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G. Quintas, B. Lendl:
"On-line FTIR detection in HPLC employing gradient elution";
Poster: International Conference on Advanced Vibrational Spectroscopy, ICAVS 4, Corfu; 10.06.2007 - 15.06.2007.

We introduce a flexible and efficient approach for background subtraction in flow cell based on-line FTIR detection in isocratic and gradient HPLC. Spectral changes in shape and intensity of the mobile phase during a chromatographic run make accurate background subtraction difficult. This especially applies for gradient based techniques, having lead to the general assumption that flow cell based techniques can only be applied in case of isocratic conditions.
In this contribution we present a new PLS-based automatic procedure for background correction that, for the first time, can be successfully applied in both isocratic and gradient on-line reversed phase LC-FTIR.
The proposed approach uses an eluent spectral reference data set from which an appropriate background spectrum is calculated which is to be used as a background for a given peak in a chromatographic run. For calculation of the background spectrum, spectral regions containing characteristic absorption of the eluents but being void of analyte absorption are used. Using a PLS procedure this is achieved with a high degree of accuracy and minimal computational cost. The method solves two problems simultaneously: the background correction in gradient HPLC and also correct possible changes in the intensity of the absorption spectra of the eluent during analyte elution.
The method was programmed in MATLAB and its use will be shown on the example of both isocratic and gradient (water : acetonitrile) on-line LC-FTIR real data sets. The elution profiles of the analytes as well as their FTIR spectra extracted from the background corrected data sets, show the potential use of the proposed approach to perform background correction in continuous liquid flow systems. This proposed approach does not require complex data pre-treatment techniques, prior knowledge, standards or reference spectra of the analytes.