Iron being an important transition metal, is the second most abundant metal on earth and possesses many oxidation states ranging from 2- to 6+. In biological systems 2+ and 3+ valances are of the greatest importance. The 2+ and 3+ oxidation states, characterized by their d6 and d5 ground state configuration respectively, are exquisitely sensitive to both pH and the nature of the ligating functionality. This sensitivity has been exploited at a cellular level, in as much as this metal can function both as an electron source as well as an electron sink [1].

Iron (II) forms a number of complexes among which most are octahedral. A large number of complexes are also formed by iron (III). Being mostly octahedral, may be considered its characteristic co-ordination polyhedron. It also forms a few tetrahedral complexes, e.g. FeCl4- [2].

Iron is the important as well as most wide spread transition metal and in living systems, it has functional roles. The chief heme proteins such as hemoglobin, myoglobin, cytochromes, and some enzymes are characterized by the presence of the heme group which is the iron containing unit [2].

Vitamins cannot be produced by the body and hence must be supplied. In addition to oxygen, water, protein, fats, carbohydrates and certain inorganic salts, a number of organic compounds are are also necessary for the life, growth and health of animals including man. These compounds are known as vitamins and are only necessary in very small amounts [3].

B-vitamins belong to the water soluble group of vitamins and it was found as a complex mixture. The B-vitamins used in the present study are niacin (B3) and pyridoxine (B6).

Niacin (nicotinic acid) was first prepared by the oxidation of nicotine. This is now used as a commertcial method, another commercial method is the vapour-phase oxidation of 3-methyl-pyridine (β-picoline) in the presence of a vanadium and iron catalyst. Still another commercial method is the oxidation of quinoline to quinilinic acid, which is then decarboxylated to nicotinic acid [3].

Examination of the ultraviolet absorption spectrum of pyridoxine showed that it is similar to that of 3-hydroxypyridine. Its synthesis is started with 5-ethoxy-4-methyloxazole, which was prepared by heating the ethyl ester of N-formyl-DL-alanine with phosphorous pentoxide in chloroform. This underwent the Diels-Alder reaction with diethyl maleate, which after treatment with acid followed by reduction with lithium aluminium hydride, was converted into pyridoxine.

Experimental

Reagents and solutions: Potassium chloride (MERCK, Germany) and hydrochloric acid (Sigma-Aldrich Laborchemikalien, GmbH) were used to prepare the buffers. Ferrous sulphate was prepared by the reaction between iron turnings and sulphuric acid (Aldrich Chemical Co Ltd, GILLINGHAM DORSET ENGLAND). Niacin (MERCK, Germany) and pyridoxine (E. Merck, Darmstadt) were employed as ligands. De-ionized water was used for preparing all the solutions as well as electrode cleaning. For purging purpose, 99.997% Nitrogen (Bangladesh Oxygen, Ltd.) was used.

Equipment

An Epsilon potentiostat developed by Bioanalytical systems, Inc. (USA) was used for the current-voltage measurements. A borosilicate glass made voltammetric cell (three electrode electrolysis system) was employed to perform this work. A Glassy carbon electrode (GCE), Ag/AgCl (satd. KCl) electrode and platinum wire were used as working electrode, reference electrode and counter electrode respectively. For the agitating of the solution an AGE (VELP SCIENTIFICA) magnetic stirrer was used.        

Study of interaction of Fe (II) with Niacinby cyclic voltammetry

Iron undergoes successful interaction with niacin in the hydrochloric acid buffer. The number of peaks remained same as that was for iron itself before interaction (Figure1) but an important change is found in case of peak current. The peak currents in both the regions are so much decreased after interaction, which is due to the complexation of iron with niacin [4].

After interaction, with respect to the uncoordinated iron, the peak positions in both the regions are changed. As a result the peak separation potential after interaction becomes reduced in comparison with the iron before interaction. This fact also makes a strong basis for the argument in favour of Fe (II)-niacin interaction [5-6].

The half wave potential, after interaction, is also changed. It becomes more positive after interaction, indicating to the successful Fe (II)-niacin interaction. It is found that after interaction the value of reduction potential achieves less negative value and becomes harder for redox process. All these facts together indicate towards successful interaction [7-10].

