(DMF) by dissolving appropriate quantity of each reagent substance separately. (i) 2,4-Dimethoxybenzaldehyde-4-hydroxybenzoylhydrazone (DMBHBH)

Transcription

1 42 CHAPTER -2 MATERIALS AND METHODS 2.1: Preparation of solutions 2.1.1: Preparation of reagent solutions Stock solutions (1 x10-2 M) of reagents were prepared in dimethyl formamide (DMF) by dissolving appropriate quantity of each reagent substance separately. (i) 2,4-Dimethoxybenzaldehyde-4-hydroxybenzoylhydrazone (DMBHBH) g of DMBHBH was transferred into a 100-ml volumetric flask. It was dissolved and diluted up to the mark using DMF solvent to get 1 x 10-2 M concentration of DMBHBH solution. (ii) 2,4-Dimethoxybenzaldehydeisonicotynoylhydrazone (DMBIH) g of DMBIH was transferred into a 100-mL volumetric flask. It was dissolved and diluted up to the mark with DMF solvent to get 1x10-2 M concentration of DMBIH solution. These reagent stock solutions were suitably diluted to get the required concentrations wherever necessary. Fresh reagent solutions were prepared every time before use : Preparation of inorganic stock solutions (i) Copper sulphate solution Requisite quantity ( g) of CuSO 4.5H 2 O (AR GSC) was dissolved in double distilled water containing a few drops of dilute sulphuric acid and made up to the mark in a 100-mL volumetric flask to get 1 x 10-2 M stock solution of Copper (II). The stock solution was standardized titrimetrically 309. (ii) Potassium dichromate solution

2 43 The stock solution of Cr (VI) (1x 10-2 M) was prepared by dissolving g of K 2 Cr 2 O 7 (AR BDH) in a 100-mL volumetric flask using double distilled water. The stock solution was standerdised 309. (iii) Mercury chloride solution The stock solution of Mercury (II) (1x 10-2 M) was prepared by dissolving g of HgCl 2 (AR BDH) in a 100-mL volumetric flask using double distilled water. The stock solution was standerdised 309. (iv) Lead acetate solution The stock solution of Lead (II) (1x 10-2 M) was prepared by dissolving g of (CH 3 COO) 2 Pb.3H 2 O (AR E.Merck) in minimum amount of dilute acetic acid and diluted up to the mark using double distilled water in a 100-mL volumetric flask. The stock solution was standerdised 309. (v) Cadmium nitrate solution The stock solution of Cadmium (II) (1x 10-2 M) was prepared by dissolving g of Cd (NO 3 ) 2.4H 2 O (AR SMC) in minimum amount of dilute nitric acid and diluted up to the mark using double distilled water in a 100-mL volumetric flask. The stock solution was standerdised 309. Working solutions of above metals were prepared by diluting the stock solutions. The solutions of various cations and anions were prepared for interference studies by dissolving appropriate amount of salt in requisite quantity of distilled water. A few drops of suitable acid were added before dilution where ever necessary to prevent hydrolysis. Solutions thus prepared were standardized if needed by standard procedures 309. The formulae, quality, molecular weight or specific gravity and molarity of stock solutions are presented in Table.2.1.

