Comprehensive two-dimensional liquid chromatography with on-line Fourier-transform-infrared-spectroscopy detection for the characterization of copolymers.

The on-line coupling of comprehensive two-dimensional liquid chromatography (liquid chromatography x size-exclusion chromatography, LC x SEC) and infrared (IR) spectroscopy has been realized by means of an IR flow cell. The system has been assessed by the functional-group analysis of a series of styrene-methylacrylate (SMA) copolymers with varying styrene content. Ultraviolet (UV) detection was used as a detection technique to verify the detection with IR. The LC x SEC-IR functional-group contour plots (comprehensive chromatograms) obtained for styrene were in agreement with the contour plots constructed from the UV signal. In addition, contour plots can be obtained from non-UV-active groups. One such plot, for the carbonyl-stretching vibration of methylacrylate (MA), is shown. Selective detection of MA proved possible using flow cell IR detection. The combination of the contour plots for styrene and MA allowed a full characterization of the copolymer and it was revealed that the present series of SMA copolymers exhibited homogeneous chemical-composition distributions (CCDs). In addition, commercially available fast-SEC columns have been assessed in this study with respect to their potential to serve as second-dimension separation columns.

Comparison of on-line flow-cell and off-line solvent-elimination interfaces for size-exclusion chromatography and Fourier-transform infrared spectroscopy in polymer analysis.

Two commercial liquid chromatography-Fourier-transform infrared spectroscopy interfaces (LC-FTIR), viz. a flow cell and a solvent-elimination interface have been assessed for use in size-exclusion chromatography (SEC) with respect to their chromatographic integrity (i.e. peak asymmetry, chromatographic resolution), quantitative and qualitative aspects. A polycarbonate/aliphatic polyester (PC/APE) blend and a polycarbonate-co-polydimethylsiloxane (PC-co-PDMS) copolymer were selected for the assessment. Both samples were successfully and selectively analyzed. The relatively large volume of the flow cell and the inherent deposition characteristics of the solvent-elimination interface led to a comparable decrease in the chromatographic resolution. The separation of oligomers was diminished in comparison with SEC-ultra-violet (UV). However, the peak asymmetry was not significantly affected by either interface. For both interfaces, a linear relationship was obtained for the FTIR response versus the injected concentration. The sensitivity was found to be higher for the solvent-elimination interface. For the current model compounds, the flow-cell interface detection limits are worse. However, the repeatability of flow-cell SEC-FTIR, evaluated by means of four SEC-FTIR analyses of polycarbonate, was considerably better than for solvent-elimination SEC-FTIR. This is probably due to the well-defined optical path length of the sample in the flow cell. By spectral subtraction, it was very well possible to obtain qualitative (functional group) information for compound identification also with flow-cell SEC-FTIR.

Fourier transform infrared spectroscopy with a sample deposition interface as a quantitative detector in size-exclusion chromatography.

The use of a state-of-the-art commercial solvent-elimination interface for liquid chromatography-infrared spectroscopy is discussed from the perspective of quantitative analysis. The effect of eluent flow-rate is investigated with respect to the homogeneity of the deposit and the trace width along the deposition trace. Low flow-rates (50 microl/min or less) turn out to be favorable for obtaining a good trace and a high sensitivity. The trace width decreased from 2.20 to 1.13 mm when the flow-rate was reduced from 500 to 25 microl/min. Preservation of chromatographic detail during deposition was evaluated at different substrate moving speeds. The additional (extra-column) band broadening that is inherent to nebulizer-deposition interfaces, causes a loss in resolution in size-exclusion chromatography (SEC)-Fourier transform infrared (FTIR) spectroscopy in comparison with SEC-UV. The repeatability of the deposition is evaluated by SEC-FTIR analyses of polystyrene standards (peak molar-masses: 1250-2,950 000 g/mol) and the RSD is found to range from 3.2 to 5.8% in response and from 0.21 to 0.47% in retention time.

