A Study on the Effects of Solvent and Temperature on 2-Amino-7-Nitro-Fluorene (ANF) Using Synchronous Fluorescence

Abstract

Synchronous fluorescence spectra are presented to investigate solute–solvent interactions in liquids. To this end, the spectra of 2-amino-7-nitro-fluorene (ANF) in six different solvents—acetic anhydride, acetone, acetonitrile, benzene, chlorobenzene, and ethyl acetate—are presented. The study also examines ANF’s synchronous fluorescence signals at five temperatures from 25 °C to 5 °C, providing a comprehensive analysis of its fluorescence characteristics in different environments and temperatures. An ANF sample dissolved in benzene at 5 °C produced a synchronous band with the largest intensity and smallest frequency shift. The results show that higher-intensity peaks are obtained at lower temperatures with solvents with a small dipole moment and dielectric constant. This suggest that the best conditions to detect ANF and similar molecules at very low concentrations are with non-polar solvents at low temperatures.

1. Introduction

Synchronous fluorescence spectroscopy (SFS) is a combination of both fluorescence excitation and emission spectroscopy. In a conventional fluorescence scan, only one of the monochromators, either the excitation or the emission monochromator, is scanned at any given time. However, in a synchronous scan, both the excitation and emission monochromators are scanned synchronously to obtain the spectrum. The monochromators are set at a constant wavelength, with the difference between the λ emission (λ_em) and λ excitation (λ_ex) scanned at a constant rate. The constant wavelength difference is represented as Δλ = λ_em − λ_ex. It can also be scanned at a constant wavenumber difference, represented as Δν = (1/λ_ex − 1/λ_em) × 10⁷. In synchronous fluorescence, the intensity of the excitation and intensity of the emission are detected simultaneously at each ∆λ and multiplied. Depending on the ∆λ chosen, the resulting signal is a narrow single fluorescent band that is characteristic of the compound. The two main advantages of SFS are band narrowing and spectral simplification [1,2]. We have used synchronous fluorescence spectra to identify toluene, aniline, naphthalene, acenaphthene, pyrene, and anthracene in a mixture of liquid hexane solutions [3], and to determine the limits of detection (LOD) of polycyclic aromatic hydrocarbons (PAHs) in water and n-hexane solutions at various concentration ranges [4,5].

Most applications of the synchronous fluorescence technique have been dedicated to the identification of molecules in mixtures. For example, there have been analyses of mixtures of PAHs [6,7,8], PAHs as environmental pollutants [9,10], the biomonitoring of marine life [11], the detection of tryptophan in water [12], the detection of tryptophan attached to the membrane of a bacteria [13], the characterization of aromatic hydrocarbons in sediments [14], and organic matter detection in the Canadian High Arctic ice [15], among other applications [16,17,18].

In this paper we used the synchronous fluorescence technique to show that solute–solvent interactions in liquids can be studied with this technique. To this end, the synchronous fluorescence spectra of 2-amino-7-nitro fluorene (ANF) dissolved in benzene, chlorobenzene, ethyl acetate, acetone, acetic anhydride, and acetonitrile were obtained. The increasing dipole moment and dielectric constant of the solvents influenced the intensity and characteristic peak wavelengths of the synchronous fluorescence bands.

In addition, the effect of temperature on the intensity of the bands was also studied.

2. Materials and Method

2.1. Materials

2-Amino-7-nitro-fluorene (99.8%, CAS No 1214-32-0), acetonitrile (CAS No. 75-05-8, 99.9% HPLC grade), acetic anhydride (CAS No. 108-24-7, 98.0% ACS reagent grade), acetone (CAS No. 67-64-1, 99.5% ACS reagent grade), ethyl acetate (CAS No. 141-78-6, 99.5% ACS reagent grade), chlorobenzene (CAS No. 108-90-7, 99.5% ACS reagent grade), and benzene (CAS No. 71-43-2, 99.0% ACS reagent grade) were used. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). For solution preparation, an appropriate amount of ANF was weighed out with an analytical balance, placed into a volumetric flask and diluted to measurement concentration. All solutions were prepared at a concentration of 10⁻⁴ M.

