Article pubs.acs.org/JPCB

Contrasting Voltammetric Behavior of Different Forms of Vitamin A in Aprotic Organic Solvents Ying Shan Tan, Dejan Urbančok, and Richard D. Webster* Division of Chemistry and Biological Chemistry, School of Physical and Mathematical Sciences, Nanyang Technological University, Singapore 637371 S Supporting Information *

ABSTRACT: Six of the major vitamers and provitamins comprising vitamin A (β-carotene, retinoic acid, retinol, retinyl palmitate, retinyl acetate, and retinal) were examined using voltammetric and controlled potential electrolysis techniques in the aprotic organic solvents acetonitrile and dichloromethane at glassy carbon and platinum electrodes. All of the compounds underwent oxidation and reduction processes and displayed a number of similarities and differences in terms of the number of redox processes and chemical reversibility of the voltammetric responses. The electrochemical properties of the compounds were strongly influenced by the functional groups on the unsaturated phytyl chains (carboxylic acid, alcohol, ester, or aldehyde groups), and not only on the fully conjugated hydrocarbon unit which is common to all forms of vitamin A. The compounds were reduced at potentials between approximately −1.7 and −2.6 vs (Fc/Fc+)/V (Fc = ferrocene) and oxidized at potentials between approximately +0.2 and +0.7 vs (Fc/Fc+)/V. The average number of electrons transferred per molecule under long time scale electrolysis experiments were found to vary between 0.4 and 4 electrons depending on the exact molecular structure and experimental conditions.

1. INTRODUCTION Vitamin A is composed of a number of different forms that all contain a 1,3,3-trimethylcyclohexene moiety but with varying oxygen containing functional groups attached to the conjugated phytyl chain (Scheme 1). Being a liposoluble compound, vitamin A is naturally absorbed and stored in mammalian fat cells and this is where many of the individual biochemical reactions of interest occur, some involving oxidation and reduction processes. In this paper, the aprotic organic solvents acetonitrile (CH3CN) and dichloromethane (CH2Cl2) were employed to study the redox reactions of the different forms of vitamin A in an environment that has some similarities to the lipophilic environment where they reside.1,2 One common association between biological activity and electrochemical reactions is that they involve electron transfer or oxidation and reduction reactions resulting in conversions between the different species.3 A number of studies have suggested the carotenoids convert to retinoids via oxidative enzymatic biological cleavage4−6 whereas other studies have concluded that they do not metabolize to one another.7−9 The existing studies on the voltammetric behavior of vitamin A have been performed on the individual vitamers in isolation and under different experimental conditions with varying solvents, electrolytes, and electrode materials.10−22 This study compares the behavior of all compounds under the same experimental conditions, which allows a better understanding of their redox properties and an assessment of their likely redox active sites. The compounds were examined using a combination of cyclic voltammetry and controlled potential electrolysis at platinum and glassy carbon electrode surfaces, in © 2014 American Chemical Society

the aprotic solvents acetonitrile and dichloromethane and at temperatures between −20 and +20 °C. The pro-vitamin A carotenoid, β-carotene,10,14,23−37 was also examined under the same conditions as the other vitamers to determine how the absence of an oxygen containing functional group affected the voltammetric behavior.

2. EXPERIMENTAL SECTION 2.1. Chemicals. β-Carotene (>97%) was obtained from TCI Japan, all-trans retinol (95%) was obtained from Acros Organics, all-trans retinal (≥98%) was obtained from SigmaAldrich, retinyl palmitate (analytical standard) was obtained from Supelco Analytical, retinoic acid (98%) was obtained from Alfa Aesar, and retinyl acetate (≥90%) was obtained from Sigma-Aldrich, and they were all stored in the dark at 277 K. Bu4NPF6 was prepared by reacting equimolar amounts of aqueous solutions of Bu4NOH (40%, Alfa Aesar) and HPF6 (65%, Fluka), washing the precipitate with hot water, and recrystallizing three times from hot ethanol.38 The electrolyte was then dried under vacuum at 433 K for 6 h and stored under vacuum. Molecular sieves of the form 1/16 inch rods with 3 Å pore size Zeolite (CAS Registry No. 308080-99-1) were obtained from Fluka. CH3CN was either high performance liquid chromatography or analytical grade obtained from (RCI Labscan) and (Tedia), respectively, and used directly from the bottles after drying over 3 Å molecular sieves using a previously Received: June 3, 2014 Revised: June 29, 2014 Published: July 1, 2014 8591

dx.doi.org/10.1021/jp505456q | J. Phys. Chem. B 2014, 118, 8591−8600

The Journal of Physical Chemistry B

Article

where Q = charge (C), n = number of electrons, (e−/mol), F = Faraday’s constant (96485 C/e−), and N = number of moles (mol).

Scheme 1. Structures of Vitamin A Derivatives: (1) all-transβ-Carotene; (2) Retinoic Acid; (3) all-trans-Retinol; (4) Retinyl Palmitate; (5) all-trans-Retinyl Acetate; (6) all-transRetinal

3. RESULTS AND DISCUSSION 3.1. Reductive Cyclic Voltammetry in CH3CN and CH2Cl2 Using GC and Pt Working Electrodes. Cyclic voltammograms performed with a 1 mm diameter GC electrode in CH3CN at a scan rate of 0.1 V s−1 for retinoic acid (2), retinol (3), retinyl palmitate (4), retinyl acetate (5) and retinal (6) showed two reductive peaks, whereas the reductive processes observed for retinol (3) had surprisingly very small peak currents (solid lines in Figure 1).

