Poster
# 55
Poster |
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6th Internet World Congress for Biomedical Sciences |
Bartolomé Quintero(1), María del Carmen Cabeza(2)
(1)(2)Dpt. Physical Chemistry. Faculty of Pharmacy. University of Granada - Granada. Spain
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[Biophysics] |
[Cell Biology & Cytology] |
Chemicals of the highest available purity (Merck and Aldrich) were used to obtain PDQ tetrafluoroborate. DTPA, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), N-t-butyl- a -phenylnitrone (PBN) and a -(4-pyridil-1-oxide)-N-t-butylnitrone (4-POBN) were purchase from Sigma. Hydroquinone (Merck) was used to synthethize quinone by causing it to react with potassium bromide in an acid medium at 80ºC. The solid obtained had a melting point of 115ºC. Aqueous solutions of quinone showed an absorption maximum at 245 nm ( e = 21500 ± 400 M-1 cm-1). PDQ tetrafluoroborate was synthesized by us. The solid obtained was stored in darkness at below -18ºC. The synthesized solid was subject to elemental analysis in a Fisons-Carlo Erba EA 1108CHNS-0 Elemental Analyzer (Scientific Instrument Center [SIC], University of Granada). IR spectra were registered at room temperature with an FT-IR Nicolet 20SXB spectrophotometer (SIC, University of Granada). For ESR measurements a Bruker ESP 300E spectrometer (SIC, University of Granada) was used. Perkin-Elmer Lambda 5 and Lambda 16 spectrophotometers were used for the spectrophotometric analysis of PDQ tetrafluoroborate. Merck-Hitachi equipment for HPLC, including a Merck L-6220 biocompatible pump and a Merck L-4500 diode array detector, were used. Aqueous media were filtered by Millipore HA filters with a pore size of around 0.45 m m. The column was a Spherisorb ODS-2 (4.6 mm x 200 mm) with a particle size of 5 m m. The mobile phases were deaereated with a Selecta P 150 W ultrasound generator, producing waves of 40 kHz. Mobile phase methanol/ammonium formiate 0.1 M (1:39) with a flow of 0.7 mL/min was routinely used. The PDQ solutions were in buffer phosphate (0.05 M, pH 6.97) with concentrations of between 12 mM and 10 m M. The possible appearance of hydroquinone was monitored by steady-state fluorescence using a Shimadzu RF-5001 PC spectrofluorimeter. A Radiometer pH M64 potentiometer with a GK2401C mixed electrode was used whenever called for. The calibrations were carried out with Crison buffer references (pH 4 and pH 7).
We checked for any effects caused by either laboratory environmental light or apparatus light sources on PDQ tetrafluoroborate degradation. The results were taken into account when designing the methodology for the spectrophotometric and spectrofluorimetric measurements. All measurements were routinely made with aliquots taken from a stock solution kept in darkness.The influence of oxygen dissolved in the solution upon the PDQ decomposition rate was noted at an early stage in our experiments and thus whenever deaereated samples were required the oxygen was purged by bubbling with argon for at least 10 min.
Synthetized PDQ tetrafluoroborate is a yellowish crystalline solid which melts in the range of 135ºC to 140ºC accompanied by a noticeable change of colour and the formation of gas. The results of the elemental analysis were C: 42.8%; H:2.76% and N:17.05%. These results agree very well with the formation of the tetrafluoroborate of the dimer of PDQ. In the IR spectrum are noticeable bands at 2189 cm-1 (N º N stretching) and 1591 cm-1 (aromatic group). From the spectrophotometric measurements a value of e = 41990 ± 220 M-1 cm-1 was obtained at the absorption maximum (348 nm) using solutions in neutral aqueous medium (phosphate buffer, pH7). Likewise, spectrophotometric measurements were used to detemine the value of the pKa correspondig to the hydroxyl dissociation in PDQ. The result obtained was 3.35 ± 0.02 at 25 ºC. Chromatographic analysis by HPLC of newly prepared PDQ aqueous solutions (phosphate buffer, pH7) showed only one signal, with a retention time of 6.66 min, associated to a spectrum with a maximum at 348 nm.
The kinetic analysis performed in the present work were made by incubating 0.4 mM PDQ solutions either in the presence or in the absence of other chemicals (DTPA, ferrous ion) kept in darkness at 37 ºC. From this solutions aliquots were taken to made PDQ 0.01 mM solutions which were used for the spectrophotometric measurements.
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[Biophysics] |
[Cell Biology & Cytology] |
