Diode array detection peak purity

H.R. Keller and D.L. Massart, Artifacts in evolving factor analysis-based methods for peak purity control in liquid-chromatography with diode array detection. Anal. Chim. Acta, 263 (1992) 21-28. 

Applications of diode array detection to peak purity determination. 

Specificity Peak Purity Degradation Products Retention Behaviour Diode array detection test Accelerated degradation Chromatogram... 

The principles of diode-array detection in HPLC and their application and limitations to peak purity are described in the literature [25-27], Examples of pure and impure HPLC peaks are shown in Figure 1. While the chromatographic signal indicates no impurities in either peak, the spectral evaluation identifies the peak on the left as impure. The level of impurities that can be detected with this method depends on the spectral difference, on the detector s performance, and on the software algorithm. Under ideal conditions, peak impurities of 0.05-0.1% can be detected. 

Fig. 3 Investigation of chromatographic peak purity by diode array detection (A) spectra of drug substance and impurity (B) coelution of a mixture containing about 10% impurity (C) coelution of a mixture containing about 0.5% impurity. The spectra were obtained in the peak maximum, at about 5% and 50% of each side of the peak. The normalization was performed with respect to the first spectrum at the peak front (a matchfactor of 1000 means identical spectra) using commercial software.

At first, one may show skepticism at the usefulness of diode-array detection because other analytical systems are more sensitive and offer similar features such as peak identification and purity checks. For these alternatives, one would have to refer to gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), and tandem mass spectrometry (MS-MS). However, one must keep in mind the significantly higher costs as a tradeoff for enhanced sensitivity. 

TABLE 1 Calculated Statistical Purity Probabilities for Individual HPLC Peaks with Single Wavelength and Diode Array Detection... 

The use of diode-array detection has greatly improved peak identification, peak purity assessment and quantification. However, there are still some problems to be solved relating to peak purity assessment (compounds possessing identical spectral characteristics may invalidate peak purity criteria) and peak quantification (poor resolved peaks cannot be quantified by the... 

Intended use User requirement specification Analysis of drug products and substances Automated analysis of up to 100 samples/day Limit of quantitation <0.05% Automated confirmation of peak purity and identity with diode-array detection Compatible with 2-mm to 4.6-mm i.d. columns... 

Fig. 8.4. Peak purity determination by spectral overlay. The HPLC signal does not indicate any impurity in either peak. Spectral evaluation (DAD) identifies the peak on the left as impure. After George [100]. Reprinted form S.A. George, in Diode-Array Detection in HPLC (L. Huher and S.A. George, eds.), Marcel Dekker Inc., New York (1993), by courtesy of...

Peak purity tests are used to demonstrate that an observed chromatographic peak is attributable to a single component. Mass spectrometry is the most sensitive and accurate technique to use for peak purity evaluation because of the specific information derived from the analysis. However, a good number of HPLC methods use mobile phase conditions that are incompatible with mass spectrometry detection. In this case, PDA spectrophotometers using peak purity algorithms may be used to support the specificity of the method. Almost all commercially available diode array detectors are equipped with proprietary software that will perform these calculations. Although this technique is more universal in application to HPLC methods, the data provided is neither particularly... 

Specific detection is an analytical determination based on specific responses related to the chemical characteristics of a molecule excited by a certain type of irradiation. In this detection method, measurement of the molecule of interest may usually be performed without separation from matrix materials or from other ingredients if appropriate instrumental adjustments are made. The need for identification and structure elucidation for newly discovered compounds drives the progress of specific detection techniques with NMR and X-ray diffraction and MS. The UV-diode array detector often aids the recognition of chromatographic peak purity, ensuring complete resolution of a separation procedure. 

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