In absorbance spectroscopy (also known as absorption spectroscopy), the intensity of light absorbed by a sample is measured as a function of wavelength. This can provide important information about the electronic structure of an atom or molecule. Depending on the sample, absorbance measurements can also give you key insight into other material properties, such as sample concentration, phase changes, or composition changes.
An optical spectrometer outputs the intensity of detected light as a function of wavelength within the visible light region. When you measure absorbance, light passes through the sample, and the spectrometer detects the transmitted light. Using these transmittance measurements, you can then calculate absorbance at various wavelengths.
- Quick Start Video: How to take an absorbance measurement
- Absorbance Theory
- How to measure absorbance?
- Equipment Set Up
- How to calculate absorbance from transmittance?
- Taking absorption measurements
- Things to remember when measuring absorbance
- Absorbance Spectroscopy: Example Uses
Band theory describes how electrons are organised within a solid and can be useful when discussing different properties of a solid (conductors, semiconductors, or metals). Within an atom, electrons can exist in regions around the nucleus known as orbitals. Depending on the electronic structure of an atom, these orbitals can be filled, partially filled, or empty. The wavelength of light that can be absorbed by a material is dependent on the different energy levels and the electronic distribution within this material.
Absorption occurs when the energy difference between the highest occupied molecular orbit (HOMO) and the lowest unoccupied molecular orbit (LUMO) of a photon is equal or greater. In this case, the photon can be absorbed by an electron in the HOMO and will be excited into a higher energy state.
The wavelengths of the absorbed light correspond to this energy difference through the equation of photon energy.
When multiple atoms come together to form molecules, this behaviour can become more complex due to the many different variables at play. For example, the types of electronic transitions that are allowed will often vary with different material properties. Therefore, measuring which wavelengths a material can absorb will tell you a lot about its electronic properties. You can measure UV-Vis absorbance to investigate the suitability of a material for a specific purpose or to determine its material properties. For example:
- Organic dyes require high absorbance over a small wavelength range so that only light of a matching colour is emitted from the dye, e.g. for a yellow dye, blue light (435 - 480 nm) must be absorbed.
- Materials that convert visible light into different forms of energy (OLEDs, solar cells, etc.) will require high absorption, ideally across the visible region. These materials frequently have desirable band gaps, which can be determined using UV-Vis spectroscopy.
- Transparent materials need to absorb as little visible light as possible. You can confirm this using UV-Vis spectroscopy.
- In organic compounds, UV-vis spectroscopy can help illuminate the amount of conjugated pi bonds in a molecule.
As light is propagated through a sample and absorbed, its intensity will fall exponentially with distance. You can determine this absorbance by the incident light, I0, and the detected light that has passed through the sample, I, through this equation.
Absorbance is a relative measurement so is therefore unitless. This absorbance, A, can be defined in a number of different ways for different samples.
Beer Lamberts Law and the Molar Attenuation Coefficient
When you measure the absorbance spectrum of a solution, it is important to notice the wavelengths where maximum absorbance is occurring, as this can help you determine certain molecular properties. It is also always important to consider the strength of absorbance at these wavelengths.
You can establish this using the molar attenuation coefficient, ε. This has also been referred to historically as the molar extinction coefficient and is also known as the molar absorptivity coefficient. It can be used if you need to calculate the electronic transitions associated with various peaks. For allowed transitions, ε > 1000 whereas for forbidden transitions, ε < 100.
You can work it out ε using Beer Lambert's Law:
Where A is absorbance, c is the molar concentration of the molecule in solution, and l is the path length through the sample (often the width of the cuvette, or the total film). You can also use this calculation to measure the concentration of a molecule in a thin film. The graphs below show the variation in absorbance intensity with concentration. We can see here that this is a linear relationship
Molar attenuation coefficient has property has dimensions of
Therefore, the SI units of the molar absorptivity coefficient is m2M-1 or cm2M-1.
