JoVE Lab Manual
Lab: Chemistry
Lab: Chemistry
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染料的紫外可见光谱
When light reaches a substance, a portion is absorbed by it, while the rest is either reflected or transmitted through it. The color of the substance, as we perceive it, depends on which wavelengths it is most likely to reflect. For example, a piece of fabric that we see as blue contains a dye that strongly reflects blue light and strongly absorbs orange and red light.
Dyes are typically conjugated compounds, meaning that they have alternating double and single bonds. Electrons can move freely within the conjugated system. Differently colored dyes must vary in the wavelengths of light that they absorb. When we look at a few examples, we see that the absorbed wavelength increases with the amount of conjugation.
So, how is wavelength related to the degree of conjugation? Let's consider molecular energy levels. We can think of delocalized electrons as occupying molecular orbitals, or MOs. A molecule absorbs light with the exact energy needed to excite an electron to a higher energy molecular orbital. The most likely transition is from the highest occupied molecular orbital, called the HOMO, to the lowest unoccupied molecular orbital, or LUMO. So, we expect that the most absorbed wavelength matches the HOMO - LUMO energy gap.
Molecules with little or no conjugation typically have a large HOMO - LUMO gap. They absorb UV light and reflect all visible light, so they appear white or colorless. Conjugated bonds stabilize molecules by lowering their energy levels, particularly at high energies. The higher the degree of conjugation, the smaller the HOMO - LUMO gap and the larger the absorbed wavelength. Metals and substitutions also affect the gap.
Let's look at an example. Retinol has a small conjugated system, while chlorophyll a has a large system with nitrogen and magnesium. Retinol absorbs at 325 nm, while chlorophyll a absorbs at both 430 and 662 nm. As expected, retinol's energy gap is larger.
We can study absorption using a UV and visible light, or UV-Vis spectrophotometer. A spectrophotometer consists of a light source, a way to control the wavelengths the sample receives, and a light detector. The sample is typically a transparent solution. Absorbance can be measured at a specific wavelength or measured over a wavelength range since compounds often absorb at more than one wavelength. Additionally, we see a range of wavelengths for each transition because the molecules are in different orientations and vibrational states.
During the measurement, the light either is absorbed, passes through without contacting any molecules, or bounces off a solvent or compound molecule. We ignore the small amount of light that bounces backward. Sometimes, light that could be absorbed by a molecule bounces off it instead. We describe how well a substance transmits a specific wavelength with a unique molar attenuation coefficient. While absorbance changes with concentration, the molar attenuation coefficient does not.
After the measurement, the spectrophotometer compares the received and original light in a ratio called transmittance. The absorbance is the negative base 10 logarithm of transmittance. If the spectrophotometer has the solvent’s absorbance, it subtracts it to show only the compound. The results are usually displayed as absorbance versus wavelength. The wavelength at which the compound absorbs the most is called lambda max. If we calculated the molar attenuation coefficient for each wavelength, it would be highest at lambda max.
The molar attenuation coefficient, the absorbance, the sample concentration, and the path length, which is the distance the light traveled through the sample, are related by the Beer-Lambert law. If we know any three variables, we can calculate the fourth.
In this lab, you will analyze the absorption characteristics of fluorescein, beta carotene, and indigo dye using a UV-Vis spectrophotometer. You'll then use the Beer-Lambert law to create a β-carotene calibration curve and then determine the concentration of the β-carotene solution.
染料的紫外可见光谱
Absorbance
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.
Dyes
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.
Spectrophotometer
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.
Beer-Lambert Law
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.
References
- Silberberg, M.S. (2012). Chemistry: The Molecular Nature of Matter and Change. Boston, MA: McGraw Hill.
- Harris, D.C. (2015). Quantitative Chemical Analysis. New York, NY: W.H. Freeman and Company.
