Modern Spectrophotometers Research Articles

Biosensing of multiple aromatic xenobiotics in water by in-house fabricated prototype device

Portable, low-cost, and accurate monitoring of hazardous mono-aromatic pollutants, such as phenol or benzene group of compounds in water is a challenging task due to the lack of suitable detectable functional groups and complex matrix of environmental samples. Here, we use a series of protein-based biosensing recognition scaffolds to enable specific detection of several mono-aromatic classes of xenobiotics. The biosensor is tuned to perform in intricate environmental conditions and is interfaced with an in-house manufactured, multi-channel device (AroTrack) capable of direct and sensitive detection of several of these aromatic contaminants, such as phenol, benzene, and 2,3-dimethylphenol (2,3-DMP) in the low ppb range (10–200 ppb). The efficiency of the prototype device was benchmarked in both simulated wastewater and real environmental samples comprising 10 times higher isostructural aromatic pollutants or ions. It was established that AroTrack is reliable for environmental sample testing with a high degree of reproducibility and efficiency comparable to that of modern spectrophotometers (<5 % error). The battery-operated device costs less than $50 to fabricate and this low cost makes it effective to be implemented in rural and low-income settings which suggests immense field deployable potential.

Measurement traceability of total radiance factors for white fluorescent materials employing incandescent-lamp Zeiss leucometer photometers and with modern spectrophotometers based on a D65 artificial illuminant

A discussion based on the theory of measurements using an upgraded Zeiss leucometer (0/d) in which the fraction of UV radiation from the incandescent lamp is controllable was used to obtain a photometric equation (appropriate for any modern leucometer-type photometer) relating the total reflectance factors measured using such an instrument to the total spectral radiance factors for white fluorescent materials measured using a modern spectrophotometer (d/0) with a D65 artificial illuminant at the leucometer effective wavelength. A review of the available experimental results using the resulting equation indicated that it was possible to obtain measurement traceability of the total spectral radiance factors for white fluorescent materials when employing Zeiss leucometer photometers with an incandescent lamp and by using modern spectrophotometers with a D65 artificial illuminant.

Practical method for correcting heterochromatic stray light in dual‐channel spectrographs for colorimetry

AbstractMeasurements by spectrophotometers more or less suffer from heterochromatic stray light generated in their spectrometers. It is divided into near‐band and off‐band stray lights. While the former appears near the spectral band of the incident flux, the latter distributes broadly across the whole range and is far more problematic from colorimetric point of view. This article presents a practical method for correcting off‐band stray light in dual‐channel array spectrographs often built in modern spectrophotometers. Unlike most correction methods using a line spread function, the presented method estimates the distribution of off‐band stray light across the array as the product of a device‐specific stray light distribution and a total stray light intensity. The latter depends on both the spectral power distribution of an incident flux and a device‐specific rate of change from the incident flux to the off‐band stray light. For further simplification, the method replaces wavelengths by several bands dividing the visible range owing to the moderate spectral dependence of the above rate of change. With those simplifications, the method is practical, effective, and robust enough to work in an inexpensive hand‐held spectrophotometer with the compact spectrograph which often suffers from off‐band stray light. The performance of the method was evaluated with two dual‐channel array spectrographs with and without 2nd‐order‐rejection filter. Specimen lights incident through ten different glass filters were measured and the errors caused by off‐band stray light in the pixel outputs from the array were successfully corrected by the method. © 2016 Wiley Periodicals, Inc. Col Res Appl, 42, 431–439, 2017

Uncertainty in modern spectrophotometers

Uncertainty in modern spectrophotometers

Alternative mathematical approaches to shade sorting

Shade sorting is the process of assigning samples of the same nominal color into groups having no significant color variation. Use of modern spectrophotometers and color measurement technology make it possible to obtain precise color differences between samples. When these color differences are viewed as distances between points, the shade sorting problem is seen to be equivalent to the clustering problem in the mathematical literature. Several mathematical techniques for clustering—complete linkage clustering, vertex labeling, and set covering—are explained and compared for their efficiency when applied to shade sorting. A particular implementation of complete linkage clustering called Clemson Color Clustering (CCC) is found to perform well as compared to the other reviewed methods. © 2000 John Wiley & Sons, Inc. Col Res Appl, 25, 368–375, 2000

Rapid Spectrophotometric Method for Analyzing Natamycin in Cheese and Cheese Rind

A rapid spectrophotometric method for determining natamycin in cheese and cheese rind has been developed. Samples are homogenized with acidified aqueous acetonitrile and homogenates are filtered. Natamycin is directly quantitated in filtered extracts on the basis of the characteristic third-derivative trough at 322.6 nm. Additional cleanup of extracts is not required because derivative transformation of the conventional analytical band at around 319 nm eliminates spectral interferences from other compounds. The analysis is simple and can be completed in 6 min. The equipment is easily accessible because most modern spectrophotometers allow instant generation of derivative spectra. The method needs small amounts of solvents and has good analytical characteristics. Overall recovery was 98.4 +/- 0.7%, and linearity was excellent (r = 0.9998) in the range examined (0.5-20 mg/kg). Quantitation and detection-limits were estimated at 0.5 and 0.25 mg/kg, respectively. Precision statistics based on within-day and between-days variations suggest an overall relative standard deviation of 1.4%.

