MARIANO MARCOS STATE UNIVERSITY

City of Batac, Ilocos Norte, Philippines

College of Medicine

Experiment 4
Experimental Determination of pH
Biochemistry Laboratory

Submitted by:
Balmaceda, Athina T.
Cerezo, Miguel Patrick I.

Dr. Gracia Urubio Urmeneta

September 21, 2015

ABSTRACT

This experiment is designed to determine the pH of a given solution through the use of
colorimetric and electrometric method.
For colorimetric method solutions such as of 0.01 N HCl, 0.01 N HOAc and 0.2% gelatin
were utilized. Approximation of pH values were based on the assigned colors yield by using the
Bogen indicator. A set of three (3) trials were set up using the following: Methyl Red- Methylene
Blue (pH range 4.5-6.0) color change includes purple to green, Methyl orange (pH range 3.1-4.4)
from red to yellow and Phenolpthalein (pH range 8.3-10.0) from colorless to red.
On the other hand, this experiment revealed that when using the electrometric method the
accurate pH of a substance tested will be determined. For instance a distilled water has a pH of
6.46 and compared to a vinegar with a pH of 2.25 which is more acidic than that of the distilled
water.
In conclusion, pH of solutions can be determined using the colorimetric and electrometric
method. However, electrometric method yields a more accurate result than the first method in
determining if the solution is a base or an acid.

INTRODUCTION
pH is a logarithmic measure of hydrogen ion concentration, originally defined by Danish
biochemist Sren Peter Lauritz Srensen in 1909.
pH = -log[H+]
where log is a base-10 logarithm and [H+] is the concentration of hydrogen ions in moles per liter
of solution. According to the Compact Oxford English Dictionary, the "p" stands for the German
word for "power", potenz, so H is an abbreviation for "power of hydrogen"
The hydrogen ion concentration in pure water around room temperature is about 1.0 107
M. A pH of 7 is considered "neutral", because the concentration of hydrogen ions is exactly
equal to the concentration of hydroxide (OH -) ions produced by dissociation of the water.
Increasing the concentration of hydrogen ions above 1.0 10-7 M produces a solution with a pH
of less than 7, and the solution is considered "acidic". Decreasing the concentration below 1.0
10-7 M produces a solution with a pH above 7, and the solution is considered "alkaline" or
"basic".
The average pH of seawater is around 8.2, slightly basic. During photosynthesis,
hydrogen atoms are used by phytoplankton and the pH will rise, becoming more basic.
Respiration and the breakdown of organic matter will lower the pH, making the water more
acidic. In seawater, the pH doesn't change very much because seawater has a natural buffer. A
buffer prevents the pH from changing because chemical reactions occur to balance changes in
the concentration of hydrogen ions.
Most organisms living in estuaries prefer a pH between 6.5 and 8.5. If the pH drops
below 5.0 or goes above 9.0, many marine organisms will have trouble surviving.

METHODOLOGY

A. Preparation of Colorimetric Buffer Standard Solutions

Prepare the colorimetric buffer standard solution assigned to you by pipetting 4.0 ml of the
buffer into a vial and adding 2 drops (small vial dropper) of the indicator for this pH. (Refer to
the preceding table). Label the vial with the pH and the initials of the indicator and submit to the
instructor.
3

B. Colorimetric Determination of pH
Determine colorimetrically the pH of 0.01 N HCl, 0.01 N HOAc and 0.2% gelatin solution.
Determine the approximate pH range of the samples by using either a universal indicator
(example: Bogen indicator) or a set of trial indicators. If Bogen indicator is being used, touch its
dropper to a cavity of your spot plate and add a drop of the solution. The colors given below will
indicate the approximate pH.
Color
Red
Orange
Yellow
Green
Blue

pH
2.0
4.0
6.0
8.0
10.0

A set of three trial indicators:

Trial Indicators
Methyl Red-Methylene Blue (MR-MB)
Methyl Orange (MO)
Phenolpthalein (H2Ph)

pH range
4.5-6.0
3.1-4.4
8.3-10.0

Color Change
Purple-Green
Red-Yellow
Colorless-Red

The pH range of the sample is first determined by the use of methyl red-methylene blue
(MR-MB). Place 2 drops of the sample in a cavity of a spot plate and add a drop of MR-MB. If
the color obtained is purple or pink, the pH of the sample is 4.5 or below. Place another 2 drops
of the sample in a cavity of a spot plate and add a drop of methyl orange (MO). If the color
obtained with MO is red, then the approximate pH range of the sample is 3.1 or below; if orange,
the pH range is between 3.1 and 4.4. If the color obtained with MR-MB is green, the pH of the
sample is 6.0 or above. Place another 5 drops of the sample in the spot plate and add a drop of
phenolphthalein (H2Ph)). If the color with H2Ph is colorless, the pH range is between 6.0 and 8.3,
if it is red or red, the pH is 8.3 or above.

