Determination Of Extinction Coefficient Research Articles

Systematically amino acid analysis: Advancements in extinction coefficient determination for therapeutic proteins

Biologicals developers often face challenges in accurately determining the extinction coefficient (EC) measurement. We have successfully improved the precision and robustness of the widely recognized amino acid analysis method for EC determination, through a stepwise optimization process. Extensive analyses based on 114 observations, covering eight proteins over three years were performed, with a maximum relative standard deviation of 1.5% among multiple analysts, and a maximum deviation of 2.8% from the theoretical EC across the eight given proteins examined.

Dense solutions’ extinction coefficients measurements strategy, based on laser sheet double structuring

Some spectroscopic measurements are limited by multiple scattering, appearing as a restricting factor, particularly in the determination of extinction coefficient for optically dense solutions. The development of experimental and computational strategies to deal with this problem seems extremely important. This could have positive effects that would impact discipline such as medicine, where the accurate determination of optical properties in biological tissues helps in-situ diagnostics. The work reported in this paper involves a double structuring of the incident radiation by diffraction gratings, thus forming moiré patterns which are then applied to an existing technique known as Structured Laser Illuminating Planar Imaging (SLIPI). After an image processing based on Fourier transforms, these strategies allowed to optimize standard SLIPI measurements by suppressing more of the multiple scattering photons for accurate calculation of extinction coefficients of dense chlorophyll solutions probed at 450 nm and 638 nm.

Interpreting weak backscattering signals: new results

ABSTRACTThe interpretation of lidar information was carried out here inverting the weak backscattering signals returned from a homogeneous atmosphere where atmospheric parameters are constant in space. It was performed interpreting weak backscattering signals using the linear approximation and the preliminary calculated value of the extinction coefficient. The analysis of the systematic errors was carried out for various distances where the backscattering signal power was measured. It was shown that the systematic errors of the extinction coefficient can exceed the systematic errors of the backscattering signal power by almost a factor of 20. There was analysed the influence of the systematic errors of the background light on the results of the lidar information interpretation. The result of the background light determination cannot be accurate enough if it is found using data obtained before sending the impulse. It was shown that the relative error of this value can be ˂1% but the relative error of the extinction coefficient determination can be ˃100%. There were analysed the experimental results and the results of computations of the scattered irradiance with absence and presence of water in the inhomogeneous coating of the aerosol particles. It was shown that the developed models adequately describe the process of scattering by a particle. It was found that the use of adequately describing models can significantly reduce the error of optical aerosol sizing.

The experimental determination of composition-dependent molar extinction coefficient of nonstoichiometric Zn–Cu–In–S nanocrystals

In this study, the different compositions of Zn–Cu–In–S quantum dots (ZCIS QDs) were prepared by a facile synthetic approach. The compositions and particle sizes of ZCIS nanocrystals were precisely controlled. The composition-dependent molar extinction coefficient of nonstoichiometric ZCIS QDs has been determined by combination of ICP-MS with UV–vis absorption and TEM. The molar extinction coefficient values were found to comply with a power function with exponents of 3.6 and 1.8 with the variation of the content of Cu or Zn, respectively. The extinction coefficient of ZCIS QDs exhibited the composition dependency and seemed to be larger than that of CuInS2 for a given size. The determination of the extinction coefficient makes it feasible to calculate the concentration of ZCIS QDs in solution and promotes its further applications in bioanalysis.

Precise determination of protein extinction coefficients under native and denaturing conditions using SV-AUC.

The accurate determination of protein concentration is an important though non-trivial task during the development of a biopharmaceutical. The fundamental prerequisite for this is the availability of an accurate extinction coefficient. Common approaches for the determination of an extinction coefficient for a given protein are either based on the theoretical prediction utilizing the amino acid sequence or the photometric determination combined with a measurement of absolute protein concentration. Here, we report on an improved SV-AUC based method utilizing an analytical ultracentrifuge equipped with absorbance and Rayleigh interference optics. Global fitting of datasets helped to overcome some of the obstacles encountered with the traditional method employing synthetic boundary cells. Careful calculation of dn/dc values taking glycosylation and solvent composition into account allowed the determination of the extinction coefficients of monoclonal antibodies and an Fc-fusion protein under native as well as under denaturing conditions. An intra-assay precision of 0.9% and an accuracy of 1.8% compared to the theoretical value was achieved for monoclonal antibodies. Due to the large number of data points of a single dataset, no meaningful difference between the ProteomeLab XL-I and the new Optima AUC platform could be observed. Thus, the AUC-based approach offers a precise, convenient and versatile alternative to conventional methods like total amino acid analysis (AAA).