3 44 Table 2.1: Salt solutions used in the qualitative and quantitative studies Ion/ Reagent Formula Quality and make Sp.Gr/ Mol.Wt Molarity of the stock solution Acetate CH 3 COONa.3H 2 O AR BDH Ascorbic acid C 6 H 8 O 6 AR SMC Bromide KBr AR Merck Chloride KCl AR Merck Citrate Na 3 C 6 H 5 O 7.2H 2 O AR CDH EDTA Na 2 C 10 H 14 N 2 O 8.2H 2 O AR Nice Fluoride NaF AR LOBA Iodide KI AR Universal Labs Nitrate NaNO 3 AR BCP Ltd Oxalate Na 2 C 2 O 4 AR FISCHER Phosphate NaH 2 PO 4.2H 2 O AR S.d.Fine Sulphate Na 2 SO 4 AR SMC Tartrate KNaC 4 H 4 O 6.4H 2 O AR S.d.Fine Thiocyanate KSCN GR Nice Thiosulphate Na 2 S 2 O 3.5H 2 O AR ADLAB Thiourea H 2 N-CS-NH 2 AR E.Merck Urea H 2 N-CO-NH 2 AR S.d.Fine Acetic acid CH 3 COOH AR S.d.Fine Ammonia NH 3 AR REIDEL Hydrochloric HCl AR Merck acid Sulphuric acid H 2 SO 4 AR Merck NH 4 NH 4 Cl AR S.d.Fine Sodium NaOH AR BDH hydroxide Sodium tera Na 2 B 4 O 7.10H 2 O AR S.d.Fine borate Triethanol N(CH 2 CH 2 OH) 3 AR Romali amine DMF HCON(CH 3 ) 2 AR Merck CTAB CH 3 (CH 2 ) 15 N(CH 3 )Br Romali % Triton X-100 C 14 H 22 O(C 2 H 4 O) n (n=9- Romali % 10) SDBS C 18 H 29 NaO 3 S Polysciences % Inc Ag(I) AgNO 3 AR S.d.Fine

4 Al(III) Al 2 (SO 4 ) 3.16H 2 O AR Nice As(III) As 2 O 3 AR Hopkin& William Ltd Ba(II) BaCl 2.2H 2 O AR S.d.Fine Bi(III) Bi(NO) 3. 5H 2 O AR GSC Ca(II) CaCl 2.2H 2 O AR SMC Ce(IV) ((NH 4 ) 2 Ce(NO 3 ) 6 ) AR LOBA Cd(II) Cd(NO 3 ) 2.4H 2 O AR SMC Co(II) CoSO 4.7H 2 O AR SMC Cr(VI) K 2 Cr 2 O 7 AR BDH Cu(II) CuSO 4.5H 2 O AR GSC Fe(II) (NH 4 ) 2 FeSO 4.6H 2 O AR S.d.Fine Fe(III) FeCl 3 AR Ranbaxy Lab. Ltd Hg(II) HgCl 2 AR BDH La(III) La(NO 3 ) 3.6H 2 O GR LOBA Li(I) Li 2 SO 4.H2O AR LOBA Mn(II) MnCl 2.4H 2 O AR S.d.Fine Mo(VI) (NH 4 ) 6 Mo 7 O 24.4H 2 O AR S.d.Fine Na(I) NaNO 3 AR BCP Ltd Ni(II) NiCl 2.6H 2 O AR GSC Pb(II) (CH 3 COO) 2 Pb.3H 2 O AR E.Merck Pd(II) PdCl 2 AR J.M & Co Ltd Ru(III) RuCl 3 AR LOBA Se(IV) Na 2 SeO 3 AR LOBA Sb(III) KSb.C 4 H 4 O 6 AR BDH Sn(II) SnCl 2.2H 2 O LR Nice Sr(II) Sr(NO 3 ) 2 AR GSC Ti(IV) TiO 2 AR Merck Th(IV) Th(NO3)4.5H 2 O AR LOBA U(VI) UO 2 (CH 3 COO) 2.6H 2 O AR William M Ltd. V(V) NH 4 VO 3 AR LOBA W(VI) Na 2 WO 2.2H 2 O AR BDH Zn(II) ZnSO 4.7H 2 O AR BDH Zr(IV) Zr(NO 3 ) 4 AR LOBA : Preparation of Buffer solutions Buffer solutions were prepared by adopting the standard procedures reported in the literature 311. The solutions employed for the preparation were presented in Table 2.2.

