From Light to Data: The Technology of Colorimetry and Spectrophotometry

What is colorimetry:
Colorimetry is simply the measurement of color. Colorimetry is the determination of the concentration of a substance by measuring the relative absorption of light relative to a known concentration of the substance. In visual colorimetry, natural or artificial white light is usually used as the light source, and measurements are usually made using a simple instrument called a colorimeter or color comparator. When photocells are used instead of eyes, the instrument is called a photoelectric colorimeter.

Colorimetric analysis is based on the principle that many substances react with each other and form a color that indicates the concentration of the substance being measured. When a substance is exposed to a beam of intensity (I 0 ), a portion of the radiation is absorbed by the molecules of the substance, and radiation of intensity (I) is emitted. This difference in intensity is used in colorimetric assays.

The amount of absorbed radiation is given by the Beer-Lambert law:
A = Ɛ•l•C
A is absorbance
Ɛ is the molar extinction coefficient [L/(mol cm)]
l is the path length (cm)
C is the concentration (mol/liter)

What is Spectrophotometry:
Spectrophotometry is a method for qualitative and quantitative analysis of the substance by measuring the absorbance of the substance at a specific wavelength or within a certain wavelength range.

Basic principles of spectrophotometry:
Matter interacts with light and has the characteristic of selective absorption. The color of a colored substance is produced by the substance interacting with light. That is, the color presented by the colored solution is due to the selective absorption of light by the substances in the solution.

Because different substances have different molecular structures, they have different absorption capabilities for different wavelengths of light. Therefore, structural groups with characteristic structures have the maximum actual wavelength for selective absorption characteristics, forming the maximum absorption peak, and producing a unique absorption spectrum.

Even the same substance absorbs light differently due to its different content. The method of using the unique absorption spectrum of a substance to identify the existence of a substance (qualitative analysis), or using the degree of absorption of a certain wavelength of light by a substance to measure the content of a substance (quantitative analysis) is called spectrophotometry.

The Lambert-Beer law is the basic principle on which the quantitative analysis of spectrophotometry is based. When a beam of monochromatic light (light of one color) passes through a uniform solution, part of it is absorbed and part of it is transmitted. Let the intensity of incident light be I0, and the intensity of transmitted light be I, then I/I0 is the transmittance, represented by T. Percent transmittance T% = (I/I0)x100%

Studies have shown that the degree of absorption of light by the solution, that is, absorbance (A) (also known as extinction E, or optical density D) and transmittance (T) has a negative logarithmic relationship, that is: A = – lgT

Lambert’s Law: The degree of light absorption (A) of a colored solution is proportional to the thickness (light path) b of its liquid layer. When the concentration of the solution is constant, the greater the thickness of the liquid layer of the solution, the greater the A value of the degree of light absorption, and the smaller the light transmittance.
That is: A = ab

Beer’s Law: The degree of light absorption (A) of a colored solution is proportional to its concentration (number of light-absorbing particles) C. That is, when the thickness of the liquid layer of the solution is constant, the greater the concentration of the solution, the greater the degree of absorption of light, and the smaller the transmittance.
That is: A = ac
Combining the above two formulas can be expressed in the following formula: A = -lgT=abc
That is, A = abc.

The above formula is Lambert-Beer’s law, which means: when a beam of monochromatic light passes through a uniform solution, the absorbance of the solution to the monochromatic light is proportional to the product of the solution concentration and the thickness of the liquid layer. [2-3]

In A =abc, the absorption coefficient a represents the sensitivity of the light-absorbing substance. The larger the value of a, the higher the sensitivity. If the unit of concentration is the concentration of the substance (unit: mol/L), the absorptivity a can be written as ε, and ε is called the molar absorptivity.

It can be seen from the above formula that when the thickness b of the solution layer and the absorption coefficient a are fixed, the absorbance A is linearly related to the concentration of the solution. In quantitative analysis, it is first necessary to measure the absorption of different wavelengths of light by the solution (absorption spectrum), from which the maximum absorption wavelength is determined, and then the light of this wavelength is used as the light source (monochromatic light) to measure the absorbance A, which is Lang Burbeer’s law is the first condition for quantitative analysis.

Benchtop Spectrophotometer (Transmittance) DSCD-910 is good performance and specially designed for testing the transparent material’s transmittance, absorbance, chromaticity value and other parameters.

DSCD-910_Benchtop Spectrophotometer (Transmittance)