t Design and preparation of buffers effective at different pHs
These experiments aimed to determine the optimum pH ranges various buffers are effective and provide opportunity for the use of the Henderson-Hasselbalch equation to prepare a buffer of a specific pH. Three different buffer systems were initially investigated; volumes of weak acid and weak bases of specified concentration were prepared and titrated against strong acid or strong base solutions with pH readings taken at frequent intervals to determine pH ranges over which the systems are most effective. The Henderson-Hasselbalch equation was used to calculate an appropriate ratio of acid:base volumes in order to prepare a buffer solution with a given pH value. The main results conveyed the optimum pH range for each buffer system, where significant changes of pH value are resisted with additions of small amounts of acid or base.
Our body cells have natural ability to resist excessive pH changes. Metabolic activity produces acid and to a lesser extent, base and they hydrogen ions associated can alter the overall charge, configuration and function of various proteins. The majority of acid results from carbohydrate and fat metabolism, producing CO2, which combines with H2O to form H2CO3, carbonic acid, dissociating to form H+ and HCO3- ions. Most bases come from metabolism of anionic amino acids and oxidation of organic anions, producing HCO3- ions. The pH changes associated are resisted by various physiological buffering systems. So preparation of a buffer system at a particular pH, as well as finding the effective pH range for different buffers, is of use as artificial buffers are therefore necessary to mimic this ability when studying biochemical processes in vitro. The Henderson-Hasselbalch equation defines the relationship between values of pH and pK and the molar ratio of conjugate acid and conjugate base concentrations.
pH = pK + log([A-]/[HA])
If pK and desired pH is known, molar ratio of acid:base can be calculated. The equation was rearranged to find the ratio necessary to produce a buffer of a given pH.
The aims of the experiment were:
* Determination of pH ranges over which the buffering systems acetic acid/acetate, Tris(base)/Tris (acid) and glycine (acid)/glycine (base) respectively are effective and plotting the associated values on a titration curve. * Use and rearrangement of the Henderson-Hasselbalch equation to prepare a buffer of specific pH. * Use and calibration of a pH meter to measure pH.
The pH meter was calibrated using solutions of pH appropriate to the titration. Volumes of glycine and Tris (0.1M) were made up and acetic acid was titrated against NaOH, Tris against HCl and glycine against NaOH. After each aliquot was ran in from the burette, pH values of the resulting solutions were measured and recorded. Hydrochloric acid was titrated against sodium hydroxide with pH measured in the same way, to give a comparison curve. The ratio of acetic acid:sodium acetate was calculated through rearrangement of the Henderson-Hasselbalch equation and a buffer solution of pH 5.2 was made through mixing the appropriate volumes of each.
Aliquots of 0.2M NaOH at intervals of 1ml were ran into a 0.1M solution of acetic acid. The pH was measured, using a pH meter, after the addition of each aliquot.
Fig 1. shows the resulting change in pH after each 1ml addition of NaOH. Raw data is presented in Table 1 in the Appendix.
A steady increase in pH is seen with the addition of up to 10ml of NaOH. The pH ranges from 2.90 – 5.82. The acetic acid/acetate buffer system is successfully resisting excessive changes in pH with the addition of alkali, this is the range over which the buffer system is most effective. A sharp change in pH is seen after 11ml of NaOH is added, increasing to 11.20. The pK value is 4.56, after addition of 5ml of NaOH.
Aliquots of 0.2M HCl at intervals of 1ml...
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