Attenuated Total Reflection Crystal Research Articles

Sophisticated Attenuated Total Reflection Correction Within Seconds for Unpolarized Incident Light at 45°.

The most common mid-infrared (MIR) attenuated total reflection (ATR) accessory has a nominal angle of incidence of 45° and does not have a polarizer. A spectrum recorded with such an accessory does not hold enough information for the sophisticated ATR correction of MIR spectra with strong peaks, which are often strongly affected by refractive index changes due to anomalous dispersion. Here we show that a 45° ATR spectrum recorded without a polarizer and the polarization angle for the same ATR Fourier transform infrared spectroscopy system provide enough information to determine the ATR s-polarized spectrum. Further analysis with an improved non-iterative Kramers-Kronig analysis immediately yields the complex refractive index function. The analysis is about two orders of magnitude faster than iterative formalism and runs within seconds on a typical office PC. The effectiveness of our advanced ATR correction formalism is showcased through its application to water, employing diamond, ZnSe, and Ge ATR crystals, along with two distinct ATR accessories. Additionally, the formalism is applied to octadecane spectra. Potential sources of errors such as incidence angle spread, dispersion of the polarization angle, and the influence of reflection at the air/ATR crystal interface are investigated by simulations.

Rapid and portable mid-infrared analysis of wet sediment samples by a novel “filter-press” attenuated total reflectance method

Mid-infrared (MIR) analysis of wet sediments or soils usually requires freezing and drying, adding considerable analysis time and cost, and promoting changes in chemical properties. A novel attenuated total reflectance (ATR) mid-infrared (MIR) method was developed to enable analysis of non-processed, aqueous sediments. Wet sediment samples from the Coorong Lagoon, South Australia, were subjected to laboratory analysis for physical and chemical properties, our new filter-press ATR method, and conventional ATR and diffuse reflectance (DRIFT) analysis of dried samples. For ATR on wet sediments, samples were pressed onto the ATR crystal with a filter-paper backing and scanned. The spectra were analyzed by principal components analysis (PCA) and partial least squares regression (PLSR) to develop multivariate models for total organic carbon (TOC). ATR spectra of as-received wet sediments were poor due to water absorption features, but filter-pressing reduced these and greatly enhanced the solids spectral component. The filter-pressed PLSR cross-validation for TOC resulted in an R2 = 0.89 and RMSECV = 0.78%. Prediction accuracies were similar to freeze-dried ATR samples and far more accurate than testing wet samples without filter-pressing. Our findings confirmed the filter-press ATR method as proof-of-concept and showed that it has the potential to remove one of the major barriers to in-field application of MIR techniques, the high and variable amounts of moisture levels commonly present in many environmental samples. Extension of the filter-press ATR method to a wider range of analytes may enable it to rapidly acquire moist or wet sediment and soil property data while in the field.

A disposable optofluidic micro-transmission cell with tailorable length for Fourier-transform infrared spectroscopy of biological fluids.

Mid-infrared Fourier-transform infrared (FT-IR) spectroscopy of liquid biological samples is limited by the high absorption of water in this spectral range, which makes conventional transmission cuvettes unsuitable as their centimeter-scale length is already too big. The most common alternative relies on the use of attenuated total reflection (ATR) accessories, which provide a small interaction path length for light along the interface between the analyte and the expensive ATR crystals. In this work, we address this issue by proposing a disposable and low-cost micro-transmission cell. Its construction relies on a simple technique, which consists of dispersing plastic spherical microparticles in a liquid sample before dispensing it between two pieces of silicon assembled one onto the other and acting as windows for the cell. Consequently, the microparticles act as a spacer of very precise height in-between the two silicon windows. This technique allows easy construction of infrared absorption cells with near-optimum optical interaction path length just by selecting the most appropriate particle size. The concept is demonstrated by measuring the concentration of glucose in aqueous solutions using microspheres of diameter 20 μm then 40 μm and analyzing the corresponding glucose absorption peaks in the wavenumber range 950-1200 cm-1. The performance is compared to that of standard ATR spectroscopy of the same samples. This resulted in a root-mean-square error of cross-validation (RMSECV) of 58.8 mg dl-1 as obtained for transmission measurements by partial least squares (PLS) regression, which is comparable to the RMSECV of 53 mg dl-1 for single-reflection diamond ATR measurements.

Infrared spectroscopy for neurochemical monitoring of alcohol and its metabolites.

