pH-dependent redox mechanism and evaluation of kinetic and thermodynamic parameters of a novel anthraquinone

Abstract

The redox behavior of 1,4-dihydroxy-2-(3-hydroxy-3-(trichloromethyl)pentyl)-8-methoxyanthracene-9,10-dione (HCAQ) was investigated at a glassy carbon electrode over a wide pH range of 3–12 by using cyclic, square wave and differential pulse voltammetry (CV, SWV and DPV). CV results of HCAQ obtained at different temperatures were used for the evaluation of thermodynamic parameters like ΔG^#, ΔH^# and ΔS^#. The values of diffusion coefficient and heterogeneous electron transfer rate constant were also determined by CV. Limits of detection and quantification were determined by SWV. The numbers of electrons and protons involved in the oxidation processes were evaluated by DPV. The value of apparent acid dissociation constant (pK_a) was obtained from the intersection of two linear segments of the E_p vs. pH plot. The effect of pH on the UV-visible spectral response was also monitored which allowed the determination of pK_a of HCAQ. The values of apparent acid dissociation constant obtained from electrochemical techniques and electronic absorption spectroscopy were found to be in very good agreement. On the basis of experimental results, a pH-dependent oxidation mechanism was proposed in order to provide useful insights into the unexplored pathways by which anthraquinones exert their biochemical action.

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1. Introduction

Anthraquinones (AQs), an important class of natural evolutionary redox molecules, have a broad range of applications.¹ AQs found in some plants and animals are used as constituents of many dyes.^2–4 A number of their derivatives play a key role in catalyzing reduction of biologically important molecules like vitamin K.⁴ Some AQs find use as algaecides and fungicides.⁵ Their derivatives, being electron acceptors, play a major role in photosynthesis and are able to transport a negative charge in biological systems.⁶ Due to the adsorption ability of AQs at a glassy carbon electrode, their derivatives are used for electrode modification.⁷ The modified electrode can be used as redox catalyst for the reduction of oxygen to hydrogen peroxide.^8–10 On complexation with macrocyclic polyethers, AQs form lumophores that can be used to selectively detect many transition metal ions in solution, thus offering an alternative photophysical detection mechanism.¹¹ Some AQs like rufigallol and aclarubicin are used as antimalarial¹² and anticancer drugs.¹³ AQs, being redox catalysts, play a vital role in the production of paper pulp.^14,15 Alizarin, an AQ derivative, is extensively used in the laboratory as pH indicator, an indicator of bone growth and a spot test reagent^16,17 due to its color variation in media of different pH. Apart from pH-dependent photophysical properties, the voltammetric behavior of AQs also depends on pH and their potential-pH diagrams can give useful information about the stability of different forms of AQs in acidic, basic and neutral conditions.¹⁸ AQs can be used for electrochemical switchable devices and pH-responsive drug delivery systems on the basis of the strong pH dependency of their redox behavior.^19–23 The broad range of applications of such compounds prompted us to investigate the electrochemical behavior of a novel AQ, 1,4-dihydroxy-2-(3-hydroxy-3-(trichloromethyl)pentyl)-8-methoxyanthracene-9,10-dione (HCAQ), in a wide pH range. The current work will assist in explaining some of the redox properties of AQs which are still unexplored by the scientific community. Although extensive literature is available about the voltammetric reduction of AQs, yet information about their electro-oxidation is meager and the pH-dependent oxidation of AQs at different temperatures is a largely unexplored area. So to bridge this gap, the pH-dependent electro-oxidation of HCAQ was investigated at different temperatures with the objectives of adding a new candidate to the literature of the AQ family and providing useful insights into the pH-dependent electrochemical fate of AQs.

