Infrared microspectroscopic imaging of plant tissues: spectral visualization of Triticum aestivum kernel and Arabidopsis leaf microstructure.

Infrared microspectroscopy is a tool with potential for studies of the microstructure, chemical composition and functionality of plants at a subcellular level. Here we present the use of high-resolution bench top-based infrared microspectroscopy to investigate the microstructure of Triticum aestivum L. (wheat) kernels and Arabidopsis leaves. Images of isolated wheat kernel tissues and whole wheat kernels following hydrothermal processing and simulated gastric and duodenal digestion were generated, as well as images of Arabidopsis leaves at different points during a diurnal cycle. Individual cells and cell walls were resolved, and large structures within cells, such as starch granules and protein bodies, were clearly identified. Contrast was provided by converting the hyperspectral image cubes into false-colour images using either principal component analysis (PCA) overlays or by correlation analysis. The unsupervised PCA approach provided a clear view of the sample microstructure, whereas the correlation analysis was used to confirm the identity of different anatomical structures using the spectra from isolated components. It was then demonstrated that gelatinized and native starch within cells could be distinguished, and that the loss of starch during wheat digestion could be observed, as well as the accumulation of starch in leaves during a diurnal period.

Stability of sugar solutions: a novel study of the epimerization kinetics of lactose in water.

This article reports on the stereochemical aspects of the chemical stability of lactose solutions stored between 25 and 60 °C. The lactose used for the preparation of the aqueous solutions was α-lactose monohydrate with an anomer purity of 96% α and 4% ß based on the supplied certificate of analysis (using a GC analytical protocol), which was further confirmed here by nuclear magnetic resonance (NMR) analysis. Aliquots of lactose solutions were collected at different time points after the solutions were prepared and freeze-dried to remove water and halt epimerization for subsequent analysis by NMR. Epimerization was also monitored by polarimetry and infrared spectroscopy using a specially adapted Fourier transform infrared attenuated total reflectance (FTIR-ATR) method. Hydrolysis was analyzed by ion chromatography. The three different analytical approaches unambiguously showed that the epimerization of lactose in aqueous solution follows first order reversible kinetics between 25 to 60 °C. The overall rate constant was 4.4 × 10(-4) s(-1) ± 0.9 (± standard deviation (SD)) at 25 °C. The forward rate constant was 1.6 times greater than the reverse rate constant, leading to an equilibrium constant of 1.6 ± 0.1 (±SD) at 25 °C. The rate of epimerization for lactose increased with temperature and an Arrhenius plot yielded an activation energy of +52.3 kJ/mol supporting the hypothesis that the mechanism of lactose epimerization involves the formation of extremely short-lived intermediate structures. The main mechanism affecting lactose stability is epimerization, as no permanent hydrolysis or chemical degradation was observed. When preparing aqueous solutions of lactose, immediate storage in an ice bath at 0 °C will allow approximately 3 min (180 s) of analysis time before the anomeric ratio alters significantly (greater than 1%) from the solid state composition of the starting material. In contrast a controlled anomeric composition (~38% α and ~62% ß) will be achieved if an aqueous solution is left to equilibrate for over 4 h at 25 °C, while increasing the temperature up to 60 °C rapidly reduces the required equilibration time.

Infrared spectroscopy with heated attenuated total internal reflectance enabling precise measurement of thermally induced transitions in complex biological polymers.

We report an improved tool for acquiring temperature-resolved fourier transform infrared (FT-IR) spectra of complex polymer systems undergoing thermal transitions, illustrated by application to several phenomena related to starch gelatinization that have proved difficult to study by other means. Starch suspensions from several botanical origins were gelatinized using a temperature-controlled attenuated total reflectance (ATR) crystal, with IR spectra collected every 0.25 °C. By following the 995/1022 cm(-1) peak ratio, clear transitions occurring between 59 and 70 °C were observed, for which the midpoints could be determined accurately by sigmoidal fits. The magnitude of the change in peak ratio was found to be strongly correlated to the enthalpy of gelatinization as measured by differential scanning calorimetry (DSC, R(2) = 0.988). An important advantage of the technique, compared to DSC, is that the signal-to-noise ratio is not reduced when measuring very broad transitions. This has the potential to allow more precise determination of the gelatinization parameters of high-amylose starches, for which gelatinization may take place over several tens of °C.

Fiber-optic infrared reflection absorption spectroscopy for trace analysis on surfaces of varying roughness. Part II: Acetaminophen on stainless steel.

Investigations of the effects of surface roughness on the utility of grazing-angle Fourier transform infrared reflection absorption spectroscopy (IRRAS) as a method for quantifying trace contamination of metal surfaces have been extended to acetaminophen, a model active pharmaceutical agent, on 316 stainless steel. The effects are more complicated than for the surfactant sodium dodecyl sulfate (SDS) on stainless steel; they include a strong surface-finish dependence of sensitivity and nonlinear behavior at surface loadings above approximately 1-2 microg cm(-2). Using data from samples in the loading range 0-0.5 microg cm(-2), unbiased partial least squared calibrations can be readily achieved for individual surface finishes with detection limits of L(D) approximately 0.15 microg cm(-2). However, as found for SDS on stainless steel, models built using data from samples of mixed surface roughness are more problematic.

Grazing-angle fiber-optic fourier transform infrared reflection-absorption spectroscopy for the in situ detection and quantification of two active pharmaceutical ingredients on glass.

Fourier transform infrared reflection-absorption spectroscopy has been used with a fiber-optic grazing-angle reflectance probe as a rapid, in situ method for trace surface analysis of acetaminophen and aspirin at loadings of approximately 0-2 microg cm(-2) on glass. Partial least-squares multivariate regression permits the loadings to be quantified, simultaneously, with root-mean-squared errors of prediction of RMSEP approximately 0.1 microg cm(-2) for both compounds. The detection limits are estimated to be LD approximately 0.2 microg cm(-2).

Fiber-optic infrared reflection absorption spectroscopy for trace analysis on surfaces of varying roughness: Sodium dodecyl sulfate on stainless steel.

The effect of surface roughness on the analytical performance of a fiber-optic grazing-angle infrared reflection absorption spectroscopy (IRRAS) system has been investigated. The instrument was used to develop calibrations to quantify trace surface loadings for sodium dodecyl sulfate (SDS) on 316 stainless steel with three different surface finishes. Partial least squares (PLS) calibrations were developed for both individual finishes and combinations of finishes. For SDS on individual surface finishes, the root mean square (rms) error of cross-validation (RMSECV) varied between 0.08 and 0.12 microg cm(-2) with values of R2 between 0.89 and 0.96. A combined model for SDS on all surfaces gave an RMSECV of 0.18 microg cm(-2) with an R2 of 0.85.