A guide for the preparation and use of buffers in biological systems

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Ionization of Water Water molecules undergo reversible ionization to yield H + and OH¯ as per the following equation. H 2 O → ← H + + OH¯ The degree of ionization of water at equilibrium is fairly small and is given by the following equation where K eq is the equilibrium constant. K eq [H + ][OH¯] = ______________ [H 2 O] At 25°C, the concentration of pure water is 55.5 M (1000 ÷ 18; M.W. 18.0). Hence, we can rewrite the above equation as follows: K eq [H + ][OH¯] = ______________ 55.5 M or (55.5)(K eq ) = [H+][OH¯] For pure water electrical conductivity experiments give a K eq value of 1.8 x 10 -16 M at 25°C. Hence, (55.5 M)(1.8 x 10 -16 M) = [H + ][OH¯] or 99.9 x 10 -16 M 2 = [H + ][OH¯] or 1.0 x 10 -14 M 2 = [H + ][OH¯] [H + ][OH¯], ion product of water, is always equal to 1.0 x 10 -14 M 2 at 25°C. When [H + ] and [OH¯] are present in equal amounts then the solution gives a neutral pH. Here [H + ][OH¯] = [H + ] 2 or [H + ] = 1 x 10 -14 M 2 and [H + ] = [OH¯] = 10 -7 M As the total concentration of H + and OH¯ is constant, an increase in one ion is compensated by a decrease in the concentration of other ion. This forms the basis for the pH scale. 3
Dissociation Constants of Weak Acids and Bases Strong acids (hydrochloric acid, sulfuric acid, etc.) and bases (sodium hydroxide, potassium hydroxide, etc.) are those that are completely ionized in dilute aqueous solutions. In biological systems one generally encounters only weak acids and bases. Weak acids and bases do not completely dissociate in solution. They exist instead as an equilibrium mixture of undissociated and dissociated species. For example, in aqueous solution, acetic acid is an equilibrium mixture of acetate ion, hydrogen ion, and undissociated acetic acid. The equilibrium between these species can be expressed as: CH 3 COOH k 1 → ← H + + CH 3 COO¯ k 2 where k 1 represents the rate constant of dissociation of acetic acid to acetate and hydrogen ions, and k 2 represents the rate constant for the association of acetate and hydrogen ions to form acetic acid. The rate of dissociation of acetic acid, -d[CH 3 COOH ]/dt, is dependent on the rate constant of dissociation (k 1 ) and the concentration of acetic acid [CH 3 COOH] and can be expressed as: ____________________ d [CH 3 COOH] dt = k 1 [CH 3 COOH] Similarly, the rate of association to form acetic acid, d[HAc]/dt, is dependent on the rate constant of association (k 2 ) and the concentration of acetate and hydrogen ions and can be expressed as: d [CH 3 COOH ] __________________ dt = k 2 [H + ] [CH 3 COO¯] Since the rates of dissociation and reassociation are equal under equilibrium conditions: k 1 [CH 3 COOH ] = k 2 [H + ] [CH 3 COO¯] or k 1 [H + ] [CH 3 COO¯] _______ = ____________________ [CH 3 COOH] and where 4 k 2 k _______ 1 k 2 [H + ] [CH 3 COO¯] K a = ___________________ [CH 3 COOH] =K a (Equilibrium constant)
Page 1 and 2: Buffers A guide for the preparation
Page 3 and 4: A Word to Our Customers We are plea
Page 6 and 7: Why Does Calbiochem® Biochemicals
Page 10 and 11: This equilibrium expression can now
Page 12 and 13: Table 1: pK a Values for Commonly U
Page 14 and 15: [H + ] x Vol = [H + ] o x Vol o whe
Page 16 and 17: concentration is dependent upon the
Page 18 and 19: Table 2: pK a and DpK a /°C of Sel
Page 20 and 21: pH Measurements: Some Useful Tips 1
Page 22 and 23: 8. Use stock solutions to prepare p
Page 24 and 25: 2. Glycine-HCl Buffer; pH range 2.2
Page 26 and 27: 8. Glycine-Sodium Hydroxide, pH 8.6
Page 28 and 29: Tris Buffered Saline (TBS), pH 7.4
Page 30 and 31: Isoelectric Point of Selected Prote
Page 32 and 33: Ionization Constants K and pK a for
Page 34 and 35: Frequently there is a linear relati
Page 36 and 37: HEPES, Free Acid, ULTROL® Grade, 1

A guide for the preparation and use of buffers in biological systems

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