5 46 Table 2.2: Preparation of Buffer solutions ph Constituents M KCl and 0.2M HCl M KCl and 0.02M HCl M Potassium dihydrogen phosphate and 0.1M HCl M Potassium dihydrogen phosphates and 0.1M Sodium hydroxide ml of 0.1 M Potassium dihydrogen phosphate add 98.4mL of 0.1 M NaOH ml of 0.1 M Disodium hydrogen phosphate and 4.5 mlof 0.1 M NaOH ml of 0.1 M Disodium hydrogen phosphate and 3.4 mlof 0.1M NaOH ml of 0.1 M Disodium hydrogen phosphate and 3.5 ml of 0.1M NaOH 2.2: A brief description of instruments employed in the present investigation 2.2.1: UV-Visible recording spectrophotometer (UV-160A) Shimadzu Corporation, Spectrometric instrument plant, Analytical instruments division, Kyoto, Japan has developed a versatile and indigenous micro processor based UV-Visible recording spectrophotometer (UV-160 A). Operational principle and constructional features UV-160A is a double beam microprocessor based spectrophotometer designed for the quantitative analysis. Its main features are Wavelength scanning system by CPU Control without using sine bar to realize high speed wavelength scanning. All in one type of spectrophotometer with CRT and Printer incorporated.

6 47 Backup mode parameters are provided so as to enable single action operation. Easy data processing, since the obtained spectrum is available by the conversation with CRO. Specifications of UV-160A spectrophotometer Measuring wavelength range Spectral bandwidth (Resolution) Wavelength readability : nm : 2 nm : 0.1 nm increment (Wavelength setting) Wavelength scanning speed : Monochromator setting speed is Nearly 3600 nm/min. Fast-nearly 2400 nm/min. Medium - nearly 1500 nm/min. Slow nearly 480 nm/min. Wavelength accuracy : ± 0.5 nm with automatic wavelength correction. Light source switching : Automatic switching according to wavelength can be selected between 295 and 364nm. Photometric system Recording mode : Double beam system. : Printout of measured data and calculated results. Multi-component : Mixed samples of up to eight Components can be determined. Light source : 50 W long life tungsten lamp (2000 hrs) and socket type

7 48 deuterium (500 hrs) with automatic control of maximum sensitivity. Monochromator : Aberration-corrected concave holographic grating with f = 4.2. Detector Recorder : A matched pair of silicon photo diode. : Computer controlled thermal graphic printer. 9-inch with graphic function 240 x 320 dots. Sample compartment : Inner size: 1100 mm wide, 230 mm deep and 105 mm high. Distance between sample and reference beam Power requirements : 100 mm. : With line voltage selector for 100,115, 220 and 240 V. Weight : 42 Kgs : Digital ph meter The ph of the buffer solutions was monitored by using ELICO digital ph meter (LI-120) manufactured by M/s. ELICO Pvt. Ltd. India. The reproducibility of the measurements is within 0.01 ph : Digital electronic balance Sartorius BS/BT 2245 model (Germany make) electronic analytical balance having maximum capacity of 220g and sensitivity of ± 0.1 mg was used for weighing purpose.

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9 49 2.3: General experimental procedures Spectrophotometric methods were developed for determination of Copper (II), Chromium (VI), Mercury (II), Lead (II) and Cadmium (II) using newly synthesized hydrazone reagents. The (substituted benzoylhydrazone) 4-hydroxybenzoylhydrazone and isonicotinoylhydrazone were used in present study. They are 1) 2,4-Dimethoxybenzaldehyde-4-hydroxybenzoylhydrazone (DMBHBH). 2) 2,4-Dimethoxybenzaldehydeisonicotynoylhydrazone(DMBIH). The reagent 2,4-Dimethoxybenzaldehyde-4-hydroxybenzoylhydrazone (DMBHBH) gave colour complexes with different intensities with Cu (II), Cr (VI) and Cd (II), in basic buffer (ph ) medium, and also 2,4-Dimethoxy benzaldehyde isonicotinoylhydrazone (DMBIH) reagent gave color complexes with Hg (II) and Pb (II) in basic buffer buffer medium. These reactions were studied systematically. The general experimental procedures and reaction conditions employed for the determination of copper, chromium, cadmium, mercury and lead are given in this section : Absorption spectra of reagent solutions and metal complexes An aliquot of reagent (usually 1 ml of 1 x 10 2 M) solution was taken in a 10-ml volumetric flask containing 3 ml of buffer solution and suitable volume of surfactant was added and the volume was made up to the mark with distilled water. The absorbance of the reagent solution was measured against buffer blank. A plot between absorbance and the wavelength was made. The following procedure was adopted for measuring the absorption spectra of complex (metal + reagent) in aqueous medium. In a 10-ml standard flask, the metal complex was prepared by taking 3 ml of buffer, suitable volume of surfactant, suitably concentration of metal ion and reagent (usually 10-fold molar excess to metal ion) solutions. The contents were diluted up to the mark with distilled water and the

