Dual-wavelength Absorption Research Articles

Analytical figures of merit of a dual-wavelength absorbance approach for real-time broad protein content monitoring for biomanufacturing

Real-time in-line broad protein content monitoring in biomanufacturing downstream unit operations enables the ability to optimize and afford consistent protein recovery. Protein determination from 2 to 400 mg/mL is demonstrated herein via real-time dual-wavelength LED photometric sensor configured at 280 and 310 nm. The figures of merit of this approach include measurement accuracy within the common acceptance criteria of 100 % ± 5 with negligible bias across the linear dynamic ranges. This work expands the utility of an LED based photometric sensor for biopharmaceutical process analytical technology (PAT) applications. It is also congruent with process digitalization and automation industry 4.0 concepts underpinned by Quality by Design (QbD) principles.

Establishing an integrated, four-dimensional quality assessment system for traditional Chinese medicine: A case study of Shuanghuanglian oral liquid

The quality of traditional Chinese medicine (TCM) is the premise to ensure its safety and effectiveness in clinical application. In this study, a complete quality control system for four-dimensional fingerprinting of TCM was innovatively constructed based on multiple detection techniques, and the quality of Shuanghuanglian oral liquid (SHL) was evaluated. Electrochemical fingerprinting (ECFP) as an emerging method without pretreatment provides rich and quantifiable information for SHL samples. The first quantitative ECFP of SHL was developed by the B-Z oscillation system. Eight characteristic parameters were analyzed and a good linear relationship was found between the oscillation lifetime and sample volume, by which the calculated values of the added sample volume (VL) showed different fluctuations between samples. What is more, high-performance liquid chromatography five-wavelength fusion fingerprint (HPLC-FWFP), GC fingerprint (GC-FP), and UV quantum fingerprint (UV-QFP) was established. Meanwhile, the purity of the peaks of the HPLC-FWFP was verified by the dual-wavelength absorption coefficient ratio spectrum (DWAR). Equal weighted ratio quantitative fingerprinting method (EWRQFM) was successfully proposed to extract all potential features for the overall quality assessment of the samples. Finally, a comprehensive evaluation strategy was proposed, namely the variation coefficient weighting algorithm (VCWA). The results of qualitative and quantitative evaluation of HPLC-FWFP, GC-FP, electrochemical quantum fingerprints (EC-QFP), and UV-QFP were integrated by this method. The established evaluation system is also a suitable strategy to control the quality of other TCM preparations.

Spectral and temporal detection of blood bilirubin level using a point-of-care device.

Bilirubin is a biomarker for liver inflammation used to assess liver functions. Its concentration in the blood has been measured using a range of techniques both in clinical and point-of-care settings. Existing point-of-care devices utilize a spectral approach, namely dual-wavelength absorption measurement, to assess the blood bilirubin concentration. This work examines a novel temporal approach based on the photodegradation of bilirubin in the blood sample. It demonstrates that combining photodegradation characteristics with dual-wavelength measurement produces a more accurate technique for measuring blood bilirubin concentration. Tracking the evolution of absorbed light as a function of time represents a low-cost and simple way of improving the accuracy of point-of-care devices for bilirubin measurements.Clinical Relevance - This work demonstrates a facile and cheap bilirubin monitoring approach that may allow bilirubin monitoring applications in homes after a patient is discharged from a hospital, which may decrease the burden on patients, families, and clinicians.

Holistically assessing the quality consistency of compound liquorice tablets from similarities of both all chemical fingerprints and the integrated dissolution curves by systematically quantified fingerprint method

