Chemiluminescence in Analytical Chemistry

is also obtained; the peaks seen from the CE run with CL detection do not show important band broadening even though the separation time is almost double that of the unlabeled tryptic digest separation. This type of phenomenon, called ‘‘chemical band narrowing,’’ has been well documented in the literature for CL detection and is based on the nature of kinetics in CL [79]. The CL kinetics allow for an intense, rapidly decaying signal to be produced when the acridinium-tagged analyte enters the reaction zone of the reactor. Once the acridinium CL is complete, the signal stops, thus decreasing the effective volume of the flow cell because a measurement is not made for the entire residence time of the analyte in the flow cell.

3.3 Chemiluminescence Reactions with Luminol

3.3.1Determination of Luminol, Derivatives, and Labeled Compounds

A successful study involving luminol CL detection was reported by Dadoo et al. in 1992 [82]. Luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) reacts with an oxidant in the presence of a catalyst in alkaline medium to be oxidized to 3- aminophthalate emitting light with a wavelength in the interval of 425–435 nm, avoiding the inconvenience typical for peroxyoxalate reactions, which require the use of organic solvents. Although a variety of oxidants can be used, including permanganate, hypochlorite, and iodine, the most commonly used agent is hydrogen peroxide. The CL emission intensity is directly proportional to the concentration of luminol, H2O2, and catalyst, and for this reason measurements of CL intensity can be used to quantitate any of these species as well as species labeled with the catalyst, peroxide, or species that may be converted into peroxide, luminol, or species labeled with luminol. For example, carboxylic acids and amines can be labeled with luminol or its derivatives [isoluminol and N-(4-aminobutyl)- N-ethylisoluminol (ABEI)], being separated and then detected, as in HPLC, after postcolumn reagent addition.

The electrophoretic apparatus used as depicted in Figure 8 is provided with an electrophoretic capillary tube (48 cm 75 m id 375 m od), a reagent capillary (75 cm 200 m 375 m od), and a reaction (outlet) capillary (65 cm 150 m 375 m od), held in place by a tee connector. A 3–4-cm section at the end of the electrophoretic capillary is etched to an outer diameter of approximately 100–120 m by placing it in concentrated hydrofluoric acid while purging the capillary with helium. The detection window, which is made on the reaction capillary by burning off the polyimide coating, is placed at the focal point of a parabolic mirror to collimate the light emitted, and subsequently focused on a PMT connected to a photon-counting system. To decrease the dark current of

Figure 8 Schematic diagram of the CE-CL detector used by Dadoo et al. (From Ref. 82, with permission.)

the PMT, the latter was cooled down to 20°C by means of a thermoelectric cooler.

This preliminary work demonstrated well the use of CL as a highly sensitive and selective detection method in CE by its application to the separation of luminol and ABEI using the same experimental conditions cited previously for the CL reaction of luminol in HPLC. Detection limits (S/N 3) of 100 amol and 400 amol were obtained for the compounds mentioned, respectively, achieving an improvement in sensitivity of 2–3 orders of magnitude with respect to the ones obtained using UV absorption for detection.

Obviously, the main purpose for the introduction of CL detection coupled to CE separations is inherent to the development and improvement of sensitive and uncomplicated devices to achieve a decrease of the band broadening caused by turbulence at the column end, together with the attractive separation efficiency of CE setups. With this purpose in mind, Zhao et al. [83] designed a postcolumn reactor for CL detection in the capillary electrophoretic separation of isoluminol thiocarbamyl derivatives of amino acids, because, like other isothiocyanates, isoluminol isothiocyanate has potential applications in the protein-sequencing area.

The separation was performed in a 50 m id 190 m od quartz capillary using a postcolumn sheath flow cuvette as the mixing chamber and a sheath stream from a syringe pump to carry the analytes away from the detection zone once emerging from the column. Mixing of the labeled analytes with hydrogen peroxide and microperoxidase as a catalyst is required because if these three components are introduced as separated streams, the peroxide will be destroyed by the catalyst before ever reacting with the analyte and hence no CL emission would be observed. In contrast to the experiments from Zare’s group [82], who added peroxide to the separation buffer and mixed the catalyst in the detection chamber, Zhao et al. [83] added microperoxidase directly to the separation buffer, producing an intimate contact between the catalyst and analyte when mixed with peroxide, avoiding the problem of bubble formation in the separation capillary, which could perturb the separations. The sensitivity of the CL detector was influenced by several parameters, rigorously controlled in the experimental work, such as microperoxidase and hydrogen peroxide concentrations and mixing distance (distance downstream from the capillary to the center of the PMT). Nevertheless, separation of labeled amino acids was improved by the addition of a low concentration of the anionic surfactant sodium dodecyl sulfate (SDS) to the running buffer, taking into account that an increase in the concentration of the micellar medium may denature the microperoxidase, destroying the catalyst and inhibiting the reaction. Using this detector, the volumetric flow of peroxide added was much larger than the one used in Dadoo’s design [82] for the same reaction. The optimal concentration of this reagent, however, was three orders of magnitude inferior.

