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Chemistry LibreTexts

3: UV Spectroscopy

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UV Spectroscopy

Which electrons can be excited? Where do they go?

In order to absorb UV-Vis light, the molecule must contain a chromophore—the part of a molecule that absorbs UV light.

An MO diagram for formaldehyde (methanol, CH2O) is shown.

clipboard_e3a979463808ef1ba691e5cdfd71e4f34.png

  • Place the proper number of electrons in the diagram.

There are 4 common types of transitions (n-\pi*, \pi-\pi*, \sigma-\sigma*, n-\sigma*) for organic compounds

  • What structural features must be present for each type of transition?
    • \sigma \rightarrow \sigma*
    • \pi \rightarrow \pi*
    • n \rightarrow \pi*
    • n \rightarrow \sigma*
  • Propose transition types for the list of functional groups below:
    • Alkane
    • Alkene
    • Alkyne
    • Alcohol
    • Carbonyl
    • Amine
    • Nitrile
  • Molecules with chromophores tend to have a lot of pi bonds. Explain why.
Table of wavelengths and extinction coefficients (\epsilon_{max}) for those transitions
Transition ~\lambda_{max} (nm) \epsilon_{max}
\sigma \rightarrow \sigma* 135 v. small
\pi \rightarrow \pi* 160-280 2,400-25,000
n \rightarrow \pi* 279-320 10-50
n \rightarrow \sigma* 160-250 100-7,000
  • Why are the \lambda_{max} values shown as ranges and not discrete numbers?
  • Using the MO energy diagram, explain the relative energies of the transitions.

clipboard_e7a3db78a14e06ecc2ce5d25da4ba9365.png

Extinction Coefficients

Beer’s law states that the absorbance of a solution is proportional to its concentration. Mathematically, Beer’s law is

A = \epsilon b c

A is the absorbance of the solution

\epsilon is the quantity called the molar absorptivity

b is the path length in cm of the solution cell

c is the solution concentration (molarity)

Absorbance spectra are usually gathered as a function of wavelength. Absorbance is dependent upon concentration.

  • Suppose that a UV absorbance spectrum was obtained for a solution of a compound. Then the concentration of the solution was doubled. Redraw the new spectrum on the same scale.

clipboard_e5232100e26909f6f3846e3971be01d76.png

Absorbance is also dependent the molar absorptivity. Molar absorptivity (\epsilon) tells you how likely an electronic transition is.

  • The more likely a particular absorbance occurs, then the [ smaller / larger ] the \epsilon will be.
  • If an electronic transition is difficult, _______ [ more / fewer ] electrons will be excited, and the \epsilon will ________ [ decrease / increase ] and the apparent intensity will be weaker.
  • \sigma \rightarrow \sigma* has a small extinction coefficient. Why?
  • n \rightarrow \pi* are the lowest energy of any transitions we observe and consequently have the longest wavelength. However, they typically have the smallest extinction coefficient. Why is the n \rightarrow \pi* transition so difficult even though the energy gap is small? Consider how much orbital overlap is present.

clipboard_ee5f83c565b1cc816a8ab937809628fcb.png

UV Spectroscopy: Analysis

UV-Vis Spectroscopy is useful in the laboratory for several purposes.

  1. Determination of structure.
  2. Determination of the concentration of a known sample

UV-Vis Spectroscopy: Determination of structure

The molecules most likely to absorb light in the UV-Vis region are structures with pi bonds. This severely limits the use of UV-Vis as a general method for determining structure.

Additionally, molecules with an isolated alkene tend to have \lambda_{max} around 170-190 nm which is not measure by most instruments.

  • With conjugation, the \pi \rightarrow \pi* orbitals are [ closer or further ] apart.

clipboard_e729ab32582a0126293da206a301de469.png

  • Conjugated systems require [ higher or lower ] energy to excite an electron.
  • However, generally moves the absorption maxima to [ longer or shorter] wavelengths.

Thus, conjugation becomes the major structural feature identified by this technique.

Determination of Structure: Predicting \lambda_{max} of Conjugated Compounds There are some standardized rules for predicting the \lambda_{max} in conjugated molecules called the Woodward-Fieser Rules.

Core Chromophore Substituent Influence

Trans diene: 215 nm

clipboard_e56354fe86eff2249a6e81d22a7a70e34.png

Alkyl +5
Alkoxy +6
Halide +10

Cis diene: 260 nm

clipboard_ead6281990cd592583018c4fbc9a424f2.png

Acyl 0
Amino 60
Another double bond +30
Phenyl +60

*Each exocyclic double bond adds 5 nm.

  • Predict the have \lambda_{max} for the following compounds.

clipboard_e0cc86e26e3cd41fd27226d3fdd7db9f8.png

Base (cis or trans): _______

5 alkyl group substituents _______

2 exocyclic bonds _______

Additional double bond _______

Total: _______ Compare to actual: 283 nm

clipboard_e43b3795eee9c6a5ad43df599c20e2b69.png

Base (cis or trans): _______

4 alkyl group substituents _______

2 exocyclic bonds _______

Additional double bond _______

Total: _______ Compare to actual: 355 nm

Determination of Structure:

Predicting \lambda_{max} of Conjugated Carbonyl Compounds The Woodward-Fieser Rules also extend to conjugated carbonyl compounds.

