Guide to pH_ pKa and pH measurement _pH meters_ by lanyuehua

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									                                        pH Meters and pH Electrodes

 When you have completed step 2 above see your instructor for a demonstration of how to calibrate a
 pH meter.

 Modern pH meters are microprocessor-based instruments and often contain liquid crystal displays that
 are spill proof. In spite of all of these advances modern pH meters still need to be calibrated with
 traceable standards to validate ones measurements.

 To calibrate a pH meter we will have to choose at least two standard pH buffers. The pH of the solution
 to be measured determines the calibration buffers needed. For example, in today's lab we want to
 produce a 0.01M sodium phosphate buffer pH 7.3. To calibrate the pH meter we must select two
 standard pH buffers that bracket pH 7.3. Therefore we will calibrate the pH meter with pH 7 and pH 10
 standard pH buffers.

 By selecting two buffers having pH values that bracket the pH of interest you will be helping your pH
 meter to respond more accurately.

 1.Place a magnetic stirring bar in the beaker containing your buffer. Then place the beaker containing
   the buffer on a magnetic stirrer near one of the pH meters.

      pH electrodes are easily broken. Handle pH electrodes carefully and do not allow the
      electrode to hit the bottom of your beaker and do not allow the stirring bar to hit the pH
      electrode.




Figure 1. Standard combination pH electrode. Do not bump the bottom of a container with the pH electrode as
the fragile glass membrane may break.

2. Raise the pH electrode out of its storage solution and, using an empty beaker, rinse the electrode gently
with a squirt bottle containing distilled water. Don't get the rinse water all over; catch it in the waste beaker.
Then blot off any water drops from the electrode with a clean paper wipe and insert the electrode into the
buffer in your beaker.

You only need to insert the pH electrode about 3-4 cm into your solution. The buffer solution must cover the
porous plug in the electrode. Locate the porous plug.

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3. To adjust the pH of the buffer to 7.3 you will need to add either 1 M NaOH or 1 M HC1 to the buffer. Use a
Pasteur pipet to add the acid or base drop-wise to the buffer.

There are two good reasons to add the acid or base to the solution slowly:

       a) the pH electrode needs 20-30 seconds to respond to each addition of acid or base
       b) 1 M NaOH and 1 M HC1 are 100 times more concentrated than the 0.01 M buffer.

4. When you have adjusted the pH to pH 7.3 give the pH meter an additional 15-30 seconds before you remove
the pH electrode from the buffer. Then remove the pH electrode from the buffer and rinse it over the waste
beaker with a squirt bottle containing distilled water. Replace the pH electrode into its storage buffer. Be careful as
the tip of the pH electrode is easily broken if it strikes an object.

5. Now, using a funnel, carefully add your buffer to your 250 mL volumetric flask and bring the volume up to the
mark with water. Do not try to add the last few mL of water by pouring from a flask or beaker. Add the last few mL
with a clean Pasteur pipet. Then place the cap on the volumetric flask and carefully invert 7 or 8 times to mix the
solution. Now you have 250 mL of buffer (0.01 M sodium phosphate pH 7.3). Dilute buffers cannot be stored for
more than a day as microorganisms will grow in them unless they are sterile filtered into a sterile container.




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                                                      Logarithms

A logarithm (or log for short) is an exponent. We will be using logs to the base 10 (we write logs to the base 10 as
log10). This means our logarithm or exponents will be in relation to the number 10.

For example:

You already know that 102 = 100 (10 times 10 = 100). The little 2 written above the ten is the exponent. The Iog10
of 100 is 2.

The Iog10 of 1000 is 3 (10 times 10 times 10 = 1000). Use your calculator to check this.

When we want to determine the logarithm to the base 10 of a number we are really asking what number can 10
be raised to that will give that same number.

For example:

Suppose our number is very big; perhaps 10,000,000. If I enter that number in my calculator and press the log key I
find that the log is 7. That means 107.

What is the number that I want the Iog10 of is less than 1.0.

For example:

What if the number is 0.0000001? If I enter this number into my calculator my answer is -7. This means I can have
negative values if the number is less than 1.

                                                    Antilogarithms

If you can find the log10 of a number it is also possible to do the reverse.

For example:

What if the log10 is 3? What is the antilog of 3 or what is the original number?

On my calculator I enter the 3 and then press the 2nd key and then the log key. The answer is 1000.

For example:

What if the number is -5? What is the antilog of-5?

If I enter the number 5 and then change the sign to negative with the +/- key and then press the 2nd key followed
by the log key I get 0.00001.