Continuous measurement of the concentrations of ethanol and its metabolites, acetaldehyde and acetic acid, in vivo is important for the alcohol research community. Most studies only measure ethanol because accurate measurement of all three compounds is challenging. Measurement inside tissue/brain is done using a microdialysis technique, followed by off-line analysis using gas chromatography (GC). To realize simultaneous measurement of ethanol and its metabolites, one can take advantage of infrared (IR) spectroscopy as a rapid and reagent-free method. Here we report a feasibility study of using IR spectroscopy to simultaneously measure ethanol, acetaldehyde and acetic acid in aqueous solution. Different concentrations in transmission mode at different optical pathlengths and using attenuated total reflectance (ATR) were measured. In vitro microdialysis was performed on the mixture of the three compounds, and the collected sample was measured using IR to demonstrate the capability of quantifying the concentrations of the three analytes simultaneously. Lastly, to overcome the limitations of the microdialysis technique, direct measurement using evanescent-field IR spectroscopy can be a potential alternative. A hydrophobic polymer coating that adsorbs ethanol and excludes water, could improve sensitivity. Sorption kinetics in polymethyl methacrylate (PMMA) and polydimethylsiloxane (PDMS) coatings on an ATR crystal were measured. Both polymers demonstrate preferential adsorption of ethanol over water.

SpectIR-fluidics: completely customizable microfluidic cartridges for high sensitivity on-chip infrared spectroscopy with point-of-application studies on bacterial biofilms.

We present a generalizable fabrication method for a new class of analytical devices that merges virtually any microfluidic design with high-sensitivity on-chip attenuated total reflection (ATR) sampling using any standard Fourier transform infrared (FTIR) spectrometer. Termed "spectIR-fluidics", a major design feature is the integration of a multi-groove silicon ATR crystal into a microfluidic device, compared with previous approaches in which the ATR surface served as a structural support for the entire device. This was accomplished by the design, fabrication, and aligned bonding of a highly engineered ATR sensing layer, which con```tains a seamlessly embedded ATR crystal on the channel side and an optical access port that matched the spectrometer light path characteristics at the device exterior. The refocused role of the ATR crystal as a dedicated analytical element, combined with optimized light coupling to the spectrometer, results in limits of detection as low as 540 nM for a D-glucose solution, arbitrarily complex channel features that are fully enclosed, and up to 18 world-to-chip connections. Three purpose-built spectIR-fluidic cartridges are used in a series of validation experiments followed by several point-of-application studies on biofilms from the gut microbiota of plastic-consuming insects using a small portable spectrometer.

Calibration model transfer in mid-infrared process analysis with in situ attenuated total reflectance immersion probes.

Process applications of mid-infrared (MIR) spectrometry may involve replacement of the spectrometer and/or measurement probe, which generally requires a calibration transfer method to maintain the accuracy of analysis. In this study, direct standardisation (DS), piecewise direct standardisation (PDS) and spectral space transformation (SST) were compared for analysis of ternary mixtures of acetone, ethanol and ethyl acetate. Three calibration transfer examples were considered: changing the spectrometer, multiplexing two probes to a spectrometer, and changing the diameter of the attenuated total reflectance (ATR) probe (as might be required when scaling up from lab to process analysis). In each case, DS, PDS and SST improved the accuracy of prediction for the test samples, analysed on a secondary spectrometer-probe combination, using a calibration model developed on the primary system. When the probe diameter was changed, a scaling step was incorporated into SST to compensate for the change in absorbance caused by the difference in ATR crystal size. SST had some advantages over DS and PDS: DS was sensitive to the choice of standardisation samples, and PDS required optimisation of the window size parameter (which also required an extra standardisation sample). SST only required a single parameter to be chosen: the number of principal components, which can be set equal to the number of standardisation samples when a low number of standards (n < 7) are used, which is preferred to minimise the time required to transfer the calibration model.

Mesoporous Zirconia Coating for Sensing Applications Using Attenuated Total Reflection Fourier Transform Infrared (ATR FT-IR) Spectroscopy.

