Unit 13: Ultraviolet-Visible (UV-Vis) Spectroscopy
Analyzing colored compounds and quantifying their concentration by measuring light absorption.
13.11 Basic Principles (Electronic Transitions, Chromophores)
Ultraviolet-Visible (UV-Vis) spectroscopy is an analytical technique that measures how much light a chemical substance absorbs. It works by passing a beam of UV or visible light through a sample and detecting the amount of light that passes through it.
The energy from UV or visible light is sufficient to cause electronic transitions, meaning it excites valence electrons from a lower energy ground state to a higher energy excited state. A molecule will only absorb light if the energy of the photon corresponds exactly to the energy difference between these electronic states.
Chromophores
Simple single bonds like C-C and C-H require very high-energy UV light to undergo electronic transitions and are not typically studied with this technique. The parts of a molecule that absorb light in the UV-Vis range are called chromophores. These are typically systems with pi ($\pi$) electrons, such as:
- Double and triple bonds (e.g., C=C, C=O, C≡C)
- Conjugated systems (alternating single and double bonds, e.g., beta-carotene)
- Aromatic rings (e.g., benzene)
- Transition metal ions with partially filled d-orbitals
The more extensive the conjugated system in a molecule, the longer the wavelength (lower the energy) of light it absorbs. If a molecule absorbs light in the visible region of the spectrum (400-700 nm), it will appear colored to our eyes. For example, beta-carotene, the molecule that makes carrots orange, has a very long conjugated system and absorbs blue-green light, reflecting orange-red light.
Solved Examples:
- What type of energy transition is measured in UV-Vis
spectroscopy?
Solution: Electronic transitions of valence electrons. - What is a chromophore?
Solution: The part of a molecule responsible for absorbing light in the UV or visible region, typically containing $\pi$ bonds. - Why is a solution of potassium permanganate ($KMnO_4$) intensely
purple?
Solution: The permanganate ion ($MnO_4^-$) is a transition metal complex that strongly absorbs light in the green-yellow region of the visible spectrum, causing it to transmit and appear purple. - Would you expect ethane ($CH_3-CH_3$) or ethene ($CH_2=CH_2$) to absorb UV
light at a longer wavelength?
Solution: Ethene. It contains a C=C double bond (a chromophore), while ethane only has C-C and C-H single bonds, which require much higher energy (shorter wavelength) UV light to excite. - Why are alkanes generally colorless?
Solution: They lack a chromophore. The energy required to cause electronic transitions in their C-C and C-H sigma bonds is very high, falling in the far-UV region, so they do not absorb visible light. - What is a conjugated system?
Solution: A system of connected p-orbitals with delocalized electrons in a molecule, which in practice means a series of alternating single and double/triple bonds. - A compound appears yellow. What color of light is it
absorbing?
Solution: It is absorbing the complementary color, which is violet/blue light (approximately 400-480 nm). - What is the relationship between the extent of conjugation in a molecule and
the wavelength of light it absorbs?
Solution: As the extent of conjugation increases, the energy gap between the ground and excited states decreases. This means the molecule absorbs light of lower energy, which corresponds to a longer wavelength. - What is an electronic transition?
Solution: The promotion of an electron from a lower energy molecular orbital to a higher energy molecular orbital by the absorption of a photon. - Would a solution of sodium chloride (NaCl) show any absorption in the
visible spectrum?
Solution: No. It is a colorless solution because the ions do not have any electronic transitions that correspond to the energy of visible light.
13.12 Beer-Lambert Law & Quantitative Analysis
While UV-Vis spectroscopy is used for some qualitative analysis, its main strength is in quantitative analysis. The relationship between the amount of light absorbed and the concentration of the substance is described by the Beer-Lambert Law.
The law states that the absorbance of a solution is directly proportional to the concentration of the absorbing species and the path length of the light through the solution.
A = εbc
- A is the Absorbance (a unitless quantity).
- ε (epsilon) is the molar absorptivity or molar extinction coefficient, a constant that is unique to the substance at a specific wavelength (units: $L \cdot mol^{-1} \cdot cm^{-1}$).
- b is the path length of the sample holder (cuvette), which is typically 1 cm.
- c is the concentration of the substance in the solution (units: $mol \cdot L^{-1}$ or $mol/dm^3$).
Since ε and b are usually constant, the law shows a direct linear relationship between absorbance and concentration (A ∝ c). This allows us to determine the concentration of an unknown solution by measuring its absorbance and comparing it to a calibration curve made from solutions of known concentrations.
Solved Examples:
- What is the primary use of the Beer-Lambert
Law?
Solution: To determine the concentration of an unknown solution by measuring its absorbance. - A solution has an absorbance of 0.5. If the concentration is doubled, what
will the new absorbance be, assuming the path length is
constant?
