Study of polystyrene-block-poly(methyl methacrylate) micelles by size exclusion chromatography/low-angle laser light scattering, 1. Influence of copolymer concentration and flow rate

A size exclusion chromatography study of the micellar system polystyrene-block-poly(methyl methacrylate) in the mixed solvent 1,4-dioxane/cyclohexane is reported. Good separation of the peaks of micelles enabled direct determination of the weight-average molecular weights of micelles with a low-angle laser light scattering detector. The values obtained were found to be in accordance with those determined independently by static light scattering. Experiments with changing flow rate and concentration of the injected sample solution show moderately fast unimer-micelles re-equalibration in the course of the separation. A strong effect of the solute trapping in the column, probably due to the adsorption of the unimer on the packing, was observed.     

Characterization of non-homogeneous polymers with size-exclusion chromatography by implementing an on-line viscometer

The viscosity distribution of a polymer sample can be obtained by using an on-line viscometer as a detector in size-exclusion chromatography. This newly defined viscosity distribution is closely related to the molecular weight distribution and expresses weight fraction times intrinsic viscosity of species i as a function of the corresponding molecular weight times intrinsic viscosity (w_i[η_i] vs. M_i[η_i]). The intrinsic viscosity ([η]) and number-average molecular weight (M?_n) can be obtained directly from a viscosity distribution. If the Mark-Houwink exponent a is known (or approximately known) for non-homogeneous polymer the M?_w/M?_n can be estimated from the viscosity distribution when the molecular weight distribution is approximated with a known distribution function. These estimates are independent of any other detector and are valid even for non-homogeneous polymer samples. The relation between the moments of the viscosity distribution and the M?_w/M?_n is presented for two widely used distribution functions, the Log-Normal and the Generalized Exponential Distributions. Polymer characterization based on the viscosity distribution is shown to be a robust technique. It is particularly attractive in characterizing non-homogeneous polymers since it is solely obtained from on-line viscometer.     

Characterization of cirrhosis and hepatocellular carcinoma using low-angle x-ray scattering signatures of serum

The diagnosis of hepatocellular carcinoma (HCC) usually occurs at late stages in the disease when there are few effective treatment options. The measurement of the concentration of tumour markers in the serum of patients is a complementary tool frequently used for the interpretation of diagnostic imaging results. It is also used as a prognostic tool for the detection of cancer. Unfortunately, the sensitivity of tumour markers is still low and many times it yields normal results for cirrhotic and HCC patients. In the current work, the detection possibility of the structural changes in serum proteins accompanying cirrhosis and HCC is investigated using a low-angle x-ray scattering (LAXS) technique. The results show that there are significant differences in the LAXS profiles of cirrhosis and HCC lyophilized serum samples compared to normal. The changes in shape, total counts and position of the first scattering peak at 4.8 degrees, which was previously reported to be sensitive to the structural changes in protein, showed the most characteristic deviations from normal serum. The present results are promising and would offer a potentially helpful complementary tool for monitoring cirrhosis and HCC.     

Characterization of peroxidase:anti-peroxidase immune complexes by capillary zone electrophoresis and high-performance size-exclusion chromatography

Determination of the molecular constituents of commercial peroxidase:anti-peroxidase (PAP) preparations is necessary for the proper interpretation of PAP applications based on competitive binding assay. Capillary zone electrophoresis with field 300 V/cm, 40 cm capillary length (20 cm effective length), and high-performance size exclusion chromatography equipped with Superose 12 HR10/30 column revealed that a PAP preparation used for Fc gamma receptor studies contained multiple sizes of immune complexes, an excess amount of free peroxidase, and little or no free anti-peroxidase antibody. The antibody:antigen ratios of the three major immune complex components were 2:2, 1:2, and 1:1. These techniques provide useful methods of qualitative, as well as quantitative analysis of PAP preparations.     

Characterization of methylalumoxane by means of gel permeation chromatography

Characterization of methylalumoxane (MAO) by gel permeation chromatography reveals the presence of different oligomeric products. Using suitable series of columns and alkylaluminium compounds with known molecular weight, it is possible to obtain the average molecular weights of most species which are present in a sample of MAO. The study of the chromatograms reveals modifications in composition of the MAO oligomers in relation to the time of residence in solution at given temperatures.     

Characterization of cellulose nitrate with gel permeation chromatography - low angle laser light scattering (GPC-LALLS), GPC-infrared,and membrane osmometry

Cellulose nitrates with nitrogen contents in the range 11,2 to 13,3 mol-% are characterized with gel permeation chromatography (GPC) using various detectors, membrane osmometry, static low angle laser light scattering (LALLS) and solution viscosity measurements. The molecular weight data from GPC-LALLS are generally in good agreement with those obtained by static LALLS and membrane osmometry. GPC-infrared is used to analyze the nitrogen content. These techniques are also used to characterize cellulose nitrate in propellants. A comparison between two different column packings in GPC-LALLS analysis is integrated into this study.     

