COLORIMETRY (Latin color color + Greek metreo to measure, measure) — the physical and chemical method of definition of intensity of coloring of solution of the analyzed substance based on visual or photo-electric measurements. The method is widely applied in a wedge., biochemical, and chemical researches for definition of concentration of various substances in solutions.
The majority unified a wedge, laboratory biochemical, methods of a research have photocolorimetric end (about - a toluidine method of definition of sugars, a glyukozooksidazny method of definition of glucose in blood, etc.). Colorimetric methods are exact and a little labor-consuming. All automated a wedge, biochemical, methods are colorimetric or spektrofotometrichesky. Photocolorimetry is widely applied at control of production of medicines in pharm, the industries, in control and analytical laboratories, century a San gigabyte. laboratories. To. use also during the determination of the size pH of solutions two-color indicators in the presence of buffer solution (or without it); in photocolorimetry use one-color indicators without buffer solution. Definition of various substances in solution by means of staining reactions was applied long ago; for the first time doctors began to do it. Analyses of mineral waters began to be manufactured by colorimetric methods in Russia since the beginning of 18 century. They were carried out by hl. obr. doctors and druggists, using juice of plants as reactants.
V. M. Severgin developed a number of colorimetric methods of the analysis of mineral waters and expanded number of the elements determined by method K.
With the help To. define either own coloring of the analyzed substance, or coloring of reaction product. Colorimetric it is possible to define from 10 - 3 to 10 - 8 mol/l. The photo cell «sees» a part of the UF-range and is applied in «ultra-violet colorimetry». An eye of the person is very sensitive to shades of flowers, but perceives only the small site of a range; besides, people have individual distinctions in such sensitivity. Use of a photo cell eliminates these defects of an eye. Absorption (absorption) of light by the painted solution in some cases submits to Buger's law — Lambert — Beera, on Krom the amount of the absorbed light depends on thickness of a layer (length of an optical way) and concentration of the painted solution (i.e. concentration of the absorbing substance). The optical density of solution is D = lg (I 0 / I), where I 0 — intensity of the light flow entering solution, I — intensity of the leaving light flow weakened by light absorption in solution. If thickness of a layer of solution b, then lg (I 0 / I) = k*b, where k — a constant. At a constant thickness of a layer of solution D = k 1 * C, where C — concentration, k 1 — constant equal (k/2,303). Combining two equations, we receive
D = lg (I 0 / I) = k 1 * b*C.
If b = 1 cm, C = 1 mol/l, then D = k 1 . Constant k 1 call a molar extinction coefficient and designate grech, a letter ε. The molar extinction coefficient depends on chemical composition, a structure and a condition of substance and on the wavelength of light passing through solution. Buger's law — Lambert — Beera is fair only for monochromatic light, i.e. a flow of light, length of waves to-rogo (λ) is identical. Size ε for various connections changes from 10 1 to 10 5 . The more size ε, the is more sensitive a method.
The relation of intensity of a monochromatic flow of the radiation which passed through the studied solution to intensity of an initial flow of the account is called transparency or a transmission of solution and designated a letter T. Size T is expressed usually as a percentage: T = 100*I 0 / I (%).
The absorption of solution designated by a letter A is expressed also as a percentage: And = 100 (I 0 - I)/I (%).
V K. it is necessary to use monochromatic light. Monochromatization is reached by use of the light filters representing the painted environments passing a light flux only with a certain wavelength however often use the light filters allocating narrow spectral ranges. The light filter passes light, additional to color of solution, i.e. corresponding to that spectral range, edges is absorbed by the analyzed solution. Light filters produce from stained glasses. Earlier used colorimeters — the colorimeter of Dyubosk and maple Autenrit's colorimeter — had no light filters that reduced the accuracy of measurements. Light absorption by solutions of many painted substances does not submit to Buger's law — Lambert — Beera; in these cases build empirical standard curves (calibration schedules).
Distinguish visual and photo-electric To. At a visual method of standard series (a method of a scale) use a set of test tubes of identical colorless glass and diameter. In test tubes pour the amounts of standard solution of the defined substance increasing in a geometrical progression and bring it to the same volume water or other suitable liquid (e.g., ethanol). The scale of colourings from the brightest to the weakest turns out. It is possible to prepare series of long-lasting standard cultivations. The analyzed solution of unknown concentration is compared to a scale of standards on intensity of coloring and find the next to it a shade. Concentration of substance thus can be defined with an accuracy of ±5%.
At a method of dilution coloring of the analyzed solution is brought to coloring of standard solution, diluting more intensively painted studied solution before coincidence to coloring of the less intensively painted standard solution, concentration of the studied substance in Krom is known. Coloring of solutions is compared visually in Valpol's comparator.
Valpol's comparator represents a box, shaped a rectangular parallelepiped, with six nests for test tubes (fig). In front and back walls there are round openings. Openings of a back wall are covered with ground glass for receiving a homogeneous background. In an average nest of the second row put a test tube with the studied solution, in two extreme — test tubes with the corresponding standard solutions; changing an average test tube, find the standard solution on coloring matching (or the closest) with the studied solution. Sometimes the examinee the solution painted more intensively is diluted with water or other solvent until its coloring is not compared to coloring of a standard sample. For measurement of volume it is convenient to use test tubes with divisions of identical diameter. Concentration is calculated by
a formula C isp = With St * V isp / V St
where With isp — concentration of the examinee of solution, With St — concentration of standard solution, V ISP — volume of the examinee of solution, V CT — volume of standard solution. The method is more exact, than previous.
The method of equalizing consists in equalizing of height of columns standard and the examinee of solutions. Heights of these solutions are equalized by moving of pogruzhatel to special ditches of the concentration colorimeter KOL-1M which is supplied a set of light filters and a lamp lighter. Concentration of the examinee of solution is calculated, how at a method of dilution.
At photo-electric To. use photocolorimeters of Federal Energy Regulatory Commission-M, Federal Energy Regulatory Commission-N-57, Federal Energy Regulatory Commission-56, Federal Energy Regulatory Commission-60. Measurement is based on equalizing of the light flows passing through the control and studied solutions and falling on photo cells. Photocolorimeters are highly sensitive, exact and objective (see. Photometry ).
Conditions of the photo-electric analysis allow to apply also extraction and photometric method when organic solvent extract only reaction product, and the painted reactant remains in an aqueous phase, without preventing definition.
For increase in accuracy of the analysis, for definition of big concentration (in pharm, the analysis), elimination of the disturbing components and influence of light absorption of a reactant apply a differential method. In this case optical density of the analyzed and standard solutions measure not in relation to pure solvent with zero absorption, and in relation to the painted solution of the analyzed substance with concentration of C0 close to concentration Sisp. Photocolorimetric titration is carried out on the FET-UNIZ device. As Kyuveta serves the glass with the magnetic stirrer and the burette over it. The flow of light penetrates contents of a glass horizontally and gets on a photo cell. Photocurrent is registered a galvanometer.
Bibliography: Grandma A. K. and Pilipenno A. T. Photometric analysis, M., 1974; Damask steels M. And. and Kalin-kin I. P. Practical guidance on photocolorimetric and to spectrophotometric methods of the analysis, L., 1972; Korenman I. M. Photometric analysis, Methods of definition of organic compounds, M., 1975.
F. M. Shemyakin.