Quantification of acid site densities on zeolites in the presence of solvents via determination of extinction coefficients of adsorbed pyridine

Fourier transform infrared (FTIR) spectroscopy is frequently used to characterize properties of catalytic sites, identify reaction intermediates, and probe interactions between adsorbates and surfaces, making it a versatile tool for elucidating reaction mechanisms in heterogeneous catalysis. While this technique is typically regarded as qualitative or semiquantitative in nature, quantitative results can be obtained by determining integrated molar extinction coefficients (IMECs), or the amount of signal per mole of adsorbed or bulk species. IMECs for vibrational modes corresponding to pyridine adsorbed onto Brønsted and Lewis acid sites have been determined in the vapor phase; however, the effect of solvents on catalytic sites cannot be captured. Liquid-phase IMECs enable quantification of adsorbates in the presence of solvents, allowing quantitative evaluations of the effect of solvents on catalysts. A method is outlined for determining IMECs of pyridine adsorbed on zeolites in the liquid phase in an attenuated total reflection (ATR) configuration. The method can also be applied to substrates other than pyridine. The IMECs for pyridine adsorbed onto Brønsted and Lewis acid sites in water, acetonitrile, and ethanol are determined. The IMEC values are shown to be dependent on solvent choice, but independent of zeolite framework.

Determination of extinction coefficients of human hemoglobin in various redox states

The role of hemoglobin (Hb) redox forms in tissue and organ toxicities remain ambiguous despite the well-documented contribution of Hb redox reactivity to cellular and subcellular oxidative changes. Moreover, several recent studies, in which Hb toxicity were investigated, have shown conflicting outcomes. Uncertainties over the potential role of these species may in part be due to the protein preparation method of choice, the use of published extinction coefficients and the lack of suitable controls for Hb oxidation and heme loss. Highly purified and well characterized redox forms of human Hb were used in this study and the extinction coefficients of each Hb species (ferrous/oxy, ferric/met and ferryl) were determined. A new set of equations were established to improve accuracy in determining the transient ferryl Hb species. Additionally, heme concentrations in solutions and in human plasma were determined using a novel reversed phase HPLC method in conjugation with our photometric measurements. The use of more accurate redox-specific extinction coefficients and method calculations will be an invaluable tool for both in vitro and in vivo experiments aimed at determining the role of Hb-mediated vascular pathology in hemolytic anemias and when Hb is used as oxygen therapeutics.

Assessment for quantification of biopharmaceutical protein using a microvolume spectrometer on microfluidic slides

Spectrophotometric procedure is a traditional method for quantifying protein concentration in solution. The benefit of this method is simple and direct, not requiring standard materials, sample incubation, and exogenous chromophore addition. There are also some drawbacks, low detection limits and high amounts of protein needed. The recent advances in fiber optics and nano-technology enable to overcome those drawbacks by using microvolume sampling and extremely short pathlengths. In this study, the utility of the microvolume system with microfluidic carriers was assessed for concentration measurement of biopharmaceutical protein using absorbance at 280 nm and also compared to a traditional instrument. The results show a high correlation of estimation by Cohen’s d effect size and Pearson correlation coefficient between two different spectrometers. For UV spectral analysis, there are no statistically significant difference between those spectra from a microvolume spectrometer and a traditional one. For the extinction coefficient determination, two different spectrometers also give quite comparable and similar values. Thus a microvolume spectrometer can be used as an alternative over a traditional one to determine the concentration of biopharmaceutical protein. Moreover, the dynamic range for protein quantification by a microvolume spectrometer is over 10-fold higher than that of a traditional one.

