Hplc Method Development

Objective

In this experiment you will be optimizing a separation of a mixture of 5 compounds (caffeine, acetone, methyl benzoate, phenatole and phenanthrene) using HPLC by varying the mobile phase composition.

Introduction

We used chromatography modeling software to assist in HPLC method development, with the goal of enhancing separations through the exclusive use of gradient time and column temperature. HPLC is accomplished by injection of a small amount of liquid sample into a moving stream of liquid (termed the mobile phase) that passes through a column packed with particles of a stationary phase. As in gas chromatography, separation of a mixture into its components depends on different degrees of retention of each component in the column. The extent to which a component is retained in the column is determined by its partitioning between the liquid mobile phase and the stationary phases. A variety of HPLC separation techniques that utilize different stationary and mobile phases have been developed.

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Reagent :

1. HPLC grade acetonitrile
2. Deionized water
3. Standard mixture of caffeine, acetone, methyl benzoate, phenatole and phenanthrene ( 100 ppm )

Procedure

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

Detecter  wavelength          : 254 nm

Flow rate                             : 1.5 mL min-1

Mobile phase                       : acetonitrile  water

1. Mobile phase effect on HPLC separation

             The instrument was set to used a mobile phase ratio of acetonitrile : water (50 : 50         v:v)  and the sample were injected . then the mobile phase were changed  to 70 : 30 .          which composition of mobile phase is suitable for the separation of these compound?

1. Component in the mixture were identified

        Each compound were injected individually to identify the component of the mixture         using the selected HPLC condition.

1. Separation using gradient elution

Based on the separation above, gradient elution separation were performed to improve the efficiency of the column.

Discussion

        In this experiment you will be optimizing a separation of a mixture of  5 compounds ( caffeine, acetone, methyl benzoate, phenatole and phenanthrene) using HPLC by varying the mobile phase composition. The mobile phase is ratio of acetonitrile : water  (50 : 50)  then the ratio were change to (70 : 30). The advantages of gradient elution is decrease the retention of the later-eluting components so that they elute faster, giving narrower and taller peaks for most components. Its also improve the peaks shape for tailed peaks. Different compounds are  eluted by increasing the strength of the organic solvent. The sample is injected while a weaker mobile phase is being applied to the system. Next, the strength of the mobile phase is later increased in increments by raising the organic solvent fraction, which subsequently results in elution of retained components. The sample and standards must be prepared carefully to minimise the errors in their concentrations. Deviations from the concentrations may affect the recorded peak areas and subsequently, the calibration curves of peak area against concentration. This may implicate the eventual results obtained.   According to safety data, caffeine is a skin irritant. Upon exposure, wash away with water. The HPLC eluent, with 60% methanol and 40% water, is a flammable mixture and an irritant. Any residue should be placed in the waste container and spillages should be washed away with plenty of water.