described method.39 Analytical grade CH2Cl2 was obtained from (Tedia) and used directly from the bottles (after drying over 3 Å molecular sieves) unless otherwise stated. Purified water, with a resistivity ≥18 MΩ cm, was obtained from an ELGA Purelab Option-Q system. The trace water content of the CH3CN in the electrochemical cells typically varied between 10−30 mM for the voltammetry experiments and 50−80 mM for the electrolysis experiments, measured by Karl Fisher coulometric titrations.39 For CH2Cl2, the trace water content did not exceed 20 mM. 2.2. Electrochemical Procedures. Cyclic voltammetry (CV) experiments were conducted with a computer-controlled Metrohm Autolab PGSTAT302N potentiostat. Working electrodes (WE) of both 1 mm diameter glassy carbon (GC) and platinum (Pt) disk (Cypress Systems) were used in conjunction with a Pt wire auxiliary electrode (Metrohm) and an Ag wire miniature reference electrode (Cypress Systems) connected to the test solution via a salt bridge containing 0.5 M Bu4NPF6 in CH3CN. Accurate potentials were obtained using ferrocene as an internal standard. Variable-temperature experiments were performed with the temperature controlled with a Julabo FP89-HL ultralow refrigerated circulator. Controlled potential electrolysis with coulometry was performed in a two-compartment cell using Pt mesh baskets as the working and auxiliary electrodes and with the Ag wire miniature reference electrode placed in the working electrode compartment solution.40 The volumes of the working and auxiliary electrode compartments were approximately 20 mL, and both solutions were continually purged with a stream of Ar gas to simultaneously stir the solutions and maintain an inert atmosphere. The number of electrons transferred during the electrolysis reactions were calculated from eq 1, n = Q /NF

Figure 1. CVs of 1 × 10−3 M retinoic acid (2), retinol (3), retinyl palmitate (4), retinyl acetate (5), and retinal (6) (from top to bottom) in CH3CN containing 0.2 M Bu4NPF6 at 295 (±2) K at a 1 mm diameter GC working electrode at a scan rate of 0.1 V s−1.

Reductive potential scans for compounds 2, 4, 5, and 6 were conducted up to the first reduction peak (dashed lines in Figure 1) to examine the chemical reversibility of the first reduction process. All first reduction processes were chemically irreversible up to a scan rate of 20 V s−1 (no reverse peaks were detected when the scan direction was reversed) except for the first reduction peak of retinal. The interesting reductive behavior of retinal has been studied more thoroughly and it has been shown to undergo a series of hydrogen-bonding and dimerization reactions.15−17,22 All of the compounds displayed purely diffusion controlled behavior shown by the linear relationship of plots of peak current (ipred) versus the square root of the scan rate (ν1/2), with no interference from adsorption effects for short time scale CV experiments. Retinal had the lowest reduction peak potential (Epred = −1.78 V vs Fc/Fc+) followed by retinoic acid, retinyl palmitate, and retinyl acetate, although it is difficult to accurately obtain the formal potentials because of the chemical irreversibility of the reduction responses (thus only the peak potential is given).

(1) 8592

dx.doi.org/10.1021/jp505456q | J. Phys. Chem. B 2014, 118, 8591−8600

The Journal of Physical Chemistry B

Article

The peak currents obtained during the reduction of retinol were very small compared to the rest of the retinoids tested, which seems unusual because retinol is believed to be one of the most important forms of vitamin A.18,21,41 The reason for the small peak currents for retinol does not arise from a much smaller diffusion coefficient, because it is of similar size to the other compounds. Instead, the difference may arise from chemical instability in solution. Different sources of retinol were examined, but similar electrochemical responses were obtained in each case, with retinol displaying abnormally small peak currents compared to the other compounds. Compounds 2, 4, 5, and 6 have very different responses in terms of the separation between the first and second reduction peak potentials. Compound 2 registers the largest peak potential separation between the first reduction process and the second, whereas 6 displays the smallest potential separation.22 However, the two reduction peaks do not represent the same processes for each compound. For retinal (6), the two electron transfer steps are associated with the first one-electron (eq 2) and then second one-electron transfer steps (eq 3) to form the anion radical and then the dianion, respectively.22 However, the other compounds display a chemically irreversible first reduction process at all scan rates (up to 20 V s−1); thus, the second reduction process does not simply involve the further one-electron reduction of the anion radical to form the dianion but instead is likely associated with the further reduction of a reaction product of the first electron transfer. R + e− ⇌ R•− •−

R



2−

+e ⇌R

Figure 2. CVs of 1 × 10−3 M β-carotene (1), retinoic acid (2), retinol (3), retinyl palmitate (4), retinyl acetate (5), and retinal (6) (from top to bottom) in CH2Cl2 containing 0.2 M Bu4NPF6 at 295 (±2) K at a 1 mm diameter GC working electrode at a scan rate of 0.1 V s−1.

(2)

When switching the working electrode to Pt, only compounds 2 and 6 display reduction responses in CH2Cl2 (Figure S2, Supporting Information). This is very similar to the result obtained for the Pt electrode when experiments were performed in CH3CN. The peak currents obtained for the reduction processes for equivalent concentrations of all compounds were higher in CH3CN compared to CH2Cl2 and higher on a GC electrode compared to Pt. A summary of the peak potentials for the reduction and oxidation processes is given in Table 1. 3.2. Oxidative Cyclic Voltammetry in CH3CN and CH2Cl2 Using GC and Pt Working Electrodes. Electrochemical oxidation of compounds 1−6 yielded a single oxidation peak in both solvents regardless of the electrode surface used (Figure 3 and Figures S3−S5 in the Supporting Information section). β-Carotene is the only compound that

(3)

β-carotene, which is one of the most abundant forms of the carotenoids in the human body,42 is insoluble in CH3CN;37 hence another set of experiments were performed to enable a comparison of the carotenoid with the retinoids under the same experimental conditions. The experiments were conducted using CH2Cl2 as the solvent. When the experiments were performed in CH2Cl2 using a 1 mm GC working electrode, compounds 2 and 6 showed two reductive peaks, whereas only one reductive peak was observed for 1 and 5. The other compounds (3 and 4) do not have any detectable reduction response within the available potential window (Figure 2). The lack of an observable peak in CH2Cl2 for 4 is likely due to its much lower current values (compared to the closely related 5) being masked by the onset of solvent reduction. Using a Pt electrode in CH3CN resulted in very different electrochemical responses for the retinoids (compared to on GC). Of all the retinoids, only 2 and 6 were found to undergo a detectable reduction reaction on a Pt electrode (Figure S1, Supporting Information). Compound 2 appears to be significantly more easily reduced than 6 when the working electrode is changed from GC to Pt. Furthermore, on Pt, 2 displays one apparently chemically reversible reduction process (but with a wide separation between the forward and reverse peaks) at −1.35 vs (Fc/Fc+)/V, instead of two reduction peaks as observed on GC. The electrochemical response of 6 on Pt is very similar to that observed on GC where two reduction processes are observed, although the first reduction process shows less chemical reversibility on Pt. All other retinoids did not show a reduction response within the potential window that is available with the Pt electrode (before hydrogen evolution occurs due to the reduction of trace water).

