The purpose of the work is to determine the concentration of substances by the colorimetric method.

I. Terms and definitions

Standard solution (sr) is a solution containing a certain amount of the test substance or its chemical-analytical equivalent per unit volume (GOST 12.1.016 - 79).

The test solution (ir) - this is a solution in which it is necessary to determine the content of the test substance or its chemical-analytical equivalent (GOST 12.1.016 - 79).

Calibration curve- graphical expression of the dependence of the optical density of the signal on the concentration of the test substance (GOST 12.1.016 - 79).

Maximum Permissible Concentration (MPC) harmful substance - this is the concentration that, with daily (except weekends) work for 8 hours or with other working hours, but not more than 40 hours a week during the entire working experience, cannot cause diseases or deviations in the state of health detected by modern research methods, in the process of work or in the long-term life of the present or subsequent generations (GOST 12.1.016 - 79).

Colorimetry - This is a method of quantitative analysis of the content of any ion in a transparent solution, based on measuring the intensity of its color.

II. Theoretical part

The colorimetric method of analysis is based on the relationship of two quantities: the concentration of the solution and its optical density (degree of color).

The color of the solution can be caused both by the presence of the ion itself (MnO 4 -, Cr 2 O 7 2- ), and the formation of a colored compound as a result of the chemical interaction of the ion under study with the reagent.

For example, a slightly colored ion Fe 3 + gives a blood-red compound when interacting with thiocyanate ions SCH - , copper ion Cu 2+ forms a bright blue complex ion 2 + when interacting with an aqueous solution of ammonia.

The color of the solution is due to the selective absorption of light rays of a certain wavelength: the colored solution absorbs those rays whose wavelength corresponds to the complementary color. For example: additional colors are called blue-green and red, blue and yellow.

An iron thiocyanate solution appears red because it predominantly absorbs green light (  5000Á) and misses the reds; on the contrary, a green solution transmits green rays and absorbs red ones.

The colorimetric method of analysis is based on the ability of colored solutions to absorb light in the wavelength range from ultraviolet to infrared. Absorption depends on the properties of the substance and its concentration. With this method of analysis, the substance under study is part of an aqueous solution that absorbs light, and its amount is determined by the light flux that has passed through the solution. These measurements are carried out using photocolorimeters. The action of these devices is based on the change in the intensity of the light flux when passing through the solution, depending on the thickness of the layer, the degree of color and concentration. The measure of concentration is optical density (D). The higher the concentration of a substance in a solution, the greater the optical density of the solution and the lower its light transmission. The optical density of a colored solution is directly proportional to the concentration of the substance in the solution. It should be measured at the wavelength at which the test substance has maximum light absorption. This is achieved by the selection of light filters and cuvettes for the solution.

Preliminary selection of cuvettes is carried out visually according to the color intensity of the solution. If the solution is intensely colored (dark), use cuvettes with a small working wavelength. In the case of weakly colored solutions, cuvettes with a longer wavelength are recommended. A solution is poured into a pre-selected cuvette, its optical density is measured, including a light filter in the path of the rays. When measuring a number of solutions, the cuvette is filled with a solution of medium concentration. If the obtained value of optical density is approximately 0.3-0.5, this cuvette is chosen to work with this solution. If the optical density is greater than 0.5-0.6, a cuvette with a shorter working length is taken; if the optical density is less than 0.2-0.3, a cuvette with a longer working wavelength is selected.

The accuracy of measurements is greatly affected by the cleanliness of the working faces of the cuvettes. During work cuvettes are taken by hand only for non-working edges, and after filling with solution carefully monitor the absence of even the smallest air bubbles on the walls of the cuvettes.

According to the law Bouguer-Lambert-Baer, the fraction of absorbed light depends on the thickness of the solution layer h, solution concentration C and the intensity of the incident light I 0

where I - the intensity of light passing through the analyzed solution;

I is the intensity of the incident light;

h is the thickness of the solution layer;

C is the concentration of the solution;

The absorption coefficient is a constant value for a given colored compound.

Taking the logarithm of this expression, we get:

(2)

where D is the optical density of the solution, is a constant value for each substance.

Optical density D characterizes the ability of a solution to absorb light.

If the solution does not absorb light at all, then D = 0 and I t =I, since expression (2) is equal to zero.

If the solution absorbs light rays completely, then D is equal to infinity and I= 0, since expression (2) is equal to infinity.

If the solution absorbs 90% of the incident light, then D = 1 and

I t =0.1, since expression (2) is equal to one.

