Ultraviolet-visible (UV-Vis) spectroscopy is one of the most popular spectroscopic techniques. It is a fast, low-cost structural characterization and identification technique. Ultraviolet-visible spectroscopy is especially important for the analysis of organic compounds that absorb radiations in the ultraviolet and/or visible region of the electromagnetic spectrum. These organic compounds also include uniquely colored compounds.
In this article, we will discuss all the fundamentals associated with UV-Vis spectroscopy, so continue reading to gain more insightful information about this very interesting spectroscopic technique.
What is UV-Vis spectroscopy
Ultraviolet-visible (UV-Vis) spectroscopy is a type of molecular spectroscopy. It is based on the interaction of ultraviolet (200-400) nm or visible (400-800) nm radiations with molecular organic compounds. The electrons present in the targeted molecules absorb energy provided by these incident radiations to undergo electronic transitions. Characteristic absorbance and/or transmittance of radiations help in the identification of molecules.
What are UV-Vis radiations
The electromagnetic spectrum consists of simultaneously oscillating electric and magnetic fields. The electromagnetic spectrum is divided into different regions of varying frequency and wavelength. The ultraviolet-visible radiations lie somewhere in the middle of the spectrum. Their wavelength varies from 10 to 800 nm while their frequency lies between 1014 to 1016 Hz.
This frequency or energy is large enough to cause the excitation of molecular electrons from their ground state to a higher energy level. 200 to 400 nm is known as the ordinary/ quartz ultraviolet region while below 200 nm is the vacuum ultraviolet region.
What are molecular electronic transitions in UV-Vis spectroscopy
According to the molecular orbital theory (MOT) of chemical bonding, the electrons of covalently bonded molecules are placed in molecular orbitals of varying energies. There are three main types of molecular orbitals namely a bonding molecular orbital, an antibonding molecular orbital, and a non-bonding molecular orbital. All these orbitals lie at different energy levels. Electrons are placed in these orbitals in an ascending energy order.
The electron-filled molecular orbital lying at the highest energy level is called HOMO while the lowest unoccupied molecular orbital is called LUMO. The unbonded electrons of heteroatoms lie at intermediate energy in the non-bonding molecular orbital. For instance, in a (CH3)2C=O molecule, there are:
- C-C, C-H, and C-O sigma bonded electrons. Therefore, it has a bonding molecular orbital (σ) and an antibonding molecular orbital (σ*).
- C=O double bond consists of a sigma bond (we discussed already) and a pi bond. The pi-bonded electrons are either placed in a bonding molecular orbital (π) or an antibonding molecular orbital (π*).
- The unbonded electrons at O are situated in the non-bonding orbital (n) (see the figure below).
In this manner, the electrons of an organic molecule are always revolving from one point to another in the molecule. This is called electronic delocalization. However, the electronic transition from one energy level to another takes place only when the electrons are provided a specific amount of energy that is equal to the energy difference (∆ E) between two respective energy levels.
The energy of UV-Vis photons often coincides with ∆ E values for conjugated organic molecules. This forms the basis of ultraviolet-visible spectroscopy.
UV-Vis absorbing conjugated organic molecules
Conjugation refers to linking delocalized electronic systems in a molecule. For example, but-1,3-diene CH2=CH-CH=CH2 is a conjugated molecule. Two adjacent double bonds are separated by a single covalent bond. Conjugated organic molecules absorb strongly in the ultraviolet-visible region of the electromagnetic spectrum.
The π to π* transitions however requires energy equivalent to 275 to 295 nm ultraviolet radiations. These electronic transitions will occur in organic molecules containing pi bonds especially conjugated organic molecules. Pi-bonded electrons are less strongly held and easy to excite as opposed to sigma-bonded electrons. That is why they undergo electronic transitions at comparatively lower energy. Thus, conjugation facilitates absorption in the ultraviolet region.
Similarly, n to π* transitions occur in molecules containing both pi-bonded electrons as well as heteroatoms containing non-bonded electrons such as in CH3-CH=CH-CO-CH3. Thus, we know that a carbonyl (CO) functional group attached to C=C bonded organic molecule further facilitates radiation absorption at a longer wavelength (lower energy). Such functional groups are called chromophores.
A chromophore is a functional group that helps an organic molecule to strongly absorb visible radiation.
A molecule absorbing radiations in the visible region (400-800 nm) of the electromagnetic spectrum consists of a large number of chromophores attached to it. A molecule absorbs a specific wavelength of ‘light’’ while transmitting the other wavelengths. In this way, it appears colored. The color of the compound is the color complementary to the wavelength absorbed.
Thus, a chromophore imparts color to the organic molecule.
Plant leaves are green in color because they consist of chlorophyll. Chlorophyll is a conjugated organic molecule; it absorbs different wavelengths of sunlight while transmitting visible radiations around 550 nm that correspond to green color.
Examples of chromophores include a carbonyl (C=O) functional group, azo (-N=N-) group, nitro (-N=O) group, etc. There are certain functional groups that are not chromophores themselves but when attached to a chromophore, they help absorption at an even lower energy (i.e., longer wavelength). These are called auxochromes.
An auxochrome is a functional group that extends the conjugation present in a molecule. It modifies the light absorbing ability of a chromophore.
For example, -OH, -NH2, -SH, and halogen functional groups do not absorb radiations above 200 nm themselves but when attached to a chromophore, these groups act as auxochromes. They have non-bonded electrons present in their heteroatoms which can extend the conjugation of an organic molecule.
Now that we have discussed the chemistry behind UV-Vis absorption, we are good to talk about how an ultraviolet-visible spectrophotometer actually functions.
