Quantitation and differentiation of bioparticles based on the measurements of light-scattering signals with a common spectrofluorometer

By simultaneously scanning both the excitation and emission monochromators of a common spectrofluorometer with same starting excitation and emission wavelength (namely, Deltalambda = 0), we obtained synchronous light scattering (SLS) signals that related to Rayleigh and Mie scatterings. It was found that the SLS signals could be applied for quantitation and differentiation of model bioparticles such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Bacillus thuringiensis and Bacillus megaterium. In PBS buffer, these model bioparticles could form colloidal suspensions or dispersions of sizes ranging from hundreds of nanometers to tens of micrometers, giving SLS signals with the intensity being proportional to the amount of bioparticles in the range from 1.7 x 10 (5) to 1.7 x 10 (9) CFU/mL. A further finding is that polarized synchronous light scattering (PSLS) signals of I 0 degrees -30 degrees against I 0 degrees -0 degrees , which could be obtained by introducing polarizing sheets accessory of the spectrofluorometer, and the derivative synchronous light scattering (DrSLS) signals, which could be obtained directly with the extension function of the spectrofluorometer, offer differentiation information of bioparticles connected with their size, shape, refractive indexes, and inner structure. Refractive indexes of spherical bacteria were then calculated based on light scattering signals.     

Thermodynamic parameters of polymer-solvent systems from light-scattering measurements below the theta temperature

As is well known, the chemical potentials of polymer and solvent in solution and, hence, the Flory-Huggins interaction parameter χ can be determined from scattered light intensities from dilute and concentrated solutions of the polymer in the solvent concerned. Preferably, measurements should be performed at temperatures as low as possible, provided the temperature exceeds the cloudpoint for the concentration used. It is shown that the lower the temperature and, consequently the higher the scattered light intensity, the better is the accuracy of the parameters obtained. At each temperature the scattered light intensity shows a maximum at some concentration. Below the theta temperature the ratio of scattered light intensity and concentrations also shows a maximum at some concentration. The values and the concentrations of these maxima for various temperatures enable the maximum of the spinodal to be determined. The spinodal itself can be determined by an extrapolation procedure of the reciprocal scattered light intensities. Measurements have been performed with three narrow-distribution polystyrene samples in cyclohexane. On the basis of the results, χ, and its dependence on concentration, temperature, and molecular weight can be determined to high accuracy.     

Investigation on hyperin-cetyltrimethylammonium bromide-fibronectin system by resonance light-scattering technique

A simple, highly sensitive assay for fibronectin (Fn) was reported using resonance light-scattering (RLS) technique based on the enhanced RLS intensity of hyperin-cetyltrimethylammonium bromide (CTMAB)-Fn system. The interaction system of hyperin-CTMAB-Fn was investigated using spectral methods. Mechanistic investigations show that the main reason of the enhanced RLS intensity of Fn is the formation of three-component complex (hyperin-CTMAB-Fn), in which CTMAB acts as a bridge between hyperin and Fn. The effects of pH, surfactant, concentration of CTMAB and hyperin, incubation time and foreign substances on the enhancement of RLS intensity were studied. Under the optimum conditions, the enhancement of RLS intensity is in proportion to the concentration of Fn in the range of 1.9-248ng/ml. The synthetic samples containing Fn were analyzed and results obtained were satisfied.     

Estimation of uncertainty in size-exclusion chromatography with a double detection system (light-scattering and refractive index)

Size-exclusion chromatography (SEC) coupled with online laser light-scattering (LS) and refractive index (RI) detection provides an excellent approach to determine the molecular weights (Mw) of proteins by the “two-detector” approach. Mw is determined only at the maximum of a peak, using either peak heights or area ratio from the two detectors. However, proper calibration of the SEC/LS/RI system is critical to obtain high precision.Today, an essential part of any analysis is to evaluate the uncertainty associated with the method. Basically, it is possible to distinguish between factors related to signal nature, precision and those due to signal processing. Given the signal of interest is the peak height or area ratio from two detectors, the signal ratio uncertainty was calculated using the random propagation of error formula. In this case, the effect of signal correlation was evaluated to avoid the uncertainty overestimation. In the second case, the sources of uncertainty affecting analytical measurement were estimated with the information from the precision assessment. For this, two designs with two-factor fully nested were followed for each method. Finally, the contributions from various uncertainty sources related with calibration are also analysed in detail. There are in fact only three main sources of measurement uncertainty: intermediate precision, calibration and repeatability. Of these, method precision is always the greatest, regardless of approach.For all proteins and peptides studied, the Mw calculated using both methods are close to the theoretical results, independently of the design, but the contributions of individual terms to combined uncertainty depend on both the design and method used. For example, the combined uncertainty varied between 223 and 813.2 Da for carbonic anhydrase, although higher values were found for human insulin and ovalbumin dimer. Other considerations that can have a significant impact on the results are discussed.The reproducibility of the two methods versus that based on ASTRA software used as reference method was performed using the concordance correlation coefficient. The methods’ reproducibility depends on the permitted losses in precision and accuracy.     