AQs are commonly used as electron transfer mediators and redox probes.²⁴ Feng and coworkers demonstrated the enhancement in power output of a microbial fuel cell by the mediator role of an AQ derivative for efficiently transferring electrons from bacteria to anode.²⁵ The results of Kumamoto et al. revealed that an oligonucleotide modified with AQ can act as a suitable redox reporter for the detection of a single-base mismatch in DNA.²⁶ Abi and Ferapontova also used an AQ as redox probe for the investigation of the electron transfer mechanism within loosely compact monolayers of DNA.²⁷ Most AQs exhibit redox behavior in the negative potential domain of glassy carbon or gold electrodes, but interestingly our AQ derivative, HCAQ, can also show redox response in the positive potential window of the electrodes. Hence, in comparison to simple AQs, HCAQ has extended applications as redox probe and electron transfer mediator.

The biological activities of AQs are related to their redox reactions.^28,29 The biological activity of hydroxyquinones is considered to correspond to their hydroxyl group.^30,31 Therefore, the presence of three hydroxyl functionalities in HCAQ is expected to lead to a broader spectrum of biological activities as compared to simple AQ or its derivatives having single or double hydroxyl groups. By modifying the position of the hydroxyl group, the redox properties of the quinonoid moiety can be altered.³² Thus, a greater alteration in the properties of HCAQ is possible due to the possibility of changing the position of the several hydroxyl groups in its structure.

2. Experimental

Analytical-grade reagent HCAQ was gifted by Prof. Dr Amin Badshah. A 2 mM stock solution of HCAQ was prepared in absolute ethanol. Fresh working solutions of HCAQ were prepared in 50% ethanol and 50% buffer. Britton–Robinson (BR) buffer of 0.2 M ionic strength with pH 3–12.0 was used as supporting electrolyte. For the preparation of BR buffer, a 0.04 M mixture of each of H₃BO₃, H₃PO₄ and CH₃COOH was used and adjusted to the desired pH with 0.2 M NaOH.³³

The solution of the analyte was purged with nitrogen gas before every voltammetric assay to remove the dissolved oxygen. All voltammetric experiments were performed using Autolab running with GPES 4.9 software (Eco-Chemie, The Netherlands). A glassy carbon electrode (GCE) having a surface area of 0.071 cm² was used as the working electrode. Pt wire and Ag/AgCl (3 M KCl) were used as counter and reference electrodes. The GCE was polished before each experiment with diamond spray of 1 μm particle size. After polishing, the electrode was thoroughly rinsed with a jet of doubly distilled water. The clean electrode was then placed in the desired buffer electrolyte and various differential pulse voltammetry (DPV) measurements were recorded until the achievement of a steady-state baseline voltammogram. This procedure ensured reproducible experimental results. The measurement cell was immersed in a water-circulating bath (I-2400, IRMECO GmbH, Germany) in order to control the temperature. The experimental conditions for DPV were pulse amplitude of 50 mV and pulse width of 70 ms. For square wave voltammetry (SWV), the experimental conditions were 50 Hz frequency and 2 mV potential increments corresponding to an effective scan rate of 100 mV s⁻¹. Absorption spectra were recorded with a Shimadzu 1601 spectrophotometer, and for pH measurements an INOLAB pH meter (model no. pH 720) was used.

3. Results and discussion

3.1. Cyclic voltammetry

To obtain a general picture of the redox behavior of the compound, cyclic voltammetry (CV) measurements of HCAQ were recorded in media of different pH using a potential window of ±1.3 V. The voltammograms obtained for pH 3.0 and 7.0 can be seen in Fig. 1. The CV response under neutral conditions (pH = 7) depicts a sharp oxidation signal, 1a, at +0.69 V, followed by a broad peak, 2a, at +1.02 V. The absence of reduction peaks corresponding to 1a and 2a in the backward scan at pH 7.0 demonstrates the irreversible nature of these electrochemical oxidation processes. The compound shows two successive one-electron reductions in the negative potential domain of the GCE. The reduced species resulting in peaks 1c and 2c gets re-oxidized at nearly the same potential as evidenced by a single broad oxidation peak, 2a*. For confirming 2a* to be the combination of two overlapping peaks, a more sensitive electrochemical technique, DPV, was employed. The differential pulse voltammograms of the analyte shown in Fig. S1† unequivocally represent the splitting of peak 2a* into two sub-peaks at lower concentration. The anodic and cathodic peak potential differences witness the first and second reductions to follow reversible and quasi-reversible processes. Unlike the behavior of the analyte for pH 7.0, the backward CV scan for pH 3.0 shows an oxidation-dependent reduction peak at +0.33 V. The different voltammetric signatures in acidic and neutral media indicate the redox behavior of HCAQ to depend strongly on the pH of the medium.

