Chemiluminescence in Analytical Chemistry

1. INTRODUCTION

In the last decade, capillary electrophoresis (CE) has become one of the most powerful and conceptually simple separation techniques for the analysis of complex mixtures. The main reasons are its high resolution, relatively short analysis times, and low operational cost when compared to high-performance liquid chromatography (HPLC). The ability to analyze ultrasmall volume samples in the picoliter-to-nanoliter ranges makes it an ideal analytical method for extremely volume-limited biological microenvironments.

Thanks to the efforts of a continuously increasing number of research groups, CE has by now been accepted as a highly efficient separation technique for qualitative purposes, with about 1500 CE-related documents appearing annually in analytical journals. However, CE has not yet been fully established as a quantification method, mainly due to the predominance of HPLC techniques applied in standard, validated analytical protocols.

Analytical techniques that involve the measurement of chemiluminescence (CL), though less commonly encountered in the literature, can be applied for quite sensitive measurements but often suffer from a lack of selectivity. Although the first observation of CL was made by Radziszewski in 1877—lophine (2,4,5- triphenylimidazol) emitting green light when reacting with oxygen in alkaline medium—investigations on the analytical use of CL have mainly been performed since the 1970s for gas-phase and from the 1980s onward for liquid-phase reactions. In addition, the rapid development of immobilization techniques has considerably enhanced the applications of chemiluminometry especially in flow injection analysis (FIA) and in liquid chromatographic systems [1].

The number of reactions producing CL cited in the literature is increasing each year, being analytically applied in chemical, biomedical, food, environmental, and toxicological disciplines [2–4]. In combination with HPLC separations several CL reactions have been used, among others, peroxyoxalate, firefly luciferase, lucigenin, and luminol. The most commonly used CL system for postcolumn detection in conventional and in microcolumn LC is the peroxyoxalate reaction [5]. The main inconvenience is related to the use of organic solvents that may cause precipitation problems with reversed-phase eluents due to the low water solubility of the average oxalate esters. In comparison with other CL reactions and in the presence of a suitable fluorophore, highest quantum efficiencies may be reached with the latter system and a more wide range of fluorophores can be analyzed.

Applying different CL systems, continuous-flow CL-based detection of several analytes has been widely applied for determination of several biological compounds and drugs. This technique has already become a highly sensitive method of detection in FIA, in liquid and gas chromatography, and in immunoassays [6–12].

With the purpose of increasing the specificity of analysis employing CLbased detection devices, several methods have been applied, including separation techniques. In this way, the combination of CL as a detection method with CE as prior separation methodology has provided a powerful analytical tool in recent years, offering excellent analytical sensitivity and selectivity and allowing the resolution and quantification of various analytes in a complex mixture. As a matter of fact, until the 1990s, chemiluminometric detection was not applied after capillary electrophoretic separation, but fast developments from some important research groups were recently noticed. Due to the advantages of CL detection and its potential when combined with the high separation ability offered by CE, research in this area has significantly increased. However, difficulties encountered when coupling the separation device with the CL detector still have to be dealt with.

In the present chapter an overview is presented about CL-based detection in CE, reviewing advances in the development of new detectors, the various CLbased reactions employed, and the applicability and usefulness of analysis to a wide range of samples.

2.CHARACTERISTICS OF CAPILLARY ELECTROPHORESIS

Tiselius originally introduced the conventional electrophoresis separation technique in 1937 [13], describing research on the separation of a protein mixture placed between buffer solutions in a tube. He observed that when applying an electric field, the sample components migrated in a direction and at a rate as determined by their charge and mobility. In this pioneering traditional work, Tiselius, who was awarded a Nobel Prize for his innovative research, recognized the separation advantages of smaller-diameter electrophoresis channels, but mentioned that detection considerations in narrow structures made the use of the technique unpractical.

