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Group 4 2B Medical Technology CHEM 600 Laboratory

The experiment consisted of different parts; preparation of standard reagents, electrometric and colorimetric determination of pH, and preparation of buffer solution. The standard reagents were prepared by diluting 60g NaOH for NaOH solution and 123mL conc. HCl for the HCl solution. Several samples were used to determine pH values using the pH meter. Acetate buffer was prepared in this experiment. It was prepared by using 1.43mL of Ch 3COOH and 0.67g of NaOh pellets. The pH of the buffer was measured electrometrically using the pH meter and was manipulated using 6.0M NaOH solution (to make it more basic) and 6.0M HCl solution (to make it more acidic) to obtain the desired pH of 5.0. For the colorimetric determination of pH, several solutions were used for each acid-base indicator. The pH values used were 4-9, and the results obtained were used for the determination of the pH of the given unknown buffer solution. The colour-change interval of an indicator is the pH range, where pronounced color change takes place and it was determined in the experiment: Thymol blue (8-9);lBromophenol blue (3-4.6); Bromocresol green (4-5), Bromocresol purple (5-6), Phenol red (6.8-8.6), Methyl orange (5-7),Phenolphthalein (8.3-10). The unknown was identified as having pH 7 based on the data on acid-base indicators.

Experiment 1 is about preparation of a buffer solution, electrometric determination of pH, and colorimetric determination of pH. Biological processes are affected by pH. The term pH refers to the measure of hydrogen ion concentration in a solution. Mathematically, pH is expressed as the negative log in base of 10 of the hydrogen ion concentration. pH = - log [H+] Our biological system cannot withstand drastic changes in pH, thus buffers are necessary. A buffer solution is a mixture of either a weak acid and its conjugate base or a weak base and its conjugate acid. Buffers resist changes in pH because of the Le Chatelier Principle governing equilibrium conditions. It resists changes in pH upon addition of small amounts of acid or base, or upon dilution. The Hendersom-Hasselbach equation for buffers relates the pH of the solution to the pK of an acid and the ration of the concentrations of the acid and its conjugate base. The electrometric determination of pH is done with the use of a pH meter while the colorimetric determination of pH is done with the use of different acid-base indicators. The objective of the experiment is to prepare different buffer solutions, and to determine the pH of the buffer solution and other samples using the electrometric and colorimetric methods.

Real Leaf Fruity Lemon Iced Tea Puas Saliva 2. Buffer Solution Glacial acetic acid NaOH pellets 3. Acid-Base Indicator Thymol blue Bromophenol blue Bromocresol green Bromocresol purple Phenol red Methyl red Methyl orange Phenolphthalein B. Procedure The following are the procedures of the various tests involved in this experiment. 1. Preparation of Reagents First, the amounts needed to prepare the reagents were computed using the dilution factor as well as the formula for getting the molar concentration. For 250mL of 6.0M NaOH solution, 60g of NaOH pellets were diluted. For 250ml of 6.0M HCl, 123mL of conc. HCl was diluted. 2. Buffer Preparation Acetate Buffer was assigned to the group. To prepare the buffer solution, the amounts of solute needed were computed. Then, 1.43 mL of CH3COOH and 0.67g of NaOH pellets were combined and diluted with distilled water. The buffer solution was then placed in a 250 mL volumetric flask and was labeled.

A. Compounds Tested 1. Standard Reagents NaOH pellets Conc. HCl 2. Electrometric Determination of pH Distilled Water Coca-cola Light Natures Spring Mineral Water

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Distilled Water Coca-cola Light Nature's Spring Mineral Water Real Leaf Fruity Lemon Iced Tea Pua's Saliva BUFFERS Acetate Buffer

8.07 2.74 4.51 3.3 4.88 5.04

5.11 x 10-9 1.82 x 10-3 3.09 x 10-5 5.01 x 10-4 1.32 x 10-5 9.12 x 10-6



= 1.43 mL NaOH = (0.0166 mole)(40 g/mole) = 0.664 g 3. Electrometric Determination of pH The pH meter was calibrated first at pH 4, 7, and 10. For the first part, the pH of distilled water was measured. The same procedure was done for all the other samples. For each of the pH measured, the [H+] were calculated using the formula 10-pH. Then, the pH of the prepared buffer solution was measured. The buffer solution was then manipulated to pH 5.0 (which is the desired pH) using the 6.0M NaOH solution (to make it more basic) and 6.0M HCl solution (to make it more acidic). 4. Colorimetric Determination of pH Bromocresol purple was assigned to our group as the acid-base indicator to be used with solutions having different pH values. Six test tubes were prepared and labeled with pH 4-9. In each test tube, 5 mL of the sample solution was used. Then, 2-3 drops of bromocresol purple was added in each. The results were then observed to determine the pH range of the indicator. Next, another set of 8 test tubes were prepared. Each contained 5mL of an unknown buffer solution. Then, the test tubes were labeled with their corresponding acid-base indicators. To determine the pH of the unknown, the results from the other acid-base indicators were used. The corresponding color changes were compared as to the color changes of the acid-base indicators in different pH values.

The value of [H ] was computed by getting the antilog of the negative pH. Mathematically, it is 10-pH. For the acetate buffer, the pH was manipulated using 6.0M HCl solution and 6.0M NaOH solution until the desired pH (pH 5.0) was obtained. Figure 1 shows the results for bromocresol purple. The color changes indicate the pH range of the indicator. For bromocresol purple the pH range is 5-6 as inferred based on the results. For the unknown buffer it was inferred that it has a pH value of 7. The inference was based on the results of the other acid-base indicators. The pH range of the acid-base indicators is determined by the pronounced color change observed. It was determined that the pH ranges are: Thymol blue (8-9); Bromophenol blue (3-4.6); Bromocresol green (4-5), Bromocresol purple (5-6), Phenol red (6.8-8.6), Methyl orange (5-7), Phenolphthalein (8.3-10).

Figure 1. Results for bromocresol purple Table 2 contains the result of the colorimetric determination of pH using different solutions having different pH value as well as the result for distilled water and the unknown buffer solution. Refer to the next page for table 2.


Table 1 contains the results of the electrometric determination of pH using different sample solutions and acetate buffer. Table 1. Electrometric Determination of pH


[1] Agregado, P. G. (2012). pH Measurement and Buffer Preparation. Retrieved December 11, 2013, from Scribd: [2] Crisostomo, A. C., et al. (2010). Laboratory Manual in General Biochemistry. Quezon City: Philippines. [3] La Rosa, J. L. (2012, January 9). Formal Report. Retrieved September 8, 2013, from Scribd.: al-Report [4] Rabara, B. P. (2012). pH Measurement and Buffer Preparation. Retrieved December 11, 2013, from Scribd: