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FR1-pH Measurement and Buffer Preparation
FR1-pH Measurement and Buffer Preparation
ABSTRACT
Phosphate buffer solutions with desired pH and concentrations were prepared by mixing a weak acid phosphate to its
conjugate phosphate base. The desired pH of the buffer was adjusted and determined by means of Electrometric and
Colorimetric methods.
Hydrochloric Acid, certain amounts of
concentrated Hydrochloric Acid (12.0M) was
INTRODUCTION diluted first. For 6.0 M Sodium Hydroxide,
Phosphate buffer solutions with different certain amount of NaOH pellets were dissolved
pH and concentration were prepared for the together with distilled water in a 500 mL
experiment. pH was introduced in 1909 by volumetric flask. Each standard was placed in
Sörensen who defined it as the negative an amber bottle and was labeled properly.
logarithm of the hydrogen ion concentration [1].
pH in the experiment is measured and 2. Buffers
determined using different kinds of methods For the buffer solutions, six phosphate
available in the laboratory. These methods are buffers were prepared instead of the original
colorimetry which uses color standards to procedure where only five phosphate buffers
determine the pH of the Buffer Solution, and and one TRIS –HCl buffer were produced. The
electrometry via pH potentiometer which uses a buffers were assigned with desired pH and
glass electrode to sense Hydrogen potential of concentration and are stated as followed.
the solution.
Table 1. List of buffer solution
1
buffers left and was noted for their color A triprotic weak acid such as phosphate has
reaction. three plateau regions in its titration curve. Every
half-neutralization of each plateau region
indicates a pka value. The pka values of Primary
RESULTS AND DISCUSSION Phosphate buffer is 2.12, Secondary phosphate is
1. Preparation of Reagents 7.21 and Tertiary Phosphate Buffer is 12.32. The
A. Prepare 6.0 M Hydrochloric Acid Henderson- Hasselbalch Equation will give the
Using the dilution formula, the experimental amount of primary phosphate and
hydrochloric acid standard was prepared by secondary phosphate buffer needed. The
adding 0.25L of distilled water to 0.25 L of phosphate buffer of interest in the preparation is
concentrated (12 M) Hydrochloric Acid. a secondary phosphate buffer with a pka value of
7.21 and a desired pH of 7.5.
Concentrated HCl = 12M
pH = pka + log [HPO42-]
Dilution: C1V1 = C2V2 [H2PO4-]
(12M)(x) = (6.0M)(0.5L)
x = (6.0M)(0.5L) 7.5 = 7.21 + log [HPO42-]
(12M) [H2PO4-]
7.5 -7.21 = log [HPO4 ]
2-
2
The group adjusted the pH of the buffer to 7.51 At Bromcresol green, the buffer also indicates
which is approximately 7.5 a blue coloration. Bromcresol green has a pH
range of 3.8-5.4 wherein the yellow is also the
below pH range and blue as above the pH range.
The pH range is now narrowed down to 5.4-8.0.
4. Colorimetric Determination of
pH For bromcresol purple, the buffer gave a
Colorimetric determination is done by using a violet coloration. The pH range of bromcresol
spot plate and an acid base indicator. The results purple ranges from 5.2-6.8. Yellow color indicates
for the colorimetric determination of pH are that it is above the pH range and pink indicates a
shown on Table 2. high pH value. The color of the buffer might have
been affected by other environmental factors
Table 2. Colorimetric Test of the Buffers hence turning pink to violet. Nevertheless, it
Acid-base pH pH pH pH pH pH Dis narrows the pH range of the buffer to 6.8-8.0.
indicator 2 3 7 7.5 8 12 H2O For Phenol Red, the pH range is 6.8-8.2. The
buffer showed a red coloration. Red for a lower
Thymol blue R YO Y Y B B YO
pH than the pH range and yellow for a higher pH
Bromphenol than the pH range.
Y YG B B BG BV GL
blue For Methyl red which has a pH range 4.4-6.2,
Bromcresol the buffer indicated a yellow coloration.
Y Y BL B BV BD B
green Methyl orange with a pH range 3.1-4.4,
Bromcresol showed orange red colorization.
Y Y V V V V Y
purple
As so as phenolphthalein which gave a
Phenol red Y Y R R RV RV Y colorless solution that indicates lower the pH
range of 8.2.
Methyl red P P Y Y Y Y P This further concludes that the pH of the
group’s secondary buffer solution with a desired
Methyl orange R RO YO O O O O
pH of 7.5 is approximately within the pH range
Phenolphthalein X X X X P RV X generalized by those Acid-Base Indicators which
is within 6.8-8.0.
Y=yellow, R=red,V=violet, O=orange, P=pink, B=blue, G=green,
X=colorless; L=light, D=dark
REFERENCES
ACID-BASE INDICATORS [1] Murray, R.K., Granner D.K., and Rodwell,
V.W. (2006). Harper’s Illustrated
The group experiment was focused on the Biochemistry. 27th ed. Singapore:
Secondary Phosphate buffer with a pH of 7.5. McGraw-Hill Companies Inc. (Asia)
[2] Ninfa A.J., Ballou, D.P., and Benore, M.
At Thymol blue, the buffer indicated a yellow (2010). Fundamental Laboratory
coloration. Thymol blue has a pH range of 1.2- Approaches for Biochemistry and
2.8 at acid range. In this range red is the color Biotechnology 2nd ed. United States of
below pH-range and yellow is color above. America: John Wiley & Sons Inc.
Thymol blue has a second pH range wherein at [3] Lehninger A. L., Nelson D.L., and Cox
pH range 8.0-9.6 it is at basic range and yellow M.M. (1993). Principles of Biochemistry 2nd
indicated below the pH range and blue is above ed. New York: Worth Publishers.
the pH range. This means the pH of the buffer is [4] Wilson K., and Walker J. (2005).
below the pH 8.0 because exceeding 8.0 would Principles and Techniques of Biochemistry
indicate a blue color. and Molecular Biology 6th ed. New York:
Cambridge University Press.
At Bromphenol blue, the buffer indicates a
blue coloration. Bromphenol blue has pH range of
3.0-4.6 and yellow indicates it is below the pH
range and blue violet indicates it is above the
range. This further narrows the pH range of the
buffer from 4.6- 8.0 as the pH range of the
Phosphate buffer.