The technique of absorbance is as old as the first alchemists. They sought to identify and understand their elixirs by looking at the color and opacity of solutions as different reagents were mixed, heated, and stirred.

Today it remains the most widely used spectroscopic technique for studying liquids and gases due to its simplicity, accuracy, and ease of use. An absorbance spectrum can be used as a qualitative tool to identify or “fingerprint” substances, or as a quantitative tool to measure the concentration of a molecule in solution.

The most common image of an absorbance measurement is a solution in a cuvette, measured in transmission with a dual-beam spectrometer – the classic introductory chemistry lab experiment. In practice, however, absorbance measurements can take many forms. They work equally well for gases as for liquids, and have found their way into consumer products and industrial applications alike. Samples no longer need to fit into the standard 1 cm pathlength cuvette, as flow cells, dip probes, micropipetters, folded gas cells, and micro-cuvettes allow the sampling optic to be customized to the sample.

Modular spectroscopy has provided infinitely more flexibility to choose the wavelength range and resolution needed, and to move between sampling optics quickly and easily for measurements in the lab or field. With our wide range of spectrometers, light sources, and accessories, we can help you to

create a flexible system to measure a wide range of solutions and concentrations. Are you ready to think outside the cuvette? Read on

Applications

• Research and Education
• Life Sciences
• Materials Identification
• Farm to Table Technologies
• Energy Technologies

Other Common Applications

• Kinetics: reaction monitoring, endpoint detection, protein and DNA thermodynamics, enzyme kinetics, on-line thermal cycling of biological particles
• Quality & process control: pharmaceutical and textile manufacturing, particle size analysis, ethylene production, polymer processing
• Chemical analysis: fluorophore characterization, phenol determination, column liquid chromatography, trace detection of metals
• Research: analysis of freshwater and marine environments, characterization of liquid crystals, study of eye tissues, photostability studies of compounds in various environments
• Environmental monitoring: SO₂ detection as a predictor of volcanic activity, fenceline monitoring near chemical plants, airborne pollution monitoring in cities, prediction of red tide events, soil contamination analysis, ozone monitoring
• Food testing: analysis of composition in dairy products, determining solids content in fruit, predicting odor and flavor suitability in wines
• Biomedical: reading microtiter plates and labs-on-a-chip, analysis of nucleic acids and proteins, clinical and in-vitro blood diagnostics

What Is Absorbance?

When light is incident on a sample in a cuvette, it can be transmitted, absorbed, or scattered. This is often written as T + A + S = 1. Transmission is the light that passes through the sample without interacting with it. Light that encounters a molecule or particle can be either absorbed or scattered. Elastic scattering occurs when the interaction changes the direction of light, but not its wavelength or energy.

When an absorption measurement is made, however, it is assumed that scatter is zero, in which case all light not transmitted to the detector is absorbed by the sample, i.e., T + A = 1.  This is true for the ideal case of an infinitely dilute solution of infinitely small particles in a transparent solvent. Luckily, it is also reasonably accurate in practice for a wider range of absorbing substances, solvents and concentrations. Absorbance occurs when the light encountering the molecule in the solvent matches the frequency of molecular vibrations or transitions in electronic energy-level states within the molecule. The chance of this happening is dependent on the cross section of the molecule for a particular energy level transition, and determines how absorptive a molecule is in solution. The more concentrated the solution, the greater the chance that a photon travelling through the solution will be absorbed. In fact, the probability of absorption increases linearly with both the pathlength and concentration of the solution, a relationship which has been quantified in the Beer-Lambert Law, also known as Beer’s Law.

What Is Beer’s Law?

Beer’s Law (also called the Beer-Lambert law) says that the absorbance of a solution will depend directly on the concentration of the absorbing molecules and the pathlength traveled by light through the solution.where

• A(λ) is the absorption of the solution as a function of wavelength            
• ε(λ) is the molar absorptivity or extinction coefficient of the absorbing molecule as a function of wavelength (in L/mol·cm)
• c is the concentration of the solution (in mol/L)
• l is the pathlength traveled by light through the solution (in cm)

But how can we determine the amount of light absorbed? By measuring the transmission through the sample. Provided the sample has low scatter (as with a relatively dilute, clean solution), almost all of the light not absorbed will be transmitted. Transmission is the ratio of incident intensity, I₀ to transmitted intensity, I, and will decrease with increasing path length or concentration.

By taking the negative log₁₀ of each side of this equation, we get a linear absorbance equation that is useful for calculations from measurements.This explains why absorbance is a dimensionless number that scales with concentration on a log scale. A perfectly transparent sample (T = 100%) will have an absorbance value of zero, while a perfectly opaque sample (T = 0%) will have an absorbance value of infinity. When units are specified, absorbance is usually described in terms of absorbance units (AU) or optical density (OD). The linearity of absorbance makes it conveniently additive.

For example, if one sample has an absorbance of 0.5 AU and another has an absorbance of 0.3 AU, then putting both samples in the light path in tandem will yield an absorbance of 0.8 AU. Similarly, if two different substances are present in the same sample, then the total absorbance will equal the sum of their individual absorbance values at that wavelength. It is important to keep in mind that many factors can affect the validity of Beer’s Law. Before using it to calculate the concentration of a solution or extinction coefficient of a substance, it is best to validate the relationship by measuring a set of standard solutions and plotting a calibration curve. The sweet spot for measuring absorbance with best accuracy is between 0.5 and 1.0 absorbance units, so aim to work in this range when choosing the pathlength of your sample cell, and create a calibration curve using concentrations across this rang