PH MEASUREMENT AND BUFFER PREPARATION

Paul Benjomin G. Agregado, Maria Kristene D. Alba, Ana Kristiana Louise A. Banzon, Rovileen M. Barroquina, Margaret Eve L. Bartolome Group 1 2F Pharmacy General Biochemistry Laboratory

ABSTRACT
The experiment was done to achieve the following: prepare different buffer solutions, determine the pH of the buffers and samples colorimetrically using different liquid indicators and electrometrically using the pH meter. Primary phosphate buffer solution was used in the experiment. It was prepared using Phosphoric acid (H3PO4) as weak acid and Primary sodium phosphate dihydrate (NaH2PO4.2H2O) as conjugate base. The pH of the buffer solution was measured electrometrically using the pH meter and manipulated using 6M HCl (to make the buffer more acidic) and 6M NaOH (to make the buffer more basic). This instrument indicates the hydrogen ion concentration in a test solution by responding to the potential developed by an electrical cell. [2] The pH meter showed accurate pH readings of the prepared buffer solution. It showed fluctuations in readings with the slightest addition of HCl and NaOH. Thus, the pH meter is more accurate in reading pH levels compared to a pH paper. The pH of the prepared buffer solution was measured colorimetrically using acid-base indicators (Thymol blue, Bromophenol blue, Bromocresol green, Bromocresol purple, Phenol red, Methyl orange, Phenolphthalein). 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. 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. The colour-change interval of an indicator is the pH range, where pronounced colour change takes place and it was determined in the experiment: Thymol blue (2-3/8-11), Bromophenol blue (3-5), Bromocresol green (3-5), Bromocresol purple (5-7), Phenol red (5-8), Methyl orange (5-7), Phenolphthalein (8-11).

INTRODUCTION
We frequently encounter acids and bases in our daily life. Fruits, such as oranges and apples, contain acids. Household ammonia, a cleaning agent, and Liquid Plumber are bases. [4] According to the 1923 definition of Thomas M. Lowry of England and Johannes N. Bronsted of Denmark, acids release protons (hydrogen ions) in their reactions whereas bases are substances wich accepts protons (hydrogen ions). [1] Strong acids release protons readily and almost completely in dilute aqueous solutions but weak acids do not so that, at equilibrium, in most cases, less than 1% of a weak acid is ionized to yield protons. Strong bases have a great capacity for accepting protons whereas weak bases are poor acceptors of protons. [1] Since weak acids dissociate only to a small extent in dilute aqueous solution, the concentration of H+ in dilute solutions of these acids is small. Frequently, the concentration of hydrogen ions in solutions of weak acids is less than 10-6 mol/L. It can be somewhat inconvenient mathematically to work with values of this small magnitude. To permit easier handling of such low values of [H+], the Danish chemist S.P.L.

Sorensen proposed in 1909 that [H+] expressed logarithmically as follows: [1] pH = -log10H+

be

The pH of solutions is important in the biomedical sciences for two main reasons. First, the proper functioning of biomolecules depends to an important degree on the control of pH. Second, changes as small as 0.1 or 0.2 pH unit can cause significant metabolic disturbances in certain cells, tissues, and organs. Because of the pH sensitivity of many biomolecules, control of pH also is important for the success of several procedures used in the biomedical laboratory. These include the separation, purification, and assay for biological activity of several biomolecules. [1] There are certain solutions that resist change in pH even when we add to them acids or bases. Such systems are called buffers. [4] Buffers resist changes in pH because of the Le Chatelier Principle governing equilibrium conditions. [4] Buffers contain relatively high concentrations of weak acids or bases and their conjugate partners which are generally present as salts of the weak acid or base. [1]

Because pH is dependent on ionic activity, a property which cannot be measured easily or fully predicted theoretically, it is difficult to determine an accurate value for the pH of the solution. [6] The pH reading of a solution is usually obtained using a pH meter or pH indicator paper/liquid. The experiment was done due to the following objectives: (1) to prepare different buffer solutions (2) to determine the pH of the buffers and samples colorimetrically using different liquid indicators and electrometrically using the pH meter and (3) to calculate the buffer capacity of ther prepared buffer solution.

weighed to contain the Primary sodium phosphate dihydrate (NaH2PO4.2H2O) powder. reagent. A mass of 1.716g Primary sodium phosphate dihydrate (NaH2PO4.2H2O) was weighed using an analytical weighing scale then was transferred to the 500mL beaker. A certain volume of distilled water was added to the beaker to make 250mL. The prepared solution was transferred to a 500mL amber bottle that was labeled properly. Computations: Molesbuffer = (0.250L)(0.10M) = 0.025 moles Henderson Hasselbach equation: pH = pKa + log 2.00 = 2.12 +log = antilog [2.00 2.12] = Mbuffer = 0.76MH2PO4-1 + 1MH3PO4 = 1.76M MolesH2PO4-1 = =

