Spectrophotometric Methodology Research Articles

Easy to use spectrophotometric method for determination of aromatic diamines in biodiesel samples

Biodiesel is the main partial substitute for petrodiesel. Some biodiesels have low oxidative stability. To solve or minimize this problem, some compounds have been added to biodiesel as antioxidants. One of the most efficient classes of compounds used for this is aromatic diamines because of their high antioxidant capacity at low concentration. Aromatic diamines can be used as artificial markers. It is sometimes necessary to determine the concentration of antioxidants in biodiesel after long storage times or to control quality. An easy to use spectrophotometric methodology was employed in this work to determine the concentration of aromatic diamines (0 to 500ppm). Five calibration curves were constructed using 3 absorption frequencies from the infrared spectra of the samples. The standard deviation, correlation coefficient, linearity range, limit of detection and limit of quantification were determined. The better results from the easy to use method (814cm−1) show a linear range of 0–100ppm with a strong correlation (R2=0.9966) and a limit of quantification of 21.6ppm.

Lab-on-a-chip spectrophotometric characterization of porcine oocytes

The method of quality assessment of the oocytes of breeding animals based on morphological criteria very often leads to unsuccessful in vitro fertilization and pure breeding result. In this paper we present a spectrophotometric methodology utilizing lab-on-a-chip techniques for optical and subjective characterization of porcine oocytes. A set of parameters describing the features of oocytes deduced from collected absorbance spectra of a single oocyte, has been proposed. Differentiation of the spectra of oocytes coming from different sizes of porcine follicle has been observed. The obtained results are the first step towards an objective quality assessment of porcine oocytes, which in the future may be extended to other breeding animals.

Field-emission light sources for lab-on-a-chip microdevices

Microfluidic devices called lab-on-a-chips utilize two kinds of characterization of a biosample, which are based on spectrofluorimetric and spectrophotometric methodologies. Lab-on-a-chips are equipped with an optical instrumentation and a software system that allow detecting of the optical signals and their processing into spectral characteristics. In the paper, technology of lab-on-a-chip (cytometer microdevice) and miniature silicon-glass field-emission light source (FELS) is described. Electrons emitted from a silicon cathode covered with carbon nanotubes excite light emission from nanocrystalline rare earth doped yttrium oxide phosphor (anode). The emitted light can be precisely tailored to the main absorption line of a fluorescent marker. The presented system has been successfully used for testing of the cow embryos as well as fluorescent-marked porcine oocytes.

Total flavonoids content in the raw material and aqueous extractives from Bauhinia monandra Kurz (Caesalpiniaceae).

The aim of this work was to evaluate the spectrophotometric methodology for determining the total flavonoid content (TFC) in herbal drug and derived products from Bauhinia monandra Kurz. Several analytical parameters from this method grounded on the complex formed between flavonoids and AlCl3 were evaluated such as herbal amount (0.25 to 1.25 g); solvent composition (ethanol 40 to 80%, v/v); as well as the reaction time and AlCl3 concentration (2 to 9%, w/v). The method was adjusted to aqueous extractives and its performance studied through precision, linearity and preliminary robustness. The results showed an important dependence of the method response from reaction time, AlCl3 concentration, sample amount, and solvent mixture. After choosing the optimized condition, the method was applied for the matrixes (herbal material and extractives), showing precision lower than 5% (for both parameters repeatability and intermediate precision), coefficient of determination higher than 0.99, and no important influence could be observed for slight variations from wavelength or AlCl3 concentration. Thus, it could be concluded that the evaluated analytical procedure was suitable to quantify the total flavonoid content in raw material and aqueous extractives from leaves of B. monandra.

Quantitative Determination of Color Coordinates for Lumninescent Materials

On the basis of the photoluminescence spectra, a spectrophotometric methodology is developed to determine the color coordinates of luminescent materials such as the red light-emitting porous silicon film, the green light-emitting aluminate-based phosphor and the blue light-emitting liquid crystal.