Sample
MR-MB

Purple (pH 4.5) Brown (pH = 4.6-5.9)

Green (pH 6.0)

MO

Red
Orange
Yellow
(pH 3.1) (pH = 3.2-4.3) (pH 4.4)

H2Ph

Colorless
Pink
Red
(pH 8.2) (pH = 8.3-9.9) (pH 10.0)

Knowing the approximate pH range, make the final pH determination by pipetting 4.0 ml of
the solution into a vial and adding 2 drops (small vial dropper) of the indicator used in the
preparation of the colorimetric buffer standards covering this pH range. Compare with the buffer
standards containing this same indicator. Use a clean piece of bond paper as background. The pH
of the solution is the pH of the buffer standard that matches closely in color intensity.
For solutions that are slightly colored or turbid like the gelatin solution, place a vial of
distilled water behind the gelatin with the indicator and a vial with gelatin behind the buffer
standard during the color comparison.
Calculate the hydrogen ion concentration of the sample tested.
C. Electrometric Determination of pH
Determine the pH of the following samples using the pH meter: soft drinks, vinegar, filtered
fresh pineapple juice, calamansi juice, urine, filtrate from soil suspension, sea water, wine.

RESULTS AND DISCUSSION

A. Colorimetric Determination of pH

B. Electrometric Determination of pH
Samples
Fresh Coconut juice
Soft drinks
Wine
Vinegar
Fresh Pineapple juice
Urine
Distilled Water
Filtrate of Soil
Sea Water

pH
1.79
2.10
2.20
2.25
3.33
5.38
6.46
7.23
7.24

On the table above, solutions that were subjected to electrometric determination of pH are
arranged from lowest to highest pH. Fresh coconut juice, soft drinks and wine among the
nine samples were the most acidic. While distilled water, filtrate of soil and sea water are
found to be more alaline. Based from the results obtained, a fresh coconut juice is more
acidic when compared to a sea water with a pH of 7.24

Questions
1. Compare the true or actual acidities of 0.01 N HCl and 0.01 N HOAc and account for the
differences, if any. How would their total or titratable acidities compare?
2. Can the colorimetric method be used to determine the pH of water? Milk? Blood? Why?
Colorimetric analysis is a method of determining the concentration of a chemical
element or chemical compound in a solution with the aid of a color reagent. It is applicable to
both organic compounds and inorganic compounds and may be used with or without
an enzymatic stage. The colorimetric method does not work when the water is already
colored because it contains dissolved organic matter or large amounts of algae. No,
colorimetric method cannot be used because the pH is dependent on the concentration of
hydrogen ions. Colorimetric method requires a change in absorbance of a particular
wavelength of light at different concentrations of the species to be determined. Hydrogen
ions are not colored and hence cannot be used in colorimetric method and water milk and
blood are composed of Hydrogen ions.

3. What physical qualities of vinegar, fruit juices, soft drinks, and wine are affected by pH?
The pH of the vinegar, fruit juices, soft drinks and wine affects the taste sugar/acid
balance and stability of these substances. Taking for example the wine, if the pH is 3.0-3.4 its
color will turn to ruby; color strength is increase; oxidation is reduced; it is more protein
stable and less bacterial growth happens. But when high pH is present (3.6-.4.0) its color is
more brown color strength is reduced; increased oxidation; but its proteins are less stable and
more bacterial growth happens. Same thing happens with the other solution change in color
will be the physical quality that will visibly change when pH is increased or decreased.
4.

CONCLUSION
pH can be determined colometircally or electrometrically. The basic principle of
electrometric pH measurement is determination of the activity of the hydrogen ions by
potentiometric measurement using a standard hydrogen electrode and a reference electrode. .
Colorimetric means to measure color. In the colorimetric method, chemicals are added to the
water sample and those chemicals react with the water to produce a color change. The color
indicates the pH of the water. The color can be measured visually or electronically. Determining
the pH of a substance is significant especially to microorganism whose survival is dependent on
the increase or decrease of their environment where they are living.

REFERENCES

1
2

http://www.123helpme.com/view.asp?id=121990] Retrieved last September 20, 2015.

http://www.chemicalforums.com/index.php?topic=21619.0 Retrieved last September 20,
2015.
3 http://omp.gso.uri.edu/ompweb/doee/science/physical/chph2.htm
Retrieved
last
September 20,2015.
4 Expressing Concentration (www.chem.purdue.edu/gchelp/solutions/character.html)
5 Different ways of expressing the concentration of solutions (chemguide.blogspot.com/2010/04/different-ways-of-expressing.html)
6 Definition
of
pH,
pOH,
"p",
sample
calculations
(www.800mainstreet.com/acid_base/definitions-ph.html)
7 Hydrolysis of salts (www.chemteam.info/AcidBase/Hydrolysis.html)
8 What is pH and why is It Important? (Alters, S and B. Alters. 2006, Biology. John Wiley
& Sons Inc., Hoboken, N.J. 755 pp, suite.io/donald-reinhardt/266b24v)