Chemiluminescence in Analytical Chemistry

reaction is therefore very important for the determination of an analyte participating in a given CL reaction.

3.1 CL Reaction of Luminol

Luminol derivatives produce emission of light by oxidation with oxygen and hydrogen peroxide under alkaline conditions. By utilizing this reaction, peroxides such as hydrogen peroxide and lipid hydroperoxides can be determined after HPLC separation. Metal ions [e.g., iron(II), cobalt(II), etc.] catalyzing the luminol CL reaction can also be determined.

Some luminol derivatives have been developed as CL labeling reagents. Analytes prelabeled with luminol derivatives are separated by HPLC, mixed with postcolumn reagents such as hydrogen peroxide and an alkaline solution of potassium hexacyanoferrate (III), and then detected by a CL detector. Highly sensitive determination is possible by optimizing the conditions to increase the CL reaction efficiency for each analyte.

3.2 CL Reaction of Lucigenin

Although acridinium derivatives including lucigenin are well-known CL compounds, few methods have been described on the use of these compounds for detection in HPLC. Differing from other CL reagents, lucigenin produces CL by the reaction with organic reductants as well as with hydrogen peroxide. Therefore, the HPLC determination of reductants i.e., ascorbic acid, glucose, etc., can be performed by using lucigenin as a postcolumn CL reagent.

3.3 CL Reaction of Aryloxalate

A CL reaction of aryloxalate is the so-called peroxyoxalate chemiluminescence (PO-CL) reaction. PO-CL is one of the most efficient and versatile CL systems for HPLC. In this system, aryloxalates or oxamides react with hydrogen peroxide to produce a high-energy intermediate 1,2-dioxetanedione. This intermediate transfers its energy to a coexisting fluorophore, and then emission of light is observed from the excited fluorophore (Fig. 2). The total emission of light is proportional to the concentration of each substrate (i.e., oxalates, hydrogen peroxide, fluorophores, or basic catalysts). Therefore, these compounds can be determined by a suitable CL detection system. By using the catalytic effects of bases in this CL reaction, various amines can also be determined by HPLC. Many compounds can be used in the PO-CL reaction system, most of which can be detected sensitively. This is one of the major reasons why the PO-CL reaction has been exclusively used for postcolumn CL detection in HPLC.

Figure 2 Aryloxalates and the PO-CL reaction. TCPO, bis(2,4,6-trichlorophenyl)oxa- late; DNPO, bis(2,4-dinitrophenyl)oxalate; DFPO, bis(2,6-difluorophenyl)oxalate; TDPO, bis[2-(3,6,9-trioxadecyloxycarbonyl)-4-phenyl]oxalate.

4. CONSTRUCTION OF HPLC-CL DETECTION SYSTEMS

An HPLC-CL detection system is constructed by considering the conditions for HPLC separation, efficiency of CL reaction, and stabilities of reagents. The three systems shown in Figure 3 are most widely employed.

System A is very simple and used for delivering a single solution of postcolumn reagent(s). This system is used in a case; after separation, hydrogen peroxide is determined by using a mixture of an aryloxalate and a fluorophore for postcolumn CL reaction.

System B is the most widely used for CL detection after an HPLC separation. In this system, two pumps are required for delivering the reagent solutions in the following cases: (1) the solutions for CL reaction are first combined and then mixed with an eluent; (2) CL reaction conditions (e.g., pH, water and organic solvent contents, and salt concentration) need to be optimized before mixing with the CL reagent.

Figure 3 CL detection systems in combination with HPLC. P, pump: I, injector; C, column; M, mixing tee; D, detector; RC, reaction coil; MC, mixing coil; RE, recorder; E, eluent; R, reagent; W, waste; IMER, immobilized enzyme reactor.

System C is used when an immobilized enzyme reactor (IMER) is introduced into system B. The analyte(s) separated by HPLC is converted to a suitable species for CL detection with an IMER, and then mixed with the CL reagent. In this system, a buffer solution as a mobile phase and an ion-exchange-type column are preferable for an enzyme reaction.

More complicated systems will be required when a gradient elution is incorporated for the separation of analyte(s). However, more simple systems are preferable in view of operations, maintenance, and running costs of analysis.

5.DESIGN TO INCREASE THE EFFICIENCY OF CL REACTIONS

To achieve a sensitive and selective determination of analyte(s), resolution of HPLC separation and sensitivity of CL detection are the major critical factors. Many attempts to increase the sensitivity, namely to improve the efficiency of the CL reaction, have been made.

5.1 Devices

5.1.1Mixing Device

In the postcolumn CL reaction, the thorough and rapid mixing of the column eluate with reagent solution(s) is an important factor to obtain a stable baseline and reproducible peak heights. In general, a T-shaped joint or other mixing devices for gradient elution are employed as mixing devices for the postcolumn CL reaction. However, several mixing devices to improve the CL reaction efficiency have also been proposed [4–7].

