Agilent Technologies Saponification of procaine Application Note

Udo Huber

Saponification of procaine: Kinetic measurements with the Agilent high throughput analysis system

Abstract

In this application note we describe how the cleavage of procaine, a

p-aminobenzoic acid ester, can be monitored using the Agilent 220

microplate sampler (MPS) with the Agilent 1100 Series LC system. The

data of the measurements is transferred to ChemStore C/S, the data-

base module of the Agilent ChemStation Plus, for data analysis. We

show that the data can then be transferred easily to a spreadsheet

program, for example Microsoft®Excel®, for further calculations such

as determination of the rate coefficient.

Application Note

Procaine is a p-aminobenzoic acid ester, which can be saponificated into p-aminobenzoic acid (PABA) and an alcohol. The reaction is shown in figure 1.

Since the reaction is first order the rate of reaction can be described as:

with:

v (rate of reaction) k (rate coefficient) [Ester] (concentration of procaine)

Integration of this formula gives:

The rate coefficient k can be determined from the slope of the straight line in the graph ln([Ester]

/[Ester]0) against time.

Introduction

Kinetic measurements play an important role in pharmaceutical chemistry. Not only for pharmacokinetics where the rate of active compound degradation has to be determined, but also for drug discovery to test the inhibition effect of a compound on an enzyme. For very fast reactions special apparatus, for example shock tubes, have to be used but slower reactions can be monitored by analyzing reaction samples at specific time intervals. This application note describes how this is achieved using the Agilent 220 MPS with the Agilent 1100 Series LC System and the Agilent ChemStation Plus software. Saponification of procaine at pH=10 was selected as a model scenario.

Figure 1 Saponification of procaine

v·-==

Esterd

][][Esterk

][

Ester Ester

][

·-=

H2N

Equipment

The system included an Agilent 1100 Series vacuum degasser, an Agilent 1100 Series binary pump, an Agilent 1100 Series thermostatted column compartment, an Agilent 1100 Series diode array detector and an Agilent 220 micro plate sampler.

The system was controlled using the Agilent ChemStation Plus (version A.07.01) and the micro plate sampling software (version A.03.01).

System Setup Overview

1. A chromatographic method for measuring procaine and PABA was developed on the Agilent 220 MPS and the Agilent 1100 Series LC system.

2. Standards for both compounds were measured, the method was calibrated and the run time was extended to 20 minutes (figure 2).

3. Three procaine samples were dissolved in 0.025 M NaH2PO

buffer adjusted to pH=10. These samples were measured with the method described before, which gives an overall run time of one hour for the three samples.

4. The measurement was repeated 24 times to give an overall study run time of 24 hours.

5. The measured data was automatically transferred to the ChemStation Plus database module were the Charts

amount against reaction time

was created.

6. To determne the rate coefficient the data was then automatically transferred to Microsoft Excel.

Time [min]

012345

Absorbance [mAU]

100

150

200

250

300

PABA

Procaine

Mobile Phases: A= 0.025M NaH2PO4in

water (pH=2.5), B = ACN

Gradient: 5 % B for 3.5 min,

flow 1 ml/min 5 % B to 50 % B in 1.5 min, flow 1 ml/min 50 % B for 0.5 min, flow 1 ml/min 50 % B to 5 % B in 0.5 min, flow 1 ml/min 5 % B, flow from 1 ml/min to

0.1 ml/min in 0.1 min 5 % B, flow 0.1 ml/min for 18.9 min 5 % B, flow 0.1 ml/min to 1 ml/min in 0.1 min 5 % B for 0.9 min,

flow 1 ml/min Stop time: 20 min Column: Zorbax SB-C18, 4.6 x 75 mm,

5 µm Column temp.: 50 ºC UV detector: DAD 204 nm/16

(reference 360 nm/100)

Figure 2 Measurement of standards

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