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Experiment No. 2 June 26, 2012 Alcohols in Buffers of Biological Systems Ma. Cristina F. Areola Joseph Dayap Vince St. D. Mesias Drexell M. Parcon

Objectives: The main goal of this experiment is to determine the effect of alcohol specifically its acidity to the buffers of biological system such as HPO4-2/H2PO4- buffer. In addition, it also aims to explain the principle behind the buffer system and its variations with respect to alcohols.

Materials: This experiment requires the use of the following apparatuses: 250-ml beaker, base burette, iron stand, spatula, watch glass, burette clamp, 10-ml graduated cylinder, 250-ml Erlenmeyer flask, stirring rod and medicine dropper. It also requires the presence of the following reagents: NaOH pellets, phenolphthalein indicator, alcohol solution (e.g. alcoholic beverages), sodium dihydrogen phosphate (NaH2PO4) and disodium monohydrogen phosphate (Na2HPO4).

Methodology: The entire process was divided into three, which are preparation of the standard NaOH solution, preparation of buffer system and the titration of buffer system with standard NaOH solution. In preparation of 250-ml of 0.5M NaOH solution, 5 grams of NaOH pellets was weighed and dissolved to 250 ml mark of volumetric flask, using water. (See calculations on discussion section) In preparation of 500 ml of 0.1 M HPO42-/H2PO4- buffer at pH=7, calculations were performed and after doing so, the said buffer system requires 2.52 grams of NaH2PO4 and 4.64 grams of Na2HPO4. The weighed quantities were dissolved in water using 500-ml volumetric flask and it was followed by further dilution up to 500-ml mark.

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In titration process, 10 ml of HPO42-/H2PO4- buffer system was titrated with 0.5 M NaOH solution. The volume of the corresponding endpoint was recorded. After doing so, the mixtures of a 10 ml buffer together with 15 ml up to 21 ml of alcohol solution (gin) with 2 volume increments (i.e. 15, 17, 19, 21) were titrated at 4 different Erlenmeyer flasks with the same NaOH solution. The volume of the corresponding end points were recorded and tabulated.

Data and Results: Titration of a HPO42-/H2PO4- buffer system mixed with various amounts of alcohols Volume of Alcoholic solution Volume of NaOH solution at corresponding endpoint 0 ml 0.75 ml 15 ml 0.8 ml 17 ml 0.9 ml 19 ml 1.0 ml 21 ml 1.0-1.10 ~ 1.05 ml

Analysis and discussion: By definition, a buffer solution results changes in pH with dilution or with addition of acids or bases. A buffer is a mixture of a weak acid and its conjugate base or a weak base and its conjugate acid. This is because the presence of a weak acid suppresses the hydrolysis of its conjugate base and the presence of the conjugate base suppresses the ionization of a weak acid. Thus, there is an enough amount of both species to react with acids or bases, thereby resists drastic change in pH. The same reason is applicable for weak base-conjugate acid buffer system. In terms of using strong acid-conjugate base pair or strong base-conjugate base pair; these systems will not work as buffer because a strong acid has a conjugate base that has negligible basicity and thus it will not react with acids, thereby the pH will dramatically decrease. On the other hand, a strong base has a conjugate acid with negligible acidity and thus it will not react with bases, thereby the pH will dramatically increase. That is the logical explanation of why weak acid-conjugate base pair or weak base-conjugate acid pair are preferred for buffer solutions. In relation to this experiment, the buffer system is composed of HPO42- and H2PO4- , thus the equilibrium reaction is NaH2PO4 + H2O H3O+ + Na2HPO4. In this case, the acid is NaH2PO4 and its conjugate base is Na2HPO4. Titration of such buffer system implies that the volume of base needs to react with NaH2PO4 is between 0.7-0.8 ml. It should be noted that this indicates its buffer capacity with respect to base. The buffering capacity is the effectiveness of a buffer solution and it is dependent on the amount of acid and conjugate base from which the

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buffer is made. The larger the amount, the larger the buffering capacity. Therefore, 0.7-0.8 ml of 0.5 M NaOH solution is required to consume all NaH2PO4 before drastic increase in pH occurs, that is when the color of the solution turned to pink. When mixtures of alcohols and buffer solution were titrated with 0.5 M NaOH solution, it was noticed that as the volume of alcohol mixed with constant volume of buffer increases, the volume of 0.5 M NaOH required to neutralize the acidity also increases. This is due to the acidic property of alcohol, when an alcohol is dissociated it will give up hydrogen ion, thus increasing the concentration of H3O+ in the buffer system (NaH2PO4 + H2O H3O+ + Na2HPO4). By Le Chateliers principle, increasing the concentration of H3O+ favors the production of NaH2PO4 and thus more amount of acid will react with NaOH. Therefore, it requires more volume (amount) of NaOH in order to reach the end point. Buffers are very important to biological systems. The pH in the human body varies greatly from one fluid to another. These pH values are crucial for the proper functioning of enzymes and the balance of osmotic pressure are maintained by buffers in most cases. The HPO42-/ H2PO4- is used to maintain the pH at intracellular fluid. Another system which is the H2CO3/HCO3- buffer for the pH of blood, this is responsible to prevent acidosis (acidic) and alkalosis (basis) of the blood. Measurement of blood pH to monitor the acid-base status is a routine in many diseases, because an uncontrolled change in pH can lead to severe consequences. Preparation of HPO42-/ H2PO4- buffer was calculated and describe as follows: 500 ml of 0.1 M H2PO4-- HPO42- buffer at pH = 7.00, pKa2 = 6.86.

Calculations: pH pKa + log = 10( )

10(pH-pKa)

10(pH-pKa) = 10(7.00-6.86) = 1.38 = = 0.05 = 1.38 0.05 = 2.38

= 1.38

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= = = =

= ( (

NaH2PO4 Na2HPO4 ) ) Na2HPO4

Weigh 2.52 g of and 4.64 g of Na2HPO4 then dissolve those quantities in a 500-ml volumetric flask. To be followed by dilution to 500-ml mark using water.

Conclusion: Addition of alcohol in HPO42-/ H2PO4- buffer system decreases its pH. However, because of the buffering capacity, minimal decrease in pH occurs. The decrease in pH is caused by the acidic property of alcohol (weak acid) and by applying the Le Chateliers principle, the reaction favors the production of NaH2PO4 because of increasing H3O+, and thus more acid will react to NaOH is needed to reach the equivalence point/end point. That is why it is increasing with respect to increasing volumes of alcohols. However, consuming all moles of an acid indicates it buffer capacity, or the maximum amount of bases that is tolerated before drastic increase in pH occurs. The effective range of this buffer is described as pK1, which implies that when the pH of the solution is out of that range, thereby buffers will not work anymore. This was demonstrated by excess moles of NaOH which is represented as the pink solution.

References: Chang, R. (2008). General Chemistry: The essential concepts. (5th edition). New York: McGraw-Hill Companies Inc. Devlin, T. (2011). Textbook of Biochemistry. (7th edition). United State: John Wiley and Sons Inc. Skoog, D., West, D., Holler, J., Crouch, S. (2004). Fundamentals of Analytical Chemistry. (8th edition). Shenton Way Singapore: Thomson Learning Asia.