Fig. 1 First-scan CVs of 1 mM HCAQ obtained at pH 3.0 and 7.0 at 100 mV s⁻¹.

An experiment based on the variation of peak current, I_p, with scan rate, ν, was used for the determination of diffusion coefficient and heterogeneous electron transfer rate constant. The scan rate effect of HCAQ was monitored for pH 7.0 at different temperatures. The voltammograms at 323 K shown in Fig. 2A demonstrate a linear increase in I_p with the square root of scan rate. Peak 1a shows a large potential shift with scan rate while the reduction peaks exhibit a slight shift in potential. These voltammetric features are characteristics of irreversible (peak 1a) and quasi-reversible redox processes (peak 2c). The values of diffusion coefficients at different temperatures were obtained from the plots of I_p (current intensity of peak 1a) versus square root of scan rate (Fig. 3) using the Randles–Sevcik equation:³⁴

where n is the number of electrons transferred, α_a is the anodic charge transfer coefficient, and A, D, C and ν are the area of the electrode in cm², diffusion coefficient in cm² s⁻¹, bulk concentration of the species in mol cm⁻³ and scan rate in V s⁻¹ respectively. The values of α_an using were determined for the irreversible process at different temperatures ranging from 308 to 328 K. An examination of Fig. 2B reveals that the effect of temperature on peak potential of the irreversible process is more pronounced as compared to the quasi-reversible process due to their respective kinetic- and thermodynamic-controlled natures. The values of D show an increasing trend with increasing temperature due to the probable decrease in viscosity of the media. The slope values of log I_p vs. log ν fall in the range of 0.50–0.53 at all temperatures, thus indicating the redox process to be limited by diffusion.³⁵

Fig. 2 Effect of (A) scan rate at 323 ± 1 K and (B) temperature at 80 mV s⁻¹ on the cyclic voltammetric behaviour of 0.7 mM HCAQ.

Fig. 3 Plots of peak current of (A) 1a and (B) 2c against the square root of scan rate using 0.7 mM solution of HCAQ.

Heterogeneous rate constant (k_s) is an important diagnostic criterion for predicting the nature of a redox process. Large values of k_s suggest that, following the application of an applied potential, equilibrium between the oxidized and reduced species gets re-established quickly. In contrast, small values of k_s indicate slow kinetics and a longer time required for equilibrium.^36,37 The values of k_s listed in Table 1 were calculated on the basis of peak 1a (Fig. 1) at different temperatures using eqn (2):^38,39

I_p = nFAk_sC (2)

where n, F, A and k_s are the number of electrons, Faraday's constant, area of the electrode and heterogeneous electron transfer rate constant.

Table 1 Diffusion coefficient and heterogeneous rate constant values of HCAQ evaluated on the basis of oxidation peak 1a at different temperatures

Peak	Temperature (K)	1/T × 10^−3 (K^−1)	αan	D/10^−7 (cm^2 s^−1)	ks/10^−4 (cm s^−1)	log ks
1a	308	3.25	0.99	0.16	2.01 (0.02)	3.698
	313	3.19	1.12	0.48	2.31 (0.02)	3.636
	318	3.14	1.25	1.17	3.66 (0.04)	3.437
	323	3.09	1.32	1.34	6.15 (0.07)	3.211
	328	3.05	1.40	4.04	7.27 (0.08)	3.139

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The heterogeneous electron transfer rate constant of HCAQ was evaluated from the peaks corresponding to the quasi-reversible redox process using the following form of the Nicholson equation:⁴⁰

where Ψ is the charge transfer parameter, D_o the diffusion coefficient of the oxidized species, υ the scan rate and a = nF/RT. The values of dimensionless parameter, Ψ, were determined from the difference of anodic and cathodic peak potential.⁴¹ The k_s values (Table 2) of a quasi-reversible redox process of HCAQ are greater than those of irreversible oxidation as expected.⁴² The values of ΔE_p at the experimental temperatures were converted to ΔE²⁹⁸_p by using eqn (4):⁴³