In recent years, traditional analytical methods have failed to meet the widespread use of multi-component Chinese pharmaceutical formulations. To solve this problem, this study proposed a comprehensive analytical strategy using compound liquorice tablets (CLTs) as an example, both in terms of chemical quality and dissolution curve consistency. Firstly, the peak purity of the two wavelengths was checked using dual-wavelength absorbance coefficient ratio spectra (DARS) to avoid the fingerprint bias caused by peak purity. Secondly, liquid-phase dual-wavelength tandem fingerprint (DWTF) of 38 batches of CLTs was established for the first time. The two analytical methods were also evaluated using the systematically quantified fingerprint method (SQFM), and the 38 batches of samples were classified into two grades with good quality consistency. Quantitative analysis of the five markers of CLTs was performed simultaneously using the standard curve method (SCM) and the quantitative analysis of multiple components by single marker (QAMS). The results showed no significant differences between the two analytical methods (p > 0.5). In addition, the in vitro dissolution of CLTs in two media (pure water and pH = 4.5 medium) was determined by the total UV fingerprint dissolution assay. The similarity of the dissolution curves was also analyzed by combining the f2 factor and the dissolution-systematically quantified fingerprint method (DSQFM). The result showed that most of the samples had f2 > 50 and Pm satisfied the range of 70–130%. Finally, a principal component analysis (PCA) model was developed to combine the evaluation parameters of chemical fingerprint and dissolution curves for comprehensive analysis of the samples. In this study, a chromatographic and dissolution-based quality analysis method was proposed, which effectively overcomes the shortcomings of previous analytical methods and provides a scientific analytical method for the quality control of natural drugs.

Nanostructured carboxylated-wood aerogel membrane for high-efficiency removal of Cu (II) ions from wastewater

Heavy-metal pollution is a significant environmental issue that has raised global concern due to its harmful impact on humans and the natural ecosystem. Heavy metal pollution poses several challenges in terms of remediation, including low adsorption capacity and efficiency, scale-up issues and non-degradability. Here, we developed an eco-friendly tunable dual-wavelength absorption mesoporous wood aerogel (TDWA) for removal of Cu2+ ions from wastewater based on wood nanotechnology and carboxylated functionalization treatment. Nanostructured pores generated from the cell walls with carboxyl groups due to the removal of most lignin/hemicellulose and 2,2,6,6-tetramethylpiperidine oxidation. Mechanical compression was parallelly applied to the transverse direction of TDWA resulted in removing micron-scale cell cavity and intercellular, which significantly improved the dynamic adsorption performance with an excellent Cu2+ adsorption capacity of 115 mg g−1. This value is superior to most wood-based adsorption membrane as wastewater mainly flowed through the continuous microfibril network in the cell wall. The double-layer mesoporous TDWA membrane (5 mm) possessed a favorable removal efficiency (99.83 %) even after 5-cyclic adsorption (92.34 %). The invented nanostructured TDWA membrane in the current study has promising potential to substitute the present plastic-based membrane in the realistic Cu2+ adsorption because of the facile technology, renewability and high removal efficient, as well as the complete biodegradability.

Dual-wavelength absorption technique for dryness measurement of wet steam.

This paper presents a dual-wavelength absorption-based approach for measuring and validating the steam dryness fraction of wet steam. A thermally insulated steam cell with a temperature-controlled measurement window (up to 200°C) is designed and fabricated to minimize condensation during water vapor measurements at different operating pressures (1-10bars). Water vapor's measurement sensitivity and accuracy are limited due to other absorbing and non-absorbing species in wet steam. The measurement accuracy is significantly improved with the proposed dual-wavelength absorption technique (DWAT) measurement method. The influence of modifying factors-namely, pressure and temperature-on water vapor absorbance is minimized by a non-dimensional correction factor. The dryness is measured with the help of the water vapor concentration and wet steam mass present in the steam cell. The DWAT dryness measurement approach is validated using a four-stage separating and throttling calorimeter combined with a condensation rig. The accuracy of the dryness measurement system using this optical method is determined to be ±1% for the range of dryness and operating pressure (1-10bars) of wet steam.

A Fast Detection Method for Pesticide Residues by Spectrometry Technique

In this paper, a fast detection method for pesticide residues was proposed, and the qualitative and quantitative detection of the pesticide could be got at once by the method of solution of multiple linear regression equations which were obtained by dual wavelength absorbance and fluorescence spectrometry with least squares method. Moreover, it could detect two kinds of mixed pesticides. Four selected pesticides, aldicarb, fenitrothion, fenvalerate, and chlorothalonil were detected by the method. The results shown that there were good linear relationship in the range of 0.01-1 ppm, and R2 ＞ 0.90. And the method could 100% discriminate the four pesticide residues, the limit of detection was below 8 ppb both single one and mixed one. The recoveries of the pesticides in cabbage samples were observed 92.12%-107.50%. And the recoveries of the mixed pesticides in mineral water samples were observed 86.44%-114.10%. The preliminary study demonstrates that the proposed method has excellent potential application for the safety inspection of food.