Figure 1: Voltammograms Of Fe (II)/Fe(III) system In The (I) presence (coordinated) Of And (Ii) absence (uncoordinated) Of niacin In hydrochloric acid buffer Of Ph 2.0

PH of maximum interaction for Fe (II)-niacin interaction

The variation in the redox behaviour of Fe (II) in the presence of niacin was examined at pH 1.2, 1.4, 1.6, 1.8, 2.0 and 2.2 using hydrochloric acid buffer. At pH 1.4, 2.0 and 2.2, it shows peaks in both the cathodic and anodic regions (Figure 2). This fact is similar to that of Fe (II) before interaction. But at pH 1.2, 1.6 and 1.8 the anodic peak becomes insignificant after interaction with niacin. This may be due to any obstacle in electron transfer or may involve an EC mechanism. This fact is not completely similar to Fe (II) before interaction.

At pH 1.4, 1.8 and 2.0, it shows sharp cathodic peaks after interaction [11-13]. But at other pH values the peaks are broad in the same region.

After interaction there are two cathodic peaks in all the cases and one anodic peak at pH 1.4, 2.0 and 2.2. The first cathodic peak correspond to the anodic peak and may involve the Fe (II)/Fe (III) redox couple. At pH 1.2, 1.4, 1.6 and 2.2 there appears an extra peak or humplike shape in the cathodic region. This situation arises only after interaction i.e. in case of Fe (II) before interaction this situation was never found. Therefore, this may be due to the simultaneous existence of co-ordinated and unco-ordinated Fe (II) [14-15]. Now, it is clear that the course before interaction. But at pH 1.2, 1.6 and 1.8 the anodic peak becomes insignificant after interaction with niacin. This may be due to any obstacle in electron transfer or may involve an EC mechanism. This fact is not completely similar to Fe (II) before interaction.

At pH 1.4, 1.8 and 2.0, it shows sharp cathodic peaks after interaction [11-13]. But at other pH values the peaks are broad in the same region.

After interaction there are two cathodic peaks in all the cases and one anodic peak at pH 1.4, 2.0 and 2.2. The first cathodic peak correspond to the anodic peak and may involve the Fe (II)/Fe (III) redox couple. At pH 1.2, 1.4, 1.6 and 2.2 there appears an extra peak or humplike shape in the cathodic region. This situation arises only after interaction i.e. in case of Fe (II) before interaction this situation was never found. Therefore, this may be due to the simultaneous existence of co-ordinated and unco-ordinated Fe (II) [14-15]. Now, it is clear that the course of complexation is more prominent at these pH values. The second cathodic peak is due to the buffer, which was also observed in case of Fe (II) in absence of niacin.

before interaction. But at pH 1.2, 1.6 and 1.8 the anodic peak becomes insignificant after interaction with niacin. This may be due to any obstacle in electron transfer or may involve an EC mechanism. This fact is not completely similar to Fe (II) before interaction.

At pH 1.4, 1.8 and 2.0, it shows sharp cathodic peaks after interaction [11-13]. But at other pH values the peaks are broad in the same region.

After interaction there are two cathodic peaks in all the cases and one anodic peak at pH 1.4, 2.0 and 2.2. The first cathodic peak correspond to the anodic peak and may involve the Fe (II)/Fe (III) redox couple. At pH 1.2, 1.4, 1.6 and 2.2 there appears an extra peak or humplike shape in the cathodic region. This situation arises only after interaction i.e. in case of Fe (II) before interaction this situation was never found. Therefore, this may be due to the simultaneous existence of co-ordinated and unco-ordinated Fe (II) [14-15]. Now, it is clear that the course of complexation is more prominent at these pH values. The second cathodic peak is due to the buffer, which was also observed in case of Fe (II) in absence of niacin.

Comparison between the peak positions of Fe (II) before and after interaction with niacin shows that after interaction the shift is towards much more positive potential at pH 1.4, 2.0 and 2.2 but at other values towards less positive potentials. It gives the indication towards complexation. In the anodic region after interaction there is no trend in peak position shift with pH.

The interaction shows that the separation in peak potential is independent of the pH values (Table1). But it is found that after interaction the peak separation potential has been decreased to a considerable extent. This gives the idea that the cathodic and the anodic peaks become more closer as a result of interaction. The main conclusion which may be drawn is that the value of peak separation potential after interaction approaches towards reversible system [16].

