BIOMEDICAL CHROMATOGKAPHY. VOL. 6.84- 87 (1992)

A Universal Peroxyoxalate-chemiluminescence Detection System for Mobile Phases of Differing pH Kazuichi Hayakawa,* Eriko Minogawa, Tohru Yokoyama and Motoichi Miyazaki Faculty of Pharmaceutical Scicnces, Kanazawa University, 13-1 Takara-machi, Kanzawa 920, Japan

Kazuhiro Imai Branch Hospital Pharmacy, University of Tokyo, 3-28-6 Mejirodai, Bunkyo-ku, Tokyo 112, Japan

A universal peroxyoxalate-chemiluminescence detection system for high performance liquid chromatography, available for a variety of mobile phases, has been developed. The system consisted of a dual-head short-stroke pump and a chemiluminescence detector. The standard conditions using bis(2,4,6-trichlorophenyl) oxalate (TCPO) as aryl oxalate were as follows. The first postcolumn solution was the mixture of 0.5 M imidazole-nitric acid (pH 7.5) and acetonitrile (1:4, v/v). The second was acetonitrile containing TCPO-hydrogen peroxide. These two solutions were delivered by the two pump-heads. After the pH of the column eluate was adjusted to the optimum range (6.5-7.5) by the first postcolumn solution, the solution was mixed with the second postcolumn solution. After flowing through a reaction coil, the chemiluminescence of the mixture was monitored. Using this system, a high sensitivity (fmol level) was obtained for perylene as an analyte with mobile phases having different pH values (2.0-8.0). Polycyclic aromatic hydrocarbons became detectable to a high sensitivity even after the column separation using an acidic mobile phase. The detection sensitivity of nitrated pyrenes after on-line electrochemical reduction using an acidic mobile phase was also increased. This system might be available for other aryl oxalates by .some modifications of the postcolumn solutions.

INTRODUCTION Peroxyoxalate-chemiluminescence (PO-CL) detection is a highly sensitive detection system for high performance liquid chromatography (HPLC) (Kobayashi arid lmai, 1980; lmai et al., 1989). This method has been used to determine fmol or sub-fmol levels of polycyclic aromatic hydrocarbons (Sigverdson and Birks, 1983), fluorescent drugs (Imai et al., 1987) and fluorophorelabelled compounds (Kobayashi et al., 1981; Mellbin and Smith, 1984; Grayeski and De Vasto, 1987; Hayakawa et al., 1989; Nakashima et al., 1989; Kawasaki et af., 1990). In these reports, two pumps were used to deliver the aryl oxalate and hydrogen peroxide. Recently, the stabilizing conditions for a mixture of these two reagents have been improved, and a single pumping system has been demonstrated. Bis[2-(3,6,9-trioxadecyloxycarbonyl)-4-nitrophenyl] oxalate (TDPO) (lmai e f al., 1986), bis(Znitropheny1) oxalate (2-NPO) (Kwakman et al., 1988) and bis(2,4,6trichlorophenyl) oxalate (TCPO) (Imaizumi et al., 1989a) have been reported as stable oxalates for this system. The CL intensity, on the other hand, is affected by several factors such as pH, salts, organic solvents, vessel materials (Kobayashi and lmai, 1980; Alvarez et al., 1986; Hanaoka et al., 1989; Imaizumi et al., 1989a; Mann and Grayeski, 1991). Among these factors, pH seems to have a quite significant effect. The CL intensity as much weaker at acidic pH than neutral pH (Honda et al., 1985). The pH effect has prevented the method from being widely used in HPLC. We have * Author to whom correspondence should be addressed. 0269 -3879/92/020084-04 $05 .OO 01992 by John Wiley & Sons, Ltd.

removed this problem by using both a buffer solution, which adjusted the pH of column eluate to the optimum range, and a dual-head short-stroke pump. The purpose of this report is to describe the usefulness of the proposed system for acidic mobile phases.

