Conventional Spectrophotometric Procedures Research Articles

Comparison of ascorbic acid content of Emblica officinalis fruits determined by different analytical methods

Emblica fruits have long been endowed with many pharmacological properties, which are attributable to their rich vitamin C content. A recent report that Emblica fruits do not contain ascorbic acid, but contain two hydrolysable low molecular weight tannins with antioxidant attributes, thus challenging the long-standing belief that amla fruits owe their potential pharmacological activity to their vitamin C content. The present study examines the vitamin C content of Emblica fruits by employing a specific enzymatic method in addition to the liquid chromatographic and the conventional spectrophotometric procedures to assess the extent of over-estimation, if any. The specific enzymatic procedure employed here indicated that Emblica fruits do not totally lack vitamin C as claimed and nevertheless, the conventional colorimetric method over-estimates the same in these fruits. A similar determination of vitamin C content in a few other conventionally rich plant sources of this vitamin indicated that over-estimation of vitamin C by colorimetric method is also evident in guava fruits ( Psidium guajava) and amaranth leaves ( Amaranthus gangeticus), but the extent was lower than that in the Emblica fruits. Thus, the discrepancy in the reported value of this nutrient is not limited to Emblica fruits alone, but can be found in other plant systems also. This study also examined the extent of retention of this vitamin after drying of the Emblica fruits and evidenced that shade drying did not improve the same as compared to sun drying. However, the amount of vitamin C retained in the dried Emblica fruits is comparable to the amounts present in several other fresh fruits.

An improved leaping detector for flow analysis applied to iron speciation in drugs

A low inner volume (ca. 64 ml) probe was built up in an injector-commutator in order to behave as a photometric leaping detector in flow analysis. It comprises a bicolour light-emitting diode (BLED), as a source of pulsed radiation in the red and green visible region, and two phototransistors as transducers. Sample injection, detector relocation, analytical signal recording, data treatment and definition of the spectral working range were computer-controlled. The feasibility of the system was initially demonstrated in the flow-injection speciation of iron, and the overall standard deviation of results was estimated as ± 1.6 and ± 1.4% for 1.6–4.0 mg l−1 Fe(II) or total iron after eightfold processing of synthetic samples. The system was further applied to drug analysis: the mean deviations of results for typical samples were estimated as ± 5.2 and ± 3.3%, and the relative standard deviation as ± 1.6 and ± 1.3% for Fe(II) and total iron, respectively. Results were compared with those obtained by a conventional spectrophotometric procedure and no statistic differences at the 95% confidence level were found. In relation to an earlier system with multi-site detection, the proposed system is more stable, presenting low drift with a relative standard deviation of 0.026% and 0.039% for measurements (n=120 during 4 h of observation) with green and red emission. It is also faster with a sampling rate of 133 h−1 and carryover problems are not found. The possibility of compensating the Schlieren noise by dual-wavelength spectrophotometry is discussed.

Protein Determination by Ponceau S Using Digital Color Image Analysis of Protein Spots on Nitrocellulose Membranes

A procedure was developed to estimate protein concentrations using color image analysis of protein spots stained with ponceau S. The method involved spotting a constant volume (2 μl) of the protein solutions on nitrocellulose paper, staining with acidic ponceau S, destaining, and air drying the paper. The image of the nitrocellulose paper was grabbed using a digital color scanner and thresholded with an optimal value to mark the area of the spot. The intensity of the color in the spot was measured in an arbitrary unit of intensity termed as inverse integrated gray value. This value showed a discernible increase with protein concentrations from 0.1 to 50 μg protein per spot (0.05–25 mg/ml). The method is simple and convenient compared to the conventional spectrophotometric procedures and allows several samples to be analyzed simultaneously. It can also be used to estimate protein concentration in the spots stained with Coomassie brilliant blue or other dyes.

Sample preparation in sequential injection analysis. Spectrophotometric determination of total phosphorus in food samples

A sequential injection (SI) procedure involving in-line sample preparation is proposed. A natural suspension or a slurry is transported together with nitric acid towards a home-made digestion bomb placed inside a microwave oven for subsequent digestion. The sample zone is stopped inside the oven and, after digestion, directed in reverse flow towards a holding coil and then towards detection. This is a good strategy because the SI system is versatile; the formed air bubbles are efficiently disposed of, thus avoiding the need for a debubbling unit, and the digestion bomb acts also as a mixing device promoting easy homogenization regardless of the number of required reagents. As application, the spectrophotometric determination of total phosphorus in foodstuffs based on the molybdenum blue method was selected. The proposed system is very robust and yields reproducible measurements, (r.s.d. usually <3%) for 20.0–400.0 mg l −1 P–PO 4. Results are in agreement with a conventional spectrophotometric procedure involving manual sample digestion.