With accurate colorimetric calculations, the change in optical density should not go beyond the range of 0.1 - 1.

For two solutions of different layer thicknesses and concentrations, but the same optical density, we can write:

D \u003d h 1 C 1 \u003d h 2 C 2,

For two solutions of the same thickness but different concentrations, we can write:

D 1 \u003d h 1 C 1 and D 2 \u003d h 2 C 2,

As can be seen from expressions (3) and (4), in practice, to determine the concentration of a solution by the colorimetric method, it is necessary to have a standard solution, that is, a solution with known parameters (C, D).

The definition can be done in different ways:

1. It is possible to equalize the optical densities of the studied and standard solutions by changing their concentration or the thickness of the solution layer;

2. It is possible to measure the optical density of these solutions and calculate the desired concentration using expression (4).

To implement the first method, special devices are used - colorimeters. They are based on a visual estimate of the intensity of the transmitted light and therefore their accuracy is relatively low.

The second method - optical density measurements - is carried out using much more accurate instruments - photocolorimeters and spectrophotometers, and it is he who is used in this laboratory work.

When working on a photocolorimeter, the method of constructing a calibration graph is more often used: the optical density of several standard solutions is measured and a graph is plotted in the coordinates D = f(C). Then the optical density of the test solution is measured and the desired concentration is determined from the calibration curve.

The equation Bouguer - Lambert - Baer valid only for monochromatic light, therefore, accurate colorimetric measurements are carried out using light filters - colored plates that transmit light rays in a certain wavelength range. For work, a light filter is selected that provides the maximum optical density of the solution. Light filters installed on the photocolorimeter transmit rays not of a strictly defined wavelength, but in a certain limited range. As a result, the measurement error on the photocolorimeter is no more than ±3 % by weight of the analyte. Strictly monochromatic light is used in special devices - spectrophotometers, in which the measurement accuracy is higher.

The accuracy of colorimetric measurements depends on the concentration of the solution, the presence of impurities, temperature, acidity of the medium of the solution, and the time of determination. This method can only analyze dilute solutions, that is, those for which the dependence D = f(C)-straight.

When analyzing concentrated solutions, they are preliminarily diluted, and when calculating the desired concentration, a correction is made for dilution. However, the accuracy of the measurements decreases in this case.

Impurities can affect the accuracy of measurements by the fact that they themselves give a colored compound with the added reagent or hinder the formation of a colored compound of the ion under study.

The method of colorimetric analysis is currently used for analysis in various fields of science. It allows accurate and fast measurements using negligible amounts of a substance, insufficient for volumetric or gravimetric analysis.

For determination, a reference solution of the analyte of known concentration is prepared, which approaches the concentration of the test solution. Determine the optical density of this solution at a certain wavelength. Then determine the optical density of the test solution at the same wavelength and at the same layer thickness. For the reference solution according to equation (17) we have:

where is the molar absorption coefficient of the test solution; - layer thickness, cm.

The optical density of the test solution is expressed by the same formula:

where is the concentration of the test solution, .

The amount of the analyte (in mg), taking into account the dilution of the solution, is found by the formula:

where is the total volume of the test solution, ; is the volume of the colored test solution, is the volume of an aliquot of the test solution taken to prepare the colored solution, .

Determination of the concentration of a substance in a solution by the value of the molar absorption coefficient

Having determined the value of the optical density of the solution at a wavelength k and knowing the value of the molar absorption coefficient . of the substance to be determined for rays of wavelength X, we find by formula (17) the value of the concentration of the substance under study:

The amount of the analyte (in g) is found by the formula:

where is the molecular (atomic) weight of the substance (ion) being determined.

The value of the molar absorption coefficient. set as follows. Prepare a reference solution of the test substance of a certain concentration and measure the value of the optical density of this solution at a wavelength k and value . calculated by the formula:

If the substance is difficult to obtain in its pure form, then you can use the table value.

Determining the concentration of a substance using a calibration curve

The functional relationship between the optical density of the solution and the concentration of the absorbing substance can be established graphically. To do this, a series of solutions of the analyte of various concentrations (reference solutions) are preliminarily prepared. Measure the values ​​of the optical density of these solutions for rays with a wavelength of X, and according to the data obtained, build a curve of dependence of the optical density of the solution on the concentration (calibration graph). The values ​​of the optical density of the reference solutions are plotted on the ordinate axis, and the corresponding values ​​of the concentrations of these solutions () are plotted on the abscissa axis. To obtain more accurate results, calculate, using the least squares method, the equation for the calibration curve.