EXPERIMENTAL
A. Compounds tested (or Samples used) Distilled water, Phosphoric acid (H3PO4), Primary sodium phosphate dihydrate (NaH2PO4.2H2O), 6M HCl, 6M NaOH, Acid-base indicators (Thymol blue, Bromophenol blue, Bromocresol green, Bromocresol purple, Phenol red, Methyl orange, Phenolphthalein) B. Procedure 1. Preparation of reagents A volume of 120mL conc. HCl (12.2M) was measured using a graduated cylinder to prepare a volume of 250mL of 6M HCl. The reagent was transferred to a suitable container that was labeled properly. Computation: (12.2M)(xL) = (6.0M)(0.250L) x = 0.12L or 120mL conc. HCl 2. Preparation of Buffer solution The buffer solution was prepared using the following guidelines: Table 1. Guidelines preparation
Weak acid H3PO4 Conjugate base H2PO4-1

= 0.011 moles Moles H3PO4= =

= 0.014 moles mL H3PO4 = 14.7M H3PO4 = = 0.000952L = 0.95mL gNaH2PO4.2H2O = (0.011M)(156g/mol) = 1.716g

for

buffer
Conc. 0.10M pH

solution
pKa H3PO4 2.12

3. Electrometric Determination of pH The pH meter was calibrated at pH 7 by soaking the electrode in neutral reagents like distilled water. The electrode was lifted out of the neutral solution and dried using a tissue paper. The electrode was then immersed in the prepared buffer solution (Primary phosphate buffer). The standard buffer should have a pH within two (2) pH units of the expected pH of the test solution. [3] The bulb of the electrode must be completely covered with solution. [3] The pH reading was

Volume 250mL

2.00

A volume of 0.95mL (19 drops) Phosphoric acid (H3PO4) was measured using a serological pipette and an aspirator. It was then dropped into a 500mL beaker. A paperbox was made and

read. If the reading was within the proper pH range of the desired, remove the electrode from the buffer solution, rinse it with distilled water using a water bottle and then dried using a tissue paper. And if the pH reading is not within the desired pH range, adjust the pH by removing the electrode from the buffer solution, cleanse and dry it then add in portions of either 6.0M HCl or 6.0M NaOH depending if you want to make the buffer solution more acidic (use former) or more basic (use latter). The process was continued until the pH of the buffer solution is within the proper range.

G EACH

+
5mL of prepared buffer Solution

+
2 drops of acid-base indicator corresponding to the labelled test tubes

Single probe combination electrode

Shake

Note color Electrode Handle

Figure 2. Colorimetric determination of pH procedure

Buffer Electronic meter

RESULTS AND DISCUSSION

1. Electrometric Determination of pH The pH meter is used to measure the electrochemical properties of liquids, pastes and semi-solids. [2] This instrument indicates the hydrogen ion concentration in a test solution by responding to the potential developed by an electrical cell. [2] The pH meter showed accurate pH readings of the prepared buffer solution. It showed fluctuations in readings with the slightest addition of HCl and NaOH. Adding the former will decrease the pH and adding the latter will increase the pH of the buffer, this is because the electrode is sensitive to change in the concentrations of [H+] and [OH-] ions. Thus, the pH meter is more accurate in reading pH levels compared to a pH paper. 2. Colorimetric Determination of pH Certain organic substances change color in dilute solution when the hydrogen ion concentration reaches a particular value. For example, phenolphthalein is a colourless substance in any aqueous solution with a hydrogen ion concentration greater than 1.0x10-8 M (pH less than 8.0). In solutions with a hydrogen ion concentration less than 1.0x10-8 M (pH greater than 8.0), phenolphthalein is red or pink. Substances like phenolphthalein, which can be used to determine the pH of a solution,

Figure 1. pH meter 4. Colorimetric Determination of pH A certain number of test tubes (7) was prepared and properly labeled with the acid-base indicators. Each test tube was filled with 5mL of the prepared buffer solution using a serological pipette and an aspirator. A certain amount (2 drops) of acid-base indicator was dropped in the corresponding labeled test tubes. The test tubes were shaken and the color was taken down. Table 2. Acid-base indicators used
Thymol blue (A) Bromophenol blue (B) Bromocresol green (C) Bromocresol purple (D) Phenol red (E) Methyl orange (F) Phenolphthalein (G)

are called acid-base indicators. Acid-base indicators are either weak organic acids, HA, or weak organic bases, BOH, where the letters A or B stand for complex organic group. [6] The equilibrium in a solution of the acidbase indicator methyl orange, a weak acid, can be represented by the equation HA red H+ + Ayellow