Table 1. Reduction and Oxidation Peak Potentials of 1 × 10−3 M Vitamin A Compounds Obtained in CH3CN Using a 1 mm Diameter GC Working Electrode at 295 (± 2) K with a Scan Rate of 0.1 V s−1 a compound (1) (2) (3) (4) (5) (6)

β-caroteneb retinoic acid retinol retinyl palmitate retinyl acetate retinal

first reduction peak potential vs (Fc/Fc+)/V

first oxidation peak potential vs (Fc/Fc+)/V

−2.12 −1.90 −2.57 −2.20 −2.28 −1.78

+0.16 +0.59 +0.47 +0.51 +0.50 +0.68

Diffusion coefficient values were estimated to be 2 × 10−6 cm2 s−1 for compound 1, 1.3 × 10−5 cm2 s−1 for compounds 2, 3, 5, and 6, and 3 × 10−6 cm2 s−1 for compound 4 at 295 K (see text). bβ-Carotene was examined in CH2Cl2. a

8593

dx.doi.org/10.1021/jp505456q | J. Phys. Chem. B 2014, 118, 8591−8600

The Journal of Physical Chemistry B

Article

final products are numerous and difficult to isolate.15−17,22 Molecular orbital calculations are useful in predating the exact location of the LUMO (for reduction) and HOMO (for oxidation). However, these calculations are not trivial for these compounds due to structural complexities in the relatively long chain polyenes making the geometry optimizations critical. Therefore, a careful assessment of the choice of theoretical models that can be employed to optimize the ground state geometries is first required.45−47 It can be observed that compounds 4 and 5, which contain the aliphatic ester functional group are both reduced at close to the same potential (and oxidized at the same potential) (Figures 1 and 3), suggesting that the ester group is primarily important to the voltammetric response. The increase in reductive and oxidative peak currents in going from compound 4 to compound 5 can be rationalized by the smaller size of 5 resulting in it having a larger diffusion coefficient. The apparent chemically reversible oxidation response that is observed for retinoic acid (2) on a Pt electrode (but not on GC) (Figures S1 and S2, Supporting Information) is similar to what has been observed during the oxidation of other carboxylic acids where the process is associated with the direct discharge of the carboxylic acid at the platinum electrode surface, forming hydrogen gas according to eqs 4−6.48−50

Figure 3. CVs of 1 × 10−3 M β-carotene (1), retinoic acid (2), retinol (3), retinyl palmitate (4), retinyl acetate (5), and retinal (6) (from top to bottom) in CH2Cl2 containing 0.2 M Bu4NPF6 at 295 (±2) K at a 1 mm diameter GC working electrode at a scan rate of 0.1 V s−1.

shows a chemically reversible oxidation peak. Compound 1 also registered the lowest oxidation potential and differed from the other compounds in that it had no heteroatom containing functional group terminating the polyene chain. Compounds 2 and 6 have higher oxidation potentials than the other compounds. For all the different conditions tested, 3 produced an unexpectedly small current (oxidative and reductive), which suggests that it does not undergo electrochemical oxidation smoothly, in contrast to some studies that had studied its mechanism in isolation of the other compounds.12,13,21,43 Considering that all compounds were studied with the same concentration of 1 mM and prepared via dilution methods to minimize error in the concentration of each compound, the small peak current suggests 3 behaves differently than the other compounds, with two possible explanations. One possibility is that the oxidation/reduction of 3 involves substantially fewer electrons than the other compounds. This could occur if the oxidized/reduced forms reacted quickly with the starting material to generate species that are not electroactive. Another more likely explanation is that 3 is decomposing or aggregating in solution and so only a small fraction of the starting material is detected in the CVs.44 3.3. Molecular Site of Oxidation and Reduction. A comparison of the voltammetric data in Figures 1−3 (and Figures S1−S5 in the Supporting Information) allow some conclusions to be drawn regarding the key redox active sites within the molecules. The substantial difference in oxidation/ reduction peak potentials observed between most of the compounds indicates that the oxygen containing functional groups (carboxylate acid, alcohol, ester and aldehyde) are involved in the oxidation and reduction reactions, and not only the fully conjugated hydrocarbon unit. This is supported by previous experiments that have shown that reduction of retinal (6) results in the loss of the aldehyde functional group, likely through an initial reversible dimerization reaction, although the

RCOOH + Pt + e− ⇌ RCOO− + Pt−H(ads)

(4)

2Pt−H(ads) → H 2 + 2Pt

(5)

RCOOH + Pt−H(ads) + e− → RCOO− + H 2 + Pt

(6)

The oxidative voltammetric behavior of β-carotene, which has no oxygen containing functional groups, differs substantially from the other vitamins, as it displays a chemically reversible two-electron process at relatively slow scan rates and at a low potential (Er1/2 = +0.1 vs (Fc/Fc+)/V) (Figure 3).23−37 For βcarotene, the oxidation is known to occur in two one-electron steps but the first and second steps are so close together in potential that they cannot be voltammetrically distinguished (the second electron transfer step may even occur at a lower potential than the first).35 The ease of oxidation for the second electron transfer step is thought to arise because of extensive delocalization of the unpaired electron within the polyconjugated chain in the initially formed radical cation and solvation effects.35 The very low formal oxidation potential observed for β-carotene (compared to the other vitamins) means that it is feasible that it can undergo an oxidative cleavage reaction to form one of the other vitamers,4−6 although it is expected that the medium used for the oxidation would be critical in the product formation. Conversely, a reductive dimerization reaction of compounds 2 or 6 to re-form β-carotene is also at least feasible because the reduction potential of β-carotene is greater (than 2 and 6) so it would not undergo further reduction itself (although this possible reaction has not been reported). It is difficult to use CV to determine the number of electrons transferred in each voltammetric process because most of the compounds display chemically irreversible voltammetric behavior. Exceptions are the voltammetric oxidation of βcarotene (1), which occurs via two-electrons35,37 and the reduction of retinal (6), which occurs in two one-electron steps.15−17,22 Therefore, coulometry experiments were applied to accurately determine electron counts for the other compounds (section 3.4). 8594

