Effect of Scan Rate and pH on Determination Amoxilline Using Screen Printed Carbon Electrode Modified with Functionalized Graphene Oxide

Graphene oxide GO was functionalized with 4-amino, 3-substituted 1H, 1, 2, 4 Triazole 5(4H) thion (ASTT) to obtain GOT. GOT characterized by FT-IR, XRD.via modification of the working electrode of the SPCE with the prepared nanomaterial (GOT) the effect of scan rate and pH on the determination of Amoxilline (AMOX) was studied using cyclic voltammetry. AMOX show various responses at pH ranging from 2 to 7 and also was observed sharp increase in the oxidation peaks in the pH 3. The formal potential (midpoint) for AMOX was highly pH-dependent. From the effect of scan rate, surface coverage concentration Γ of electroactive species the values of the electron transfer coefficient and the electron transfer constant rate ket was obtained as 5.39×10-10 molcm-2, 0.5, and 2.45×10-3 cm.s-1respectively. Keyword: screen printed carbon electrode, cyclic voltammetry, Thiocarbohydrazide, Graphene oxide,Amoxilline.


Introduction
Graphene oxide is one layer of a polycyclic hydrocarbon network and is partially aromatic, has various oxygen functional groups (CO, OH, and COOH) prepared from the oxidative treatment of bulk graphite (Hummer method) [1] [2].Presence of high density electronegative oxygen atoms on the GO basal plane, gives rise to an energy gap in the electron density of states [3] making GO non-conductive.The hydroxyl and the epoxide groups lay on the surface sheet while carbonyl and carboxyl groups attached at the edge.Carboxylic groups were used to react with thiocarohydrizde (TCH) by cyclocondensation reaction.This could be allowed to prepare GO-4-amino,3-substituted 1H,1,2,4 Triazole 5(4H) thion(ASTT) (GOT) Individual layers of GO can be obtained by sonication and by stirring aqueous suspension for long enough [4].Electrochemical sensor based Graphene oxide was often used to determin various drugs and biological molecules such as dihydronicotinamide adenine dinucleotide (NADH) [5]and diethylstilbestrol(DES) [6].This work is concerned with study of voltammetric behavior of Amoxicillin (AMOX) at screen printed carbon electrode modified withGraphene oxide functionalized at the edge of sheet and studies their impact to determine Amoxilline(AMOX).Amoxicillin (2S, 5R, 6R)-6{[(2R)-2-amino-2-(4-hydroxylphenyl)-acetyl] amino}-3, 3dimethyl-7-oxo-4-thia-1-azabicyclo [3.2.0] heptane-2-carboxylic acid, belongs to a class of antibiotic, called the Penicillin contains a β-lactam ring figure (1) which is responsible for its anti-bacterial against Gram-positive and Gram-negative bacteria.

Apparatus
Electrochemical measurement was recorded on a portable potentiostat Stat 200 (Drop Sens S.L. Oviedo, Spain).The principle function of a potentiostatis controlling potential and measuring current; which was connected via a USB connection to laptop computer installed with the measurement software Drop View (DropSens DRP-110).The measurement was made with screen printed carbon electrode (SPCE) having, carbon counter electrode CE, silver/silver chloride reference electrode RE and a carbon working electrode WE (4 mm diameter) printed on a ceramic surface [8],a small area enables very small sample to perfome the measurement.Volumes smaller than 10 μl deposited directly on the electrode surface [9] are adequate for the analysis.All of the pH measurements were made with a PH-meter BP3001.Functionalized GO was dispersed with an ultrasonic instrument type (soniprep 150).Powder XRD analysis was carried out by using powder diffractometer (Japan) XRD Shimadzu 6000 with an incident Cu-Kα radiation of 1.54 ° 40.0Kv and 30mA; scan range (2θ= 5-80°) and; scan speed: 10 (deg/min).).FTIR spectra were obtained on Shimadzu IR affinity 8400s, Japan.Scan range 4000-400 cm −1 with a resolution of 4 cm −1 .Infrared spectrophotometer using potassium bromide disc.

Preparation of Graphene oxide
Graphite oxide (GTO) was synthesized by hummers' method [2] .The GTO mixture formed was filtered and washed with 5% aqueous solution HCL and distilled water until the pH of the rinsing water became (6-7).The product was dried at 55 c  for 48h.The Graphite oxide GTO aqueous solution mixture was exfoliated by sonication and stirring for 25 min [4] to obtain aqueous colloidal of Graphene oxide GO sheet.

Preparation of Thiocarbohydrazide
Thiocarbohydrazide TCH was prepared [10] by reaction of hydrazine N2H4 with carbon disulfide CS2 at 10 c  with stirring until formation of yellow precipitate to produce hydrazinium dithiocarbazinate HDTC as in equation 1.

→ . 1
The resultant mixture refluxed for half hour to remove hydrogen sulfide H2S as in equation 2.

. → 2
The reaction mixture was filtrated to separate the crystalline precipitate of TCH then washed with ethanol and water.The TCH crystals were dried with vacuum oven for 6 hours at 40-50 c  .

