Classical Spectrophotometric Methods Research Articles

Development of a green chromatographic method for determination of colorants in food samples

A green chromatographic analytical method for determination of Tartrazine, Brilliant Blue and Sunset Yellow in food samples is proposed. The method is based on the modification of a C18 column with a 0.25% (v/v) Triton X-100 aqueous solution at pH 7 and in the usage of the same surfactant solution as mobile phase without the presence of any organic solvent modifier. After the separation process on the chromatographic column, the colorants are detected at 430, 630 and 480 nm, respectively. The chromatographic procedure yielded precise results and is able to run one sample in only 8 min, consuming 15.0 mg of Triton X-100 and 38.8 mg of phosphate. When the flow rate of the mobile phase is 1 ml min −1 the retention times are 2.1, 3.6 and 7.0 min for Tartrazine, Brilliant Blue and Sunset Yellow, respectively; and all peak resolutions are ca. 2. The analytical curves present the following linear equations: area = 7.44 10 5 + 2.71 10 5 [Tartrazine] ( R = 0.998, n = 7); area = 1.09 10 5 + 3.75 10 5 [Brilliant] ( R = 0.9995, n = 7) and area = −7.34 10 4 + 2.33 10 5 [Sunset] ( R = 0.998), n = 7) and, the limits of detection for Tartrazine, Brilliant Blue and Sunset Yellow were estimated as 0.125, 0.080 and 0.143 mg l −1. When the proposed method is applied to food samples analysis, precise results are obtained (R.S.D. < 5%, n = 3) and in agreement with those obtained by using the classical spectrophotometric method. The traditional usage of organic solvent as mobile phase in HPLC is not used here, which permits to classify the present method as green.

Amperometric sensor for glutathione reductase activity determination in erythrocyte hemolysate

The development of an amperometric sensor for glutathione reductase (GR) activity in erythrocyte hemolysate to contribute to oxidative stress evaluation is presented. In this assay, the reduced form of glutathione, the product of the GR reaction, reacts with 5,5 ′-dithiobis(2-nitrobenzoic acid), producing GSTNB, which is easily reduced in the electrode surface. The current was recorded during 180 s after the sample addition, applying a potential of −300 mV. The sensor presented a suitable sensitivity, a good operational range, and precision. The effects of pH variations and specific uncompetitive inhibitor (safranin-O) in the enzyme activity were also evaluated. The GR activity determination in human erythrocyte hemolysate using this method has provided results that are statistically equal to those obtained by the classical spectrophotometric method, with 95% of confidence. The advantages of this method are the saved time, reagents, and samples and the possibility of its use in the field.

A size-exclusion HPLC method for the determination of sodium chondroitin sulfate in pharmaceutical preparations

A size-exclusion HPLC method for the determination of sodium chondroitin sulfate (SCS) in pharmaceutical preparations has been developed and validated. The most important feature of this method compared with the previously reported assay methods was improved economical and determinative applications through direct analysis of SCS from pharmaceuticals. The linearity, precision, specificity, and accuracy of the method were established and validated. The intra- and inter-day precision was satisfactory with relative standard deviation lower than 1.0%. The recovery of SCS from multi-components pharmaceutical preparations were from 93.38 to 100.46%. Comparing our HPLC assay results with classical spectrophotometric methods, the developed method was considerably easy, simple and reproducible. As a result, the present method was supposed to be successfully applied to the assay of SCS for the routine quality control in pharmaceutical preparations.

Precise Equilibrium Constants from Spectrophotometric Data: BrCl in Br2/Cl2 Gas Mixtures

The equilibrium constant for the reaction, Br2(g) + Cl2(g) ⇄ BrCl(g), is estimated using the classic spectrophotometric method with precise data and a multispectrum fitting approach. Analysis of spectra for 18 Cl2:Br2 mixing ratios yields K° = 9.1 at 22 °C, with a nominal statistical error (1 σ) of 0.04. This error translates into a remarkable precision of 0.4 cm-1 in the dissociation energy of BrCl, easily beating that for the current best spectroscopic estimate (±5 cm-1). However, a sensitivity analysis shows that K° is susceptible to small systematic errors and minor changes in the least-squares fit model, leading to a more conservative estimate of 2% error. The derived K° is consistent with statistical mechanical estimates that employ the current value for the BrCl dissociation energy, but it differs considerably from some recently measured and used values, which range from 6.4 to 10.4. The revised estimate of the BrCl dissociation energy is &#x1d49f;e = 18 248 ± 2 cm-1.

