Guillermo Quintás

Automated sample preparation and analysis using a sequential-injection-capillary electrophoresis (SI-CE) interface

A fully automated sequential-injection-capillary electrophoresis (SI-CE) system was developed using commercially available components as the syringe pump, the selection and injection valves and the high voltage power supply. The interface connecting the SI with the CE unit consisted of two T-pieces, where the capillary was inserted in one T-piece and a Pt electrode in the other (grounded) T-piece. By pressurising the whole system using a syringe pump, hydrodynamic injection was feasible. For characterisation, the system was applied to a mixture of adenosine and adenosine monophosphate at different concentrations. The calibration curve obtained gave a detection limit of 0.5 microg g(-1) (correlation coefficient of 0.997). The reproducibility of the injection was also assessed, resulting in a RSD value (5 injections) of 5.4%. The total time of analysis, from injection, conditioning and separation to cleaning the capillary again was 15 minutes. In another application, employing the full power of the automated SIA-CE system, myoglobin was mixed directly using the flow system with different concentrations of sodium dodecyl sulfate (SDS), a known denaturing agent. The different conformations obtained in this way were analysed with the CE system and a distinct shift in migration time and decreasing of the native peak of myoglobin (Mb) could be observed. The protein samples prepared were also analysed with off-line infrared spectroscopy (IR), confirming these results.

Analytical potential of mid-infrared detection in capillary electrophoresis and liquid chromatography: a review.

Literature published in the last decade concerning the use of mid-infrared spectrometry as a detection system in separation techniques employing a liquid mobile phase is reviewed. In addition to the continued use of isocratic liquid chromatographic (LC) techniques, advances in chemometric data evaluation techniques now allow the use of gradient techniques on a routine basis, thus significantly broadening the range of possible applications of LC-IR. The general trend towards miniaturized separation systems was also followed for mid-IR detection where two key developments are of special importance. Firstly, concerning on-line detection the advent of micro-fabricated flow-cells with inner volumes of only a few nL for transmission as well as attenuated total reflection measurements enabled on-line mid-IR detection in capillary LC and opened the path for the first successful realization of on-line mid-IR detection in capillary zone electrophoresis as well as micellar electrokinetic chromatography. Secondly, concerning off-line detection the use of micro-flow through dispensers now enables to concentrate eluting analytes on dried spots sized a few tens of micrometers, thus matching the dimensions for sensitive detection by mid-IR microscopy. Finally in an attempt to increase detection sensitivity of on-line mid-IR detection, mid-IR quantum cascade lasers have been used. Applications cover the field of food analysis, environmental analysis and the characterization of explosives among others. Best detection sensitivities for on-line and off-line detection have been achieved in miniaturized systems and are in the order of 50 ng and 2 ng on column, respectively.

Simultaneous determination of Folpet and Metalaxyl in pesticide formulations by flow injection Fourier transform infrared spectrometry

Fourier transform infrared (FTIR) spectrometric methodology has been developed for the simultaneous determination of Folpet and Metalaxyl in pesticide formulations. The method involves the extraction of both active principles by sonication of the sample with CHCl3 and direct measurement of the absorbance using peak height values at 1798 cm −1 corrected with a baseline defined at 1810 cm −1 for Folpet, and peak area data established from 1677 to 1667 cm −1 corrected using a baseline from 1692 to 1628 cm −1 for Metalaxyl. Limits of detection (3 s) values of 17 and 16 gg −1 were found for Folpet and Metalaxyl, respectively, with results comparable with those found by liquid chromatography with UV detection. The new method involves a considerable decrease in solvent consumption. The automation of the procedure by incorporating on-line dissolution and filtration of the samples allows complete mechanisation of the method and improves the safety of operation.

Background Correction and Multivariate Curve Resolution of Online Liquid Chromatography with Infrared Spectrometric Detection

The use of multivariate curve resolution-alternating least-squares (MCR-ALS) in liquid chromatography-infrared detection (LC-IR) is troublesome due to the intense background absorption changes during gradient elution. Its use has been facilitated by previous removal of a significant part of the solvent background IR contributions due to common mobile phase systems employed during reversed phase gradient applications. Two straightforward background correction approaches based on simple-to-use interactive self-modeling mixture analysis (SIMPLISMA) and principal component analysis (PCA) are proposed and evaluated on reversed phase gradient LC-IR data sets obtained during the analysis of carbohydrate and nitrophenol mixtures. After subtraction of the calculated background signal, MCR-ALS provided improved signal-to-noise ratios, removed remaining mobile phase and background signal contributions, and resolved overlapping chromatographic peaks. The present approach tends to enable easy-to-use background correction to facilitate the use of MCR-ALS in online LC-IR, even in challenging situations when gradient conditions are employed and only poor chromatographic resolution is achieved. It, therefore, shows great potential to facilitate the full exploitation of the advantages of simultaneous quantification and identification of a vast amount of analytes employing online IR detection, making new exciting applications more accessible.

New cut-off criterion for uninformative variable elimination in multivariate calibration of near-infrared spectra for the determination of heroin in illicit street drugs

A new cut-off criterion has been proposed for the selection of uninformative variables prior to chemometric partial least squares (PLS) modelling. After variable elimination, PLS regressions were made and assessed comparing the results with those obtained by PLS models based on the full spectral range. To assess the prediction capabilities, uninformative variable elimination (UVE)-PLS and PLS were applied to diffuse reflectance near-infrared spectra of heroin samples. The application of the proposed new cut-off criterion, based on the t-Students distribution, provided similar predictive capabilities of the PLS models than those obtained using the original criteria based on quantile value. However, the repeatability of the number of selected variables was improved significantly.