10 50 absorbance of the complex was measured against the reagent blank prepared identically. A plot between absorbance and the wavelength was plotted from which the analytical wavelength was selected : Effect of ph on the absorbance of the metal complexes To examine the effect of ph on the intensity of the colour, the metal complex in solution was prepared using buffer solutions having different ph values. In a set of 10-mL standard flasks, 3 ml of buffer (different ph values 1.0 to 11.0) solution, constant amount of metal ion, reagent (usually 10-fold molar excess to metal ion) solution and required quantity of surfactant were taken, made up to the mark with distilled water. The absorbance of each solution (metal complex) was measured at a selected wavelength ( max ) against corresponding reagent blank prepared accordingly. A plot was made between absorbance and ph from which the working ph was selected : Effect of reagent concentration on the absorbance The following procedure was employed to find optimum amount of reagent required for full colour development. Varying and known aliquot of reagent solution was taken in a set of 10 (10-mL) calibrated flasks, each containing 3 ml of buffer solution, fixed amount of metal ion and adequate volume of surfactant. The contents were diluted up to the mark with distilled water and absorbance of the solution in each flask was measured at a selected wavelength against suitable reagent blank prepared identically. From this experiment, required number of folds of the reagent necessary for full color development was ascertained : Effect of time on the absorbance of reaction mixture and stability In a 10-ml calibrated flask, 3 ml of buffer solution, 0.5 ml of metal ion (usually in Beer s law range) and 0.5 ml of reagent (usually 10-fold molar excess) and required quantity of surfactant were taken and diluted up to the mark with

11 51 distilled water. The absorbance of the colored complex solution was measured at a selected wavelength in different time intervals against reagent blank prepared similarly. From this experiment, the time stability of the complex and time interval required for full color development was known : Effect of surfactants on the metal complexes Surfactant micellar can enhance sensitivity and can bring about changes in solubility, pk a, chemical equilibrium, stability, reaction rates and mechanisms of some chemical process. Hence, the effect of surfactants (CTAB, Triton X-100 and SDBS) on the solubility, stability and absorbance of metal complexes were studied and suitable surfactant was selected. In order to arrive at the optimum volume of surfactant required to retain the metal complexes stable in solution state, the following method was adopted. To different 10-mL volumetric flasks each containing 3 ml of buffer solution, various known aliquots of surfactants, appropriate amount of metal ion and required aliquots of reagent solution were added. The contents of flasks were made up to the mark with distilled water and the absorbance of the solution in each flask was measured at the selected wavelength against reagent blank prepared similarly. The surfactants such as Triton X-100 (neutral), Cetyltrimethyl ammoniumbromide (CTAB) (cationic) and Sodiumdodecylbenzene sulphonate (SDBS) (anionic) were used in the present study of metal complexes in solution state. From this experiment the required quantity of surfactant was ascertained.

12 : Applicability of Beer s law To ascertain the sensitivity of the colour reactions and to explore the possibility of determining micro amounts of metal ions, the following procedure was used in the present study. In a set of twelve (10-ml) volumetric flasks, 3 ml of buffer solution, varying and known aliquots of metal solution of appropriate concentration, suitable aliquot of surfactant and excess amount of reagent solution were taken and diluted to the mark with distilled water. Then the absorbance of all solutions was measured at a selected wavelength (λ max ) against blank solution. A plot between absorbance and amount of metal ion ( g/ml) was constructed. The slope and intercept of the plot was computed. Various analytical parameters are calculated using the slope of calibration plot : Effect of foreign ions on the absorbance of reaction mixture In order to assess the applicability of the proposed methods for the analysis of real or synthetic samples containing the metal ions, the effect of the presence of various foreign ions which were generally found associated with the test metal ions on the absorbance of the complex solution was studied by adopting the following procedure. Fixed amount of metal ion solution was taken in 10-mL volumetric flasks containing 3 ml of buffer solutions, required quantity of surfactant. Then appropriate amount of foreign ion was added to all the flasks except one. The reagent solution was added to all at the end. The contents were made up to the mark with distilled water. The absorbance of the complex in each flask was measured at a selected wavelength (λ max ) against reagent blank. From this absorbance the tolerance limit of the foreign

