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Ben Paolo C. Rabara, Andrea Bernadette Reyes, Jhona Marie Sana, Suzette Pamela Santos, Patricia Sigua, Fatima Tabi Group 7 2F Pharmacy Organic Chemistry Laboratory

A 500mL Phosphate buffer was prepared using 33.5g of HPO4-2 and 2.76g of H2PO4-. The pH was adjusted to the desired value of 8.0 by adding 1.0M HCl using a pH meter to monitor the pH. The buffer was then tested for Colorimetric determination using different acid-base indicators.

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. Mathematically, pH is expressed as the negative log in base of 10 of the hydrogen ion concentration. pH = -log [H+] Buffer solutions are solutions that resist changes in pH (by resisting changes in hydronium ion and hydroxide ion concentrations) upon addition of small amounts of acid or base, or upon dilution. They usually consist of a weak acid and its conjugate base, or, less commonly, a weak base and its conjugate acid. Henderson-Hasselbalch equation is use to relate the pH of the solution to the pK of an acid and the ratio of the concentrations of the acid and its conjugate base. [1] The objectives for this experiment are: 1) To prepare different buffer solutions; 2) To determine the pH of the buffers and samples colorimetrically using different liquid indications and electrometrically using the pH meter; and

3) To calculate the buffer capacity of the prepared buffer solutions.

A. Compounds tested Distilled water, Primary sodium phosphate monohydrate, Secondary sodium phosphate heptahydrate and the Acid-base indicators (Thymol blue, Bromphennnol blu, Bromcreson green, Bromcresol purple, Phenol red, Mthyl red, Methyl orange and Phenophthalein). B. Procedure 1. Preparation of Reagents 250mL of 0.5M HCL was prepared from 1.0M HCl. (1.0M HCl)(500 250mL HCl ml)(0.5M HCl) =

2. Buffer Preparation A buffer solution was prepared using the following guidelines: Volume (L) = 0.500 Concentration (M) = 0.5 Buffer Solution = Phosphate Desired pH = 8.0 Primary sodium phosphate monohydrate and Secondary sodium phosphate heptahydrate were used in preparing the buffer.

pH= pKa + log ([WA]/[CB]) pH= 7.21 + log ([HPO4-2] / [H2PO4-]) log-1 (8-7.21) = [HPO4-2] / [H2PO4-] 6.17/1 = [HPO4-2] / [H2PO4-] Total theoretical moles of buffer = 6.17 Total actual moles of buffer = (.05M)(0.25L) = 0.125 moles Actual Moles [HPO4-2]: (6.17/6.17) (x/0.125 moles) = 0.125 moles HPO4-2 Actual Moles [H2PO4-]: (1/6.17) (x/0.125 moles) = 0.020 moles H2PO4Grams of HPO4-2: 0.125 moles x 268 Grams of H2PO4-: 0.020 moles x 138 = =

Actual moles x MW = g/mol = 33.5g HPO4-2 Actual moles x MW = g/mol = 2.76g H2PO4for Buffer

Figure 2. A pH meter used Electrometric Determination


Figure 1. Computation Preparation

3. Electrometric Determination of pH The pH meter was calibrated at pH 8. 20mL portion of the buffer solution was adjusted to the desired pH by adding 1.0M HCl in portions (dropwise). 4. Colorimetric Determination of pH Prepare 6 test tubes and label it with the pH of the buffer and acid-base indicator to be added. Pipet 5 mL of a buffer and add 2 drops of an acid-base indicator. Shake the mixture and note down its color. Repeat this procedure using a different acid-base indicator.

Using the Phosphate Buffer as the sample the following results were obtained through Colorimetric Determination of pH: Table 1. Results of Colorimetric Determination of pH for pH 8.0 of Phosphate Buffer Acid Base pH 8.0 Buffer Indicator Thymol Light yellow Bromophenol blue Lavender Bromocresol green Faded blue Bromocresol purple Purple Phenol red Pinkish red Methyl orange Yellow orange Phenolphthalein Colorless Colorimetric determination of pH showed the varying color changes acidbase indicator undergoes when added to a solution of a certain pH. It could also show molecular characteristics of a substance such as color changes due to electron confinement.


The buffer assigned was tested using Electrometric Determination of pH and adjusted to the desired pH by adding 1.0M HCl.

Table 2. Results of Colorimetric Determination of pH aside from pH 8.0 Acid Base 2.0 3.0 5.0 7.0 7.5 Indicator Light Light Thymol Dull Pink Dull Yellow Dull Yellow Yellow Yellow Bromophenol Dull Yellow Yellow Lavender Blue Aqua blue Bromocreson Light Dull Yellow Light Blue Blue Aqua green Yellow Bromocresol Bright Yellow Yellow Purple Purple purple Yellow Yellow Dull Light Phenol red Fuchsia Yellow Orange Orange Orange Orange Methyl orange Neon Pink Red Orange Orange Red Phenolphthalein Colorless Colorless Colorless Colorless Colorless

12.0 Dark Blue Light Blue Light Blue Purple Dark Pink Orange Red Violet

Crisostomo, Angelica C., et al. (2010). Laboratory Manual in General Biochemistry. Quezon City: C & E Publishing, Inc. Campbell, M.K., Farell, S.O.(2009). Biochemistry. 6th ed. Philippines: Cengage Learning AsiaPte. Ltd Kahn, G. and Stokes, J. The Comparison of the Electrometric and Colorimetric Methods for Determination of the pH of Gastric Contents. df 1/4/2012