# Absorbance

## 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.

## ON-DEMAND WEBINAR

#### 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:

Register now

• ## 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.

• ## 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.

• ## 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:

• ## 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.”

— Karl Ross-Petersen

• ## 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:

• ## 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.

• ## 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:

• eBook

## ELISA 101

ELISA methods have become widely used across various fields of biology, from healthcare and food safety to cancer research and vaccine development. Whether you need to measure gluten in…

• Publications

## The Best Microplate Readers According to our Product Reviewers

According to Biocompare, “one of the most overlooked pieces of equipment in any lab is the microplate reader. While they play an important role in generating data from a wide range of a…

• Application Note

## Detect endotoxin with the Kinetic-QCL Kinetic Chromogenic LAL Assay

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,…

• eBook

## Virology and Vaccine Research Microplate Reader Solutions

We understand what a challenging time this is for those who are working to get accelerated testing and medicines to the market as soon as possible and are committed to supporting scient…

• Flyer

## Become compliant with SoftMax Pro GxP Software

SoftMax Pro GxP Software is the latest, most secure software to achieve full FDA 21 CFR Part 11 and EudraLex Annex 11 compliance with streamlined workflows to ensure data integrity. Eve…

• Blog

## COVID-19 Timeline: Diagnostics, Vaccines, and Therapeutic Antibody Development

Global research efforts are focused on understanding the SARS-CoV-2 virus in order to develop potential therapies for COVID-19. Join us as we explore a scientific timeline of key…

View Blog

View Blog

• eBook

## The Ultimate Guide to Microplate Reader Solutions

Evaluating microplate readers doesn’t have to be overwhelming. First, consider your application needs. If your budget is modest, a single-mode reader dedicated to your main application…

• Publications

## Project Profile: Organoid Innovation Center

Lab Manager speaks to Dan O’Connor, vice president, drug discovery, Molecular Devices, about the company’s Organoid Innovation Center in San Jose, CA. The center is 180 sq. ft., within…

• Publications

## Shaping the Future of Organoid Research

Molecular Devices, a provider of innovative life science technology, recently unveiled a brand new, first-of-its-kind Organoid Innovation Center. Situated at the company’s global headqu…

• Blog

## FDA 21 CFR Part 11 and the importance of regulatory compliance in GMP and GLP labs

The regulations for food and drug in the United States, described in the Title 21 of the Code of Federal Regulations, and the EudraLex Annex 11 in EU, are critical in ensuring safe…

View Blog

View Blog

• Blog

## Celebrating International Women’s Day

Monday, March 8, 2021 is International Women’s Day (IWD). The theme of this year’s IWD is #ChooseToChallenge--encouraging us to call out gender bias and inequality and to seek out…

View Blog

View Blog

• Publications

## Multiple Steps from Microplate-Reader Workstations

Scientists take many roads to overcoming bottlenecks in workflows. For example, a workstation including a microplate reader can handle multiple steps, which improves efficiency and adds…

• Blog

## The history of ELISA from creation to COVID-19 research

Over the past three decades, ELISA or enzyme-linked immunosorbent assay, has become vital to many areas of research and has shown to have many applications—from detecting food and…

View Blog

View Blog

• eBook

## Streamline beer, wine, and food quality control and safety analyses

Absorbance microplate readers are widely used in research, drug discovery, bioassay validation, quality control, and manufacturing processes in the pharmaceutical, biotech, food and bev…

• eBook

## Streamline absorbance assays for nucleic acid & protein quantitation

Absorbance microplate readers are widely used in basic research, drug discovery, bioassay validation, quality control, and manufacturing processes in pharmaceutical, biotech, food and b…

• Application Note

## Quantifying gluten in beer using an ASBC-approved ELISA method

With the recent rise in the prevalence of celiac disease, monitoring gluten levels in food and beverage has become increasingly important as more people strive to avoid gluten.

• Application Note

## Assess cell viability and proliferation with colorimetric readouts

Learn how absorbance microplate reader provides a colorimetric readouts for cell viability or proliferation.

