Wavelength accuracy

Definition and Uses of Standards. In the context of this paper, the term "standard" denotes a well-characterized material for which a physical parameter or concentration of chemical constituent has been determined with a known precision and accuracy. These standards can be used to check or determine (a) instrumental parameters such as wavelength accuracy, detection-system spectral responsivity, and stability (b) the instrument response to specific fluorescent species and (c) the accuracy of measurements made by specific Instruments or measurement procedures (assess whether the analytical measurement process is in statistical control and whether it exhibits bias). Once the luminescence instrumentation has been calibrated, it can be used to measure the luminescence characteristics of chemical systems, including corrected excitation and emission spectra, quantum yields, decay times, emission anisotropies, energy transfer, and, with appropriate standards, the concentrations of chemical constituents in complex S2unples. 

Calibration. In general, standards used for instrument calibration are physical devices (standard lamps, flow meters, etc.) or pure chemical compounds in solution (solid or liquid), although some combined forms could be used (e.g., Tb + Eu in glass for wavelength calibration). Calibrated lnstr iment parameters include wavelength accuracy, detection-system spectral responsivity (to determine corrected excitation and emission spectra), and stability, among others. Fluorescence data such as corrected excitation and emission spectra, quantum yields, decay times, and polarization that are to be compared among laboratories are dependent on these calibrations. The Instrument and fluorescence parameters and various standards, reviewed recently (1,2,11), are discussed briefly below. 

For ease of use and wavelength accuracies of 1-2 nm, organic materials or inorganic ions in solution have been recommended as standards (Table II). However, these must be used carefully because (a) the peak maxima are matrix dependent, (b) narrow Instrumental bandpasses are necessary, (c) impurities may affect peak location, and (d) the peak wavelength values have generally not been certified (11). 

Esquivel, J. B., Wavelength accuracy testing of UV-visible detectors in liquid chromatography, Chromatogmphia, 26, 321, 1988. 

UV detector wavelength accuracy, absorbance linearity, and sensitivity ... 

Waters Symmetry CIS (150x3.9 mm i.d., 5 pm) used in most tests except wavelength accuracy and compositional accuracy tests. 

FIGURE 6 Automated 205/273 nm wavelength stepping results for wavelength accuracy test, using caffeine standard solution. Courtesy of PerkinElmer LAS, Shelton, CT. 

On some detectors, a holmium oxide filter or special holmium oxide cell is integrated into the detector these can then be moved into the optical path during calibration mode. The detector itself or associated control software may then be able to check for wavelength accuracy. Note If the filter or cell is made of glass, and not quartz, 241 nm may not be used. 

Experiment 4 is intended to check the detector s wavelength accuracy at 205nm and 273nm. Using the same caffeine solution as for the OQ test, this test is performed and calculated as in the procedure described earlier under subsection (i) of Detector in section (j) of the OQ guidelines. 

It is recommended that OQ test the following on an HPLC system flow accuracy, pump compositional accuracy, pressure pulsations, column oven temperature accuracy/stability, detector noise/drift and wavelength accuracy, autosampler injection precision and carryover. 

The protein analyzer tests response linearity (absorbance/fluorescence), wavelength accuracy and UV linearity, dynamic noise, drift, and the zero offset. 

The parameters that require qualification for a UV absorbance detector are wavelength accuracy, linearity of response, detector noise, and drift. These determine the accuracy of the results over a range of sample concentrations and the detection limits of the analysis. 

The wavelength accuracy and detector linearity and detector noise have the same effect on laser-induced fluorescence, as those of a UV absorbance detector. 

Linearity problem Broken optical fiber Wavelength accuracy Detector linearity Noise... 

Laser-induced Wavelength accuracy problems Incorrect analysis... 

Wavelength accuracy and reproducibility reproducibility of sensitivity/response factors Heteroscedasticity... 

Reproducible slope ( = sensitivity, response factor) linearity Wavelength accuracy Efficiencies, peak shapes... 

The detection method can also be a source of potential variations from instrument to instrument. For example, UV detectors usually specify the wavelength accuracy to within +/-1-3 nm, and a change of this value could prove significant. Another, often ignored, factor is the time constant of the detector. Too low a setting of this factor can show significant noise levels and too high a value can distort the peak shape. 

UV-Vis detector The detectors should operate with a wavelength range from 200 to 800 nm. The detector should have a wavelength accuracy of +2nm. The detector response should be linear with a correlation coefficient r2 not less than 0.999 over the full dynamic range. The linear range should be up to 2.0 Absorbance Unit Full Scale (AUFS). 

Pump flow rate accuracy and gradient accuracy Detector linearity of response, noise, drift, and wavelength accuracy Injector precision, linearity, and carryover Column heater temperature accuracy... 

Robustness for UV-Vis Analysis. Wavelength accuracy, wavelength repeatability, diluting solvent (i.e., pH, concentration), solution stability, and bubble formation by the sipper can be investigated during validation of the analytical component. 

Detector linearity of response, noise, drift, and wavelength accuracy... 

Wavelength accuracy and reproducibility, stray light, resolution, photometric accuracy and reproducibility, noise, baseline flatness, stability, and linearity... 

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