Solution: Since absorbance is directly proportional to concentration, the new absorbance will be $0.5 \times 2 = 1.0$. - What is a calibration curve in the context of UV-Vis
spectroscopy?
Solution: It is a graph of absorbance versus concentration for a series of standard solutions of known concentrations. By measuring the absorbance of an unknown sample, its concentration can be found by reading it off the curve. - A solution of a compound with a molar absorptivity of 2000 L mol⁻¹ cm⁻¹ is
placed in a 1 cm cuvette. Its absorbance is measured as 0.4. What is its
concentration?
Solution: Using A = εbc: $0.4 = (2000)(1)(c)$. Therefore, $c = 0.4 / 2000 = 0.0002$ mol/L. - What is absorbance?
Solution: Absorbance is a logarithmic measure of the amount of light absorbed by a sample as the light passes through it. - Why is the path length (b) usually 1
cm?
Solution: Using a standard path length of 1 cm simplifies calculations and allows for easy comparison of molar absorptivity values between different experiments and labs. - If you dilute a sample by a factor of 10, what will happen to its
absorbance?
Solution: The concentration will decrease by a factor of 10, and therefore the absorbance will also decrease by a factor of 10. - What does the molar absorptivity constant (ε)
represent?
Solution: It is a measure of how strongly a substance absorbs light at a particular wavelength. A high ε value means the substance is a very strong absorber. - A student creates a calibration curve that is not a straight line. What is a
possible reason?
Solution: The Beer-Lambert Law works best for dilute solutions. At very high concentrations, the relationship between absorbance and concentration can become non-linear. - What is a cuvette?
Solution: It is the small, transparent sample holder with a fixed path length (usually 1 cm) that is placed inside the spectrophotometer.
13.13 Applications of UV-Vis Spectroscopy
UV-Vis spectroscopy is a versatile and widely used technique in many fields:
- Quantitative Analysis: Its most common application is determining the concentration of an analyte in a solution, such as the concentration of transition metal ions in water or the amount of active ingredient in a pharmaceutical tablet.
- Purity Determination: It can be used to check for the presence of impurities in a sample if the impurity has a different absorption spectrum from the main component.
- Reaction Kinetics: By monitoring the change in absorbance of a reactant or product over time, the rate of a chemical reaction can be determined.
- Biochemistry: Used to determine the concentration of proteins and nucleic acids (DNA/RNA) in biological samples.
- Environmental Monitoring: Used to measure the concentration of pollutants, such as nitrates or phosphates, in water samples.
Solved Examples:
- How can a hospital lab use UV-Vis spectroscopy to measure hemoglobin levels
in blood?
Solution: A blood sample can be treated to convert hemoglobin into a stable, colored derivative. The absorbance of this solution is then measured and compared to a calibration curve to determine the hemoglobin concentration. - A chemist is studying a reaction where a colorless reactant turns into a
colored product. How can UV-Vis spectroscopy
help?
Solution: They can monitor the reaction by measuring the increase in absorbance at the wavelength where the product absorbs light. This allows them to calculate the rate of the reaction. - Why is UV-Vis a good technique for analyzing drinking
water?
Solution: It is a sensitive technique that can quickly and accurately measure the concentration of various contaminants, such as transition metal ions or organic pollutants, at very low levels. - Can UV-Vis spectroscopy be used to identify an unknown
compound?
Solution: It can provide some clues (e.g., the presence of conjugation), but it is not a definitive identification tool like IR or NMR because the absorption bands are very broad and often not unique. - What is the main advantage of UV-Vis for quantitative
analysis?
Solution: It is simple, fast, sensitive, and non-destructive. - How is DNA concentration typically measured in a biology
lab?
Solution: By measuring the absorbance of the DNA solution at a wavelength of 260 nm using a UV spectrophotometer. - A beverage company wants to ensure every bottle of its orange soda has the
same color. How can UV-Vis help?
Solution: They can use it as a quality control tool. By measuring the absorbance of the orange dye in each batch at a specific wavelength, they can ensure the concentration is consistent. - Can UV-Vis spectroscopy be used to analyze a solid
sample?
Solution: Not directly. The sample must first be dissolved in a suitable transparent solvent. - What is a key limitation of UV-Vis
spectroscopy?
Solution: It is only useful for analyzing substances that have a chromophore, meaning it cannot be used for many simple saturated compounds like alkanes. - Why must the cuvette be made of a material that is transparent to the light
being used?
Solution: So that the instrument only measures the absorbance of the sample inside the cuvette, not the absorbance of the cuvette material itself. Quartz cuvettes are used for UV measurements, while glass or plastic can be used for visible measurements.