Determination of pharmaceuticals by High Performance Liquid

Chromatography: Determination of Caffeine in Beverages

Objectives:

1. To explain the principle of High Performance Liquid Chromatography (HLPC)

2. To identify significant information from the chromatogram
3. To carry out quantitative procedure in HLPC

Introduction:

High-performance liquid chromatography is a technique in analytical chemistry used to

separate, identify, and quantify each component in a mixture. It relies on pumps to pass a pressurized
liquid solvent containing the sample mixture through a column filled with a solid adsorbent material.
Each component in the sample interacts slightly differently with the adsorbent material, causing
different flow rates for the different components and leading to the separation of the components as
they flow out of the column. HLPC has been used for manufacturing legal, research, and medical
purposes. Chromatography can be described as a mass transfer process involving adsorption. HLPC
relies on pumps to pass a pressurized liquid and a sample mixture through a column filled with
adsorbent, leading to the separation of the sample components. The active component of the column,
the adsorbent, is typically a granular material made of solid particles, 2-50 micrometres in size. The
components of the sample mixture are separated from each other due to their different degrees of
interaction with the adsorbent particles. The pressurized liquid is typically a mixture of solvents and is
referred to as a mobile phase. Its composition and temperature play a major role in the separation
process by influencing the interactions taking place between sample components and adsorbent. These
interactions are physical in nature, such as hydrophobic, dipole-dipole and ionic, most often a
combination. Caffeine is an alkaloid that occurs naturally in the leaves, seeds and fruit of tea, coffee,
cocoa, kola trees and more than 60 other plants. The widespread occurrence of caffeine in a variety of
plants played a major role in the long-standing popularity of caffeine-containing products. The most
important sources of caffeine are coffee, tea, guarana, mate, cola nuts and cocoa. The amount of
caffeine found in these products varies- the highest amounts are found in guarana, followed by tea
leaves, mate tealeaves, coffee beans, cola nuts and cocoa beans. Recreationally, caffeine is used to
provide a boost of energy or a feeling of heightened alertness. It is often used to stay awake longer.
The pleasant stimulant feeling, which often occurs at low doses, may be replaced by psychological
symptoms, which resemble anxiety and depressive neuroses at high doses.