Having determined the value of the optical density of the test solution at the same layer thickness, it is possible to find the concentration of the analyte using the obtained calibration curve. If the solution does not obey the Bouguer-Lambert-Beer law, then the straight-line dependence is violated on some part of the curve or on the entire curve. In this case, it is necessary to increase the number of standard solutions. The concentration of standard solutions is usually expressed in . The amount of analyte in milligrams is determined by formula (23).

Determination of the concentration of a substance by the "equalization" method or by changing the thickness of the absorbing layer

The optical density of the test solution is determined by the formula:

where is the molar absorption coefficient of the test solution; - concentration of the analyte, ; - layer thickness, cm.

The device of the immersion colorimeter (Dubosque colorimeter) is based on the use of this equality, in which color identity is achieved by changing the thickness of the solution layer. The optical scheme of the immersion colorimeter is shown in Fig. 96. One light flux from mirror 1 passes through the layer of the test solution in cuvette 2, cylinder 4, prism 6, lenses 8 and 9 and enters the eyepiece, illuminating the right half of the optical field. Another light flux passes through the standard solution layer in cell 3, cylinder 5, prism 7, lenses 8 and 9, enters the eyepiece, illuminating the left half of the optical field. Cuvettes 2 and 3 are mounted on holders, which move vertically with the help of gears and racks. Glass cylinders 4 and 5 with polished ends are fixed. By moving the cuvettes 2 and 3 vertically, the height of the solution columns is changed and the interfaces in the eyepiece of the optical field disappear. The heights of the columns of the reference solution and the test solution are counted on a millimeter scale.

Optical density

D, a measure of the opacity of a layer of matter to light rays. Equal to the base 10 logarithm of the radiant flux ratio (See radiant flux) F 0 incident on the layer to a stream weakened as a result of absorption and scattering F passing through this layer: D=lg( F 0 /F), otherwise, O. p. is the logarithm of the reciprocal of the Transmission coefficient of the substance layer: D= lg(1/τ). (The decimal logarithm lg is replaced by the natural logarithm logarithm logarithm logarithm lg, which is sometimes used.) The concept of a natural limit was introduced by R. Bunsen; it is used to characterize the attenuation of optical radiation (light) in layers and films of various substances (dyes, solutions, colored and milky glasses, and many others), in light filters and other optical products. Densitometry is especially widely used for the quantitative evaluation of developed photographic layers in both black-and-white and color photography, where methods for measuring it form the content of a separate discipline, densitometry. There are several types of optical radiation, depending on the nature of the incident radiation and the method of measuring the transmitted fluxes of radiation ( rice. ).

The O.P. depends on the set of frequencies ν (wavelengths λ) that characterizes the initial flow; its value for the limiting case of one single ν is called monochromatic op. rice. , a) the monochromatic O. p. of a layer of a non-scattering medium (without taking into account corrections for reflection from the front and rear boundaries of the layer) is 0.4343 k ν l, where k ν - natural absorption index of the environment, l- layer thickness ( k ν l= κ cl- indicator in the equation of Bouguer - Lambert - Beer law a; if scattering in the medium cannot be neglected, kν is replaced by the natural Weakening index). For a mixture of non-reacting substances or a set of media arranged one after the other, the OD of this type is additive, i.e., it is equal to the sum of the same OD of individual substances or individual media, respectively. The same is true for regular nonmonochromatic optical radiation (radiation of a complex spectral composition) in the case of media with nonselective absorption (independent of ν). Regular non-monochromatic The opp of a set of media with selective absorption is less than the sum of the opp of these media. (For devices for measuring O. p., see the articles Densitometer, Microphotometer, Spectrozonal aerial photography, Spectrosensitometer, Spectrophotometer, Photometer.)

Lit.: Gorohovsky Yu. N., Levenberg T. M., General sensitometry. Theory and practice, M., 1963; James T., Higgins J., Fundamentals of the Theory of the Photographic Process, trans. from English, M., 1954.

L. N. Kaporsky.

Types of optical density of the medium layer depending on the geometry of the incident and the method of measuring the transmitted radiation flux (in the sensitometric system adopted in the USSR): , which retained the original direction; b) to determine the integral optical density D ε, a parallel flow is directed perpendicular to the layer, the entire past flow is measured; c) and d) two measurement methods used to determine two types of diffuse optical density D ≠ (incident flux - ideally scattered). The difference D II - D ε serves as a measure of light scattering in the measured layer.

Great Soviet Encyclopedia. - M.: Soviet Encyclopedia. 1969-1978 .

