High-Performance Liquid Chromatography (HPLC): Quantitative Applications and Representative Method

 Quantitative Applications

HPLC is routinely used for both qualitative and quantitative analyses of environ- mental, pharmaceutical, industrial, forensic, clinical, and consumer product samples. Figure 12.30 shows several representative examples.




Preparing Samples for Analysis 

Samples in liquid form can be analyzed di- rectly, after a suitable clean-up to remove any particulate materials or after a suitable extraction to remove matrix interferents. In determining polyaromatic hydrocarbons (PAH) in wastewater, for example, an initial extraction with CH2Cl2 serves the dual purpose of concentrating the analytes and isolating them from matrix interferents. Solid samples must first be dissolved in a suitable sol- vent, or the analytes of interest must be brought into solution by extraction. For example, an HPLC analysis for the active ingredients and degradation products in a pharmaceutical tablet often begins by extracting the powdered tablet with a portion of mobile phase. Gases are collected by bubbling through a trap contain- ing a suitable solvent. Organic isocyanates in industrial atmospheres can be de- termined in this manner by bubbling the air through a solution of 1-(2- methoxyphenyl)piperazine in toluene. Reacting the isocyanates with 1-(2-methoxyphenyl)piperazine serves the dual purposes of stabilizing them against degradation before the HPLC analysis while also forming a derivative that can be monitored by UV absorption.

Quantitative Calculations 

Quantitative analyses are often easier to conduct with HPLC than GC because injections are made with a fixed-volume injection loop in- stead of a syringe. As a result, variations in the amount of injected sample are mini- mized, and quantitative measurements can be made using external standards and a normal calibration curve.

Representative Method

Although each HPLC method has its own unique considerations, the following de- scription of the determination of the fluoxetine in serum provides an instructive ex- ample of a typical procedure.

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