A validated and fast procedure for FTIR determination of Cypermethrin and Chlorpyrifos.

A FTIR methodology has been developed for the simultaneous determination of Cypermethrin and Chlorpyrifos in pesticide commercially available formulations. The method involves the extraction of both active principles with CHCl(3) and direct measurement of the peak area values between 1747 and 1737cm(-1) corrected with a baseline defined at 2000cm(-1) for Cypermethrin and peak height values established at 1549cm(-1) corrected using a baseline situated at 1650cm(-1) for Chlorpyrifos. The limits of detection achieved were of the order of 0.7 and 0.4% (w/w), and the relative standard deviation 0.4 and 0.2% for Cypermethrin and Chlorpyrifos, respectively. The developed procedure provided statistically comparable results with those obtained by HPLC, for a series of commercial samples, which validated the FTIR method. The procedure developed reduces organic solvent consumption, per sample preparation, from 51ml CH(3)CN required for HPLC to 2.5ml CHCl(3), and reduces waste generation also increasing the sample measurement frequency, from 3 to 30 samples/h, as compared with the HPLC-UV reference method.

Univariate method for background correction in liquid chromatography–Fourier transform infrared spectrometry

An univariate method is proposed for background correction in on-line gradient liquid chromatography-Fourier transform infrared (LC-FTIR) spectrometry using acetonitrile:water as mobile phase components. The method is based on the calculation of the ratio of absorbances (AR) at two characteristic wavenumbers for each spectrum. This parameter is subsequently used to locate the most appropriated eluent spectrum within a reference spectra matrix (RSM) to be subtracted from each spectrum included in the sample chromatogram. To correct minor changes in eluent spectra intensity during the elution of analytes, a correction factor (Kf), defined as the ratio of the absorbance of the sample and the selected eluent spectrum at a defined wavenumber was determined. The performance of the procedure was evaluated by correcting an on-line gradient LC-FTIR injection of a mixture of two pesticides (Atrazine and Diuron). Using the AR of the absorbance at 2248.6 and 2256.3 cm(-1) and a Kf at 2248.6 cm(-1), the correlation factors between FTIR spectra extracted at the peak apex from the LC-FTIR chromatogram and those obtained from pure pesticide standards were 0.975 and 0.94 for Atrazine and Diuron, respectively.

A rapid method for the differentiation of yeast cells grown under carbon and nitrogen-limited conditions by means of partial least squares discriminant analysis employing infrared micro-spectroscopic data of entire yeast cells

This paper shows the ease of application and usefulness of mid-IR measurements for the investigation of orthogonal cell states on the example of the analysis of Pichia pastoris cells. A rapid method for the discrimination of entire yeast cells grown under carbon and nitrogen-limited conditions based on the direct acquisition of mid-IR spectra and partial least squares discriminant analysis (PLS-DA) is described. The obtained PLS-DA model was extensively validated employing two different validation strategies: (i) statistical validation employing a method based on permutation testing and (ii) external validation splitting the available data into two independent sub-sets. The Variable Importance in Projection scores of the PLS-DA model provided deeper insight into the differences between the two investigated states. Hence, we demonstrate the feasibility of a method which uses IR spectra from intact cells that may be employed in a second step as an in-line tool in process development and process control along Quality by Design principles.

New background correction approach based on polynomial regressions for on-line liquid chromatography-Fourier transform infrared spectrometry.

In the present study a new approach for the chemometric background correction in on-line gradient LC-FTIR is introduced. For this purpose, the spectral changes of the elution mixture during gradient elution were analyzed applying 2D correlation spectroscopy. The fundamentals of the new background correction algorithm, based on polynomial fits calculated from a reference spectra matrix (Polyfit-RSM method) are explained. The Polyfit-RSM approach was applied on blank gradient runs as well as on LC-FTIR data obtained from the injection of a soft drink sample using acetonitrile:water as eluent. Results found were critically assessed and compared to those obtained by two previous background correction methods which are likewise based on the use of a reference spectra matrix (RSM). The Polyfit-RSM method provided lower noise levels throughout the whole spectral range than other alternative background correction methods, an excellent recovery of analyte spectra as well as chromatograms with a low noise level and also free from baseline shifts. A significant finding, which implies a major advantage for the practical applicability of the algorithm, is that the size of the RSMs can be reduced without affecting the accuracy of the correction method.

On-Line Fourier Transform Infrared Spectrometric Detection in Gradient Capillary Liquid Chromatography Using Nanoliter-Flow Cells

A capillary liquid chromatographic system has been successfully interfaced with a mid-IR Fourier transform infrared (FT-IR) spectrometer. Spectra were recorded on-line using a micromachined transmission CaF(2) cell (internal volume of 7.5 nL) that was placed on a dedicated beam condenser attached to the spectrometer. Linear gradients were run from (50:50) to (35:65) water (0.05% TFA)/acetonitrile in 15 min for the separation of standard solutions of four nitrophenols (4-nitrophenol, 3-methyl-4-nitrophenol, 2,4-dinitrophenol, and 2-nitrophenol) in a reversed phase system, providing limits of detection between 35 and 94 ng on-column. The changing background absorption due to gradient elution was successfully corrected by using a dedicated algorithm implemented in Matlab. When this chemometric data treatment was used, highly characteristic analyte spectra could be recorded as indicated by correlation coefficients between 89 and 95.8%, obtained when comparing mid-IR spectra of standard solutions and the spectra extracted from the chromatogram.