For bulk solids, you can define absorbance in terms of an absorption coefficient, α(λ). This is a property of a solid material and is wavelength dependent. Therefore, using it will produce a spectrum across a range of wavelengths. The absorption coefficient, α(λ) relates to absorbance measurements through the following equation:
Here, d is the distance travelled into the bulk solid. In the following diagram, d represents the thickness of the film and the substrate.
The SI units of the absorption coefficient of a bulk solid are m-1 or cm-1.
|Absorption Coefficient||m-1||Bulk Solid|
|Molar Absorptivity Coefficient||m2M-1||Molecule in solution|
How to Measure Absorbance?
You can use a spectrometer to obtain an absorbance spectrum by directing a light source through your solution (or thin film) and then through the spectrometer. It processes this data by splitting the light into its constituent wavelengths and calculating the relative intensity of light at each wavelength.
This initial measurement will tell you how much light has been transmitted through the sample and into a spectrometer. The transmittance value, T, of a sample is defined as the ratio of the intensity of light incident on the sample, I0, to the intensity of light emerging from the other side of the sample, I:
I0 can be measured by directing the incident beam towards the spectrometer with no sample present.
The transmission of a sample is wavelength-dependant. You can use an optical spectrometer to measure the transmission of a sample for many wavelengths simultaneously. This data can then be converted to give you a measurement of the absorbance.
Calculating Absorbance from Transmittance
You can calculate the absorbance, A, of the sample by taking the base 10 logarithm of the transmission:
The factor of 0.43 comes from our taking the base-10 logarithm of the transmission rather than the natural logarithm. In some scientific fields, the absorbance is defined by the natural logarithm of the transmission. Within the study of liquids and solids, you would generally use the base-10 logarithm. From the equation above, you can calculate the absorbance of a sample by first calculating the transmission, and then simply taking the negative logarithm.
The table below shows the relationships between different values of transmission and absorbance.
Absorbance can be measured directly on the Ossila Optical Spectrometer by selecting the "Absorption" setting. The software will require a background measurement and a direct beam measurement be taken, then you can measure the absorption.
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Taking Absorption Measurements
Recording an absorbance spectrum requires you to measure three separate spectra. These are:
1. A background spectrum
The detector in the spectrometer will record a signal even when there is no light falling on it. This is due to the thermal generation of electron-hole pairs, which produce a "dark" signal. Because this background is unrelated to the sample, you will need to subtract it from all subsequent spectra in order to account for these effects. You can take a background spectrum by turning off the illumination source and recording a spectrum.
2. A reference spectrum, I0
This spectrum will measure how much light is incident on the sample. You can do this by turning on our illumination source and recording a spectrum without the sample present. It is often necessary to use a reference sample in place of your actual sample. For example, if you are interested in the transmission of a thin film that is deposited on a glass substrate, you may wish to exclude the effect of the glass in the measurement. In this case, place a blank glass substrate in the light path for the reference measurement. Or, if you are measuring the transmission of molecules in solution inside a cuvette, you may want to exclude the effects of both the solvent and the cuvette. In this case, use a cuvette containing just the solvent as the reference sample. The background should be subtracted from the measured spectrum.
3. A measurement spectrum, I
To record this, place the sample in the light path and then take the measurement spectrum. You should then subtract the background from this spectrum.
If you select the 'Absorption' measurement type in software, the background subtraction and this conversion is performed automatically. From these measurements of transmission, an absorbance spectrum is produced.
Things to Remember when Measuring Absorbance
Note that there is a large caveat to calculating absorbance from transmission: it assumes that no light is reflected from the sample surface and no light is scattered and lost in the sample. In addition to absorption, it is these additional loss mechanisms that prevent light from passing through the sample.
As a result, we do not recommend that you use this technique for measuring the absorption of turbid/rough media where high scattering losses are expected, or of media with a high refractive index where significant reflection can occur at the interface with air.
