Understanding Absorbance & Transmittance

Why measure absorbance?

In biology and chemistry, absorbance is used to measure the concentration of molecules in solution, since many biomolecules absorb light at specific wavelengths.

For example, Nucleic acids and proteins absorb UV light, chlorophyll absorbs light of blue and orange/red and hemoglobin absorbs yellow-green light. These analytes can be quantified without further procession, at least if they are found in a solution with low background absorbance.

However, absorbance quantification is not limited to a handful of absorbing molecules. Chemical reactions assist in producing absorbing molecules for quantification dependant on a specific substrate or on an enzyme. Hence, color development (and absorbance) is more intense, the more of the compound/enzyme is found in the sample. Further below, many examples for absorbance measurements are given, from measuring cell viability to protein concentration with protein assays.

The parameters needed to measure absorbance

Light source

The measurement of light transmission / absorption requires light. Different light sources can be used for absorbance measurements differing in the spectral range they cover, their intensity and the stability of the light they emit.

Tungsten Halogen lamps cover the range from 360 nm to more than 1000 nm and are often used due to their cost-effectiveness. Xenon flashlamps, however, cover the spectral region from 220 nm to 1000 nm and cover the UV-range of the spectrum, making the detection and quantification of nucleic acids (260 nm) and proteins (280 nm) possible. BMG LABTECH absorbance microplate readers are equipped with a xenon flashlamp for absorbance measurements and hence provide maximum flexibility to measure any wavelength between 220 and 1000 nm, or all the whole spectrum of wavelengths.

Sample

Samples in liquid form need to be transferred either to a clear cuvette or to a microplate in order to measure their absorbance. Various materials are commonly used and differ from one to another. Polystyrene is most often used and may be altered to provide specific binding characteristics, either for cell culture or ELISA applications. However, they are non-transmissive in the UV-range which requires the use of special plates of cyclic olefin copolymer to measure absorbance below 400 nm. 

Sample volume

A second aspect to consider is the sample volume that is needed for reliable absorbance measurements. Standard cuvettes require approximately 4.5 ml whereas semi-micro volume cuvettes allow reducing the volume down to 1.5 ml and micro-volume cuvettes get along with 70 µl. The cuvette measurement is horizontal: light is directed from one side of the sample with the and on the opposite side the transmitted light is detected on the opposite side. Therefore, the geometry of cuvettes provides a standardized path length of 1 cm. This is one of the major differences to microplate measurements as they run vertically (top to bottom or bottom to top). Accordingly, the path length also changes with the sample volume and requires the use of equal volumes for one experiment. Additionally, meniscus effects play an important role for the path length in microplate absorbance measurements,as explained in detail in the HowTo Note: How to deal with path length and meniscus in microplates. Measurements performed in the middle of a microplate well suffer from a much shorter path in presence of a strong meniscus compared to a measurement with no meniscus. This is of specific importance when applying Beer-Lambert or when having highly variable menisci in an experiment. In aqueous solutions it is possible to correct for meniscus-dependent path length changes by exploiting the absorbance of water (water-peak path length correction).

Standard 96 well plates are typically used with sample volumes ranging between 100 - 300 µl, translating to an approximate path length if 2.9 to 7.4 mm. It might be necessary to further reduce the volume of precious sample without compromising in path length. To this end, half area plates or higher density plates may be used. They provide a smaller well area but the same height as standard 96-well plates and, thus, providing high path lengths with lower volumes.

Detector of absorbance measurements

After the transmitted light passes through the sample it needs to be detected. Two different types of detectors are commonly used in microplate readers: either a photomultiplier tube (PMT) or a CCD spectrometer. PMT- based systems require the selection of wavelength before the detection. This is done by optical filters or monochromators that select the desired wavelength before it is guided to the sample.

The transmitted light of the desired wavelength then reaches the PMT, which amplifies the signal from the transmitted light and results in a voltage indicative of its intensity. In order to scan absorbance over a range of wavelengths, PMT-based systems use a monochromator that selects the desired wavelength for each point in the spectrum and measures them sequentially.

This is the main difference to the second type of absorbance detectors, the spectrometer. It splits the transmitted light into different wavelengths and these are directed onto a CCD detector, capturing the intensity of all wavelengths at once. This way, spectra as well as single or few wavelength measurements can be acquired in a similar amount of time. For instance, BMG LABTECH instruments employ UV/vis Spectrometer for absorbance measurements that capture a whole spectrum (220 1000 nm) in less than a second.

Calculating absorbance versus transmission

Detection path in cuvettes and microplates

A solution containing an analyte with known absorbance characteristics is placed in the cuvette, and the reader sends light of known intensity through the sample while detecting the light that passes through it. Any light that does not reach the detector has either been absorbed or scattered. The scattered component is measured separately using appropriate blanks and subtracted to obtain the true absorbance of the analyte.

