Analysis of Aromatic Amines in Surface Waters Receiving Wastewater from a Textile Industry by Liquid Chromatographic with Electrochemical Detection

Abstract

A high performance liquid chromatography (HPLC) method with electrochemical detection (ED) was developed for the determination of benzidine, 3,3‐dimethylbenzidine, o‐toluidine and 3,3‐dichlorobenzidine in the wastewater of the textile industry. The aromatic amines were eluted on a reversed phase column Shimadzu Shimpack C₁₈ using acetonitrile+ammonium acetate (1×10^?4 mol L^?1) at a ratio 46:54 v/v as mobile phase, pumped at a flow rate of 1.0 mL min^?1. The electrochemical oxidation of the aromatic amines exhibits well‐defined peaks at a potential range of +0.45 to +0.78 V on a glassy carbon electrode. Optimum working potentials for amperometric detection were from 0.70 V to +1.0 V vs. Ag/AgCl. Analytical curves for all the aromatic amines studied using the best experimental conditions present linear relationship from 1×10^?8 mol L^?1 to 1.5×10^?5 mol L^?1, r=0.99965, n=15. Detection limits of 4.5 nM (benzidine), 1.94 nM (o‐toluidine), 7.69 nM (3,3‐dimethylbenzidine), and 5.15 nM (3,3‐dichlorobenzidine) were achieved, respectively. The detection limits were around 10 times lower than that verified for HPLC with ultra violet detection. The applicability of the method was demonstrated by the determination of benzidine in wastewater from the textile industry dealing with an azo dye processing plant.     

Speciation of Organotin Compounds by Capillary Electrophoresis Coupled with Hydride Generation Atomic Fluorescence Spectrometry

Abstract

Capillary electrophoresis (CE) coupled with hydride generation atomic fluorescence spectrometry (HG‐AFS) was developed for the speciation analysis of organotin compounds. The four organotin cations of trimethyltin (TMT), monobutyltin (MBT), dibutyltin (DBT), and tributyltin (TBT) were completely separated by CE in a 50 cm×75 µm i.d. fused‐silica capillary at 15 kV and using a mixture of 50 mmol l^?1 H₃BO₃?50 mmol l^?1 Tris‐5% v/v methanol (pH 7.10) as electrolyte. 0.008 mmol l^?1 cetyltrimethylammonium bromide (CTAB) added to the electrolyte suppressed the adsorption of the organotin cations on the inner wall of capillary. The generated hydride species were detected on‐line with AFS. The precisions (RSD, n=5) were in the range of 1.7–3.1% for migration time and 3.8–4.7% for peak area response for the four organotin species. The detection limits ranged from 1–10 µmol l^?1 (as Sn).     

Application of Multicriteria Methodology in the Development of Improved RP-LC-DAD for Determination of Rizatriptan and Its Degradation Products

The aim of this study was to establish an improved isocratic RP-LC-DAD method for separation and determination of rizatriptan benzoate and its two degradation products, L-749.019 and L-783.540 in tablets. Since the chromatographic behavior of target substances can be influenced by various experimental parameters, the whole study was carried out by employing experimental design methodology. The investigation included the influence of mutual changes of the mobile phase composition (methanol amount in the range 3–7% and pH of the water phase from 5.0 to 6.0) and the temperature (from 20 to 30 °C). The response surface design by means of Box-Brehnken design was used to obtain a predictive model which describes the changes in the response within the experimental domain. Additionally, several different target responses were evaluated and Derringer’s desirability function was used for reaching a suitable compromise among the responses. This multi-criteria decision making approach is based on constructing a desirability function for each individual response and afterwards establishing the overall desirability function. Such methodology provided us with the best operating conditions, satisfactory resolutions between the analytes and the shortest possible total analysis time. The experiments were performed on C₁₈ XTerra (150 mm × 3.9 mm), 5 μm column with the mobile phase consisting of a mixture of methanol, TEA and 0.01 mol L^?1 KH₂PO₄ (6:9.4:84.6 v/v) pumped at a flow rate of 1.2 mL min^?1, pH of the water phase adjusted to 6 with 85% orthophosphoric acid, a column temperature of 20 °C and detection at 225 nm. Afterwards, the new method was validated and subsequently applied in analysis of commercially available rizatriptan tablets.     

