Absorbance Measurement

Keywords: Absorbance, Measurement, Absorbing, UV, NIR

Absorbance is a process of light interacting with matter. When light hits something that takes some of the light we talk about absorbance. The light that is absorbed is not lost; it is transformed to heat or chemical energy as the absorbing molecule gets excited. Absorbance is a physical process we come across every day as we see colors: White light such as sunlight contains all colors of visible light. When it shines on the green grass, the grass absorbs all colors but green. The green light is emitted, and this is what gives the grass its color.

From UV to NIR and longer wavelengths, absorbance measurements provide valuable information about the chemical composition of materials in all states of matter in biology and chemistry. 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. These reactions depend 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

Light incident on a sample can be transmitted, absorbed or scattered. Transmission is the light that passes through the sample; light that encounters a molecule can be absorbed or scattered. Absorbance can be used as a qualitative tool to identify substances, or as a quantitative tool to measure the concentration of a molecule in solution.

Advantages of Absorbance

• Non-destructive measurements: Unless your sample is photosensitive, the measurement can be repeated without altering the sample.
• Quantitative results: Use absorbance to determine the concentration of solutions.
• Accuracy and repeatability: With proper tools and methods, absorbance can be quantified with great measurement-to-measurement precision.

Ocean Insight has the tools and expertise to configure spectral systems for hundreds of different absorbance applications. Configuring a system for general-purpose absorbance begins with a spectrometer like the Ocean HDX or the Flame, which both cover the UV-Vis range, or NIRQuest+ for the NIR. 

What is typically measured by absorbance?

Protein quantification

Many absorbance measurements aim at determining the concentration of proteins in solution as wekk as measuring whole protein content of a sample.

Nucleic quantification

Nucleic acids and their components show natural absorbance at range of wavelength. Without the need of any sample preparation or staining procedure absorbance measurements can be exploited to determine DNA/RNA concentration fast and easily as well as evaluate the nucleic acid purity.

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 viability.

Absorbance Video Tutorial

How to Set Up an Absorbance Measurement Watch how to set up our equipment and take an absorbance measurement. Featured products include the Flame spectrometer, a light source, sampling accessories and OceanView software. 

Using UV Absorbance for Continuous, Online Monitoring of Proteins 

Learn more about continuous, online monitoring of proteins using UV absorbance. See how we use an Ocean HDX high-sensitivity spectrometer to capture real-time protein concentration values, and then convert the data into answers that are meaningful to the user.