An investigation to the peak current in the forward scan shows that after interaction the decrease in peak current with respect to that before interaction is of a high order at all pH values (Table 1). This decrease due to interaction is also pH independent but more prominent at pH 1.6. This type of current decrease only happens, when there is a shortage of free electroactive species, i.e. when a large amount of uncoordinated iron becomes coordinated, they lose their electroactive ability and as a result due to the shortage of the number of free iron, the amount of current decreases [17-19].

The consideration for the half wave potential (E_1/2) after complexation in the cathodic region exhibits that they shift towards less positive potential in some cases and towards more positive potential [17-19] in other cases, without showing any trend. When these are compared to the E_1/2 of uncomplexed Fe (II), it is found that there is a huge shift, which alarms to the successful complexation at all the pH values.

The reduction potential of the complexed situation is only measurable at pH 1.4, 2.0 and 2.2, which follows no significant trend against pH (Table 1). But compared to the uncomplexed Fe (II), E^° values are of less negative after interaction, i.e. after complexation the redox process becomes more harder and at pH 2.2 it is the hardest [17-18].

Therefore the above description tells about the successful complexation at almost all the pH values. But the formation of a sharp complexed iron peak together with a uncomplexed iron peak and the highest decrease in peak current after interaction may give the decision that pH 1.6 is the pH of highest interaction between Fe (II) and niacin.

Table 1: Current-Potential Data for Fe (II)-Niacin Interaction (1:1) in Hydrochloric Acid Buffer at Scan Rate 100 mVs^-1 at GCE

Figure 2: Effect of Ph on The Behaviour of Fe (II)/Fe (III) System in Presence of Niacin At Scan Rate 100 Mvs-1 and Ph (a) 1.2, (b) 1.4, (c) 1.6, (d) 1.8, (e) 2.0 and (f) 2.2

Composition of Maximum Interaction for Fe (II)-Niacin Interaction

Study for the composition of maximum interaction was also accomplished at the scan rate 100 mVs^-1 and at pH 1.6 for Fe (II)-niacin interaction. For this a fixed amount of Fe (II) was mixed with different concentrations of niacin. Compared to the free iron, the cathodic and anodic peaks shift towards each other, which can be understood from the peak separation potential after interaction. These values also approach towards the reversible value of peak separation potential [20-22]. It is also found that compared to the uncoordinated Fe (II), with the increase in ligand ratio, the decrease in the cathodic peak current becomes gradually larger (Table 2). It happens due to a decrease in the number of uncoordinated Fe (II). Table 2  shows that there are about three ranges of the concentration of niacin, in which the decrease in peak current remains almost constant. This indicates towards the formation of more than one species by Fe (II)-niacin interaction [20-22].

The consideration of half wave potential also shows that there are different regions of ligand concentrations, which gives nearest values. This also indicates the formation of more than one species [17-18]. But in all the cases the half wave potential is more positive compared to that of the uncoordinated Fe (II) at that specific pH value.

An interesting feature is that the peak in the anodic region is clearly absent in two regions of ligand concentrations. The fact behind this may be that the absence of sufficient amount of uncoordinated Fe (II) for further complexation to occur to the maximum efficiency. It is also found that compared to the uncoordinated Fe (II), with the increase in ligand ratio, the decrease in the cathodic peak current becomes gradually larger (Table 2). It happens due to a decrease in the number of uncoordinated Fe (II). Table 2 shows that there are about three ranges of the concentration of niacin, in which the decrease in peak current remains almost constant. This indicates towards the formation of more than one species by Fe (II)-niacin interaction [20-22].

The consideration of half wave potential also shows that there are different regions of ligand concentrations, which gives nearest values. This also indicates the formation of more than one species [17-18]. But in all the cases the half wave potential is more positive compared to that of the uncoordinated Fe (II) at that specific pH value.

An interesting feature is that the peak in the anodic region is clearly absent in two regions of ligand concentrations. The fact behind this may be that the absence of sufficient amount of uncoordinated Fe (II) for further complexation to occur to the maximum efficiency.