Mid-infrared attenuated total reflection (ATR) spectroscopy is a powerful tool for in situ monitoring of various processes. Mesoporous silica, an extensively studied material, has already been applied in sensing schemes due to its high surface area and tunable surface chemistry. However, its poor chemical stability in aqueous solutions at pH values higher than 8 and strong absorption below 1250cm-1 limits its range of applications. To circumvent these problems, a mesoporous zirconia coating on ATR crystals was developed. Herein, the synthesis, surface modification, and characterization of ordered mesoporous zirconia films on Si wafers and Si-ATR crystals are presented. The modified coating was applied in sensing schemes using aromatic and aliphatic nitriles in aqueous solution as organic pollutants. The mesoporous zirconia coating shows strong chemical resistance when kept in alkaline solution for 72h. The success of surface modification is confirmed using Fourier transform infrared (FT-IR) spectroscopy and contact angle measurements. Benzonitrile and valeronitrile in water are used as model analytes to evaluate the enrichment performance of the film. The experimental results are fitted using Freundlich isotherms, and enrichment factors of 162 and 26 are calculated for 10mgL-1 benzonitrile and 25mgL-1 valeronitrile in water, respectively. Limits of detection of 1mgL-1 for benzonitrile and 11mgL-1 for valeronitrile are obtained. The high chemical stability of this coating allows application in diverse fields such as catalysis with the possibility of in situ monitoring using FT-IR spectroscopy.

Rheo-IR: A combined setup for correlating chemical changes via FTIR spectroscopy and rheological properties in a strain-controlled rheometer

Fourier-transform infrared (FTIR) spectroscopy is a common tool for determining, both qualitatively and quantitatively, the chemical composition of a material in the solid, liquid, or gas phase. It is additionally used as a technique to monitor the rate of chemical changes. These chemical changes can have rheological relevance, e.g., polymerization kinetics, rubber crosslinking, or epoxy curing, just to mention a few. We report the design of an IR transparent upper-plate rheological geometry used as an attenuated total reflectance (ATR) sampling tool. Additionally, we describe the technical and methodological adaption of the ATR crystal into an ARES G2 rheometer for combined FTIR and rheological measurements. In this new setup, a strain-controlled rheometer is combined with an ATR crystal and the IR beam is guided through two off-axis parabolic mirrors to the quasi-static upper plate of the rheometer to gain maximum IR sensitivity. Thereby online and directly correlated real-time FTIR spectra can be acquired while simultaneously conducting rheological measurements. This allows for in situ correlation of macroscopic rheological properties with microscopic and molecular chemical changes. These experiments are conducted for a material under controlled conditions having exactly the same sample time evolution for both measurements. To demonstrate its potential, this newly developed method was applied to correlate the polymer network formation for a free-radical copolymerization of acrylic acid and methylenebis(acrylamide) as a crosslinking agent via IR spectroscopy and the respective mechanical time evolution, in a dilute water-based solution.

Three different Fourier-transform mid-infrared sampling techniques to characterize bio-organic samples.

In recent years, there has been a surge in the number of applications of Fourier-transform mid-infrared (FTIR) spectroscopy for the characterization of environmental samples and prediction of some of their properties whose measurement has traditionally involved time-consuming and costly methods. However, there are several different mid-infrared techniques available, and there is a gap in knowledge regarding the best-suited technique for recording informative spectra of different types of environmental samples. This study compared the three most widespread FTIR techniques using solid and liquid samples. A total of 11 environmental samples belonging to four categories were analyzed with attenuated total reflectance (ATR), photoacoustic (PAS), and diffuse reflectance (DR) FTIR spectroscopy. Overall, PAS-FTIR was the best technique, providing a greater amount of information, especially for opaque samples (i.e., organic waste, biochar, and soil), than ATR-FTIR and DR-FTIR spectroscopy. Attenuated total reflectance FTIR provided the best spectra for soft samples, such as plant materials, probably due to their ability to achieve good optical contact with the ATR crystal. Finally, DR-FTIR performed relatively well for most samples but was found to be more sensitive to moisture in the samples, resulting in noise in specific areas, and was less sensitive in bond vibrations related to Si.

A pocket-sized 3D-printed attenuated total reflection-infrared filtometer combined with functionalized silica films for nitrate sensing in water

Strong anion-exchange materials carry a positive charge that allows them to trap and concentrate anions while releasing other anions. Here, we introduced a quaternary ammonium anion exchange group into mesoporous silica films coated on attenuated total reflection (ATR) crystals to enrich nitrate from aqueous phase in the volume probed by the evanescent field. The ion-exchange and enrichment capabilities of the films were characterized using FTIR spectroscopy. Thereby fast analyte enrichment and full sensor recovery were observed. In addition, high enrichment factors of up to 1600 were achieved. After characterization, these coated ATR crystals were used in a dedicated ATR-IR filtometer comprising a Fabry-Pérot filter detector unit and a miniaturized thermal emitter with a overall dimensions of only 80 mm × 120 mm × 70 mm. The filter covered the spectral region between 1250–1800 cm−1 allowing for recording IR spectra of nitrate enriched into the mesoporous silica film. The sensor was calibrated using the Langmuir adsorption model as calibration function. From this a limit of detection of 1.2 mg L−1 was derived for the ATR-IR filtometer. This emphasizes the high potential of functionalized mesoporous silica films combined with low-cost filtometers for portable water sensors.