EXPERIMENTAL ~

~~~~~~~~

Apparatus. Schematic diagrams of the HPLC and Cow injection analysis (FIA) systems used are shown in Fig. 3 . The HPLC system consisted of a Jasco (Tokyo, Japan) BIP-1 pump (PI), a Rheodyne (Cotati, CA, USA) model 7125 injector (I) with a 20 FL loop, a guard column (GC) and an analytical column (AC). The FIA system consisted of the same apparatus as the HPLC. except a damper coil (DC, PTFE, 10 m x 0.25 mm i.d.) was used instead of the column. The flow rate of all mobile phases (M)was 1.0mL/min in both systems. The CL detection system used in conjunction with HPLC and FIA consisted of a Sanuki (Tokyo, Japan) DMX 2200-T dual-head short-stroke pump (P2), a mixing coil (MC, PTFE, 6Ocm X0.25 mm i.d.), a reaction coil (RC. PTFET, 10 cm X 0.25 mm Ld.), a Soma (Tokyo, Japan) S-3400 luminescence detector (CL) with an 100p.L flow cell and a Shimadzu (Kyoto, Japan) chromatopak C-R3A integrator (IG). Flow rates of the two postcolumn solutions were each 0.5 mllmin. All experiments were carried out at room temperature (20 & 2°C). Other conditions are described in the Results and Discussion section. Chemicals and preparation of solutions. The chemicals used were all of analytical reagent grade. Standard solutions of polycyclic aromatic hydrocarbons were prepared by dissolvReceived 18 March 1991 Accepted 10 May I991

PO-C'L IIETEC'TION FOR VARIOUS MOBILE PHASES

fl M

the same system as i n ( A )

+

Waste Waste

Figure 1. Schematic diagrams of (A) HPLC and (6) FiA equipped with a peroxyoxalate-cherniluminescence detection system.

For abbreviations see text.

8.5

limit of the optimum pH range, the pH of the buffer (pH,) was adjusted to 7.5 with nitric acid. Taking the mixing efficiency into consideration, the buffer was mixed with acetonitrile (1 :4, v/v). We have operated the system efficiently under the following mobile phase conditions: salt (or acid) concentration, less than 6 0 m ~ pH, ; higher than 2; organic solvent to buffer ratio, over 1 : l (v/v); flow rate, l.OmL/rnin. These conditions cover a wide variety of mobile phases used in reversed phase HPLC, although there are several exceptions. If the expected p H of the mobile phase (pH,) is to bc 6.5 after the addition of the above postcolumn solutions, the necessary final concentrations of imidazole (C,) and nitric acid (CJ are given by the following equations, where x is the final concentration of the acid species which comes from the mobile phase (Perrin and Dempsey, 1974).

ing them in acetonitrile or the mobile phases used.

Postcolumn solutions were as follows. The buffer solution (R,) was a mixture of 0 . 5 ~imidazole buffer (adjusted to pH7.S with nitric acid) and acetonitrile (1:4, v/v). The CL reagent solution (RJ was an acetonitrile solution containing 1.0mM TCPO and 20 m M hydrogen peroxide.

RESULTS AND DISCUSSION

Universal conditions It is important in a simple system that the number of pumps can be reduced. An acetonitrile solution containing TCPO and hydrogen peroxide can be delivered by a single pump as described above. Therefore, TCPO was used as an aryl oxalate in the following experiments. The CL reaction, on the other hand, requires both an optimum p H (5.5-7.5) and a catalyst (Honda et al., 1985). Imidazole (pK 6.9) is an effective buffer in the above pH range as well as a catalyst for the CL reaction (Imai et al., 1986). When imidazole was added to a mixture of TCPO and hydrogen peroxide, TCPO was easily hydrolysed. This result suggests that two pump-heads are necessary to keep the TCPO away from the imidazole. Recently, a dual-head short-stroke pump has been used in HPLC-CL systems becausc of its high mixing efficiency and ability to deliver different solutions (Hayakawa et al., 1991). This pump was used to deliver the two postcolumn solutions in this work. The order in which the buffer and the CL reagent solution are mixed with the eluent is an important factor for maximizing the detector response. When