Samples Raman shift standards

In some on-line cases, the Raman-shift standard may be left in place to validate the total calibration of the system using the other Raman lines of cyclohexane. Alternatively, if the sample position is not physically accessible, then another standard (e.g., a process solvent if this material has known, defined bands which are stable) can be used instead. By comparing the Raman shifts and amplitudes of the measured Raman peaks with the stored shifts and amplitudes, it can be quickly established if the unit is within accepted calibration values. 

Feedback correction must be applied to every sample measurement without operator intervention and without degrading the operating speed of the system. Of the calibration observations that are needed, all except the white-light spectrum need to be recorded simultaneously or nearly simultaneously with every sample measurement. To deal with laser position shift, a Raman-shift standard spectrum can be obtained through the sample channel—more on this later. 

The primary tool is measurement of the Raman signal produced by a standard substance, perhaps sapphire or diamond, both of which have strong and simple spectra. The Raman shift is known and the wavelength is measured by the procedure that is described earlier. One aspect of the compensation routine is to include a measurement of the Raman-shift standard in the system self-test that is run at the start of each measurement session under the conditions that will obtain when samples are being run. A Raman-inactive substance that scatters is used as a sample. This produces a strong shift standard spectrum that permits unambiguous identification of the position of its signal. Its intensity provides an observation of the condition of the laser. 

Returning to the dispersive case, it is far more reliable to use many calibration lines than to use only one. Ideally, a large number of accurately known frequencies would be dispersed across the spectrum, then observed under the same conditions as the sample. Assuming the optical and data acquisition conditions are precisely reproduced for the standard and the sample, the sample frequencies may be accurately calculated from the standard spectrum. As noted earlier, Raman shifts may then be determined from the equally accurately known laser frequency. 

Table 10.4. Raman Shifts of Standard Samples (ASTM E1840-96)"...

For the case of Raman shift accuracy, the simplest criterion for tracking instrument performance is the standard deviation of several observed Raman shifts for standard samples. The standard should be examined under the same experimental conditions as subsequent experimental samples, and cover at least as large a Raman shift range. For example, 4-acetamidophenol has peaks covering the Raman shift range of most common samples (see Fig. 5.7), and ASTM shift values are available with standard deviations <1.0cm (Table 10.4). If the user needs to qualify an instrument for very low or very high Raman shift, or for greater precision, different standards must be used. 

The example is illustrated by the results of Table 10.5. The Raman shift range from 400 to 2000 cm was calibrated with the 4-acetamidophenol shift standard, and the calibrated spectrum was recorded and stored on disk. Then calcium ascorbate was observed, with and without recalibration between spectra. Finally, spectra of calcium ascorbate were obtained approximately daily (each after recalibration) over a period of 2 months. The 769- and 1582 cm peaks were chosen for analysis, and their peak frequencies were determined by a center-of-gravity criterion included in the data analysis software (GRAMS 32). It is important that these qualification spectra duplicate the instrumental conditions to he used for real samples, at least as far as optical geometry, sampling mode, and calibration procedure. The objective is to provide an accurate indication of instrument performance in the intended application. 

Figure 10.8. Schematic of procedure for correction of spectra for instrumental response variation. R represents the response function 0z. and

In Raman spectroscopy the intensity of scattered radiation depends not only on the polarizability and concentration of the analyte molecules, but also on the optical properties of the sample and the adjustment of the instrument. Absolute Raman intensities are not, therefore, inherently a very accurate measure of concentration. These intensities are, of course, useful for quantification under well-defined experimental conditions and for well characterized samples otherwise relative intensities should be used instead. Raman bands of the major component, the solvent, or another component of known concentration can be used as internal standards. For isotropic phases, intensity ratios of Raman bands of the analyte and the reference compound depend linearly on the concentration ratio over a wide concentration range and are, therefore, very well-suited for quantification. Changes of temperature and the refractive index of the sample can, however, influence Raman intensities, and the band positions can be shifted by different solvation at higher concentrations or... 

TERS on DNA bases was demonstrated for the hrst time in 2004 by Watanabe et al. In that work, experiments and density-functional theory (DFT) calculations on adenine molecules in a nanocrystal were presented. From the acquired spectra, which differed from standard Raman spectra, it was concluded that those crystals were mechanically deformed in contact-mode TERS [74]. Consequently, band shifts were observed and attributed to interactions of molecule and metal tip. Comparing SER and TER spectra, a band shift could be observed, too, mainly caused by more specihc interactions of adenine and metal. In SERS of adenine on silver island hims, molecules were evenly attached to the rough surface by the amino group and its adjacent nitrogen atoms (N1 and N7). In contrast, in contact-mode TERS experiments (i.e., tip always touches the sample), the silver tip was constantly moved over the molecules with a force of - 5 pN per molecule. Based on theoretical calculations, the authors concluded that the TERS probe was selectively pressed to one nitrogen atom (namely N3). Later it was shown that in contact-mode TERS, an adsorption-desorption process of the molecule at the tip could be responsible for the band shift [62]. 

The curing of four UV-curable clearcoats was studied by confocal Raman microscopy. The disappearance of the C C line near 1636/cm provided the signature for the curing of the samples, but quantification of the degree of cure by standard peak fitting and baseline subtraction methods did not work well because of sample fluorescence, baseline shifts and overlapping peaks. A smoothed second-derivative processing approach overcame all of these difficulties and provided a simple. 

---