Transformation of aqueous protein attenuated total reflectance infra-red absorbance spectroscopy to transmission.

Infrared (IR) spectroscopy is increasingly being used to probe the secondary structure of proteins, especially for high-concentration samples and biopharmaceuticals in complex formulation vehicles. However, the small path lengths required for aqueous protein transmission experiments, due to high water absorbance in the amide I region of the spectrum, means that the path length is not accurately known, so only the shape of the band is ever considered. This throws away a dimension of information. Attenuated total reflectance (ATR) IR spectroscopy is much easier to implement than transmission IR spectroscopy and, for a given instrument and sample, gives reproducible spectra. However, the ATR-absorbance spectrum varies with sample concentration and instrument configuration, and its wavenumber dependence differs significantly from that observed in transmission spectroscopy. In this paper, we determine, for the first time, how to transform water and aqueous protein ATR spectra into the corresponding transmission spectra with appropriate spectral shapes and intensities. The approach is illustrated by application to water, concanavalin A, haemoglobin and lysozyme. The transformation is only as good as the available water refractive index data. A hybrid of literature data provides the best results. The transformation also allows the angle of incidence of an ATR crystal to be determined. This opens the way to using both spectral shape and spectra intensity for protein structure fitting.

Non-invasive identification of polymers in cultural heritage collections: evaluation, optimisation and application of portable FTIR (ATR and external reflectance) spectroscopy to three-dimensional polymer-based objects

The conservation of polymer-based cultural heritage is a major concern for collecting institutions internationally. Collections include a range of different polymers, each with its own degradation processes and preservation needs, however, they are frequently unidentified in collection catalogues. Fourier transform infrared (FTIR) spectroscopy is a useful analytical tool for identifying polymers, which is vital for determining storage, exhibition, loan and treatment conditions. Attenuated Total Reflection (ATR), and External Reflection (ER) are proven effective FTIR sampling techniques for polymer identification and are beginning to appear in conservation labs. This paper evaluates and optimises the application of these two FTIR techniques to three-dimensional plastic objects in the museum context. Elements of the FTIR measurement process are investigated for 15 common polymers found in museum collections using both authentic reference sheets, and case study objects to model for surface characteristics. Including: use of the ATR and ER modules, the difference between clamping and manually holding objects in contact with the ATR crystal, use of the Kramers–Kronig Transformation, signal-to-noise ratios for increasing number of co-added scans, resultant time taken to collect each measurement, associated professional, health and safety considerations, and the use and availability of reference materials for polymer identify verification. Utilising this information, a flowchart for applying FTIR spectroscopy to three-dimensional historic plastic objects during museum collection surveys is proposed to guide the conservation profession.

Pore Size-Dependent Structure of Confined Water in Mesoporous Silica Films from Water Adsorption/Desorption Using ATR-FTIR Spectroscopy.

The local structure of water on chemically and structurally different surfaces is a subject of ongoing research. In particular, confined spaces as found in mesoporous silica have a pronounced effect on the interplay between the adsorbate-adsorbate and adsorbate-surface interactions. Mid-infrared spectroscopy is ideally suited to quantitatively and qualitatively study such systems as the probed molecular vibrations are highly sensitive to intermolecular interactions. Here, the quantity and structure of water adsorbed from the gas phase into silica mesopores at different water vapor pressures was monitored using mid-infrared attenuated total reflection (ATR) spectroscopy. Germanium ATR crystals were coated with different mesoporous silica films prepared by evaporation-induced self-assembly. Quantitative analysis of the water bending vibration at 1640 cm-1 at varying vapor pressure allows for retrieving porosity and pore size distribution of the mesoporous films. The results were in excellent agreement with those obtained from ellipsometric porosimetry. In addition, different degrees of hydrogen bonding of water as reflected in the band position and shape of the stretching vibrations (3000-3800 cm-1) were analyzed and attributed to high-density, unordered bulk, low-density, and surface-induced ordered water. Thereby, the progression of surface-induced ordered water and bulk water as a function of water vapor pressure was studied for different pore sizes. Small pores of 5 nm diameter showed a number of two-ordered monolayers, whereas for pores >12 nm diameter, the number of ordered monolayers is significantly larger and agrees with the number observed on planar SiO2 surfaces.