Table 2 Heterogeneous electron transfer rate constant values of HCAQ evaluated on the basis of quasi-reversible redox peaks at different temperatures

Temperature (K)	ks/10^−3 (cm s^−1)
313	1.21 (0.04)
318	7.60 (0.19)
323	7.79 (0.16)

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The k_s values (listed in Table 1) corresponding to the redox process labelled as 1a were used for the determination of thermodynamic parameters. ΔG^# was evaluated by eqn (5):⁴⁴

ΔG^# = 5778.8(5.096 − log k_s) (5)

With an increase in temperature, heterogeneous rate constant increases and ΔG^# decreases. The Arrhenius equation was used for the calculation of enthalpy and entropy changes of activation. E_a obtained from the slope of a plot of log k_s against 1/T (Fig. 4) allowed the evaluation of ΔH^# and ΔS^# according to eqn (6) and (7). An examination of Table 3 reveals an increase in ΔS^# and a slight decrease in ΔH^# values with increasing temperature.

ΔH^# = E_a − RT (6)

ΔG^# = ΔH^# − TΔS^# (7)

Fig. 4 Plot of log k_s as a function of 1/T.

Table 3 Thermodynamic parameters of HCAQ^a

Peak	Temperature (K)	ΔG^# (kJ mol^−1)	Ea (kJ mol^−1)	ΔH^# (kJ mol^−1)	ΔS^# (J K mol^−1)
a Although estimated errors in ΔG^# and Ea are ±2%, additional significant figures are given to allow more accurate calculation of ΔH^# and ΔS^#.
1a	308	51.00	62.66	60.09	29.54
	313	50.37		60.06	30.95
	318	49.31		60.01	33.67
	323	48.09		59.97	36.77
	328	47.51		59.93	37.87

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The positive ΔH^# value is responsible for the non-spontaneity of the oxidation reaction. The slight decrease in ΔH^# with increasing temperature shows the tendency of the electrode process towards spontaneity. The trend of ΔS^# shown in Table 3 indicates that, at high temperature, the oxidized product of HCAQ diffuses quickly from the electrode surface towards the solution. The trend is the same as predicted by transition state theory .⁴⁵ Thus, at higher temperature the increase in ΔS^# and decrease in ΔH^# are responsible for higher rate constant values.

3.2. Differential pulse voltammetry

The DPV technique is associated with the minimizing of charging current and consequent enhanced sensitivity.⁴⁶ Therefore, the redox behavior of HCAQ was investigated by DPV in media of different pH. The DPVs shown in Fig. 5A and S2† indicate a shift in peak potentials with changing pH. This behavior corresponds to the involvement of protons during the electron transfer processes. Moreover, the shift of E_pa towards lower potentials with increasing pH shows facile electron abstraction under basic conditions. The differential pulse voltammograms in the potential domain of 0 to +1.3 V show two oxidation peaks at low pH and three at high pH. DPV was also employed to study the reduction of HCAQ. The potential scan in the negative direction has a couple of peaks corresponding to the two-step reduction of HCAQ. The DPV scan in the potential domain of −1.3 to 0 V following scanning from 0 to −1.3 V without cleaning the electrode surface shows a single reverse oxidation peak. Peak 1a with a width at half peak height (W_1/2) of 54 mV indicates the involvement of two electrons in this oxidation step.⁴⁷ For the determination of the number of protons involved in the oxidation process, peak potential was plotted against pH.

The slope of the plot shown in Fig. 5B shows that the oxidation (corresponding to peak 1a) occurs with the involvement of two protons and two electrons. The intersection of the two linear segments of the E_p vs. pH plot indicates HCAQ to have an apparent acid dissociation constant value of 10.4. In contrast to peak 1a, peak 2a is broad indicating one electron loss during this oxidation step. The plot of peak potential versus pH shown in Fig. S3† demonstrates the removal of an electron to be accompanied by a proton. Similar behavior was also observed for peak 2a′.