Highly sensitive semi-quantitative field test for the determination of chromium (VI) in aqueous samples

A method for the semi-quantitative colorimetric determination of chromium(VI) at sub μg/L levels after sorptive preconcentration is presented. The method is based on the retention of the reaction product (preformed in liquid phase) between Cr(VI) and diphenylcarbazide on membrane embedded cation exchange material. The color intensity of the membrane can be correlated to Cr(VI) concentrations in the range 0.05–50 μg/L (i.e. almost three orders of magnitude lower than the conventional spectrophotometric procedure) with a detection limit of about 10 ng/L (using 50 mL of sample volume). Due to the visual inspection mode and comparative color detection the precision is only 30–80% rsd which, however, is regarded as sufficient for screening purposes. Analysis of real samples including different kinds of waters and extracts of soil and filter collected airborne particulate matter demonstrated the applicability of the method for fast and species selective screening. Recovery experiments generally gave reasonably good results, yet also revealed the risk of the conversion of chromium species during sample pretreatment procedures due to redox reactions.

A comparative evaluation of thiobarbituric acid methods for the determination of malondialdehyde in biological materials

A comparative evaluation was made of the conventional spectrophotometric procedure and three published high performance liquid chromatographic (HPLC) procedures for the determination of malondialdehyde (MDA) as the thiobarbituric acid (TBA) derivative when applied to liver, fish meal, serum, and urine. Except for urine, spectrophotometric analysis overestimated MDA content. Purification of the TBA-MDA complex obtained from liver and fish meal on reverse phase cartridges was found to entail a loss of complex bound to residual peptides in the trichloracetic acid (TCA) extract. Mincing as opposed to homogenizing liver samples led to a doubling of values for MDA content. Hexanal was a major TBA reactant, in addition to MDA, in all the samples. Acid hydrolysis and heat were necessary for the release of MDA bound to the amino groups of proteins and other amino compounds. Methods for free MDA have limited application to biological materials except short term in vitro preparations such as peroxidizing microsomes, in which free MDA accumulates. On the basis of these and other observations, a modified HPLC procedure for the determination of MDA as the TBA-MDA complex is proposed.

Thermal lens spectrometry in trace metal analysis

A dual-beam thermal lens spectrometer was used for the determination of trace amounts of Co, Bi, Fe, Ni and Mo. Its characteristics have been tested on model systems and the geometry of the optical scheme optimized to give increased sensitivity and a wider linear calibration range. The systematic methodological approach to the adaptation of conventional spectrophotometric procedures to thermal lens spectrometry has been elaborated. It is based on the specific demands of thermal lens spectrometry and includes an additional study of the original technique, the step-by-step elaboration of the procedure for thermal lens spectrometry on the model system and then on the spectrophotometric reaction with final testing on real samples. Various liquids were studied to ascertain their suitability as thermo-optic media. The mixed water–organic solvents proved to be the most suitable because they combine the convenience of water at the sampling stage with the high thermo-optic parameters of organic liquids. Thermal lens techniques are based on well known spectrophotometric routines (Co with 2-nitroso-1-naphthol, Ni with α-dioximes, Mo with thiocyanate ions, Fe with 1,10-phenanthroline and Bi with iodide ions). The application of thermal lens spectrometry allowed us to increase the sensitivity of these analytical procedures by up to 103–105 times. The limits of detection (3 × S/N) are: Bi, 10 pg cm–3; Co, 0.17 pg cm–3; Fe, 50 pg cm–3; Mo, 10 pg cm–3, and Ni, 9 pg cm–3.

An extrinsic marker for higher plant cell walls

Cytochromec was ionically bound to cell walls as a useful extrinsic marker. The marker binds rapidly, can readily be removed by salt treatment, and can be quantitated by conventional spectrophotometric procedures. The use of the marker allows each step of a cell wall purification to be monitored qualitatively and, furthermore, it provides a rapid, simple quantitative method to determine the yield of purified cell walls once isolated from the cell.

