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To ascertain whether a low-angle laser scattering photometer under development (76) would give absolute molecular weights for biological macromolecules, it was necessary to obtain specific refractive index increments for light of wavelength 632.8 nm (He-Ne laser). Such data were unavailable for polymers although calibration data have been reported (27) for 589.3 and 632.8 nm for a series of electrolytic solutions. Anderson ( 7 ) also calibrated a commercial differential refractometer resembling that of Brice and Halwer ( 5 ) using the data obtained on salt solutions interferometrically by Kruis (20), and more recently Block ( 4 ) described a technique for using 632.8-nm radiation from a secondary source in differential refractometry. None of these investigators determined specific refractive index increments of polymer systems. We report refractive index increments for a number of polymers determined with the commercially available Brice-Phoenix differential refractometer at four wavelengths from 632.8 to 435.8 nm. Calibration measurements were made at 24.2 f 0.1"C by use of potassium chloride and sucrose, followed by studies of polystyrene, bovine serum albumin, D-glyceraldehyde phosphate dehydrogenase, polydimethylsiloxane, amylopectin, and deoxyribonucleic acid (DNA).

If the counterion of a polyelectrolyte is not identical with any of the ions of a low molecular weight electrolyte added to the solution, the system may be regarded as a four-component system. Relations for the refractive index increments have been derived which allow the determination of the coefficient of selective sorption of the added electrolyte from the refractive index increments of the components independent of the original counterion of the polyelectrolyte. Equilibrium dialysis and differential refractometry were used to study the interaction of KCl, KBr and NaI with poly[-1(2-hydroxyethyl)pyridiniumbenzenesulfonate methacrylate] or with an analogous polymer containing a toluenesulfonate counterion in aqueous solutions. The coefficient of selective sorption increases in the series Cl− < Br− < I− from strongly negative to strongly positive values; the specific interaction of these counterions with the macroion increases in the same order.

In the use of refractometry for polymer characterization, it is shown that interpretation of data through use of a molar refractivity as defined by the Lorenz-Lorentz equation is more accurate than by use of the parachor. The disagreement between experimental and calculated values of the unit molar refractivity may be attributed to the arbitrary definition of the residue unit and the method of summation of group refractivities. In general, refractive index should depend on molecular weight and the conditions of polymerization, but this may depend on the dielectric properties and volume fraction of the domains comprising chain ends, initiator fragments, and free volume. The effect of polymer conformation on the molar refractivity of polyisoprene is found to be slight. For copolymer systems, there is a linear dependence of refractive index on weight fractional composition only where there is little order. The refractive index of a copolymer is not independent of the nature of the distribution of the residues along the chain.

A simple, rapid and versatile method for the separation and determination of sugars via high performance liquid chromatography is described. Virtually any combination of monosaccharides, disaccharides and/or higher oligomers can be separated. Detection via differential refractometry simplifies quantitative measurements and permits collection of emerging sugars for identifying tests. As little as 20 μg of an individual sugar can be determined and preliminary data indicate excellent precision and accuracy. Aqueous solutions of sugars, including samples containing substantial salts or other impurities, can be analyzed directly without derivatization or pretreatment.

SUMMARY We have described a technique by which a portable interference refractometer may be used to determine the accumulation of nitrogen in a totally closed anaesthetic circuit. The inspired mixture contained halothane, oxygen, nitrous oxide and nitrogen. The concentrations of the first two were controlled with u.v. and paramagnetic analysers respectively. It was then possible to determine the percentage of nitrous oxide replaced by nitrogen by measuring the refractive power of the mixture.

The principles underlying a new method of refractometry of living cells are discussed. The method was evolved from the chance observation that the amoebocytes of the blood of the earthworm, examined in their own blood, appeared bright instead of dark by positive phase-contrast microscopy. This was shown to be due to the presence of dissolved haemoglobin which raised the refractive index of the medium above that of the cytoplasm. In order to determine the refractive index of the latter it was only necessary to dilute the blood until the cytoplasm became virtually invisible. Non-pigmented proteins and other high molecular weight substances have now been substituted for haemoglobin. The nature of the initial observations suggested that if the cell could be regarded to a first approximation as being composed entirely of proteins, the cytoplasmic protein concentration could be equated to the protein concentration of the immersion medium which made the cell appear with minimum contrast. This would only be true if equal concentrations of different proteins in solution had the same refractive index. The nature of refractive index and its relationship to density are discussed and it is shown that for nearly all unconjugated proteins so far investigated the specific refraction increments (i.e. the increase in refractive index per 1 per cent, increase in concentration) have almost the same values (.00185±2 per cent.). The effects of many factors such as pH, salts, temperature, wavelength, concentration, and nature of the solvent are discussed. Since living cells contain substances other than proteins the specific refraction increments of protein derivatives, lipides, carbohydrates, and salts are considered and it is shown that the presence of moderate amounts of such substances is unlikely to affect the refractive index of cells to any great extent. It is suggested that the mean specific refraction increment of protoplasm should be taken as .0018 and that this value can be used in order to calculate the solid and water content of protoplasm from values of refractive index.

Urinary excretion of amphetamine has been examined in a number of male albino rats of the Wistar strain after intraperitoneal administration of d-amphetamine-HCl. The collected urine was rendered alkaline, extracted with chloroform, washed with water, dried with anhydrous magnesium sulfate, and evaporated down to 10 ml; the excretion of unchanged drug was determined using (a) silica-gel thin-layer chromatography and a number of solvents and (b) interference refractometry. The effect on the excretion of the amine when urine was acidic or alkaline was also measured. Quantities smaller than 1 mg were measured with these techniques.

In this Paper a generalised formula for the refraction of light through a prism is obtained, and the particular cases pertaining to practical methods of refractometry deduced from it. The sensitivity of various methods for various prism angles and refractive indices is shown in a series of curves, as is also the liability to error due to errors in auxiliary constants.

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The specific refractive index increments of a number of polymer/solvent systems have been measured by means of an interference refractometer. The technique described permits rapid and precise evaluation of dn/dc, as well as eliminating the difficulties normally associated with interferometric measurements in organic solvents. Die spezifischen Brechungsindex-Inkremente fur einige Polymer/Losungsmittel-Systeme wurden mit Hilfe des Interferenzrefraktometers gemessen. Die beschriebene Mestechnik erlaubt schnelle und genaue Auswertung von dn/dc und eliminiert die Schwierigkeiten, welche normalerweise mit interferometrischen Messungen in organischen Losungsmitteln verbunden sind.

SYNOPSIS Accurate and reproducible interference microscopy of unstained sections requires (a) the use of a shearing system, preferably with a half-shade eye-piece; (b) complete removal of embedding wax from the section; (c) complete replacement of one mounting medium by any successive medium; (d) accurate measurement of the refractive index (RI) of the mounting media at the temperature and wave-length used for microscopy. The optical path through a specimen mounted in water is consistently lower than expected from measurements in non-polar fluids—this is attributed to swelling of the tissues in water. Cellulose, glycogen and some mucins have a relatively low RI, and secretory granules and cytoplasmic chromidial substance a relatively high one. The highest RI observed was in calcified cartilage matrix. The “effective thickness” of tissue components, especially certain mucins, is often considerably less than the geometrical thickness of the section. This is attributed to the presence in the tissues of substantial spaces containing mounting medium. The relation of the RI and the effective thickness of tissue components in sections to their permeability to histological dyes is discussed. It is concluded that both the RI and the effective thickness may be of some value in predicting the ease with which dye particles of a given size can penetrate the tissue.

Differential refractometry has been used to obtain accurate values of the specific refractive index increments for the following polymers in several solvents: polystyrene, poly(methyl methacrylate), poly(4-vinyl pyridine), block copolymers of styrene with methyl methacrylate and of styrene with 4-vinyl pyridine. Examples of the application of the dn/dc parameter to determine the composition of copolymers and to detect differences in stereochemical configuration of polymers are also given. The Gladstone—Dale equation is shown to be a convenient method to predict dn/dc from the refractive index of the solvent and values of the refractive index of the polymer and its specific volume.

1. The acetone-extractable substances from different natural rubbers have an appreciable influence on the index of refraction of their hydrocarbons. This influence is different in the case of Hevea rubbers from that in the case of African rubbers, for example. 2. The indices of refraction of different kinds of raw rubber after extraction likewise differ appreciably. These differences may be attributed in part to the different quantities of benzene-insoluble non-rubber substances, in part to differences in the degree of polymerization of the different hydrocarbons. 3. A study of the influence of mastication, depolymerization by heat and oxidation on the magnitude of the light refractive exponents of rubber and gutta-percha shows that there are characteristic differences between the two kinds of hydrocarbons, at least as far as the initial stage of oxidation is concerned. 4. From the purely practical and laboratory-testing points of view, it is possible to detect quickly and easily differences in the same kinds of raw rubber which are originally present or result from mastication, and to follow the course of mastication. 5. Furthermore it appears possible to draw conclusions regarding the constitution of rubber and gutta-percha hydrocarbons from differences in their viscosimetric and osmotic behavior. Accordingly the refractometry of rubber and gutta-percha hydrocarbons may furnish a new way of gaining a deeper insight into the structure of these natural substances.

The solid content of several halophilic bacteria was estimated by immersion refractometry. Values for extreme halophiles ranged from 28 to 32%, and for moderate halophiles from 31 to 45%. The solid content of these organisms changed with variation in the salt content of the environment. It is thought that these differences are the result of changes in the water content of the cell. Spheres (spheroplasts) had a slightly lower solid content than the parent rods. Nuclear and other inclusion bodies, mostly of lower refractive index than the cytoplasm, were noted.

The Paper treats of the conditions of pressure, discharge, &c., most suitable for the production of a bright hydrogen spectrum, such as is required for refractometry and similar purposes. The rapid deterioration of the tubes with use is shown to be caused by a rise of pressure due to the evolution of hydrogen by the electrodes. The trouble may be obviated by sealing an auxiliary bulb of 1½ or 2 litres capacity to the discharge tube. This reduces the rate of pressure variation and prolongs the useful life of the tube nearly a hundredfold. The use of capacity and inductance is shown to be very helpful with partially deteriorated tubes.

1. 1. A method for measuring the refractive index of sectioned tissue is described. It entails phase-contrast observation of sections mounted in fluids of known refractive index. 2. 2. The refractive indices of structures such as intestinal and renal epithelial brush borders, nuclei and cytoplasm were investigated. 3. 3. The epithelial cells of the small intestine and their brush borders showed a gradient of refractive index passing from villus tip to base of crypt. 4. 4. The cytoplasmic inclusions in the zona glomerulosa of the suprarenal, which have the same staining reactions, comprised two groups with widely different refractive indices. 5. 5. The refractive index of fixed structures varied according to the fixative employed. For instance, with Champy's fluid, it was high; with mercuric-chloride formol, it was low. 6. 6. The refractive index of tissue sections was altered by washing them in water. The refractive index of the nuclear membrane fell, but that of brush borders and peritubular basement membrane rose. 7. 7. These findings are discussed and tentative explanations suggested.

Some deuterium peroxide containing less than 0.3% ordinary hydrogen was produced both by the persulphate and the electric discharge methods. By means of fractional distillation, solutions of deuterium peroxide in deuterium oxide of various concentrations up to 90% were obtained, of which the following properties were measured: refractive index at 25°C, density, viscosity and surface tension at 0°C. and 20°C. Measurements of the last two properties were also extended to solutions of ordinary hydrogen peroxide. The physical constants of the two isotopic peroxides are compared and discussed. The density, viscosity and surface tension of supercooled heavy water at 0°C. were also measured for the first time.

A description is given of methods and apparatus used for the refractometric study of stationary and non-stationary radical polymerization kinetics. Evaluation of these methods and apparatus by studying photochemical polymerization of styrene, methyl methacrylate and vinyl acetate showed that they can be applied to determine rate constants of elementary stages and activation energies.

New data are presented on volume-osmotic pressure relationships at equilibrium in human erythrocytes. The inadequacy of previous theoretical treatments of the subject is pointed out and a new theoretical approach is presented. The refractive index of the erythrocyte gives a direct measure of its solid concentration, and the volume is estimated as the reciprocal of concentration. The erythrocyte volume was found to be linearly related to the reciprocal of the osmotic pressure over a range from 300 m-osm. down to a lower limit of between 130 and 200 m-osm. (depending on the pH of the immersion medium). The gradient of the regression line was not, however, that expected from the simple Boyle-van’t Hoff law. The isotonic water content of the erythrocyte derived from the gradient on the basis of the simple Boyle-van’t Hoff Law was found to be less than the actual water content obtained by direct refractometry, i.e. the value of Ponder’s R was found to average 0.95. Below a point between 130 and 200 m-osm. it was found that the erythrocyte volume did not increase in proportion to the fall of osmotic pressure. This was attributed to incipient haemolysis. The isotonic haemoglobin concentration of erythrocytes was found to vary from 38.5% at pH 6.9 to 30.3% at pH 5.6 in the same subject. The thermodynamic basis of the Boyle-van’t Hoff law is examined. It is explained that a cell will obey the simple Boyle-van’t Hoff law only if the osmotic coefficients of all fractions of the cell solute do not change significantly within the range of intracellular concentrations produced in osmotic experiments. From the fact that the osmotic coefficient of haemoglobin changes rapidly with concentration, it is concluded that the erythrocyte will not obey the simple Boyle-van’t Hoff law; swelling in hypotonic solutions will be less than that predicted from the law. The degree of the discrepancy from the Boyle-van’t Hoff law is calculated from known osmotic coefficients of haemoglobin, and is shown to result in an expected value of Ponder’s R less than 1.0, and ranging from 0.925 to 0.975 depending on the corpuscular haemoglobin concentration. The observed values of R in different experiments are compared with the expected values and agreement is found within the limits of error of the experimental method.

The results of a preliminary study of the feasibility of using recording differential refractometry for total serum protein determination indicate that the method has some merit.

SUMMARY: Refractive index measurements were made on the spores and vegetative cells of strains of Bacillus cereus, B. cereus var. mycoides and B. megaterium by phase contrast and interference microscopy with protein immersion. The refractive indices of the spores were found to be very high and comparable with that of dehydrated protein, suggesting that they contained very little water. Those of the vegetative cells were much lower, and indicated a solid content of about 30%, w/v.

SUMMARY Quantitative microscopical methods are now of outstanding importance in the diagnosis and assessment of mammalian red blood cells. After release from the erythropoetic organs, all red blood cells in the peripheral blood of mammals pass through the reticulocyte stage before becoming mature erythrocytes. Therefore, the determination of reticulocyte number is used as an index of erythropoetic activity in clinical haematology. The corpuscular solid concentration, the corpuscular dry weight, the osmotic resistance and the size of red blood cells can be determined by use of microscopical methods. For the determination of the corpuscular solid concentration of erythrocytes, cell refractometry by the use of phase-contrast microscopy has proved to be a very sensitive and accurate method. The dry weight of single erythrocytes may likewise be obtained by the use of interference microscopy with relatively high accuracy. Both of these methods might also be of use in clinical haematology. In the experimental work described in this paper, both methods mentioned above are used to answer the question: whether and under what conditions the refractive index as a criterion of measurement of the solid concentration and the relative dry weight can be determined separately for reticulocytes and erythrocytes from the same blood sample by use of microscopical methods alone. The frequency distributions of refractive indices and relative dry weights determined on human reticulocytes and mature erythrocytes show significantly lower mean values for the reticulocyte generation in accordant with findings published by other authors and obtained by use of sometimes entirely different methods. The additional information gained by the determination of the frequency distribution of refractive index and dry weight of reticulocytes and mature erythrocytes separately in a blood sample and the necessity for simplifying and automating such methods for haematological diagnosis are discussed.

The experiments described have for their object the application of critical angle methods for the refractometry of liquids in the ultra-violet. A thin film of liquid can be held between two quartz hemispheres which are traversed centrally by an approximately parallel beam; the film receives the radiation at the varying angles of incidence resulting on rotation of the system. In this way, analysing the transmitted radiation with the aid of a quartz spectrograph, the critical angles for definite wave-lengths are measured, from which refractive indices can be calculated. The procedure necessary in seeking precise results is discussed, and a series of measurements on glycerine-water mixtures likely to be useful for immersion fluids in ultra-violet microscopy is given. A set of interesting phenomena of the extinction bands is described and explained.

Precise measurement of changes in air refractivity is provided by the high-frequency (h.f.) refractometer at relative humidities below 50%. Above this limit, the indicated refractivity may exceed the true refractivity by an amount that increases with humidity. The experimental techniques of gravimetry, ellipsometry, and photomicrography are combined with refractometer observations in a controlled environment to study the source of this anomaly. It is found that the anomaly is due to the added polarization of a layer that forms progressively upon the sensor surfaces, that the layer growth is insensitive to the h.f. electric field, and that the layer density lies between that of moist air and of bulk water while its depth increases to some 1500 Å near saturation humidity. At humidities above 90%, microscopic water droplets also form on the sensor surfaces. These observations are attributed to vapor adsorption and condensation on the sensor, with the latter related to surface asperities; the relative contribution of these two components in the layer structure depends upon the method by which the environment has been humidified.

Some applications of a viscosimetric detector in gel permeation chromatography In this paper a detector which permits direct measurement of the viscosity of the eluate, leading to a more quantitative use of gel permeation chromatography, is described. If the elution volume and the concentration of polymer (determined by refractometry) are known, it is possible, with the use of a gel permeation chromatography universal calibration curve, to determine the molecular weight of each fraction, thus giving rapid, complete and accurate characterization of an unknown polymer. This technique has been applied to: (1) the study of linear polymers and the relation between viscosity and molecular weight; (2) the characterization of branched polymers and the determination of the degree of branching as a function of molecular weight; (3) the study of copolymers of different composition. Here the composition of each fraction was determined with the aid of a third detector, allowing evaluation of polydispersity in weight as well as in composition of the copolymers.

The difficulties involved in the direct measurement, by eyepiece-micrometer or from photomicrographs, of small microscopic objects such as living bacteria are discussed. The accuracy with which this can be done is limited by the numerical aperture of the optical system and the wavelength of light used. With visible light it is scarcely possible to determine the dimensions of an object more accurately than to the nearest 0.4µ. It also seems probable, from the nature of the diffraction pattern at the edges of images of objects of circular cross-section such as bacteria, that direct measurement of the width of the image will tend to give an underestimate of the true width of the object. An interference microscope enables thickness measurements to be made that are not subject to these particular limitations, because with it, the phase-change in the light passing through the middle of a bacterium can be measured very accurately. This phase-change is proportional to the product of the refractive index of the bacterium minus that of the mounting medium, and its true thickness. Two methods were used to determine the mean thickness of the living bacilli in a number of different cultures of Lactobacillus bulgaricus. With the first, the mean refractive index of the bacilli was measured directly by the method of immersion refractometry first used by Barer and Ross (1952), and phase-change measurements were made on the bacilli mounted in dilute saline. Their mean thickness was calculated from these measurements. With the second method, phase-change measurements were made on the bacilli mounted in saline and also mounted in protein solutions with refractive indices ranging from 1.365 to 1.376; and, from these, both their mean thickness and their mean refractive index were calculated. The phase-change measurements were made with a Smith interference microscope and half-shade eyepiece (manufactured by Messrs. Charles Baker). The values for the mean thickness of the living L. bulgaricus from 14 different cultures obtained by the first method ranged from 1·13 µ to 1·23 µ; and those from 9 different cultures obtained by the second method ranged from 1·02 µ to 1·14 µ. The mean refractive indices of the latter calculated by the second method agreed very closely with that obtained by immersion refractometry, and differed by a maximum of 0.009 in all the cultures measured. It therefore seems unlikely that the mean thickness measurements obtained by either method are wrong by more than about ±0.1 µ.

CONTENTS I. Introduction?53 II. Origins of the refractometric method of physicochemical analysis and its development in the first half of the 20th century?53 III. The additivity of refractive indices?56 IV. Relationship between the deviations from additivity of refractive indices, densities, and specific refractions?58 V. Unique points on refractive-index diagrams?60 VI. Classification of refractive-index isotherms?62 VII. The use of refractometry for the study of interaction between components in binary liquid systems?63

Summary The present study has confirmed the reliability of total serum protein determinations as measured by the quick, simple technique of hand refractometry. The applicability of capillary blood samples for this purpose provides a means by which determination of total serum proteins becomes a rapid, bedside procedure, performed easily by either physician or nurse.

光学的方法によって行なわれた自由拡散の解析に際して, 不均一性にもとづく拡散曲線の変化については, 従来具体的定量的な知見にとぼしかった。著者は実験から得られた拡散曲線を正準座標に変換して, その頂点座標と成分の拡散定数や濃度との関係を2成分系, n成分系および対数分布関数系について理論的に考察し, 直接的な知見をうるに便利な関係を見いだした。とくに 2成分系について, その成分の拡散定数や濃度割合の変化にともなう正準拡散曲線の頂点座標の変化を明示し, その実質を明らかにした。またここで得られた諸関係を用いて 1) 塩化カリウムと庶糖, 2) 庶糖とグリシン, 3) ウシ血しょうアルブミンと庶糖の各2成分系についての実測値と理論値との比較から, 前述の考察の有用性を示した。

Swelling of LOEB-type membranes has been investigated using light-reflexion (for{qtλ} = 0.580 μ) differential refractometry and densitometry. These membranes are highly unsymmetrical and exhibit on the evaporated solvent side a face with a lower water content (38 per cent w/w) than the remaining (61.8 per cent). This zone is presumably made of a homogeneous non-porous gel. The water content increases continuously from the surface inwards, probably very quickly in the surface region, more slowly, but not negligeably, in the deeper regions.

In this paper the authors discuss the possibilities, for work of high precision, of the critical angle method of measuring the refractive index of a prism. For general work the method is inferior in convenience and accuracy to the method of minimum deviation, but by a suitable arrangement of the apparatus it can be used to determine refractive indices near 1.53 without the use of a spectrometer, and serves as a useful check on more general methods which depend on the accuracy of a spectrometer circle. In the second part of the paper the errors of setting in critical angle refractometry are discussed, and a simple method of determining the necessary correction is described.