IMPROVEMENT OF MULTIVARIATE UV ANALYSIS BY SELECTING WAVELENGTHS AND INSTRUMENTAL PARAMETERS

ABSTRACT An empiric procedure of successive approximations for the selection of the most convenient wavelengths and instrumental parameters to execute an UV multivariate analysis is described. The selection criterion was according to the minimum PRESS value. For the identification of the best wavelength intervals a tree-fraction scheme has been used, starting from the full spectrum and going down to 3 nm intervals. General considerations for applying this procedure on any multicomponent mixture are finally reported. The study has been developed on a mixture of propyphenazone, paracetamol and caffeine, present in several pharmaceuticals, whose spectra so totally overlap to make inconsiderable the classical spectrophotometric methods. The obtained results were enthusiastic demonstrating a very high improvement of accuracy and precision. In fact the mean error on propyphenazone, paracetamol, caffeine passed respectively from 12.8, 5.49, 41.88 %, reading the full spectrum at the default conditions, to 1.06, 0.52, 1.18 %, employing the selected conditions.

Pathologie des enzymes de la glycolyse érythrocytaire

Since Carson et al. [8] described G6PD deficiency in 1956, more than twenty different hereditary enzyme deficiencies have been found in the red blood cell. Most of these lead to nonspherocytic haemolytic anemia. Studies in molecular biology have recently altered the method of diagnosis of these enzymopathies. Two groups of deficiencies can be isolated: the first leading to an impairment of ATP production (type: pyruvate kinase), the second an impairment in the oxidoreduction system of the red blood cell (type: G6PD). Biological diagnosis can be done in four steps: haemolysis characterization, exclusion of auto-immun diseases, haemoglobinopathies and red blood cell membrane disorders,enzymatic activity measurement on haemolyzed red blood cells using classical spectrophotometric methods with NAD, NADP, mutation identification by molecular biology. Results of enzymatic measurement is often difficult due to the great heterogeneity of these deseases.

Selective Differential Spectrophotometric Methods for Determination of Niclosamide and Drotaverine Hydrochloride

ABSTRACT Differential (ΔA), second derivative (ΔD2) and third derivative (ΔD3) differential ultraviolet spectrophotometric methods have been presented for the quantitation of niclosamide and drotaverine hydrochloride in pure forms and in their pharmaceutical formulations. For niclosamide, the method is based on measuring ΔΔ A, Δ ΔD2, A ΔD3 of niclosamide in alkaline solutions against their neutral ethanolic solutions as blanks. For drotaverine hydrochloride, the acidic solutions of the drug are measured against their alkaline solutions as blanks. The proposed methods are sensitive, highly specific and advangageous over the conventional UV assays, since the interference of the excipients, impurities, degradation products or other accompanying drugs is nullified. Accuracy of the analysis with the proposed procedures is significantly greater than the classical spectrophotometric methods.

Comparison of Electrochemical and Spectro-Photometric Detection in Hrp-Based Elisa for Tobacco Mosaic Virus

