A semiautomated method combining intensity normalization with effective elimination of the solvent signal and non-Raman background is presented for Raman spectra of biochemical and biological analytes in aqueous solutions. The method is particularly suitable for rapid and effortless preprocessing of extensive datasets taken as a function of gradually varied physicochemical parameters, e.g. analyte and/or ligand concentration, temperature, pH, pressure, ionic strength, time, etc. For intensity normalization, the strong Raman OH stretching band of water in the range of 2700-3900 cm −1 recorded together with the analyte spectrum in the fingerprint region below 1800 cm −1 is employed as internal intensity standard. Concomitant dependences of the solvent Raman spectra are taken into account and, in some cases, turned into advantage. Once the Raman spectra of the solvent are acquired for a particular range of the parameter varied, solvent contribution can be subtracted correctly from any analyte spectrum taken within this range. The procedure presented can be efficiently applied only for the analytes having their own Raman signal in the range of OH stretching vibrations much weaker than that of the solvent. However, this is the case for a great number of biochemical and biological samples. Accuracy, reliability and robustness of the method were tested under the conditions of spontaneous Raman, resonance Raman and surface-enhanced Raman scattering. Serviceability of the method is demonstrated by several real-world examples.
In SERS-active systems, when a free base porphyrin is adsorbed at a roughened metal surface, the metal ion may be incorporated into the porphyrin core. We have investigated the metalation kinetics of 5,10,15,20-tetrakis(1methyl-4-pyridyl)porphyrin (TMPyP), reÑected in the time evolution of its SERRS spectra. To establish proper metalation Raman markers, resonance Raman spectra of the free base and chemically Ag metalated TMPyP forms were measured under di †erent conditions. The increase in intensity of the 395 cm-1 line, the decrease in intensity of the 329 cm-1 line, the reduction of the 1337 + 1360 cm-1 doublet to one strong line at 1340 cm-1 and the vanishing of the 1140 cm-1 medium line (the last exceptionally for 514.5 nm excitation) were found to be suitable markers for quantitative analysis. All other spectral changes related to metalation may coincide with e †ects caused by other variations of the porphyrin state. Time evolution of the porphyrin metalation was studied in various Ag colloid systems, with or without phosphate anions, citrate and/or Triton X-100. Time-dependent SERRS spectra of TMPyP were recorded within periods from minutes to several hours. Factor analysis of the SERRS spectra proved that the spectral changes were induced by only one phenomenon (metalation). The SERRS spectra were decomposed into the spectrum of the free base TMPyP and that of its Ag metalated form, and the metalation kinetics were determined by means of the time dependence of the metalated form portion in the SERRS spectrum. The results show a remarkable reliance on the state of the metal surface.
Processes of adsorption and metalation of a cationic water-soluble free base porphyrin, i.e., 5,10,15,20-tetrakis-(1-methyl-4-pyridyl)porphyrin (TMPyP) on Ag colloids have been monitored by surface enhanced resonance Raman spectroscopy (SERRS) as a function of time and porphyrin concentrations. Ag colloids employed were prepared either by laser ablation or by chemical reduction (by sodium borohydride and citrate) of Ag salts. SERRS intensities of TMPyP depend on the morphology of the colloidal aggregates in each system; aggregation, in turn, is governed by a balance between the so-called "diffusion limited aggregation" and "contact limited aggregation" processes, strongly influenced by the TMPyP concentration and the initial state of the colloidal particles (i.e. the residuals ions stuck at the substrate surface). The time evolution of the overall SERRS intensities is thus a function of the colloid preparation procedure. On the other hand, the kinetics of metalation reflects primarily the accessibility of the substrate surface for porphyrin adsorption. Consequently, it is highly sensitive to porphyrin concentration, the covering of the colloid surface by porphyrin molecules being the main qualitative limiting factor. Moreover, the process of metalation is controlled by residual ions at the substrate surface and therefore completely different for laser-ablated and chemically prepared Ag colloids. In the former, the amount of the metalated species is only limited by the adsorbate concentration while in the latter it is also mediated by the removal of residual ions from the colloidal surface by porphyrin molecules.
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