HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
SEPARATION AND ANALYSIS OF MIXTURES
Increasingly, the determination of low concentrations of active ingredients (either desired or undesired) in complex mixtures, sold for human consumption, has become more necessary. Federal regulations have imposed strict limits on the type and concentrations of a host of substances sold as foods or drugs. Such requirements demand analytical techniques that are fast and reliable and combine the separation (to alleviate interferences) and analysis steps in a single operation.
Chromatography is the most widely used technique for the analysis of non-inorganic mixtures. Gas chromatography (where the sample must be volatilized) and liquid chromatography (where the sample can be determined in the liquid state) are the most common methods in general use. High Performance Liquid Chromatography (HPLC) is the method of choice whenever the sample cannot easily be converted to the gas phase.
In this laboratory, you will use the technique of HPLC to determine either the concentration of caffeine in a soft drink, coffee or tea, or caffeine in one of a variety of analgesic (pain relief) formulations.
Figure I depicts the main components of a modern HPLC system and their interrelationships. In HPLC, a solution containing the compound(s) of interest is injected into a loop which has been calibrated to contain a specified volume (a 20 L loop injector is a commonly used size). The valve switch is then rotated, allowing a sample stream of mobile phase (the eluent) to sweep the sample from the loop onto a column, packed with a suitable stationary phase, where the separation occurs. The eluent is delivered from a pump at a constant rate, (on the order of 1 mL/min) at a pressure sufficiently high to overcome the backpressure of the column. Pressures of 1000-2000 psi are commonly necessary. An upper limit of 4000 psi is normally set on the instrument. Recall that the separation efficiency is inversely proportional to the particle size of the column packing material. High pressures are required to force a liquid through a tightly-packed column filled with small particle material, and the availability of high pressure solvent delivery systems is directly responsible for the "high performance".
Assuming that a suitable column has been chosen for the separation of interest, all components should pass through the column and "elute" at different times (differential migration). This time differential is due to the differences in the distribution (partitioning) of the various components between the mobile phase (eluent) and stationary phase (column packing), which arise from the physical/chemical differences among the components of the mixture. Thus, each component will pass through the detector and be identified separately.
The time for a substance to pass through the column, termed the retention time, is therefore, related to the identity of the compound and is the basis for qualitative analysis. Quantitative information is obtained from the area or height of the peak produced by the detector.
Figure I : Diagram of a Typical HPLC
Several different approaches to HPLC detection exist. Perhaps most common is a detector based on the absorbance of ultraviolet or visible light, a UV/Vis absorbance detector. These detectors are, in reality, miniature UV/Vis spectrophotometers, equipped with a flow-through cell, allowing continuous monitoring of the eluent. The wavelength selected corresponds to the region of the electromagnetic spectrum where the compound(s) of interest and/or their associated chromophores absorb light. In the linear dynamic range of the calibration curve the absorbance is proportional to the concentration of the compound of interest. The data is recorded and presented using a chromatographic integrator. The absorbance measured by the detector produces a peak with a...
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