Chapter-2 Materials and Methods
2 Experimental Sections
A comprehensive account of the materials used in synthesis and relevant particulars of the instruments/equipments that has been used for the synthesis and characterization of all compounds are described in this chapter.
2.1Materials
All the chemicals and solvents used for the present work were for analytical grade quality and were used without purification unless otherwise specified. Triethanol amine, thionyl chloride, 1-naphthol, 2-naphthol, tris-(2-aminoethyl) amine (tren), 8-hydroxyquinoline, sodium metabisulfite, sodium nitride, imidazole, 1-napthyl amine, aniline, p-Me aniline, p- OMe aniline, p-Cl aniline, p-Br aniline, p-I aniline, different metal salts, different organic acids and esters were obtained from Aldrich (US) and used as received. Inorganic acids were obtained from Merck (India) and used as received. All the solvents used for spectroscopic studies were of spectroscopy grade and further purified (dried and distilled) by following standard procedure.2.1
2.2 Particulars of Instruments/Equipment used for the following physiochemical studies
pH Measurement
pH values of the reaction solutions were recorded with a Systronics Type 355 digital pH meter and also by using Merck pH indicator paper.
Electrical Conductance Measurement
Solution electrical conductance measurements were measured on a Systronics Type 304 direct digital reading conductivity meter. Solution strength was maintained at 10-3 M in appropriate solvents by using 0.01N KCl solution as calibrate.
Infrared Spectroscopy
Infrared spectra of the compounds were recorded as KBr pellets or as thin films using a Nicolet Impact- 410 Fourier Transform Infra Red Spectrophotometer, or on a Perkin- Elmer Spectrophotometer (spectrum one)
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Chapter-2 Materials and Methods
Electronic Absorption Spectroscopy
UV-visible spectra were recorded, by dissolving a calculated amount of the sample in appropriate solvents, on a Hitachi UV-visible U-2001 Spectrophotometer or on a Perkin Elmer Lambda 25 UV-Visible Spectrophotometer.
1H and 13C NMR Nuclear Magnetic Resonance Spectroscopy
1H and 13C NMR spectra were recorded on a Varian 400 MHz spectrometers using tetramethylsilane (TMS) as internal standard.
Thermal Studies
Thermogravimetry (TG) and Differential Scanning Calorimetry (DSC) were conducted on a Metallo-Toledo TGA/SDTA 851e and 821e instruments. Experiments were done using either aluminum or platinum crucibles. Pure N2 gas was used as the flow gas.
Powder X- ray Diffraction (PXRD)
The powder XRD was recorded on BRUKER-D8 ADVANCE with CuKα source (λ = 154 Å ) on a glass surface of air dried sample.
Magnetic susceptibility
Solid-state magnetic susceptibility of the complexes at room temperature was recorded using Sherwood Scientific balance MSB-1.
Elemental analysis
Elemental analyses were carried out on a Perkin-Elmer 2400 automatic carbon, hydrogen and nitrogen analyzer.
Mass Spectrometry
HRMS spectra were recorded in WATERS LC-MS/MS system, Q-Tof PremierΤΜ.
Electrochemistry
Cyclic voltammetric measurements were carried out using a CH Instruments make CHI660C electrochemistry system. The cell contained a glassy carbon working electrode, a Pt wire auxiliary electrode and a saturated calomel electrode (SCE) as reference electrode. A salt bridge (containing supporting electrolyte, tetra-n-butyl ammonium perchlorate (TBAP) dissolved in dry MeCN) was used to connect the SCE with the
Chapter-2 Materials and Methods
electrochemistry solution.2.2 All experiments were carried out under a dinitrogen atmosphere at RT and were uncorrected for junction potentials. Under our experimental conditions, the E1/2 values (in Volts) for the couple Fc+/Fc were 0.45 in MeCN vs. SCE.
EPR Spectroscopy
X-Band EPR spectra were recorded with a Jeol JES-FA series spectrometer fitted with a quartz dewar for measurements at liquid nitrogen temperature. The spectra were calibrated with with an internal manganese marker.