Flow‐Injection Spectrophotometric Determination of N‐acetylcysteine in Pharmaceutical Formulations with On‐Line Solid‐Phase Reactor Containing Zn(II) Phosphate Immobilized in a Polyester Resin

Abstract

A flow‐injection spectrophotometric procedure was developed for determining N‐acetylcysteine in pharmaceutical formulations. The sample was dissolved in deionized water and 400 µl of the solution was injected into a carrier stream of 1.0×10^?2 mol l^?1 sodium borate solution. The sample flowed through a column (70 mm length×2.0 mm i.d.) packed with Zn₃(PO₄)₂ immobilized in a polymeric matrix of polyester resin and Zn(II) ions were released from the solid‐phase reactor because of the formation of the Zn(II) (N‐acetylcysteine)₂ complex. The mixture merged with a stream of borate buffer solution (pH 9.0) containing 5.0×10^?4 mol l^?1 Alizarin red S and the Zn(II)Alizarin red complex formed was measured spectrophotometrically at 540 nm. The analytical curve was linear in the N‐acetylcysteine concentration range from 3.0×10^?5 to 1.5×10^?4 mol l^?1 (4.9 to 24.5 µg ml^?1) with a detections limit of 8.0×10^?6 mol l^?1 (1.3 µg ml^?1). The relative standard deviations (RSDs) were smaller than 0.5% (n=10) for solutions containing 5.0×10^?5 mol l^?1 (8.0 µg ml^?1) and 8.0×10^?5 mol l^?1 (13.0 µg ml^?1) of N‐acetylcysteine, and the analytical frequency was 60 determinations per hour. A paired t‐test showed that all results obtained for N‐acetylcysteine in commercial formulations using the proposed flow‐injection procedure and a comparative procedure agreed at the 95% confidence level.     

Optimization and validation of an ion‐pair RP‐HPLC‐UV method for the determination of total free iodine in rabbit plasma: application to a pharmacokinetic study

An ion‐pair reverse‐phase high performance liquid chromatographic method with UV–vis detection has been developed for the determination of total free iodine in rabbit plasma after vaginal administration of povidone–iodine (PVP‐I). Sample preparation was done by protein precipitation with acetonitrile in 96‐well format and aspirin was used as the internal standard. The 100 µL sodium thiosulfate solution (5 g L^?1) was added to 100 µL plasma sample before protein precipitation, to convert the total free iodine in plasma to iodide (I^?). Separation was performed on a C₁₈ column (200 × 4.6 mm i.d., 5 µm). The mobile phase consisting of a mixture of water phase (containing 10 mmol L^?1 18‐crown‐6 ether, 5 mmol L^?1 octylamine and 5 mmol L^?1 sodium dihydrogen phosphate, pH adjusted to 6.0 with phosphoric acid) and acetonitrile in the ratio 70:30 (v/v) was delivered isocraticly at a flow rate of 1.0 mL min^?1. The method was sensitive with a lower limit of quantification of 0.005 µg mL^?1, with good linearity (r² > 0.9990) over the linear range of 0.005–2 µg mL^?1. All the validation data, such as linearity, accuracy and precision, were within the required limits. The method was successfully applied to study the pharmacokinetic of PVP–I in rabbits after vaginal administration. Copyright © 2009 John Wiley & Sons, Ltd.     

A new quinoline-based chemosensor in ratiometric sensing of Hg2+ ions

A new quinoline-based chemosensor 1 has been designed and synthesised. Its metal ion-binding properties have been documented in organic and aqueous organic solvents. While chemosensor 1 recognises Hg²⁺ ions (K _a = 2.15 × 10⁴ M^? 1) by exhibiting ratiometric change in emission in CHCl₃/CH₃OH (1:1, v/v), under similar condition both Zn²⁺ and Cd²⁺ ions are sensed by significant non-ratiometric increase in emission with measurable red shift. In DMSO/H₂O (5:95, v/v), the sensor 1 exhibits a greater selectivity towards Hg²⁺ ions (K _a = 9.20 × 10³ M^? 1) over the other metal ions examined.     

