Refractometry

REFRACTOMETRY (from Latin refringo - I refract), a method of measuring the refraction (see) of a light ray passing from one medium into another. When determining the refractive index with a refractometer, i.e., an instrument measuring refraction, air almost always serves as the first medium. The measurement of refraction by the refractometers described below is based on measuring the limiting angle of refraction. The largest angle of incidence α (Fig. 1) of a ray traveling in the first medium (the less dense one) is 90°—ray 4; it enters tangentially to the surface separating both media. In this case, the angle of refraction β < 90°; the denser medium is illuminated only from the perpendicular of incidence 1 to the angle of refraction corresponding to an angle of incidence of 90° (k); in Fig. 1, A, it is called the limiting angle of refraction. When the angle of incidence is increased beyond 90°, the ray travels in the second medium and is no longer refracted, but undergoes total internal reflection; in the field of view of the measuring instrument, a sharp line separating one half (A) from the other (B) is not obtained. Consequently, the limiting angle of refraction is at the same time the limiting angle of total

test medium

Figure 1

Figure 2. Abbe refractometer.

nor reflection. For each refractive medium, the limiting angle represents a strictly defined value. The angle β is determined as follows: sin 90° / sin β = N (N is the refraction in the second medium), sin β = 1/N. By measuring the angle β, the sought refractive index N is determined. When measuring with a refractometer, it is necessary to take into account the temperature and the source of the light used (its wavelength); usually, the measurement is referred to the D-sodium flame. Measuring instruments. In practice, two apparatuses are most common: the Abbe refractometer (Figure 2) and the immersion refractometer (Fig. 3). In the former, it is possible to determine the refractive index in the range of 1.30–1.70; it is also possible to determine it in viscous liquids and solid substances.

Pulfrich. Accuracy is up to 1–2 units in the 4th decimal place. The immersion refractometer allows measurement of n_D = 1.325–1.492; it is simpler and gives clearer results than the Abbe instrument; it is very good for physiological studies of liquids. Accuracy is ±3.7 units in the 5th decimal place. The path of light rays entering the refractometer is shown in Fig. 4. In the case of small quantities of liquid, auxiliary prisms (Fig. 3) are used, on the surface of which a few drops of liquid are placed. There is a series of variable auxiliary prisms with different refractive indices. To maintain a constant temperature, a special device with a heating coil is used; its construction is understandable

from Fig. 5 (the same device is used for other refractometers). The data obtained by reading the scale are converted using special tables. Abbe refractometer (Fig. 2). Daylight falling on the mirror passes through a double prism into a telescope connected to a sector on which divisions are marked. Arrows indicate the direction of the water flow maintaining a uniform temperature (see above). - The Wollny butter refractometer (Fig. 6) for determining the refractive index of solid and liquid oils is a simplified Abbe refractometer. It consists of a heated Abbe double prism and a telescope closely connected to it. The colored boundary line of the limiting angle of reflection formed in the field of view is achromatized for observing oils

by a special construction of the glass prism. The refractive index of liquid oils is usually determined at 25°; that of solid ones, predominantly at 40°; fats that solidify at a higher temperature are determined at the minimum temperature that keeps the fats in a liquid state; the observation result is then converted to determinations at 25° or 40°. - The refractometer for sugar and liquid oils (Fig. 7) is used for mass studies in production of all kinds of sugar juices of various concentrations and for liquid oils. To protect against external influences in production, the telescope, with the exception of the eyepiece, is enclosed in a case. With this refractometer, it is possible to determine the refractive index in the range from 1.330 to 1.540.

Pulfrich refractometer (Fig. 8). With the help of this instrument, it is possible 1) to determine the refraction and dispersion of liquid and solid substances at room temperature, 2) to measure at a higher temperature the refraction of both liquids and solid substances that become liquid at a higher temperature, 3) to measure the differences in refraction and dispersion of liquids and solid substances that differ little from each other in terms of their optical properties (use as a differential refractometer). Measurement accuracy is up to the 4th decimal place for the refractive index and ±1–2 in the fifth decimal place for dispersion and all difference measurements. The principle of determination is the same—measurement of the angle of total reflection. To determine dispersion, measurements are performed with a sodium flame and with the red, green, and blue lines of a hydrogen Geissler tube. Interference refractometer, see Interference. G. Yu. Gefter. The number of rays passing through a layer of liquid depends on its refractive properties, resp. on its refractive index. The degree of refraction in each liquid is determined by the quantity, size, and physical state of the particles dissolved in it. In biological fluids (serum, tissue juices, transudates and exudates), the determining factor for refraction is the amount of proteins as high-molecular substances. To salts and dissolved crystalloids belongs

Figure 5. Device for heating the refractometer.

