Stability testing

Aim:

To evaluate the acid hydrolysis of the ester methyl acetate (due to its fast rate of
hydrolysis hence enabling you to see quick results. The principle of what happens to
methyl acetate is the same as what might happen for an ester drug, however for drugs the
process of hydrolysis could be much longer (i.e. months or even years).

Introduction

Drugs may be decomposed by a number of routes including hydrolysis, oxidation or

polymerisation. Many drugs are esters or amides and therefore are susceptible to
hydrolysis which might be catalysed by hydrogen ions (acid catalysis) or hydroxyl ions
(base catalysis) (Equation 1.1).

Ester compound Carboxylic acid Alcohol

Equation.1.1. Ester hydrolysis in an aqueous environment.

The rate of hydrolysis of many susceptible drugs is likely to follow first order kinetics (i.e.
being dependent on the concentration of the material which hydrolyses):

d(C0 - Ct ) dCt  kC
Rate   t
dt dt
where:
co is the initial drug concentration
ct is the drug concentration at time t
k is the hydrolysis rate constant
2.303 C0 kt
By integration, the following equation is produced: t   log  log C  log C 
t 0
k Ct 2.303

A plot of Log Ct against t should produce a straight line (Fig.1.1).

Fig.1.1. A first order reaction showing the remaining amount of active against time.
During the hydrolysis of methyl acetate, acetic acid is produced (Equation.1.2).

CH3COOCH3 + H2O + H+  CH3COOH + CH3OH + H+

Methyl acetate Water Acetic acid Methanol

Equation 1.2. Acid-catalysed hydrolysis of methyl acetate.

The resultant acid can be quantified by titration with an appropriate base such as sodium
hydroxide (NaOH) (Equation 4.3):

NaOH + HCl → NaCl + H2O

Sodium hydroxide Hydrochloric acid Sodium chloride Water

Equation 1.3. A typical equation for acid-based titration.

IMPORTANT
Lab coats, safety glasses and gloves should be worn at all times.

Experimental Method

Ask for help/advice if you are unsure about any of the experimental
procedures.

1. You have four thermostatic water baths set up at 25°C, 45°C and 65 °C.

2. In a clean 250 ml Erlenmeyer (conical) flask, place 50 ml HCl (0.2 M) using a

pipette.

3. Add water (45 ml) and properly mix with the acid. Place the flask in the specified
water bath and leave for 10 min to equilibrate.

4. Pipette methyl acetate (5 ml) (MW 74 g/mol; density 0.934 g/ml) into the flask and
mix rapidly. Start counting the time using a stop watch. Immediately take 5 ml of the
mixture using a clean pipette (not the one you used before) and place it in a conical
flask containing 45 ml deionised water.

5. Titrate the contents immediately and accurately with NaOH (0.2 M) using
phenolphthalein as an end-point indicator.

6. Repeat this procedure in the following time intervals: 10, 20, 30, 40 and 50 min
using clean flasks containing 45 ml deionised water. Remember that the original
flask must be immersed in the water bath all the time.
Results:

From the titration data, calculate the concentration of acids in the solutions and so
the concentration of residual ester.

To do this:
 Calculate the initial concentration C0 of methyl acetate.

[Density  Volume (ml) ]  MW

Concentration of methyl acetate (C0 ) 
Total volume of methyl acetate solution (L)

 Calculate Moles of NaOH used: Moles = Concentration (M) × Volume (L)

 Calculate Moles of acetic acid reacted. Remember: this is = moles of NaOH.

 Calculate the Moles of acetic acid at different titration times.

 At 0 minutes only HCl is present. Between 10 minutes and 50 minutes,

both HCL and acetic acid are present. Hydrochloric acid remains constant
for all titration samples, therefore moles of acetic acid at 0 minutes minus
the moles of acetic acid reacted at different temperatures equals the
moles of acetic acid.

 Calculate the concentration of acetic acid at different titration times:

Moles of acetic acid (moles)

Concentration of acetic acid(M) 
Volume (L)

 Calculate the concentrations of methyl acetate (Ch) hydrolysed at different times.

Remember: there is a 1:1 mole ratio.

 Calculate the concentration of methyl acetate (C t) remaining at different times.

Ct = C0 - Ch
Table 1.1. Results at Temperature (X) oC.

NaOH volume Time (min)

(ml) 0 10 20 30 40 50
First (ml)
Final (ml)
Amount
required for
titration (ml)
Moles of
NaOH
Moles of
acetic acid
Concentration
of acetic acid
(M)
Concentration
of residual
methyl
acetate (M)
Log Ct
k
t1/2