Rapid Determination of Coumatetralyl in Human Serum by High‐Performance Liquid Chromatography Coupled with Electrospray Ionization Tandem Mass Spectrometry

Abstract

A rapid, sensitive, and selective high‐performance liquid chromatography‐tandem mass spectrometric method (HPLC‐MS‐MS) for the determination of coumatetralyl in human serum using warfarin as an internal standard has been developed and validated. Coumatetralyl and the internal standard were extracted from the human serum samples by liquid‐liquid extraction with ethyl acetate, followed by separation on a XDB C₁₈ reversed‐phase column (150 mm×2.1 mm i.d., 5 µm) using a mobile phase consisting of acetic acid‐ammonium acetate (5 mmol/L, pH=4.5)/methanol (20:80, v/v) at a constant flow rate of 0.40 mL/min. Coumatetralyl and the internal standard were ionized by negative ion pneumatically assisted electrospray and detected in the multiple‐reaction monitoring mode using precursor→product ion combinations at m/z 291→247 and 307→161, respectively. The calibration curve was linear (r²=0.9945) in the concentration range of 0.5～100.0 ng/mL, with a lower limit of quantification of 0.5 ng/mL in human serum. Intra‐ and inter‐day relative standard deviations were less than 6.3 and 11.0%, respectively. The mean extraction recovery was 87.9% for coumatetralyl and 90.1% for the internal standard. This method is found to be able to determine trace coumatetralyl in human serum and can be used for the diagnosis of poisoned human beings.     

Square‐Wave Voltammetry Determination of Aspartame in Dietary Products Using a Boron‐Doped Diamond Electrode

Abstract

The use of square‐wave voltammetry in conjunction with a cathodically pretreated boron‐doped diamond electrode for the analytical determination of aspartame in dietary products is described. In this determination, the samples were analyzed without previous treatment in a 0.5 mol l^?1 H₂SO₄ solution. A single oxidation peak at a potential of 1.6 V vs. Ag/AgCl (3.0 mol l^?1 KCl) with the characteristics of an irreversible reaction was obtained. The analytical curve was linear in the aspartame concentration range 9.9×10^?6 to 5.2×10^?5 mol l^?1 with a detection limit of 2.3×10^?7 mol l^?1. The relative standard deviation (n=5) obtained was smaller than 0.2% for the 1.0×10^?4 mol l^?1 aspartame solution. The proposed method was applied with success to the determination of aspartame in several dietary products and the results were similar to those obtained using an HPLC method at 95% confidence level.     

Supramolecular inclusion of a pillared double-layered host by an anion-directed second-sphere coordination

In this paper, we have illustrated the utilisation of a second-sphere coordination approach to construct supramolecular inclusion solids with varieties of guest molecules. A flexible molecule N,N,N′,N′-tetra-p-methylbenzyl-ethylenediamine (L1) bearing doubly protonated H-bond donors was designed, capable of forming N–H…Cl hydrogen bonds with a crystallographically unique chloride anion, to construct an anion-directed ligand. The pillared double-layered host framework was constructed by an anion-directed ligand and primary coordination sphere [CoCl₄]^2 ?  through weak C–H…Cl hydrogen-bonding interactions. A variety of guest molecules, such as p-anisaldehyde, 1,4-dimethoxy-2,5-bis(methoxymethyl)benzene, can be included, leading to the formation of novel supramolecular inclusion solids: [L1]·4[H]⁺·[CoCl₄]^2 ? ·2Cl^? ·1.5[C₈H₈O₂]·0.25[CH₃OH] (1) and [L1]·4[H]⁺·[CoCl₄]^2 ? ·2Cl^? ·1.5[C₁₂H₂₀O₄]·0.5[CH₃OH] (2).

We have presented herein the utilisation of a second-sphere coordination approach to construct supramolecular inclusion solids with a variety of guest molecules. A novel type of a pillared double-layered host framework was constructed by a second-sphere coordination between the anion-directed ligand (L1 = N,N,N′,N′-tetra-p-methylbenzyl-ethylenediamine) and [CoCl₄]^2 ?  through weak C–H…Cl hydrogen-bonding interaction, and a variety of guest molecules, such as p-anisaldehyde, 1,4-dimethoxy-2,5-bis(methoxymethyl)benzene, can be included, leading to the formation of supramolecular inclusion solids: [L1]·4[H]⁺·[CoCl₄]^2 ? ·2Cl^? ·1.5[C₈H₈O₂]·0.25[CH₃OH] (1) and [L1]·4[H]⁺·[CoCl₄]^2 ? ·2Cl^? ·1.5[C₁₂H₂₀O₄]·0.5[CH₃OH] (2)