Development and Validation of the UV Spectrophotometric Method for Simultaneous Determination of Paracetamol and Pseudoephedrine in Bulk and Combined Tablet Dosage Form

Paracetamol (PARA) and Pseudoephedrine (PSE) are co-formulated drugs that and widely used over the world for the treatment of symptoms associated with common cold. In the present study a specific, rapid and simple UV spectrophotometric method with good sensitivity was developed and validated for the simultaneous quantification of PARA and PSE in bulk and tablet dosage form using methanol as diluent. This method is based on the direct measurement of absorbance at 248 nm wavelength for PARA solution, whereas dual-wavelength absorption measurement is applied for PSE solution (i.e. subtraction of absorbance at 217 nm from that at 201 nm, in which PARA absorbance subtraction is equal to zero). The calibration curves were linear (R2 = 0.9988 and 0.9976 for PARA and PSE, respectively) in a concentrations range of 2 – 10 μg/mL for both drugs. Limit of detection is LOD = 0.4159 μg/mL and 0.29679 μg/mL and the limit of quantification is LOQ = 1.26 μg/mL and 0.8994 μg/mL for PARA and PSE, respectively. The RSD% values for all levels of precision (repeatability, intra-day and inter-day) are within the specified limit for both drugs. The accuracy of PARA determination was ascertained by standard addition method, and the percentage recovery for 50, 100, and 150 % of standard were 97.93, 100, and 101.39%, respectively (RSD% = 1.7). The accuracy of PSE determination was studied by percentage recovery (n = 3), and the results were 100.7, 101.16, and 99.57% (RSD% = 0.6648). The proposed spectrophotometric method was successfully applied to the analysis of commercial tablets and the obtained results were satisfactory.

Tunable dual-wavelength absorption switch with graphene based on an asymmetric guided-mode resonance structure.

We propose a tunable dual-wavelength absorption (TDWA) switch based on an asymmetric guided mode resonance (AGMR) structure. A TDWA switch consists of a graphene layer and an AGMR structure sandwiched by cap and slab layers on a buffer/silicon substrate. The AGMR structure adds a smaller grating unit cell next to a larger one, exciting a second resonance close to but distinct from the first resonance. For switching, the TDWA between an absorptive or reflective mode with each on-/off-state, the chemical potential of graphene is tuned from 0.0 eV to 0.6 eV. For the absorptive mode, two absorption peaks of ≥ 96.2% are separated by 23 nm, both having an on-off ratio of ∼15.52. For the reflective mode, two reflectance peaks of ≥ 93.8% are separated by 23 nm, having on-off ratios of 15.56 dB and 18.95 dB. The maximum on-off ratios of 39.98 dB and 34.55 dB are achieved near the reflectance peaks. Both the period of the AGMR and the cap thickness alters the two peak wavelengths linearly, while the grating width of the AGMR varies nonlinearly from 17 nm to 28 nm. The buffer excites a weak Fabry-Perot resonance, which interacts with the TDWA structure, the result of which is the two absorption peaks are varied. Finally, as the incidence angle of light increases up to 5.3°, the distance of the two peak wavelengths is tuned from ∼22 nm to ∼77 nm with ≥ 96% absorption or ≥ 93% reflectance in each mode.

A new validated high-performance liquid chromatography method for standardization of rosmarinic acid in Salvia extracts