Approach for Increased Information from the Second-Derivative Spectra in UV-Vis Absorption Spectroscopy

The resolution of overlapping bands in the UV-Vis absorption spectra leading to determination of their basic parameters ([Formula: see text]) provides important information about the energies and probabilities of the electronic transitions. In the analysis of UV-Vis absorption spectra recorded linearly in wavelength by means of modern spectrophotometers with holographic gratings, the analytical shape describing individual bands is asymmetric. This factor leads to certain limitations in determining their number with the use of the second-derivative spectra and decreased effectiveness of the computing procedure. It was found that the loss of information in d2 A/dλ2 is due to its long-wavelength attenuation in comparison with d2 A/dν˜2. An analytical equation connecting d2 A/dλ2 with d2 A/dν˜2 is proposed, which restores the information from the second derivative in the long-wavelength region.

A semi-micromethod for the determination of the extinction coefficients of duplex and single-stranded DNA

We have developed a rapid and convenient procedure for the determination of concentrations and extinction coefficients of oligo- and polynucleotides. It offers significant advantages over other methods in terms of precision and the ability to detect artifactual or erroneous results. Samples are first completely digested with appropriate enzymes to mononucleotides and nucleosides. Using the multicomponent linear regression capabilities of commonly available spreadsheet programs, the absorbance spectrum of the digest can be analyzed as a linear combination of the contribution of the possible constituent monomers. If all the spectral components present have been included, the analysis yields the concentration of each of the monomer species whose sum is the concentration (in monomer units) of the original undigested sample. When combined with the predigest absorbance spectrum, the extinction coefficients of the intact sample can then be calculated. The analysis also yields the fractional base composition of the oligomer or polymer. The extensive spectral data provided by digital readouts of modern spectrophotometers permit the application of sensitive tests of the goodness of fit, thus facilitating the detection of artifacts and sample inhomogeneity. Both single-strand and duplex structures can be analyzed comfortably in sample sizes of 25 to 35 nmol (total) of mononucleotides with a precision of 1%. The concentrations obtained by this method agree, on the average, within 0.2% with those determined by phosphate analysis of the same sample. The method also yields the base composition with an accuracy of ca. 5% for high-molecular-weight polymers and 2% for short oligomers (15–20 bp) when compared to the predicted values.

The use of a spectrophotometer for the optical monitoring of thin films

A spectrophotometer can be employed for the optical monitoring of thin films at a single wavelength by using the direct turning-value method. This approach allows periodic in situ measurements of the photometric properties of coatings to be made over a wide spectral range. This method is successful provided that efficient transfer of the light beam from the spectrophotometer to the sample in the vacuum chamber and back again can be ensured. Thus the advantages of modern spectrophotometers (high resolution, wide spectral range, baseline correction, computer interface etc.) can be utilized for monitoring and in situ measurement. The use of optical fibres enables the optical signal to be transmitted efficiently, and the consequent small size of the device makes it suitable for use in r.f. sputtering systems. Thus commercial spectrophotometers equipped with appropriate accessories can be used for optical monitoring and for an additional check on the coating performance in the wavelength range of interest.

Half-time analysis of the integrated Michaelis equation. Simulation and use of the half-time plot and its direct linear variant in the analysis of some alpha-chymotrypsin, papain- and fumarase-catalysed reactions.

