Absolute Response Calibration

In principle, one could calculate an absolute Raman cross section from the response of an instrument calibrated with a standard radiometric source. This approach is difficult but has been used to provide the cross sections in Table 2.2. If the relative response function is calibrated accurately, however, it is much simpler to determine cross sections by comparison to standards. Provided the sample positioning and optics permit quantitative Raman signal reproducibility, cross sections of liquids may be determined by comparing the response-corrected peak area to a band with known absolute cross section, such as the benzene 992 cm band. For response-corrected spectra, the ratio of the peak areas under identical experimental conditions equals the ratio of the absolute cross sections. 

---

Calibration is used here to describe whatever process is used to relate observed spectral frequencies and intensities to their true values, and validation is a procedure to verify the calibration and determine the magnitude of experimental error. Raman spectroscopy is a demanding technique in terms of reproducibility and accuracy and involves a variety of instrumental configurations. Calibration is often the source of irreproducibility and inconsistency in reported Raman spectra. This chapter is divided into four general sections frequency calibration (10.2), response function calibration (10.3), absolute response calibration (10.4), and a summary of procedures (10.5). For each section, standards and procedures for instrument validation are considered. 

There is no widely used detector that has predictable absolute response factors. This is why predicted rather than calibrated response factors are only used when calibration is not practical, such as for unknowns, or when a closely related compound has been calibrated, allowing extrapolation to the desired response factor. 

In summary, it can be said of atmospheric oxidant that, on the average, 75 25% can be identified as ozone. When properly standardized, the techniques discussed yield essentially the same response to the smog oxidant. However, the rather large differences among the laboratory response curves to ozone indicate the urgent need to clarify the chemistry of these systems. In particular, there is real need to have available an absolute ozone calibration method in the range of 0 to 100 p.p.h.m. 

Calibration of the instrument is the first essential step of a quantitative procedure. Like any other analytical instrument, the response of a mass spectrometer is not absolute and might deviate with time. In addition, the sample matrix has a variable influence on the mass spectrometry response. Calibration involves determination of the correlation between a known concentration of the analyte and the resulting mass spectrometry signal. In ideal situations, the sample and the analyte standards are both analyzed under identical experimental conditions. Depending on the levels of accuracy and precision that are required, the calibration might be performed by one of the methods described below. 

The external standard method uses absolute response factors, the internal standard method is calibrated in terms of response ratios. In both calibration methods, each peak is calculated independently. In external standard methods, the amount of sample injected must be highly reproducible. The method is well suited for automatic mechanical methods of injection. Optimum system performance must be maintained by frequent checks and regular recalibration. The internal standard method is independent of sample size and compensates for any slight instrumental drift. When used properly, it is the most accurate... 

External Standardization Technique (EST). This method requires the preparation of calibration standards. The standard and the sample are run as separate injections at different times. The calibrating standard contains only the materials (components) to be analyzed. An accurately measured amount of this standard is injected. Calculation steps for standard (1) for each peak to be calculated, calculate the amount of component injected from the volume injected and the known composition of the standard then (2) divide the peak area by the corresponding component weight to obtain the absolute response factor (ARE) ... 

Analytical standards are prepared for two purposes for fortifying control matrices to determine the analytical accuracy and for calibrating the response of the analyte in the mass spectrometer detector. The purity of all standards must be verified before preparation of the stock solutions. All standards should be refrigerated (2-10 °C) in clean amber-glass bottles with foil/Tefion-lined screw-caps. The absolute volume of the standard solutions may be varied at the discretion of the analyst, as long as the correct proportions of the solute and solvent are maintained. Calibrate the analytical balance before weighing any analytical standard material for this method. 

Sample preparation, injection, calibration, and data collection, must be automated for process analysis. Methods used for flow injection analysis (FLA) are also useful for reliable sampling for process LC systems.1 Dynamic dilution is a technique that is used extensively in FIA.13 In this technique, sample from a loop or slot of a valve is diluted as it is transferred to a HPLC injection valve for analysis. As the diluted sample plug passes through the HPLC valve it is switched and the sample is injected onto the HPLC column for separation. The sample transfer time typically is determined with a refractive index detector and valve switching, which can be controlled by an integrator or computer. The transfer time is very reproducible. Calibration is typically done by external standardization using normalization by response factor. Internal standardization has also been used. To detect upsets or for process optimization, absolute numbers are not always needed. An alternative to... 

Collection of multiple data sets for each time span, with frequent alternation of the polarization, is an essential feature of our protocol. This provides some protection against the effects of drifts in laser power, photomultiplier quantum yield, and absolute calibration of the TAC, photochemical decomposition of the dye, and any other long-term processes that may alter the measured fluorescence response curves. Separate analysis of each data set is necessary to provide an indication of the uncertainty in run-to-run reproducibility and to detect and delete the rare spurious data set. 

The mean-centering operation effectively removes the absolute intensity information from each of the variables, thus enabling snbsequent modeling methods to focus on the response variations about the mean. In PAT instrument calibration applications, mean-centering is almost always nsefnl, because it is almost always the case that relevant analyzer signal is represented by variation in responses at different variables, and that the absolute values of the responses at those variables are not relevant to the problem at hand. 

Direction of the gas chromatographic effluent into a vessel containing activated carbon attached to an automatic recording electromicrobalance is the basis for the device known as the Brunei mass detector (47). This is an absolute analytical method and requires no calibration, and in fact, can be used to calibrate other detectors which have unpredictable responses. The sensitivity of the detector is in the same range as the thermal conductivity detector. 

As for the second condition, usually the spectral response, which is provided by the instrument manufacturers, is used. For the erythemal broadband detectors, it has been demonstrated that regular testing of their spectral sensitivity is needed, in addition and prior to their absolute calibration. This is because their spectral sensitivity is determined fiom a series of optical filters and other components (e.g. the phosphor layer) that may degrade with time or with environmental conditions (e.g. humidity), changing therefore its characteristics. 

The effect of the membrane length upon the blank and the calibration slope are shown for a 1 nM Hg2(N03)2 solution in Figure 13. Both blank and response increase with increasing membrane length butjplateaus after about 0.5 m. The response/blank ratios for the 0.1, 0.2, 0.3, 0.5 and 1.0 m lengths are 0.44, 0.53, 0.61, 0.73 and 0.73 in arbitrary units respectively. Since the blank noise was found to be approximately related to the absolute value of the blank, we chose a length of 0.5 m. 

Three calibration blank standards should be analyzed to establish a representative blank level, after which the calibration standards are analyzed. After calibration, the quality control standard should be analyzed to verify the calibration. The sample introduction system is flushed with rinse blank, and the blank solution is analyzed to check for carry-over and the blank level. If the blank level is acceptable, the samples can be analyzed. If the blank values are too high, the flushing of the sample introduction system and analysis of the blank solution should be repeated until an acceptable blank level is reached. The calibration blank value, which is the same as the absolute value of the instrument response, must be lower than the method s detection limit. 

The availability of an on-line radioisotope detector for CE is especially appealing for several reasons. First, state-of-the-art radiation detection technology offers extremely high sensitivity. Second, radioisotope detection affords unrivaled selectivity because only radiolabeled sample components yield a response at the detector. Third, the radiolabeled molecule possesses the same chemical properties as the un-labeled molecule, thereby permitting tracer studies. Fourth, radioisotope detection can be directly calibrated to provide a measurement of absolute concentration of the labeled species. Finally, a capillary electrophoresis system in which radioactivity detection is coupled with more conventional detectors adds extra versatility to this new separation technique. 

---