Real time FTIR spectroscopy of tissue smears from gynecologic cancer tissues: A preliminary study.

e17089 Background: Women with suspected Gynecologic cancer undergo surgical procedures during which tissue from suspected areas is excised. Fast histopathology analysis is performed intra-operatively using frozen section (FS) analysis, results of which are available within less than an hour. However, the accuracy of the FS test ranges between 75% and 100% when compared to final histopathology diagnoses. Fourier transform Infrared (FTIR) spectroscopy, utilized for classification of tissue samples into malignant and benign tumors, has shown comparable results to those of FS histopathology analysis. However, the sample preparation time and the effects of tissue preparation on the measured spectra have been a concern for the utilization of this technique in clinical practice. In this study we used attenuated total reflection (ATR) FTIR spectroscopy to examine fresh tissue impression smears as an alternative to the FS technique for rapid classification of tissue samples obtained during surgery. Methods: The study was approved by relevant ethics committees and was conducted in accordance with the Declaration of Helsinki. All patients provided written, informed consent. In total, 23 biopsies (ovarian and uterine) were extracted from suspected tumor sites during surgical procedures and sent to the histopathology laboratory for both pathological and FTIR analyses. Results of the histopathology analysis classified 15 samples as benign and 8 samples as malignant. Prior to the histopathologic analysis, tissue samples from these tumors were lightly pressed against the surface of an ATR crystal, leaving on its' surface impression smears. These smears were air dried for ~5 minutes. Mid-IR absorbance spectra were collected using an ATR-FTIR spectrometer. Machine learning techniques (PCA-LDA and SVM) were utilized to build discrimination models from the absorbance data of the measured smears. Sensitivity and specificity were calculated. Results: IR absorbance spectra of malignant smears were consistently higher from spectra of benign smears in the 850cm-1 to 1450 cm-1 range and they were consistently lower in the 3200cm-1 to 3600cm-1 range. The PCA-LDA discrimination model correctly classified the samples with a sensitivity and specificity of 100%, and the SVM showed a training accuracy of 100% and a cross validation accuracy of 91.3%. Conclusions: These preliminary results suggest that ATR-FTIR spectroscopy of tissue smears may have an important role in the development of next-generation techniques for intra-operative tumor classification.

Linear Scanning ATR-FTIR for Chemical Mapping and High-Throughput Studies of Pseudomonas sp. Biofilms in Microfluidic Channels.

A fully automated linear scanning attenuated total reflection (ATR) accessory is presented for Fourier transform infrared (FTIR) spectroscopy. The approach is based on the accurate displacement of a multibounce ATR crystal relative to a stationary infrared beam. To ensure accurate positioning and to provide a second sample characterization mode, a custom-built microscope was integrated into the system and the computerized work flow. Custom software includes automated control and measurement routines with a straightforward user interface for selecting parameters and monitoring experimental progress. This cost-effective modular system can be implemented on any research-grade spectrometer with a standard sample compartment for new bioanalytical chemistry studies. The system was validated and optimized for use with microfluidic flow cells containing growing Pseudomonas sp. bacterial biofilms. The complementarity among the scan positioning accuracy, measurement spatial resolution, and the microchannel dimensions paves the way for parallel biological assays with real-time control over environmental parameters and minimal manual labor. By rotating the channel orientation relative to the beam path, the system could also be used for acquisition of linear biochemical maps and stitched microscope images along the channel length.

Time Variations in the FTIR Spectra of Liquids Measured in the ATR Configuration

Attenuated total reflection (ATR) is often employed in the measurements of FTIR spectra. Such a configuration is convenient for experiments with liquid samples, since pressing them to the surface of the ATR crystal is not necessary. Spectral changes that occur in the course of measurements with liquids are revealed. It is shown that significant variations in the concentrations of components may occur in the ATR-FTIR measurements of solutions. Redistribution of components of a solution in the vicinity of the ATR crystal may lead to inhomogeneity of the sample.

