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pH Measurement and Buffer Preparation

Tinuviel M. Esguerra, Zyra Fem C. Flores, Kherson Gandola,

Dionne Audrey G. Go and Ma. Ellah Patricia Gutierrez
Group 4 2C Biochemistry Organic Chemistry Laboratory

In this experiment, pH measurement and buffer preparation are involved. The main objectives of this experiment are:
a) prepare different buffer solutions b) compute the buffer capacity of the buffer solutions made c) determine the pH
of the buffers and samples colorimetrically using liquid indicators and electrochemically using the pH meter. Different
buffer solutions were prepared by different groups. In this group, the assigned buffer for the group was 0.50 M
phosphate buffer at pH 7. Before the group preceded to the actual computation, it was important to know the starting
materials and compute for the amount of each component in preparation of the buffer solution. The Henderson-Hassle
Balch equation was used in order to prepare it. A pH meter was used to determine the pH of the buffer. Then, 6M HCl
and 6MNaOH was used to adjust its pH. Three samples were used in order to perform the experiments. These samples
were distilled water, sprite, and the buffer solution at pH 7.00. In the Electrometric Determination of pH, the pH of
Distilled water was found to be at 6.31. In sprite sample, it is 3.35. While in the buffer prepared, it is 6.96. In the
Colorimetric Determination of pH, the use of acid-base indicators were involve. Same samples were used. Yet,
different pH of buffer solutions were also involved. At pH 6.96, a drop of Thymol Blue appeared to be pale yellow. In
Bromophenol Blue, it became indigo. In Bromocresol Green, it is blue. While in Bromocresol Purple, it appeared as
pale indigo. In Phenol Red, it appeared as orange yellow. While it is pale yellow in Methyl Red and yellow orange in
Methyl Orange. However, it appeared as colorless in the Phenolphthalein indicator. The sprite sample was compared in
a buffer solution with a pH of 2.99 because of its reaction with the indicators. On the other hand, the distilled water
was found to be at pH 5.02 with the same reason as the sprite sample.

The main objectives of this experiment are: a)
pH is a critical part of biochemistry because all prepare different buffer solutions b) compute the
biological processes are affected by it. pH is defined as buffer capacity of the buffer solutions made c)
the measurement of hydrogen-ion which determines determine the pH of the buffers and samples
the acidity and basicity in a sample solution. A pH colorimetrically using liquid indicators and
which is greater than 7 is considered as a electrochemically using the pH meter. The assigned
basic/alkaline solution while pH less than 7 is labeled buffer for the group was 0.50 M phosphate buffer at
as an acidic solution. However, a pH of 7 alone is pH 7.
considered as a neutral solution. A good example of it
is pure water. pH of a sample solution can be EXPERIMENTAL
determined by the used of pH meter. A. COMPOUNDS TESTED
Moreover, acid-buffer system is one of the bodys Distilled Water
pH control mechanisms [1]. Buffer is defined as the Sprite
mixture of a weak acid and its conjugate base or a Buffer solution
weak base and its conjugate acid. It is capable of Thymol Blue
resisting and stabilizing the sudden change in pH when Bromophenol blue
an acid or base is added in a solution. It plays a major Bromophenol green
role in a human body system because it is the one
Bromocresol purple
responsible for the maintenance of pH of the fluids of
Methyl red
an organism. In a human body, alkaline/ basic buffers
Methyl orange
are being released in the bloodstream when the blood
is acidic however, if the blood is too basic/alkaline, Phenolphthalein
acidic buffer is the one being released into the Phenol red
bloodstream to maintain the normal activity of the
body [2]. In addition, the most significant buffer agent B. PROCEDURE
in the body is the bicarbonate. ELECTROMETRIC DETERMINATION OF pH
Henderson-Hasselbalch equation which is also known The pH meter was calibrated at pH 4, 7 and 10. The
as the buffer equation is normally used in computing pH of 20 mL portion of sprite and distilled water was
the pH of a sample solution containing the amount of measured and [H+] was calculated. The pH of the
acid and its conjugate base. prepared buffer solution was adjusted to desired pH by
adding either 6.0 M HCl or 6.0 M NaOH and monitored
by the pH meter.