Spectrophotometric distances to Galactic H ii regions

We present a near-infrared study of the stellar content of 35 H ii regions in the Galactic plane, 24 of which have been classified as giant H ii regions. We have selected these optically obscured star-forming regions from the catalogues of Russeil, Conti & Crowther and Bica et al. In this paper, we have used the near-infrared domain J-, H- and Ks-band colour images to visually inspect the sample. Also, we have used colour-colour and colour–magnitude diagrams to indicate ionizing star candidates, as well as the presence of young stellar objects such as classical T Tauri stars and massive young stellar objects (MYSOs). We have obtained Spitzer Infrared Array Camera images for each region to help further characterize them. Spitzer and near-infrared morphology were used to place each cluster in an evolutionary phase of development. Spitzer photometry was also used to classify the MYSOs. A comparison of the main sequence in the colour–magnitude diagrams for each observed cluster was used to infer whether or not the cluster kinematic distance is consistent with brightnesses of the stellar sources. We find qualitative agreement for a dozen of the regions, but about half the regions have near-infrared photometry that suggests they may be closer than the kinematic distance. A significant fraction of these already have spectrophotometric parallaxes that support smaller distances. These discrepancies between kinematic and spectrophotometric distances are not a result of the spectrophotometric methodologies, as independent non-kinematic measurements are in agreement with the spectrophotometric results. For instance, the trigonometric parallaxes of star-forming regions were collected from the literature and show the same effect of smaller distances when compared to the kinematic results. In our sample of H ii regions, most of the clusters are evident in the near-infrared images. Finally, it is possible to distinguish among qualitative evolutionary stages for these objects.

A New Methodology for Pharmaceutical Analysis Based on the Competitive Aggregation in an Amphiphilic System

In this work, a simple spectrophotometric methodology based on the competitive aggregation in a dyesurfactant- drug system is described for the determination of pharmaceutical compounds. This is a modification of recently established surfactant to dye binding degree method. Eriochrome Blue Black R (EBBR) and Didodecyldimethylammonium bromide (DDABr) were the dye and surfactant used, respectively. In the proposed method, the anions of EBBR bind to the cationic DDABr molecules to form dye-surfactant aggregates. Upon adding an amphiphilic drug to the mixture, the drug competes with the dye to interact with the surfactant, which results in a decrease in dye-surfactant aggregates formation. This causes a change in UV-Vis absorption properties of the dye. The measurement parameter is the difference between the absorption of the dye in the presence and absence of the drug. In the appropriate experimental conditions the absorbance differences are directly proportional to the drug concentration. Piroxicam and folic acid are used as two model drugs to develop the methodology. The influence of variables such as pH, concentrations of buffer, EBBR and DDABr on the measurement parameter was studied. Under the optimum conditions, the calibration graphs were linear up to 1.6 and 2.7 ig ml-1 for piroxicam and folic acid, respectively, with the corresponding detection limits of 0.05 and 0.09 ig ml-1. The method was applied to the determination of the drugs in pharmaceutical formulations. Keywords: Surfactant, Mixed aggregate, Dye-surfactant aggregate, Piroxicam, Folic acid

Preparação de eletrodos de hidroxiapatita por diferentes metodologias de síntese e sua aplicação na determinação de fosfito em fertilizantes líquidos

Phosphite determination in fertilizing products is very important for the quality control of these products. Classical methods of analysis include gravimetric or spectrophotometric methodologies, which create toxic chemical residues. This paper describes an alternative hydroxyapatite-based electrode for phosphite determination. A linear electrochemical response was obtained from a phosphite concentration of 1 x 10-5 to 1 x 10-1 mol L-1. The results obtained the by proposed method are in accordance with those obtained by the gravimetric method at a 95% confidence level for direct determination in liquid fertilizing products.