Fig. 5 (A) DPVs of 0.7 mM HCAQ recorded at 5 mV s⁻¹ in 50% ethanol and 50% BR buffer of pH 3–11. (B) Plot of E_p versus pH using DPV data of peak 1a.

3.3. Square wave voltammetry

Square wave voltammetry (SWV), being one of the most sensitive and fast electrochemical techniques,⁴⁸ was employed to determine the limit of detection (LOD) and limit of quantification (LOQ) of HCAQ. From the calibration curves (Fig. S4A†), LOD and LOQ with values of 0.08 and 0.26 mmol L⁻¹ were determined according to the previously reported eqn (8) and (9):⁴⁹where S is the standard deviation of the intercept and m the slope of the plot of peak current versus concentration shown in Fig. S4B.†

3.4. Electronic absorption spectroscopy

UV-visible spectroscopy was carried out for the purpose of photometric characterization and confirmation of the apparent acid dissociation constant obtained from electrochemical techniques. Simple anthracenedione has been reported to give four electronic absorption signals.⁵⁰ The signal at 207 nm is attributed to n–σ* transition of the carbonyl group while the others are assigned to π–π* transition. The signal at 252 nm is allocated to π–π* transition of the benzenoid system and those at 272 and 326 nm are assigned to π–π* transition of the quinonoid system. The assignment of signals is made on the basis of comparison with the UV spectrum of anthracene.⁵⁰ The UV-visible spectra of HCAQ show five signals (Fig. 6A). The signal at 207 nm (n–σ* transition of carbonyl group) is absent in the spectrum of HCAQ due to blocking of the lone pair on C=O because of possible hydrogen bonding between OH at 1 and 4 positions. The signals at 232 and 252 nm are assigned to π–π* transitions of the benzenoid system while that at 287 nm is assigned to π–π* transition of the quinonoid system.^50,51 The splitting of the benzenoid band may be due to the existence of mesomeric structures or conjugation breakage. The broad band (at 425–550 nm) lacking in the absorption spectrum of simple anthraquinone⁵² is present in the spectrum of HCAQ. As the hydroxyl group is electron donating and gives an absorption band in the visible region as a result of a charge transfer (CT) mechanism,^51,53 so the spectral band displacements are related to the increase in length of the chromophore by additional ring formation due to intermolecular hydrogen bonding.^53,54

Fig. 6 (A) UV-visible spectra of different concentrations of HCAQ obtained at pH 7. (B) Absorption spectra of 16 μM HCAQ recorded at pH 3–11.

The absorption spectra of HCAQ in media of different pH are shown in Fig. 6B. Observation of the spectra reveals that the absorption band due to CT mechanism is strongly affected by the pH as compared to other peaks. The band corresponding to CT mechanism of the orange-colored solution appears in the range of 470–500 nm at pH < 9 and gets shifted to 552–590 nm at pH ≥ 9 due to deprotonation of HCAQ. The drastic color variation (see Fig. 7) of HCAQ from pH 10 to 11 unequivocally indicates that HCAQ ionizes at pH ∼ 10. The isosbestic point at 299 nm indicates that HCAQ and its mesomeric form exist in equilibrium with the same value of molar extinction coefficient. The presence of OH groups at positions 1 and 4 is responsible for the mesomeric form of HCAQ. Another isosbestic point at 516 nm represents HCAQ and its deprotonated form existing in equilibrium and absorbing the same amount of light at this wavelength. Isosbestic point typically indicates the presence of only two species.^55–57 The occurrence of isosbestic points for multicomponent solutions is almost impossible because the likelihood of identical absorbance of three or more compounds at any wavelength is negligibly small.⁵⁵

Fig. 7 Color variation of 16 μM HCAQ at pH 3–12.

From the slopes of the plots of absorbance versus concentration, the molar extinction coefficients with values shown in Fig. 8A were calculated at the respective wavelengths. On the basis of the peaks at 475 and 554 nm, the apparent acid dissociation constant of HCAQ with a value of 10.1 was obtained from the plot of absorbance versus pH as shown in Fig. 8B. This apparent acid dissociation constant value is in close agreement with that obtained from DPV and the drastic color variation shown in Fig. 7.

