the desired ionic strength and the buffering capacity required.  If the pH is expected to decrease during the experiment, choose a buffer with a pKa slightly below the working pH.  Conversely, if the pH is expected to increase during the experiment, select a buffer with a pKa slightly above the working pH.  Having decided on the total buffer concentration, you must adjust the ratio of the protonated and unprotonated forms of the buffer in your solution so as to give the desired pH.  Typically, buffers are composed of  weak acids and their salts, or weak bases and their salts.  If the protonated form is uncharged, it is an acid (like acetic acid), and its unprotonated form is a salt (e.g., sodium acetate).  Conversely, if the unprotonated form is uncharged it is a base (like Tris base), and its protonated form is a salt (e.g., TrisHCl).

Four practical ways to make a buffer are described below:

 (1) The Slow and Stupid Method - To avoid adding extra salt to a solution, prepare a buffer composed of an acid and its salt by dissolving the acid form of the buffer in about ~60% of the water required for the final solution volume.  Adjust the pH using a strong base, such as NaOH.  When preparing a buffer composed of a base and its salt, start with the base form and adjust the pH with strong acid, such as HCl.  After the pH is correct, dilute to just under the final solution volume.  Check the pH and correct if necessary, then add water to the final volume.
    Advantages: Easy to understand.
    Disadvantages: Slow.  May require lots of base (or acid).  If the base (or acid) is concentrated, it is easy to overshoot the pH. If the base (or acid) is dilute, it is easy to overshoot the volume.  Ionic strength will be unknown. Adding a strong acid or base can result in temperature changes, which will make pH readings inaccurate (due to its dependence to temperature) unless the solution is brought back to its initial temperature. 

(2) The Mentally Taxing Method - Using the buffer pKa , calculate the amounts (in moles) of acid/salt or base/salt present in the buffer at the desired pH.  If both forms (i.e., the acid and the salt) are available, convert the amount required from moles to grams, using the molecular weight of that component, and the weigh out the correct amounts of  both forms.  If only one form is available, you can prepare the buffer by adding all of the buffer as one form, and then adding acid or base to convert some of the added buffer to the other form.  Decide what the total concentration of buffer will be in the solution, and convert the concentration to amount (in moles) using the volume of solution, and then to grams, using the molecular weight of the buffer form available.  Then calculate the amounts (in moles) of each form that will be present in the final solution, using the buffer pK and the desired pH.   Then calculate how much strong acid or base must be added to convert enough of the buffer form added to the other form,  to give the correct amounts of each form at the pH of the final solution.  Dissolve the buffer and strong acid or base in slightly less water than is required for the final solution volume.  Check the pH and correct if necessary.  Add water to the final volume.
    Advantages: Fast.  Easy to prepare.  Additional pH adjustment is rarely necessary, and when necessary, the adjustment is small. Ionic strength easily calculated.
    Disadvantages: Requires the buffer pKa and solving two equations (see Theory ).

(3)  The Two Solution Method - Make separate solutions of  the acid form and base form of the buffer, both solutions having  the same buffer concentration (and ionic strength, if required) as the concentration of total buffer in the final solution.  To obtain the desired pH, add one solution to the other while monitoring the pH with a pH meter.
    Advantages:  Easy to do.
    Disadvantages: Requires both forms of buffer.  The required solution volumes are proportional to the ratio of buffer components in the final solution at the desired final pH, so making equal amounts of each form may waste a lot of one solution.

(4) The Completely Mindless Method - Find a table of the correct amounts of acid/salt or base/salt required for different pH's, and dissolve the components in slightly less water than is required for the final solution volume.  Check that the pH and correct if necessary. Add water to the final volume.
    Advantages: Easy to do (with appropriate table).  Convenient for frequently prepared buffers.
    Disadvantages: May be impossible to find table.  Table may be incorrect.  Requires both forms of buffer.  Component amounts from table will need to be adjusted to give the buffer concentration and volume in your solution. Ionic strength is unknown.


 Return to top

Return to Biochemistry 353