Optical density D, a measure of the opacity of a layer of matter to light rays. Equal to the decimal logarithm of the ratio radiation flux F 0 incident on the layer to a stream weakened as a result of absorption and scattering F passing through this layer: D=lg( F 0 /F), otherwise, the O. p. is the logarithm of the reciprocal of transmission coefficient material layer: D= lg(1/t). (The decimal logarithm lg is replaced by the natural logarithm logarithm logarithm lg, which is sometimes used.) Bunsen ; it is used to characterize the attenuation optical radiation (light) in layers and films of various substances (dyes, solutions, colored and milk glasses, etc.), in light filters and other optical products. OP is especially widely used for the quantitative evaluation of developed photographic layers in both black-and-white and color photography, where methods for measuring it form the content of a separate discipline - densitometry . There are several types of optical radiation, depending on the nature of the incident radiation and the method of measuring the transmitted fluxes of radiation ( rice. ).

The O.P. depends on the set of frequencies n (wavelengths l) characterizing the initial flow; its value for the limiting case of one single n is called the monochromatic O. p. Regular ( rice. , a) the monochromatic O. p. of a layer of a non-scattering medium (without taking into account corrections for reflection from the front and rear boundaries of the layer) is 0.4343 k n l, where k n - natural absorption rate environment, l- layer thickness ( k n l= k cl- indicator in the equation Booger - Lambert - Bera Law ; if scattering in the medium cannot be neglected, k n is replaced with natural weakening indicator ). For a mixture of non-reacting substances or a set of media arranged one after the other, the OD of this type is additive, i.e., it is equal to the sum of the same OD of individual substances or individual media, respectively. The same is true for regular nonmonochromatic optical radiation (radiation of a complex spectral composition) in the case of media with nonselective (independent of n) absorption. Regular non-monochromatic The opp of a set of media with selective absorption is less than the sum of the opp of these media. (On devices for measuring O. p., see the articles Densitometer , Microphotometer , Spectrozonal aerial photography , Spectrosensitometer , Spectrophotometer , Photometer .)

Lit.: Gorohovsky Yu. N., Levenberg T. M., General sensitometry. Theory and practice, M., 1963; James T., Higgins J., Fundamentals of the Theory of the Photographic Process, trans. from English, M., 1954.

Great Soviet Encyclopedia M.: "Soviet Encyclopedia", 1969-1978

Colorimetry

Of the optical methods of analysis in the practice of analytical laboratories, colorimetric methods are most widely used (from lat. color- color and Greek. μετρεω - I measure). Colorimetric methods are based on measuring the intensity of the light flux passing through a colored solution.

In the colorimetric method, chemical reactions are used, accompanied by a change in the color of the analyzed solution. By measuring the light absorption of such a colored solution, or by comparing the color obtained with that of a solution of known concentration, the content of the colored substance in the test solution is determined.

There is a relationship between the color intensity of the solution and the content of the colored substance in this solution. This dependence, called the basic law of light absorption (or the Bouguer-Lambert-Beer law), is expressed by the equation:

I = I 0 10 - ε c l

where I is the intensity of light passing through the solution; I 0 - the intensity of the light incident on the solution; ε is the coefficient of light absorption, a constant value for each colored substance, depending on its nature; C is the molar concentration of the colored substance in the solution; l is the thickness of the light-absorbing solution layer, see

The physical meaning of this law can be expressed as follows. Solutions of the same colored substance at the same concentration of this substance and the thickness of the solution layer absorb an equal amount of light energy, i.e., the light absorption of such solutions is the same.

For a colored solution enclosed in a glass cuvette with parallel walls, it can be said that as the concentration and thickness of the solution layer increase, its color increases, and the intensity of light I transmitted through the absorbing solution decreases compared to the intensity of the incident light I 0 .

Fig.1 Passage of light through a cuvette with a test solution.

The optical density of the solution.

If we take the logarithm of the equation of the basic law of light absorption and reverse the signs, then the equation becomes:

The value is a very important characteristic of the colored solution; it is called the optical density of the solution and is denoted by the letter A:

A = ε C l

It follows from this equation that the optical density of the solution is directly proportional to the concentration of the colored substance and the thickness of the solution layer.

In other words, with the same layer thickness of a solution of a given substance, the optical density of this solution will be the greater, the more it contains a colored substance. Or, conversely, at the same concentration of a given colored substance, the optical density of the solution depends only on the thickness of its layer. From this, the following conclusion can be drawn: if two solutions of the same colored substance have different concentrations, the same color intensity of these solutions will be achieved with their layer thicknesses inversely proportional to the concentrations of the solutions. This conclusion is very important, since some methods of colorimetric analysis are based on it.

