Pathlength calibration

The amplitude of the waveform will vary from 2 to 15%, depending on the state of the windows. The relationship between the pathlength of the cell, L (in cm), and the peak-to-peak fringes is given by the following relationship  

If the spectrometer is calibrated in wavelengths then the above equation can be converted into a more convenient form, as follows  

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The method of pathlength calibration by using interference fringes was outlined. 

Sensitivity The sensitivity of a molecular absorption analysis is equivalent to the slope of a Beer s-law calibration curve and is determined by the product of the analyte s absorptivity and the pathlength of the sample cell. Sensitivity is improved by selecting a wavelength when absorbance is at a maximum or by increasing the pathlength. 

Cuvettes are matched if they are identical in terms of pathlength and reflective-refractive properties. Cuvettes used for calibration and the analysis of samples after calibration must be matched so that absorption readings are due solely to concentration effects and not cuvette differences. 

A vernier adjustment (scale expansion) between fixed ranges is also available for calibration of absorbance and transmittance. The dual flow cells have a capacity of 8 /il and a 10-mm optical pathlength. 

Results and Discussion. The 2-ethyl polyaniline concentration in the silica gel film was determined by constructing a Beer s law calibration curve from solutions of known concentration. Assuming an average molecular weight of 5000, the 2-Et PANi concentration in the silica gel was found to be 9.6 x 10 4 M. The refractive indices of CS2 and 2-Et PANi SiC>2 were estimated to be 1.6 and 1.4 at 1.06 im, respectively. The emeraldine base doped silica gel was found to have low losses due to scatter, and exhibited good transparency at 1.06 im. Spectrophotometric measurements at 1.06 fim yielded absorption coefficients of 0.1 cm-1 (> 99% T over 1 mm pathlength) for the CS2 reference and 4 cm 1 (96% T over 1 mm pathlength) for the 2-Et PANi doped silica film. 

What NIR spectral region will be targeted for the calibration development for example, combination region, first combination/overtone (FCOT), second combination/overtone (SCOT) Each region will imply a different transmission pathlength, and hence cell or probe design. 

Calibration Determine the absorbance of each Standard Solution in a 1-cm pathlength cell at 490 nm against the water blank. Calculate the slope of the curve obtained by plotting absorbance versus micrograms per milliliter of lactose. The slope of the curve is the absorptivity (a) of the lactose-reagent product. 

As determined from HPLC, the purity of the peptide was greater than 90%. Steady-state fluorescence spectra of this peptide were collected from 310-480 nm with an excitation wavelength of 264 nm. A 1 cm pathlength cuvette was used with concentrations of 8.6 pM. The emission quantum yields were determined relative to N-acetyl-L-tryptophanamide at pH = 6.9 (f = 0.13) (17). Steady-state circular dichroism spectra were obtained using a 0.5 mm pathlength cylindrical cell and concentrations of 0.26 mM. The mean molar ellipticity [0] (deg cm dmol" ) was calibrated with (+)-10-camphorsulfonic acid. Concentrations of the solutions were determined by measuring the absorbance of 4-methylaminobenzoic acid. 

As the absorbance of a component in solution depends not only on its concentration but also on the cell pathlength (equation (13)), for quantitative work it is necessary to measure the pathlength accurately otherwise, the quantitative methods developed will be specific to the cell used for the calibration. The pathlength of a transmission cell can be determined by using the interference fringes that appear in the spectrum of the empty cell (Figure... 

Since AAS is t5q)ically a trace technique, it might happen that the analyte content of certain samples are above the linear range of the response. The initial temptation to simply dilute the offending samples should be resisted in favour of a more efficient procedure. By simply rotating the burner head somewhat a portion of the flame is removed from the optical beam. The net effect is that the pathlength of the sample within the optical beam is shortened. Standards are simply rerun under the modified conditions and a new calibration plot is established. Since all manipulations of the sample(s) and/or standards increase uncertainty, it is to be anticipated that the linear range of the analyte response will be increased appreciably but that the anticipated precision should not be adversely affected. By contrast, having to dilute samples will probably decrease the precision as well. 

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