N-Cyclohexyl-2-aminoethanesulfonic acid (CHES), with p K a of 9.3, is one of the

Question

N-Cyclohexyl-2-aminoethanesulfonic acid (CHES), with pKa of 9.3, is one of the buffers that biochemists use to maintain a constant pH during enzymatic reactions that need a slightly alkaline pH. A CHES buffer is usually made by titrating a solution of the acid form of CHES (Mw = 207.29 g/mol) with NaOH (Mw = 40.00 g/mol).

Calculate the mass of CHES that shall be weighed out in order to prepare 90 mL of 350 mM CHES buffer stock solution at pH 8.6. Express the answer in grams. The allowed error is 2%

Explanation / Answer

CHES buffer: molecular wt = 207 g/mol

given: pKa = 9.3, Buffer pH = 8.6

Step 1:

Use Henderson-Hasselbach eq:

pH = pKa + log [A-]/[HA]------------(1)

HA = acid (CHES)

A- = conjugate base of CHES

8.6 = 9.3 + log [A-]/[HA]

log [A-]/[HA] = 8.6 - 9.3 = - 0.7

A-/HA = 10^-0.7 = 0.2

[A-] = 0.2[HA]----------- (2)

Step 2:

Given: Total buffer conc M = 350 mM = 0.35M

Volume V = 90 ml = 0.09L

Total number of moles = n = n[A-] + n[HA] = M*V = 0.35 * .09 = 0.0315 moles--------(3)

As per eq(2)

M[A-] = 0.2*M[HA]

n[A-]/V = 0.2* n[HA]/V

i.e n[A-] = 0.2*n [HA]--------------(4)

substituting eq (4) in eq (3)

1.2*n[HA] = 0.0315

n[HA] = 0.0315/1.2 = 0.1575 moles

n[HA] = mass of HA/mol wt of HA = mass of CHES/Mol wt of CHES

mass of CHES = 0.1575 * 207 = 32.6 g

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