Chemiluminescence detection in liquid chromatography: Applications to clinical, pharmaceutical, environmental and food analysis—A review

Abstract

Chemiluminescence (CL)-based detection has become in the last years quite a useful detecting tool in liquid chromatography (HPLC) due to its simplicity, low cost and high sensitivity and selectivity, and the development in instrumentation. Minimal instrumentation is required and no external light source is needed; thus, the optical system is quite simple. As a consequence, a wide variety of analytical methods have been developed in clinical, pharmaceutical, environmental and food analysis. In this review, applications of the HPLC–CL coupling in those different fields have been included and classified in relation to the different CL systems employed (namely peroxyoxalate reaction, tris(2,2′-bipyridine) ruthenium (II) reaction, luminol system and direct oxidations) and also sub-classified according to the group of analyte. The review covers the literature from 2000 until the end of 2008.

Introduction

Chemiluminescence (CL) is defined as the production of electromagnetic radiation (ultraviolet, visible or infrared) observed when a chemical reaction yields an electronically excited intermediate or product, which either luminesces (direct CL) or donates its energy to another molecule responsible for the emission (indirect or sensitized CL). As advantages inherent to CL techniques, it is possible to remark the basic instrumentation required and the simplification of the optical system because no external light source is needed. CL is often described as a dark-field technique: the absence of strong background light level, such as found in absorptiometric techniques, reduces the background signal and leads to improved detection limits. For over 30 years, the phenomenon of CL in the liquid phase has provided a well-established and widely applied spectrometric branch of Analytical Chemistry [1], [2], [3]. During the last years, several books, chapters and reviews have been published about the CL detection, principles and analytical applications in the liquid phase [4], [5], [6], [7], [8], [9], [10], [11], including developments in instrumentation [12], and the use of CL as detection mode in flow injection analysis (FIA) [13], [14], HPLC [14], [15], and capillary electrophoresis (CE) [14], [15], [16], [17].

High performance liquid chromatography (HPLC) coupled to CL detection represents an interface between the selectivity of a powerful separation method and the sensitivity of this detection technique. The application of CL as detection system in HPLC requires the generation of CL emission by a post-column reaction of the analytes in the column eluent with the reagent(s). This combination provides a high efficiency in separation and low detection limits inherent to CL systems. As a drawback, this assembly requires additional pump(s) to deliver post-column CL reagent or the incorporation of devices for a rapid mixing of the column eluate with the reagent solution(s) to obtain a stable baseline. Also, considering that the CL reactions used in HPLC are very rapid and are accompanied by intense CL, it is necessary to use a reaction coil between the mixing device and the detector, in which the length and diameter must be adjusted in order to optimize the CL reaction time. This will allow the measurement of the CL intensity at the time that the maximum CL emission is observed, by using the maximum S/N ratio as a criterion, in some cases, in presence of a large background signal. Different configurations have been considered in order to achieve a sensitive and selective determination of analytes, related with the good resolution of the HPLC separation, the adequate efficiency of the CL system, the stability of the reagents or the compatibility of the CL reaction conditions and the requirement for the HPLC separation, most of them in a post-column way [18]. The most important problem of this HPLC–CL coupling is the compatibility between the chemical environments required for an efficient chromatographic separation (mainly the composition of the mobile phase) and those required for an efficient and sensitive CL emission (reaction temperature, pH, solvent, nature of the CL precursor and coexisting compounds such as catalyst and enhancer affecting the CL reaction yield). Thus, careful optimisation of all the variables involved in the HPLC–CL coupling is an important task.

This review includes recent analytical applications (from 2000 until now) of the coupling HPLC with CL detection, mainly focused on the wide range of applications developed in this period related to the following fields: clinical and pharmaceutical, environmental and food analysis. Each group of applications has been divided according to the used CL system and according to the different families of analytes involved. An extensive table (Table 1) with a summary of the relevant aspects of the included papers, concerning the CL system or the chromatographic conditions as well as the applications and the sensitivity of the methods has been included. First of all, a brief introduction about the principles and characteristics of the CL systems included in this review will be presented.