Modern high-performance capillary electrophoresis (HPCE) is an instrumental approach to electrophoresis in which the components of a sample placed between two buffer solutions are separated in an open capillary tube with an inside diameter ranging from 2 to 200 m and a length usually between 10 and 100 cm. The separation is based on the electrophoretic mobility of the analyte species induced by the large potential applied across the capillary (generally 10– 30 kV). Separation efficiency in free solution, as performed initially, was limited by thermal diffusion and convection phenomena. To overcome these problems, various traditional stabilizing media were used, such as polyacrylamide, cellulose powder, glass wool, paper, or silica gel. Likewise, the use of tubes with small internal diameters showed numerous advantages because these narrow capillaries are themselves anticonvective allowing the performance of free-solution (or

open-tube) electrophoresis, the gel media not being essential for this purpose. Moreover, their high electrical resistance allows the use of very high electrical fields with only minimal heat generation and efficient heat dissipation due to the large surface-area-to-volume ratio.

The separation of small ions, neutral molecules, and large biopolymers in modern CE can be carried out using different modes of operation [14, 15], including free (capillary) zone electrophoresis (CZE), isotachophoresis (CITP), isoelectric focusing (CIEF), micellar electrokinetic chromatography (MEKC), and gel electrophoresis (CGE). CZE is the most commonly used technique owing to the simplicity of operation and versatility. The separation mechanism is based on differences in solute size and charge at a given pH using fused silica capillaries only filled with buffer, the latter chosen based on UV transparency, electrical inertia, and durability properties. These capillaries contain surface silanol groups that may become ionized in the presence of the electrophoretic buffer, showing three layers in the interface between the fused silica capillary wall and the running buffer: the negatively charged silica surface due to the anionic form of silanol groups (at neutral or alkaline pH), an immobile layer (Stern layer), and the diffuse layer of solvated cations in the surface of the silica built up to maintain charge balance and creating a potential difference very similar to the wall capillary, called zeta potential. When the high voltage is applied across the capillary, this layer of cations migrates toward the cathode producing a migration bulk flow of liquid through the capillary called electroosmotic or endoosmotic flow (EOF). As the EOF in fused silica capillaries is normally greater than the electrophoretic mobility rates of the individual analytes in the sample, the different charged species are moved in the same direction (usually from the anode to the cathode), being then separated in the same run. In function of the highest charge/mass ratio, the migration of the different species is produced, being cations migrating fastest, neutral species moving at the velocity of the EOF but not being separated from each other, and anions with the greatest electrophoretic mobilities migrating last. Oncolumn or postcolumn detectors can be used, the ‘‘retention time’’ being the time required for a solute to migrate to the point where detection occurs based on different modes, depending on specific solute properties [16].

By controlling the pH of the buffer medium it is possible to change the cited charge/mass ratio of the analyte, affecting ionization and electrophoretic mobility. The electroosmotic velocity can be adjusted by means of an adequate selection of several parameters inherent to the buffer, such as pH (if more silanol groups are ionized, the bulk flow is increased), viscosity (as viscosity increases the velocity decreases), the concentration and ionic strength (decreasing the zeta potential and EOF when increased), the intensity of the electric field (flow increasing proportionally to voltage), and the dielectric constant. According to the charge, hydrophobicity, size, and stereochemical configuration of the analytes,

an increasing range of applications can be carried out by CE, such as inorganic determinations, peptide and protein separations, environmental and food analysis, DNA sequencing, oligosaccharide separation, and single-cell assays. Several books and specific reviews include the fundamentals and applications of this technique [17–30].

Successfully validated CE methods are now routinely applied in many pharmaceutical quality control laboratories where applications include purity testing, quantitative assays, separation of enantiomers, and the determination of drug stoichiometry [31, 32]. Also, there has been a significant increase in publications dealing with the application of CE to clinical diagnosis and with biological sample preparations prior to CE analysis [33–38]. The application of CE in forensic sciences shows its capability of providing information about a wide range of chemical species and matrices, including gunshot and explosive residues, drug and DNA identification, to name a few. Due to the demonstrated superiority and taking into account that CE methodology preserves the requirement of legal systems based on the use of minute sample sizes only, CE has become a technique rigorously applied to analyze evidence and to help the criminal justice system to find correct conclusions [39].