the buffer solution was introduced after mixing the CL reagent solution and the eluent, no response was observed. When both solutions were mixed before introduction into the eluent, the response was small. The fast decomposition of TCPO was considered as the reason for these results. Among several possible configurations, the largest response was observed when the CL reagent was added after mixing the buffer solution and eluent, as shown in Fig. 1. Flow rates of the two postcolumn solutions were each 0.5 mL/min according to our previous report (Hayakawa et ul., 1991). Both the pH and imidazole concentration of the buffer depend on the mobile phase. Using the upper

From the above equations, the values of C, and C, are and 2.30 X lo-' M , respectively. 6.10 X l W 3M Consequently, a 0.46 M imidazole-0.122 M nitric acid solution is necessary as the aqueous component of the buffer solution. Taking this into consideration, 0.5 M imidazole-nitric acid (pH 7.5): acetonitrile (1:4, v/v) was used as a postcolumn buffer solution in the following experiments. If other aryl oxalates are used in the system, pH, should be changed to their optimum values. It has been reported that imidazole concentration affects T,,, (the time taken to reach the maximum C L intensity) (Hanaoka et al., 1988). The imidazole concentration calculated above is higher than the optimum concentration added to mobile phases in our previous report (Hayakawa et uf., 1991). Therefore, we examined other optimizing conditions, namely TCPO and hydrogen peroxide concentrations and reaction coil length. When perylene was used as an analyte, the largest signal-to-noise ratio was observed when concentrations of TCPO and hydrogen peroxide were 1.0 mM and 20 m M , respectively, and the reaction coil (RC in Fig. 1) wa5 10cm. When several acetonitrile: aqueous buffer solutions (9:1, v/v) were used as carriers for FIA, without a postcolumn pH buffer, the detector response decreased with decreasing carrier solution pH and finally was not observed at pH lower than 4.0. This result correlates with the report that the signal of dipyridamol at pH 2-3 was less than Ul00 that at p H 7 for a given mobile phase (Imai el al., 1990)). Using a buffer solution, the final mobile phase pH values increased to over 6.5 and the detector response increased significantly. Table 1 shows relative values of the signal and the signal-tonoise ratio for 14 aqueous solutions using the present system. Relative signals were all over 0.61 and relative signal-to-noise ratio were over 0.77. This result suggests that this system is effective for acidic mobile phases. The decrease in signal found in the tris(hydroxymethy1)aminomethane (Tris) buffers might be attributable to a shortened T,,,,, due to the catalytic effect that Tris has on the CL reaction.

K. HAYAKAWA E T A L

d6

Table 1 shows another interesting result, that hydrochloric acid is available to control pH to some extent, although halogen ions have been shown to decrease the CL intensity significantly (Honda et al., 1983).

A

2 1 3

1

Applications Several polycyclic aromatic hydrocarbons and related compounds are well known mutagens and carcinogens. As a highly sensitive and selective determination method for these compounds, HPLC with CL detection has attracted much attention. However, the mobile phase pH has sometimes restricted the application of the method. This problem has been resolved effectively by the present detection system. Figure 2 shows typical chromatograms of pyrene, perylene and benzo[a]pyrene with 0.1 M trifluoroacetic acid (the pH adjusted to 2.0 with sodium hydroxide): acetonitrile (1 :9, v/v) as the mobile phase. No peak was observed in the previous detection system without a buffer solution (B). A similar result was reported in the determination of dipyridamol (lmai et al., 1990). However, using the present detection system with a postcolumn buffer solution, all three peaks were observed (A). From Fig.

