PH MEASUREMENT AND BUFFER PREPARATION

Anna Katrina M. Donato, Abigail O. Dy, Keith Brian T. Enriquez, Debbie Marie R. Fermin

Group 3 2E- Medical Technology Biochemistry Laboratory

ABSTRACT

A 500 ml Phosphate buffer solution of pH 7.0 with a molar concentration of 5M was prepared
using 10.67057611 g of Primary sodium phosphate monohydrate (NaH2PO4.H2O) and
12.77743171 g of Secondary sodium phosphate heptahydrate (Na2HPO4.7H2O). The pH of the
buffer was adjusted to the desired value by adding in either portions of 1.0M HCl or 1.0 M NaOH
while being monitored by a pH meter. The buffers prepared with varying pH were subjected in
Colorimetric determination using different acid-base indicators. The sample, distilled water with
pH 3, was also subjected to Colorimetric determination giving the result colors of yellow-orange
for Thymol blue, light green for Bromphenol blue, blue for Bromcresol green, yellow for
Bromcresol purple, yellow for Phenol red, pink for Methyl red, orange for Methyl orange, and
colorless for Phenolphthalein.

INTRODUCTION

The term pH refers to a measure of the hydrogen ion concentration of a solution. Solutions
with a high concentration of hydrogen ions have a low pH and solutions with low concentrations
of H+ ions have a high pH. Therefore, pH is also used as a measure of the acidity or basicity of a
solution. Mathematically, pH is expressed as the negative log in base 10 of the aquated
hydrogen ion concentration.
pH = -log [H+]

One way of measuring pH is by using a device called pH meter. A pH meter consists of a pair
of electrodes connected to a meter capable of measuring small voltages, on the order of
millivolts. A voltage, which varies with the pH, is generated when the electrodes are placed in a
solution. This voltage is read by the meter, which is calibrated to give the pH.

A buffer is a solution which contains a weak conjugate acid-base pair that can resist drastic
changes in pH upon the addition of small amounts of a strong acid or base. A buffer resists
changes in pH because it contains both an acidic species to neutralize OH- ions and a basic one
to neutralize H+ ions. It is a requirement though that the components of a buffer must not
consume each other, that’s why buffers are often prepared by mixing a weak acid or a weak
base with a salt of that acid or base.
EXPERIMENTAL

A. Compounds tested (or samples used)

Distilled water, Primary sodium phosphate monohydrate (NaH2PO4.H2O), Secondary sodium

phosphate heptahydrate (Na2HPO4.7H2O), Acid- base indicators ( Thymol blue, Bromphenol blue,
Bromcresol green, Bromcresol purple, Phenol red, Methyl red, Methyl orange, Phenophthalein)

B. Procedure

1. Preparation of Reagents

250 ml of 0.5 M HCl was prepared from 1.0 M HCl. The container was labeled properly.

(10 M HCL)(500 ml) x (0.5 M HCL) (x ml)

X= 250 ml HCl

Figure1.Calculation of volume of 0.5 M HCl

1. Preparation of buffer

The buffer solution was prepared using the following guidelines:

Table 1. Guideline for buffer preparation

Volume Concentration Buffer Solution Desired pH

(L) (M)

0.500 0.5 Phosphate 7.0

Primary sodium phosphate monohydrate (NaH2PO4.H2O) and Secondary sodium phosphate

heptahydrate (Na2HPO4.7H2O) were used in preparing the buffer. The container was labeled
properly.

Given:

pH= 7 Weak acid= HPO4-2

0.5 M Conjugate base= H2PO4-

250 ml pKa= 7.21

Handerson-Haselbach:

pH= pKa + log [WA]/ [CB]

pH = 7.21 + log [HPO4-2]/ [H2PO4-]

log-1 (7-7.21)= [HPO4-2]/ [H2PO4-]

0.616595001/ 1= [HPO4-2]/ [H2PO4-]

Total theoretical moles of buffer= 1.616595001

Total actual moles of buffer= (0.5M)(0.25L)= 0.125 moles

Actual moles [HPO4-2]: (0.616595001/1.616595001) = ( x/ 0.125)

= 0.047676984 moles HPO4-2

Actual moles [H2PO4-]: (1/ 1.616595001)= (x/ 0.125)

= 0.077323015 moles H2PO4-

Grams of HPO4-2:

Actual moles x MW = 0.047676984 moles x 268 g/mol

= 12.77743171 g HPO4-2

Grams H2PO4-:

Actual moles x MW= 0.077323015 moles x 138 g/mol

= 10.67057611 g H2PO4-

Figure 2.Computation for buffer preparation

 1. Electrometric Determination of pH

The pH meter is calibrated. The pH of 20 ml portion of distilled water was measured and the
+
[H ] was calculated. The pH of the buffer solution was adjusted to the desired pH by adding in
portions of either 1.0 M HCl or 1.0 M NaOH while being monitored by the pH meter.

2. Colorimetric Determination of pH

a. Preparation of Color Standards Using the Buffer Solutions

Six vials/ test tubes were prepared and labeled with the pH of the buffer and acid-base
indicator to be added. 5 ml of a buffer of a certain pH and 2 drops of an acid-base indicator was
added into the vial. The mixture was shaken and the resulting color was noted. This procedure
was performed on all the buffers prepared. Another set of 6 vials/ test tubes were prepared. The
procedure was repeated but another acid-base indicator was used.

