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Intense luminescence backgrounds cause significant problems in quantitative Raman spectroscopy, particularly in multivariate analysis where background suppression is essential. Taking second derivatives reduces the background, but differentiation increases the apparent noise that arises on spectra recorded with CCD detectors due to random, but fixed, variations in the pixel-to-pixel response. We have recently reported a very general method for correcting CCD fixed-pattern response in which spectra are taken at two or more slightly shifted spectrometer positions and are then subtracted to give a derivative-like shifted, subtracted Raman (SSR) spectrum. Here we show that differentiating SSR data (which has inherently higher S/N than the undifferenced data) yields spectra that are similar to those that are obtained from the normal two-step differentiation process and can be characterized as pseudo-second-derivative, PSD, spectra. The backgrounds are suppressed in the PSD spectra, which means they can be used directly in multivariate data analysis, but they have significantly higher S/N ratios than do simple second derivatives. To demonstrate the improvement brought about by using PSD spectra, we have analyzed known samples, consisting of simple binary mixtures of methanol and ethanol doped with laser dye. When the background levels of all samples included in the models were < or =10x greater than the intensity of the strongest Raman bands, partial least-squares calibration of the PSD data gave a standard error of prediction of 3.2%. Calibration using second derivatives gave a prediction error which was approximately twice as large, at 6.5%; however, when data with background levels . approximately 100x larger than the strongest Raman bands were included, the noise on the second-derivative spectra was so large as to prevent a meaningful calibration. Conversely, the PSD treatment of these samples gave a very satisfactory calibration with a standard error of prediction (3.3%) almost identical to that obtained when the most fluorescent samples were excluded. This method clearly has great potential for general purpose Raman analytical chemistry, because it does not depend on specialized equipment, is computationally undemanding, and gives stable and robust calibrations, even for samples in which the luminescence background level fluctuates between the extremes of being practically zero and completely dominating the Raman signal.

The Stein Collection in the British Library contains the Diamond Sutra, the world's oldest, dated, printed document. The paper of the Diamond Sutra and other documents from the Stein collection is believed to be dyed yellow by a natural extract, called huangbo, from the bark of Phellodendron amurense, which contains three major yellow chromophores: berberine, palmatine, and jatrorrhizine. Conservation of these documents requires definite information on the chemical composition of the dyes but no suitable, completely noninvasive analytical method is known. Here we report resonance Raman studies of a series of pure dyes, of plant materials and extracts, and of dyed ancient and modern paper samples. Resonance Raman spectroscopy is used to enhance the spectra of the dyes over the signals from the paper matrixes in which they are held. The samples all give resonance Raman spectra which are dominated by intense fluorescence, but by using SSRS (subtracted shifted Raman spectroscopy) we have obtained reliable spectra of the pure dyes, native bark from the Phellodendron amurense, modern paper dyed with huangbo extracted from this bark, and ancient paper samples. For both ancient paper samples whose pigment bands were detected, the relative intensities of the bands due to berberine and palmatine suggest that the ancient paper is richer in berberine than its modern counterpart. This is the first nondestructive in situ method for detection of these pigments in manuscripts, and as such has considerable potential benefit for the treatment of irreplaceable documents that are believed to be dyed with huangbo but documents on which conservation work cannot proceed without definite identification of the chemical compounds that they contain.