     

Spectrophotometric Determination of Gold in Water and Ore with 2‐Carboxyl‐1‐Naphthalthiorhodanine

Abstract

This paper reports on the synthesis of a new chromogenic reagent, 2‐carboxyl‐1‐naphthalthiorhodanine (CNTR). A high sensitive, selective, and rapid method for the determination of gold based on the rapid reaction of gold with CNTR and the solid phase extraction of the colored chelate with a reversed phase polymer‐based C₁₈ cartridge was developed. In the presence of 0.05–0.5 mol L^?1 of phosphoric acid solution and emulsifier‐OP medium, CNTR reacts with gold to form a red chelate of a molar ratio 1∶3 (gold to CNTR). This chelate was enriched by the solid phase extraction with a polymer‐based C₁₈ cartridge and the retained chelate was eluted from the cartridge with dimethyl formamide (DMF). The enrichment factor of 100 was achieved. In the DMF medium, the molar absorptivity of the chelate is 1.35×10⁵ L · mol^?1 · cm^?1 at 540 nm. Beer's law is obeyed in the range of 0.01～2 µg mL^?1 in the measured solution. The relative standard deviation for 11 replicates sample of 0.5 µg L^?1 level is 2.05%. The detection limit, based on three times the standard deviation is 0.02 µg L^?1 in the original sample. This method was applied to the determination of gold in water and ore with good results.     

Voltammetric Determination of L‐Dopa Using a Carbon Nanotubes‐Nafion Modified Glassy Carbon Electrode

Abstract

A method for determination of L‐dopa by the adsorption stripping voltammetry (ASV) using a multiwalled carbon nanotubes (MWNTs)–Nafion modified glassy carbon electrode (GMGCE) was proposed. This chemically modified electrode (CME) shows a better stability. A sensitive oxidation peak was observed and the anodic peak potential is ca. 0.374V (vs. SCE). The influences of various experimental parameters on the current peak were completely studied. Under the optimized condition, the method has been applied to the determination of L‐dopa in samples. There is a good linear relationship between the peak current (i_p) and L‐dopa concentration in the range of 3.5×10^?7～1.5×10^?5 mol/L, with the limit of detection 5.0×10^?8 mol/L.     

Voltammetric Determination of Rosiglitazone in Pharmaceutical Preparations and Human Plasma

Abstract

The voltammetric behavior of rosiglitazone was studied using direct current (DC_t), differential pulse (DPP), and alternating current (AC_t) polarography. The drug manifests cathodic waves over a pH range of 2–11.2. In Britton‐Robinson buffer (BRb; pH 4), the diffusion current–concentration relationship was found to be rectilinear over a range of 4–24 µg · mL^?1 and 0.1–16 µg · mL^?1 using DC_t and DPP modes, respectively, with minimum limits of detection (LOD) of 0.15 µg · mL^?1 and 0.07 µg · mL^?1 using the DC_t and DDP modes, respectively. The diffusion‐current constant (I _d) was 6.63±0.03 (n=5). The proposed method was successfully applied to the determination of the studied compound both in pure form and in formulations. The mean percentage recoveries in tablets were 100.09±1.18 and 100.85±0.88 (n=5) using DC_t and DPP modes, respectively. Furthermore, the proposed method, adopting the DPP mode, was applied to the determination of rosiglitazone in spiked human plasma and the obtained mean percentage recoveries were 99.14±3.29 (n=4).     

Comparison of Ion-Pairing and Reversed Phase Liquid Chromatography in Determination of Sulfamethoxazole and Trimethoprim