Transmission and absorption measurements are particularly susceptible to noise. Each of the three spectra involved in the calculation will have its own associated noise, which will be added when the calculations are completed. There are some approaches to help you reduce noise and achieve a clean transmission or absorption spectrum.
- Choose the right light source (see spectroscopy light sources). You should ensure a high intensity of light over the wavelength range being measured. If there is no light, there is nothing to be absorbed by the sample and nothing to be measured by the spectrometer; therefore, your data will be meaningless.
- Use the correct integration time. To maximise the signal, you should use an integration time that gives a clear signal for the reference spectrum. About 90% of the spectrometer's saturation values are ideal. If you use a light source with higher intensities, you'll be able to use shorter integration times.
- Use spectral averaging. By averaging several spectra, you can reduce the noise in the average spectrum by the square root of the number of samples. We recommend that you use a bright source, as this means you can have short integration times and, therefore, many spectra can be recorded and averaged quickly.
- Be aware of limitations and prepare the sample accordingly. If you use a highly absorbent sample, almost no light will be able to pass through. This leads to a similar situation as when using a dim light source, i.e., there will be no signal (or the signal will be obscured by noise), and so you will have nothing to measure. Ideally, you want the transmission of the sample to remain above 0.05 for all wavelengths (i.e., the absorption is below 1.3). Practically, you can achieve this by reducing the concentration of molecules in your solution or reducing your film thickness. If this is not possible, the points above will be particularly important, as reducing noise will encourage the maximum measurable absorption. If your spectra shows very high absorption, it may not be accurate.
Absorbance Spectroscopy: Example Uses
There is not a single answer to the question, "What can we tell from absorbance spectroscopy?" There are so many material properties that you can measure with this technique. Additionally, the amount of information you can gain from these readings will depend entirely on your sample. In particular, UV-Vis absorbance spectroscopy is particularly versatile as it can be used to characterise materials with allowed energy transitions between 1-4 eV. You can therefore use optical spectroscopy to study chemical dyes, chromophores, conjugated organic materials, or materials whose optical properties in the visible region are of interest, such as solar cells or OLEDs. Additionally, if there is a significant absorbance peak in the visible wavelengths for your sample, you can also use optical spectroscopy to measure the concentration of that specific material.
Practically everything has a recorded absorbance spectrum. Therefore, you can use an absorbance spectrum of your material to identify different molecular structures within a solution or film. You can also use it to help determine the ratios of various molecules within a solution or a film.
In the above example, you can see peaks that align with P3HT. You can also see some peaks that align with o-IDTBR in the mixed polymer blend. The location and intensity of these peaks can help you work out how much of each material is in a film and how these materials have interacted with one another.
Conjugated organic molecules are molecules with alternating single and double carbon bonds. This means they have conjugated pi-bonds associated with them, and therefore have additional pi and anti-pi energy levels. Only certain electronic transitions can be detected using optical absorbance spectroscopy (π → π, Σ → π) and these different energy transitions will lead to different absorbance peaks.
Optical spectroscopy is incredibly useful if you want to determine the amount of pi-conjugation within a molecule. An increase in the amount of conjugation in a molecule will cause a bathochromic shift. You can measure the levels of aggregation of pi-conjugation in a solution or a material non-invasively using an optical spectrometer.
Measuring Material Properties of Semiconductors
You can determine a semiconductors band gaps using absorbance spectroscopy via the Tauc equation.
Where A is an optical constant, h is Planck's constant, α is absorption coefficient, Eg is the material's band gap, and v is the frequency of the photon. γ is a factor that varies depending on the nature of the electronic transition. For direct bandgap transitions, it is equal to 0.5. For indirect bandgap transitions, it is equal to 2. Other transition gamma factors are defined in the table below.
For direct band gaps semiconductors, you can find the band gap of a material using optical absorption by plotting (αhν)2 against hv. By applying a linear fit to the absorbance edge of this plot, the x-intercept will give you the material band gap.
