SPEKTROFOTOMETRIYA — the area of the measuring equipment developing methods and devices for definition of spectral characteristics of objects. In medicobiological researches the greatest value has the analysis of molecular and atomic absorption spectrums (see. Spectral analysis , Spectroscopy ). Define by S. in various biol. tests the content of enzymes, hormones, proteins, vitamins, many inorganic matters, analyze qualitative and quantitative structure of blood smears etc. The Page
is the cornerstone registration of extent of weakening of a monochromatic beam of light at its passing through substance (Buger's law — Lambert — Beera: lg (I 0 / I) = E*c*d, where I 0 and I — intensity of light respectively before its passing through a layer of substance; with — concentration of the substance absorbing light; d — thickness of a layer; E — the coefficient depending on the wavelength of incident light and the nature of substance). The relation of intensity of last light and intensity falling (T = 1/10) call a transmission. Usually this size is expressed percentage of intensity of incident light. In practice also the size of a logarithm of a transmission taken with the return sign is often used (D = - lgT = E*c*d) — the so-called optical density, size a cut is linearly connected with concentration of substance. If size with is expressed in moths on liter, d — in centimeters, then E call a molar absorbtion coefficient or a molar extinction coefficient. It characterizes probability of absorption by substance of electromagnetic energy and the equal 1 mol on 1 l is in number equal to the optical density of solution (D) 1 cm thick and with concentration of substance.
Buger's law — Lambert — Beera is fair only for a plane-parallel bunch of monochromatic light and during the performance of a number of conditions. In practice often it is necessary to face deviations from this law. Can be carried to number of reasons for rejection physical. - chemical properties of the analyzed substance or all solution (dissociation, fluorescence, etc.)» tool factors (e.g., lack of due degree of monochromaticity of a beam of light), the factors caused by heterogeneity of a body of interest in a beam of light (especially clearly it is shown at a mikrospektrofotometriya of objects) etc.
The important principle C. is the principle of additivity (i.e. summations) optical density, according to the Crimea the size of optical density of mix of the connections submitting to Buger's law — Lambert — Beera and not entering chemical interaction with each other, is equal to the sum of optical density of these connections.
Quantification of the test containing one substance includes the following operations: 1) registration of a full range of absorption of substance (D measure as function of wavelength λ), the choice of analytical wavelength (λ anat ); 2) preparation of 6 — 7 reference (standard) solutions covering all expected range of concentration of the defined substance in the analyzed solutions and measurement of optical density (D St ) these solutions at λ anat . Then build the schedule of dependence of size of optical density of D St from concentration of reference solution. The most exact results are yielded by measurements in the range of D within 0,05 — 1,50. If the constructed dependence, i.e. D St = f (with St ), it is linear (the defined substance submits to Buger's law — Lambert — Beera), definition of concentration of substance is carried out analytically, using a formula with = with St * (D/D St ). At non-compliance with the law of Buger — Lambert — Beer quantification will be seen off a graphical method (by means of the calibration schedule). The optical density of the analyzed solution is measured in all cases concerning solution of comparison (a direct spectrophotometric method). As solution of comparison both pure solvent, and the solution containing all components of the analyzed solution except for the defined substance can be used. In some cases as solution of comparison it is more reasonable to use solution of the defined substance of the known concentration (usually lower, than in the analyzed tests). During the carrying out such measurements speak about differential S.
Neredko carry out registration of derivative absorption spectrums. Derivative S. is applied generally in qualitative analysis for the purpose of identification of strongly blocked spectrum lines, during the definition of exact provision of a maximum of indistinct strips of absorption, identification of malopogloshchayu-shchy impurity, establishment of structure of organic compounds etc.
S. carries out in infrared (personal computer), UF and seen spectral ranges. Also spectrophotometers of combinational dispersion belong to the devices working in the visible range (KR-spectrophotometers). By means of S. of combinational dispersion study vibrational energy levels of molecules (see. Spectral analysis , Molecule ), to-rye can be used for definition of number S — S-bonds in proteins, numbers of the coupled and not coupled bases in nucleinic to-takh, abundance alpha and beta spiralizovannykh sites in polypeptides, etc.
by S. of microobjects, or a mikrospektrofotometriya — rather independent area of a research (see. Microspectral analysis ). Microspectrophotometers, as well as mikrospektroflyuorimetra, are usually intended for work in the visible range of a range, is more rare in the UF-area and are carried out according to one beam scheme. For registration of distribution of the absorbing substance on the area of a microobject use different scanners: system of optical probes, subject little tables moving according to the set program, television (electronic) development of the image and so forth. Devices sovr. designs are supplied with the microcomputers which are automatically processing the obtained information and allowing to analyze a form of microobjects (e.g., in the automatic analysis of blood smears), etc. In flowing mikroflyuorimetra the high-speed analysis of fluorescent characteristics of the individual blood cells painted by the corresponding dyes is carried out. The tendency to development of the highly specialized microspectrophotometers and mikrospektroflyuorimetr intended for work in conditions a wedge, laboratories was outlined.
Atomic absorption spectrums study by means of flame spectrophotometers (see. Photometry ). Atomic and absorbing methods give the chance of definition practically of all elements of a periodic system and differ in sharp selectivity and sensitivity (to 10 - 14 г).