Abstract

Two simple, rapid, and sensitive HPLC methods have been developed for the simultaneous determination of sulfamethoxazole and trimethoprim in their pure and dosage forms, one utilizing reversed phase HPLC and the other ion-pair HPLC. In the reversed phase HPLC method (A) the mobile phase consists of 0.05% aqueous solution of formic acid with pH adjusted to 4.5±0.2 with triethylamine : acetonitrile:tetrahydrofuran 50 : 49 : 1 (v/v), and the mobile phase pumped at flow rate of 1.0 ml min^?1. An Appolo LC18 column (5.0 µm), 250 mm length × 4.6 mm diameter, was utilized as the stationary phase. Detection was affected spectrophotometrically at 254 nm. In the ion-pair HPLC method (B) the mobile phase consisted of methanol : buffer 35 : 65 (v/v) with the buffer composed of potassium dihydrogen phosphate 0.3 M and sodium heptan sulfonic acid 5.0 mM. To 500 ml of buffer was added 2.0 ml triethylamine, and then the pH was adjusted to 5.0 with phosphoric acid, and the mobile phase was pumped at a flow rate of 1.2 ml min^?1. A Hypersil C₁₈ column (5.0 µm), 150 mm length × 4.6 mm diameter, was utilized as the stationary phase. Detection was affected spectrophotometrically at 254 nm. Linearity ranges for sulfamethoxazole and trimethoprim were 1.0–110 and 1.5–98 µg ml^?1, respectively, with method A and 0.5–100 and 1.0–125 µg ml^?1, respectively, with method (B). Minimum detection limits obtained were 0.1969 and 0.3451 µg ml^?1 for sulfamethoxazole and trimethoprim, respectively, with method A, and 0.1377 and 0.2454 µg ml^?1 with method (B). The proposed methods were further applied to the analysis of tablets containing the two drugs, and the results were satisfied.     

Spectrophotometric Determination of Zinc in Pharmaceutical and Biological Samples with Di‐2‐pyridyl Ketone Benzoylhydrazone: a Simple,Fast, Accurate and Sensitive Method

Abstract

A simple, fast, accurate, and sensitive spectrophotometric method was developed to determine zinc(II). This method is based on the reaction of Zn(II) with di‐2‐pyridyl ketone benzoylhydrazone (DPKBH), at pH=5.5 and 50% (v/v) ethanol. Bee?s law was obeyed in the range 0.020–1.82 µg mL^?1 with a molar apsorptivity of 3.64×10⁴ L mol^?1 cm^?1, and a detection limit (3δ) of 2.29 µg L^?1. The action of some interfering ions was verified and the developed method applied to pharmaceutical and biological samples. The results were then compared with those obtained by using a flame atomic absorption technique.     

Veterinary antimicrobials and antiparasitics in fee-fishing ponds: analytical method and occurrence*

ABSTRACT

The aim of this work was to develop and validate a method using online solid-phase extraction and ultra-high-performance liquid chromatography coupled to tandem mass spectrometry to determine residues of 22 veterinary drugs including sulfonamides, amphenicols, fluoroquinolones, benzimidazoles, trimethoprim (TMP) and oxytetracycline (OTC) in water from fee-fishing ponds. The optimal analytical conditions were as follows: XBridge C8 SPE column, Acquity UPLC CSH C18 analytical column, sample loading with water:methanol (98:2, v/v), mobile phase of water with 0.1% acetic acid:methanol (with gradient elution) and eluent flow rate of 0.3 mL min^?1. Quantification was performed in selected reaction monitoring mode and sulfadimethoxine-d₆, ciprofloxacin-d₈, florfenicol-d₃ and albendazole-d₃ were used as internal standards. Water samples collected from 11 fee-fishing ponds showed the presence of residues of FF (0.42–0.74 µg L^?1), albendazole (0.05–0.31 µg L^?1) and thiabendazole (0.45 µg L^?1). Thiamphenicol and TMP were detected at concentrations lower than the limits of quantification of the method (0.1 and 0.001 µg L^?1, respectively).     

Development and Validation of Liquid Chromatography Method for the Separation of Valdecoxib and its SC‐77852 Impurity

Abstract

A reversed‐phase liquid chromatography method has been developed for the separation of valdecoxib and impurity SC‐77852. The best results were achieved using a mobile phase—methanol: 1% water solution TEA (52∶48 v/v), pH 7.35 (adjusted with 85% orthophosphoric acid), column temperature 24°C. Separation was carried out on XTerra? RP₁₈ (150 mm×4,6 mm), particle size 5 µm, flow rate 1 ml/min, using detection on 220 nm. The method was statistically validated for its selectivity, linearity, precision (repeatability), and robustness. Quantitation and detection limits were determined for both valdecoxib and SC‐77852. Method robustness was further evaluated by performing full factorial design experiment. Validated method was used for assay of valdecoxib and SC‐77852 in Bextra^® film‐coated tablets.     