Kinetic study of the formation reaction of colloidal silica spheres by transmitted-light-intensity and dynamic light-scattering measurements

Kinetic analyses of the formation reaction of colloidal silica spheres which are synthesized from ethyl silicate (EtSi), ammonia and a trace of water in ethanol are made by the transmitted-light-intensity and dynamic light-scattering methods. Sphere size versus time profiles from the two methods agree well especially at the beginning of the reaction. The polymerization starts after a certain induction time (t _i) ranging from several tens of seconds to several minutes. t _i increases as the concentrations of NH₃, EtSi and/or H₂O decrease. The apparent rates of the reaction, v ^′ are estimated from the reciprocal periods between the intersections of the linear line with the initial and final horizontal lines in the cube root of the absorbance versus time plots. Log v ^′ increases linearly with slopes of 1, 2 and 0.5 as the logarithms of the concentrations of EtSi, NH₃ and/or H₂O increase, respectively. These results are consistent with the assumption proposed earlier that the polymerization mechanism of the formation of the small preliminary particles is followed by their coalescence to form large silica spheres. Received: 10 November 1998 Accepted in revised form: 12 January 1999     

Densitometric DNA Analysis in Agarose Electrophoretic Gels

JPC – Journal of Planar Chromatography – Modern TLC - Agarose gel electrophoresis is a basic separation tool used in molecular biology, mostly for qualitative DNA analysis. There are...     

Densitometric Determination of Mebhydrolin Napadisylate in Tablets

JPC – Journal of Planar Chromatography – Modern TLC - A simple and rapid high-performance thin-layer chromatography (HPTLC) densitometric method has been developed for the determination...     

Densitometric Determination of Embelin in Lysimachia punctata

JPC – Journal of Planar Chromatography – Modern TLC - Athin layer chromatographic method with densitometric UV detection at λ = 285 nm has been developed for quantification of...     

Amperometric measurements with ion-selective electrode membranes in a flow system

Calcium and salicylate sensitive electrode membranes based on plasticized PVC were used for selective amperometric detection in a flow injection system. The peak current was found to depend linearly on concentration and the working range extended well below the potentiometric detection limit.Dedicated to Professor W. Simon on the occasion of his 60th birthday     

Study on the resonance light-scattering spectrum of lysozyme-DNA/CdTe nanoparticles system

The interactions of lysozyme and calf thymus DNA (ctDNA) or thioglycolic acid (TGA) modified CdTe nanoparticles in aqueous solution have been studied by resonance light-scattering (RLS) spectroscopy. At pH 7.2 Britton-Robinson (BR) buffer solution and pH 7.4 phosphate buffered saline (PBS), the RLS signals of ctDNA and TGA modified CdTe nanoparticles were greatly enhanced by lysozyme in the region of 220-750 nm characterized by the peak around 306 and 353 nm, respectively. Under optimal conditions, the increase of RLS intensity of the two systems is proportional to the concentration of lysozyme. The linear range is 0.1-25 microg/ml for the lysozyme-ctDNA system, and 0.2-10.7 microg/ml for the lysozyme-TGA modified CdTe nanoparticles system. The detection limit is 0.041 microg/ml for the lysozyme-ctDNA system, and 0.083 microg/ml for the lysozyme-TGA modified CdTe nanoparticles system, respectively. Meanwhile lysozyme can also be used as a probe to determine the ctDNA. The increase of RLS intensity of the system is also proportional to the concentration of ctDNA. The linear range is 0.078-13 microg/ml. The detection limit is 0.024 microg/ml. Three kinds of samples were analyzed with satisfactory results.     