Thus, in order to determine the concentration (C) of a colored solution, it is necessary to measure its optical density (A). To measure the optical density, the intensity of the luminous flux should be measured.

The color intensity of solutions can be measured by various methods. There are subjective (or visual) methods of colorimetry and objective (or photocolorimetric).

Visual methods are such methods in which the assessment of the color intensity of the test solution is done with the naked eye.

With objective methods of colorimetric determination, photocells are used instead of direct observation to measure the color intensity of the test solution. The determination in this case is carried out in special devices - photocolorimeters, from which the method was called photocolorimetric.

Visual Methods

Visual methods include:

1) standard series method;

2) duplication method (colorimetric titration);

3) adjustment method.

Standard series method. When performing analysis by the standard series method, the color intensity of the analyzed colored solution is compared with the colors of a series of specially prepared standard solutions (with the same thickness of the absorbing layer).

Solutions in colorimetry usually have an intense color, so it is possible to determine very small concentrations or amounts of substances. However, this may be accompanied by certain difficulties: in this way, samples for preparing a series of standard solutions can be very small. To overcome these difficulties, standard solution A is prepared at a sufficiently high concentration, for example 1 mg/ml. After that, by dilution from solution A, a standard solution B of a much lower concentration is prepared, and from this, in turn, a series of standard solutions is prepared.

To do this, the required volumes of reagent solutions in the required sequence are added to test tubes or cuvettes of the same size and the same color of glass with a pipette. It is advisable to add portions of solutions of the analyte from the burette, because their volumes will be different to provide different concentrations in a series of standard solutions. In this case, the initial solution must contain all components, except for the analyte. (zero solution). Solutions of the necessary reagents are added to the test solution. All solutions are brought to a constant volume, and then the color intensity of the test solution is visually compared with the solutions of a series of standard solutions. It is possible to match the color intensity with any solution of the series. Then it is considered that one hundred test solution has the same concentration or contains the same amount of the analyte. If the color intensity seems to be intermediate between neighboring solutions of the series, the concentration or content of the analyte is considered the arithmetic mean between the solutions of the series.

Colorimetric titration (duplication method). This method is based on comparing the color of the analyzed solution with the color of another solution. - control. To prepare a control solution, prepare a solution containing all components of the test solution, with the exception of the analyte, and all the reagents used in the preparation of the sample, and add the standard solution of the analyte from the burette to it. When so much of this solution is added that the color intensities of the control and analyzed solutions are equal, it is considered that the analyzed solution contains the same amount of the analyte as it was introduced into the control solution.

Equalization method. This method is based on equalizing the colors of the analyzed solution and a solution with a known concentration of the analyte - a standard solution. There are two options for performing a colorimetric determination by this method.

According to the first option, the equalization of the colors of two solutions with different concentrations of the colored substance is carried out by changing the thickness of the layers of these solutions at the same strength of the light flux passing through the solutions. In this case, despite the difference in the concentrations of the analyzed and standard solutions, the intensity of the light flux passing through both layers of these solutions will be the same. The ratio between the thicknesses of the layers and the concentrations of the colored substance in the solutions at the time of equalization of the colors will be expressed by the equation:

l 1= C2

where l 1 is the thickness of the solution layer with the concentration of the colored substance C 1 , and l 2 is the thickness of the solution layer with the concentration of the colored substance C 2 .

At the moment of equality of colors, the ratio of the thicknesses of the layers of the two compared solutions is inversely proportional to the ratio of their concentrations.

Based on the above equation, by measuring the thickness of the layers of two identically colored solutions and knowing the concentration of one of these solutions, one can easily calculate the unknown concentration of the colored substance in the other solution.

To measure the thickness of the layer through which the light flux passes, glass cylinders or test tubes can be used, and for more accurate determinations, special devices - colorimeters.

According to the second option, to equalize the colors of two solutions with different concentrations of a colored substance, light fluxes of different intensity are passed through layers of solutions of the same thickness.

In this case, both solutions have the same color when the ratio of the logarithms of the intensities of the incident light fluxes is equal to the ratio of the concentrations.

At the moment of achieving the same color of the two compared solutions, with an equal thickness of their layers, the concentrations of the solutions are directly proportional to the logarithms of the intensities of the light incident on them.

According to the second option, the determination can be performed only with a colorimeter.