2.2. Method

The fluorescent measurements were carried out with a Shimadzu RF-6000 spectrofluorometer (Missouri City, TX, USA). The instrument has dimensions of 565 × 610 × 274 mm³. For the data collection and instrument operation, LabSolutions RF software was used. The software provides instrument control, spectral data acquisition, and analysis capabilities for fluorescence, bioluminescence, chemiluminescence, and electro-luminescence, in addition to their respective spectrum modes. The measurements in this work were performed under fluorescence excitation and the synchronous spectrum mode. For the spectroscopic measurements of both the excitation and emission fluorescence, the slit width (entrance and exit aperture ) of the monochromator was set to 5 nm. The instrument has a wavelength scanning speed of between 60,000 and 20 nm/min with a 9-step selection. The experiment was conducted with a resolution of 1 nm at a 600 nm/min scanning speed. A total of 30 scans were taken and averaged using the in-built data processing functions available in the software. The spectrum was auto-corrected for Rayleigh scattering with the Spectrum-Correction function available in the software.

For the light source, a 150 W Xenon arc lamp composed of metal (tungsten, brass, nickel-plated brass, stainless steel, and nickel-plated zinc alloy) and Quartz glass with a self-deozonating lamp house was used. The wavelength range of the source was in the range of 200–900 nm. For the light source compensation method, a monochromatic light-monitoring comparison operation method was employed.

The dimensions of the sample compartment were 280 × 305 × 110 mm. Housed inside the sample compartment was set a 10 mm rectangular-type cell holder with screw fixing to the base. The sample chamber was also fitted with a sample cover, which was equipped with an open/close sensor. The detector protection function activated if the sample chamber cover was open. During the spectroscopic measurements the sample chamber cover was kept closed to prevent stray background light from entering the spectrometer. The monochromator consisted of concave mirror optics with blazed holographic grating, having a groove density of 1300 grooves per mm. The excitation side of the monochromator was an off-plane optical system with a blaze wavelength of 350 nm, while the emission side was an in-plane optical system with a blaze wavelength of 400 nm. The monochromator had a wavelength accuracy and repeatability of ±1 nm and ±0.2 nm, respectively. The instrument was equipped with two separate detectors. The monitoring side was equipped with a silicon photodiode, while the emission side was equipped with a R928 photo-multiplier tube (PMT) detector with the highest signal-to-noise ratio of 1000:1 (RMS) or 300:1 (P-P).

The absorption measurements were performed with a Cary 50 Bio UV-Visible spectrometer from Varian (Agilent Technologies, Santa Clara, CA, USA) with a 15 W Xenon lamp and using Cary WinUV software (version 5.3). For the absorption measurements, the scan rate was set at 60 nm/minute and a 1 nm resolution. A 10 mm pathlength Quartz cuvette from Sterna was used for all the spectroscopic measurements. No correction was implemented for the instrumental response.

3. Results and Discussion

3.1. Absorption Bands

The absorption bands from our investigation are presented in Figure 1. The absorption bands of ANF in benzene, ethyl acetate, and acetonitrile have been reported [19], and the values are within 3 nm or less of our reported values. The absorption bands of ANF are identified by the name of the solvent in each spectrum. It is important to notice from the absorption spectra that all the bands have a peak absorption at or above 390 nm, except for the spectrum of ANF in acetic anhydride, which is around 360 nm. This result will be discussed later, and has a possible explanation related to the reactivity of ANF in the presence of acetic anhydride.