dx.doi.org/10.1021/jp505456q | J. Phys. Chem. B 2014, 118, 8591−8600

The Journal of Physical Chemistry B

Article

Diffusion coefficient values are also difficult to obtain for those compounds where there are uncertainties in the number of electrons transferred on the short voltammetric time scale. The diffusion coefficient (D) for retinal (6), where one-electron is transferred in the first reduction wave, has previously been estimated to be 1.3 × 10−5 cm2 s−1,22 via application of the Randles−Sevcik equation (eq 7), where ip is the peak current, n is the number of electrons transferred, A is the electrode area, c is the concentration, and ν is the scan rate. Compounds 2, 3, and 5 are similar in size and structure to retinal and would thus be expected to have very similar diffusion coefficient values. Assuming that retinyl acetate and retinyl palmitate undergo oxidation/reduction by the same number of electrons, and by comparing the relative voltammetric peak heights of 4 and 5 under the same experimental conditions, allows the diffusion coefficient of retinyl palmitate (4) to be estimated to be 3 × 10−6 cm2 s−1. For β-carotene and using an n-value of 2 for the oxidation process, the D value obtained from eq 7 was calculated to be 2 × 10−6 cm2 s−1 (in CH2Cl2). i p = 2.686 × 105n3/2AcD1/2ν1/2

Figure 4. (a) CV of 1 × 10−3 M β-carotene (1) in CH2Cl2 containing 0.2 M Bu4NPF6 at 295 (±2) K at a 1 mm diameter GC WE at a scan rate of 0.1 V s−1. (b) CV of 1 × 10−3 M β-carotene (1) in CH2Cl2 containing 0.2 M Bu4NPF6 at 253 K at a 1 mm diameter Pt WE at a scan rate of 0.1 V s−1: (solid line) before and (dotted line) after the reductive transfer of two electrons per molecule in a controlled potential electrolysis cell.

(7)

3.4. Electrolysis and Coulometry. Controlled potential bulk electrolysis experiments with coulometry were conducted to measure the number of electrons that each of the oxidation and reduction processes involved over long times. From the CV compilation results discussed previously, the best experimental conditions in achieving the optimal oxidation and reduction responses was when using CH3CN, except for β-carotene (1) which was performed in CH2Cl2 due to solubility constraints. Most compounds displayed intense color changes during the electrolysis reactions, which are shown in the Supporting Information. 3.4.1. β-Carotene. Coulometry measurements made during the exhaustive electrolysis of 1 at room temperature indicated that the reduction involved the transfer of 2e− per molecule (Figure S6, Supporting Information). However, when conducting the reductive electrolysis at 253 K, it was possible to record up to the transfer of 4e− per molecule, which indicates that the temperature has an effect on the electrochemical reduction of 1.33,37 There has been some controversy as to how many electrons are involved in the reduction reaction of 1. Earlier studies by Takahashi and Tachi10,11 and Kuta and Ju51,52 suggested a 4e− reduction process, while Mairanovsky et al.,23 Valentin Pfund et al.,24 and Park14 suggested a 1e− process. These reported differences might have arisen due to the different experimental conditions that were used as well as the potential that was used for the electrolysis. It was previously stated that the first reduction consisted of a 1e− process because it was being compared to the peak height of a known oxidation process of another compound which was a 2e− process.23 However, in the experimental conditions used in this paper for 1, estimating the peak height from the oxidative current relative to reductive current at the same scan rate does not agree with the assumption of a 1e− reduction process, because the oxidation process is known to involve the transfer of two electrons and appears smaller than the reduction process (Figure 4a). However, it is possible that the reduction process that is quite broad involves closely overlapping individual electron transfer steps, and by variation of the experimental conditions, the individual steps may be better separated. For example, when the temperature was lowered to 253 K, the reduction wave was

considerably broadened and appeared to involve multiple processes (Figure 4b). Therefore, when the electrolysis was carried out at 253 K with the potential held at ∼−2.10 vs (Fc/ Fc+)/V, the reaction appeared to involve the transfer of just two-electrons. Further square-wave voltammetry (SWV) experiments were performed at several temperatures to see if the reduction wave could be differentiated into individual processes. It was apparent that the stepwise process is visible when the SWVs are conducted at 213 K and the peaks slowly merge up to one single response as the temperature increases to 295 (±2) K (Figure S7, Supporting Information). CV experiments performed on 1 showed that it undergoes a chemically reversible oxidation process in CH2Cl2 (Figure 3).25−37 Bulk controlled potential coulometry confirmed that the oxidation occurred via 2e− (Figure 5b). After an exhaustive electrolysis was performed at 253 K, CV scans were conducted to see if the two-electron oxidized product survived on the electrolysis time scale. The dashed line in Figure 5a shows evidence of some of the dication surviving (∼30%). The partial survival of the primary oxidized compound was mainly due to the lower temperature, which helped to lengthen its lifetime under electrolysis conditions compared to at room temperature where the dication decayed within a few seconds. New reaction products were also detected that could be oxidized at a higher potential at ∼+0.55 vs (Fc/Fc+)/V as well as being reduced at a lower peak potential of ∼+0.35 vs (Fc/Fc+)/V (Figure 5a). Experiments were conducted to determine if β-carotene was able to undergo oxidative conversion into any of the other vitamers, with compounds 2, 3, 5, and 6 considered as possible products. Because the starting material and potential products were available as standards in pure form, it was possible to use liquid chromatography−mass spectrometry (LC−MS) to test for the existence of possible reaction products. β-Carotene was exhaustively electrolyzed in a CH3CN:CH2Cl2 1:4 ratio solution containing 5% of methanol and the final electrolyzed solution analyzed by LC-MS. However, LC−MS analysis did not lead to the detection of any of the other vitamins, indicating 8595