Sequential spectrophotometric determination of chromium(III) and chromium(VI) using flow injection analysis

A flow injection analysis (FIA) procedure for the speciation of Cr(III) and Cr(VI) using the 1, 5-diphenylcarbazide (DPC) method is presented. As Cr(III) does not interfere in the Cr(VI)-DPC reaction, both Cr(VI) and total chromium [after the on-line oxidation of Cr(III) by Ce(V)] are sequentially determined. Cr(III) is obtained by difference. Under the experimental conditions described, the calibration graphs are linear up to 2 µg ml–1 of Cr(VI) and 4 µg ml–1 of Cr(III). The detection limits found were 18 ng ml–1 for Cr(VI) and 55 ng ml–1 for Cr(III), at a signal to noise ratio of 3. The common interfering elements in the Cr(VI)-DPC reaction were investigated under dynamic FIA conditions. The FIA method was also compared with the conventional spectrophotometric procedure.

Kinetic studies of sepharose‐and CH‐sepharose‐immobilized dihydrofolate reductase

Dihydrofolate reductase, purified to homogeneity from amethopterin-resistant Lactobacillus casei, was immobilized by coupling to cyanogen bromide-activated Sepharose or carbodiimide-activated CH-Sepharose. Coupling yields were determined by amino acid analysis following the hydrolysis of the gel. Enzyme activity was measured by the conventional spectrophotometric procedure, thus permitting the facile characterization of the immobilized enzyme. The pH optimum of the immobilized enzyme was shifted to 5.8 compared with pH 5.5 for the soluble enzyme. The immobilized enzyme retained greater than 90%of the initial activity over a six-month period and could be reused as many as ten times without loss of activity. As observed with the soluble enzyme, the activity of immobilized enzyme, which was lost on denaturation with 4M guanidine hydrochloride, was recovered rapidly and completely by washing the gel with buffer. The K(m) (app) values for dihydrofolate and NADPH for the immobilized enzyme were increased 15-164-fold over the K(m) values measured for soluble dihydrofolate reductase. Scatchard analysis of the interaction of amethopterin with the immobilized enzyme yielded linear plots and a K(d) (app) value of 0.56 x10(-8)M, and revealed that all of the immobilized enzyme molecules were capable of binding the ligand.

Determination of malonaldehyde in biological materials by high-pressure liquid chromatography

An HPLC method is described for the determination of malonaldehyde in biological materials. The procedure involves extracting the sample with trichloracetic acid, heating the extract with thiobarbituric acid, separating the thiobarbituric acid-malonaldehyde complex on a μBondapak C 18 column, and measuring the absorbance using a 546-nm interference filter. The method was found to be specific for malonaldehyde in several food and feed samples. Under routine assay conditions, a coefficient of variability of 7.0% was obtained for samples containing 1–2 μg of malonaldehyde per gram (instrument detection limit 1 ng). This procedure yields lower values for the concentration of malonaldehyde in food samples than the conventional spectrophotometric procedure based on absorbance of the thiobarbituric acid-malonaldehyde complex at 532 nm.

23] Measurement of binding of gaseous and nongaseous ligands to hemoglobins by conventional spectrophotometric procedures

This chapter presents an overview of the measurement of binding of gaseous and nongaseous ligands to hemoglobins by conventional spectrophotometric procedures. Ferrous hemoproteins bind a number of gaseous and nongaseous ligands specifically at the heme group. The reactions are fully reversible and may be described, at equilibrium, by standard thermodynamic relationships involving the application of the law of mass action. Equilibrium studies of the interaction of hemoglobin with heme ligands have been simplified by spectrophotometric procedures relating the changes in absorption spectrum to the degree of saturation of hemoglobin with ligands. The main assumption on which the spectrophotometric methods are based is that, within a tetramer partially saturated with a ligand, the spectral properties of the free and of the bound hemes correspond to those of the fully unliganded protein and of the fully liganded protein, respectively. Thus, in the quantitative evaluation of the ligand binding isotherm, linearity of the relationships between combination with ligand and spectral changes is assumed. The chapter describes gaseous ligands and nongaseous ligands.

Hepatic malonyl-CoA levels of fed, fasted and diabetic rats as measured using a simple radioisotopic assay.

A simple radioisotopic assay for malonyl-CoA is described. The method is based on the malonyl-CoA-dependent incorporation of labeled acetyl-CoA into palmitic acid catalyzed by fatty acid synthetase in the presence of NADPH. Its main advantages over the more conventional spectrophotometric procedure is that it is extremely sensitive and allows the simultaneous determination of picomole quantities of malonyl-CoA in multiple tissue extracts. It should prove particularly suitable for studies on the regulation of lipid metabolism in isolated hepatocytes where the quantity of tissue available for analysis is frequently very small. Application of the method to the measurement of malonyl-CoA in livers from fed, fasted, and diabetic rats yielded values that were consistent with the recently postulated role of malonyl-CoA in the regulation of hepatic ketone body production.