3.2. Fluorescence Bands

The fluorescence excitation, emission, and SF spectra of ANF in separate solutions with benzene, chlorobenzene, ethyl acetate, acetone, acetic anhydride, and acetonitrile as the solvents are presented in Figure 2. The ANF concentration is 1 × 10⁻⁴ M. The spectra are identified with the name of the solvent at the top. In each spectrum, (a) corresponds to the fluorescence excitation (blue) and fluorescence emission (red). Although the excitation and emission spectra were recorded at five different temperatures, only the spectra at 5 °C are shown. The synchronous fluorescence spectra (b) are shown at different temperatures from 5 to 25 °C. The intensity of the SF bands increased as the temperature decreased, with a maximum value at 5 °C. The only exception was ANF in chlorobenzene, which decreased in intensity as the temperature was lowered.

Table 1. Wavelength of observed excitation spectra (λobs); excitation wavelength (λexcit.) for emission; observed peak wavelength for absorption (λ_abs), fluorescence excitation (λ_exc), fluorescence emission (λ_em), synchronous fluorescence (λ_SF), and ∆λ for SF of ANF in six different solvents. Full width at half maximum, fwhm. All values in nm.

ANF	λobs/λexcit.	Absorption	Excitation	Emission	Synchronous
Solvents	nm	λabs	λex	λem/(fwhm)	λSF/(fwhm)	∆λ
Benzene	519/389	394	429	513/(94)	519/(41)	90
Chlorobenzene	555/393	397	414	549/(97)	555/(62)	141
Ethyl acetate	584/398	400	445	575/(111)	584/(49)	139
Acetone	631/403	408	454	629/(122)	629/(61)	177
Acetic anhydride	537/357	360	403	534/(120)	534/(43)	134
Acetonitrile	658/399	404	446	650/(133)	658/(63)	212

summarizes the absorption and fluorescence results of Figure 1 and Figure 2. The first column identifies the six solvents. The second column shows the wavelengths (nm) at which the ANF fluorescence excitation spectra (blue) were observed and the wavelengths that were used to excite the ANF to obtain the fluorescence emission spectra. The third column shows the peak wavelengths of the absorption spectra in Figure 1. The fourth column shows the wavelengths of the peak maximum of the fluorescence excitation spectra. The benzene excitation band (blue) in Figure 2 shows three different vibrational transitions to the first electronic excited state. The chlorobenzene excitation band in Figure 1 shows one band that could be two close bands that overlap. The ethyl acetate blue band shows three different vibrational transitions to the first electronic excited state. The acetone blue band shows two different vibrational transitions. The acetic anhydride fluorescence excitation shows two different vibrational transitions, one shoulder and one strong band. The acetonitrile fluorescence excitation spectrum (blue) shows at least five different vibrational transitions to the first electronic excited state. The emission wavelengths in column five show a single peak for all the solvents; in addition, the full width at half maximum (fwhm) of each emission band is shown in parentheses in the same column to compare it with the fwhm of the synchronous fluorescence band. The peak wavelengths of the synchronous fluorescence bands are shown in column six, with the fwhm in parentheses, which in general is approximately half the value of the fwhm of the fluorescence emission bands. Column seven shows the ∆λ used to obtain the SF bands.

3.3. Ground and Excited State of ANF

The absorption and fluorescence emissions of ANF and other molecules in polar solvents were originally studied by Lippert [20,21] and Mataga [22]. Ultraviolet absorption by ANF in a liquid solution produces a transition from the ground state (S_o) to the first excited state (S₁), causing intramolecular charge transfer from the amino donor to the nitro acceptor group, as shown in Figure 3 [19,20,21,22].

The dipole moment was determined to be 7 D in the ground state [20] and 32 D in the excited state [23]. The excitation of ANF in 1,4-dioxane at around 400 nm by Czekalla et al. [24] determined a dipole moment of 6.4 D in the electronic S_o state and 20.3 D in the S₁ state. The X-ray molecular structure of the ground state was reported [19]. The calculated structures of the ground and excited states using density functional theory (DFT) were published [25]. Fu et al. performed the calculations using DFT and Zerner’s Intermediate Neglect of Differential Overlap (ZINDO) method [26]. The dipole moment change of 25 D upon excitation explained the large shift in fluorescence in acetonitrile [27].

