CHM160L Biochemistry 1 Laboratory 4th Quarter SY 2010-2011

BUFFERS
Sevilla, Ureah Thea A. , De Ramos, Mariano Cornelio G.
1Professor,

School of Chemical Engineering, Chemistry and Biological Engineering, Mapua Institute of Technology; 2Student (s), CHM160L-A41, School of Chemical Engineering, Chemistry and Biological Engineering, Mapua Institute of Technology

ABSTRACT The experiment was done in order for the students to learn and familiarize the preparation of buffers, to prepare different buffers to be used in the succeeding experiments in the laboratory, to determine the action of buffers in some commercial products and to determine the pKa and isoelectric pH of buffers. As we all know, a buffer solution is an aqueous solution consisting of a mixture of a weak acid and its conjugate base or a weak base and its conjugate acid. In this particular experiment, we used acetic acid and sodium acetate for a buffer stock solution that would be used for different samples. pKa values were also determined graphically in the second part of this experiment and the isoelectronic pH of the amino acid was calculated.
Keywords: pH, pI, Buffer Capacity

INTRODUCTION 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. Buffer solutions are used in industry for chemical manufacturing and fermentation processes, and to set the proper conditions for dyeing fabrics. In research laboratories, buffers are used for chemical analyses, syntheses, and calibration of pH meters. In living organisms, these solutions maintain the correct pH for many enzymes to work. Blood plasma contains a buffer (of carbonic acid and bicarbonate) to maintain a pH of approximately 7.4. The main component of a buffer solution, such as a weak acid or weak base, may be used as a buffering agent. The function of a buffering agent is to drive an acidic or alkaline solution to a certain pH and maintain it at that pH. As pH managers, they are important in many applications, including agriculture, food processing, medicine, and photography. This article discusses buffer solutions prepared with water, but not other solvents. Also, these solutions are presented in terms of the Brnsted-Lowry notion of acids and bases, not the Lewis acid-base theory. The ability of a buffer solution to resist changes in pH is the result of the equilibrium between a weak acid (HA) and its conjugate base (A): HA(aq) + H2O(l) H3O+(aq) + A(aq) Any alkali added to the solution is consumed by the hydronium ions. These ions are mostly regenerated as the equilibrium moves to the right and some of the acid dissociates into hydronium ions and the conjugate base. If a strong acid is added, the conjugate base is protonated, and the pH is almost entirely restored. This is an example of Le Chatelier's principle and the common ion effect. This contrasts with solutions of strong acids or strong bases, where any additional strong acid or base can greatly change the pH. This may be easier to see by comparing two graphs: When a strong acid is titrated with a strong base, the curve will have a large gradient throughout, showing that a small addition of base/acid will have a large effect; by comparison, a weak acid/strong base titration curve will have a smaller gradient when the pH is close to the pKa value. Buffer capacity can be defined in many ways. You may find it defined as "maximum amount of either strong acid or strong base that can be added before a significant change

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in the pH will occur". This definition - instead of explaining anything - raises a question "what is a significant change?" - sometimes even change of 1 unit doesn't matter too much, sometimes - especially in biological systems - 0.1 unit change is a lot. Buffer capacity can be also defined as quantity of strong acid or base that must be added to change the pH of one liter of solution by one pH unit. Such definition - although have its practical applications - gives different values of buffer capacity for acid addition and for base addition (unless buffer is equimolar and its pH=pKa). This contradicts intuition - for a given buffer solution its resistance should be identical regardless of whether acid or base is added. The isoelectric point (pI), sometimes abbreviated to IEP, is the pH at which a particular molecule or surface carries no net electrical charge. Amphoteric molecules called zwitterions contain both positive and negative charges depending on the functional groups present in the molecule. The net charge on the molecule is affected by pH of their surrounding environment and can become more positively or negatively charged due to the loss or gain of protons (H+). The pI is the pH value at which the molecule carries no electrical charge or the negative and positive charges are equal. Surfaces naturally charge to form a double layer. In the common case when the surface charge-determining ions are H+/OH-, the net surface charge is affected by the pH of the liquid in which the solid is submerged. Again, the pI is the pH value of the solution at which the surfaces carries no net charge.