Table 1. FIA detector response of perylene using carrier solutions with differing pH values' Aqueous solution

Signal (relative)

Water

1

1

200 mM Nitric acidsodium hydroxide (pH 2.0)

1.01 k 0.09

0.93

200 mM Perchloric acidsodium hydroxide (pH 2.0)

1.01 iO.10

0.93

100 mM Trifluoroacetic acidsodium hydroxide (pH 2.0)

1.02 2 0.1 1

0.90

10 mM Sulfuric acidsodium hydroxide (pH 2.0)

1.05 20.03

0.77

10 mM Glycinehydrochloric acid (pH 2.0)

0.93 f_ 0.02

0.86

10 mM Glycinenitric acid (pH 2.0)

1.03 k 0.06

0.92

100 mM Acetic acidsodium hydroxide (pH 4.0)

0.61 & 0.05

0.80

100 mM Phthaiic acidsodium hydroxide (pH 4.0)

0.94 5 0.05

0.81

10 mM Citric acidsodium hydroxide (pH 4.0)

1.OO k 0.02

0.81

10 mM Phosphoric acidsodium hydroxide (pH 4.0)

0.98 ? 0.04

0.77

100 mM Tris( hydroxymethy1)aminomethane-hyrochloric acid (pH 8.0)

0.65 f0.02

0.84

100 mM Tris(hydroxymethy1)aminomethane-nitric acid (pH 8.0)

0.73 f 0.01

0.78

10 mM Boric acidsodium hydroxide (pH 8.0)

1.OO k 0.03

Sig nal-to-noise (relative)

I

I

5

10

0.77

15

I

I

I

I

0

5

10

15

Time/min

Figure 2. Typical chromatograms of (1) 2 . 5 ~lo-'' mol pyrene, (2) 2 . 0 ~ mol perylene and (3) 5.0 x mol benzo[alpyrene by peroxyoxalate-chemiluminescence detection with (A) the present system and (6)the previous system. Column: lnertsil ODs-2 ( 2 5 0 ~ 4 . 6mm i.d.). Mobile phase: 100 mM trifluoroacetic acid-sodium hydroxide (pH 2.0):acetonitrile (1:9, v/v). Postcolumn solutions of the present system: R1, 0.5 M imidazole-nitric acid (pH 7.5):acetonitrile (1 :4, v/v); Rp, acetonitrile containing 1.0 mM TCPO and 20 mM hydrogen peroxide. Postcolumn solution of the previous system: acetonitrile solution containing 0.5 mM TCPO and 150 mM hydrogen peroxide. For other conditions see text.

2(A) the detection limit (S/N=3) of perylene was calculated as less than 1fmol. This value is comparable to the limit reported previously (Imaizumi et al., 1989b). We previously reported a HPLC-CL determination method for fmol levels of dinitro-, nitro-, nitroso- and amino-pyrenes (Imaizumi et al., 1990). After HPLC separation, dinitro- nitro- and nitroso-pyrenes were electrochemically reduced to their amino derivatives at an optimum pH of 4.75. However, this pH is lower than that required for optimum CL detection. When we used the present detection system with a small modification in the TCPO and hydrogen peroxide concentrations, all signals and signal-to-noise ratios increased by factors of 2.4-5.9 and 1.8-4.4, repsectively (Table 2). The present TCPO system is effective for many mobile phases without any change in conditions. Small modifications of postcolumn solutions should allow an even wider range of possible mobile phases. The same configuration of the postcolumn system as described in this report might also be effective for other aryl oxalates such as TDPO and 2-NPO.

Table 2. Comparison of signals and signal-to-noise ratios of nitropyrenes by the previous and the present detection systems" Compound

Signal lpresent/previous)

Signal-ta-noise (present/previous)

1.8-Dinitropyrene 1.6-Dinitropyrene 1-Nitropyrene I-Nitrosopyrene

3.0 3.6 5.9 2.4

2.3 2.7 4.4 1.8

Column: Nucleosil ODS (250 x 4.6 m m i.d.). Mobile phase: 2.0 mM imidazole-perchloric acid (pH 4.75):acetonitrile (20:80, v/v). Postcolumn solutions of the present system: R1, 0.5 M imidazole-nitric acid (pH 7.5):acetonitrile (1 :4, vlv); RZ, acetonitrile containing 0.05 mM TCPO and 10 mM hydrogen peroxide. Postcolumn solution of the previous system : acetonitrile solution containing 0.5 mM TCPO and 150 mM hydrogen peroxide. Injection amount:each 1 pmol. For other conditions see text. a

Injection amount, 100 fmol. Relative signal is meanfS.D. ( n = 3 ) . Carrier solution, aqueous so1ution:acetonitrile (1:9, v h ) . For other FIA conditions see text.

a

I

0

PO-CL DETECTION FOR VARIOUS MOBILE PHASES

87

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