Wavelength setting

An hplc assay was developed suitable for the analysis of enantiomers of ketoprofen (KT), a 2-arylpropionic acid nonsteroidal antiinflammatory dmg (NSAID), in plasma and urine (59). Following the addition of racemic fenprofen as internal standard (IS), plasma containing the KT enantiomers and IS was extracted by Hquid-Hquid extraction at an acidic pH. After evaporation of the organic layer, the dmg and IS were reconstituted in the mobile phase and injected onto the hplc column. The enantiomers were separated at ambient temperature on a commercially available 250 x 4.6 mm amylose carbamate-packed chiral column (chiral AD) with hexane—isopropyl alcohol—trifluoroacetic acid (80 19.9 0.1) as the mobile phase pumped at 1.0 mL/min. The enantiomers of KT were quantified by uv detection with the wavelength set at 254 nm. The assay allows direct quantitation of KT enantiomers in clinical studies in human plasma and urine after adrninistration of therapeutic doses. 

A quality control laboratory had a certain model of HPLC in operation. One of the products that was routinely run on the instrument contained two compounds, A and B, that were quantitated in one run at the same detector wavelength setting. At an injection volume of 20 /tL, both compounds showed linear response. The relatively low absorption for compound B resulted in an uncertainty that was just tolerable, but an improvement was sought. 

The problem could have been resolved by running two injections, either with different wavelength settings and/or with different dilutions injected, but this would have appreciably increased the workload of the technicians and the utilization factor of the instrument. Without an additional instrument, the laboratory would have lost much of its flexibility to schedule additional analyses at short notice. Another solution would have been to utilize a programmable detector that switches wavelengths between peaks, but that only works if the weakly detected component strongly absorbs at some other wavelength. 

Figure 19. Procedure of measuring reflectrometric interference spectroscopy in a parallelized setup. Instead of white light, the wavelength obtaineds from some filters are used, and the interference spectra is constructed based on these supporting wavelengths. For each wavelength, all the measurement spots are obtained simultaneously, the total time for one wavelength set is less than 10 seconds.

Mass spectrometry detection gained the acceptance of bioanalytical scientists primarily based on its higher selectivity compared to detection that relies on UV/visible absorbance. Absorption spectra of aqueous solutions usually appear as broad absorbance bands. The selectivity provided by UV/visible absorbance for a colorless analyte is usually very low. To detect a colorless analyte, a wavelength setting below 210 nm is usually used. UV absorbance in this region is not specific because most compounds containing hetero-atoms and multiple bonds absorb UV below 200 to 210 nm. 

An impurities analytical procedure should be described adequately so that any qualified analyst can readily reproduce the method. The description should include the scientific principle behind the procedure. A list of reagents and equipment, for example, instrument type, detector, column type, and dimensions, should be included. Equipment parameters, for example, flow rate, temperatures, run time, and wavelength settings, should be specified. How the analytical procedure is carried out, including the standard and sample preparations, the calculation formulae, and how to report results, should be described. A representative chromatogram with labeled peak(s) should be included in the procedure. 

The use of a line source and the ratio method (i. e. 7/7) tend to minimize errors in AAS. Thus, if the wavelength setting is seriously incorrect, it is unlikely that any absorption will be observed. If the wavelength is incorrectly tuned, the effects on the value of7 will roughly equal those on the value of I , and the error may not be too serious. 

Turn the monochromator wavelength setting to 285 nm using the coarse adjustment, then use the fine wavelength control to tune in to the line maximum at 285.2 nm. 

If a spectrofluorometer is used, set the excitation wavelength to 280 nm and the emission wavelength to between 320 and 350 nm. If the exact emission wavelength is not known, determine it empirically by scanning the standard solution with the excitation wavelength set to 280 nm. If the instrument is a fdter fluorometer, use an excitation cutoff filter <285 nm and an emission fdter >320 nm. 

There is no inherent sample size restriction, large or small, but is fixed by the optical components used in the instrument. The diffraction limit of light, roughly a few cubic micrometers depending on the numerical aperture of the optics used and the laser s wavelength, sets the lower bound.7 In a process application, the type of fiber optics used also affects sample volume examined. Macroscopic to microscopic samples can be measured with the appropriate selections of laser wavelength, laser power, and optics. 

Figure 10. Luminescence spectrum of calcined vanadyl porphyrin on LaY at 77K with the excitation wavelength set at 468 nm.

Confirm wavelength setting. Confirm wavelength accuracy. 

Normal 2. Wrong detector wavelength 2. Confirm wavelength setting. 

In the monochromator, the diffraction grating produces a spectrum in the plane of the exit slit. The exit slit serves as a window to isolate the particular line (wavelength) of interest. When the wavelength setting of the monochromator is adjusted, the grating slowly rotates and the spectrum moves sideways across the exit slit. This adjustment may be done manually, or sometimes, in more expensive automated instruments, under microprocessor control via a stepper motor. 

The main variables associated with the monochromator are the slit width and wavelength setting. 

Apparatus Use a suitable spectrophotometer (Perkin-El-mer Model 6000, or equivalent), a graphite furnace containing a L vov platform (Perkin-Elmer Model HGA-500, or equivalent), and an autosampler (Perkin-Elmer Model AS-40, or equivalent). Use a lead hollow-cathode lamp (lamp current of 10 mA), a slit width of 0.7 mm (set low), the wavelength set at 283.3 nm, and a deuterium arc lamp for background correction. 

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