Figure 6. Wollny butter refractometer with an open double prism.

a significantly smaller role. Thus, practically, the refractive index of serum, given the relative constancy of its salt composition, testifies quite accurately to the quantitative content of proteins in it. A necessary condition for refractometric studies is the constancy of the temperature of the liquid being studied during the entire time of the test. Technique. In physiology and pathology, for the determination of the refractive index in small quantities of liquid, the Pulfrich immersion refractometer (Figs. 3 and 4) has received the greatest application. The diagram shows how the rays that have passed through the liquid are collected by a prism and read with the help of an optical system Figure 7. Refractometer for sugar and liquid located inside the instrument

instrument. In the presence of a second "auxiliary prism" (Fig. 3), it is possible to determine the refraction in a liquid layer 1 mm thick, for which only one drop of serum is sufficient. This device allows for serial studies (5–6 or more in a row) within a short period of time. The test liquid, together with the lower part of the refractometer, is placed in a water bath with a constant temperature (usually 17.5°). Individual divisions of the scale visible in the eyepiece of the instrument correspond to the op

Figure 7. Refractometer for sugar and liquid located inside the instrument

Figure 8. Pulfrich refractometer (old model).

determined index of refraction. Reis compiled tables of protein content in plasma, serum, and other fluids based on their refractive index. These data are suitable for practical purposes, however they are not absolutely accurate, since the index of refraction depends not only on the quantity of proteins in percent, but also on the ratio of their individual fractions (globulins, albumins, fibrinogen) (see table). 78 Table for listing units of the immersion refractometer scale in % protein (at t°=17.5°). (According to Reis.) Serum * Ex- and transudates Index of re- fraction I Scale numbers Protein in % Scale numbers ¹ Protein: in % 1 1.31275 40 3.91 45 0.77 | 1.31313 4.16 0.97 1.31350 4.33 1.18 1.31388 4.60 1.58 . 1.34125 4.81 1.59 1.34163 5.03 1.80 1.34500 4; 5.25 2.0i 1.31537 5.17 2.21 1.31575 5.68 2.42 1.34612 5.90 2.62 1.31653 6.12 2.83 1.31587 6.31 3.01 1.34724 6.55 3.21 1.34751 6.77 3.45 1.31793 6.98 3.65 1.34835 7.20 3.85 1.34873 5> 7.12 4.07 1.34910 7.63 4> 4.27 1.31917 7.85 4.48 1.34981 53 8.06 4.68 1.35021 8.28 4.89 1.35058 8.49 5.10 1.35095 8.71 5.30 1.35132 8.92 5.50 1.35169 9.14 5.70 ' 1.35205 9.35 5.90 1.35212 9.57 5L 6.11 1.35279 9.78 6.31 1.35316 9.99 5! 6.51 1.35352 10.20 6.71 1.35388 10.41 6.91 * nD of dest. water = 1.33320; nD of non-protein. guest, elements = 0.00277; nD 1% protein = = 0.00172. Determination by means of a refractometer of the quantity of proteins due to its speed and comparative accuracy has found wide application. In particular in physiol. experiment and in various diseases (nephritis, nephroses, myxedema, edematous disease, heart decompensations, anemias, diabetes insipidus) it appears important to repeatedly study albumin-emia, for which a refractometer is used. The quantity of protein in serum is also a direct derivative reflection of its water content. Refractometry therefore became one of the most common methods for determining the degree of hydremia (see.) in the study of water exchange in the body. In normal conditions the refractometric index of serum fluctuates between 55 and 63 on the Pulfrich refractometer scale, which corresponds to protein content from 7.2 to 8.9%. An extremely low index (down to 44 and less) is observed mainly in true hypalbuminemias (for example in nephrotics) and in relative dilutions of blood (Congestive edema, abundant drinking). High protein content is mainly observed in blood thickenings (dry eating, diabetes insipidus), however it is sometimes also observed in nephritides. Refractometry can also be used for rapid recognition of exudates and transudates, as well as for determining proteolytic strength, resp. quantity of pepsin, trypsin-kinase, etc. (see also Blood-proteins).

m. vovei. Various food substances are also refractometrically investigated: milk with respect to fat, milk sugar, degree of dilution of milk with water; fats and oils, beer, malt extracts, etc. In technical ! chemistry oils, tar, petroleum, petroleum products, etc. are refractometrically investigated; | in the sugar industry methyl alcohol, ethyl alcohol.-In analytical laboratories-for precise determination of concentration of various aqueous solutions, for investigation of crystals. R. became a method in the study of the structure of organic compounds. By processing tissues according to the Spalteholz method, it is possible to determine the index of refraction in them. This makes it possible to study more finely histological relationships in normal tissues, as well as changes in various organs in pathological conditions.