As shown in Figure 4, a mixing device with a small vessel, in which two flowing solutions are caused to rotate (A), was developed [6]. Another rotating flow mixing device (B) having three directional inlets, in which three solutions are mixed and flowed out through an outlet at the center of the top of the vessel, was also reported [7]. By using these devices, the CL reaction conditions were investigated from the standpoint of the improvement on the signal-to-noise (S/N) ratio and peak broadening.

5.1.2Reaction Coil

In general, the CL reaction for HPLC requires rapid reaction accompanied by intense CL. Taking into account the sensitivity, it is preferable to measure CL at the time that the maximum CL intensity is observed. However, it should be considered that a large background signal may be observed simultaneously. Therefore, the time for measuring CL should be set to give the maximum S/N ratio. The adjustment of length and diameter of the reaction coil that is introduced between a mixing device and a detector is effective to obtain an optimized CL reaction time.

5.1.3Electronic Device to Improve S/N Ratio

Electronic devices that improve the S/N ratio by filtering electronic signals from the CL detector are commercially available. Devices using both the analog and digital technologies are currently obtainable. These devices are inserted between a detector and a recorder, and treat electronic signals from a detector to give a

Figure 4 Rotating flow mixing devices having two (A) and three (B) directional inlets.

chromatogram with an improved S/N ratio. Typical results are shown in Figure 5 [8]. The S/N ratio increased 5–10 times by using this device for the detection of hydrogen peroxide in PO-CL.

5.1.4Flow Cell and Flow Rate

A spiral coil of Teflon, or a spiral groove on a stainless steel surface covered with quartz, is used as a flow cell in the CL detector. These cells are placed in front of a photomultiplier tube (PMT), which detects photons emitted by the CL reaction. The cell volumes are generally in the range of 60–120 L.

A joining part of the spiral coil or the quartz part that is in contact with stainless steel in the flow cell might not withstand high levels of pressure (generally smaller than 10 kg/cm2); hence careful operation to prevent excess flow is

Figure 5 Chromatograms of hydrogen peroxide obtained without (A) and with (B) a signal cleaner. HPLC conditions: peak (2.5 pmol of hydrogen peroxide on column); eluent, 50 mM imidazole-HNO3 (pH 7.0)/CH3CN ( 87:13, v/v); postcolumn CL reagent, 0.6 mM TDPO in CH3CN/ 0.5 M in CH3CN ( 50:50, v/v); column, TSK-gel ODS-80TM (250 4.6 mm id). (From Ref. 8.)

required. It should be noted that precipitation of salts in a mobile phase or postcolumn CL reagents might increase the pressure to cause breakage of the flow-cell. A suitable flow rate of a mobile phase and postcolumn CL reagent(s) must be determined under a user-specified pressure to prevent damage of the flow cell by considering a mixing efficiency and a CL reaction yield.

5.1.5Pump

The use of the pumps capable of delivering pulseless flows is desired for the mobile phase and CL reagent(s) to obtain a stable and reproducible chromatogram. For this purpose, a dual-piston pump with minor pump pulsation or a sy- ringe-type pump without pulsation are preferable. Pumps for an HPLC separation with high-performance ability are often used to deliver postcolumn CL reagent(s). However, these pumps are rather expensive and plural pumps will be required when delivering many reagent solutions. To overcome this, attempts to effectively delivering two reagent solutions by using an inexpensive dual-head reciprocating piston pump with a short stroke (4.9 l per stroke) were made [9].

5.2 Optimization of Reaction Conditions

To achieve highly sensitive detection, optimization of various factors affecting the CL reaction is required. Reaction temperature, pH, solvent, nature of CL compounds, and coexisting compounds such as a catalyst and an enhancer affect the CL reaction yield.

5.2.1Choice of the CL reagent

The choice of a CL reagent has a serious influence on the detectability of analytes. Therefore, applicability of a CL reagent to a given analyte and method, stability, and ease of preparation as well as CL reaction efficiency should be considered when selecting a reagent. Although novel CL reagents such as adamantyl dioxetane derivatives have been developed, only a few of them can be combined with HPLC.

Some derivatives of luminol and acridine are currently used for the HPLCCL detection. They are prepared by changing a substituent or introducing a new functional group into the luminol or acridine skeleton to give better CL efficiency or reactivity with analyte(s).

In the PO-CL system, efforts to increase the solubility of aryloxalate in the mobile phases employed for HPLC and to improve the fluorescence quantum yield of the fluorophore have been made. Many fluorophores have been developed for the PO-CL system and applied to various analytes [10].

5.2.2Temperature

The fluctuation of the temperature in the mixing device, reaction coil, and flow cell affects the CL reaction velocity and emission duration thus affecting the sensitivity and reproducibility. Therefore, it is preferable to keep a constant temperature [11].