Influence of ripeness grade on accumulation and extractability of grape skin anthocyanins in different cultivars

The accumulation of anthocyanins in grape skin and their extractability evolution along ripening were evaluated in Galician red cultivars during 2005 and 2006 harvests. Seed tannin maturity and total phenolic compounds were also investigated through seed phenolic maturity and total polyphenol indexes, respectively. Furthermore, statistical correlations between phenolic composition and classical parameters were established through ripening in order to find a more rapid and simpler methodology, which permits to predict the maximum anthocyanin concentration in red wines. So, statistical correlation was found for total polyphenol index and total acidity in all the cultivars studied, while the greatest anthocyanin accumulation can only be estimated from titrable acidity for the most coloured cultivars. The cultivars studied were characterised on the basis of their phenolic composition (anthocyanins, proanthocyanidins, catechins) in order to know their oenological potential. Principal component analysis allowed a first approximation to the characterisation and differentiation of the red cultivars studied on the base of their phenolic composition in vintage, as measured by spectrophotometric methodologies. Simple phenolic determinations, which can be carried out in winery, provided fundamental information for the oenologist during the planning and management of pressing and maceration processes.

The quality of our drinking water: Aluminium determination with an acoustic wave sensor

A new methodology based on an inexpensive aluminium acoustic wave sensor is presented. Although the aluminium sensor has already been reported, and the composition of the selective membrane is known, the low detection limits required for the analysis of drinking water, demanded the inclusion of a preconcentration stage, as well as an optimization of the sensor. The necessary coating amount was established, as well as the best preconcentration protocol, in terms of oxidation of organic matter and aluminium elution from the Chelex-100. The methodology developed with the acoustic wave sensor allowed aluminium quantitation above 0.07 mg L −1. Several water samples from Portugal were analysed using the acoustic wave sensor, as well as by UV–vis spectrophotometry. Results obtained with both methodologies were not statistically different ( α = 0.05), both in terms of accuracy and precision. This new methodology proved to be adequate for aluminium quantitation in drinking water and showed to be faster and less reagent consuming than the UV spectrophotometric methodology.

PARAFAC2 and MCR-ALS quantification of Diltiazem antihypertensor based on a kinetic spectrophotometric methodology

A Diltiazem kinetic spectrophotometric UV–Vis method, based on a reaction of the Diltiazem with hidroxylamine and a ferric salt, was used for the quantification of Diltiazem in different pharmaceutical formulations. This method is based on the acquisition of three-way data structures [wavelength (nm) × time (s) × concentration (mg/L)] followed by chemometric analysis by an appropriate PARAFAC2 or MCR-ALS second-order calibration model. The results obtained are compared with those obtained by direct determination, at maximum wavelength, and by the United States Pharmacopeia (USP) standard chromatographic method. For all the pharmaceutical formulations analysed good quantification results were found with PARAFAC2 and MCR-ALS second-order calibration models. For bulk drug analysis, detection limits of 6 and 2 mg/L, and for pharmaceutical formulations analysis, an average detection limit of 41 and 39 mg/L were found, respectively with PARAFAC2 and MCR-ALS.

Sequential Injection Spectrophotometric Determination of Metoclopramide in Pharmaceutical Preparations

In this work, a spectrophotometric methodology for the determination of metoclopramide in pharmaceutical preparations is proposed. The approach was based on the reaction of metoclopramide with Folin–Ciocalteu reagent and was implemented on a sequential injection analysis automated flow system. An in‐line mixing chamber was used to promote reaction zone homogenization, to increase reaction time, and to attenuate the high refractive index gradient generated by the utilization of concentrated solutions, thus enabling the minimization of the Schlieren effect. Linear calibration plots for metoclopramide concentrations up to 100 mg L−1 were obtained, with good precision (RSD<3%, n=10). The detection limit was 2.0 mg L−1 and the quantification limit was 6.8 mg L−1.