From the above discussions and considerations it may be confirmed that at pH 1.6, Fe (II) forms more than one species with niacin.

Table 2: Current-Potential Data for Fe (II)-Niacin Interaction with Different Ligand Concentrations in Hydrochloric Acid Buffer (Ph 1.6) At Scan Rate 100 Mvs^-1 at GCE

Study of the Interaction of Fe (II) with Pyridoxine by Cyclic Voltammetry

Fe (II)-pyridoxine interaction was also studied in the same reaction conditions. It exhibits clear peak in the cathodic region. The peak in the anodic region is absent. The peak current also drops to a considerable amount after interaction. This may be due to the large extent of complexation.

Study of the Interaction of Fe (II) with Pyridoxine by Cyclic Voltammetry

Fe (II)-pyridoxine interaction was also studied in the same reaction conditions. It exhibits clear peak in the cathodic region. The peak in the anodic region is absent. The peak current also drops to a considerable amount after interaction. This may be due to the large extent of complexation.

Again after interaction with pyridoxine the peak positions are also changed to a great extent. The cathodic peak is shifted towards less positive potential. And the peak in the anodic region is disappeared. This fact again confirms the fact of complexation.

Moreover, the half wave potential, after interaction is shifted towards the less positive potential. This also conclude about the successful complexation. Again due to the absence of anodic peak, the peak separation potential and the reduction potential are beyond calculation. All these discussions also give signal towards successful interaction [7-10].

PH of maximum interaction for Fe (II)-pyridoxine interaction 

The redox behaviour of Fe (II)-pyridoxine interaction (1:1) was also examined in hydrochloric acid buffer of same pH values as those for Fe (II)-niacin interaction. Similar to the uncoordinated Fe (II), these also exhibit one cathodic signal for Fe (II) and a second one due to the buffer. The anodic peaks were absent at all the pH values. But at lower pH values (1.2 and 1.4), a slight humplike shape was observed in the anodic region. This may be due to the co-existence of coordinated and uncoordinated iron. In all the cases after interactions the peaks become broader. These characters are a little bit different from the Fe (II)-niacin interaction.

The change in peak current after interaction gives a clear picture of complexation. The peak current decreases to a considerable extent compared to the Fe (II) before interaction at almost all the pH values. The fact behind this is that when Fe (II) was uncoordinated, the concentration of electroactive species was higher and peak current was high but when they become coordinated, they lose their ability to carry current i.e. number of electroactive species decrease and so peak current decrease. This means that complexation occurs at all the pH values and it is more prominent with at pH 1.6, 1.8 and 2.2. This also gives an idea that successful complexation occurs at higher pH values.

The values of peak current decrease show that the decrease is maximum at pH 2.2. It means that here the number of electroactive species is the minimum [17-19]. Therefore, it may be said that at pH 2.2 this Fe (II)-pyridoxine interaction is feasible, i.e. pH 2.2 is the pH of maximum interaction for this interaction.

The half-wave potential (E_1/2) exhibits that at all the pH values the half-wave potential also shifts in comparison with Fe (II), alarming to the successful interaction.

Therefore these discussions about the peak number, peak shape, peak current and half wave potential altogether may give the decision that pH 2.2 is the maximum pH, at which Fe(II) reacts most successfully with pyridoxine.

Composition of maximum interaction for Fe (II)-pyridoxine interaction

At pH 2.2, the study for the composition of maximum interaction in case of Fe (II)-pyridoxine interaction was also done, which is the pH of maximum interaction for this couple and the observations are done at scan rate 100 mVs^-1. It shows an absence of peak in the anodic region because of the higher consumption of the Fe (II) by the ligand at all the concentrations of pyridoxine. Therefore the peak separation potential data cannot be calculated. This confirms that significant interaction occurs at all ligand concentrations. It also may declare the system to be irreversible. Compared to niacin, pyridoxine interacts more strongly with Fe(II), since in case of niacin the anodic signal is absent at the higher concentrations but for pyridoxine, it is absent at all the concentrations of ligand. Again due to the absence of the anodic peak the reduction potential also cannot be calculated (Figure 3).

The half-wave potential shows a significant character. But a matter of great interest is that at all the ligand concentrations, the half-wave potentials shift to the less positive potential after interaction at pH 2.2.