dx.doi.org/10.1021/jp505456q | J. Phys. Chem. B 2014, 118, 8591−8600

The Journal of Physical Chemistry B

Article

electron reduction itself undergoes reduction in another multielectron process. The voltammetric oxidation process of retinoic acid (2) appears as a single chemically irreversible peak, which was found to be a 2e− process via coulometry conducted at 295 (±2) K in CH3CN. The CV of the reaction solution conducted after the bulk transfer of 2e− shows that the peak at +0.61 vs (Fc/Fc+)/V disappears while a new peak at slightly more positive potential appears. No other peaks were detected for oxidation products (Figure S9, Supporting Information). 3.4.3. Retinol. Electrolysis experiments on retinol (3) were not performed because of the very small oxidation and reduction peaks seen on both electrode surfaces during CV experiments. 3.4.4. Retinyl Palmitate. Exhaustive bulk reductive electrolysis of 4 resulted in the transfer of 2e− per molecule. The CV recorded at the completion of the electrolysis showed that the first reduction peak had completely disappeared while the second reduction peak remained the same size as prior to the electrolysis (Figure S12, Supporting Information). Overall, it appears that the long-term product of the reduction is the species that is detected at the second reduction process (during CV experiments on the starting material), similar to the result that was obtained for retinoic acid. Therefore, the reaction can either involve the two-electron reduction to form a dianion which then reacts quickly to form a new product (EEC process, where “E” represents each electron transfer and “C” represents a chemical step), or alternatively there is an initial electron transfer to form a radical anion that reacts quickly to form another compound that is further reduced (in an ECE or ECEC type process). Retinyl palmitate displays a chemically irreversible oxidation process with a relatively small peak current compared to the rest of the compounds tested (except for retinol). However, unlike retinol, the small peak current for 4 is mainly a result of it having a much lower diffusion coefficient due to it being larger than the other compounds. Exhaustive oxidative electrolysis of 4 involved the transfer of just 0.4e− per molecule (Figure 7). The low number of electrons transferred during the electrolysis suggests that the initially formed oxidized product is able to react with the starting material, thereby removing some of the starting material from the solution. CVs conducted after the electrolysis using a 1 mm GC electrode do not show any evidence of a new product which could be reduced or oxidized within the available potential window. 3.4.5. all-trans-Retinyl Acetate. Reductive electrolysis with the potential held slightly more negative than the first reduction peak of (5) resulted in the transfer of 2e− per molecule. A CV obtained at the end of the electrolysis showed that the peak associated with the first reduction process was no longer present (Figure S15, Supporting Information). The second reduction peak remained unchanged, except for a shoulder that appeared at a slightly more positive potential. A new oxidative peak was detected at a positive potential which presumably corresponds to the oxidation of the generated reduced products. The color of the solution during the electrolysis reaction changed from initially colorless to orange at the end of a 2e− bulk electrolysis process (Figure S16, Supporting Information). This color is very similar to the color obtained during the reductive electrolysis of retinyl palmitate (4) (Figure S13, Supporting Information).

Figure 5. (a) CV of 1 × 10−3 M β-carotene (1) in CH2Cl2 containing 0.2 M Bu4NPF6 at 253 K at a 1 mm diameter GC WE at a scan rate of 0.1 V s−1: (solid line) before and (dotted line) after the transfer of two electrons per molecule in a controlled potential electrolysis cell. (b) Current/coulometry versus time data obtained during the oxidative electrolysis of β-carotene at +0.30 vs (Fc/Fc+)/V.

that electrochemical oxidation of β-carotene in the aforementioned medium does not lead to the direct synthesis of the oxygenated vitamers. 3.4.2. Retinoic Acid. The electrolysis potential was held at −1.90 vs (Fc/Fc+)/V to test the number of electrons transferred during the first broad reduction process, and it was found to be a 1e− step (Figure 6b). A CV conducted after the electrolysis showed that the second reduction peak had increased from a peak current of 3.21 μA at the beginning of electrolysis to 9.32 μA at the end of the 1e− reduction process (Figure 6a). Therefore, it is clear that the product of the one-

Figure 6. (a) CV of 2 × 10−3 M retinoic acid (2) in CH3CN containing 0.2 M Bu4NPF6 at 283 K at a 1 mm diameter GC WE at a scan rate of 0.1 V s−1: (solid line) before and (dotted line) after the transfer of one electron per molecule in a controlled potential electrolysis cell. (b) Current/coulometry versus time data obtained during the reductive electrolysis of retinoic acid at −1.90 vs (Fc/Fc+)/ V. 8596

dx.doi.org/10.1021/jp505456q | J. Phys. Chem. B 2014, 118, 8591−8600

The Journal of Physical Chemistry B

Article

electrolysis was completed did not show any new product which could be oxidized or reduced within the potential range scanned. Lowering the temperature to 253 K did not appear to help in stabilizing any of the intermediate species that might have been formed, as there are still no new processes detected via CV after the electrolysis procedure was complete (Figure S18, Supporting Information) and also resulted in the transfer of 0.5e− per molecule. 3.4.6. all-trans-Retinal. Reductive electrolysis was performed at 253 and 293 K in CH3CN for 6 and a total of 1e− was achieved at the end of the electrolysis at −1.84 vs (Fc/ Fc+)/V at both temperatures (Figure 9). In this instance, both

Figure 7. (a) CV of 1 × 10−3 M retinyl palmitate (4) in CH3CN containing 0.2 M Bu4NPF6 at 295 (±2) K at a 1 mm diameter GC WE at a scan rate of 0.1 V s−1: (solid line) before and (dotted line) after the transfer of 0.4 electrons per molecule in a controlled potential electrolysis cell. (b) Current/coulometry versus time data obtained during the oxidative electrolysis of retinyl palmitate at +0.54 vs (Fc/ Fc+)/V.

Oxidative electrolysis of retinyl acetate was conducted by holding the potential at +0.53 vs (Fc/Fc+)/V, which led to the transfer of 0.5e− for the exhaustive oxidation in CH3CN. After 0.5e− had been passed, the oxidative current decreased to a very low level, suggesting the completion of the electrolysis (Figure 8). The low number of electrons transferred is very similar to that obtained for compound 4, which also contains the ester functionality. Therefore, it appears that for compounds 4 and 5, the initially oxidized products react with the starting material, resulting in a lower count of electrons as the starting material is depleted from further reaction. The CV performed after the

Figure 9. (a) CV of 2 × 10−3 M retinal (6) in CH3CN containing 0.2 M Bu4NPF6 at 253 K at a 1 mm diameter GC WE at a scan rate of 0.1 V s−1: (solid line) before and (dotted line) after the transfer of one electron per molecule in a controlled potential electrolysis cell. (b) Current/coulometry versus time data obtained during the oxidative electrolysis of retinal at −1.84 vs (Fc/Fc+)/V.