The elimination of turbulent mixing in the flow chamber and the short residence time of the reaction mixture in the detection chamber provided a high separation efficiency of 100,000 theoretical plates for labeled amino acids, obtaining good resolution in comparison with previous CL detectors [79, 82]. For the isoluminol thiocarbamyl derivative of valine, a detection limit of 500 amol was reported; however, it was not possible to separate all 20 isoluminol-deriva- tized amino acids.

To facilitate the implementation of a CL-based detector in the CE system, Dadoo et al. focused their efforts in 1994 on the design of a simpler detector interface in which the signal is generated at the column outlet [84]. Based on the same CE system previously used [82] and removing a 1–3-mm section of the polyimide at the outlet end of the capillary by burning, the modified CL apparatus was coupled to the CE setup as shown in Figure 9. The outlet end of the separation capillary is immersed in the reservoir containing the electrolyte and the reagent for the CL reaction. When the analytes emerge from the column they react with the CL reagents in a reservoir producing visible light, which is transported by a fiberoptic (perpendicular to the capillary and with an end immersing in the solution) to a PMT tube. Immersing a platinum wire for the grounding electrode in the reservoir completes the CE electrical circuit. As in previous work, the same

Figure 9 End-column CL detector for CE proposed by Zare’s group. (From Ref. 84, with permission.)

reaction was used by the authors for separation of luminol and ABEI and also for separation of arginine and glycine derivatized with ABEI. In this separation of luminol and ABEI, the efficiency obtained was between 10,000 and 20,000 theoretical plates, indicating an improvement with respect to the one obtained for the previous design but with a detection limit for luminol (500 amol) lower than the one previously reported. The factors responsible for the decrease in sensitivity include the inefficient light collection as obtained using the fiberoptic in comparison with the application of the parabolic reflector and, also, the absence of cooling of the PMT. Although this end-column detector produces band broadening as a result of the mixing at the column outlet, and in spite of the slow CL reaction kinetics and a large detection zone, which provides relatively low numbers of theoretical plates, the simplicity and the ability to obtain detection limits in the nanomolar range make the setup suitable for routine work. The authors propose future modifications for achieving increased sensitivity, including cooling of the PMT tube to lower the dark current and the use of additional fiberoptics to increase light collection.

Based on the reaction of luminol and hydrogen peroxide, detection by electrogenerated CL (ECL) was also applied in CE [85]. In this detection technique, which has been used until now in LC and in FIA, the production of light is followed by an oxidation or reduction reaction at an electrode that serves the

Figure 10 Separation of ABEI-DSC-derivatized n-octylamine (2) and n-propylamine

(3) with ECL detection. Remaining ABEI (1) and ABEI DSC (not shown) are also detected. Conditions: 20% methanol in 5 mM sodium borate separation buffer, pH 10.9; 5-s injection at 25 kV, 1.0 10 6 for each labeled amine; 25-kV separation potential; 10-mm platinum wire electrode. (From Ref. 85, with permission.)

nescently active analyte that produces interference or suppression of a given CL reaction, the analyte being detected indirectly as an inverted peak, where the CL intensity decreases from a normally high background level. Liao et al. [86, 87] demonstrated for the first time the feasibility of this indirect CL detection technique in CE. Their contribution shows the utility of the Cu(II)-catalyzed luminol CL system for determination of five amino acids without previous preor postcolumn derivatization, making possible the detection of a wide range of biomolecules able to strongly and rapidly complex with Cu(II) as amines, catechol, catecholamines, and proteins. To carry out the separation and detection, the interface used is slightly modified with respect to the one proposed by Dadoo et al. [82]. Figure 11 illustrates the resolution obtained in the separation of five amino acids. Cu(II) catalyzes the luminol CL reaction, the CL emission intensity being proportional to the concentration of free Cu(II). In the presence of amino acids, the catalytic activity of free Cu(II) is decreased owing to the postcapillary formation of Cu(II)–amino acid complexes, and the CL intensity is considerably reduced. Good resolution efficiency is achieved by choosing an adequate electrophoretic