Introduction: Tea bags or infuses of Salvia species from Lamiaceae family are traditionally used for the treatment of cough, and throat inflammations. They are known for antioxidant properties mainly related to the presence of rosmarinic acid (RA). Therefore it is necessary to develop a reliable analytical method for RA assay for standardization of Salvia species and also other plants containing RA like Melissa, Origanum, Lavandula, Rosmarinus, Thymus, Zataria, Mentha, Perovskia, Zhumeria, and Satureja species. In this study using a suitable extraction method by removing unwanted components present in crude methanol extract, phenolic content containing RA was extracted from dry powders of six Salvia species. Then, a suitable high-performance liquid chromatographic (HPLC) method was optimized for quantification of RA in Salvia species. Methods: HPLC analysis was done on a Waters system, equipped with 515 HPLC pump and waters 2487 dual wavelength absorbance detector. The column was a Nova-Pak C18 (3.9 × 150 mm), and Millenium software was used for the determination of the compounds and processing the data. The method was validated according to USP 32 requirements. Results: Among the investigated 6 species, S. virgata was the richest in RA level, demonstrating 3.50 ± 0.12 mg/g, followed by S. sclarea and S. chloroleuca showing 1.65 ± 0.08 and 1.65 ± 0.21 mg/g. S. ceratophylla with 0.10 ± 0.01 mg/g of RA in dried plant powder was the poorest. Conclusion: The validated HPLC method allows determination of amounts as low as 2.5 µg/ mL of RA and linearity in the ranges of 2.5-25 µg/mL and 100–600 µg/mL, which is suitable for standardization of Salvia species in traditional and pharmaceutical formulations

Intracardiac light catheter for rapid scanning transmural absorbance spectroscopy of perfused myocardium: measurement of myoglobin oxygenation and mitochondria redox state.

Absorbance spectroscopy of intrinsic cardiac chromophores provides nondestructive assessment of cytosolic oxygenation and mitochondria redox state. Isolated perfused heart spectroscopy is usually conducted by collecting reflected light from the heart surface, which represents a combination of surface scattering events and light that traversed portions of the myocardium. Reflectance spectroscopy with complex surface scattering effects in the beating heart leads to difficulty in quantitating chromophore absorbance. In this study, surface scattering was minimized and transmural path length optimized by placing a light source within the left ventricular chamber while monitoring transmurally transmitted light at the epicardial surface. The custom-designed intrachamber light catheter was a flexible coaxial cable (2.42-Fr) terminated with an encapsulated side-firing LED of 1.8 × 0.8 mm, altogether similar in size to a Millar pressure catheter. The LED catheter had minimal impact on aortic flow and heart rate in Langendorff perfusion and did not impact stability of the left ventricule of the working heart. Changes in transmural absorbance spectra were deconvoluted using a library of chromophore reference spectra to quantify the relative contribution of specific chromophores to the changes in measured absorbance. This broad-band spectral deconvolution approach eliminated errors that may result from simple dual-wavelength absorbance intensity. The myoglobin oxygenation level was only 82.2 ± 3.0%, whereas cytochrome c and cytochrome a + a3 were 13.3 ± 1.4% and 12.6 ± 2.2% reduced, respectively, in the Langendorff-perfused heart. The intracardiac illumination strategy permits transmural optical absorbance spectroscopy in perfused hearts, which provides a noninvasive real-time monitor of cytosolic oxygenation and mitochondria redox state.NEW & NOTEWORTHY Here, a novel nondestructive real-time approach for monitoring intrinsic indicators of cardiac metabolism and oxygenation is described using a catheter-based transillumination of the left ventricular free wall together with complete spectral analysis of transmitted light. This approach is a significant improvement in the quality of cardiac optical absorbance spectroscopic metabolic analyses.

Simultaneous imaging of temperature and concentration of ethanol–water mixtures in microchannel using near-infrared dual-wavelength absorption technique

This paper presents a simultaneous imaging method of temperature and ethanol concentration of ethanol–water mixtures in microfluidic channels. The principle is based on the facts that the absorbance at a wavelength of 1905 nm is dependent on the temperature of water and that the absorbance at 1935 nm is independent of the temperature but strongly dependent on the molar concentration of water, which is reciprocal to the molar concentration of ethanol in the mixture. The absorbance images at the two wavelengths were acquired alternately, each at 50 frames per second, by an alternate irradiation system and near-infrared (NIR) camera, and then converted to the temperature and concentration images by a linear regression model. The imaging method was applied to a dilute ethanol–water mixture with an ethanol concentration of 0.43 M and water flowing side by side in a temperature-controlled Y-channel. The concentration images clearly showed differences between the mixture and water streams, and that the transverse concentration gradient between the two streams decreased downstream by mutual diffusion. It was also confirmed that the mutual diffusion coefficient increased as the temperature increased. The temperature images showed that uniform distributions were immediately formed due to heat transfer between the fluid and channel materials.