Optical Microscopy
Optical micrograph images were taken in Zeiss-Axio Cam-MRc microscope fitted with the digital camera of air-dried samples on glass micro slides.
Steady state Fluorescence
The steady state fluorescence spectra were recorded on a Varian Cary-Bio spectro- fluorimeter and corrected for emission. Fluorescence quantum yield was determined in each case by comparing the corrected spectrum with that of naphthalene (ΦF = 0.23) 2.3 in ethanol by taking the area under the total emission using the following equation .2.4
Φ
S =Φ
R (FSAR/FRAS)(η
S/η
R)2where ΦS and ΦR are the radiative quantum yields of the sample and the reference, FS and FR are the area under the fluorescence spectra of the sample and the reference, AS and AR
are the absorbance of the sample and the reference (at the excited wavelength), ηS and ηR are the refractive indices of the solvent used for the sample and the reference. The quantum yield of naphthalene was measured using quinine sulfate in 1N H2SO4 as reference at λex of 350 nm (ΦF = 0.54). The quantum yield of naphthalene was measured using quinine sulfate in 1N H2SO4 as reference at λex of 350 nm (ΦF = 0.54).
Time-resolved Fluorescence Spectroscopy
Time-resolved intensity decays of the proteins were measured using a Life Spec II spectrofluorimeter (Edinburgh instrument). The sample was excited by Pico-quant 290 nm laser source and the decay was measured through 50 ns time scale at a time resolution of 0.0122 ns/channel. The decay curves were analyzed by FAST software using discrete
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Chapter-2 Materials and Methods
exponential method, provided by Edinburgh instrument along with the fluorescence instrument. The generated curves for intensity decay were fitted in the functions:
( )
iexp( )
i i
I t − t
= α
∑ τ
Where, τi is the initial intensity of the decay component i, having a lifetime αi. The mean lifetime (τm) of BSA in different experimental condition was calculated following the equation2.5
i i i m
i i
τ = α τ
α
∑ ∑
X-ray Crystallography
The intensity data were collected using a Bruker SMART APEX-II CCD diffractometer, equipped with a fine focus 1.75 kW sealed tube Mo Kα radiation (λ = 0.71073 Å) at 273(3) K, with increasing ω (width of 0.3° per frame) at a scan speed of 3 s/frame. The SMART software was used for data acquisition. Data integration and reduction were undertaken with SAINT and XPREP2.6 software. Multi-scan empirical absorption corrections were applied to the data using the program SADABS.2.7 Structures were solved by direct methods using SHELXS-97 and refined with full-matrix least squares on F2 using SHELXL-97.2.8 All non-hydrogen atoms were refined anisotropically. The hydrogen atoms were located from the difference Fourier maps and refined. Structural illustrations have been drawn with ORTEP-3 for Windows.2.9
References:
2.1. Armarego, W. L. F.; Perin, D. D. Purification of Laboratory Chemicals, Fourth addition, 1997.
2.2 Sawyer, D. T.; Roberts, J. L. Jr, Experimental Electrochemistry for Chemists, Wiley, New York, 1974.
2.3 Birks, J. B. Photophysics of Aromatic Molecules, Wiley-Interscience, New York, 1970.
2.4 Uchiyama, S.; Matsumura, Y.; de Silva. A. P.; Iwai, K. ; Anal. Chem., 2003, 75, 5926.
2.5 Swaminathan, R.; Krishnamoorthy, G.; Periasamy, N. Biophys J., 1994, 67, 2013.
2.6 SMART, SAINT and XPREP, Siemens Analytical X-ray Instruments Inc., Madison, Wisconsin, USA, 1995.
2.7 Sheldrick, G. M., SADABS: software for Empirical Absorption Correction, University of Gottingen, Institute fur Anorganische Chemieder Universitat, Tammanstrasse 4, D-3400 Gottingen, Germany, 1999–
2003.
2.8 Sheldrick, G. M.; SHELXS-97, University of Gottingen, Germany, 1997.
2.9 Farrugia, L. J. Appl. Crystallogr., 1997, 30, 565.