the first and second reduction peaks disappeared when a CV was run at the end of the electrolysis and a new oxidized peak appeared at −0.94 vs (Fc/Fc+)/V. The new oxidation peak has been proposed to be from a dimerized product from the radical anion generated after the first reduction, although the species does not survive for long enough time to be fully characterized.22 Oxidative electrolysis of retinal indicated that the reaction involved the transfer of 4e− (Figure S21, Supporting Information), which is equivalent to the number of electrons that was reported earlier by Park14 who conducted the experiment using THF as the solvent. Table 2 gives a summary of the number of electrons involved in the oxidation and reduction processes for both carotenoid and retinoids at various temperatures. The identification of the long-term products of the electrolysis is difficult due to the low concentration of electrolyzed compounds (reactions were conducted at ≤2 mM of analyte), difficulty in separating the much higher concentration of supporting electrolyte, and light sensitivity of the compounds. Nevertheless, the electrolysis experiments have allowed accurate electron counts to be determined for both oxidation and reduction processes and demonstrated that the temperature and exact applied potential also have a strong

Figure 8. (a) CV of 1 × 10−3 M retinyl acetate (5) in CH3CN containing 0.2 M Bu4NPF6 at 295 (±2) K at a 1 mm diameter GC WE at a scan rate of 0.1 V s−1: (solid line) before and (dotted line) after the transfer of 0.5 electrons per molecule in a controlled potential electrolysis cell. (b) Current/coulometry versus time data obtained during the oxidative electrolysis of retinyl acetate at +0.53 vs (Fc/ Fc+)/V. 8597

dx.doi.org/10.1021/jp505456q | J. Phys. Chem. B 2014, 118, 8591−8600

The Journal of Physical Chemistry B

Article

Table 2. Number of Electrons Transferred and Potential Applied during Oxidative and Reductive Controlled Potential Bulk Electrolysis of Compounds 2−6 in CH3CN and Compound 1 in CH2Cl2 reduction compound

applied potential/vs (Fc/Fc+)/V

(1) β-carotene

(2) (3) (4) (5) (6) a

retinoic acid retinol retinyl palmitate retinyl acetate retinal

−2.14 −2.10 −2.60 −1.90 NDa −2.24 −2.30 −1.84

oxidation

no. of electron(s) (temperature) 2e− (295 2e− (253 4e− (253 1e− (283 NDa 2e− (295 2e− (295 1e− (253

± 2 K) K) K) K) ± 2 K) ± 2 K) K)

applied potential/vs (Fc/Fc+)/V

no. of electron(s) (temperature)

+0.30

2e− (253 K)

+0.61 NDa +0.54 +0.53 +0.62

2e− (295 ± 2 K) NDa 0.4e− (295 ± 2 K) 0.5e− (295 ± 2 K) 4e− (295 ± 2 K)

ND = not determined.

(0.4−0.5), which again suggests a similar oxidative reaction pathway. Retinol (3), which has a much shorter chain length then retinyl palmitate and differs from retinal by the terminating group, shows much smaller oxidative and reductive current responses from all of the other compounds during CV measurements. It appears to be the most sensitive to the external environment, which causes it to lose its electroactivity and hence only display very small oxidative and reductive peak currents. Therefore, experiments conducted in isolation on retinol need to consider the possibility that the compound is spontaneously undergoing transformations in solution.

effect on the number of electrons transferred and so affect the electrochemical pathway for some of the compounds.

4. CONCLUSION A series of voltammetric experiments have shown similarities and differences in the electrochemical behavior of the different forms of vitamin A. Differences in the voltammetric responses were also detected when experiments were conducted at GC and Pt electrodes, in CH3CN and CH2Cl2 solvents and at variable temperatures between 253 and 295 K. The results indicated that retinal is the only vitamer that displays a chemically reversible reductive process when electrochemical experiments were conducted in CH3CN using GC as the working electrode. Although the other compounds are all able to be reduced, they display chemically irreversible voltammetric processes up to the maximum scan rate measured of 20 V s−1. The oxidation and reduction processes occurred at the sites of the molecules encompassing the oxygen containing functional groups, except for β-carotene, which was oxidized at a much lower potential than the other compounds with the reaction occurring within the polyene chain. β-Carotene (1) was also the only vitamer that displayed a chemically reversible oxidation process. The oxidation of 1 occurs via two electrons and at low temperatures (253 K) the dication partially survives during controlled potential electrolysis experiments and can be detected in the bulk solution. The voltammetric results indicate that it is difficult to study these vitamers using a Pt working electrode because most of the compounds do not give a clear response within the available potential window (due to the hydrogen evolution reaction on Pt). For example, when studying β-carotene, the reduction process on a Pt electrode displays the overlapping peaks of the analyte with the background. On Pt, retinoic acid does not display its second reduction process that is otherwise detectable on a GC electrode. Retinyl palmitate (4) and retinyl acetate (5) show interesting similarities in their voltammetric behavior. Compounds 4 and 5 are both esters but the structure of retinyl palmitate differs from retinyl acetate by the presence of a long saturated chain. The voltammetric behavior of both compounds is very similar except for retinyl acetate always registering a larger peak current than retinyl palmitate due to differing diffusion coefficients. The number of electrons involved in the reduction process for both compounds was close to two, and the colors of the solutions after the bulk electrolysis were the same. It is likely that both species undergo very similar reductive reaction pathways. The bulk oxidation of both compounds led to the exhaustive transfer of a relative low number of electrons per molecule



ASSOCIATED CONTENT

S Supporting Information *

Additional cyclic voltammograms and results from controlled potential electrolysis experiments. This material is available free of charge via the Internet at http://pubs.acs.org.



AUTHOR INFORMATION

Corresponding Author

*R. D. Webster. E-mail: [email protected] Telephone: +65 6316 8793. Notes

The authors declare no competing financial interest.