Functionalization of GO with TCH
Carboxylic groups in GO can be used as starting point to react with TCH at its melting temperature in cyclocondensation reaction.This could be allowed to prepare GO-4-amino,3substituted 1H,1,2,4 Triazole 5(4H) thion(ASTT),( GOT) as shown in Fig. (2).The product GOT was cooled, washed with distilled water to dissolve the non-reacted TCH and dried at 60c  .

Fabrication of the modified electrode
1 mg of purified GOT andGO was dispersed into 10 ml of redistilled water and 3µL Nafion and for 1 h sonicated.The modified electrode was made by coating the WEby dropping of 4 µL from the above mixture.Then 50µL from freshly prepared solution of AMOX was dropped on reservoir area to cover modified working electrode, counter and reference electrodes for at least one minute.

Pretreatment of SPCEs
At scan rate 0.1 V s −1 the potential was swept between −0.5 and +1.0 V in 0.1 M H2SO4 in order to pretreat the SPCE and to get a reproducible voltammogram increase sensitivity of sensors as well as to obtain a stable baseline for long term experiments [11].Then, the SPCE was washed with double distilled water and dried at room temperature 24-25 .

Characterization of modifiers GO and GOT FT-IR of thiocarohydrizde (TCH)
Figure (3) shows FT-IR spectrum of prepared TCH.The peaks at 3273.2, 3209.5, 3305.9 cm - 1 corsspondes to N-H and NH2 stretching vibrations respectively.The NH2 bending and wagging vibrations contributed to the two peaks at 1643.35 and 1143.79 cm-1 respectively [12].The characteristic peaks 1531 and1500.6 cm-1 assigns to the coupled modes N-H wagging and C-N stretching vibrations [13].The C=S stretching contributes to two peaks at 1288 cm-1 and 935 cm-1 also these peaks contain contributions of other vibration such as C-N stretching and C-N-N bending vibration .4) Shows the FT-IR spectrum of GO.The stretching vibration of (C-OH, COOH, and residue of H2O) appeared at 3401 cm -1 with broad and strong band.The peak at 1587cm -1 was assigned to unoxidized graphitic domain.

FT-IR of GO
The two bands at 1221 cm -1 and 1400 cm -1 were corresponded to (C-O) stretching vibration of epoxide groups (C-O-C) and (C-O-H) bending vibration of COOH group or C-OH group respectively.The OH bending vibration of COOH groups can be used to evaluate the amount of COOH groups [14].Vibration at 1060 cm -1 is assigned to alkoxy (C-OH) groups.Besides, the band at 1721 cm -1 might refer to not only the carbonyl group stretching vibration of COOH situated at the edges but also to ketones or quinone [15].

FT-IR of functionalize GO with TCH
The infrared spectrum of GO functionalized with Thiocarbohydrazide (GOT) shown in figure (5) the peaks in the region 3000-3500 referred to stretching vibration of NH2 groups.These peaks were not appeared in the FT-IR spectra of GO and they were suggested to the formation of functionalization of GO with TCH.A band in the 1590-1650 cm -1 region is characteristic of the NH2 scissoring vibration; additionally, absorption band at 750-850cm -1 assigns to NH2 twisting and wagging deformations.Also , absorption bands at 1333.32, 1501.4,1175cm -1 that appeared in spectrum of (GOT) correspond to stretching vibration of (C=S) , C=N, and N-N respectively which denotes to stretching vibration that had been introduced to GO by functionalized with TCH.

XRD
XRD of GOA powder sample of GO was examined with x-rays diffract meter.Figure (6) shows the XRD pattern of GO.It is found that GO exhibits a strong diffraction at 11.95 o corresponding to d-spacing of 0.73 nm [16,17] which is higher than that of graphite (0.336 nm) indicates introduced functional groups (carboxylic acid, carbonyl, hydroxyl and epoxy) on the basal and edges planes of GO sheets [18] Moreover, the increase in d-spacing proved the oxidation of graphite flakes (GT) Since the degree of oxidation is proportional to the interlayer spacing (d) of GO [19].

Surface area study
The effective area of the electrodes a bare SPCE ,GO-SPCE and GOT-SPCE was calculated in a solution of 0.5mM 3[Fe(CN)6] -3 in 0.1M KCl, using Randles Sevick equation [20] at different scan rates ranging from 0.01 to 0.1V.s - =2.69×10 5 Α 3/2 1/2 1/2 (3) Where (Ip) refers to the peak current, (A) is the surface area of electrode cm2, n is the number of electrons ,DR is diffusion coefficient, C is the concentration of K3 [Fe (CN)6] -3 and υ refers to the scan rateVs-1.For Potassium ferricyanide , DR = 7.6 × 10 -6 cm 2 s -1 [21],and n=1.The redox behavior of 0.5mM K3[Fe(CN)6] -3 in 0.1M KCl was investigated by cyclic voltammetry.The redox peak currents at bare SPCE, GO-SPCE and GOT-SPCE increased linearly with scan rate ranging from 0.01 to 0.1V.s -1 as shown in figures ( 7), ( 8) and ( 9) respectively.The apparent electroactive area values was found for the electrodes bare SPCE, modified electrode GO-SPCE and GOT-SPCE to be 0.04 cm 2 , 0.05 cm 2 and 0.06 cm 2 respectively.This enlargement in the effective surface area proved the modification of bare SPCE with GO and GOT.