• Application Note

## Measure total protein in cell lysates with SpectraMax ABS Plus Microplate Reader

Quantification of protein concentrations from cell lysates is a key step for many downstream applications, such as western blots and enzyme-linked immunosorbent assays (ELISAs).

• Application Note

## Streamline BCA-based protein quantitation on the SpectraMax iD5 reader

Learn how to quantitate a cellular protein sample with BCA protein assay kits on the SpectraMax® iD5 Multi-Mode Microplate Reader.

• Publications

## Molecular Devices introduces enterprise-level SoftMax Pro 7.1 GxP Software

Built on its current, industry-leading data acquisition and analysis software, SoftMax Pro 7.1 GxP Software expands its system audit trail functions to include date and time stamps; ena…

• Application Note

## Advanced kinetic analysis of a bacterial growth assay

Difficult-to-kill bacteria have become a problem in hospital-acquired infections. Identifying compounds which kill these bacteria is of interest to many pharmaceutical companies.…

• Application Note

## Quantitate interleukin-8 concentrations on the SpectraMax ABS Plus Microplate Reader with the SimpleStep ELISA kit

Learn how to run the Human IL-8 SimpleStep ELISA on the SpectraMax® ABS Plus Microplate Reader using a convenient preconfigured protocol.

• Application Note

## Low-volume, high-throughput DNA and protein detection on the SpectraMax ABS Plus Microplate Reader

Nucleic acid and protein quantitation are essential measurements upstream of many sophisticated assays in genetics and molecular biology. Various methods have been developed to…

• Application Note

## Measure long-term cell growth using a discontinuous kinetic reading

Many biological experiments require monitoring cell growth or measuring enzymatic changes over long periods of time (hours, days or even weeks). In addition, certain model organisms…

• Application Note

## DNA and RNA absorbance measurements using SpectraMax Microplate Readers

Ultraviolet (UV) measurements in microplates became possible when Molecular Devices introduced the first UV-capable microplate reader. Since then, microplate measurements of DNA, RNA,…

• Application Note

## Maximizing performance of the SpectraDrop Micro-Volume Microplate for DNA quantitation

Molecular Devices SpectraDrop™ Micro-Volume Microplate allows users to read up to 64 samples per plate on SpectraMax® Microplate Readers, with sample volumes as low as 2 µL. The…

• Application Note

## High-throughput melamine detection with Romer Labs AgraQuant Melamine Assays

The organic base melamine is used to make a number of products, including plastics, flame retardants, pigments, and fertilizers. The practice of adding melamine to animal feed and…

• Application Note

## Optical density measurements automatically corrected to a 1-cm pathlength with 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 (cross…

• Application Note

## Protein quantitation with the EMax Plus Microplate Reader

Endpoint readers are prolific in the laboratory since absorbance has become the detection of choice for many applications. Examples include ELISAs for quantitation of cytokines and…

• Application Note

## Microplate-based endotoxin testing in GLP/GMP environments using a colorimetric assay

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,…

• Application Note

## Phenolic compounds measurement in red wines

Measurement of tannin, iron-reactive phenolics, anthocyanin and polymeric pigment in red wine is an important part of quality control in the wine industry. Precise and reliable…

• Application Note

## Direct protein quantitation using the SpectraDrop Micro-Volume Microplate

Quantitation of protein using UV absorbance is a rapid, nondestructive method that relies on the absorbance of near-UV wavelengths of light by the tryptophan and tyrosine residues in a…

• Application Note

## Multitask kinetic measurements of IPTG’s effects on protein expression and cell growth

Measuring several different signal outputs simultaneously over time is especially useful for studying a protein or compound’s effect on cellular growth or gene expression. In this…

• Application Note

## Nucleic acid quantitation and analysis using the QuickDrop Spectrophotometer

Spectrophotometry is a well-established technique used to quantitate and analyze biological substances. Of these substances, nucleic acids are one of the most routinely measured in…