New Instrumental and Chemometric Developments form the on-line hyphenation of Liquid Chromatography and Infrared Spectroscopy

Abstract

The coupling of liquid chromatography (LC) to infrared (IR) spectrometry is an interesting analytical tool, because high resolution provided by LC is combined with the non-destructive and molecular specific information of IR spectrometry. However, the potential of on-line LC-IR employing a flow cell interface, characterized by its technical simplicity, cannot yet be fully exploited due to the limited sensitivity of IR detection and difficulties in on-line measurements arising from the dominating absorption of most of the commonly used mobile phase components. Accordingly, the objective of this Thesis was to promote the use of the on-line hyphenation of LC and IR spectroscopy through the development of instrumental and chemometric tools to overcome existing limitations, thus increasing the applicability of this technique. Chapter 2 describes innovations in the field of isocratic separation. A method for the determination of lecithin and soybean oil in dietary supplements using on-line Gel Permeation Chromatography – Attenuated Total Reflectance – Infrared (GPC-ATR-IR) was developed including a simple procedure to select the optimum wavenumber used for the extraction of characteristic elution profiles. For the monitoring of polymerized triglycerides in deep-frying oil, an approach employing on-line GPC-IR spectrometry and the Science Based Calibration (SBC) multivariate approach for chromatogram-extraction were reported. An on-line LC-IR procedure for the determination of glycolic acid in cosmetics employing the rapid scan data acquisition mode was proposed. Chapter 3 deals with different methods for background correction in on-line LC-IR using reference spectra matrices (RSM). The determination of the critical eluent composition for polyethylene glycols using methanol as organic modifier was discussed employing the absorbance ratio as identification parameter for background correction. For the determination of fructose, glucose, sucrose and maltose in beverages employing acetonitrile as organic modifier, again the absorbance ratio was used as identification parameter. A Partial Least Squares (PLS) procedure as well as the application of point-to-point matching algorithms for automated background correction were discussed for background correction in acetonitrile:water gradients using different examples of applications. Chapter 4 deals with the compensation of the background contribution employing column-wise techniques. Polynomial regressions modelling the background absorbance at each wavenumber throughout the gradient run were applied for acetonitrile:water mobile phase gradients. The use of cubic smoothing splines could be demonstrated for background correction under reversed phase gradient conditions employing different alcohols as organic modifiers. In Chapter 5, background correction based on factor analysis and reference spectra matrices was discussed. The contribution of the background absorption to overall LC-IR signals could be compensated using two approaches based on Principal Component Analysis (PCA) and simple-to-use interactive self modeling analysis (SIMPLISMA), obtaining good results in acetonitrile:water systems. Subsequently, multivariate curve resolution-alternating least squares (MCR-ALS) was applied improving peak and spectral resolution and eliminating remaining signal variation due to background absorption and detector drift. The chemometric extraction of ‘analyte-specific’ chromatograms in on-line LC-IR was discussed in Chapter 6. Obtained results confirm that the SBC method is particularly well suited for recovering ‘analyte-specific’ signals from on-line LC-IR chromatograms. The last chapter describes instrumental developments in on-line LC-IR. On-line IR detection in gradient capillary LC using micromachined nanoliter-flow cells was discussed. Four model compounds were separated and identified using an acetonitrile:H2O gradient with limits of detection in the concentration range of 35-94 ng μL-1, representing an increase in mass sensitivity by a factor of approximately 30 as compared to standard LC systems. The last chapter also focuses on the development of LC with on-line dual Quantum Cascade Laser (QC-laser) detection. Compared to state-of-the-art FTIR spectrometers, the use of the developed QC-laser based system provided a significant improvement in both, sensitivity and data acquisition frequency.

The coupling of liquid chromatography (LC) to infrared (IR) spectrometry is an interesting analytical tool, because high resolution provided by LC is combined with the non-destructive and molecular specific information of IR spectrometry. However, the potential of on-line LC-IR employing a flow cell interface, characterized by its technical simplicity, cannot yet be fully exploited due to the limited sensitivity of IR detection and difficulties in on-line measurements arising from the dominating absorption of most of the commonly used mobile phase components. Accordingly, the objective of this Thesis was to promote the use of the on-line hyphenation of LC and IR spectroscopy through the development of instrumental and chemometric tools to overcome existing limitations, thus increasing the applicability of this technique. Chapter 2 describes innovations in the field of isocratic separation. A method for the determination of lecithin and soybean oil in dietary supplements using on-line Gel Permeation Chromatography – Attenuated Total Reflectance – Infrared (GPC-ATR-IR) was developed including a simple procedure to select the optimum wavenumber used for the extraction of characteristic elution profiles. For the monitoring of polymerized triglycerides in deep-frying oil, an approach employing on-line GPC-IR spectrometry and the Science Based Calibration (SBC) multivariate approach for chromatogram-extraction were reported. An on-line LC-IR procedure for the determination of glycolic acid in cosmetics employing the rapid scan data acquisition mode was proposed. Chapter 3 deals with different methods for background correction in on-line LC-IR using reference spectra matrices (RSM). The determination of the critical eluent composition for polyethylene glycols using methanol as organic modifier was discussed employing the absorbance ratio as identification parameter for background correction. For the determination of fructose, glucose, sucrose and maltose in beverages employing acetonitrile as organic modifier, again the absorbance ratio was used as identification parameter. A Partial Least Squares (PLS) procedure as well as the application of point-to-point matching algorithms for automated background correction were discussed for background correction in acetonitrile:water gradients using different examples of applications. Chapter 4 deals with the compensation of the background contribution employing column-wise techniques. Polynomial regressions modelling the background absorbance at each wavenumber throughout the gradient run were applied for acetonitrile:water mobile phase J. Kuligowski | NEW INSTRUMENTAL AND CHEMOMETRIC DEVELOPMENTS FOR THE ON-LINE HYPHENATION OF LIQUID CHROMATOGRAPHY AND INFRARED SPECTROSCOPY II gradients. The use of cubic smoothing splines could be demonstrated for background correction under reversed phase gradient conditions employing different alcohols as organic modifiers. In Chapter 5, background correction based on factor analysis and reference spectra matrices was discussed. The contribution of the background absorption to overall LC-IR signals could be compensated using two approaches based on Principal Component Analysis (PCA) and simple-to-use interactive self modeling analysis (SIMPLISMA), obtaining good results in acetonitrile:water systems. Subsequently, multivariate curve resolution-alternating least squares (MCR-ALS) was applied improving peak and spectral resolution and eliminating remaining signal variation due to background absorption and detector drift. The chemometric extraction of ‘analyte-specific’ chromatograms in on-line LC-IR was discussed in Chapter 6. Obtained results confirm that the SBC method is particularly well suited for recovering ‘analyte-specific’ signals from on-line LC-IR chromatograms. The last chapter describes instrumental developments in on-line LC-IR. On-line IR detection in gradient capillary LC using micromachined nanoliter-flow cells was discussed. Four model compounds were separated and identified using an acetonitrile:H2O gradient with limits of detection in the concentration range of 35-94 ng μL-1, representing an increase in mass sensitivity by a factor of approximately 30 as compared to standard LC systems. The last chapter also focuses on the development of LC with on-line dual Quantum Cascade Laser (QC-laser) detection. Compared to state-of-the-art FTIR spectrometers, the use of the developed QC-laser based system provided a significant improvement in both, sensitivity and data acquisition frequency.