Purification of turkey coronavirus by Sephacryl size-exclusion chromatography

Sephacryl S-1000 size-exclusion chromatography was used to purify turkey coronavirus (TCoV) from infected turkey embryo. TCoV was propagated in the 22-day-old turkey embryos. Intestines and intestinal contents of infected embryos were harvested and homogenized. After low speed centrifugation, the supernatant was concentrated by ultracentrifugation through a cushion of 30 or 60% sucrose solution, or by ammonium sulfate precipitation. The purification methods included sucrose gradient and Sephacryl S-1000 size-exclusion chromatography. Ultracentrifugation through a cushion of 60% sucrose solution was better than the other two methods for concentration of TCoV from intestinal homogenate. The most effective method for purifying TCoV and removing extraneous materials was size-exclusion chromatography as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. More spike-rich particles were observed in the sample purified by chromatography than those purified by sucrose gradient as examined by electron microscopy. Differentiation of turkey anti-TCoV antiserum from normal turkey serum was better achieved by ELISA plates coated with TCoV preparation purified by size-exclusion chromatography than that purified by sucrose density gradient. The results indicated that Sephacryl S-1000 chromatography was useful for purification of TCoV.     

Studies of compact hard tissues and collagen by means of Brillouin light scattering

A measure of the elastic properties of tissue can be found from the propagation of sound in the tissue. Longitudinal sonic velocities were measured for mineralized turkey leg tendon (density 1.50 g/cc), deer antler (1.77 g/cc) and cow tibia (2.05 g/cc) in the 10 GHz frequency regime by means of Brillouin light scattering using a nine pass Fabry-Perot interferometer. Wet, air dried, mineralized and demineralized specimens were tested. Sonic velocity in each tissue increased with mineral content and decreased when the tissue was wet. All wet values are higher than for wet rat tail tendon collagen, axially and radially, but with considerably less anisotropy. The results are interpreted to indicate that bone matrix collagen is more highly crosslinked than tail tendon collagen. The loss of anisotropy is taken to correspond to a much higher crosslinking density between adjacent collagen molecules in mineralized tissue compared to rat tail tendon. The axial sonic velocity of dried rat tail tendon is almost that for low density dried mineralized tissue and greater than the radial sonic velocity of these tissues, but the radial sonic velocity for dried rat tail tendon is much lower, again corresponding to less crosslinking in this tissue. Longitudinal modulus, K, is defined as the tissue density times the square of the velocity. The compliance, 1/K, was found to be a linear function of density for each of the four conditions. It suggests that a Reuss formalism describes the elastic properties. Since the difference between the compliance for wet and dry tissue is also a linear function of density, the effect of water on the compliance is additive. The axial sonic velocity for cow bone is essentially constant over a frequency range spanning 10 orders. Presumably the axial sonic velocity is controlled by the continuity of the collagen fibers lying along the bone axis. The radial velocity decreases by 30% over this frequency range, probably due to the many levels of structure observed in long bone like osteons, Haversian canals and blood vessels, as well as internal surfaces like cement lines and between lamellae. The sonic anisotropy of hard tissues decreases considerably with increasing frequency. While rat tail tendon collagen is very anisotropic both sonically and optically, hard tissues whether wet, dry, mineralized or demineralized show much less anisotropy. The optical index of refraction, both axially and radially, was found by Brillouin scattering for the air dried demineralized tissues. A close match was found between optical and sonic anisotropy for all the demineralized tissues.     

Blood cell counting and classification by nonflowing laser light scattering method

We present a nonflowing laser light scattering method for automatically counting and classifying blood cells. A linear charge-coupled device (CCD) and a silicon photoelectric cell (which is placed behind a pinhole plate on the CCD) form a double-detector structure: the CCD is used to detect the scattered light intensity distribution of the blood cells and the silicon photoelectric cell to complete the focusing process. An isotropic sphere, with relative refractivity near 1, is used to model the blood cell. Mie theory is used to describe the scattering of white blood cells and platelets, and anomalous diffraction, red blood cells. To obtain the size distribution of blood cells from their scattered light intensity distribution, the nonnegative constraint least-squares (NNLS) method combined with the Powell method and the precision punishment method are used. Both numerical simulation and experimental results are presented. This method can be used not only to measure the mean and the distribution of red blood cell size, but also to divide the white blood cells into three classes: lymphocytes, middle-sized cells, and neutrocytes. The experimental results show a linear relationship between the blood cell (both white and red blood cells) concentration and the scattered light intensity, and therefore, the number of blood cells in a unit volume can be determined from this relationship.     

