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Experiment+01+_+v+W12

# Experiment+01+_+v+W12 - 1-1 Experiment 1 BIOLOGICAL BUFFERS...

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1-1 Experiment 1 BIOLOGICAL BUFFERS Introduction A large fraction of the constituents of cells are weak acids, and some are weak bases; for example: proteins and amino acids, nucleic acids and nucleotides, fatty acids, and most metabolic intermediates. Since the acquisition of a proton can cause an uncharged-base to take on a positive charge ( ie NH 3 + H + NH 4 + ) or can neutralize a negative charge ( ie RCOO + H + RCOOH), the ionic forms of the many molecules that exist in a cell are very much dependent on the intracellular pH. For experiments in vitro , the pH must be set and maintained at a value that will assure appropriate levels of the "biologically active" ionic form(s) of the molecule(s) being examined. The maintenance of pH is accomplished by the introduction of a buffer into the biochemical solution. Buffers resist changes in pH by fixing the ratios of protonated and unprotonated forms of all ionizable groups within the solution. The relation between pH and the ratios of the protonated and unprotonated forms of weak acids and weak bases is described by the Henderson-Hasselbalch equation. The Henderson-Hasselbalch equation is revisited here because it is not only experimentally important in the design of buffers, but also is central to the understanding of many laboratory procedures; for example, separating and identifying molecules, determining pKa values, moderating chemical reactivities, etc. Objectives 1. Prepare buffers by three methods, measure their pHs. 2. Examine buffer capacity. 3. Determine the pKa of a pH indicator dye. Theory The dissociation of a Bronsted (protonic) general acid, HA , is represented by the chemical equation: HA Z H + + A Z-1 (1) where z and z-l are the net charges on the conjugate acid HA Z , and, A Z-1 , its conjugate base. Examples of Bronsted acids are: Note, especially in the case of zwitterionic glycine, that z is the algebraic sum of the charges on that species. The z superscripts have been omitted from equations (2) to (5) for clarity. The Law of Mass Action establishes a quantitative relationship between the chemical activities of an acid and its dissociation products: Ka = (a H + ) (a A ) = ([H + ] γ H + ) ([A] γ A ) (2) a HA [HA] γ HA Bronsted Acid z value NH 4 + +1 CH 3 COOH 0 H 3 N + CH 2 COO - 0 HPO 4 -2 -2

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