Colorimetry

COLORIMETRY, COLORIMETERS (from Latin color - color and Greek metron - measure). Colorimetry is a method of quantitative analysis based on determining the amount of a substance by the intensity of the color inherent to the substance itself or to some product of a reaction occurring between the given substance and a specific reagent. Accordingly, one can distinguish between direct and indirect colorimetry. In practice, the latter is of the greatest importance, as direct colorimetry is applicable only when determining substances that possess a sufficiently intense inherent color. Such substances in technology are various dyes, and in biology, various pigments (chlorophyll, hemoglobin, bile pigments, etc.). In essence, colorimetry is close to spectrophotometry.

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In the latter case, the absolute quantity of light rays of a specific part of the spectrum absorbed during passage through a layer of the tested colored substance of a certain thickness is determined. If the absorption coefficient (Bunsen-Roscoe) for a given substance is known, the quantity of the latter can be calculated. In colorimetry, one does not use the measurement of absolute brightness or light absorption, but rather compares the light absorption by the tested solution with the absorption by a colored standard. The latter can be either a solution of the same substance of a known concentration or an artificially prepared standard (colored solution, glass, gelatin film, etc.). All methods of colorimetry are based on the premise that a specific relationship exists between the amount of colored substance and the intensity of the color. In a number of cases and within certain concentration limits, this relationship has the character of simple proportionality, i.e., when the concentration of the substance causing the color is doubled, the intensity of the color also increases twofold, and so on. For other cases, this condition does not hold, and regularities of a different order are observed. In the latter case, one can judge the amount of substance based on a comparison of solutions of different color intensity only by using a formula specifically established for the given case, expressing the relationship between color intensity and concentration. It is simpler and more reliable in such cases not to compare the color intensity of solutions of different concentrations, but to find the concentration of the standard solution that yields the same color intensity as the tested solution. The comparison is performed at a constant thickness of the liquid layer. In practice, this is carried out as follows: a series of test tubes of exactly the same diameter is chosen (this is established either by means of a gauge or by pouring the same volume of water into all test tubes and selecting those in which the water stands at the same level). In these test tubes, a series of dilutions of the standard solution of different concentrations is prepared with the calculation that the limits of the taken concentrations lie, on one side, higher, and on the other side, lower than the assumed concentration of the tested solution. A series of dilutions is prepared according to the principle of a geometric progression, i.e., so that the content of the standard substance from one test tube to another changes by a certain number (the ratio of the series), and not by the same amount. The provided table gives examples of such series. Ratio of the series: 3, 2, 1.5, 1.25. Number of members: 4, 5, 7, 11. Concentration limits: 9, 7, 1:27, 1:16, 1:11.4, 1:9.3, 1:0.1, 1:0.09. Series of dilutions: 1; 3; 9; 27; 1; 2; 4; 8; 16; 1; 1.5; 2.25; 3.38; 5.1; 7.6; 11.4; 1; 1.25; 1.56; 1.95; 2.44; 3.05; 3.81; 4.76; 5.95; 7.44; 9.3; 0.75; 0.56; 0.42; 0.32; 0.24; 0.18; 0.13; 0.1; 1; 0.67; 0.45; 0.3; 0.2; 0.13; 0.09. First, a series with a high ratio is set up, two test tubes are found between which the color of the tested solution lies, and for a more precise reading, a new series of dilutions is placed between these two test tubes, taking the concentrations of the solutions in these test tubes as the extreme members of the series and placing a series with a smaller ratio between them. Thus, the dilution whose color coincides as much as possible with the color of the tested liquid is found. The concentration of the sought substance in the latter will be equal to its concentration in the corresponding standard test tube. The volumes of liquid and the amount of reagent must be the same both in all standard test tubes and in the test tube with the tested solution. If the tested liquid is colored itself, then to exclude the influence of its own color, the principle of the Walpole compensation comparator is used. This comparator is a wooden block in which two (or three) pairs of vertical depressions and three through horizontal holes intersecting the paired vertical depressions are drilled (Fig. 1a and 1b). A test tube with the standard solution is placed in the 1st hole, the tested liquid in the 2nd, the tested liquid but without the addition of the color-inducing reagent (instead, the same volume of water is added) in the 3rd, and a test tube with water in the 4th. Thus, when viewed through the horizontal holes (Figure 1b), the color of the standard solution is summed with the inherent color of the liquid being examined, just as it occurs in test tube 2; the shades are equalized, and the influence of the inherent color is compensated. With uncolored liquids, when there is no need to use the Walpole principle, one can, instead of preparing a series of standard solutions of different concentrations, simply gradually increase the concentration of the standard solution by adding the latter to the reagent diluted with water.

Knowing the concentration of the standard solution, one can calculate the content of the sought substance using the formula x = n * (v_x / v_st), where x is the amount of the sought substance in the volume of liquid taken for determination, n is the amount of substance contained in the added volume of the standard solution, v_x is the final volume of liquid in the test tube with the tested solution, v_st is the same in the test tube with the standard solution. It is also possible to perform colorimetry in such a way that a reagent is added to the tested and standard solutions, and then water is added to the darker solution until the colors are equalized. Based on the ratio of the final volumes, the content of the substance in the tested solution is calculated. Both of the latter methods are not very reliable, because the intensity of the color is very often influenced by the method of mixing the reagents, and upon dilution, the color may change not only depending on the volumes but also as a result of secondary processes. Truly accurate results can be obtained by colorimetry only when using special instruments—colorimeters. The latter allow for the equalization of the color intensity of solutions of different concentrations by changing the thickness of the solution layer. For the majority (but by no means for all and not at all concentrations) of colored solutions, the rule known as "Beer's Law" is valid, according to which the color intensity of two solutions turns out to be identical when the products of the concentrations and the thickness of the liquid layer are equal, i.e., when C_1 * H_1 = C_2 * H_2, where C_1 is the concentration of one solution, H_1 is the thickness of its layer, and C_2 and H_2 are the same values for the second solution. Knowing the thickness of the layers and the concentration of one of the solutions (the standard), it is easy to find the concentration of the tested one using the formula C_x = C_st * (H_st / H_x), where C_x and H_x are the concentration and layer thickness of the unknown solution, and C_st and H_st are the same for the standard. The simplest colorimetric instrument built on this principle is the so-called Hehner cylinders (Fig. 2),

