High Performance Liquid Chromatography (hplc): Method Development

Introduction

        The fundamental basis for HPLC consists of passing a simple (analyte mixture) in a high pressure solvent (mobile phase) through a column packed with sorbents (stationary phase). The mobile phase acts as a carrier for the sample solution. As the analytes pass through the column they interact between the two phases, mobile and stationary, at different rates. The difference in rates is primarily due to different polarities for the analytes. The analytes that have the least amount of interaction with the stationary phase or the most amount of interaction with the mobile phase will exit the column faster. Therefore, changes in the polarity of the mobile phase can affect the interactions of the sample and the stationary phase and thus changes the efficiency of the HPLC separation. Changes in mobile phases can be done either by isocratic elution or gradient elution.

        Isocratic elution is the mobile phase composition remains unaltered during the separation. The mobile phase may comprise of a single solvent or a pre-mixed mixture of solvents. On the other hand, gradient elution is the composition of the mobile phase is changed during the separation. Two or more solvents that differ in polarity are employed. After sample introduction, the ratio of these solvents is programmed to vary either continuously or in steps.

        The objective of this experiment is to optimize a separation of a mixture of three compounds which are caffeine, methyl benzoate and phenatole using HPLC by varying the mobile phase composition. Then, in this experiment, we have to identify the components in the mixture by using the selected HPLC conditions.

Procedure

1. Instrument set-up (may vary depending on instrument)

Detector wavelength        : 254 nm

Flow rate                : 1.5 mL min-1

Mobile phase                : acetonitrile:water

1. Effect of mobile phase on HPLC separation

The instrument was set to use a mobile phase ratio of acetonitrile:water (50:50 v:v) and the sample was injected. Then, the mobile phase composition was changed to 70:30. The composition of mobile phasa was determined which is suitable for the separation of these compounds.

1. Identification of components in the mixture

Each component was injected individually to identify the components of the mixture using the selected HPLC conditions.

1. Separation using gradient elution

A gradient elution was performed based on the separation above to improve the efficiency of the column.

Results and calculations

1. Caffeine

100 ppm = [pic 1]

           =  ×  × 50 ml[pic 2][pic 3]

                           = 5 mg in 50 ml volumetric flask

1. Methyl benzoate

100 ppm =  ×  ×× × 50 mL[pic 4][pic 5][pic 6][pic 7]

                             = 4.6296×10-3 mL

                4.6296×10-3 mL ×  × [pic 8][pic 9]

                           = 4.6296 μL

1. Phenatole

100 ppm =  ×  ×  ×  × 50 mL[pic 10][pic 11][pic 12][pic 13]

           =5.1760×10-3 mL