Pitfalls in absorbance measurements

Absorbance measurements are widely used to quantify substances in solution, but they are not always straightforward. Results can be affected by sample turbidity, interfering compounds, inaccurate path length, and instrument limitations, which can make it difficult to obtain a reliable signal.

Using the wrong plate

When measuring in the UV-range, as it is required for nucleic acids or NADH, it is absolutely required to use UV-transparent plates. Otherwise the absorbance measurement will result in maximum absorbance in all samples.

Comparing cuvette with microplate measurements

The path length in cuvettes is normalized to 1 cm but in microplates it changes depending on volume and plate format. Therefore, absorbance acquired in microplates is typically lower than the absorbance of the same solution measured in cuvettes. If aqueous solutions are measured the microplate measurement can be normalized to 1 cm by using the water-peak path length correction. Determination of the path length further allows to calculate analyte concentrations with Beer-Lambert law.

Using different volumes

For measurements in a microplate itis recommended to always use the same volume to generate the same path length for all samples. However, in samples with a meniscus, the path length may be different despite using the same volume. Again, for aqueous solutions the water-peak path length correction can be used to overcome the problem or to determine the path length for Beer-Lambert calculations.

Disturbance of the light path

Anything which is in the light path will increase the measured absorbance. Often these are air bubbles in the sample, condensation on a lid, dust, scratches or fingerprints on the bottom of the plate. Hence, checking the plate just before measurement is recommended.

What is typically measured by absorbance?

Absorbance is commonly used in applications that measure the concentration of specific substances in solution. It is particularly valuable in assays for proteins, nucleic acids, pigments, and other analytes that either absorb light directly or generate a measurable color change.

Protein quantification

Many absorbance measurements aim at determining the concentration of proteins in solution. Several colorimetric methods are available to measure the whole protein content of a sample. This is required to normalize protein samples before downstream applications such as western blots or immunoprecipitations. In our blog article “Protein measurement: find a suitable method” you will find detailed descriptions of Bradford, BCA and other methods and tips to help you choose.

Nucleic quantification

Nucleic acids and their components show natural absorbance at 260 nm. Without the need of any sample preparation or staining procedure absorbance measurements at this wavelength can be exploited to determine DNA/RNA concentration fast and easy. By taking additional measurements at other wavelengths such as 230 nm, 280 nm or 340 nm, nucleic acid purity can be evaluated on top.

ELISAs

Using absorbance, it is as well possible to quantify a specific protein in solution. A popular method is the enzyme-linked immunosorbent assay (ELISA). In brief, an antibody immobilized in the microplate well binds specifically to the protein of interest and captures it in the plate. A second antibody that is likewise specific for the protein of interest binds to the captured protein and can be recognized by secondary enzyme-bearing antibodies. Accordingly, the more protein of interest is present in the sample, the more enzymes will be bound in the microplate well. A substrate converted by the enzyme to a chromophore can finally be measured by absorbance and report on the amount of the specific protein present in the sample³.

Cell viability

Colorimetric assays that report on cell health are popular as they are quick and cost effective. When employing a microplate, many conditions can be tested at once. Absorbance-based methods mainly rely on the capability of metabolically active cells to reduce substrates such as MTT or Resazurin. Their reduced forms display absorbance at specific wavelengths and thus report on the metabolic activity of a cell sample, a characteristic often affected by cell viability⁴. Our cell viability blog post describes in detail colorimetric and other methods to measure cell health.

Microbial growth

The absorbance read mode of microplate readers and photometers determines the multiplication of bacteria and yeast by measuring the “optical density at 600 nm” (OD₆₀₀). Strictly speaking, it is light scattering⁵ and not absorbance that is being measured in this measurement mode for this application. The particles (bacteria or yeast) in microbial suspensions scatter light: the more microbes are present in solution, the more light is scattered. This also means less light reaches the detector and the measured transmission is lower. In this way, higher absorption is calculated for increased numbers of microbes, although this change is caused solely by an increase in light scattering instead of specific absorbance at 600 nm.

Frequently asked questions

References

1. IUPAC, Compendium of Chemical Terminology, 2nd ed. (the "Gold Book") (1997). Online corrected version:  (2008) "Absorbance". doi:10.1351/goldbook.A00028
2. IUPAC, Compendium of Chemical Terminology, 2nd ed. (the "Gold Book") (1997). Online corrected version: (2014) "Beer–Lambert law (or Beer–Lambert–Bouguer law)". doi:10.1351/goldbook.B00626
3. Crowther, J.R. (1995). Chapter 2: Basic Principles of ELISA. ELISA: Theory and Practice. Humana Press. pp. 35–62. ISBN 0896032795.
4. Riss, T. L. (2013) Cell Viability Assays. Assay Guidance Manual. Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences. https://www.ncbi.nlm.nih.gov/books/NBK144065/
5. Keiran Stevenson et al. (2016) General calibration of microbial growth in microplate readers. Scientific Reports volume 6, Article number: 38828 (2016) https://doi.org/10.1038/srep38828