■ ■

ACKNOWLEDGMENTS This work was supported by an A*Star SERC Public Sector Funding (PSF) Grant (112 120 2006). REFERENCES

(1) Webster, R. D. Voltammetry of the Liposoluble Vitamins (A, D, E and K) in Organic solvents. Chem. Rev. 2012, 12, 188−200. (2) de Abreu, F. C.; Ferraz, P. A. D; Goulart, M. O. F. Some Applications of Electrochemistry in Biomedical Chemistry. Emphasis on the Correlation of Electrochemical and Bioactive Properties. J. Braz. Chem. Soc. 2002, 13, 19−35. (3) Dryhurst, G. Electrochemistry of Biological Molecules; Academic Press: New York, 1977; pp 1−5. (4) Mernitz, H.; Wang, X.-D. The Bioconversion of Carotenoids into Vitamin A: Implication for Cancer Prevention, in: Loessing, I. T. (Ed.), Vitamin A: New Research; Nova Biomedical Books: New York, 2007; pp 39−57. (5) Mathews, C. K.; Van Holde, K. E.; Ahern, K. G. Biochemistry, 3rd ed.; Addison Wesley Longman Inc.: San Francisco, 2000; pp 696−697. (6) Nagao, A. Oxidative Conversion of Carotenoids to Retinoids and Other Products. J. Nutr. 2004, 134, 237S−240S. (7) Willet, W. C.; Stampfer, M. J.; Underwood, B. A.; Taylor, J. O.; Hennekens, C. H. Vitamins A, Vitamin E, and Carotene: Effects of

8598

dx.doi.org/10.1021/jp505456q | J. Phys. Chem. B 2014, 118, 8591−8600

The Journal of Physical Chemistry B

Article

Supplementation on their Plasma Levels. Am. J. Clin. Nutr. 1983, 38, 559−566. (8) Constantino, J. P.; Kuller, L. H.; Begg, L.; Redmond, C. K.; Bates, M. W. Serum Level Changes After Administration of a Pharmacologic Dose of β-Carotene. Am. J. Clin. Nutr. 1988, 48, 1277−1283. (9) Johnson, E. J.; Suter, P. M.; Sahyoun, N.; Ribaya-Mercado, J. D.; Russell, R. M. Relation Between β -Carotene Intake and Plasma and Adipose Tissue Concentrations of Carotenoids and Retinoids. Am. J. Clin. Nutr. 1995, 62, 598−603. (10) Takahashi, R. Polarographic Reduction-Waves of Vitamin A, βCarotene and Vitamin D. Rev. Polarogr. 1961, 9, 247−248. (11) Takahashi, R.; Tachi, I. Polarographic Studies on Fat Soluble Vitamins in Nonaqueous Media. Part II. Polarography of Vitamin A and its Related Compounds in Acetonitrile-Benzene Mixture as Solvent. Agric. Biol. Chem. 1962, 26, 771−777. (12) Atuma, S. S.; Lindquist, J.; Lundström, K. The Electrochemical Determination of Vitamin A: Part I. Voltammetric Determination of Vitamin A in Pharmaceutical Preparations. Analyst 1974, 99, 683−689. (13) Atuma, S. S.; Lundström, K.; Lindquist, J. Electrochemical Determination of Vitamin A: Part II. Further Voltammetric Determination of Vitamin A and Initial Work on the Determination of Vitamin D in the Presence of Vitamin A. Analyst 1975, 100, 827− 834. (14) Park, S.-M. Electrochemical Studies of β-Carotene, All-transRetinal and All-trans-Retinol in Tetrahydrofuran. J. Electrochem. Soc. 1978, 125, 216−222. (15) Powell, L. A.; Wightman, R. M. Electroreduction of Retinal. Formation of Pinacol in the Presence of Malonate Esters. J. Am. Chem. Soc. 1979, 101, 4412−4413. (16) Powell, L. A.; Wightman, R. M. Spectroelectrochemistry of Retinal. Electrodimerization in the Presence of Proton Donors. J. Electroanal. Chem. 1980, 106, 377−390. (17) Powell, L. A.; Wightman, R. M. Mechanism of Electrodimerization of Retinal and Cinnamaldehyde. J. Electroanal. Chem. 1981, 117, 321−333. (18) Wring, S. A.; Hart, J. P.; Knight, D. W. Voltammetric Behavior of All-trans-Retinol (Vitamin A1) at a Glassy Carbon Electrode and its Determination in Human Serum Using High-Performance Liquid Chromatography with Electrochemical Detection. Analyst 1988, 113, 1785−1789. (19) Nelson, A. Voltammetry of Retinal in Phospholipid Monolayers Adsorbed on Mercury. J. Electroanal. Chem. 1992, 335, 327−343. (20) Wang, L.-H. Simultaneous Determination of Retinal, Retinol and Retinoic Acid (All-trans and 13-cis) in Cosmetics and Pharmaceuticals at Electrodeposited Metal Electrodes. Anal. Chim. Acta 2000, 415, 193−200. (21) Ziyatdinova, G.; Giniyatova, E.; Budnikov, H. Cyclic Voltammetry of Retinol in Surfactant Media and its Application for the Analysis of Real Samples. Electroanalysis 2010, 22, 2708−2713. (22) Tan, Y. S.; Yue, Y.; Webster, R. D. Competing HydrogenBonding, Decomposition, and Reversible Dimerization Mechanisms During the One- and Two-Electron Electrochemical Reduction of Retinal (Vitamin A). J. Phys. Chem. B 2013, 117, 9371−9379. (23) Mairanovsky, V. G.; Engovatov, A. A.; Ioffe, N. T.; Samokhvalov, G. I. Electron-Donor and Electron-Acceptor Properties of Carotenoids. Electrochemical Study of Carotenes. J. Electroanal. Chem. 1975, 66, 123−137. (24) Valentin Pfund, B.; Bond, A. M.; Hughes, T. C. Simple Voltammetric Method for the Determination of β-Carotene in Brine and Soya Oil Samples at Mercury and Glassy Carbon Electrodes. Analyst 1992, 117, 857−861. (25) Grant, J. L.; Kramer, V. J.; Ding, R.; Kispert, L. D. Carotenoid Cation radicals−Electrochemical, Optical, and Electron-ParamagneticRes Study. J. Am. Chem. Soc. 1988, 110, 2151−2157. (26) Khaled, M.; Hadjipetrou, A.; Kispert, L. D. Simultaneous Electrochemical and Electron-Paramagnetic Resonance Studies of Carotenoid Cation Radicals and Dications. J. Phys. Chem. 1991, 95, 2438−2442.