Characterization of lysosomal contribution to whole-cell light scattering by organelle ablation

Angularly resolved light scattering measurements made at visible wavelengths have the ability to quantify subcellular morphology, with particular sensitivity to organelles the size of mitochondria and lysosomes. We have recently reported on a lysosome-staining-based method that provides scattering contrast between stained and unstained cells, and through the use of appropriate models, we extracted a size distribution and contribution to cellular light scattering that we attributed to lysosomes. We provide an independent measurement of the lysosomal size distribution and contribution to cellular light scattering by exploiting photodynamic ablation of lysosomes and observing its effect on angularly resolved light scattering measurements. From these measurements, we conclude that lysosomes scatter approximately 14% of the light from EMT6 cells at 633 nm and that their size distribution has a mean and standard deviation of 0.8 and 0.4 microm, respectively.     

Aggregation of amphiphilic pullulan derivatives evidenced by on-line flow field flow fractionation/multi-angle laser light scattering

Size-exclusion chromatography (SEC) is a useful steric separation technique for the analysis of water-soluble polysaccharides in aqueous solution. However, in the case of amphiphilic derivatives, the usefulness is limited because of interactions between hydrophobic segments and the stationary phase. Alkyl-bearing pullulans differing from the extent and the length of alkyl groups were characterized using flow-field flow fractionation with on-line coupling multi-angle laser light scattering (F4/MALLS). Comparison of SEC and F4 is presented and the interest of F4 in the field of amphiphilic derivatives is demonstrated.     

Diffusion coefficients and hydrodynamic radii of three spherical RNA viruses by laser light scattering

The hydrodynamic radii of three spherical RNA viruses have been deduced from their diffusion coefficients as measured by laser light beating spectroscopy. In two cases these radii were found to be significantly larger than those measured by other techniques.     

Automated laser light scattering system for assessemnt of sperm motility

An automated apparatus for the objective estimationin vivo of human sperm motility using quasielastic light scattering is described. Computer control removes tedious and repetitive aspects of such studies. Typical results demonstrate the potential of such a system for routine assessment of percentage motility and sperm swimming speed.     

Determination of albumin and myoglobin in dialysate and ultrafiltrate samples by high-performance size-exclusion chromatography

A high-performance size-exclusion chromatographic method was developed, validated and implemented for simultaneous and quantitative determination of albumin and myoglobin along with inulin, vancomycin and creatinine in dialysate and ultrafiltrate samples from in vitro hemodialysis experiments. The experimental parameters including mobile phase pH, ionic strength, detection wavelength, flow-rate, injection volume were first optimized for the determination of albumin, myoglobin, inulin, vancomycin and creatinine. The peak height ratio and detection limits of the proteins were then comparatively studied at 210, 254 and 280 nm by UV and diode array detection. The method was further validated by evaluating the linearity, precision and accuracy of the proteins. The assay was finally implemented to the simultaneous and quantitative determination of the proteins in dialysate and ultrafiltrate samples.     

Determination of long-chain branching distribution in polyethylene by combination of gel permeation chromatography and viscometry

Direct measurement of intrinsic viscosity on gel permeation chromatography effluent was combined with use of the universal calibration concept in order to determine long-chain branching in polyethylene. The experimental procedure, involving an axial dispersion correction for the limited resolution of our chromatographic system, is presented. Results obtained for three branched polyethylene samples are compared with those provided by the well-established method of preparative fractionation by column elution and examination of the fractions by viscometry and light scattering.     

Purification of poliovirus 14 S subunits by sucrose gradient ultracentrifugation and high-performance size-exclusion chromatography

Purification of 14 S subunits from extracts of poliovirus-infected HeLa cells was achieved by a combination of sucrose gradient ultracentrifugation and high-performance size-exclusion chromatography. The particles were free of admixtures of other subviral particles, of nonstructural viral proteins, and of host cell proteins. The purified material retained the physical and antigenic properties of native 14 S subunits fully, as well as their ability to assemble to empty capsids in vitro.     

Characterization of layered scattering media using polarized light measurements and neural networks

Measurements of the spatial distributions of polarized light backscattered from a two-layer scattering medium are used to train a neural network. We investigated whether the absorption coefficients and thickness of the layer can be determined when the scattering properties are known. When determining the absorption of the upper layer or the layer's thickness, polarized light measurements provide better performance than unpolarized measurements, demonstrating the sensitivity of polarized light to superficial tissue. Determination of the lower layer's absorption coefficient is not improved by polarized light measurements. Prior knowledge of the tissue under investigation is also beneficial because errors are reduced if the range of absorption or thickness is restricted.     

Determination of safe exposure levels of He/Ne laser light to the human body by means of erythrocyte irradiation studies

He/Ne laser irradiation produces various shape changes and haemolysis in erythrocytes. Under total absorption conditions, after correcting for the reflection from the surface, blood samples were exposed to various intensities of laser light. The upper limit of these intensities, at which the erythrocytes maintain their normal characteristics, has been used to develop safety levels for the various organs of subjects of varying complexions, assuming that even the cells below the skin layer are not damaged by laser exposures. Depending on the complexion these levels show considerable variations for various organs.