Colorimeter principle
When monochromatic light passes through a solution with the same thickness and a small concentration, according to the Lambert-Beer law, the degree to which light is absorbed by the solution, which is called absorption, is proportional to the concentration of the solution and is proportional to the thickness of the solution. A = εCL, where: A is the absorbance, C is the concentration of the solution, L is the thickness of the solution, and ε is the extinction coefficient.
According to the Lambert-Beer law, when a beam of monochromatic light passes through a solution, the intensity of the light is reduced because the solution absorbs a portion of the light energy. If the concentration (or thickness) of the solution is constant, the greater the thickness (concentration) of the solution, the more pronounced the weakening of the light intensity.
The standard solutions and test solutions prepared by the same method have the concentrations of C1 and C2, respectively, which are the same for the same solution ε, and when the thickness is also the same:
A1=εC1L
A2=εC2L
C2=(A2/A1)*C1
In the formula, A1 and A2 can be directly read by the colorimeter, and C1 is the known concentration of the standard solution, from which the concentration of the solution to be tested can be calculated.
Lambert-Beer law
The solution of many chemical substances has a color (colorless compounds can also add color reagents to react to form colored substances). When the solubility of a colored solution changes, the depth of color also changes. The greater the concentration, the darker the color. Therefore, the concentration of the colored solution can be determined by comparing the color of the solution. This method is called colorimetric analysis.
First, Lambert-Beer law
When a beam of monochromatic light passes through a colored solution, part of the incident light is reflected back by the vessel, part of it is absorbed by the solution, and the other part passes through the solution, as shown in the figure. They have the following relationship:
Io=Ia+Ir+It 1-1
Where: Io - incident light intensity, Ia - absorbed light intensity, Ir - reflected light intensity, It - transmitted light intensity
Because in actual measurement, the cuvettes used are all of the same specifications. The intensity of the reflected light is a certain value and does not cause measurement errors, so the influence of reflected light can be ignored. The above formula can be simplified as:
Io=Ia+It 1-2
It can be seen from Formula 1-2 that when the incident light intensity Io is constant, the larger the absorbed light intensity Ia is, the smaller the transmitted light intensity It is. In other words, the reduction of light intensity is only related to the absorption of light by a colored solution.
So, what are the factors related to the absorption of light by the solution? Experiments have shown that the greater the concentration C of the solution, the thicker the liquid layer thickness L (ie, the longer the path the light travels in the solution), the more light is absorbed by the solution. The relationship between them is determined by the following formula:
Lg = KCL 1-3
This formula is the Lambert-Beer law.
K in the formula is called the extinction coefficient and it represents the absorbance of the colored solution at unit concentration and unit thickness. In the condition of the wavelength of the incident light, the type of the solution and the temperature, K is a constant value. Absorptivity is one of the important characteristics of colored compounds and is of great significance in colorimetric analysis. The larger the K value, the stronger the light absorption ability of the substance, and the more significant the change in absorbance when the concentration is changed, the higher the sensitivity is in the colorimetric measurement.
Lambert-Beer law means that the absorption of a certain intensity of light by a colored solution is proportional to the product of the thickness of the liquid layer and the concentration of the colored material in the solution. Lambert's law states the relationship between light absorption and thickness; Beer's law states the relationship between light absorption and concentration.
Application of Lambert-Beer Law in Photoelectric Colorimeter
Assume that there are two colored solutions, one of which is a standard solution of known concentration and the other is a solution to be tested. According to the formula:
In the standard solution: As=KsCsLs 1-4
In the solution to be tested: Ax=kxCxLx 1-5
Dividing Formula 1-4 by Formula 1-5 yields:
= 1-6
If the above two solutions have the same liquid layer thickness and the same temperature and two different concentrations of the same substance, the wavelength of the monochromatic light selected for the measurement is also the same:
Ls = Lx, Ks = Kx, substituting 1-6 can be obtained:
= 1-7
It can be seen that the absorbance is proportional to the concentration under the above conditions. This relationship is the design basis of the photoelectric colorimeter and one of the basic calculation formulas of colorimetric analysis. The concentration Cs of the standard solution in the formula is known, As and Ax can be measured by the photoelectric colorimeter, then the concentration Cx of the solution to be tested can be obtained:
Cx = × Cs 1-8
Since in the actual measurement, the standard solution and the solution to be tested are diluted, and when the results are reported, they are usually expressed as 100 ml (or 1000 ml). Therefore, in the actual calculation, it is necessary to multiply the dilution factor in the above formula.
The method for finding the concentration of the test solution is: direct comparison method (calculation method), factor method and standard curve method. These methods are described in the "Biochemical and Biochemical Test Technology" course.
Wavelength selection:
Since the colored solution is selective for light absorption, the color filter must be selected for colorimetric determination, otherwise the sensitivity is low, resulting in inaccurate measurement results. The general principle of filter selection is that the maximum light transmitted by the filter should be the maximum absorbed light of the solution. From the color point of view, the color of the filter and the color of the solution to be tested should be "complementary colors."
What is the complementary color? When two colors are added together to obtain white color, the two colors are called "complementary colors." The two colors directly opposite to each other in the figure are complementary colors.
Why should the color of the filter be a complementary color to the solution to be tested when choosing a filter? This is because the filter and the colored solution have similar light-transmitting characteristics, and the same colored light as the self-colored light can be transmitted to the maximum. The colors that are complementary to their own colors can be maximally absorbed.
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