Application of response surface methodology for modelling the enzymatic assay of hydrogen peroxide by Emerson–Trinder reaction using 4-iodophenol

In this work, an initial-rate spectrophotometric method and response surface methodology (RSM) were combined for modelling and optimizing the experimental parameters of the enzymatic Emerson–Trinder reaction, for the determination of hydrogen peroxide. This spectrophotometric indicator reaction is currently used in biotechnology for the determination of phenolic compounds (e.g. in industrial samples) and also for determination of various substrates (e.g. in clinical chemistry). Using 4-iodophenol as a hydrogen donor in this reaction, the quality of the generated second-order polynomial response model equation was checked by the kinetic assay of H2O2 in real samples (e.g. cosmetic and human pooled serum samples), where their resulting satisfactory analytical characteristics were reported.

Dynamic Assessment of Apoptosis for In Vitro Design of Bortezomib Combination Therapies for Lymphoid Malignancies.

Conventional techniques for assessing drug response and apoptosis induction rely on static assessment of cellular changes at predetermined time points (e.g. detection of exposed membrane phospholipids by Annexin V). The Kinetics of Optical Response assay (KOR) is a new technique that detects induction of apoptosis dynamically. It employs a spectrophotometric methodology to detect changes in optical density associated with membrane blebbing related to growth and death, allowing detection of apoptosis in real time. The KOR assay has already predicted the response to cytotoxic agents of AML cell lines and primary samples. This study uses the KOR assay in lymphoid malignancy and shows sensitivity to apoptosis induction by conventional and novel agents including bortezomib. The lymphoma cell line DOHH2 (t(14;18)), U266 (myeloma), K562 (CML) and primary CLL cells were used in this study with HL60 (AML) as a control. Cells were seeded in 96 well plates and treated with a variety of drugs alone or in combination (cytarabine, fludarabine, doxorubicin, daunorubicin, etoposide, melphalan, bortezomib) at multiple concentrations. Measurements were made at 5 min. intervals for up to 48 hrs and analysed using KORSoft™ software to generate apoptotic response curves. To validate this approach conventional techniques were used for comparison (Alamar Blue for cytotoxicity and flow cytometric analysis of cell cycle and apoptosis using propidium iodide and Annexin V staining respectively). The KOR assay can show changes in growth characteristics, induction of apoptosis and necrosis in response to drugs permitting a continuous analysis for maximum sensitivity (Smax). DOHH2 was found to be dose responsive to four of the drugs used, with the Smax for 10μM daunorubicin at 6 hours (48%), 1μM doxorubicin at 8 hours (38%), 100μM etoposide at 8 hours (52%), and minimally to 100μM cytarabine at 16 hours (21%). There was no effect from fludarabine. The addition of bortezomib increased Smax to 89% with etoposide and to a lesser degree with the other cytotoxic drugs. U266 showed a similar spectrum of results with greatest Smax with 100μM melphalan at 9 hours (57%) enhanced to 78% with the addition of bortezomib. There was minimal response to cytarabine and fludarabine. Parallel flow cytometric analysis using Annexin V and PI showed similar results to those from the KOR assay confirming the assessment of apoptosis to be valid. Cell cycle analysis showed an increased sub-G1 peak in keeping with apoptosis at times of Smax assessed by the KOR assay. The Alamar Blue cytotoxicity assay showed a dose dependent decrease in cell proliferation in response to increasing drug dose again paralleling other apoptosis measurements implying an apoptotic effect due to drug action and correlate well with those from the KOR assay. Primary CLL samples following CD19 selection were cultured with and without IL4 and exposed to the KOR assay with cytotoxics and bortezomib. Culture with IL4 alone gave good growth characteristics and revealed the combination of etoposide and bortezomib to provide the best induction of apoptosis (Smax 82%) compared to etoposide (26%) or bortezomib (32%) alone. The KOR assay is a microtitre approach to the assessment in real time of apoptosis. This study suggests the combination of bortezomib and etoposide is effective for lymphoma. Such approaches can accelerate the development of effective clinical trials.