5 components of a UV-Vis spectrophotometer
A UV-Vis spectrophotometer consists of the following main components:
1. Radiation source
- A hydrogen-discharge lamp or a deuterium-discharge lamp is used for providing radiation in the ultraviolet region.
- A tungsten filament lamp is used for illuminating the sample with visible radiations.
- The monochromator is a prism or a diffraction grating that splits the polychromatic radiation into its constituent wavelengths.
- Only a single wavelength is selected to pass through the sample at a time.
3. Beam splitter
- The monochromatic radiant beam is split into two beams of equal intensity as it passes through the beam splitter.
4. Reference and sample cells
- The cells are made up of quartz as the quartz material itself is transparent to both ultraviolet and visible radiations.
- 0.1 cm to 10 cm cylindrically shaped cells are used in UV-Vis spectroscopy.
- Blank solvent is placed in the reference cell.
- The sample solution is prepared in the same solvent and filled in the sample cell, up to 3/4th of the cell volume.
- Photomultiplier tubes are often used as detectors in UV-Vis spectroscopy.
- The detector receives the transmitted radiation. It is connected to a computer software that calculates the difference between the intensities of the incident (I0) and transmitted (It) radiations.
- An electrical signal is consequently generated which is fed to the recorder.
- The recorder plots the UV-Vis spectrum as a plot of absorbance (A) versus wavelength (λ).
What is the basic principle of UV-Vis spectroscopy
- Equal intensity incident radiations are passed through the sample and the reference cells. The blank solution which does not contain the UV-Vis absorbing analyte transmits all the radiation as such.
- Contrarily, the organic molecules present in the sample solution absorb a specific wavelength. The electrons present in these organic molecules undergo excitation from a lower to a higher energy level by absorbing these radiations.
- All the wavelengths which are not absorbed are transmitted out of the sample.
- The transmitted radiations are recorded, and a difference is calculated between the incident and transmitted radiations to determine the wavelengths absorbed by the sample.
- A UV-Vis spectrum is plotted. The highest intensity peak marks the lambda max ( λmax ) value. This is the wavelength absorbed by the largest number of analyte molecules. Thus, it is characteristic of the organic functional groups present in the target molecule.
- This helps in the structural elucidation of the molecule under study. This is called qualitative analysis using UV-Vis spectroscopy.
- Quantitative analysis can also be performed using Beer Lambert’s law.
The significance of Beer Lambert’s law in UV-Vis spectroscopy
Beer Lambert’s law states that radiation absorption is directly proportional to the sample concentration.
A stands for the absorbance, c is sample concentration in mol dm-3, l is pathlength which can be determined from the sample cell size in cm while ε represents molar absorptivity which is constant for a specific solute.
Beer Lambert’s law is derived from equations 1 and 2.
T= It/I0…… Equation 1
This equation relates transmittance (T) with radiant intensity. It is the intensity of transmitted light while I0 is the incident light intensity, as we saw earlier in the article.
A=log (I0/It) or A=log(1/T)………… Equation 2
This equation relates absorbance with transmittance. By using the two equations, we can infer that the greater the amount of light absorbed by the sample, the lesser will be its transmittance and vice versa. Absorbance and transmittance have no units because both are just ratios.
Different dilutions of a sample solution can be prepared, and the absorbance of each dilution can be determined at the λmax value to draw a straight-line calibration curve. This calibration curve can then be used to find the concentration of an unknown sample solution.
Read more about quantitative analysis using UV-Vis spectroscopy here.
Interpreting a UV-Vis spectrum
A UV-Vis spectrum can be interpreted using the following λmax values at which an organic functional group absorbs radiations maximally.
|Functional group||λmax (nm)|
|Alkene (C=C )||171|
|Carboxylic acid (COOH)||208|
|Acetyl chloride (COCl)||220|
These λmax values can be used to determine the suspected functional groups present in a molecule. All this data can be combined to reveal the entire molecular structure of the target compound, as shown in the figure below.
Different wavelength shifts in a UV-Vis spectrum
- Red-shift or Bathochromic effect: Shift to a longer wavelength. A red shift occurs in the presence of chromophores which makes the molecule absorb lower energy radiations i.e., longer wavelengths. Red color has the longest wavelength in the visible region thus the name red-shift is given.
- Blue-shift or hypsochromic effect: Shift to a shorter wavelength in the absence of chromophores and/or by the removal of conjugation. The name blue shift comes from the blue color which lies at the start of the visible region (refer to a rainbow set).
- Hyperchromic effect: The increase in absorption intensity at a particular point is called the hyperchromic effect. The greater the analyte molecules present in a sample; the higher will be the hyperchromic effect.
- Hypochromic effect: The decrease in absorption intensity at a specific point in the UV-Vis spectrum is called the hypochromic effect. This is the reverse of the hyperchromic phenomenon.
The Woodward-Fieser rule is an empirical rule that further helps in predicting how a structural feature controls the radiation absorbing ability of a molecule.
For reading why UV-Vis spectroscopy is important in analytical chemistry, you may like our article: What are the uses of analytical spectroscopic techniques in chemistry.
Here are two video tutorials for you to revise the concepts we talked about in this article:
1. Akash, M. S. H. and K. Rehman (2020). Ultraviolet-Visible (UV-VIS) Spectroscopy. Essentials of Pharmaceutical Analysis. Singapore, Springer Nature Singapore: 29-56.
2. Förster, H. (2004). UV/VIS Spectroscopy. Characterization I:H. G. Karge and J. Weitkamp. Berlin, Heidelberg, Springer Berlin Heidelberg: 337-426.
3. M.Younas (2017). Organic Spectroscopy and Chromatography, 11-33.