Owing to the ultrasmall sample volumes introduced in the system and because of the small internal-diameter requirements in CE, together with the fact that in general the analytes of interest are present in low concentrations, poor detection limits are encountered, limiting the usefulness of the technique. For this reason, relatively concentrated analytical solutions, online preconcentration methods, and a variety of sample injection techniques for the preconcentration of analytes have been developed to improve sensitivity [38, 40]. Researchers have developed specific and nonspecific bioaffinity and molecular recognition CE methods for preconcentrating and characterizing analytes present in a wide concentration range in diluted liquids, fluids, or complex matrices. The methods combine the low as well as the high binding selectivities of the sorbing molecules with the efficient resolution abilities of CE. Detection limits of parts-per-trillion or -quadrillion can be reached, which allow a significant impact in forensic, environmental, biomedical, clinical, food, and pharmaceutical analysis [41, 42].

The selection of detection techniques capable of providing detection improvements has been a principal issue of research. A wide range of methods applied to meet detection limitations in CE have been taken mainly from liquid chromatographic techniques with only minor modifications, including ultraviolet (UV) absorption, fluorescence, mass spectrometry, conductivity, and electrochemistry principles.

It is clear that the main performance criteria that must be taken into account when selecting a detector for a particular determination are selectivity and sensitivity, followed by linearity of signal response, linear detection range, and repro-

ducibility. These performance criteria are related to the independence of the detector response of buffer composition and of the physical devices (cells, joints, fittings, connectors, etc.), which must not contribute to extracolumn zone broadening, being compatible with the separation conditions.

Depending on the architecture of the detection cell, on-column or off-col- umn detection can be carried out. Oncolumn detection is most commonly used as in this case the detection cell makes part of the electrophoretic capillary, thus eliminating the broadening effect mentioned above and producing high separation efficiencies. However, using offcolumn detectors, the band broadening is generally increased.

Another classification of detectors is based on the specificity of the detection principle, being divided into ‘‘bulk-property’’ and ‘‘specific-property’’ series [43]. The former evaluate differences between a physical property of the solute relative to the buffer alone, such as the refractive index, conductivity, and the application of indirect methods. In spite of their universal character, lower sensitivities and dynamic ranges are obtained. Specific-property detectors measure inherent physicochemical properties of species, for example UV absorption, fluorescence emission, and mass spectral behavior. In this sense, only the analytes showing these properties are detected, minimizing background signals and increasing sensitivity and width of linear ranges for these determinations. Detection may occur in the migration process (UV, fluorescence, conductivity, and refractive index) or as the components elute from the capillary (postcolumn derivatization before detection, and electrochemical and mass spectrometric methods).

UV (and much less frequent, visible) absorbance detection is the most widely used detection technique for CE owing to its ability to detect nearly all species without derivatization and to the easy adaptability of UV detectors originally designed for HPLC work, though several types of commercially available CE equipment offer some basic types of absorbance detectors [15]. High-quality fused-silica capillaries having a cutoff of approximately 170 nm are suitable for the UV detection of a wide range of compounds by applying fixedor variablewavelength instruments. The easiest way to perform oncolumn UV or luminescence detection is by making a window in the polyamide coating of the fused silica capillary, removing a small section ( 1 cm) by burning off the polyamide coating, although alkaline etching or mechanical scraping can also be used. The main limitations of UV detection are its relatively low sensitivity and dynamic ranges. The concentration limits of detection (typically 0.1–1 M) are usually limited by the short pathlength of oncolumn detection systems (inner diameter of 25 m or greater) and the limited time available to observe the sample as it passes the detector. Various methods have been employed to increase the pathlength for optical detection in small capillaries, including the use of axial illumination, Z-shaped flow cells, multireflection cells, and the use of tubing with noncircular cross-section. Diode-array detectors provide additional spectral infor-

mation from the separated analytes that can be used to assist in peak purity assessment, analyte identification, and prerun screening to determine the wavelength setting for optimum detection sensitivity.

Fluorescence detection is the second mode quite frequently applied in CE analysis because of the low detection limits—not strictly pathlength dependent— and the easy adaptability of fluorescence detectors to the smaller diameter capillary [44–46]. The high sensitivity obtained is due to the fluorescence emission inherently at higher wavelengths than the excitation wavelength, yielding low background signals. Moreover, a single fluorescent analyte may emit multiple photons. As with UV detection, depending upon the needs of each specific application, direct and indirect detection methods can be considered. Also, a wide variety of reagents exist for pre-, post-, and oncolumn derivatization in CE to convert analytes into products with more favorable detection characteristics [47].