ABSTRACT An electrochemical method was compared with a spectrophotometric method using a horseradish peroxidase (HRP)-based indirect enzyme-linked immunosorbent assay (ELISA) with direct antigen coating (DAC) technique for the determination of tobacco mosaic virus (TMV). In substrates for a spectrophotometric HRP-based ELISA method, was selected as the substrate for the electrochemical method as well as the spectrophotometric method. 2, 3-Diaminophenazine is the product of the oxidation of OPD with H2O2 catalyzed by HRP in the selected conditions. It is electroactive and has a sensitive voltammetric response at -0.93 V (vs. Ag/AgCl) in alkaline solution. The detection limit of free HRP, by second-order derivative linear-sweep voltammetry, is 1.1 mU/L. This method can be further used to detect TMV antigen. The peak current is linear with TMV concentration in the range of 0.25-5.0 ng/mL. The detection limit for TMV is 0.25 ng/mL and the highest dilution ratio detected for the infected leaf sap is 1:102400. Compared with the classical OPD ELISA spectrophotometric method, the detection limits of the electrochemical method for the clarified TMV and the infected leaf sap detection are about ten and four times lower, respectively.

Flow injection analysis-amperometric determination of ascorbic and uric acids in urine using arrays of gold microelectrodes modified by electrodeposition of palladium

Uric acid (UA) and ascorbic acid (AA) present in urine were rapidly determined by the amperometric method in association with flow injection analysis. An array of gold microelectrodes modified by electrochemical deposition of palladium was employed as the working electrode. Uric and ascorbic acids were quantified in urine using amperometric differential measurements at +0.75 and +0.55 V, respectively. This method is based on three steps involving the flow injection of: (1) the sample spiked with a standard solution, (2) the pure sample, and (3) the enzymatically treated sample. The enzymatic treatment was carried out with ascorbate oxidase, uricase, and peroxidase at pH 7. The calibration curves for freshly prepared ascorbic and uric acid standards were very linear in the concentration ranges of 0.44–2.64 mg l −1 (AA) and of 0.34–1.68 mg l −1 (UA) with a relative standard deviation (RSD) <1 %. For uric acid, the real sample analyses where compared with the classical spectrophotometric method, showing deviations between 3.1 and 8.6% ( n = 9).

An improved microdiffusion method with solid‐phase detection for the determination of total cyanogens in fresh cassava. Comparison to the method of Cooke (1978)

A microdiffusion method with solid-phase detection for the determination of total cyanogens (=cyanogenic potential, CNp) in fresh cassava roots was developed and evaluated against the classical spectrophotometric method of Cooke (J Sci Food Agric 29 (1978) 345–352). Using seven different cassava cultivars, a significant difference (approx 15%) in the determined CNp was observed only for one of these. The new method offers several advantages over the earlier described spectrophotometric methods. Filtration of homogenates is not necessary, neither is cooling nor heating. The developed colour is stable on the reaction sheet, so that levels may be measured immediately or when appropriate, and be filed for documentation purposes. Further the solid-phase reaction may be measured in different ways according to the instrumentation available, ie reflectometry as used in this study, or absorption of transmitted light using a TLC-densitometer or a microplate reader. The amount of enzyme (linamarase) used per analysis is lower than for the spectrophotometric assays, giving economic advantages to the present assay. In addition to the extract, only three solutions are used when performing the assay, ie the buffer, the substrate solution for standards, and the enzyme solution. The assay may be run either according to a ‘slow’ overnight protocol or to a ‘fast’ protocol at 40°C. © 1998 SCI.

An improved microdiffusion method with solid-phase detection for the determination of total cyanogens in fresh cassava. Comparison to the method of Cooke (1978)

A microdiffusion method with solid-phase detection for the determination of total cyanogens (=cyanogenic potential, CNp) in fresh cassava roots was developed and evaluated against the classical spectrophotometric method of Cooke (J Sci Food Agric 29 (1978) 345–352). Using seven different cassava cultivars, a significant difference (approx 15%) in the determined CNp was observed only for one of these. The new method offers several advantages over the earlier described spectrophotometric methods. Filtration of homogenates is not necessary, neither is cooling nor heating. The developed colour is stable on the reaction sheet, so that levels may be measured immediately or when appropriate, and be filed for documentation purposes. Further the solid-phase reaction may be measured in different ways according to the instrumentation available, ie reflectometry as used in this study, or absorption of transmitted light using a TLC-densitometer or a microplate reader. The amount of enzyme (linamarase) used per analysis is lower than for the spectrophotometric assays, giving economic advantages to the present assay. In addition to the extract, only three solutions are used when performing the assay, ie the buffer, the substrate solution for standards, and the enzyme solution. The assay may be run either according to a ‘slow’ overnight protocol or to a ‘fast’ protocol at 40°C. © 1998 SCI.

