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Absorbance Measurements

Absorbance measurements are crucial in many areas of scientific research. In an absorbance measurement, light over a broad wavelength range passes through a sample and a spectrometer measures the light that is transmitted. Using these transmittance measurements, you can calculate the amount of absorbance at different wavelengths. This article describes how to take an absorbance measurement using an optical spectrometer.

Equipment Setup

Optical spectrometers output light intensity as a function of wavelength within the visible light region. To measure absorbance, align your optical spectrometer, light source and sample holder so light can travel straight through your sample and into the spectrometer. You can align these components using a breadboard, or you use optical fibers to direct the light.

This initial measurement will tell you how much light has been transmitted through the sample and is directly related to absorbance.

Calculating Absorbance from Transmittance

Transmittance (T) is a measure of how much light passes through a sample. It's determined by the ratio of the intensity of light entering the sample (I0) to the intensity of light exiting the sample (I):

You can calculate the absorbance spectrum, A, of the sample by taking the base 10 logarithm of the transmission spectrum:

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 factor of 0.43 comes from 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.

The table below shows the relationships between different values of transmission and absorbance.

Transmission Absorbance
0.01 2
0.02 1.7
0.05 1.3
0.1 1
0.2 0.7
0.5 0.3
1 0

Absorbance can be measured directly using 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.

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 should place a blank glass substrate in the light path for the reference measurement. This will account for any optical effects.

If you are measuring the transmission of molecules in solution inside a cuvette, you should exclude the optical properties 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

Once you have measured the background and reference spectra, 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 Spectroscopy Software, this background subtraction and the conversion from transmission to absorbance spectra happens automatically. This allows you to easily get an absorbance spectra.

Tips for Absorbance Measurements

Loss of Light

When calculating absorbance from transmission, you must assume no light is reflected from the sample surface and no light is scattered by the sample. These additional loss mechanisms prevent light from passing through the sample, so you must reduce these effects as much as possible.

This technique is not appropriate for measuring the absorption through turbid/rough media where high scattering losses are expected, or of media/solutions with a high refractive index where significant reflection can occur at the interface with air.

Reducing Noise

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.

1. Choose the right light source. 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.
2. 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.
3. 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.
4. 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.

References

• Rocha, F. S., Gomes, A. J., Lunardi, C. N., Kaliaguine, S., & Patience, G. S. (2018). Experimental methods in chemical engineering: Ultraviolet visible spectroscopy-UV-vis. The Canadian Journal of Chemical Engineering, 96(12), 2512–2517. DOI:10.1002/cjce.23344
• 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
• Makuła, P., Pacia, M., & Macyk, W. (2018). How to correctly determine the band gap energy of modified semiconductor photocatalysts based on UV–vis spectra. The Journal of Physical Chemistry Letters, 9(23), 6814–6817. DOI: 10.1021/acs.jpclett.8b02892

Contributing Authors

Written by

Dr. Mary O'Kane

Application Scientist