What is absorbance?
Absorbance (A), also known as optical density (OD), is the quantity of light absorbed by a solution. Transmittance is the quantity of light that passes through a solution. Absorbance and % transmittance are often used in spectrophotometry and can be expressed by the following:
Absorbance equation
A = Log10 (I0/I)
where I0 is the intensity of the incident light, and I is intensity of that light after it passed through the sample
T = I/I0 and %T = 100 (T)
The equation that allows one to calculate absorbance from % transmittance is
A = 2 - log10 (%T)
Determine concentration using the Beer-Lambert Law
The concentration of a sample can be calculated from its absorbance using the Beer–Lambert law, which is expressed as follows:
A = ε * c * p
Where ε is the molar absorptivity, or molar extinction coefficient, in L mol-1 cm-1
c is the concentration of the solute in solution, in mol/L
p is the path length of the sample, in cm, for example 1 cm for a cuvette

Ultraviolet (UV) measurements in microplates became possible when Molecular Devices introduced the first UV-capable microplate reader. Since then, the microplate measurements of DNA, RNA, and proteins that this enabled have become very popular. Learn more about how absorbance is measured, and some key applications that utilize absorbance.
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Exploring absorbance based assay applications: from virus to cannabis research
Absorbance reading with SpectraMax® microplate readers and SoftMax® Pro Software enables optical density measurement of samples in microplate or cuvette format. The powerful analysis tools in the software generate a standard curve and calculated data. For the GMP/GLP regulated labs, the systems come equipped with our SoftMax Pro GxP compliance software.
Key highlights:
- How does absorbance detection work?
- Detect immunoglobulins (Ig) with ELISA – More ways to investigate infectious diseases and immune responses
- Streamline analysis of beer and wine measuring absorbance
- Determine total aflatoxin in cannabis using an ELISA

Absorbance Overview
Absorbance definitions
Absorbance applications
- Cell Viability, Cytotoxicity, Cell Proliferation (MTT, XTT, MTS)
- DNA/RNA Quantitiation
- Enzyme Kinetics, Bacterial / Microbial Growth
- Endotoxin Assays
- ELISA / Immunoassays (Quantifying antigens, cytokines, etc.)
- Micro-volume Applications
- Optical density measurements using PathCheck Technology
- Protein Quantitation (BCA, Bradford, Lowry assay)
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How does absorbance detection work?
Spectrophotometers and absorbance plate readers measure how much light is absorbed by a sample. Microplate readers that are capable of detecting light in the ultraviolet (UV) range can be used to determine the concentration of nucleic acids (DNA and RNA) or protein directly, without the need for sample labeling. Light of a certain wavelength, dependent on the material being measured, is passed through a sample, and a detector on the other side of the microplate well measures how much of the original light was absorbed by the sample in the well.
What is absorbance measured in?
Absorbance is measured using a spectrophotometer or microplate reader, which is an instrument that shines light of a specified wavelength through a sample and measures the amount of light that the sample absorbs.
Spectrophotometer vs. Plate Reader
A standard spectrophotometer measures absorbance one sample at a time, typically placed in a cuvette through which light is sent horizontally. An absorbance plate reader offers higher throughput and can measure the absorbance of samples in microplates, usually 96-well or even 384-well, by sending light through each well vertically.
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How to run an absorbance ELISA protocol?
An ELISA, or enzyme-linked immunosorbent assay, is a method used to quantitatively detect an antigen within a sample. Most ELISAs are run in microplates, with the bottom of the microplate wells serving as the solid surface to which an antigen of interest attaches, either directly or via an antibody. An enzyme conjugate, which reacts with substrate to produce a colored solution, is used to detect the antigen. A microplate washer is used to wash away non-specific, unbound material in the wells, and an absorbance ELISA plate reader detects the color change produced when target antigen is present.
How does stray light affect my optical density (OD) reading?