Modeling the attenuated total reflectance infrared (ATR-FTIR) spectrum of apatite

Attenuated total reflectance (ATR) infrared spectra were measured on a synthetic and a natural fluorapatite sample. A modeling approach based on the computation of the Fresnel reflection coefficient between the ATR crystal and the powder sample was used to analyze the line shape of the spectra. The dielectric properties of the samples were related to those of pure fluorapatite using an effective medium approach, based on Maxwell–Garnett and Bruggeman models. The Bruggeman effective medium model leads to a very good agreement with the experimental data recorded on the synthetic fluorapatite sample. The poorer agreement observed on the natural sample suggests a more significant heterogeneity of the sample at a characteristic length scale larger than the mid-infrared characteristic wavelength, i.e., about 10 micrometers. The results demonstrate the prominent role of macroscopic electrostatic effects over fine details of the microscopic structure in determining the line shape of strong ATR bands.

Specular reflectance (SR) and attenuated total reflectance (ATR) infrared (IR) spectroscopy of transparent flat glass surfaces: A case study for soda lime float glass

This paper describes the optical principles of specular reflection (SR) and attenuated total reflection (ATR)-infrared (IR) spectroscopy, both of which are useful methods for glass surface analyses. It should be noted that the shape, position, and relative intensity of peaks in reflectance spectra vary drastically depending on the IR incidence angle as well as the probe method (SR vs. ATR). For example, in SR-IR analyses of soda lime glass, the Si–O–Si stretch band shows a blue-shift from its original position and a new peak grows at ~1200cm−1 as the IR incidence angle increases. In contrast, the Si–O–Si stretch band appears significantly red-shifted from its original position in the ATR-IR spectra of soda lime glass. SR-IR spectra of thin and flat samples can contain transmission spectral features, due to the reflection from the backside, in the region where the bulk absorption coefficient is low. These artifacts are due to the complex nature of refractive index and should not be interpreted as new peaks or new chemical states of soda lime glass surfaces. An ATR-IR analysis to probe hydrous species in glass surface is demonstrated using two ATR crystals with different refractive indices.

Determination of lidocaine in urine at low ppm levels using dispersive microextraction and attenuated total reflectance–Fourier transform infrared measurements of dry films

IR spectra provide valuable information about biological systems and can be obtained with compactable and affordable instruments, but the lack of sensitivity of this technique hampers its use in the determination of drugs in clinical fluids. Taking lidocaine as a target molecule, in this paper we introduced a methodology for determining drugs in urine samples using infrared spectroscopy. The lack of sensitivity of the IR was compensated with the combination of an effective and straightforward dispersive liquid–liquid microextraction and the measurement of the dry film of the organic extracts through attenuated total reflectance (ATR). The method developed improves the sensitivity by eliminating the solvent and preconcentrating the analyte in the surface of the ATR crystal. Urine samples were taken from 15 volunteers, and 9 samples were spiked at 6 concentration levels ranging from 0 to 33mgL−1. Multivariate models based on partial least squares regression and science-based calibration were performed and validated with a separated set of spiked urine samples from other patients, obtaining ratio prediction to deviation (RPD) values higher than 3.5. The limit of detection values obtained were 0.5mgL−1 for the univariate calibration and 1.7mgL−1 for a reliable multivariate calibration. Therefore, the procedure is limited to only levels of lidocaine higher than 2mgL−1 but serves as an untargeted, fast, and versatile screening tool, which maintains all the advantages of the widespread application of the IR spectroscopy to the clinical analysis, such as simplicity, compactability, and minimum use of reagents and solvents.

Control of Lignin Solubility and Particle Formation Modulates Its Antioxidant Efficiency in Lipid Medium: An In Situ Attenuated Total Reflectance FT-IR Study

Lignin is an abundant plant polymer usually regarded as waste material. In the present work, antioxidant properties of lignin preparations with differing lipid solubility were studied using biodiesel as a convenient lipid test substrate. In place of formerly used assays, we used attenuated total reflectance (ATR) FT-IR spectroscopy to follow in situ biodiesel autoxidation on a heated ATR crystal as a function of time. The study demonstrates that a complex balance between intrinsic (chemical) efficiency, solubility, and particle formation controls the antioxidant efficiency of differently prepared lignin fractions. It was found that solubility and particle formation of lignin preparations strongly modulate its antioxidant efficiency and that these properties might depend on the presence of lipid components within the original lignin source.