(27) Jeevarajan, A. S.; Khaled, M.; Kispert, L. D. Simultaneous Electrochemical and Electron Paramagnetic Resonance Studies of Carotenoids: Effect of Electron Donating and Accepting Substituents. J. Phys. Chem. 1994, 98, 7777−7781. (28) Jeevarajan, A. S.; Khaled, M.; Kispert, L. D. Simultaneous Electrochemical and Electron Paramagnetic Resonance Studies of Keto and Hydroxy Carotenoids. Chem. Phys. Lett. 1994, 225, 340−345. (29) Jeevarajan, J. A.; Jeevarajan, A. S.; Kispert, L. D. Electrochemical, EPR and AM1 Studies of Acetylenic and Ethylenic Carotenoids. J. Chem. Soc., Faraday Trans. 1996, 92, 1757−1765. (30) Jeevarajan, J. A.; Kispert, L. D. Electrochemical Oxidation of Carotenoids Containing Donor/Acceptor Substituents. J. Electroanal. Chem. 1996, 411, 57−66. (31) Jeevarajan, J. A.; Wei, C. C.; Jeevarajan, A. S.; Kispert, L. D. Optical Absorption Spectra of Dications of Carotenoids. J. Phys. Chem. 1996, 100, 5637−5641. (32) Kispert, L. D.; Gao, G.; Deng, Y.; Konovalov, V.; Jeevarajan, A. S.; Jeevarajan, J. A.; Hand, E. Carotenoid Radical Cations, Dications and Radical Trications. Acta Chem. Scand. 1997, 51, 572−578. (33) He, Z.; Kispert, L. D. Effect of Electrolytes and Temperature on Dications and Radical Cations of Carotenoids: Electrochemical, Optical Absorption, and High- Performance Liquid Chromatography Studies. J. Phys. Chem. B 1999, 103, 10524−10531. (34) Liu, D. Z.; Gao, Y. L.; Kispert, L. D. Electrochemical Properties of Natural Carotenoids. J. Electroanal. Chem. 2000, 488, 140−150. (35) Hapiot, P.; Kispert, L. D.; Konovalov, V. V.; Savéant, J.-M. Single Two-Electron Transfers vs Successive One-Electron Transfers on Polyconjugated Systems Illustrated by the Electrochemical Oxidation and Reduction of Carotenoids. J. Am. Chem. Soc. 2001, 123, 6669−6677. (36) Kildahl-Andersen, G.; Konovalova, T. A.; Focsan, A. L.; Kispert, L. D.; Anthonsen, T.; Liaaen-Jensen, S. Comparative Studies on Radical Cation Formation from Carotenoids and Retinoids. Tetrahedron Lett. 2007, 48, 8196−8199. (37) Tan, Y. S.; Webster, R. D. Electron-Transfer Reactions between the Diamagnetic Cation of α-Tocopherol (Vitamin E) and β-Carotene. J. Phys. Chem. B 2011, 115, 4244−4250. (38) Kissinger, P. T.; Heineman, W. R. Laboratory Techniques in Electroanalytical Chemistry, 2nd ed.; Marcel Dekker Inc: New York, 1996; pp 55−60. (39) Hui, Y.; Webster, R. D. The Absorption of Water into Organic Solvents Used for Electrochemistry Under Conventional Operating Conditions. Anal. Chem. 2011, 83, 976−981. (40) Webster, R. D.; Bond, A. M.; Schmidt, T. The Electrochemical Reduction of Some 2,6-Disubstituted Pyridine Based Esters and Thioic S-Esters in Acetonitrile. J. Chem. Soc., Perkin Trans. 2 1995, 1365− 1374. (41) Senoo, H. Vitamin A-Storing Cell (Stellate Cell) System. In Vitamin A: New Research; Loessing, I. T., Ed.; Nova Biomedical Books: New York, 2007; pp 59−70. (42) Khachik, F.; Beecher, G. R.; Goli, M. B.; Lusby, W. R. Separation, Identification, and Quantification of Carotenoids in Fruits, Vegetables and Human Plasma by High Performance Liquid Chromatography. Pure Appl. Chem. 1991, 63, 71−80. (43) MacCrehan, W. A.; Schönberger, E. Determination of Retinol, α-tocopherol, and β-Carotene in Serum by Liquid Chromatography with Absorbance and Electrochemical Detection. Clin. Chem. 1987, 33, 1585−1592. (44) Collins, C. M.; Leventis, N.; Sotiriou-Leventis, C. Relative Reactivity of Vitamin A Versus a Mixture of β-Carotene Geometric Isomers with Electrochemically Generated Superoxide and Hydroperoxyl Radicals. Electrochim. Acta 2001, 47, 567−576. (45) Silva López, C.; Nieto Faza, O.; López Estévez, S.; De Lera, A. R. Computation of Vertical Excitation Energies of Retinal and Analogs: Scope and Limitations. J. Comput. Chem. 2006, 27, 116−123. (46) Walczak, E.; Szefczyk, B.; Andruniów, T. Geometries and Vertical Excitation Energies in Retinal Analogues Resolved at the CASPT2 Level of Theory: Critical Assessment of the Performance of 8599

dx.doi.org/10.1021/jp505456q | J. Phys. Chem. B 2014, 118, 8591−8600

The Journal of Physical Chemistry B

Article

CASSCF, CC2 and DFT Methods. J. Chem. Theory Comput. 2013, 9, 4915−4927. (47) Macernis, M.; Sulskus, J.; Malickaja, S.; Robert, B.; Valkunas, L. Resonance Raman Spectra and Electronic Transitions in Carotenoids: A Density Functional Theory Study. J. Phys. Chem. A 2014, 118, 1817−1825. (48) Treimer, S. E.; Evans, D. H. Electrochemical Reduction of Acids in Dimethyl Sulfoxide. CE Mechanisms and Beyond. J. Electroanal. Chem. 1998, 449, 39−48. (49) Treimer, S. E.; Evans, D. H. Electrochemical Reduction of Acids in Dimethyl Sulfoxide. Comparison of Weak C−H, N−H and O−H Acids. J. Electroanal. Chem. 1998, 455, 19−28. (50) Lauw, S. J. L.; Ganguly, R.; Webster, R. D. The Electrochemical Reduction of Biotin (Vitamin B7) and Conversion into its Ester. Electrochim. Acta 2013, 114, 514−520. (51) Kuta, E. J.; Yu, M. Polarography of Conjugated Unsaturated Lipids. Lipids 1967, 2, 411−418. (52) Kuta, E. J. Polarographic Investigation of Conjugated FatSoluble Vitamins. Science 1964, 144, 1130−1131.

8600

dx.doi.org/10.1021/jp505456q | J. Phys. Chem. B 2014, 118, 8591−8600

Contrasting voltammetric behavior of different forms of vitamin A in aprotic organic solvents.

Six of the major vitamers and provitamins comprising vitamin A (β-carotene, retinoic acid, retinol, retinyl palmitate, retinyl acetate, and retinal) w...
1MB Sizes 1 Downloads 3 Views