Substitution of half-time parameters in the integrated form of the Michaelis-Menten equation for any enzyme-catalysed reaction yields an equation that gives a linear relationship between the half-time of the reaction and the substrate concentration at that point of the reaction. The logarithmic term of the integrated equation becomes a constant as a result of the substitution, which means that the use of the half-time plot of the equation requires calculation only of half-time and substrate-concentration values at various stages of the reaction. The half-time method is both simple and exact, being analogous to an [S(0)]/v(i) against [S(0)] plot. A direct linear form of the half-time plot has been devised that allows very simple estimation of Michaelis parameters and/or initial velocities from progress-curve data. This method involves no approximation and is statistically valid. Simulation studies have shown that linear-regression analysis of half-time plots provides unbiased estimates of the Michaelis parameters. Simulation of the effect of error in estimation of the product concentration at infinite time [P(infinity)] reveals that this is always a cause for concern, such errors being magnified approximately an order of magnitude in the estimate of the Michaelis constant. Both the half-time plot and the direct linear form have been applied to the analysis of a variety of experimental data. The method has been shown to produce excellent results provided certain simple rules are followed regarding criteria of experimental design. A set of rules has been formulated that, if followed, allows progress-curve data to be acquired and analysed in a reliable fashion. It is apparent that the use of modern spectrophotometers in carefully designed experiments allows the collection of data characterized by low noise and accurate [P(infinity)] estimates. [P(infinity)] values have been found, in the present work, to be precise to within +/-0.2% and noise levels have always been below 0.1% (signal-to-noise ratio approximately 1000). As a result of the considerations above, it is concluded that there is little to be feared with regard to the analysis of enzyme kinetics using complete progress curves, despite the generally lukewarm recommendations to be found in the literature. The saving in time, materials and experimental effort amply justify analysis of enzyme kinetics by progress-curve methods. Half-time plots linear to >/=90% of reaction have been obtained for some alpha-chymotrypsin-, papain- and fumarase-catalysed reactions.

Differentiation of spectra as a means of resolving spin-forbidden bands: iron(III) complexes

Calculated second and fourth differentials of iron(III) spectra in molten salts (an extreme case, where temperature-broadened spin-forbidden transitions barely show up on an intense charge-transfer band edge) have resolved out expected transitions, plus some more which are tantatively assigned; this approach, which requires no prior knowledge of the correct number of bands present or their mathematical shape, is thus capable of resolving and subseuently assigning ligand-field spectra, but cannot be directly performed on modern spectrophoto-meters without due care being taken.

An Evaluation of Two Simple Methods for Calibrating Wavelength and Absorbance Scales of Modern Spectrophotometers

Two simple methods have been evaluated for calibrating wavelength and absorbance scales of modern spectrophotometers, namely, a method for wavelength scales which is based on the locations of the maxima and minima of the absorbance wave of a pair of transparent closely-spaced parallel plates, and a method for absorbance scales which is based on the absorbances of properly oriented screens of square-woven single strands of brass wire. The evaluations were carried out by means of a Beckman D.U., a Cary, and a Hardy-GE spectrophotometer. It is our hope that the simple, convenient methods will encourage more frequent calibration of spectrophotometers, especially when the wavelengths and values of absorbance extremes are of importance. (This paper deals only with methods and should not be construed as an evaluation of the performance of the spectrophotometers employed.)

Societies and Academies

LONDON.Royal Society, April 6.-Sir J. J. Thomson, president, I in the chair.-J. H. Jeaas; The instability of the pear-j shaped figure of equilibrium of a rotating mass of ' liquid. The form of the pear-shaped figure of equilibrium was calculated so far as the second order of small quantities by Sir G. Darwin, who believed he had shown it to be stable. In a recently published paper (Phil. Trans., A, 215, p. 27) it was shown that othe stability could only be finally decided upon after the figure had been calculated to terms of the third order. In the present paper these third-order terms are evaluated, and the pear-shaped figure is definitely shown to be unstable.-Sir William Kamsay: A hypothesis of molecular configuration in three dimensions of space.-J. Proudman: The motion of solids in a liquid possessing vorticity. This paper contains investigations on the motion of a homogeneous frictionless liquid by the methods of theoretical hydrodynamics. The principal subjects considered are two-dimensional motion with uniform vorticity and three-dimensional motion with varying vorticity, the positions of the solids being specified by generalised co-ordinates. The general work consists in reducing solutions to those of Neumann's potential problems.-Dr. S. J. Lewis; The ultra-violet absorption spectra of blood sera. The work described in this preliminary paper has for its object the investigation of the absorption spectra of blood sera in the ultra-violet region of the spectrum. Modern spectrophotometers are used to determine the absorption values on passing ultra-violet light through a prescribed layer or solution of serum. With these values as ordinates and wave-lengths as abscissa; an absorption curve is drawn. With normal serum the general characters of the curve are constant, and there is very little variation in detail. With certain pathological sera the curves show much greater modifications, and some of these are well defined and appear to be peculiar to given diseases. It is found that the major part of the absorption is due to the proteins.- G. W. Paget and R. E. Savage: The growth-rings on herring scales. This communication brings forward morphological evidence as to the structure and significance of the so-called “ growth-rings” on herring scales. At present the interpretation of 'these rings as rings of growth depends, in the main, upon statistical data. Morphological evidence of a differential growth-rate of the scale as a whole is altogether lack-. ing. The present observations place upon a sure foundation the view that the transparent rings do indeed, mark a recurring period of minimum growth.