Devices for a spectral analysis are completed with electronic processing devices and managements, blocks of the automatic delivery of tests, recorders, blocks of the digital-press, devices of automatic development of a range, allow to define several elements in one test.
The page is widely applied in biol. researches. It is used for quantitative definition of the most various biol. connections: enzymes, vitamins, hormones, proteins and other nitrogenous substances, nucleinic to - t, carbohydrates, alcohols, aldehydes, phenols, ketones, organic to - t, lipids, pigments, a number of inorganic matters (e.g., sodium, potassium, calcium, iron, zinc, chlorine, sulfur), etc.
the Main nodes of spectrophotometers usually are: source of radiation; the monochromator intended for allocation from a radiation spectrum of a source of narrow spectral intervals; receiver of radiation; otschetny device. Spectrophotometers are subdivided on one-beam and dual-beam, not registering and registering. The widespread in medical laboratories one-beam not registering spectrophotometers represent rather simple and cheap devices with optics from optical glass or quartz. Use of quartz optics gives the chance to take measurements in the area 200 — 1100 nanometers, covering ultra-violet, visible and near infrared sites of a range. The studied sample and a standard consistently enter into a light bunch. Results of comparison of bunches (in %) or optical density (D) are specified in sizes of a transmission on the arrow or digital device.
Optical scheme of spectrophotometers most often autocollimation. Usually the hydrogen lamp (for work in a spectral range of 220 — 320 nanometers) or a filament lamp is a light source (for work in a spectral range of 320 — 1100 nanometers). The ray of light through system of mirrors gets on the dispersing prism, edges decomposes it, forming a range. Rotating a prism, it is possible to receive light of waves of various length at the exit of the monochromator; then beams pass a standard (sample) and get on registrar — a photosensitive layer of a photo cell. Assessment is made or by means of the calibration schedule, or on the basis of creation of functional dependence between sizes of optical density and wavelength.
Spectrophotometers Federation Council-4, Federation Council-4A, SFD-2, the Federation Council-16, the Federation Council-26 belong to the one-beam not registering spectrophotometers. In particular, the spectrophotometer Federation Council-26 has expanded in ultra-violet area (to 186 nanometers) the spectral range, high-res in an ultra-violet spectral range and provides the best convergence of results of measurement of coefficients of a transmission in comparison with analogs. Range of measurement of coefficient of a transmission from 0 to 100%. The main error of graduation of a scale of waves in ultra-violet area of 0,1 nanometers, in the visible range of 0,5 nanometers, in near infrared area of 5,0 nanometers.
In the dual-beam registering spectrophotometers the flow from a source of radiation is divided into two bunches: the main and bunch of comparison (reference). In the main bunch the studied sample, in a bunch of comparison — a standard is established. At measurements of coefficient of a transmission of substances in solution two identical ditches, one of usually use to-rykh another is filled with the studied sample, and a standard.
Principle of operation of two beam devices (fig.) it is based on a zero method. The receiver of radiation is lit serially modulated (by means of mirror sector disks — modulators) with the beam of light which passed through the studied sample (or reflected from it), and a bunch of comparison. If light flows generally and reference channels are equal, then illumination of the receiver of radiation will be constant at any moment and the signal of alternating current on an entrance of intensifying system will not be. At absorption by a sample of a part of a light flow the total light flux on the receiver (costs at the exit of the scanning monochromator) will change with a frequency of modulation, on an entrance of the amplifier the signal of the same frequency will appear. Tension of a signal amplifies and moves on the servomotor, setting in motion the device weakening a bunch of comparison, working until the difference of light flows disappears i.e. will not disappear a signal on an entrance of the amplifier yet. Weakening of a bunch of comparison is carried out by turn of the polarizing prism which is in a bunch or introduction to it of the compensating optical wedge. Along with the movement of the compensating device the tracer according to the form moves. The automatic turn of the disperser (most often prisms) allows to register these sizes continuously.
Devices of a visible band of a range of the Federation Council-8, the Federation Council-10, the Federation Council-14, the Federation Council-18 and infrared spectrophotometers one beam IKS-12, IKS-21, and also dual-beam IKS-14, IKS-22, IKS-29, IKS-31 belong to the registering spectrophotometers. Infrared spectrophotometers with replaceable prisms allow to measure coefficient of a transmission of substances in a spectral range of 1 — 25 micron and further. They provide automatic recording of a range with high precision and resolving power. However at a research medical - biol. objects infrared spectrophotometers are used restrictedly because of strong absorption of IK-radiation by the water which is the main component of living tissue.
Bibliography: Agroskin L. S. and Papayan G. V. Cytophotometry, the Equipment and methods of the analysis of cells on light absorption, L., 1977; Asatiani B.C. New methods of biochemical photometry, M., 1965; Grandmother's A. A., etc. Methods of a spectral analysis, M., 1962; Berstein I. Ya. and Kamensk Yu. L. A spectrophotometric analysis in organic chemistry, L., 1975; Introduction to quantitative cytochemistry, the lane with English, under the editorship of V. Ya. Brodsky and N. I. Polyakov, M., 1969; 3 and y-del A. N., Ostrovskaya G. V. and Ostrovsky Yu. I. Equipment and practice of spectroscopy, M., 1976, bibliogr.; P e y with and x with about I. V. Optik's N of spectral devices, L., 1975.
R. R. Lidemang; I. M. Arefyev (tekhn.).