Experimental and Density Functional Theoretical Studies on the Spectra of <i>N</i>, <i>N'</i>-di[3-hydroxy-4-(2-benzothiazole)phenyl]urea

The experimental infrared (IR), nuclear magnetic resonance (NMR), and ultraviolet (UV) spectra, and density functional theory (DFT) calculations of the novel compoundN,N′-di[3-hydroxy-4-(2-benzothiazole) phenyl]urea (4-DHBTU) are presented. Compared with the UV spectra of the 2-(4-amino-2-hydroxyphenyl) benzothiazole (4-AHBT) monomer, the experimental spectra of 4-DHBTU, a dimer of 4-AHBT, show dual-wavelength absorption with significantly enhanced absorption intensity and an obvious red shift of the maximum absorption peak. Analysis of the experimental spectra and the DFT calculations shows that the structures ofcis-C11 andtrans-C11 are the two most stable conformers, and that the main reason for the different UV spectral properties of the dimer and monomer is the coexistence ofcis-C11,trans-C11,cis-C22, and trans-C22 in the 4-DHBTU sample. In addition, the DFT calculations indicate that a hydrogen-bonding interaction between 4-DHBTU and the dimethyl sulfoxide (DMSO) solvent leads to a large1H NMR chemical shift for atoms 15H and 16H in 4-DHBTU.

Development of a potassium-selective optode for hydroponic nutrient solution monitoring

Highly efficient and reliable plant growth such as that required in biological life support systems for future space-based missions can be better achieved with knowledge of ion concentrations within the hydroponic nutrient solution. This paper reports on the development and application of ion-selective bulk optodes to plant growth systems. Membranes for potassium-selective sensing are reported that have been tailored so that their dynamic range is centred on potassium activities within typical nutrient solution recipes. The developed sensors have been shown to exhibit a potassium activity measuring range from 0.134 to 117mM at pH 6.0. These bulk optodes show full scale response on the order of several minutes. They show minimal interference to other cations and meet worst-case selectivity requirements for potassium monitoring in the considered half strength Hoagland solution. When continuously immersed in nutrient solution, these sensors demonstrated predicable lifetimes on the order of 50h. The developed instrument for absorption-based measurements including light source, mini-spectrometer and optode probe is presented. Custom instrument control and monitoring software including a spectral normalization procedure, use of a dual-wavelength absorbance ratio technique and automatic adjustment for pH variation result in an instrument that is self-calibrating and one that can account for effects such as light source fluctuations, membrane thickness variations and a variety of other factors. The low mass, low volume nature of bulk optode sensing systems, make them a promising technology for future space-based plant production systems. Their low-cost and technology transfer potential suggest that they could provide terrestrial growers a new and reliable mechanism to obtain ion-selective knowledge of their nutrient solution, improving yields, reducing costs and aiding in compliance to continually more stringent environmental regulation.

Functional characterization of permuted enhanced green fluorescent proteins comprising varying linker peptides.

New variants of green fluorescent protein (GFP) can be engineered by circular permutation of their amino acid sequence. We characterized a series of permuted enhanced GFP (PEGFP) with new termini introduced at N144-Y145 and linkers of 1, 3, 5 and 6 residues inserted between G232 and M1, as well as a variant with an extended 7-residues linker between K238 and M1. A minimum linker length of 3 residues was necessary for a functional chromophore to be formed, and linkers exceeding 4 residues yielded almost the same fluorescence quantum yield as enhanced GFP (EGFP). PEGFP exhibited dual-wavelength absorption and fluorescence excitation with peaks at 395 and 490 nm but single-wavelength emission at 512 nm. Fluorescence emission increased with increasing pH for all excitation wavelengths with a pKa of 7.7. Between the pH values of 6 and 8 optical absorption showed an isobestic point at 445 nm. PEGFP rapidly denatured in urea between 50 and 60 degrees C. Renaturation proceeded with a short (approximately 29 s) and a longer (> 150 s) time constant. Transient transfection of HEK293 and HeLa cells revealed the expression dynamics of PEGFP to be similar to that of EGFP. Laser-scanning microscopy of HeLa cells demonstrated that the PEGFP are particularly well suited as fluorescent indicators in two-photon imaging.