Reagentless Sensors for Acetaldehyde

A reagentless acetaldehyde sensor is designed based on the co-entrapment of aldehyde dehydrogenase (AIDH), NADH oxidase and high molecular weight NADs (NAD-dextran and NAD-PEG) in a polyvinyl alcohol) bearing stryrylpyridinium groups (PVA-SbQ) matrix. The amperometric detection is based on hydrogen peroxide oxidation by applying a potential difference of 550 mV between two platinum electrodes, equivalent to ca. 600 mV vs. SCE. The sensitivity is close to 2 mA 1 mol−1, the linear range extends from 0.5 μM to 330 μM acetaldehyde and the response time is lower than 3 minutes. More than 80 reproducible measurements can be performed without cofactor addition, indicating a good operational stability. Acetaldehyde concentration in alcoholic beverages is determined and validation of the results is made using a classical spectrophotometric method.

Kinetic study of double-helix formation and double-helix dissociation of polyadenylic acid

The changes in the secondary structure of polyadenylic acid [poly(A)], caused by variations of pH at constant temperature, have been investigated. The position of the equilibrium 2 single-strands ⇌ double-strand is shifted towards the left-hand side by an increase in the ionic strength of the solution, and for constant values of the latter, by the addition of small amounts of divalent transition metal ions.The kinetics of the conformational changes have been investigated by T-Jump, stopped-flow and classical spectrophotometric methods. With the latter two techniques the reactions were initiated by changes in solution pH. The process of double-helix dissociation was found to involve at least three first-order steps. The process of double-helix formation begins with a second-order step leading, via a labile steady-state intermediate, to a multitude of mismatched double-strands. These, in turn, evolve to the final, completely formed double-helical form according to a series of first-order steps. The value of the second-order rate constant (105 dm3 mol–1 s–1) is similar to those found for the double-helix formation reactions of oligonucleotides. Increases in pH from 4 to 5.9 were used to study the conversion of a metastable double-stranded form of poly(A) into a form that is to be considered the most stable under the experimental conditions. This rearrangement process occurs through a series of three first-order steps, like the (much faster) process of double-helix dissociation.

Activity of class I and II alcohol dehydrogenase in the sera of alcoholics.

The activity of class I and II alcohol dehydrogenases in the sera of alcoholics was estimated using specific and highly sensitive fluorogenic substrates. It was found that serum class I activity was increased about 2 fold above that of the control group. No such increase was observed in the activity of class II, and we did not find a significant difference in non-alcoholics. The total alcohol dehydrogenase activity measured by classical spectrophotometric methods at pH 9.2 confirmed the increase of class I alcohol dehydrogenase. Additionally, the activity of liver injury markers were detected. Only gamma-glutamyltransferase activity exceeded the normal range. Based on the results of the present study, we would suggest that the significant elevated serum activity of class I alcohol dehydrogenase may be derived not from liver cells, because they did not display the obvious enzymatic markers, but from different organs, especially the gastrointestinal tract. The tissue distribution of alcohol dehydrogenase isoenzymes and gamma-glutamyltransferase would help to confirm this possibility.

Potentiometric Determination of Glutathione and Glutathione Transferase Activity

Abstract A new method of determination of Glutathione Transferase activity (GST) and Glutathione (GSH) in solution has been developed. The determination was performed by detection of ions (H+, Cl−, F−) produced during the GST catalyzed reaction using ion-selective electrodes. The Michaelis constants of GST π from human placenta toward GSH, 1-Chloro-2, 4-dinitrobenzene (CDNB) and 1-Fluoro-2, 4-dinitrobenzene (FDNB) obtained potentiometrically were similar to those calculated with the classical spectrophotometric method. The use of pH electrode gave the best results for GST activity (1 pH unit slope per 0.1 unit of enzyme in 1 ml of solution; detection limit of 0.001 unit/ml). Different purification steps of GST TI from human placenta followed by this method gave results comparable to the spectrophotometric one. The best calibration curve for GSH (2 × M detection limit) was obtained following the production of F- ions with a fixed amount of enzyme at a concentration of 0.2 u/ml. A calibration obtained performing the measurements in filtrated human serum showed the same behaviour.