Stray light is a general term for unwanted light reaching the detector of an instrument. The effect of stray light on an absorbance reading is often an unexpectedly low OD; the absorbance measured is lower than the true absorbance of the sample. The impact is usually on linearity and is greatest when measuring ODs above 2.0-2.5. Stray light is not user-correctable and is usually not caused by user error, with possible causes including optical components like degraded excitation filters. Spectrophotometers and absorbance plate readers typically incorporate features to minimize stray light.
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Cell Viability, Cell Proliferation, Cytotoxicity Assays (MTT, XTT, MTS)
Colorimetric assays using tetrazolium salts like MTT, XTT, and MTS can be used to measure cell proliferation and cytotoxicity. For example, the MTT assay depends on the reduction of MTT by enzymes present in viable cells to form a blue formazan product that can be quantified by measuring the absorbance. The choice of assay may be based on the desired workflow and time required.
Read the application note to learn about some of these methods:
ELISA / Immunoassays (Quantifying antigens, antibodies, cytokines...)
Enzyme-linked immunosorbent assays (ELISAs) are used to measure the amount of a specific protein, typically in a microplate format, and results are most often detected via absorbance in the visible wavelength range.
Here are several application notes about ELISAs you may find of interest:
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DNA/RNA Quantitation
The absorbance of a DNA sample measured at 260 nm on a spectrophotometer or microplate reader can be used to calculate its concentration. Absorbance quantitation of DNA works on samples ranging from about 0.25 µg/mL to about 125 µg/mL in a microplate format.
Learn how absorbance is measured on our absorbance microplate readers with our featured app notes:
Customer story: Absorbance plate reader tests DNA/RNA-synthesizers
OligoMaker ApS uses the SpectraMax 190 reader to test DNA/RNA-synthesizers
"We use the SpectraMax 190 as an important step in testing the DNA/RNA-synthesizers we build. We have to quantify the primers and probes to determine how much DNA/RNA is synthesized on the DNA/RNA Synthesizer.”
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Endotoxin assays
Monitoring for contaminants is a critical step during the production process in the pharmaceutical and medical device industries. A frequent contaminant, endotoxin, can cause fever, inflammation, headache, nausea, and even death. Fortunately, endotoxins can be readily monitored using turbidimetric, colorimetric, or fluorometric assays.
These application notes describe the different types of endotoxin assays:
Enzyme kinetics, bacterial / microbial growth
Many biological experiments require monitoring bacterial growth or measuring enzymatic changes over long periods of time (hours, days or even weeks).
Here are a few featured application notes illustrating how to set up kinetic assays to measure bacterial growth using our instruments and software:
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Customer story: Absorbance plate readers used in biofilm research
Université Catholique de Louvain uses our SpectraMax absorbance and multi-mode readers to help fight biofilms
“Another great advantage” says Wafi Siala of the UCL “is the large choice of microplate sizes from 6 to 384 wells which the SpectraMax M series offers.”
Micro-volume Applications
Quantitation of many sample types is accomplished through absorbance measurements in a cuvette or microplate. However, when material is scarce or samples are precious, quantitation of very low sample volumes is required. Instruments and accessories are available that enable quantitation in volumes as low as 2 µL, or even 0.5 µL.
Learn more about our micro-volume instruments and techniques:
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Optical density measurements using PathCheck Technology
UV/VIS spectrophotometers and microplate readers differ fundamentally in their beam geometry. In spectrophotometers, samples are read through cuvettes or tubes with a horizontal, 1-cm light path, making assays based on extinction coefficients straightforward. In microplate readers, the vertical light beam results in a pathlength that depends on the volume of fluid in each well. This problem has been remedied with Molecular Devices PathCheck Technology, which automatically corrects optical density (OD) measurements to a 1-cm pathlength.
These application notes discuss the principles and uses of PathCheck Technology:
Protein Quantitation (BCA, Bradford, Lowry assays)
Protein concentration can be measured directly, via absorbance at 280 nm in a UV spectrophotometer, or indirectly, using colorimetric methods such as BCA or Bradford assays.
Here are a few application notes on protein quantitation you may find of interest:
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