Temperature and pH dependence of the metarhodopsin I-metarhodopsin II equilibrium and the binding of metarhodopsin II to G protein in rod disk membranes

The equilibria between metarhodopsins I and II (MI and MII) and the binding of MII to retinal G protein (G) were investigated, using the dual wavelength absorbance response of rod disk membrane (RDM) suspensions to a series of small bleaches, together with a nonlinear least-squares fitting procedure that decouples the two reactions. This method has been subjected to a variety of theoretical and experimental tests that establish its validity. The two equilibrium constants, the amount of active G protein (that can bind to and stabilize MII) and the fraction bleached by the flash, have been determined without a priori assumptions about these values, at temperatures between 0 and 15 degrees C and pHs from 6.2 to 8.2. Binding of G to MII in normal RDM exhibits 1:1 stoichiometry (not cooperative), relatively weak, 2-4 x 10(4) M-1 affinity on the membrane, with a pH dependence maximal at pH 7.6, and a low thermal coefficient. The reported amount of active G remained constant even when its binding constant was reduced more than 10-fold at low pH. The method can readily be applied to the binding of MII to other proteins or polypeptides that stabilize its conformation as MII. It appears capable of determining many of the essential physical constants of G protein coupled receptor interaction with immediate signaling partners and the effect of perturbation of environmental parameters on these constants.

Temperature and pH dependence of the metarhodopsin I-metarhodopsin II equilibrium and the binding of metarhodopsin II to G protein in rod disk membranes.

The equilibria between metarhodopsins I and II (MI and MII) and the binding of MII to retinal G protein (G) were investigated, using the dual wavelength absorbance response of rod disk membrane (RDM) suspensions to a series of small bleaches, together with a nonlinear least-squares fitting procedure that decouples the two reactions. This method has been subjected to a variety of theoretical and experimental tests that establish its validity. The two equilibrium constants, the amount of active G protein (that can bind to and stabilize MII) and the fraction bleached by the flash, have been determined without a priori assumptions about these values, at temperatures between 0 and 15 degrees C and pHs from 6.2 to 8.2. Binding of G to MII in normal RDM exhibits 1:1 stoichiometry (not cooperative), relatively weak, 2-4 x 10(4) M-1 affinity on the membrane, with a pH dependence maximal at pH 7.6, and a low thermal coefficient. The reported amount of active G remained constant even when its binding constant was reduced more than 10-fold at low pH. The method can readily be applied to the binding of MII to other proteins or polypeptides that stabilize its conformation as MII. It appears capable of determining many of the essential physical constants of G protein coupled receptor interaction with immediate signaling partners and the effect of perturbation of environmental parameters on these constants.

Absorbance measurements in turbid media by the photon correlation method

Optical absorption in highly turbid media was quantified by the time shift of the electric field autocorrelation function of diffused photons. The intensity autocorrelation function was analyzed by the third-order cumulant expansion and a linear relationship between the time shift and the absorber concentration was observed. The slope of the fitted line gave the molecular extinction coefficient of the absorber. The absorption spectra were also obtained from the time shift. Applicability to dual-wavelength absorption measurement is also discussed. We demonstrate for the first time, as far as we know, the feasibility of absorbance quantification in turbid media by the photon correlation method.

Comparison of the analytical capabilities of an amperometric and an optical sensor for the determination of nitrate in river and well water

The analysis of nitrate in water has been studied using novel amperometric and optical sensors. A flow-injection analysis system with amperometric detection has been developed in which nitrate is determined as nitrite after reduction in a cadmium column. The working electrode is glassy carbon modified with a crosslinked redox polymer. The linear range is 0.1 to 190 mg l NO 3-N ( r⪢0.999) and the limit of detection (LOD) is 50 μg l NO 3-N. A fibre optic sensor based on a dual wavelength absorption approach has also been developed. A signal at 210 nm where nitrate absorbs was referenced against a signal at 275 nm where nitrate does not absorb. Its linear range is from 0.4 to 30 mg l NO 3-N and its LOD is 400 μg l NO 3-N. These diverse methods have been applied to the analysis of the same river water samples and good correlations have been observed between the two measurement techniques and a standard ion chromatography method.

Thermal gradient microbore liquid chromatography with dual-wavelength absorbance detection

Thermal gradient microbore liquid chromatography with dual-wavelength absorbance detection