3.4. Correlation of Solvent Properties with ANF Absorption and Fluorescence Emissions

Table 2. Peak wavenumber (cm⁻¹) of absorption and fluorescence emissions. Difference between absorption and emission wavenumbers (ν_ab − ν_em) of ANF. Dipole moment (Debye). Dielectric constant ($ϵ$), index of refraction (n), and ∆f of the solvents.

Solvent	νabs (cm−1)	νem (cm−1)	νab − νem (cm−1)	Dipole Moment (D)	$$\mathbf{Dielectric}\mathbf{Constant}\left(\mathit{ϵ}\right)$$	Index of Refraction (n)	∆f
Benzene	25,380.7	19,493.2	5877.6	0.0	2.3	1.5011	0.001642
Chlorobenzene	25,188.9	18,214.9	6973.9	1.54	5.62	1.5248	0.142936
Ethyl acetate	25,000	17,391.3	7608.7	1.88	6.02	1.3724	0.199281
Acetone	24,509.8	15,898.3	8611.6	2.69	20.7	1.3587	0.284307
Acetic anhydride	27,777.8	18,726.6	9051.2	2.8	21	1.3901	0.273457
Acetonitrile	24,752.5	15,384.6	9367.9	3.44	37.5	1.3414	0.305416

shows the peak wavenumber (cm⁻¹) of the absorption and fluorescence emissions calculated from the corresponding peak wavelengths presented in Table 1. It also shows the difference between the absorption and emission wavenumbers (ν_ab − ν_em) of ANF. In addition, a summary of the dipole moments (Debye), dielectric constants, and indexes of refraction of the solvents was reported [28]. The value of the orientation polarizability (∆f) was calculated from Equation (1):

$$\mathsf{\Delta }f=\left({\displaystyle \frac{ϵ-1}{2ϵ+1}}-{\displaystyle \frac{n^2-1}{2n^2+1}}\right)$$

(1)

$\mathsf{\Delta }f$ involves the dielectric constant (ε) and refractive index (n) of the solvent. An approximation that correlates the (ν_ab − ν_em) with the (∆f) is the Lippert–Magata equation:

$${\nu }_{ab}-{\nu }_{em}={\displaystyle \frac{2}{hc}}{\displaystyle \frac{{\left({\mu }_{\mathrm{S}1}-{\mu }_{\mathrm{S}0}\right)}^2}{a^3}}\left(\mathsf{\Delta }f\right)+constant$$

(2)

In this equation, h (= 6.6256 × 10⁻²⁷ ergs) is Planck’s constant, c (=2.9979 × 10¹⁰ cm/s) is the speed of light. The difference between the wavenumbers (cm⁻¹) of the absorption and fluorescence emissions, ${\nu }_{ab}-{\nu }_{em}$, is proportional to the difference in the dipole moments of the excited state (${\mu }_{\mathrm{S}1}$) and the ground state (${\mu }_{\mathrm{S}0}$) of the fluorescent molecule, and $a^3$ is the volume of the spherical cavity where the molecule resides.

Using the Lippert–Mataga formalism [20,21,22], a linear plot of the (ν_ab − ν_em) versus ∆f values from Table 2 produces a positive slope, as shown in Figure 4, with a very good correlation for all the solvents.

3.5. Synchronous Fluorescence Intensity and Polarity

The intensity of the bands in the six different solvents is shown in Figure 5a. Figure 5b shows the peak intensity as a function of the dielectric constant of the solvent. Figure 5c shows the peak intensity as a function of the dipole moment of the solvent in Debye (D) units. The intensity of the SF decreases as the dielectric constant, or the dipole moment of the solvent molecules, increases. The large dipole moment change upon the excitation of ANF from 7 D in the ground state to 32 D in the excited state explains the change in the intensity of the bands. The solvent molecules surrounding the ANF molecules show an increase in attraction forces, from the lowest to the highest dielectric constant and/or dipole moment of the solvent. The fluorescence quenching by the solvent is the largest for the compound with the largest dipole moment (acetonitrile), compared to the compound with lowest dipole moment (benzene).