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). [7] Colorimetric Analysis uses the variation as a means of determining the pH since the intensity of the color of a solution changes with its concentration or pH. The color may be due to the inherent property of a substance in the solution, or the formation of a product as a result of the addition of a suitable reagent or acid-base indicator. The pH of a solution can be determined by comparing the color intensities of the solution with unknown pH with the intensities of the solutions with known pH. [6] Table 3. Color-change intervals of acidbase indicators used (from resources)
Acid-base indicator Color in the more acidic range pH range (colorchange interval) Color in the more basic range

The anion of methyl orange is yellow, and the non-ionized form is red. If acid is added to the solution, the increase in the hydrogen ion concentration shifts the equilibrium toward the red form in accordance with the law of mass action.

The indicator's colour is the visible result of the ratio of the concentrations of the two species A- and HA. For methyl orange:

Thymol Blue Thymol blue Bromphenol blue Bromocresol green Bromocresol purple Phenol red Methyl orange Pheolphthalein

Pinkish red Yellow Yellow Yellow Yellow Orange Red colorless

1.2-2.8 8.0-9.6 3.0-4.6 4.5-5.5 5.2-6.8 6.8-8.4 3.1-4.4 8.0-9.8

Yellow Blue Violet Blue Purple Red Yellow Pink

When [H+] has the same numerical value as Ka, the ratio of [A-] to [HA] is equal to 1, meaning that 50% of the indicator is present in the red acid form and 50% in the yellow ionic form, and the solution appears orange in colour. When the hydrogen ion concentration increases to a pH of 3.1, about 90% of the indicator is present in the red form and 10% in the yellow form, and the solution turns red. No change in colour is visible for any further increase in the hydrogen ion concentration. [6] Addition of a base to the system reduces the hydrogen ion concentration and shifts the equilibrium toward the yellow form. At a pH of 4.4 about 90% of the indicator is in the yellow ionic form, and a further decrease in the hydrogen ion concentration does not produce a visible colour change. The pH range between 3.1 (red) and 4.4 (yellow) is the colour-change interval of methyl orange; the pronounced colour change takes place between these pH values. In general, the colour-change interval of an indicator is the pH range, where pronounced colour change takes place; the borders of this interval can be estimated by pKa-1 and pKa+1.[6]

Table 4. Results of the colorimetric determination of pH

Acid-base indicator Thymol blue Bromphenol blue Bromocresol green Bromocresol purple Phenol red Methyl orange Phenolphthalein 2.0 Dull pink Dull yellow Light yellow Yellow Yellow orange Neon pink Colorless 3.0 Dull yellow Yellow Dull yellow Bright yellow Fuchsia Orange red Colorless 5.0 Light yellow Lavender Light blue Yellow Yellow Red Colorless pH 7.0 Dull yellow Blue violet Blue Purple Dull orange Orange Colorless 7.5 Light yellow Blue Blue Purple Light orange Orange Colorless 8.0 Light yellow Lavender Faded blue Purple Pinkish red Yellow orange Colorless 11.0 Dark blue Blue violet Light blue Purple Dark pink Orange Red violet

REFERENCES
From books: [1]Cecil, J.R. (1995). Basic Biochemical Laboratory Procedures and Computing with Principles, Review Questions, Worked Examples, and Spreadsheet Solutions. (1st ed.). New York: Oxford University Press. Pages 40-65. [2]Bernas, G.C., Ysrael, M.C., & Bernaldez, A.T. (1994). Basic Laboratory Studies in Biochemistry (3rd ed.). Manila: UST Publishing House. Pages 59 and 17. [3]Boyer, R.F. (2006). Biochemistry Laboratory: Modern Theory and Techniques. (1st ed.). San Francisco: Pearson Education Inc. Pages 55-69. [4]Bettelheim, F.A., & Landesberg, S.M. (2010). Laboratory Experiments for Introduction to General, Organic, and Biochemistry. (7th ed.). USA: Brooks/Cole. Pages 207-210. [5]Crisostomo, A.C., et. al. (2010). Laboratory Manual in General Biochemistry. Quezon City: C & E Publishing, Inc. Pages 1-4. From the internet: [6]images.kimme08.multiply.multiplycontent.co m/attachment/0/ (12/31/11) [7]http://www.scribd.com/doc/25375272/PhMeasurement-and-Buffer-Preparation (12/31/11)