Quantitative determination by high-performance liquid chromatography of acetylsalicylic acid and related substances in tablets

High-performance liquid chromatography on a Zorbac C 8 7-μm column (25 cm × 0.46 cm I.D.) with methanol-water-1 M phosphoric acid (59:36:5) as the mobile phase has been used for the analysis of several naturally aged batches of fourteen brands of acetylsalicylic acid tablets. The extraction solvent is methanol, containing 2% v/v of formic acid. Salicylic acid is the main impurity. Acetylsalicylsalicylic cid is the second most important impurity, and the corresponding salicylsalicylic acid is rarely present. Buffered or dispersible tablets contain relatively more of the latter two impurities and eventually also the corresponding higher oligomers. Acetylsalicylic anhydride is always a minor impurity. Comparison is made with classical spectrophotometric methods, which are observed to be selective for salicylic acid.

PH-Stat titration method for dihydrofolate reductase activity measurements: Application to determination of substrate Michaelis constant and antifolate inhibition constant

A pH-Stat titration method was developed for measuring dihydrofolate reductase (DHFR) activity; this method permits detection of very low DHFR activities corresponding to 100 pmol of substrate reduced per minute. This value is about ten times lower than those observed using the classical spectrophotometric method. This sensitivity makes it possible to measure the DHFR in crude tissue extracts. With beef liver DHFR, Michaelis constants for the cofactor NADPH and the natural substrate determined by this method were 1.9 ± 0.3 × 10 −5 and 8.5 ± 0.5 × 10 −7 m, respectively. The inhibition constant of methotrexate, a competitive inhibitor of dihydrofolate, was 3.4 ± 1.3 × 10 −11 m.

Separation and automatic spectrophotometric determination of low concentrations of cyanide in water

Semi-automatic methods are described for the routine determination of cyanide in water. Membrane diffusion and isothermal distillation are examined for the separation/concentration of cyanide; the isothermal distillation procedure is optimized for routine use. An air-segmented flow analyzer is used to quantify cyanide. Two classical spectrophotometric methods are adapted and compared. The method based on reaction with picric acid is applicable at cyanide concentrations exceeding 1 mg l −1. A modified Aldridge method is far better for lower concentrations. Combination of isothermal distillation with the automatic version of the Aldridge method is suitable for the determination of cyanide in waters in the concentration range 0.01–10 mg l −1. Interference by sulphide and sulphite and their removal are described.

A spectrophotometric study of the complex formation between cobalt(III) and trans-1,2-cyclohexanedinitrilotetraacetic acid (CyDTA)

An intriguing and unique system has been observed during the otherwise routine study of the cobalt(III) complex of trans-1,2-cyclohexanedinitrilotetraacetic acid (CyDTA). Using classical spectrophotometric methods to determine the stoichiometry and the stability of a complex, significant deviations from the predicted 1:1 complex were observed in a system buffered at pH = 4.6. It is postulated that in addition to the usual 1:1 complex, the propensity of the reactants to form complexes and the strong oxidizing conditions used in this investigation result in the formation of a second, higher order complex between Co(III) and CyDTA. When the concentration of CyDTA exceeds that of Co(III), the metal:ligand ratio for this complex is 1:2. A structure is proposed, and approximate stability constants of both complexes are discussed.

Determination of rhodanese activity using a pH-STAT apparatus

A new method for the assay of the rhodanese activity is described. The method uses a pH-STAT apparatus to monitor the rates of enzyme catalyzed reaction under controlled conditions. A standard kinetic analysis of the bovine kidney enzyme, including pH and temperature dependence, gave results which are in agreement with those obtained by the classic spectrophotometric method.