For several composite semiconductors, band gap changes can indicate changes in material composition. For example, in perovskite solar cells, incorporation of bromine into the crystal lattice will shift the band gap to a lower wavelength. This will indicate an increase in the band gap.
Determing Solution Concentration and Optical Density
Here, we will be measuring the absorbance variation at 533 nm, where there is a large absorbance peak. By plotting the absorption coefficient against concentration, we can create a standard graph. We can then use this to determine the direction of molar concentration from the absorbance coefficient.
You can also use optical spectroscopy to track the growth of bacteria in a cell culture. If you use a clear medium, as the amount of bacteria increases, the absorbance of your culture will increase massively. By plotting a bacterial growth curve, you can follow these stages of bacterial growth. This is especially useful if you are working on any antimicrobial technology.
Identifying Purity of Materials
Absorbance spectroscopy can be an easy way to check the purity of any material if it has absorbance peaks within the region of your spectrometer. To do this, take an absorbance spectrum of your materials, either in solution or spin-coated into a film. By comparing the absorbance peaks with a reference spectrum, you will be able to pick out any peaks from material impurities.
Optical absorbance is often used for this in industrial applications, acting as a quick and easy way to check the quality of metal alloys. It can also be used for biological applications, where risks of contamination can threaten sterile environments.
Idenitifying Aggregates and Complexes
At a low enough concentration, using UV-Vis spectroscopy can help you explore the formation of complexes in solution or even aggregates in films. Measuring the amount of coordination in solutions will inform you about whether the material you are measuring is interactive. It can also provide information about how other molecules are interacting within the sample you are measuring.
Examples where complexes and coordination are studied using UV-Vis spectroscopy include:
- At low wavelengths, the types of Pb-halide co-ordination can be determined using UV-Vis spectroscopy. This can be very informative when you want to recognise the influence of other materials (solvents/organic cations) on crystal structure.
- The formation of H/J aggregates in polymer morphology can be detected using optical spectroscopy. If H aggregates are forming, you will see blue shift in your absorption spectra towards lower wavelengths compared to the monomer. However, J aggregation will lead to the formation of a sharp peak that is redshifted compared to the monomer peak. [ref N Hestand]
- In determining how nickel(II) and cobalt(II) benzhydrazone complexes interact with DNA protein receptors. This can also be detected at low wavelengths (<400 nm) using UV-Vis spectroscopy.
Characterising Device Layers
Most optical or optoelectronic devices require multiple layers in order to work effectively.
The optical properties of these layers will depend entirely on their function. You will need to characterise this, as well as the entire device, independently. Additionally, supplementary layers can be introduced into a device to improve its optical properties.
Examples of these layers and their desired optical characterisation include:
- Antireflective layers which can be placed on top of devices that absorb light, with the aim of increasing the amount of light that can be absorbed. This should result in an increase in the absorbance peaks.
- Charge transport layers that surround a device shouldn't be parasitically absorbing light. Absorbance measurements can ensure that these layers do not absorb in the same region as the absorbing molecules.
- Light shifting molecules which can be placed in the path of light entering or leaving a device to get rid of unwanted light. For example, luminescent, down-shifting layers can absorb UV-light and convert it into visible light. This will increase the amount of usable light that reaches a solar cell.
Other Uses for Absorbance Spectroscopy
Other useful ways which you can use absorbance spectroscopy:
- To determine the glass transition temperature of polymer films.
- To track reactions in your solution using universal indicators.
- To test electrochromic materials (which change colour depending on the electrical stimulus).
- To measuring the size of certain nanoparticles.
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- Hestand, N. J., & Spano, F. C. (2018). Expanded theory of H- and J-molecular aggregates: The effects of vibronic coupling and intermolecular charge transfer. Chemical Reviews, 118(15), 7069–7163. DOI: 10.1021/acs.chemrev.7b00581
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