3.6. Synchronous Peak Wavelength and Polarity

The wavelengths of the SF peak intensities were also investigated. The bands in Figure 5a were normalized and are presented in Figure 6a.

Figure 6b shows the wavelength at the SF peak as a function of the dielectric constant. (c) shows the wavelength at the SF peak as a function of the dipole moment. The peak wavelength of the SF band increases as a function of the increase in the dielectric constant and the dipole moment, except for acetic anhydride, which does not follow the pattern. This behavior for acetic anhydride could be explained as a possible chemical reaction between the amine part of ANF and acetic anhydride. The reaction is a well-known acetylation reaction and could occur as

$$C_4{H}_6{O}_3+H_{2}N{C}_{13}{H}_{8}N{O}_2\to C{H}_{3}COOH+\left(C{H}_{3}CO\right)NH{C}_{13}{H}_{8}N{O}_2$$

(3)

Acetic anhydride is an acetylating agent that has been found to react with organic amines at temperatures between 60 °C and 70 °C in the absence of a catalyst or another solvent. The reaction takes place during a time interval between 7 and 20 h [29]. This could explain the anomalous peak absorption shown in Figure 1, and the different behavior as a function of the dielectric constant and the dipole moment of the solvent shown in Figure 6b,c. Studies to test the possibility of acetylation are under investigation in our laboratory. The change in the wavelength of the fluorescence emission and synchronous fluorescence is also associated with the lowering of the energy level for emission with respect to the absorption level. A large solvent dipole moment will lower the level more than a non-polar molecule, increasing the wavelength of the emission or decreasing the energy of the photon emitted.

3.7. Synchronous Intensity and Temperature

The temperature dependence of the SF bands is shown in Figure 7a,b. Except for ANF in chlorobenzene, the signal increases as the temperature decreases.

The intensities of ANF increased gradually as the temperature decreased in all the solvents (Figure 7a) except for chlorobenzene (Figure 7b), where the signal intensity oscillated until 15 °C and then decreased more rapidly. This solvent was the only one that showed this behavior. Looking at the SF intensities from Figure 5a, that of ANF in benzene is the largest and that of ANF in acetonitrile is the smallest. For any given temperature, the quenching by the solvent was the largest for the compound with the largest dipole moment (acetonitrile), compared to that for the compound with the lowest dipole moment (benzene). Figure 7a could indicate that as the temperature decreased from 25 °C to 5 °C, the viscosity of the solvent increased, slowing the reorientation process around ANF [30]. This process decreased the solvent quenching rate. As a result, the intensity of the signal increased. More detailed studies are needed to explain the anomalous behavior of ANF in chlorobenzene, because the signal intensity increased from 5 °C to 15 °C, but oscillated from 15 to 25 °C.

4. Conclusions

The synchronous fluorescence bands of ANF are narrower (fwhm) than the fluorescence emission bands, allowing for the better separation of them in different solvents. The intensity of the ANF bands decreased as a function of the increase in the dielectric constant and/or dipole moment of the solvent. Except for chlorobenzene, larger intensity peaks were obtained at lower temperatures with solvents with a small dipole moment and dielectric constant. The large dipole moment change upon the excitation of ANF explains the shift in the SF and fluorescence emission with the increasing dielectric constant and/or dipole moment of the solvent. An acetylation chemical reaction is probably responsible for the peak absorption and SF wavelength associated with acetic anhydride. This suggests that the best conditions to detect ANF and similar molecules at very low concentrations are with non-polar solvents at low temperatures.