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What is the log of 1,000,000? _______________________

What is the log of 0.000000001?____________________

What is the log of 0.0015? __________________________

What is the antilog of 2.3? _________________________

What is the antilog of 0.1? __________________________

What is the antilog of 10? ___________________________




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                                                           pH

The pH of a solution influences many reactions: Effects on enzymes and other proteins, the concentrations of
certain ion species in solution, preparation of buffers, manipulating the rate of chemical reactions, solubility of
some compounds may be dependent on pH, production of some commercial products including DNA fragments,
anything requiring enzymes, and cell culture media to name just a few.

pH is commonly defined as follows: pH = -log [H3O+].

pH measurement is quick and easy; it allows us to determine an important part of the environment in a solution by
reference to the pH scale. A pH of 7 is said to be neutral because the activities of both hydrogen ions and
hydroxide ions are the same.

What are the most common errors in the measurement of pH?

          Forgetting to calibrate or incorrectly calibrating a pH meter is most likely the number one source of pH
           measurement errors.

          Too high a concentration of a reagent or a high concentration of certain ions like Na+.

          The failure to compensate for temperature can also cause significant errors in pH measurement. Some
           buffer solution i.e., tris are sensitive to temperature.

Some terms to learn

Reference junction

Liquid junction potential

Reference filling solution

Electrode storage:

Maintenance and Cleaning pH Electrodes: always refer to manufacturers’ instructions.
The electrode fill hole: most combination pH electrodes require addition of the correct electrolyte through the fill
hole when the level of fluid inside is low.

Rinsing

Stirring

pH buffer standards

Temperature

Calibration




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Web Sites for pH and buffers.
The Biology Project developed at The University of Arizona.
http://www.biology.arizona.edu/biochemistry/problem_sets/ph/ph.html
This is an excellent web site. Be sure to work through the tutorials and pH & Buffer Questions.

Do the following for homework.
1. How much dibasic sodium phosphate 12 H2O would one have to weigh out to produce 1 liter of a 1M
         solution?____________.




2.    How much dibasic sodium phosphate 7 H2O would one have to weigh out to produce 1 liter of a 1M solution?




3.    Do both of the solutions above have the same number of phosphate molecules?




4.    What is the final concentration that results when you dilute 100 mL of 0.2M sodium phosphate to a final volume of
       1000 mL ?_________.




5.    How would you make up 250 mL of a 0.05M sodium phosphate buffer pH 6.8?




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                                         Dissociation, pKa, Buffers and pH
                                                      Dissociation


Let’s begin with the dissociation of an acid that is abbreviated HA. We may write the dissociation as
                                 + -
                 HA             HA

Notice there is two arrows in the equation above. The two arrows indicate that the reaction is reversible. If nearly all
                                             + -
of the HA dissociates to form the ions H A then we say that the equilibrium lies far to the right. An acid whose
equilibrium lies far to the right is called a strong acid.
                                           +   -
If more HA remains in the solution than H A then we say that the equilibrium lies far to the left. Such an acid is
called a weak acid.

We may say that the product of the concentrations of reactants on the right side of the equation divided by the
product of the reactants on the left side of the equation is equal to some constant Ka
          +    -
or Ka = [H ] [A ]
          HA

If Ka is very big then the acid is a strong acid (in reality the dissociation of a strong acid so complete that the
concentration of any undissociated acid can't be determined accurately). If the Ka is very small then it is a weak
acid. The Ka values for weak acids have been published and are available.

                                                          pKa

Weak acids are commonly chosen in the preparation of buffers. For example, acetic acid is a weak acid. Its Ka is 1.8
    -5                                                           +
X 10 . This is a small Ka which means that the concentration of H that dissociate in a solution of acetic acid is
small.

The pKa value is useful when trying to determine which buffer to select. One can convert the Ka to the pKa as
follows:

        PKa. = -logKa
                                                                                   +       -
It is important to remember that the pKa is related to the concentrations of HA, H , and A present in solution.

                                                    Buffers and pH

Selecting a good buffer may seem daunting when one sees the list of possible buffers. The list can be narrowed by
selecting a buffer whose pKa value is close to the desired pH value. Below is a short list of weak acids. Match the
best weak acid on the left with the following pH values: 3.5, 5.0, 8.0 and 9.0

Acetic acid                      pKa = 4.75        ___________________
Formic acid                      pKa = 3.75        ___________________
Glycine amide hydrochloride      pKa = 8.2         ___________________
Boric acid                       pKa = 9.24        ___________________




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Buffers are typically produced by selecting a weak acid and the salt of the weak acid or by selecting a weak base
and the salt of the weak base. For example if one selected acetic acid as the weak acid then sodium acetate would
be selected as the salt of the weak acid. The pKa for acetic acid is = 4.75. At pH.4.75 an acetic acid sodium acetate
buffer would contain equal amounts of acetic acid and acetate ion. This greatest buffer capacity for any buffer
occurs over a two pH unit range centered about the pKa value of the weak acid or weak base i.e., acetate buffer is
most effective from pH 3.75 to pH 5.75.

Things to remember about buffers:

1.    Choose a buffer whose pKa value is close the desired pH.

2.    Prepare buffers at the temperature and concentration you expect to work at.




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