Chemical analysis using second-order data collected on hyphenated instruments has proven advantages over first-order or zero-order techniques due to what is known as the second-order advantage. The primary second-order advantage is the ability to perform analysis in the presence of unknown interferences. This work demonstrates another key advantage of second-order chemical analysis, that is, the ability to standardize data sets of a second-order chromatographic analyzer under conditions which result in retention time variations along the chromatographic asis, An objective technique to standardize second-order chromatographic-spectral data is both theoretically and experimentally developed and tested, This method corrects for retention time shifts that occur between the analysis of the calibration sample and "unknown" samples, When this technique is combined with bilinear data analysis techniques like generalized rank annihilation method (GRAM), standardization and quantitation can be performed in the presence of unknown interferences with a single calibration sample, Most signal inconsistencies in second-order chromatographic data are confined to shifts of the time axis in the chromatographic profile. This retention time shift correction method is objective because it relies upon spectral signal shape and an understanding of the instrumentation. Retention time correction of this type would mot be objective for first-order chromatographic analysis because retention time is the only qualitative information present, In one example of experimental evaluation, quantitation single analyte in a sample of five chemical components is performed using liquid chromatography with absorbance detection (LC/UV-vis). Both the chromatographic and spectral signals of these five chemical components are highly overlapped, In this example, a retention time shift between the calibration and "unknown" data sets of 0.2 s resulted in a 20% quantitation error prior to standardization., After alignment of the data sets using second-order chromatographic standardization, quantitative error was reduced to nearly 1%, Theoretical simulations which evaluate the performance of this technique as a second-order chromatographic retention time correction method were performed for a wide range of resolution and signal-to-noise values, In simulations where chromatographic resolution was 0.3 or below, quantitative precision improvements resulting from second-order chromatographic standardization ranged from 3-fold to 10-fold, The standardization method presented should be generally applicable to chromatography hyphenated with ail forms of spectroscopic detection, such as gas chromatography/mass spectrometry.