Fast sample preparation for analysis of tablets and capsules: the ball-mill extraction method.

A new ball-mill extraction method for solid dosage forms was developed. It was used for tablets, and compared with a conventional (powdering and sonication) method applied in pharmaceutical analysis of solid dosage forms. The ball-mill sample preparation procedure is both quantitative and fast. No powdering, weighing and sonication steps are needed in the sample preparation. The complete procedure takes 2 min (milling and extraction) and 5 min (centrifugation), respectively, much less than the conventional method in which sample preparation takes approximately 45-90 min. The samples are centrifuged in the mill vial, which saves time and avoids evaporation of solvent. Stainless steel extraction vials with different diameters were fabricated to enable the use of various extraction volumes. The extraction recovery was tested using various types of tablets (small, large and extended release tablets) with active compounds at low and higher concentrations, recoveries were comparable with the conventional method. The relative small investment and simplicity of the method makes it excellently suited for use in various pharmaceutical (development and quality assurance) laboratories.

Determination of pesticides in vegetables using large-volume injection column liquid chromatography-electrospray tandem mass spectrometry.

Direct injection of a large volume (900 microl) of a sample extract onto a liquid chromatographic (LC) column, LC separation and electrospray tandem mass spectrometric detection were used for the quantitative analysis of a wide polarity range of pesticides in carrots and potatoes. Rapid sample preparation involved extraction of a small amount of sample (2 g) with a small volume of organic solvent (3 ml), clean-up over a filter and dilution of the organic extract with the aqueous LC eluent. The extraction efficiency for the selected pesticides was studied using methanol, acetone and acetonitrile as solvents. Evaluation of the performance of the overall method, using extraction with acetonitrile and detection in the selected-reaction-monitoring mode, showed excellent linearity in the range of 2-100 microg/kg with limits of detection of 0.5-2 microg/kg for both types of vegetable. With relative standard deviations of the MS peak area measurements of less than 6.5% (n=8) the repeatability of the method was fully satisfactory.

Analyte identification in capillary electrophoretic separation techniques.

A review on applications of on-line hyphenation in capillary electrophoresis and capillary electrochromatography for the identification of migrating analytes is presented. There is an urgent need for unambiguous analyte identification by combining spectral information and observed migration times, because the parameters influencing the migration times and separation efficiencies in these separation techniques are not easily controlled, especially when real samples containing unknown interferences have to be analyzed. The spectrometric techniques covered here are ultraviolet and visible radiation (UV/Vis) absorption, fluorescence including fluorescence line-narrowing spectroscopy, Raman spectroscopy, nuclear magnetic resonance and mass spectrometry. Attention is essentially confined to literature reports in which the extra information provided by the detector is really used for identification purposes, especially in real-life samples, while the interfacing as such and analyte detectabilities in standard solutions are only briefly discussed. This article covers an extensive fraction of the literature published on this topic until the beginning of 1998.

Capillary electrophoresis of the collagen crosslinks HP and LP utilizing absorbance, wavelength-resolved laser-induced fluorescence and conventional fluorescence detection.

A capillary electrophoretic (CE) method is presented for the determination of the collagen crosslinks hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP). Various detection techniques are compared, i.e. UV-Vis diode-array absorbance detection (DAD) and fluorescence detection both in the laser-induced fluorescence (LIF) and the conventional fluorescence mode. LIF detection was performed using a frequency-doubled Rhodamine dye laser pumped by an excimer laser, for excitation at 290 and 325 nm. The emission was measured with an intensified diode-array detector mounted on a spectrograph to obtain wavelength-resolved spectra. Relevant concentration detection limits were achieved only by using LIF detection, i.e. 200 nM of HP and LP in a 30 mM phosphate buffer (pH 2.0). Linear calibration curves were obtained from the detection limits up to the maximum concentration available, 23 microM for HP and 4.2 microM for LP, respectively for both fluorescence modes. The identity of the migrating compounds was confirmed by on-line recording of both the absorption and the fluorescence spectra.