+2 drops of acid-base indicator to a spot

+10 drops Phosphate buffer to each spot

mix note color

Figure 3. Colorimetric determination using spot plate

Table 2. Acid-base indicators used in Colorimetric determination of pH

Acid-base Indicators

Thymol blue

Bromphenol blue

Bromcresol green

Bromcresol purple

Phenol red

Methyl red

Methyl orange
 Phenolphthalein

b. Determination of the pH of samples

Two drops of an acid-base indicator was added to 5 ml of distilled water. The mixture was
shaken and the resulting color was noted. This procedure was repeated using each acid-base
indicator.

RESULTS AND DISCUSSION

1. Electrometric Determination of pH

The pH meter showed accurate readings of the pH of the buffer. It showed fluctuations in
readings with the slightest addition of HCl and NaOH. It displayed sensitivity to a small amount
of [H+] and [OH-] ions. The pH meter therefore, is more accurate in reading pH levels compared
to a pH paper.

2. Colorimetric Determination of pH
Table 3. Results of Calorimetric Determination of pH
Acid-Base pH Distilled
indicator Water
2.0 3.0 7.0 7.5 8.0 12.0

Thymol blue red yellow- yellow yellow yellow blue yellow-

orange orange

Bromophenol yellow yellow- blue blue blue blue- light

blue green violet green

Bromcresol yellow dark blue blue blue- blue- blue

green yellow green green

Bromcresol yellow yellow violet violet blue- violet yellow

purple violet

Phenol red yellow yellow yellow- red red red yellow

orange violet

Bogen indicator ------ ------ ------ ------ ------ ------ ------

Methyl red pink pink yellow yellow yellow pink pink

Methyl orange red red- yellow- orange orange orange orange

orange orange

Phenolphthalein colorless colorless colorless colorless light- red- colorless

 pink violet

pH 3.0

For pH 2.0, 3.0, 7.0, 7.5, 8.0, 12.0, and distilled water, the following are the color results for
acid-base indicator Thymol blue: red, yellow-orange, yellow, yellow, yellow, blue, and yellow-
orange.

For pH 2.0, 3.0, 7.0, 7.5, 8.0, 12.0, and distilled water, the following are the color results for
acid-base indicator Bromophenol blue: yellow, yellow-green, blue, blue, blue, blue-violet, and
light green.

For pH 2.0, 3.0, 7.0, 7.5, 8.0, 12.0, and distilled water, the following are the color results for
acid-base indicator Bromcresol green: yellow, dark-yellow, blue, blue, blue-green, blue-green,
and blue.

For pH 2.0, 3.0, 7.0, 7.5, 8.0, 12.0, and distilled water, the following are the color results for
acid-base indicator Bromcresol purple: yellow, yellow, violet, violet, blue-violet, violet, and
yellow.

For pH 2.0, 3.0, 7.0, 7.5, 8.0, 12.0, and distilled water, the following are the color results for
acid-base indicator Phenol red: yellow, yellow, yellow-orange, red, red, red-violet, and yellow.

For pH 2.0, 3.0, 7.0, 7.5, 8.0, 12.0, and distilled water, the following are the color results for
acid-base indicator Methyl red: pink, pink, yellow, yellow, yellow, pink, and pink.

For pH 2.0, 3.0, 7.0, 7.5, 8.0, 12.0, and distilled water, the following are the color results for
acid-base indicator Methyl orange: red, red-orange, yellow-orange, orange, orange, orange, and
orange.

For pH 2.0, 3.0, 7.0, 7.5, 8.0, 12.0, and distilled water, the following are the color results for
acid-base indicator Phenolphthalein: colorless, colorless, colorless, colorless, light-pink, red-
violet, and colorless.
 Colorimetric determination of pH showed the varying color changes an acid-base indicator
undergoes when added to a solution of certain pH. This property of an acid-base indicator can
therefore be used to identify different substances by narrowing their pH range. For example:
Using Bromophenol blue as an acid-base indicator, a solution turned yellow-green. By such
observation, one can say that the pH of the solution is 3.0. This can help in the identification of a
substance since different substances exhibit different pH levels. Acid-base indicators can also be
used to narrow down the pH range of a substance. For example: A resulting color of blue-violet
using acid-base indicator Bromcresol green indicates a pH>8.0, and a resulting color of violet in
acid-base indicator Bromcresol purple indicates a pH<7.5. Therefore, we can estimate that the
pH of the substance must be between 7.5 and 8.0.

Acid-base indicators also show molecular characteristics of a substance. Color changes in

molecules can be caused by changes in electron confinement. More confinement makes the light
absorbed bluer (darker), and less makes it redder (lighter).

REFERENCES

From books:

[1] Bursten, B.E., Brown, T.L., LeMay, H.E.(2004). Chemistry: The Central Science. 9th ed.
Singapore: Pearson Education Inc.

[2] Campbell, M.K., Farell, S.O.(2009). Biochemistry. 6th ed. Philippines: Cengage Learning Asia
Pte. Ltd.

From the internet:

[1] Biology online

http://www.biology-online.org/dictionary/Buffer 1/10/ 10

[2] Brooklyn academic

http://academic.brooklyn.cuny.edu/biology/bio4fv/ page/ph_def.htm 01/10/10

[3] Harper College

http://www.harpercollege.edu/tm-ps/chm/100/dgodambe/thedisk/ph/abind.htm 01/12/10