Effect of pH
The influence of pH on the oxidation peak current and potential of 0.5mM AMOX in PBS pH range from2 to 8 at the GOT-Nafion modified SPCE was investigated by CV at scan rate 0.1V.s - .AMOX shows significant response figure (10).The redox peaks current varied with pH values.The redox peak at pH3 was appeared with higher oxidation and reduction current values.There was no response of blank PBS in GOT-Nafion/ SPCE in all pH range indicated that the current and potential value of the oxidation and reduction were due to the AMOX only.
The anodic peak current values of AMOX at the GOT-SPCE were plotted versus pH values of the supporting electrolyte (PBS).Figure (11) reveals that the sharp increase in the oxidation peaks in the pH range of 2-3 this may be due to fast electron process then decreased in the range of 3 to 4 this can be related to change of AMOX interaction with surface of modified SPCE (GOT) [22].Gradually increase in current was observed as the pH values increased from 4 to 6 due to oxidation ability of AMOX, where the electrochemical response of AMOX in pH7 was low.Considering all these points, the pH3 was with highest current value and show clear redox peak than the other pH values.
For a chemically reversible half reaction, the pH dependence of a PET couple is given by eq (8) [23].
,°= ° ( / )−2.303 ∕ (8) ,° was an effective formal potential, the potential midway between the peaks for redox peaks for A and B. The dependence formal redox potential ° of AMOX on the pH was investigated by cyclic voltammetry.The ° value was obtained from the average value of anodic potential and cathodic potential.Fig. (12) shows that the formal potential (midpoint) of AMOX was highly pH-dependent.However, It was found that the E º values shifted to more negative values (decrease linearly) with increase in pH buffer solution.Between pH 2 and 3, a slope has value agreement with the theoretical value of 0.06V [24,25] according to the equation.°=−0.065+0.26 (9) These results of the electrochemical oxidation of Amoxicillin cconfirm the involving of equal number of electrons and protons [26] then the potential returned to be shifted to more positive values between pH 3-7 with a slope 0.0315 V/pH according to the equations.°=0.0315 −0.0375 (10) This behavior may be due to the involving of two electrons and one proton in electrochemical oxidation process of AMOX in this pH range [27].The intersection of the curve was located at pH 3 corresponding to the apparently pKa.The value of pKa is in agreement with A.T.suji et al [28] and GN.Rolinson [29]  The cyclic voltammograms for bare SPCE, GO-SPCE and GOT-Nafion/SPCE in 0.1M blank phosphate buffer solution (pH 3.0) and in the presence of 0.5 mM AMOX is shown in figure (13).The potential was swept from -0.5V to 0.6V at scan rate 0.1V.s - .When the SPCE coated with GO (5 curve) redox peaks for AMOX were observed at 0.12 V with anodic peak current (13.186μA) and at -0.05V with cathodic peak current (15.904μA).The cyclic voltammogram of GOT-Nafion/SPCE in blank 0.1 M phosphate buffer solution (pH 3.0) showed broad anodic and cathodic peaks (4curves).But in the presence AMOX (3 curves), the anodic peak current was appeared at about 0.08 V (anodic current16.443μA),with cathodic peak on the reverse scan at -0.3V (cathodic current 17.393μA).The increase in redox signal may be attributed to the functionalization of GO with 4-amino-3mercapto1, 2, 4-triazol at the edge of GO sheet which facilitate the electron movement between AMOX and the electrode surface and also could be attributed to GOT which has higher effective surface area.
Based on this, GOT-Nafion/SPCE can be used as electrochemical sensing for sensitive determination of AMOX.At the bare SPCE in presence AMOX (1 curve) no redox peak can be seen indicating oxidation process of AMOX hard to occur due to surface of SPCE has small amounts of oxygenated functionalities [30].On the other hand, the bare SPCE have no electrochemical response in blank PBS (2 curve).

Fig. (
Fig. (4) Shows the FT-IR spectrum of GO.The stretching vibration of (C-OH, COOH, and residue of H2O) appeared at 3401 cm -1 with broad and strong band.The peak at 1587cm -1 was assigned to unoxidized graphitic domain.The two bands at 1221 cm -1 and 1400 cm -1 were corresponded to (C-O) stretching vibration of epoxide groups (C-O-C) and (C-O-H) bending vibration of COOH group or C-OH group respectively.The OH bending vibration of COOH groups can be used to evaluate the amount of COOH groups[14].Vibration at 1060 cm -1 is assigned to alkoxy (C-OH) groups.Besides, the band at 1721 cm -1 might refer to not only the carbonyl group stretching vibration of COOH situated at the edges but also to ketones or quinone[15].