Detection wavelength, robustness

Table 1 also contains some peak measurement/analysis parameters that might be investigated in a robustness test (see Section III.C). ° However, except from the detection wavelength, these parameters are hardly ever evaluated in a robustness test, even though they can have a large influence on the electropherogram. 

For validation the following robustness factors should be considered different lots of the capillary, temperature ( 2°C), applied voltage/current ( 2% relative), buffer electrolyte concentration ( + 10% relative), pH ( 0.1), concentration of additives, e.g., organic modifiers or chiral additives ( 10% relative), injection time ( 0.5s), detection wavelength ( + 2nm), batch-to-batch variation of chiral selectors ( 2—3 different lots), CE instruments (two instruments of two different manufacturers preferentially). 

A typical example of HPLC method development and validation was provided by Boneschans et al. [9]. They developed an HPLC method for piroxicam benzoate and its major hydrolytic degradation products, piroxicam and benzoic acid. The authors utilised a robust stationary phase (Phenomenex Luna, Cig), with an optimised mobile phase comprising of acetonitrile/water/acetic acid (45/7/8 v/v), and a flow rate of 1.5 ml/min. The operating pH of the mobile phase (pH 2.45) was selected on the basis that it is ca. 2 pH units from the pKa of the drug, and hence reasonably insensitive to changes in mobile-phase preparation. The injection volume was 20 pi with a detection wavelength of 254 nm. They utihsed... 

For determining the robustness of a method a number of parameters, such as extraction time, mobile-phase pH, mobile-phase composition, injection volume, source of column lots and/or suppliers, temperature, detection wavelength, and the flow rate, are varied within a realistic range and tlie quantitative influence of the variables is determined. If the influence of a parameter is within a previously specified tolerance, this parameter is said to be witliin the robustness range of the method. These method parameters may be evaluated one factor at a time or simultaneously as part of a factorial experiment. 

Besides a physical improvement of the separation, it can also be optimized visually if the spectra of the two concerning peaks are different. Then, a suitable wavelength can be selected to suppress interferences. However, such an approach has to be balanced with respect to the QL and the robustness of the quantitation if the detection wavelength does... 

Detection Most pharmaceutical compounds have UV chromophores. UV detector is the most commonly used detector. Wavelengths at Xmax or X ai are usually selected for method robustness. For example, three wavelengths at 265 (Xmax), 226 (Xvai), and 210 nm (Xmax) can be potentially used for a compound with the UV profile shown in Figure 1.2. However, sensitivity is the highest at 265 nm, which is why 265 nm should be selected for best detection. In addition, UV profiles of impurities in the sample should be considered. A detection wavelength should be selected to ensure that all components have acceptable sensitivity, not just the major component. 

Photometers are more sensitive than spectrophotometers, are cheaper and more robust and are well suited to routine work where monitoring at 254 nm or some other fixed wavelength is acceptable. Spectrophotometers, however, allow tuning to the most favourable wavelength either to maximize sensitivity for a particular solute or to detune the response to other solutes. By allowing monitoring down to 190 nm, weakly absorbing or saturated compounds can be detected. 

The imaging detectors, whether for point mapping, line scanning, or array detection, can be coupled with different types of spectrometers. Instrument types are classified by wavelength selection modality into imaging Fourier transform (FT) and tunable filter (TF) spectrometers, both of which are presented below, and dispersive spectrometers. FT imaging systems are classical laboratory instruments while TF spectrometers are compact and robust systems for chemical imaging. 

Sensors based on fluorescence are quite robust because the wavelength and the orthogonal detection geometry of the incident and emitted radiation results in a high signal-to-noise ratio. The sensors described here utilize quenching of fluorescence. Thus, the analyte is the quencher Q and the indicator is a fluorescing dye F, which when excited to F, emits fluorescence with a characteristic decay time. 

---