Densitometric HPTLC Analysis of Aloenin in Aloe Pharmaceuticals

JPC – Journal of Planar Chromatography – Modern TLC - A thin-layer chromatographic method has been established for quantification of aloenin in aloe (Aloe arborescens Mill.)...     

Densitometric TLC Analysis of Azaarenes in Grilled Meat

JPC – Journal of Planar Chromatography – Modern TLC - A TLC method with densitometric detection is described for qualitative and quantitative analysis of seven azaarenes —...     

Dynamic light scattering in turbid colloidal dispersions: a comparison between the modified flat-cell light-scattering instrument and 3D dynamic light-scattering instrument

It remains a challenge to measure dynamics in dense colloidal systems. Multiple scattering and low light-transmission rates often hinder measurements in such systems. One of the well-established techniques for overcoming the problem of multiple scattering is cross-correlation techniques such as 3D dynamic light scattering (3D-DLS). However, a high degree of multiple scattering, i.e., vanishing single-scattering contribution in the signal, limits the use of the 3D-DLS technique. We present another approach to measure turbid media by way of upgrading our flat-cell light-scattering instrument (FCLSI). This instrument was originally designed for static light-scattering (SLS) experiments and is similar to a Fraunhofer setup, which features a flat sample cell. The thickness of the flat sample cell can be varied from 13 mum to 5 mm. The small thickness increases the transmission, reduces multiple scattering to a negligible amount, and therefore enables the investigation of dense colloidal systems. We upgraded this instrument for DLS measurements by the installation of an optical single-mode fiber detector in the forward scattering regime. We present our instrumentation and subsequently test its limits using a concentration series of a turbid colloidal suspension. We compare the performances of our modified flat-cell light-scattering instrument with that of standard DLS and with that of 3D-DLS. We show that 3D-DLS and FCLSI only have a comparable performance if the length of the light path in the sample using the 3D-DLS is reduced to a minimum. Otherwise, the FCLSI has some advantage.     

Rheological measurements and light-scattering experiments of dilute solutions of high molecular polystyrene. Light-scattering of flowing polymer solutions

We describe the behavior of dilute polymer solutions by means of light-scattering under shear flow. Solution properties of polystyrene in benzene over a wide range of molecular weight has been studied to determine the coefficientsa andK of the Mark-Houwink relationship and to estimate the rheological conditions with regard to light-scattering experiments of flowing polymer solutions. The investigations were carried out to measure the shear-rate dependence of macromolecules in solution, e.g., to observe an orientation and changing of the mean-square radius of gyration.     

Densitometric Thin-Layer Chromatography of Protease Inhibitors in Pharmaceutical Preparations

JPC – Journal of Planar Chromatography – Modern TLC -     

Laser light-scattering characterization of gelatin in formamide

Laser light scattering (LLS) including angular dependence of absolute integrated scattered intensity (static LLS) and of the spectral distribution (dynamic LLS) has been used successfully to characterize gelatin in formamide at room temperature. In static LLS, the use of formamide as a single solvent instead of an aqueous salt solution avoids the well-known problem of preferential sorption of salts in the domain of gelatin molecules. Therefore the true weight-average molecular weight M_w, the z-average radius of gyration, and the second virial coefficient have been determined. In dynamic LLS, precise measurements of the intensity-intensity time correlation function permit a Laplace inversion to obtain an estimate of the normalized characteristic linewidth distribution which could be reduced to a translational diffusion coefficient distribution, G(D). This report shows that the calibration between D and M can be established from M_w and G(D) by using only two broadly distributed gelatins instead of a set of narrowly distributed gelatin standards. After establishing a calibration between D and M, we were able to estimate the molecular weight distribution of gelatin from G(D). © 1994 John Wiley & Sons, Inc.     

Evaluation of a TLC Densitometric Method for Analysis of Azole Antifungal Agents

A new method for identification and quantitative determination of the azole antifungal agents; ketoconazole, bifonazole, fluconazole and itraconazole is described using TLC with densitometric detection. Validation of the method was carried out to confirm its precision (%RSD ranged between 1.31 and 3.45), recovery (99.7–102.4%) and linearity (r  = 0.99287–0.99722) within the concentration range under investigation. The experimental conditions obtained enable the method to be used for both qualitative and quantitative pharmaceutical analysis.