Antioxidant activities of five different mulberry cultivars in Korea

Five major mulberry [Pachungsipyung (M-1), Whazosipmunja (M-2), Suwonnosang (M-3), Jasan (M-4) and Mocksang (M-5)] cultivated in Korea were assessed for their polyphenolic composition using spectrophotometric methodology, and tested for antioxidant potential by some different assays. The total polyphenol (TP) was found from 2235 to 2570 μg/g gallic acid equivalents, total anthocyanin (TA) content to vary from 1229 to 2057 μg/g, coloured anthocyanins (CA) from 126 to 190 μg/g, and total flavanol (TF) from 16.4 to 65.4 μg/g catechin equivalents except Mocksang (M-5). The ethanolic extract from mulberry fruit shows a rapid and concentration-dependent increase of antioxidant activity. Especially, the antioxidant activities of M-2 and M-4 are higher than those of the others in a hemoglobin-induced linoleic acid system. The reducing power compared with BHT was observed to high value in M-2, M-3 and M-4 extracts. Effectiveness in reducing powers was in a descending order of M-4>M-2>M-3>M-1>M-1. The DPPH-scavenging ability of the ethanolic extract from mulberry fruit was 60.0% at 200 and 212 μg of M-2 and M-4, respectively. M-2 and M-4 also showed sharply increase of hydroxyl scavenging ability with concentration of the extracts. IC 50 values in scavenging abilities on hydroxyl radicals were 30 mg and in a descending order of M-5>M-3>M-1>M-4>M-2. Superoxide anion-sacavenging activities of M-2, M-3 and M-4 showed 17.1, 14.5 and 14.8 SOD unit/mg, respectively.

Electrochemical sensor for amino acids and albumin based on composites containing carbon nanotubes and copper microparticles

This work reports on the analytical performance of composites obtained by dispersing copper microparticles and multi-wall carbon nanotubes within a mineral oil binder (CNTPE-Cu) for the determination of amino acids and albumin. The strong complexing activity of amino acids towards copper makes possible an important improvement in the sensitivity for the determination of amino acids and albumin. This new electrode permits the highly sensitive amperometric detection of amino acids, even the non-electroactive ones, at very low potentials (0.000 V) and physiological pH (phosphate buffer solution pH 7.40). The response of the electrode is highly dependent on the amount of copper, demonstrating the crucial role of the metal in the analytical performance of the sensor. The best analytical performance is obtained for the electrode containing 6.0% (w/w) copper. The resulting sensor shows a fast response (7 s) and a sensitivity that depends on the nature of the amino acid. The electrode surface demonstrates an excellent resistance to surface fouling, with R.S.D. of 4% for the sensitivities of 10 successive calibration plots. Albumin is determined with CNTPE-Cu using a protocol based on the accumulation of the protein for 10 min at −0.100 V, followed by the square-wave voltammetric analysis. The quantification of albumin concentration in lyophilized control serum gives excellent agreement with the classical spectrophotometric methodology and with the value informed for the supplier.

Fourth Order Derivative Spectrophotometric Determination of Benzyl Alcohol in Piroxicam Injections

AbstractPiroxicam is a drug with analgesic and anti‐inflammatory properties. It is present in numerous pharmaceutical preparations. Injectable forms usually contain benzyl alcohol as an excipient, which is used as a blocking anesthetic (4%) and an antiseptic (4–10%). In this work, spectrophotometric methodology was used in order to determine benzyl alcohol in piroxicam injectable formulations by applying the fourth derivative method adopting the zero‐crossing technique. The results obtained show that the method has significant advantages over other reported methods and is appropriate for routine pharmaceutical analysis. The method showed excellent linearity in the range of 2–100 μg mL−1 with limit of detection (S/N = 3) 0.07 μg mL−1 (6.47 × 10−7 M). The proposed method could be applied successfully for the determination of benzyl alcohol in injectable formulations with average % recovery of 100 ± 0.61.