5.2.3Solvent

The nature of the employed organic solvents affects the efficiency of the PO-CL reaction. Organic solvents such as acetonitrile and methanol utilized frequently for reversed-phase HPLC are well suited for a PO-CL reaction. Generally, a higher content of these solvents in the mobile phase gives more intense CL. Effects of organic solvents’ nature on the stability of postcolumn reagents were investigated and it was found that a mixture of hydrogen peroxide and bis(2,4,6- trichlorophenyl)oxalate (TCPO) was most stable in acetonitrile. When bis[4-ni- tro-2-(3,6,9-trioxadecylcarbonyl)phenyl] oxalate (TDPO) and hydrogen peroxide dissolved in a mixture of ethyl acetate and acetonitrile were used, the addition of phthalic acid esters to this solution decreased the noise level of CL [7].

5.2.4Catalyst

Many compounds are known to catalyze CL reactions. As these catalysts act at trace amounts, the CL reaction can be applied to determination of these substances. Since most of the CL reactions concern oxidation reactions, compounds catalyzing the oxidation process have been well investigated.

For example, peroxidase catalyzes the reaction of luminol derivatives with hydrogen peroxide and results in an increase of the CL reaction velocity and CL intensity. Therefore, intense CL can be obtained from the analyte labeled with luminol derivatives after HPLC separation, followed by reaction with peroxidase.

On the other hand, several oxidases are known to generate hydrogen peroxide, acting as an oxidant in the CL system, from corresponding substrates. IMERs in which the oxidases are immobilized on adequate supporting materials such as glass beads have been developed. IMERs are often used for flow injection with CL detection of uric acid and glucose, and are also applicable to the CL determination of acetylcholine, choline, polyamines, enzyme substrates, etc., after online HPLC separation.

5.2.5Enhancer

As compounds exhibiting enhancing effects on CL reactions, a variety of phenols, e.g., firefly luciferin and 6-hydroxybenzothiazole derivatives [12, 13], 4-iodophe- nol [14], 4-(4-hydroxyphenyl)thiazole [15], 2-(4′-hydroxy-3′-methoxy-benzyli- dene)-4-cyclopentene-1,3-dione (KIH-201) [16], and 2-(4-hydroxyphenyl)-4,5- diphenylimidazole (HDI) and 2-(4-hydroxyphenyl)-4,5-di(2-pyridyl)imidazole (HPI)[17] (Fig. 6A), and phenylboronic acid derivatives, e.g., 4-phenylylboronic acid [18], 4-iodophenylboronic acid [19], and 4-[4,5-di(2-pyridyl)-1 H-imidazol- 2-y1]phenylboronic acid (DPPA) [20] (Fig. 6B), in the luminol/hydrogen peroxide/peroxidase system are well known. Rhodamine B and quinine are used as sensitizers in the CL-emitting reaction between cerium (IV) and thiol compounds. This CL reaction was successfully applied to the sensitive determination of various thiol drugs [21–32].

Fluorophores having lower oxidation potentials also enhance the PO-CL reaction. The addition of these compounds to the postcolumn reagent is very effective so as to increase the sensitivity. The attempt to enhance CL intensity using micelles of surfactants has also been reported [33], but it has yet not been applied to HPLC.

5.2.6Buffer and pH

Ion species present in the CL reaction mixture sometimes affect the CL intensity. In the PO-CL system, phosphate buffer has a tendency to decrease the CL intensity, but borate and imidazole buffers provide more intense CL.

Figure 6 Representative (A) phenol-type and (B) phenylboronic acid-type enhancers for luminol/hydrogen peroxide/peroxidase system. KIH-201, 2-(4′-hydroxy-3′-methoxy- benzylidene)-4-cyclopentene-1,3-dione; HDI, 2-(4-hydroxyphenyl)-4,5-diphenylimida- zole; HPI, 2-(4-hydroxyphenyl)-4,5-di(2-pyridyl)imidazole; DPPA, 4-[4,5-di(2-pyridyl)- 1H-imidazol-2-yl]phenylboronic acid).

In general, halogen ions have a decreasing effect upon the CL intensity. Salt concentration and buffer pH are also known to affect the CL reaction. The choice of buffer and its pH suited to each CL reaction system are hence very important to obtain intense CL.

6. APPLICATIONS

6.1 HPLC-CL Detection Using Luminol Derivatives

The structures of luminol derivatives used for HPLC-CL detection are shown in Figure 7A. Analytes labeled with luminol derivatives can be detected using hydrogen peroxide and potassium hexacyanoferrate(III) under alkaline conditions after HPLC separation (Table 1). For example, ibuprofen in saliva [34], saturated

Figure 7 (A) Luminol-type CL reagents and (B) derivatization reactions for DPH and 6-AMP.