UV-Vis Spectroscopy of Dyes

Concepts Instructor Prep Student Protocol

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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.

Print Procedure Steps

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Source: Lara Al Hariri and Ahmed Basabrain at the University of Massachusetts Amherst, MA, USA

Absorbance Wavelength Comparison
In the first part of the lab, you'll use ultraviolet and visible light absorption spectroscopy, or UV-Vis absorption spectroscopy, to analyze the absorption characteristics of fluorescein, β-carotene, and indigo dye. To perform UV-Vis spectroscopy, you'll place a solution between a light source and a photodetector. The molecules will absorb light at wavelengths that correspond to the energies needed to excite their electrons and scatter or transmit the rest.

An absorption spectrum represents the variation in the number of photons of each wavelength that reach the detector. A higher absorbance corresponds to fewer detected photons of that wavelength. You'll take a spectrum of the pure solvent first, which the instrument will subtract from the spectrum of the dye solution to show you just the absorbance data from the dye. This reference is called a solvent blank.
- Before you start, put on a lab coat, safety glasses, and gloves. Note: The β-carotene and indigo solutions use hexane and dimethylformamide, respectively, so you will work with the dyes in a fume hood. DMF and hexane will degrade thin nitrile gloves, so change your gloves if you get solvent on them.
- Now, obtain a clean quartz cuvette. Cuvettes typically have two transparent sides and two textured or opaque sides. Always hold the cuvette by the textured sides to keep the transparent sides clean.
- First, you'll analyze a solution of fluorescein in water. To make your solvent blank, fill your cuvette about 3/4 full with deionized water.
- Now, clean the transparent sides of the cuvette with a laboratory wipe. Check the cuvette for air bubbles and gently tap the cuvette to dislodge them.
- Then, bring the cuvette to the spectrometer. Open the sample chamber of the spectrometer and clean your cuvette one last time. Smudges and dust can absorb or reflect light, which will introduce errors into the data.
- Now, insert the cuvette into the sample holder so that the transparent sides are in line with the light source. Close the sample chamber door completely. Follow the instructions for your spectrometer software to set up a scan of absorbance from 200 – 800 nm and scan your cuvette as the solvent blank.
- When the scan finishes, empty the cuvette and use compressed air to remove large drops of water.
- Then, bring it to the shared hood and use a clean Pasteur pipette to fill the cuvette 3/4 full of the provided 3 µM fluorescein solution.
- Check the cuvette for air bubbles and remove them as needed before bringing the cuvette to the spectrometer. Remember to clean the transparent sides one last time before placing the cuvette in the sample chamber. Then, scan the cuvette as your sample.
- When the scan finishes, identify the wavelengths that fluorescein absorbed. The wavelength with the highest absorbance is called λ_max.
- Record the wavelengths of the absorbance peaks and the overall absorption range in your lab notebook and then save the data.
  Table 1: Absorbance and λ_max of dyes
  
  Absorbance range (nm) λ_max (nm)
  Fluorescein
  β-carotene
  Indigo
  Click Here to download Table 1
- Now, empty your cuvette into the aqueous waste and rinse it with water to remove traces of fluorescein.
- You'll analyze β-carotene next, so you need a hexane solvent blank. Rinse the cuvette with hexane a few times to remove leftover water before filling it 3/4 full of pure hexane.
- Remove air bubbles, clean the sides of the cuvette, and place it in the spectrometer. Make sure that the scan is set for absorbance from 200 – 800 nm, and then scan the cuvette as a solvent blank.
- When the scan finishes, empty the cuvette into the organic waste and dry it with compressed air.
- Use a clean pipette to fill the cuvette 3/4 full of the provided 3 µM β-carotene solution and return to the spectrometer.
- Clean the transparent sides of the cuvette, place it in the spectrometer, and scan it as your sample. Write the wavelengths of the peaks in your lab notebook and save the data.
- Now, analyze the indigo dye. The indigo solution is in DMF, so empty your cuvette into the organic waste and rinse out the leftover β-carotene and hexane with pure DMF.
- Fill the cuvette 3/4 full of pure DMF, place it in the spectrometer, and make sure that the scan settings are the same as before. Run the solvent blank scan, and then empty the cuvette into the organic waste and dry it with compressed air.
- Use a clean pipette to fill the cuvette 3/4 full of the provided 3 µM indigo dye solution and scan it as your sample. Write down the absorbance information and save your data.
- Lastly, empty your cuvette into the organic waste and rinse it with acetone.
β-Carotene Calibration Curve
In the second half of the lab, you'll use UV-Vis spectroscopy to relate absorbance intensity and concentration for β-carotene by measuring the absorbances of 5 β-carotene solutions with different concentrations. This will let you make a calibration curve of absorbance versus β-carotene concentration and derive the equation describing that relationship.
- To begin, make a table in your lab notebook with columns for β-carotene concentration and absorbance at 450 nm.
  Table 2: Absorbance of β-carotene
  
  Test Concentration (µM) Absorbance at λ_max (450 nm)
  1 1.9
  2 3.7
  3 7.5
  4 11
  5 15
  Click Here to download Table 2
- Then, get a clean cuvette and fill it 3/4 full of hexane as your solvent blank.
- Remove any air bubbles, clean the transparent sides of the cuvette, and place it in the spectrometer. Set up an absorbance scan from 200 – 800 nm and scan the cuvette as the solvent blank.
- When the scan finishes, empty the cuvette into the organic waste and dry it with compressed air.
- Then, use a clean pipette to fill the cuvette 3/4 full of the 1.9 µM β-carotene solution, which is the lowest concentration solution.
- Clean the sides of the cuvette, insert it into the spectrometer, and scan it as a sample.
- When the scan finishes, display the absorbance intensity of the peak at 450 nm. Record this in your lab notebook as the absorbance for the 1.9 µM solution.
- Now, empty the cuvette into the organic waste, rinse it with hexane, and dry it with compressed air.
- Use a clean pipette to fill the cuvette 3/4 full of the 3.7 µM β-carotene solution. Clean the sides of the cuvette, insert it into the spectrometer, and scan it as another sample.
- When the scan finishes, write down the absorbance of the peak at 450 nm. Repeat this for the remaining solutions, working from lowest to highest concentration. Save your data when you're done.
- Lastly, empty the cuvette into the organic waste and clean it with acetone.
Results
First, look at the spectra of the three dyes. Fluorescein in water absorbs blue and purple light, with the maximum absorbance at 490 nm. It does not absorb red light, and it only absorbs some yellow and green light. Consistent with this, solid fluorescein is red, and fluorescein solutions are usually yellow to green.

β-carotene also absorbs blue and purple light. The maximum absorbance of β-carotene in hexane is 450 nm, and you'll see another large peak at 478 nm. The strong absorption of purple light is part of why β-carotene appears orange.

Indigo in DMF absorbs UV light and red, orange, and yellow light, with a distinct peak at 611 nm. Thus, indigo dye reflects primarily blue and purple light, giving it its characteristic color.
- Now, make the calibration curve for β-carotene concentration. Plot the β-carotene absorbance values at 450 nm from the second half of the lab against the concentrations of the solutions.
- Then, apply a trend line and find the linear equation that fits the data. According to the Beer-Lambert law, in this equation, y is absorbance, x is concentration, and the slope is the product of the relevant molar attenuation coefficient and the pathlength.
- Check your equation. Rearrange it to solve for concentration, and fill in the β-carotene absorbance at 450 nm from the first part of the lab. The calculated concentration should be close to 3 µM/L.