The fact in the case of peak position shows that compared to the uncoordinated Fe (II), the cathodic peak after interaction has shifted towards less positive potential. And remains at almost constant position in two ranges of the concentration of pyridoxine, which gives the indication of the formation of more than one species.

It is found that the decrease in peak current becomes higher with increasing ligand concentrations. This means that at higher concentration of pyridoxine, the extent of interaction increases. But the decrease is more than the Fe (II)-niacin interaction. It achieves an almost constant value in two regions of ligand concentrations. This also indicates the formation of more than one species [11,21-22].

From the above discussions it may be said that at pH 2.2, Fe (II) forms more than one species with pyridoxine.

Figure 3: Plots of Q Vs T1/2 and -Q_r Vs Θ For Fe (II)/Fe (III) System in Presence of Pyridoxine at Ph (i) 1.2, (ii) 1.4, (iii) 1.6, (Iv) 1.8, (V) 2.0 And (vi) 2.2 in Hydrochloric Acid Buffer

Studies of the Interactions of Fe (II) with Niacin and Pyridoxine by Chronoamperometry and Chronocoulometry

Chronoamperometry was also adopted to examine the Fe (II)-niacin and Fe (II)-pyridoxine interaction. Similar to the Fe (II)-niacin interaction, there is also a decrease in the spike height for Fe (II) after interaction with pyridoxine [23,11]. This also signifies that after interaction the rate of electrolysis has been decreased due to the shortage of free electroactive species (Fe (II)) [4,23].

The chronocoulometric study, which is the integrated form of chronoamperometry, was also observed for both of the interactions. For Fe (II)-pyridoxine interaction, the charge at τ increases for Fe (II) after interaction compared to Fe (II) before interaction. This behaviour is exactly opposite to that of for Fe (II)-niacin interaction.

Presence of pyridoxine, the plots of Q vs t^1/2 and -Q_r vs θ on the same graph shows the same facts which is found for Fe(II) in the absence of those. In both the cases the two plots do not intersect at Q = 0 axis as well as they do not have equal slope. Therefore it may be said that in both of these cases adsorption occurs [4,23].

The chronoamperometric study for Fe (II)-pyridoxine interaction supplies current response, which shows that with the increase in pH, the current spike height decreases and at pH 2.2, the height is the minimum. Similar is the case for niacin and after pH 1.6 the spike height is almost same. 

Therefore with the increase in pH, the rate of electrolysis [4-23] decreases.

The charge response found from the chronocoulometric analysis does not show any established character for both of these interactions [4,23,11].

The plots of Q vs t^1/2 and -Q_r vs θ on the same graph was also done at all the pH values. The plots for Fe (II)-Pyridoxine interaction are displayed in Figure3. These show a general qualitative feature at all the pH values. In all the cases the two plots do not intersect at Q = 0 axis as well as they do not have equal slope. Therefore it may be said that at all the pH values, adsorption occurs [4,23,11]. Similar is the case for Fe (II)-niacin interactions.

From the above discussions and together with the cyclic voltammetric data, it may be said that pH of maximum interaction for iron-pyridoxine interaction is 2.2 and for iron-niacin interaction is 1.6.

Fe (II)-niacin interaction shows sharp peak at two pH values. Fe (II)-pyridoxine interaction also exhibits it at two pH values but these are not too sharp as those of niacin. Simultaneous existence of the coordinated and uncoordinated peaks are found for Fe (II)-niacin interactions in the cathodic region at four pH values, but for Fe (II)-pyridoxine interaction in the anodic region at lower pH values. The peak separation potential and the reduction potential cannot be calculated at all the pH values for both of these interactions. The half wave potential for Fe (II)-pyridoxine interaction and Fe (II)-niacin interaction does not show any specific character with pH. Fe (II)-niacin interaction shows highest decrease in peak current at pH 1.6 but Fe (II)-pyridoxine at pH 2.2. This means that maximum interaction of iron occurs with niacin at lower pH and with pyridoxine at higher pH. Fe (II)-pyridoxine interaction is very much strong at all the concentrations of the ligand than those in the Fe (II)-niacin interactions. In both the Fe (II)-niacin and Fe (II)-pyridoxine interactions, an information about the formation of more than one species is found.

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