Fig. 8 (A) Plots of absorbance versus concentration using UV-visible spectroscopic data obtained at pH 7. (B) Absorbance of 16 μM μM HCAQ as a function of pH.

3.5. Redox mechanism

The oxidation of HCAQ exhibited two peaks in acidic and three in basic media (see Fig. 5). The oxidation corresponding to peak 1a was found to occur by the transfer of two electrons as determined from the average W_1/2 value of 54 mV. The E_pa vs. pH plot indicated the oxidation to occur with the involvement of two protons. On the basis of the results obtained from DPV and SWV it is proposed that the hydroxyl groups of HCAQ, after the loss of two electrons and two protons, presumably form oxygen radicals³⁸ that may lead to conversion to 2-(3-hydroxy-3-(trichloromethyl)pentyl)-8-methoxyanthracene-1,4,9,10(4aH,9aH)-tetraone⁵⁸ as shown in Scheme 1.

Scheme 1 Proposed oxidation mechanism of HCAQ corresponding to peak 1a.

Fig. 5 shows that peak 2a is pH dependent in acidic conditions while independent of pH in basic media and thus the mechanistic pathway gets switched from an EC to a CE mechanism (see Schemes 2A and B). The average W_1/2 value and slope of E_pa vs. pH plot of peak 2a show a one-electron and one-proton oxidation process. This peak is assigned to the hydroxyl group of the pentyl substituent because the trichloromethyl is an electron-withdrawing group, and therefore this hydroxyl group is oxidized at high potential. The DPVs in basic media show the appearance of a third pH-dependent peak 2a′. The half peak potential value and shifting of peak position with pH show the involvement of one electron and one proton in this process. On the basis of these results, a CEC mechanism is proposed as shown in Scheme 3. The results of peak width at half peak current and E_pc versus pH plots revealed the two-step reduction of HCAQ to occur with the involvement of one electron and one proton in each step. The mechanism proposed is shown in Scheme 4.

Scheme 2 (A) Proposed mechanism corresponding to peak 2a showing proton-coupled electron loss in acidic conditions. (B) Suggested CE (chemical reaction followed by electron transfer reaction) type oxidation mechanism of HCAQ corresponding to peak 2a in neutral and alkaline conditions.

Scheme 3 Proposed oxidation mechanism of HCAQ corresponding to peak 2a′ in neutral and alkaline conditions.

Scheme 4 Proposed redox mechanism of HCAQ corresponding to peaks appearing in the negative potential window of GCE.

4. Conclusion

The compound HCAQ was found to oxidize in a pH-dependent irreversible manner. The irreversibility of oxidation was confirmed by the absence of a corresponding reduction peak in the cyclic voltammetric response and the value of rate constant. The effect of temperature was studied for the evaluation of various kinetic and thermodynamic parameters of the compound. The temperature-dependent peak shift was found more pronounced for the irreversible process as compared to the reversible process. Thermodynamic parameters revealed the endothermic nature of the redox processes. Increase in electron transfer rate constant showed the trend of redox process towards reversibility at higher temperatures. The positive enthalpy change showed the redox behavior to be of an endothermic nature. Decreased values of Gibbs free energy with increase in temperature indicated that the redox processes become facile at elevated temperature. The pH-dependent results of three electroanalytical techniques helped in the proposal of a redox mechanism of the compound. The electronic absorption spectroscopy results showed the charge transfer mechanism to depend strongly on the pH of the medium. The values of apparent acid dissociation constant determined by electronic absorption spectroscopy, DPV and drastic change in solution color were found to be in very good agreement.

Acknowledgements

The authors gratefully acknowledge the financial support of Higher Education Commission of Pakistan through project number 3070, Quaid-i-Azam University, the University of Toronto Scarborough, NSERC and Deanship of Scientific Research at King Saud University through the research group project number RGP-VPP-345.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra04462b

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