A. Preparation of color standards using the
buffered solution
Six test tube was prepared and labeled with the pH Thymol Blue Pinkish 1.2-2.8 Yellow
of the buffer and acid-base indicator to be added. In a red
vial, 5 mL buffer with a certain pH and 2 drops of acid-
base indicator was added. The mixture was shaken and Thymol blue Yellow 8.0-9.6 Blue
noted the color observed. The procedure was done to
all the prepared buffers. Another six set test tube was Bromophenol Yellow 3.0-4.6 Violet
prepared. The procedure was repeated but different blue
acid-base indicator was used.
B. Determination of the pH of samples Bromocresol Yellow 4.5-5.5 Blue
Two drops of acid-base indicator was added to green
5mL-distilled water/sprite. The mixture was shaken
Bromocresol Yellow 5.2-6.8 Purple
and noted the color observed. The same procedure
was done but with another acid-base indicator.
Phenol red Orange 6.8-8.4 Red
A. Electrometric Determination of pH Methyl orange Red 3.1-4.4 Yellow
The pH meter was used to determine the
electrochemical properties of liquids, pastes and semi- Phenolphthalei colorless 8.0-9.8 Pink
solids. However, the pH meter used has an electrode n
only suitable for liquids. The instrument used indicates
the hydrogen ion concentration in the test solution by B. Colorimetric Determination of pH
responding to the potential of the electrical cell. The Colorimetric analysis is a method of determining the
instrument also gave accurate pH readings from the concentration of a chemical element or chemical
prepared buffer solutions. However, the buffer compound in a solution with the aid of a color reagent.
solutions made was a little lower than the desired pH The color reagent was the buffer samples that are
thus, adding 6M NaOH to make the solutions to have dropped with the indicators aforementioned. Those
the desired pH which is pH 7. If the solution has a would be the standard pH colors that will be used to
higher pH than the desired one, addition of 6M HCl determine the pH of distilled water and the sprite
must be done rather than the 6M NaOH. The pH meter sample. The pH of a solution can be determined by
is sensitive when it comes to reading the concentration comparing the color intensities of the solution with
of hydrogen and hydroxide ion making it accurate than unknown pH with the intensities of the solutions with
the handheld pH meter and pH paper. As shown in standard pH. As shown on table 3, the distilled water
table 1, the pH of distilled water is 6.31, the sprite showed close similarities with pH 5.02 while the sprite
sample 3.34 and the buffer prepared is 6.96. These sample showed close similarities with pH 2.99.
three was used and will be further discussed on the
next topic. Table 3.1 Colorimetric Determination of pH

Table 1. Electrometric Determination of pH Indicator pH 2.02 pH 2.99 pH 5.02

Samples pH [H+] Thymol pale pink pale pale

Blue yellow yellow
Distilled Water 6.31 4.9x10-7M
Bromophe pale pale pale
Sprite Sample 3.34 4.9x10-4M nol blue yellow yellow indigo
Buffer 6.96 1.1x10-7M Bromocre pale light pale blue
Prepared sol Green yellow yellow

Bromocre light yellow pale

Colorimetric Determination of pH
sol Purple yellow yellow
This part of the experiment was just like using the
pH paper in determining the pH of one solution. There
were certain Acid-Base indictors used to determine the Phenol pale yellow yellow
pH level of the sample as shown in table 2. There are Red orange
also ranges and color intervals for a specific indicator
used. The color changes in the molecules of a solution Methyl light pink pink pale pink
using an indicator can be caused by electron Red
Methyl light red scarlet orange
Table 2. Indicators used
Acid-base Acid- Acid- Acid- Phenolpht colorless colorless colorless
indicator base base base halein
indicator indicato indicator
Table 3.2. Colorimetric Determination of pH
Indicator pH 6.96 pH 7.53 pH 7.96 of the solutions that were mixed with different
Thymol pale pale pale Since this is a comparative experiment, there
Blue yellow yellow yellow are some similar results with other pH levels. Some
factors that may cause discrepancy in the pH is
Bromophe indigo indigo indigo miscalculation of the concentration of the sample,
nol blue accuracy in the pH meter, etc. Overall, the group
managed to compare the experimental results with the
Bromocre blue blue blue calculate pH of the samples.
sol Green
Bromocre pale purple purple [1], [2] (n.d) (2016). The importance of pH balance.
sol Purple indigo Retrieved from
balance 2/26/17
Phenol orange pale pale
Red yellow scarlet yellow Bathan, G.I. , Crisostomo, A.B.C., Daya, ML., De Guia,
R.M., Farrow, F.L., Gabana, M.G., Guevarra, L.A.Jr.,Lui,
Methyl pale pale pale red n a,G.T., Pen
n a, L.L., Santiago, L.A.,
M.L.D., Pen
Red yellow yellow
Santiago, M.R., Sarile, A.S., Torres, P.C., Vargas, A.G.,
Methyl yellow yellow yellow Ysrael, MC., (2017) Retrieved from Laboratory Manual
Orange orange orange orange In General Biochemistry Second Edition. 2/26/17

Phenolpht colorless colorless colorless


Table 3.3. Colorimetric Determination of pH

Indicator pH 12.03 Distilled Sprite
Water Sample

Thymol pale blue pale pale

Blue yellow yellow

Bromophe indigo pale pale

nol blue indigo yellow

Bromocre indigo pale light

sol Green green yellow

Bromocre purple pale yellow

sol Purple yellow

Phenol pink yellow yellow


Methyl pale light pink pink

Red yellow

Methyl yellow orange scarlet

Orange orange

Phenolpht dark pink colorless colorless


pH = 5.02 2.99

The pH of the samples distilled water, sprite
soda, and the buffers solutions were measured with a
pH meter. The group was given the instruction to
prepare a buffer with pH 7.0. 8 groups have prepared
different buffer solutions and the results of the
colorimetry method were compared. The objective of
the colorimetric determination is to compare the colors