Figure 11 Electropherogram of a mixture of five amino acids using indirect CL detection. Conditions: 21-kV separation voltage, and 2 s at 21 kV for sample injection; sample concentration 0.5 mM of each amino acid. Peak identities: (1) arginine; (2) leucine; (3) serine; (4) cysteine; (5) aspartic acid. (From Ref. 86, with permission.)

buffer compatible with the detector reaction. In this case, to avoid the formation of microparticulates of Cu(II) hydroxide or carbonate, which will interfere with the CL detection, a small amount of tartaric acid must be added to the Cu(II) solution because it provides complexation with Cu(II) until the amino acid is introduced. This new detection system in CE is simpler than direct CL detection and reaches higher sensitivities, showing detection limits for the tested amino acids in the range 100–400 fmol, two orders of magnitude higher than obtained with CL detection in CE for labeled amino acids, as described in previous work [83].

Other studies in this specific area are also based on the catalytic effect of a variety of metal ions such as copper (II), cobalt (II), nickel (II), iron (III), and manganese (II) on the luminol–hydrogen peroxide reaction providing a rapid and efficient detection mode for these five ions, when an online CL detector is used before separation by CE [88]. This contribution combines capillary ion analysis (CIA) and CL detection by means of a postcapillary reactor similar to the one originally developed by Rose and Jorgenson [80] and finally modified by Wu

and Huie [78] for CL detection. The reaction capillary is situated just in front of the PMT without the use of optical fibers to transport light, avoiding light loss by 10% and increasing the detection sensitivity due to the use of a sample injection volume lower than 20 nL. The most effective mixing mode to improve the sensitivity of this determination is by using luminol as one of the electrophoretic components and introducing only hydrogen peroxide solution in a postcapillary way. Thus, luminol, hydrogen peroxide, and the metal ions are placed at the detector window simultaneously, and the fast kinetic rate will produce a light response detected at the same time. It is clear that the pH must be conveniently selected because the EOF and the speciation of the metal ions are strongly influenced by this parameter. In the separation process, the net mobility of the ions is the sum of EOF and electrophoretic mobility, this last component being very similar for the transition metals and lanthanides, which do not allow an adequate separation. To overcome this problem, which impedes a good resolution, it is necessary to selectively alter the mobilities by means of a complexation process with a weak chelating reagent such as 8-hydroxyquinoline-5-sulfonic acid (HQS) or α-hydroxyisobutyric acid (HIBA). The net electrophoretic mobilities will be the weighted averages of the mobility of each free metal ion and its complexes and logically depend on the degree of complex formation obtained, using an optimal concentration of chelating agent and a pH value of 4.54. The latter represents a compromise between the optimum pH for the separation of metal ions and the required acidic medium to avoid hydrolysis effects, making it then possible to carry out the CL reaction. The detection limits of Co(II), Cu(II), Ni(II), Fe(III), and Mn(II) were 20 zmol, 2 amol, 80 amol, 740 amol, and 100 amol, respectively, obtaining a sensitivity considerably improved in comparison with common techniques for detection in CIA such as UV absorption and electrochemical detection.

Recently, this group has carried out for the first time CL detection with CE for rare-earth metal ion analysis [89]. Employing CL detection with CE as an oncolumn analysis method, dual effects of rare ions on the CL reaction of luminol with H2O2 were first reported. Under static conditions, rare-earth ions can complex with luminol and inhibit CL emission but on the other hand, they could catalyze the CL reaction second transformation from luminol free radical to aminophthalate and enhance CL emission in CE with online CL detection. Using the CE-CL detection system with a coaxial reactor similar to that shown in Figure 5 [79], in which the separation capillary is filled with electrophoretic buffer (10 mM phosphate buffer, pH 4.5), CL reagents (H2O2 and luminol) and Co2 are siphoned into the tee and flowed into the detection window in incessant stream, and hence there frequently existed fresh reagent to react. The sample is injected by electroinjection for 10 s at a voltage of 10 kV. The background CL emission is constantly produced. As the background CL tends to be stable, a constant concentration of luminol free radical is present. When a rare-earth ion migrates into the detection window from the separation capillary, it reacts with