Laser-induced fluorescence (LIF) has also been utilized as a highly sensitive detection principle for CE [48–51]. However, while the LIF detector is now able to achieve zeptomole (10 21) detection limits, conventional derivatization techniques are inefficient at these exceptional levels [52]. Also, CE has successfully been coupled with mass spectrometry (MS) [53], nuclear magnetic resonance (NMR) [54, 55], near-infrared fluorescence (NIRF) [56, 57], radiometric [58], flame photometric [59], absorption imaging [60], and electrochemical (conductivity, amperometric, and potentiometry) [61–63] detectors. A general overview of the main detection methods is shown is Table 1 [64].

A most powerful detection mode under actual investigation in CE is CL. Because CL detection does not require a light source for excitation—the required energy being produced by a suitable chemical reaction—problems in baseline stability limiting detection limits are overcome, providing excellent sensitivities due to the low background noise. Recently, some review articles have been produced in this field [46, 65–68].

3.CHEMILUMINESCENT SYSTEMS FOR DETECTION IN CAPILLARY ELECTROPHORESIS

For CL purposes, only minimal instrumentation is required and because no external light source is needed, the optical system is quite simple. Hence strong background light levels are excluded, as occurring in absorption spectroscopy, reducing the background signal, and leading to improved detection limits. For this reason, CL has been defined as a ‘‘dark-field technique’’ as this technique produces a signal against a dark background, making it easier to detect and thus to acquire the CL signal by a photomultiplier tube (PMT), which must obviously be sufficiently sensitive in the spectral region of interest.

However, several problems that may limit the application of CL techniques should be considered. First, as a principle any fluorescing substance can be measured after suitable chemical excitation, which implies that a CL reagent is not limited to just one unique analyte. This lack of selectivity is thus brought along fundamentally in FIA applications, where selective reactors must be incorporated before the CL reaction can occur, which for HPLC purposes is not a real problem. Another disadvantage is the dependence of the CL emission on several environmental factors mentioned above, which should be dealt with during the HPLC or CE separation procedure as well as during FIA analysis. Hence a compromise between the required and optimized separation and detection conditions should be worked out for each analytical procedure. Finally, since CL emission is not constant but varies with time (light flash composed of a signal increase after reagent mixing, passing through a maximum, then declining to the baseline), and this emission-versus-time profile can widely vary in different CL systems, care must be taken to detect the signal in the flowing stream at strictly defined periods.

In the absence of analyte, many CL systems show a low emission background level. Hence, in flow systems, as the CL intensity is proportional to the analyte concentration, the emission appears as a sharp peak superimposed on a low constant blank signal, which is measured when the mixture of analyte and CL reagents passes through the detector cell. Because only a small portion of CL emission is measured from this time profile, nonlinear calibration curves may be obtained for reactions with complex kinetics [1].

It is clear that the need to obtain improved detection technology is now related to the general trend in analytical chemistry to reduce the waste volumes of organic solvents by using more aqueous systems and to study smaller samples at increasingly lower concentrations. As the CL technique may provide improvement in these areas, in terms of low detection limits, wide dynamic range, and high sensitivity, the instrumentation for the measurement of CL and the coupling with CE devices is being progressively developed using different indirect and direct CL systems. All the CL systems used in CE that will be described in this section are summarized in Table 2.

3.1 Chemiluminescence Reactions with Peroxyoxalates

Peroxyoxalate-based CL reactions are related to the hydrogen peroxide oxidation of an aryl oxalate ester, producing a high-energy intermediate. This intermediate (1,2-dioxetane-3,4-dione) forms, in the presence of a fluorophore, a charge transfer complex that dissociates to yield an excited-state fluorophore, which then emits. This type of CL reaction can be used to determine hydrogen peroxide or fluorophores including polycyclic aromatic hydrocarbons, dansylor fluores- camine-labeled analytes, or, indirectly, nonfluorescers that are easily oxidized (e.g., sulfite, nitrite) and quench the emission. The most widely used oxalate