PH MEASUREMENT AND BUFFER PREPARATION

Kriziaoumo P. Orpia, Michael Gavin G. Quinto, Nico Joy C. RidulmeKathryn Chemaine L. Samorano and Ryza Ruba

Group 8 2E Medical Technology Biochemistry Laboratory

ABSTRACTPhosphate 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.

INTRODUCTIONPhosphate buffer solutions with different

pH and concentration were prepared for the experiment. pH was introduced in 1909 by Sörensen who defined it as the negative logarithm of the hydrogen ion concentration [1]. pH in the experiment is measured and determined using different kinds of methods available in the laboratory. These methods are colorimetry which uses color standards to determine the pH of the Buffer Solution, and electrometry via pH potentiometer which uses a glass electrode to sense Hydrogen potential of the solution.

EXPERIMENTALI. Compounds Used

1. For Buffer Preparation Sodium Primary Phosphate

Monohydrate (NaH2PO4.H2O) Sodium Secondary Phosphate

Heptaphydrate (Na2HPO4.7H2O) Concentrated Phosphoric Acid

(H3PO4)2. Acid-Base Indicator Thymol blue Bromphenol blue Bromcresol green Bromcresol purple Phenol red Methyl red Methyl orange Phenolphthalein3. Standard Reagent Preparation Conc. HCl Conc. NaOH

II. Procedure1. Preparation of

ReagentsThe reagents to be prepared were 6.0M

Hydrochloric Acid (HCl) and 6.0M Sodium Hydroxide (NaOH). In preparing 500mL 6.0M Hydrochloric Acid, certain amounts of concentrated Hydrochloric Acid (12.0M) was diluted first. For 6.0 M Sodium Hydroxide, certain amount of NaOH pellets were dissolved

together with distilled water in a 500 mL volumetric flask. Each standard was placed in an amber bottle and was labeled properly.

2. BuffersFor the buffer solutions, six phosphate

buffers were prepared instead of the original procedure where only five phosphate buffers and one TRIS –HCl buffer were produced. The buffers were assigned with desired pH and concentration and are stated as followed.

Table 1. List of buffer solution

VOLUME

CONCENTRATION BUFFER pH

0.5L 0.5M Phosphate 2.00.5L 0.5M Phosphate 3.00.5L 0.5M Phosphate 7.00.5L 0.05M Phosphate 7.50.5L 0.5M Phosphate 8.00.5L 0.5M Phosphate 12.0

3. Electrometric Determination of pHIn measuring pH via electrometry, the

apparatus used was a pH meter. The pH meter was calibrated was then calibrated at pH 4, 7 and 10. After calibrated, the pH of the six buffer solutions were measured and adjusted to their desired pH using the prepared standards, Hydrochloric Acid (HCl) and Sodium Hydroxide (NaOH).

4. Colorimetric Determination of pHUsing a spot plate, small amounts of the

primary phosphate buffer where placed on eight spots of the spot plate. The first spot was treated with the first acid-base indicator, Thymol blue, the second spot was treated with bromphenol blue. Each spot was treated with different acid-base indicator, one different from the other. Each spot was noted for the color it produced after the acid-base indicator treatment. It was also done to the other five buffers left and was noted for their color reaction.

RESULTS AND DISCUSSION1. Preparation of Reagents

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A. Prepare 6.0 M Hydrochloric AcidUsing the dilution formula, the

hydrochloric acid standard was prepared by adding 0.25L of distilled water to 0.25 L of concentrated (12 M) Hydrochloric Acid.

Concentrated HCl = 12M

Dilution: C1V1 = C2V2

(12M)(x) = (6.0M)(0.5L)x = (6.0M)(0.5L)

(12M)

V1 = 0.25L of conc. HCl

B. Preparation of 6.0 M Sodium HydroxideFor the Sodium hydroxide standard, it was

prepared by dissolving 120g of Sodium Hydroxide pellets to 0.5L of distilled water.

6.0M = (x)mol 3.0 mol = 40g 0.5L 1 mol

X = 3.0 mol m=120g NaOH pellets

2. Buffers Each of the buffers was assigned to two

groups. Secondary Phosphate buffer with a desired pH of 7.5 and 0.05M concentration was assigned to the group. Buffers are aqueous systems that tend to resist changes in their pH when small amounts of acid (H+) or base (OH-) are added [3]. A buffer consists of a weak acid (proton donor) and its corresponding conjugate base (proton acceptor). For the experiment, the weak acid is the Primary Phosphate ion in Sodium Primary Phosphate Monohydrate (NaH2PO4.H2O) and its conjugate base is the Secondary Phosphate in Sodium Secondary Phosphate Heptaphydrate (Na2HPO4.7H2O).

