UV-Vis Spectroscopy of Dyes

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.