Development of α-Amino Acid Detection with Peroxyoxalate Chemiluminescence by Using Water-Acetonitrile-Ethyl Acetate Mixed Solution

Peroxyoxalate chemiluminescence was, for the first time, examined by using ternary mixed solutions of water-hydrophilic/hydrophobic organic solvent. Eosin Y as a model fluorescence compound was dissolved with the ternary solutions of water (1.0 mM carbonate buffer, pH 9.0)-acetonitrile-ethyl acetate, water-rich of 15:3:2 volume ratio and organic solvent-rich of 3:8:4 volume ratio, to which bis(2,4,6-trichlorophenyl) oxalate and hydrogen peroxide chemiluminescence reagent were added. The chemiluminescence observed with the ternary solutions, especially the organic solvent-rich solution, showed a larger signal than that observed with the water only solution or water-acetonitrile mixed solution. Chemiluminescence in the presence of twenty types of α-amino acid was similarly examined by using the ternary organic solvent-rich solution. The chemiluminescence of three α-amino acids with fluorescence properties was enhanced with the ternary solution. The data reported here may contribute to development of a new, sensitive peroxyoxalate chemiluminescence detection system.


Introduction
Various types of aqueous-organic solvent mixed solutions are used in dissolu-How to cite this paper: Kan, H., Mizutani, R. and Tsukagoshi, K. (2020) Development of α-Amino Acid Detection with Peroxyoxalate Chemiluminescence by Using Water-Acetonitrile-Ethyl Acetate Mixed Solution. American Journal of Analytical Chemistry, 11, 15-24. tion [1], cleaning [2], preservation [3], and as reaction solvents [4]. Such mixed solutions are also useful in separation science, as extraction solvents and in liquid chromatography. We reported unique behavior of ternary mixed solutions of water-hydrophilic/hydrophobic organic solvent, such as water-acetonitrile-ethyl acetate mixed solution [5] [6] [7]. For example, the ternary mixed solutions were confirmed to work as a two-phase separation solution. The mixed solutions separated into upper and lower phases through the phase transformation in a batch vessel. In contrast, when the mixed solution was fed into a microspace, such as capillary tubes and microchannels on a microchip, annular flow with inner and outer phases was generated under certain conditions; we call this "Tube Radial Distribution Flow" (TRDF). The specific microfluidic flow, TRDF, was applied to capillary liquid chromatography [8] and micro-extraction [9].
The mixing behavior of ternary water-hydrophilic/hydrophobic organic solvent solutions was investigated using a Y-type microchannel. These two mixed solutions, water-acetonitrile and acetonitrile-ethyl acetate, were individually fed into two separate microchannels of a microchip that combined to form a single channel in a Y-type microchannel, affording a ternary mixed solution in the single channel. Unique microfluidic behavior was observed in the single channel.
We termed it "microfluidic inverted flow" for the ternary mixed solutions [10].
Denaturation was examined in a ternary mixed solution of water-hydrophilic/ hydrophobic organic solvent using λ-DNA and a plasmid as models. The experimental data indicated that λ-DNA changed from a double helix structure to a single helix structure and that the plasmid partially transformed to generate a denaturation bubble in the structure. The novel idea of using the ternary mixed solution first enabled the interaction of the hydrophobic organic solvent molecule with the double helical structure of DNA [11].
In our previous study, we examined luminal chemiluminescence (CL) behavior in a ternary mixed solvent solution of water-acetonitrile-ethyl acetate, and the mixed solution was found to enhance luminol CL in time and intensity under certain conditions [12]. In this study, we examined CL behavior with four kinds of mixed solutions of water only, water-acetonitrile, and ternary mixed solutions of water-acetonitrile-ethyl acetate for peroxyoxalate reagent, instead of luminol reagent [13] [14], in the presence of Eosin Y as a model fluorescence compound. The peroxyoxalate CL of Eosin Y was found to be most enhanced by the ternary mixed solutions.
The CL detection methods of α-amino acids without labeling procedures have been incorporated in flow injection analysis [15], high-performance liquid chromatography [16], and capillary electrophoresis [17]. They provided simple and procedures with high sensitivity. We tried to examine the peroxyoxalate CL response of α-amino acids in the present batch-type CL measurement system with a ternary mixed solution in order to get basic data for developing a flow system in the future.

CL Measurement System
The CL profiles and CL counts were examined with a batch-type detection sys- The preparation of CL regent solution and the mixing procedure of the reagent solution and the analyte solution containing fluorescent compounds, such as eosin Y, were carried out as a reference of our previous paper [18], as follows:

CL of Eosin Y with the Four Solutions
The phase diagram of water-acetonitrile-ethyl acetate is shown in Figure 1   reaction system was similar to that reported in our previous paper [12], where we examined the influence of the ternary mixed solution on a luminal CL reaction system. The effect of the ternary mixed solution on peroxyoxalate CL is discussed in the next section.

CL of Eosin Y with Various Compositions of the Ternary Mixed Solution
We examined the CL profiles by using various compositions of the ternary mixed solution, the water rich and organic solvent-rich, as shown in the phase diagram of Figure 4. The compositions of the water-rich mixed solutions in  solvent-rich solution of (D) provided a larger CL than the water-rich solution of (C). Although the reason is not yet clear, the organic solvent-rich solution may inhibit the hydrolysis reaction of TCPO. Similar CL behavior based on the luminol reaction was reported and discussed in our previous paper [12].

CL Profiles of Eosin Y and α-Amino Acids with the Organic Solvent-Rich Ternary Solution
The CL profiles of Eosin Y and phenylalanine (1 mM each) were examined by using the organic solvent-rich ternary solution of (D). The obtained data are shown in Figure 6. Although the CL profile of phenylalanine was examined with a detection window about 5 times larger than that of Eosin Y, we could observe a sufficient CL signal even for phenylalanine as an α-amino acid by using the specific mixed solution.
The CL counts of twenty types of α-amino acids were examined by using the organic solvent-rich solution of (D), together with solution (A) as a reference. As alanine was not dissolved with solution (A) and (D), we could not examine it. The obtained data are shown in Figure 7, where the CL counts are the average values of 3 -5 measurements. The relative standard deviations (n = 5) of phenylalanine, tryptophan, and tyrosine for their CL counts were 1.32%, 1.20%, and 1.91%, respectively. As can be seen in the figure, solution (D) provided larger CL counts for all α-amino acids than solution (A), in particular for phenylalanine, tryptophan, and tyrosine, the fluorescent α-amino acids. An enhanced effect for peroxyoxalate CL could be observed in the presence of fluorescence α-amino acids, similar to co-existing Eosin Y. α-Amino acids other than fluorescent α-amino acids also showed little CL, compared to the blank values that signified CL counts obtained in the absence of α-amino acids. As mentioned above, solu-

Conclusion
We tried to develop a new peroxyoxalate CL system by using the ternary mixed solution of the water-acetonitrile-ethyl acetate. The CL of Eosin Y and three α-amino acids (phenylalanine, tryptophan, and tyrosine) were enhanced with the ternary organic solvent-rich solution, compared to the CL observed with the water only and water-acetonitrile mixed solutions. The CL counts of α-amino acids by using the ternary mixed solution of the water-acetonitrile-ethyl acetate (volume ratio 3:8:4) is almost two times than by using the buffer only solution.
So it will be easier to do the detection in FIA, TRDC or other analytical systems by using the ternary mixed solution of the water-acetonitrile-ethyl acetate (volume ratio 3:8:4). The information obtained here may have important implications for future applications.