New Zn(II) complexes based on biologically active substituted acetic acid and nitrogen donor ligands: synthesis,crystal structure and biological applications

The complexes [Zn(phenylacetato)₂(2-aminopyridin)₂] (3), [Zn(phenylacetato)₂(1,10-phenanthroline)]·H₂O (4), and [Zn(phenylacetato)₂(2,9-dimethyl-1,10-phenanthroline)]·0.5 H₂O (5) were prepared and characterized by IR-, UV–Visible, ¹H and ¹³C NMR spectroscopy, and single crystal X-ray diffraction. BNPP hydrolysis of the complexes and their parent nitrogen ligands showed that the hydrolysis rate of bis-(4-nitrophenyl) phosphate (BNPP) was 1.7 × 10⁵ L mol^?1 s^?1 for 3, 3.1 × 10⁵ L mol^?1 s^?1 for 4 and 4.3 × 10⁴ L mol^?1 s^?1 for 5. Antibacterial activities show the effect of complexation on activity against Gram-positive (S. epidermidis, S. aureus, E. faecalis, M. luteus and B. subtilis) and Gram-negative (K. pneumonia, E. coli, P. mirabilis and P. aeruginosa) bacteria using the agar well diffusion method. Complex 4 showed good activity against G? bacteria except P. aeruginosa, and against G+ bacteria except E. ferabis. Complex 5 showed no activity against G? bacteria, low activity against M. luteus and B. subtilis bacteria and high activity against S. epidemidis and S. aureus. Complex 3 did not show any activity against G? or G+ bacteria.     

Affinity Adsorption Solid Substrate‐Room Temperature Phosphorimetry for the Determination of Alkaline Phosphatase

Abstract

In the presence of Pb(Ac)₂, the silicon dioxide nanoparticle containing rhodamine 6G (R‐SiO₂) can emit strong and stable solid substrate‐room temperature phosphorescence (SS‐RTP) signal on the surface of acetyl cellulose membrane (ACM) at λ_ex/λ_em=482/649 nm. It was found in the research that specific affinity adsorption reaction between triticum vulgare lectin (WGA) (which was labeled with luminescent silicon dioxide nanoparticle) and alkaline phosphatase (AP) can be carried out on the surface of ACM. The product of the reaction can emit stronger SS‐RTP signal. A new method of SS‐RTP for the determination of AP was established, based on an affinity adsorption reaction between AP and WGA labeled with nanoparticles containing rhodanime 6G luminescent molecules. The linear range of this WGA‐AP‐WGA‐R‐SiO₂ method is 1.00–360.00 ag AP spot^?1 (sample volume: 0.40 µL spot^?1, corresponding concentration range: 2.50–900.00 fg mL^?1). The regression equation of working curve is ΔIp=16.24+0.8856 m_AP (ag spot^?1), r=0.9993. Detection limit of this method calculated by 3S_b/k is 0.14 ag spot^?1. After 11‐fold replicate measurements, RSD are 3.9% and 3.1% for the systems containing 1.00 and 360.00 ag AP spot^?1, respectively. Compared with R‐SiO₂‐WGA‐AP method (detection limit: 0.45 ag spot^?1, corresponding concentration range: 2.00–320.00 ag spot^?1), the sensitivity of WGA‐AP‐WGA‐R‐SiO₂ method was obviously improved and the linear range was wider. The sensitivity, accuracy, and precision of this method are high. It has been successfully applied to determine AP in human serum.     

Experimental and computational investigations on the high binding-selectivity of pyrimidine derivatives by a pillar[5]arene

The binding selectivity of an adenine-monofunctionalized pillar[5]arene (H) with a series of pyrimidine derivatives were investigated through ¹H NMR experiments and density functional theory (DFT) study. High binding-selectivity was demonstrated. Typically, H displayed very strong binding strength with 6-(2,4-dioxo-3, 4-dihydropyrimidin-1 (2H)-yl)hexanenitrile (G1) [K_a >10⁵ M^?1], up to about 3000-fold as compared with 1-hexylpyrimidine-2,4(1H, 3H)-dione (G5) [K_a = 31 M^?1]. The strong binding ability of H with G1 was due to the cooperative multiple hydrogen bond, dipole-dipole, C-H···π and π···π interactions. The high binding-selectivity was also verified by calculation results. The calculated interaction energy (ΔE_i) of G1?H was ?12.92 Kcal·mol^?1 while that of G5?H was ?2.85 Kcal·mol^?1.