H2PO4- HPO4

2- + H+

(1° Phosphate) (2° Phosphate) (Hydrogen Ion)

Phosphate Buffers have three pka values (acid dissociation constant) making it a triprotic acid [4]. pka is needed in the Henderson Hasselbalch equation which is.

pH = pka + log [HPO42- ]

[H2PO4-]

A triprotic weak acid such as phosphate has three plateau regions in its titration curve. Every half-neutralization of each plateau region indicates a pka value. The pka values of Primary Phosphate buffer is 2.12, Secondary phosphate is 7.21 and Tertiary Phosphate Buffer is 12.32. The Henderson- Hasselbalch Equation will give the experimental amount of primary phosphate and secondary phosphate buffer needed. The phosphate buffer of interest in the preparation is

a secondary phosphate buffer with a pka value of 7.21 and a desired pH of 7.5.

pH = pka + log [HPO42- ]

[H2PO4-]

7.5 = 7.21 + log [HPO42- ]

[H2PO4-]

7.5 -7.21 = log [HPO42- ]

[H2PO4-]

0.29 = log [HPO4

2- ] [HPO4

2-]

100.29= [HPO42- ]

[HPO42-]

1.9498446 = [HPO42- ]

1 [HPO42-]

The theoretical mole of the buffer is 2.9498446 moles. Actual Moles of the buffer was calculated from 0.05 M and 0.5 L in volume to be 0.0025 moles. By ratio and proportion, 0.228 g of Primary Phosphate and 0.2271g of secondary phosphate must be used to from the buffer.

3. Electrometric Determination of pHThe buffer was prepared by manipulating the

amount of weak acid and its conjugate base in the solution. In electrometric Determination of pH, the pH of the buffer was read by means of a pH meter. A pH meter is a potentiometer. It consist of a Glass electrode which acts as the cathode and a Reference electrode in this case Standard Calomel Electrode which acts as the anode. Calomel electrode is a platinum electrode in contact with a paste of mercury, mercurous chloride and potassium chloride [4]. The pH of each buffer prepared in the laboratory was measured and adjusted to their desired pH using the pH meter.

If the actual pH was less than the desired pH, the buffer would be treated with a small amount of the Standard Sodium Hydroxide (NaOH). And if the actual pH was greater than the desired pH, the buffer would be treated the same but with a small amount of the Standard Hydrochloric Acid. The group adjusted the pH of the buffer to 7.51 which is approximately 7.5

4. Colorimetric Determination of pHColorimetric determination is done by using a

spot plate and an acid base indicator. The results for the colorimetric determination of pH are shown on Table 2.

Table 2. Colorimetric Test of the Buffers

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Acid-baseindicator

pH2

pH3

pH7

pH7.5

pH8

pH12

Dis H2O

Thymol blue R YO Y Y B B YO

Bromphenol blue

Y YG B B BG BV GL

Bromcresol green

Y Y BL B BV BD B

Bromcresol purple

Y Y V V V V Y

Phenol red Y Y R R RV RV Y

Methyl red P P Y Y Y Y P

Methyl orange R RO YO O O O O

Phenolphthalein

X X X X P RV X

Y=yellow, R=red,V=violet, O=orange, P=pink, B=blue, G=green, X=colorless; L=light, D=dark

ACID-BASE INDICATORS

The group experiment was focused on the Secondary Phosphate buffer with a pH of 7.5.

At Thymol blue, the buffer indicated a yellow coloration. Thymol blue has a pH range of 1.2-2.8 at acid range. In this range red is the color below pH-range and yellow is color above. Thymol blue has a second pH range wherein at pH range 8.0-9.6 it is at basic range and yellow indicated below the pH range and blue is above the pH range. This means the pH of the buffer is below the pH 8.0 because exceeding 8.0 would indicate a blue color.

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.

At Bromcresol green, the buffer also indicates 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.

For bromcresol purple, the buffer gave a violet coloration. The pH range of bromcresol purple ranges from 5.2-6.8. Yellow color indicates that it is above the pH range and pink indicates a high pH value. The color of the buffer might have been affected by other environmental factors hence turning pink to violet. Nevertheless, it narrows the pH range of the buffer to 6.8-8.0.

For Phenol Red, the pH range is 6.8-8.2. The buffer showed a red coloration. Red for a lower

pH than the pH range and yellow for a higher pH than the pH range.

For Methyl red which has a pH range 4.4-6.2, the buffer indicated a yellow coloration.

Methyl orange with a pH range 3.1-4.4, showed orange red colorization.

As so as phenolphthalein which gave a colorless solution that indicates lower the pH range of 8.2.

This further concludes that the pH of the group’s secondary buffer solution with a desired pH of 7.5 is approximately within the pH range generalized by those Acid-Base Indicators which is within 6.8-8.0.

REFERENCES[1] Murray, R.K., Granner D.K., and Rodwell,

V.W. (2006). Harper’s Illustrated Biochemistry. 27th ed. Singapore: McGraw-Hill Companies Inc. (Asia)

[2] Ninfa A.J., Ballou, D.P., and Benore, M. (2010). Fundamental Laboratory Approaches for Biochemistry and Biotechnology 2nd ed. United States of America: John Wiley & Sons Inc.

[3] Lehninger A. L., Nelson D.L., and Cox M.M. (1993). Principles of Biochemistry 2nd ed. New York: Worth Publishers.

[4] Wilson K., and Walker J. (2005). Principles and Techniques of Biochemistry and Molecular Biology 6th ed. New York: Cambridge University Press.

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