Parametric investigation on thermo-hydraulic performance of wire screen matrix packed solar air heater

This paper presents performance results of a wire screen matrix packed solar air heater based on a mathematical model developed to investigate the effect of various system and operating parameters on the thermo-hydraulic performance. A computer programme is developed in C++ to estimate the temperature rise of the entering air for evaluation of effective efficiency by solving the governing equations numerically using relevant correlations for heat transfer coefficient for packed bed systems. The mathematical model developed is compared with the experimental results as well as previous model and the results are found fairly in agreement. Further, a correlation is developed for determination of extinction coefficient based on the geometrical parameter of matrix. Comparative study of thermo-hydraulic performance of high and low porosity matrices indicate the superiority of high porosity matrices as maximum effective efficiency for high porosity matrix is 76% while for low porosity it is found to be 50%, showing an overall enhancement of about 26%. The effective efficiency value clearly indicates that there exists an optimum duct depth (0.015–0.035m), duct length (2–5m) and width (0.2–0.6m) which results in best thermo-hydraulic performance for various mass flow rates and the maximum effective efficiency is obtained for duct depth of 0.015m, length 5m and width 0.5m.

Methodology for materials analysis using swept-frequency feedback interferometry with terahertz frequency quantum cascade lasers.

Recently, we demonstrated an interferometric materials analysis scheme at terahertz frequencies based on the self-mixing effect in terahertz quantum cascade lasers. Here, we examine the impact of variations in laser operating parameters, target characteristics, laser-target system properties, and the quality calibration standards on our scheme. We show that our coherent scheme is intrinsically most sensitive to fluctuations in interferometric phase, arising primarily from variations in external cavity length. Moreover we demonstrate that the smallest experimental uncertainties in the determination of extinction coefficients are expected for lossy materials.

The determination of extinction coefficient of CuInS2, and ZnCuInS3 multinary nanocrystals

A pioneering work for determining the extinction coefficient of colloidal semiconductor nanocrystals (NCs) has been cited over 1500 times (W. Yu, W. Guo, X. G. Peng, Chem. Mater., 2003, 15, 2854-2860), indicating the importance of calculating NC concentration for further research and applications. In this study, the size-dependent nature of the molar extinction coefficient of "greener" CuInS(2) and ZnCuInS(3) NCs with emission covering the whole visible to near infrared (NIR) is presented. With the increase of NC size, the resulting quantitative values of the extinction coefficients of ternary CuInS(2) and quaternary ZnCuInS(3) NCs are found to follow a power function with exponents of 2.1 and 2.5, respectively. Obviously, a larger value of extinction coefficient is observed in quaternary NCs for the same size of particles. The difference of the extinction coefficient from both samples is clearly demonstrated due to incorporating ZnS with a much larger extinction coefficient into CuInS(2) NCs.

The influence of spectral slit width on the absorption of visible or ultra-violet light by pharmacopoeial substances.

Abstract The importance of narrow slits in the spectrophotometric determination of extinction coefficients has been demonstrated. Care is needed in the B.P. 1958 spectrophotometric tests or assays of apomorphine hydrochloride, chloroquine phosphate, chloroquine sulphate, diiodohydroxyquinoline, naphazoline nitrate, papaverine hydrochloride and especially procyclidine hydrochloride, to avoid spuriously low results.

Determination of extinction coefficients of ion pairs formed by multicharged ions

The reasons for discrepancies between reported values of extinction coefficients of ion pairs formed by multicharged ions are analysed and approaches to a correct description of such systems are suggested.

Refractive index and extinction coefficient determination of an absorbing thin film by using the continuous wavelet transform method

We present the continuous wavelet transform (CWT) method for determining the dispersion curves of the refractive index and extinction coefficient of absorbing thin films by using the transmittance spectrum in the visible and near infrared regions at room temperature. The CWT method is performed on the transmittance spectrum of an a - Si(1-x)C(x):H film, and the refractive index and extinction coefficient of the film are continuously determined and compared with the results of the envelope and fringe counting methods. Also the noise filter property of the method is depicted on a theoretically generated noisy signal. Finally, the error analyses of the CWT, envelope, and fringe counting methods are performed.

Improvements in the determination of extinction coefficients of a thin film using an envelope method

The algorithm used in the envelope method has been integrated with a variational method, for accurate determination of the refractive index (n) and extinction coefficient (k) of a weakly absorbing film deposited onto a weakly absorbing thick substrate. The proposed algorithm leads to significant improvement in the degree of accuracy of k values in the medium and weak absorption region down to k~5 × 10−5.

