Experiment 1 Determination of pH

pH

The pH of a solution is the common logarithm of the reciprocal of the

hydrogen ion concentration expressed as :

1
pH = log -----
H+

pH is detected and measured by using a pH meter, but the most common detectors are
the color changes of acid-base indicators. An acid-base indicator is a weak organic
acid. The ionized and unionized forms of the indicator have different colors:

Hln + H2O <===> H+ + ln-

acid color basic color

When the indicator is placed in an acid solution the equilibrium is shifted to

the left and the Hln from predominates. In an alkaline colution, the ln - form
predominates. The color of the solution depends upon the ration of the Hln
to ln and is related to pH in the following equation:

[ln-]
log10 --------- = pH – pKa
[Hln]

The color of the indicator solution is therefore a measure of the pH.

Buffers

A buffer solution is one that resists a change in pH when a small amount of

acid or base is added. A buffer solution contains a weak Bronsted acid, HA
and its conjugate base, A -. An example is the mixture of acetic acid and
sodium acetate.

Buffers are significant in Biochemistry. An excellent example is its effect in

blood. Blood contains phosphates, carbonates, and proteins as buffering
components and has a constant pH between 7.35 and 7.45 even though
there are large amounts of acids and bases constantly introduced by
digestion of foods, absorption, muscular activity and respiration.

Procedure for pH determination:

1. Preparer the following samples:

a. Defibrinated blood
b. Fresh milk
c. Freshly voided urine

2. Determine the approximate pH of the sample using the pH

paper by following these steps.
a. Dip a piece of pH paper in the sample for about 10
seconds. Remove the paper and place it on a watch
glass.
b. Match the color produced in the pH paper with the
color chart to determine the pH of the sample.
c. Record the results.

3. Determine the accurate pH of the samples using the pH

meter.
Buffer Solution Preparation:

Each group will be assigned to prepare phosphate buffered saline solution

(PBS) that has to be adjusted to different designated specific pH.

Follow the procedure given in the laboratory for the PBS preparation.

N.B. Students will be appropriately supervised in using the pH meter for

their laboratory exercise.
QUESTIONS:
1. What is the H+ concentration of a phosphate buffer solution if its
pH is 7.4?
pH= -log [H+]
(log) 7.4= [H+]
0.869 or 0.87=H+

2. What is the significance of buffer systems in humans?

 Buffering in blood is crucial to our survival. The pH of blood must be kept

constant for normal body functions to work. If blood becomes too acidic, or too
basic, then enzymes and proteins are unable to function.

3. What is the Henderson-Hasselbalch equation? Explain.

 The Henderson Hasselbalch equation is an approximate equation that shows the

relationship between the pH or pOH of a solution and the pKa or pKb and the
ratio of the concentrations of the dissociated chemical species. In order to use
the equation, the acid dissociation constant must be known.

 There are multiple ways to write the equation. Two of the most common are:

pH = pKa + log ([conjugate base]/[weak acid])

pOH = pKa + log ([conjugate acid]/[weak base])

4. What is the normal pH of blood? Urine? Gastric juice? Why is it

necessary to know the pH of these physiologic fluids?

 Blood is normally slightly basic, with a normal pH range of about 7.35 to 7.45.
Usually the body maintains the pH of blood close to 7.40. The normal values
range for urine is from pH 4.6 to 8.0. The normal volume of the stomach fluid is
20 to 100 mL and the pH is acidic (1.5 to 3.5).

 For the blood, it is necessary because to help the body's metabolic processes
and other systems to work well. These processes produce acids, so the body has
a complex system of feedback and regulation to maintain healthy pH levels.

 For the urine, if your urine is at an extreme on either the low or high end of pH
levels, you can adjust your diet to reduce the likelihood of painful kidney stones.
 In short, your urine pH is an indicator of your overall health and gives your doctor
important clues as to what's going on in your body.

 For the gastric fluid, it is critical to the digestion of many nutrients and acts as
the first line of defense against harmful bacteria and viruses. The stomach is a
reservoir of strong acid, with a much lower pH than in any other part of our
digestive system.

5. What is the biochemical relevance of pH?

 pH is biologically important because it affects the structure and activity of
macromolecules. ... Extreme pH levels can denature enzymes, completely
disrupting their function. Other proteins are also destabilized by extreme pH
levels. The pH and pOH of a solution are related such that: pH+pOH=14.