Optimization of the Emerson–Trinder enzymatic reaction by response surface methodology

The Emerson–Trinder reaction has been optimized in this work using an initial rate spectrophotometric method and response surface methodology (RSM). In this investigation, the variation range of critical variables along with the fixed parameters were selected based on a preliminary ‘one at a time’ (OVAT) procedure for the subsequent RSM chemometric analysis as follows: pH (6–10), buffer concentration (50–250 mM), 4-aminoantipyrine (4-AAP) concentration (1–5 mM), temperature (25–45°C). The optimum values of fixed parameters were: 4-fluorophenol (4-FP, 30 mM), horseradish peroxidase (HRP) enzyme activity (0.12 U mL−1), and the fixed concentration of the H2O2 in the chemometric experiments was 11.4 µM. The non-linear nature of the experimental response of the reaction system was explained by a second-order polynomial equation, which revealed the impact of the experimental factors, their interactions and also their optimum values. The results of the reported RSM analysis proved to be quite appropriate for the design and optimization of this reaction, as illustrated by the relatively high value of the determination coefficient (R2=96.7%) for the fitting of quadratic model, along with the satisfactory results generated by the analysis of variance (ANOVA). All the evaluated analytical characteristics of this method: typical reaction progress curves, resulting linear calibration curve, within-day precisions at low and at high levels, and the upper and lower detection limits were, also, reported. In addition, to check the quality of the optimization and validity of the model, the assay of H2O2, in pooled serum matrix and in cosmetic samples, was performed.

Novel analytical applications of porphyrin to HPLC post-column flow injection system for determination of the lanthanides

A novel indirect substitution spectrophotometric methodology using porphyrin is developed for the determination of some metals that do not react with porphyrin directly. The methodology is concerned with a multistep reaction system, which consists of 3 complexation reactions that occur in a sequence of EDTA with metal, EDTA with Cu(II) and porphyrin with Cu(II). The complexation reaction of Cu(II) with a cationic porphyrin, meso-tetrakis (4- N-trimethylaminophenyl)-porphine (ttmapp), which has a high molar absorptivity about 5 × 10 5 l mol −1 cm −1, serves for spectrophotometric detection. This methodology was verified through individual determinations of 14 species of lanthanides following a batchwise procedure, which was further developed to an automatic procedure on a HPLC post-column flow injection system for simultaneous analysis of the lanthanides. Compared with the conventional post-column derivatization method using 4-(2-pyridylazo) resorcinol (PAR), the analytical sensitivity was greatly improved in this method and the results also showed good linearity.

Simultaneous Spectrophotometric Determination of Benzyl Alcohol and Diclofenac in Pharmaceutical Formulations by Chemometrics Method

Diclofenac sodium (DS) is a drug with analgesic, antipyretic, and anti-inflammatory properties. It is present in numerous pharmaceutical preparations. In injectable forms, it is usually accompanied by benzyl alcohol (BA) as an excipient, which is used as a blocking anesthetic (4%) and an antiseptic (4–10%). In this work a spectrophotometric methodology was applied in order to determine benzyl alcohol and diclofenac in injectable formulations by applying a multivariate calibration method. By a multivariate calibration method such as partial least squares (PLS), it is possible to obtain a model adjusted to the concentration values of the mixtures used in the calibration range. In this study, the concentration model is based on absorption spectra in the 230–320 nm range for 25 different mixtures of benzyl alcohol and diclofenac. Calibration matrix contains 10–95 and 1–50 μg mL−1 for benzyl alcohol and diclofenac, respectively. The root mean square errors of prediction (RMSEP) for benzyl alcohol and diclofenac were 3.0776 and 1.7557, respectively. The proposed method was validated by using a set of synthetic sample mixtures and subsequently applied to simultaneous determination of benzyl alcohol and diclofenac in two different pharmaceutical formulations.