Cavity Ring-Down and Cavity-Enhanced Detection Techniques for the Measurement of Aerosol Extinction

An instrument employing cavity ring-down (CRD) and cavity-enhanced detection (CED) for the local measurement of aerosol extinction is described and demonstrated. CRD measures the lifetime of photons in a high-quality optical cavity and thereby determines the sum of sample extinction between the cavity mirrors and that due to mirror losses. CRD systems can be calibrated with a single gas for the determination of extinction. A green laser emitting subnanosecond pulses is used as a light source, facilitating measurements free from optical interference in the cavity. The addition of a low-frequency chopper allows for the determination of extinction coefficients with simple linear fitting procedures and also facilitates CED measurements by providing laser power modulation for phase-sensitive detection. CED measures the average power transmitted by the optical cavity. After calibration with two gases, CED allows for the independent measurement of extinction with very high dynamic range and for an independent co...

Extinction Coefficient Determination of Polymeric Materials and Their Use in the Optical Characterization of Infrared Microscopes: A Preliminary Investigation into Quantitative Infrared Microspectroscopy

A comparison of infrared spectra collected in both a macro sample configuration and on a microscope accessory was conducted. The results of this comparison demonstrated that as the microscope aperture was reduced, absorbance values increased for a given polymer film by as much as 33% as compared to the same data collected in the corresponding macro experiment. A detailed evaluation of the optical pathlength for both the micro and macro configurations was conducted. Results of this evaluation and that of detector non-linearity showed that the latter effect was most problematic. During the course of the investigation the average angle of light traversing through the objective was determined for several infrared microscopes. Further analysis indicated that changing the aperture size of the microscope can have significant influence on the absorbance values obtained. In addition, extinction coefficients of poly(methyl methacrylate), polystyrene, poly(vinyl acetate), polycarbonate, and poly(ethylene terephthalate) were determined using macro FT-IR spectroscopy.

Antioxidant properties of melatonin: a pulse radiolysis study

Various one-electron oxidants such as OH , tert-BuO , CCl 3OO , Br 2 − and N 3 , generated pulse radiolytically in aqueous solutions at pH 7, were scavenged by melatonin to form two main absorption bands with λ max = 335 nm and 500 nm. The assignment of the spectra and determination of extinction coefficients of the transients have been reported. Rate constants for the formation of these species ranged from 0.6-12.5 × 10 9 dm 3 mo l −1 s −1. These transients decayed by second order, as observed in the case of Br 2 − and N 3 radical reactions. Both the NO 2 and NO radicals react with the substrate with k = 0.37 × 10 7 and 3 × 10 7 dm 3 mol −1 s −1, respectively. At pH ∼2.5, the protonated form of the transient is formed due to the reaction of Br 2 − radical with melatonin, pK a ( MelH ⇌ Mel + H +) = 4.7 ± 0.1. Reduction potential of the couple (Mel /MelH ), determined both by cyclic voltammetric and pulse-radiolytic techniques, gave a value E 1 7 = 0.95 ± 0.02 V vs. NHE. Repair of guanosine radical and regeneration of melatonin radicals by ascorbate and urate ions at pH 7 have been reported. Reactions of the reducing radicals e aq − and H atoms with melatonin have been shown to occur at near diffusion rates.

Quantitative FTIR Determination of Extinction Coefficients of Adsorbed CO

A combined IR and MS technique was used to determine extinction coefficients of adsorbed CO on the 4% Rh/SiO2 catalyst. An isotopic exchange experiment has been made to obtain accurate integrated absorption intensities. The experimental procedure provides a fast and accurate, as well as a highly reproducible, method for the determination of extinction coefficients of adsorbed surface species. Integrated absorption intensities for Rh+(12CO) and Rh+(13CO)2 were determined at 373 K and 0.1 MPa to be 1.35 and 8.02 cm/μmol, respectively.

Concentration and Extinction Coefficient Determination for Oligonucleotides and Analogs Using a General Phosphate Analysis

A general phosphate analysis (GPA) is developed which assays the concentration of nucleic acid oligomers and their analogs based on stoichiometric phosphorus in the sequence. The method involves complete digestion of the oligomer sample to orthophosphate using acid at high temperature and subsequent colorimetric analysis by phosphomolybdate complex formation. GPA is applied to oligomers having phosphodiester, methylphosphonate, and phosphorothioate backbone linkages. Given the absorption spectra of oligomers having these backbones, extinction coefficients are obtained and compared to other quantitative and predictive methods. In addition to sequences having the usual nucleoside residues found in naturally occurring nucleic acids, oligomers having base analog residues can be readily quantified by GPA.