13 53 ion was determined. The amount of foreign ion which brings about a change in absorbance by ± 2% was taken as its tolerance limit. Cations were added mostly as chlorides, sulphates or nitrates and the anions were added as sodium or potassium salts. Some of the cations interfering were masked by using adequate quantity of suitable masking agents : Applications The present methods were applied for the determination of Cu (II), Cr (VI), Cd (II), Hg (II) and Pb (II) in biological samples, real samples, alloy samples, commercial samples, water samples, soil samples and liver samples. Where ever original samples are not available, synthetic samples were prepared accordingly and used : Grapeleaves The extract of the leaf sample was obtained from Andhra Pradesh Agricultural Research Institute (APARI) Hyderabad. The sample solution was prepared following the procedure described by Piper 318. The sample solution thus prepared was diluted appropriately with distilled water and analyzed for copper, 712 µg/ml flouride solution was added to mask Iron (III). The suitable aliquots of sample was analyzed and the results obtained are presented in Table : Real Samples: The real samples of tannery effluent, chrome liquor samples, For which 100 ml of grab sample is taken from tannery effluent, filtered and then treated with 5 ml of HNO 3 to convert the Cr (III) into Cr (VI), so that total Cr (VI) content may be estimated. Another grab sample of chrome liquor is treated with HNO 3 so that Cr (VI) could be formed. Both these samples are diluted to three times, so that it comes under detection limit for Cr (VI) by UV-Visible spectrophotometer.

14 : Alloy Samples Certified samples of bearing metal alloy samples were not available. Therefore, synthetic mixtures whose composition corresponds to bearing metal alloy were prepared. An accurately weighed amount of steel sample (0.5g) was dissolved completely in minimum amount of aquaregia by slow heating on sand bath and then heated to fumes of oxides of nitrogen. After cooling 5-10mL of 1:1 H 2 0:H 2 SO 4 mixture was added and evaporated to dryness. Sulphuric acid treatment was repeated three times to remove all the nitric acid. The residue was dissolved in 20 ml of distilled water and filtered and the filtrate was made up to 100 ml in a calibrated volumetric flask with distilled water. The present developed was applied to the determination of Cd (II) in synthetic mixtures : Cigarette tobacco solution The tobacco of cigarettes were dissolved in 2 ml of AR grade concentrated sulphuric acid and heated on a hot plate for 20 min. The contents were diluted with 20 ml of water and filtered. The filtrate was collected in 50 ml slandered flask with distilled water. The absorbance values were referred to the pre determined calibration plot to compute the amount of cadmium : Preparation of water, soil and liver samples Water samples: Each water sample (250 ml) filtered with whatman No.40 was mixed with 10 ml of concentrated nitric acid in a 500 ml distillation flask. The sample was digested in the presence of an excess potassium permanganate solution according to the method recommended by Fifield et al 319. The solution was cooled and neutralized with dilute NH 4 OH solution. The digest was transferred in to a 25 ml calibrated flask and diluted up to the mark with deionized water. Soil samples: 2 gm of soil, 5 to 7 ml of concentrated H 2 SO 4 and an excess of KMnO 4 are mixed in conical flask equipped with a reflux condenser. The crystals of