The pKas were determined and based on these values, the isoelectric ph (pI) were calculated.

RESULTS Table 1 pH of Pure Samples SAMPLES Buffer Egg White Fruit Juice 1M HCl 1M NaOH Table 1.1 Buffer Capacities VOLUME in mL 1 2 Beaker Buffer 15 15 Fruit Juice Egg White 1M HCl 0.1 1M NaOH 0.1 pH 4.73 4.94 BC 0.025 0.111 Beaker Buffer Fruit Juice Egg White 1M HCl 4 15 5 15 0.1 pH 5 9.39 4.37 2.36 14.08

MATERIALS AND METHODS The materials that were used are: 0.2 M Acetic acid, 0.2 M Sodium acetate, Volumetric Flask, 1 whole egg, Fresh fruit juice, pH meter, 1 M HCl, 1 M NaOH and 0.005 M Leucine. A 250-ml of Buffer stock solution was prepared using 0.2 M of the acid and 0.2 M of the salt mixed together to form the aqueous solution. pH values of different samples were monitored using the pH meter and the buffer capacities were computed. On the second part of the performance, 0.005 M of Leucine was titrated with 0.1 M NaOH with 0.5 ml increments. pH values were recorded. pH vs. Volume NaOH were plotted.

3 15 0.1 4.26 0.061 6 15 -

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1M NaOH pH BC

0.1 4.59 0.03

8.79 0.111

0.1 9.69 0.022

  

  

  

For the case of fruit juice: Table 2 Determination of pKa Values Volume NaOH added 0 0.5 1 1.5 2 2.5 3 Figure 1 Volume NaOH added vs. pH pH 1.5 1.57 1.76 2.4 11.28 11.82 12.02 No. Of equivalents = (1 M HCl)(0.001 L) = 1 x 10-4 moles Change in pH = |4.94-4.37| = 0.111 So, the buffer capacity of the fruit juice is:

BC = 0.025 This means 0.025 L of the acid is the maximum volume that can be added in the solution before a significant change in pH will occur. Same thing applies in the other samples. The buffer capacities were indicated in the table. The values were computed in the same manner. On the second part, the pH values of Leucine were recorded every addition of 0.5 ml of the base. The volume of NaOH added were plotted vs. the pH that were measured by the ph meter. Figure 1 illustrates the sudden large increase in the pH of Leucine upon addition of 2.0 ml of the base. pKas were determined graphically. pK1 is approximately 1.90 and pK2 was estimated 11.80. The isoelectric pH was computed by getting the average of the two pKa values and computed as 6.85. The pI value can affect the solubility of a molecule at a given pH. Such molecules have minimum solubility in water or salt solutions at the pH that corresponds to their pI and often precipitate out ofsolution. Biological amphoteric molecules such as proteins contain both acidic and basic functional groups. Amino acids that make up proteins may be positive, negative, neutral, or polar in nature, and together give a protein its overall charge. At a pH below their pI, proteins carry a net positive charge; above their pI they carry a net negative charge. Proteins can, thus, be separated according to their isoelectric point (overall charge) on a polyacrylamide gel using a technique called isoelectric focusing, which uses a pH gradient to separate proteins.

LEUCINE
15 10 5 0 1 2 3 4 5 6 7 pH

DISCUSSION In the first part of this experiment, 1 M HCl and 1 M NaOH were added in different samples and their pH values were measured. Of course, upon the addition of the acid, the pH of the samples decreases compared to that of their original ph values. The same thing happened upon the addition of the base, higher pH values were recorded from the pH meter. After determining the pH values, the buffer capacities were calculated using the equation:

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CONCLUSIONS pH of buffers may be affected by different factors like addition of neutral salts, dilution, wherein a decrease in ionic strength may be encountered which may lead to a decrease in the reserve acidity or basicity of the buffer, and also temperature. So upon preparing your buffer solutions, take note of these factors for you to attain a good buffer solution. .REFERENCES
http://www.chembuddy.com/?left=pHcalculation&right=pH-buffer-capacity http://en.wikipedia.org/wiki/Isoelectric_point http://en.wikipedia.org/wiki/Buffer_solution

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