in which the change in the thickness of the liquid layer is achieved by simply draining part of the solutions through taps. The cylinders have a glass bottom; they are placed on a white backing, light passes from below, and the color of the liquids is compared by looking into the cylinders from above. An important condition, which extremely increases the accuracy of colorimetric studies, is bringing the compared colored fields of view as close as possible so that, upon equality of color, they would merge or be separated only by a very thin line. This is achieved by using various optical devices; some of them are schematically depicted in Fig. 4. Figure 2 depicts the Wolff colorimeter, based on the principle of Hehner cylinders. A double plate (Fig. 4a) is used in Autenrieth colorimeters.

Figure 1.

Figure 2. A and B - Hehner cylinders.

Figure 3. Diagram of the path of rays in the Bürker colorimeter: O-ocular; A-Hüfner-Albrecht body; Cm-vessel with standard solution; S-vessel with compensating liquid; T-test solution; H_%-compensating liquid; M-mirror. In earlier Duboscq colorimeters, Fresnel prisms were usually used (Fig. 46). In the newest models, the so-called Hüfner-Albrecht body (Fig. 4b) or Lummer-Brodhun body (Fig. 4c) are more often used. Observation is made through a suitable ocular. In addition to the Autenrieth and Duboscq colorimeters (see Autenrieth colorimeter and Duboscq colorimeter), a new modification of the latter by Bürker should be mentioned here, which combines the precision of a precision optical instrument with the advantages of Walpole's compensation principle. When using this colorimeter, neither the inherent color of the test liquid nor the color of the reagent interferes, moreover the path of the rays in both halves of the field of view is exactly symmetrical (Fig. 3). In 1928, the Leitz firm introduced universal colorimeters that allow the use of both the ordinary Duboscq principle and the Bürker principle, and are also adapted for comparing turbidities; their microattachments (Fig. 5) allow measurements to be made with only 1-2 cm³ of liquid. For particularly precise work, the Krüss polarizing colorimeter has a number of advantages, in which when the intensities of the color are unequal, the hue itself also changes, which significantly increases the accuracy of setting. Generally speaking, the accuracy of colorimetric measurements when using a good instrument can be very significant and lies within several tenths of a percent up to 0.5%. To achieve this accuracy, it is necessary to take a number of precautions. Both fields of view should be illuminated with exactly the same brightness; this is verified by placing the same liquid in both vessels of the colorimeter; in this case, the scale readings should be the same. For the greatest reliability of results, it is recommended to change the solutions places after the reading has been made; the ratios of the heights should remain the same. It is necessary to make a series of readings (at least five) and take their arithmetic mean. The smaller the difference in concentrations of the standard and test solutions, the higher the accuracy of the determinations; the ratio of concentrations should not exceed 1:2. In addition to uniformity, it is also necessary to maintain constant illumination intensity; therefore it is better to use an artificial source of light; new models have an illuminator with an electric bulb attached to the colorimeter stand (Fig. 6). It is best to use light of medium brightness; insufficient or too strong brightness reduces the accuracy of readings and causes rapid eye fatigue. During colorimetry, one should as much as possible protect the eye from stray light; therefore for precise work it is better to use a darkened room.

The accuracy of colorimetry increases if possible to exclude those rays of light that are weakly or not at all absorbed by the test solution, i.e., first of all, the rays corresponding to the color of the solution itself and therefore not stopped by it. This is achieved by using appropriate light filters (e.g., yellow for blue solution color and vice versa). This not only increases the accuracy of comparing the intensity of colors but also facilitates the setting, since along with the intensity the hue of the color also changes.

Figure 5.

The solutions to be compared should be as close as possible in temperature; the permissible difference should not exceed 3°. It is always necessary to preliminarily verify that for a given system and for given concentrations, Beer's law remains valid. Among the advantages of C., which quickly won this method wide practical application, in addition to simplicity and speed, along with sufficient accuracy for most practical purposes, belongs the extreme sensitivity of the method, which significantly expands the limits of measurement and allows one to get by with minimal amounts of substance, which is especially important in biological research, e.g., in blood analysis; on the other hand, when using colorimetric methods, it is possible to quantitatively determine such substances that are inaccessible to ordinary chemical methods for direct determination. Thus, it is possible to determine colorimetrically with an error of not more than 1-2% thousandths of a milligram of arsenic, nitrous acid, phosphorus, nitrogen, iodine, etc. These features of colorimetric methods have led to the widespread application of C. in medical and physiological laboratory techniques, so that at the present time for most substances important in clinical relations, simple, convenient and accurate colorimetric methods have been developed (as examples one can mention the determination of nitrogen by the Nessler method, the determination of uric acid, sugar, creatinine, phosphorus, cholesterol, bilirubin, lactic acid, etc.).

Methods for determining Hb are based on C. C. finds wide application in water analysis. A completely special field of application of C. is the measurement of active reaction (see Indicator method).