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When light interacts with a substance, a portion of the light is absorbed while the rest is either reflected or transmitted through it. Substances that we perceive as having a color reflect light in the visible range. The color of the substance that we are able to see depends on which wavelength of light is reflected. A substance that we perceive as blue reflects light in the blue range (430 – 480 nm) of the visible spectrum. According to the color wheel, the same substance absorbs light that is complementary to the reflected light. So, the blue substance absorbs light in the orange region (590 – 630 nm) of the visible spectrum. Not all compounds absorb in the visible region, and as a result, they appear as colorless to the human eye.
Light is defined by its energy, E, and its wavelength, λ. Here, h is Planck's constant, and c is the speed of light.
The wavelength of light is inversely proportional to its energy. So, higher energy light has a shorter wavelength.
Different colored dyes vary in the wavelength of light that they absorb. Most dyes are conjugated compounds with alternating double and single bonds and typically absorb light in the visible region.
The conjugated part of the dye molecule can be very short, meaning that there is a low degree of conjugation and few alternating double and single bonds, or long, meaning that there is a high degree of conjugation with many alternating double and single bonds. These alternating double bonds do not necessarily only have to be between two carbons. These conjugated bonds can include the carbonyl groups and the double bonds between carbon and oxygen. The degree of conjugation determines the wavelength of light the compound absorbs. For example, compounds with a high degree of conjugation absorb a longer wavelength than compounds with a lower degree of conjugation.
Based on molecular orbital theory, delocalized electrons occupy molecular orbitals. The highest occupied molecular orbital, or HOMO, is the highest energy orbital with an electron. The lowest unoccupied molecular orbital, or LUMO, is the lowest energy orbital with no electron. Molecules with little or no conjugation typically have a large energy gap between the HOMO and LUMO. However, conjugated molecules have a smaller energy gap between the HOMO and LUMO.
To excite an electron from a lower energy level to a higher energy level, or from the HOMO to LUMO, the molecule must absorb light with energy equal to the energy gap between the two orbitals. For this reason, molecules with a large energy gap require higher energy light, such as UV light, to excite an electron. Dyes, however, have a smaller energy gap and require lower energy light, such as visible light, to excite an electron.
For this reason, molecules with a large energy gap require higher energy light, such as UV light, to excite an electron. Dyes, however, have a smaller energy gap and require lower energy light, such as visible light, to excite an electron.
Recall that the energy of light is inversely proportional to the wavelength. So, higher energy light has shorter wavelengths than lower energy light that has longer wavelengths.
Experimentally, light absorbance is measured using a UV-Visible (UV-Vis) spectrophotometer. This instrument utilizes a light source that is transformed by a monochromator into specific wavelengths of light that will pass through a sample and into a detector at the other end. The samples must be in a liquid, so a solvent is required if the organic compound is a solid. This solution is held in a sample holder known as a cuvette. Depending on the sample, the cuvette may be made of quartz crystal, glass, or plastic, and has a particular pathlength. This pathlength is the distance the light must travel through the sample. Since the solvent will also absorb light, a sample blank of the solvent alone is required. Therefore, when the instrument captures the absorbance spectrum of the sample compound, it can subtract the background spectrum of the solvent to display absorbance caused by only the sample. Transmittance, T, is the fraction of the original light that passes through the sample.
Here, P0 is the irradiance, or the energy per second per unit area, of the light beam prior to striking the sample. P is the irradiance of the light beam striking the detector. P is typically lower than P0, as some of the light is absorbed by the sample.
Absorbance, A, is defined as the negative log of transmittance.
Absorbance has a value range between 0 (no absorption) and 2 (99% absorption). When no light is absorbed, P0 is equal to P, and the transmittance is equal to one. Thus, absorbance is zero. If 90% of the light is absorbed, then 10% is transmitted and T is equal to 0.1. This results in an absorbance equal to 1. If 99% of the light is absorbed, then 1% is transmitted (T= 0.01), and absorbance is equal to 2.
The spectrum obtained is a plot of the absorbance versus the wavelength. For a UV-Vis spectrophotometer, this range is between 200 and 800 nm.
Transmittance and absorbance of a particular compound is related to the concentration of the compound in solution. This relationship is described by the Beer-Lambert law.
The absorbance of the sample is equal to the product of the concentration of the compound, the path length, and the molar attenuation coefficient. This coefficient is unique to each compound and will vary by wavelength. However, if the wavelength is held constant, the molar attenuation coefficient will be the same irrespective of changes in concentration. The wavelength that corresponds to the highest absorbance of the sample, known as λmax, also will have the largest molar attenuation coefficient.