15 55 KMnO 4 is added slowly in small portions, while stirring. It is heated until vapours of SO 3 are evolved. After cooling down,10 ml of distilled water is added. The excess of KMnO 4 and manganese oxides are eliminated by adding H 2 O 2. Iron is isolated by precipitation as hydroxide. After filtration the solution is transferred in to 25 ml standard flask and the volume is brought to the mark with distilled water. Liver sample: 2 to 5 gm of dried liver sample was taken in a 250 ml beaker. 6 ml of 1:1 nitric acid and perchloric acid were added. The contents were digested for one hour and repeatedly treated with 6 ml portions of nitric acid and perchloric acid mixture until the solution becomes colorless. The acid solution was evaporated to dryness and the resulting white residue was dissolved in minimum volume of 1 M nitric acid and made up to the volume in a 100 ml volumetric flask : Real water samples Each filtered (with whatman No. 40) river water sample (250 ml) was mixed with 10 ml of concentrated nitric acid in 500 ml distillation flask. The sample was digested in the presence of excess potassium permanganate solution according to the method recommended by Fifield et.al 319, the solution was cooled and neutralized with a dilute NH 4 OH solution. The digest was transferred into a 25-mL standard flask and diluted upto the mark with distilled water : Biological samples The accuracy and applicability of the proposed method has been applied to the determination of lead in National Institute for Environmental Studies (NIES) No.1 tea leaves, No.2 Human Hair, No.3 Pond sediment. A 0.1 g sample was taken in a beaker and dissolved in concentrated nitric acid ( 5 ml) with heating. The solution was cooled, diluted and filtered. The filtrate was made to 100 ml with water in a calibrated flask. NIES, No.4 Vehicle Exhaust Particulates (1 g) was dissolved in 18 ml of concentrated nitric acid, 18 ml of concentrated perchloric acid and 2 ml of

16 56 concentrated hydrofluoric acid in a 100 ml Teflon beaker, evaporated to a small volume, filtered through a filter paper and made up to 100 ml with distilled water. An aliquot (10 50 ml) of the sample solution was taken individually and lead was determined by the general procedure : Composition and Stability of the complex The composition of the complex was determined by Job s continuous variation and mole ratio methods. The stability constant of complexes were calculated by using the data obtained from Job s plot : Job s continuous variation method In a set of (nine) 10-mL volumetric flasks, 3 ml of buffer solution and sufficient quantity of surfactant were added to each flask. Equimolar solutions of metal ion and reagent solutions were added to each flask in such a proportion that the total volume of the solution was 10 ml. The contents in each flask were made up to the mark with distilled water. The absorbance of the coloured complex in each flask was measured at a selected wavelength against a corresponding reagent blank. A plot between mole fraction of the metal ion (V M /V M +V L ) and the absorbance was made from which the composition of the complex was computed : Molar ratio method In a set of (ten) 10-ml calibrated flasks, 3 ml of buffer solution, required quantity of surfactant, constant amount of metal ion and known (varying) aliquots of the reagent solutions were added. The contents of each flask were made up to the mark with distilled water. The absorbance of the coloured complex in each flask was measured at a selected wavelength against a reagent blank prepared under identical conditions. The composition of the complex was ascertained by plotting a graph between absorbance and volume of reagent.

17 Determination of the stability constant of the complex The spectrophotometric data obtained in the Job s method was used to calculate the stability constant of the complex species under investigation. For the complex formation reaction, the stability constant in terms of experimentally obtained absorbance value was given by the general equation. mm + nl M m L n m m n A/ A n [(1 A/ A m m n m )] [ C] m n 1 Where A m = absorbance corresponding the point of intersection of extrapolated lines A = observed absorbance at concentration, C C = concentration corresponding to the point of intersection = stability constant for 1:1 complex 1:2 complex (1 ) (1 ) C 4 C Where Am A Am, where = degree of dissociation Derivative spectra The following procedure was adopted for recording the first and second derivative spectra of complex in aqueous medium. In a 10-mLstandard flask, the metal complex was prepared by taking 3 ml of buffer, suitable volume of surfactant, suitable concentration of metal ion and reagent (usually 10- fold molar excess to metal ion) solutions. The contents were diluted up to the mark with distilled water. From the zero order spectrum the derivative (first and second order) spectra were recorded with 9 degrees of freedom, scan speed fast in a wave length region nm against the reagent blank prepared identically. The derivative amplitude was measured by peak zero method.