Core Chromophore Substituent \alpha-Influence \beta-Influence

Acyclic enone: 215 nm

clipboard_e0f7ca4cd3d5a8defe466bd39631a3529.png

Alkyl +10 +12
Alkoxy/Hydroxyl +35 +30

Cyclic hexenone: 215 nm

clipboard_e144a11c19832d3c49df1915c2139d5d1.png

Chloro +15 +12
Bromo +25 +30
Acyl +6 +6

Cyclic pentenone: 202 nm

clipboard_e5d4c61aa6c43ce0427630e8c572f904d.png

Another double bond +30  
Phenyl +60  

*Each exocyclic double bond adds 5 nm.

** In water, add 8 nm.

  • • Predict the have \lambda_{max} for the following compounds.

clipboard_ebc550a19eead18955bd8fffc8736e114.png

Base (cis or trans): _______

1 alpha alkyl group substituents _______

2 beta alkyl group substituents _______

Additional double bond _______

Total: _______ Compare to actual: 249 nm

clipboard_ea781de51c4e8dcc247d84e7e9e805297.png

Base (cis or trans): _______

alpha bromo group substituents _______

2 beta alkyl group substituents _______

Additional double bond _______

Total: _______ Compare to actual: 251 nm

There are lots of tables for use in predicting values. There are also databases of known UV spectra for direct comparison.

UV-Vis Spectroscopy: Determination of the concentration of a known sample

Beer's Law: A = \epsilon b c

A is the absorbance of the solution

\epsilon is the quantity called the molar absorptivity

b is the path length in cm of the solution cell

c is the solution concentration (molarity)

  • Explain how you could determine the concentration of a sample using Beer’s Law. Show the math.

The literature value of ε for 1,3-pentadiene in hexane is 26,000 (mol / L)-1cm-1 at its maximum absorbance at 224 nm. You obtain a 10 mL sample and take a UV spectrum in a cell with a 1 cm path length, finding that A224 = 0.850.

  • What is the concentration (molarity) of the solution? Show your work.
  • How many grams of 1,3 pentadiene (molar mass = 68 g/mol) are in the solution?

UV Spectroscopy: Solvent Choices

Solvents used for UV Spectroscopy Samples

  • What solvents (list solvents with structures) would interfere with UV analysis?
Solvent Structure
Acetic acid (AcOH) clipboard_e521d2d4308faae7df311901e95c97447.png
Acetone clipboard_ed9362dfee4f39476d4ca229279a9732d.png
Benzene (PhH) clipboard_e66b4d22c98166221f7f525a20df3098a.png
tert-Butyl methyl ether clipboard_eb7e33ca1255552a4b69a0b4ec4228c29.png
Chloroform clipboard_ec48494cd553c0d7c6cf59c62f9436e23.png
Diethyl ether clipboard_e080af901badc8dbd4c47cf18dae9b9da.png
Dimethyl formamide clipboard_e6a21f4aea7369c7d91d0024859469212.png
Dimethyl sulfoxide (DMSO) clipboard_eeeb9d656081feb00055994f5e47b1a1e.png
Ethyl acetate (EtOAc) clipboard_ec59380423bab322c82779474b809bc49.png
Hexane clipboard_e1cff34ef7ea13a999298a91b2d09d220.png
Octane clipboard_ea28bfe8dae4594a3362ae87ea80b1cfd.png
Tetrahydrofuran (THF) clipboard_e2ce65742c4bee79fff991c4f4816d21e.png
Toluene (PhMe) clipboard_edf412ac83b868edb9262eb3c61d7f6fb.png
Water clipboard_e01d875548f589dad77401b5c078360e3.png
  • Not everything is soluble in hexane/octane, what range of wavelengths can you measure using methanol as your solvent?

Practice Problem

  1. It is often important to be able to quantify the amount of protein in a solution. One method is based on the fact that aromatic amino acids tyrosine (Tyr) and tryptophan (Trp) absorb ultraviolet light maximally at ~280 nm. The amino acid cysteine does not absorb at this wavelength; however, two cysteines may react with each other to form a disulfide (S-S) linkage which is called cystine and this structural feature does. The general method is to just take a solution of unknown protein sample, stick it into a spectrophotometer, and read the A280.

Based on a sample of measured molar extinction coefficient (ε) values for 80 proteins, the ε at 280 nm of a folded protein in water, ε(280), can best be predicted with this equation:

\epsilon(@ 280) (\text{M$^{-1}$cm$^{-1}$}) = (\#Trp)(5,500) + (\#Tyr)(1,490) + (\#cystine)(125)

Of course, it is necessary to know the structure of the protein so that the number of each type of amino acid residue can be entered into the equation.

  • Examine the equation and predict the type of absorption that each of the three amino acids in the equation undergoes at 280 nm.
  • Draw the side chains of these three amino acids.
  • Does this match your prediction? Explain.

This page titled 3: UV Spectroscopy is shared under a not declared license and was authored, remixed, and/or curated by Kate Graham.

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