Ocean Optics
Worldwide Headquarters
Largo, Florida, USA

+1 727-733-2447

info@oceanoptics.com

Sales, Service
& Support Facility
Duiven, The Netherlands

+31 26-319-0500

info@oceanoptics.eu

Ocean Optics GmbH Sales,
Service & Support Facility
Ostfildern, Germany

+49 711-34-16-96-0

info@oceanoptics.eu

Sales Support
for the
United Kingdom

+44 1865-811118

info@oceanoptics.eu

Sales, Service
& Support Facilities
Shanghai, PRC – Beijing, PRC

+86 21-6295-6600

asiasales@oceanoptics.com

Close
Google Translate:  Chinese (Simplified)EnglishFrenchItalianSpanish

Absorbance

AbsorbanceThe 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.

Advantages

  • Non-destructive: Unless the sample is photo-sensitive, the measurement can be repeated without altering the sample. Can be performed in-situ or within process flows.
  • Quantitative: Allows determination of solution concentration or the extinction coefficient of a substance.
  • Accurate: Can quantify absorbance to within 0.001 absorbance units when implemented properly.

Applications

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.

T + A + S = 1

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. Beers Law 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.

Transmission as a function of 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. Absorbance as a function of concentration 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 range.

Application Note:

Technical Notes:

Featured Products for Absorbance in Gases:

Absorbance of Gases

Maya2000 Pro This high-resolution spectrometer is configured for UV absorbance in the sample setup. We use Grating #H7 set for 200-300 nm, with a 5 µm slit as entrance aperture and a detector collection lens for increased sensitivity.
D-2000 Deuterium source produces continuous output from ~215-400 nm
CUV-UV-10 Cuvette holder for 10-cm pathlength cuvettes
CV-Q-100 Cylindrical quartz cell with Teflon stopper; has volume of 28.2 mL
QP455-025-XSR-BX Pair of extreme solarization-resistant patch cord assemblies, 455 µm diameter, 0.25 m length
OceanView Spectroscopy software

Featured Products for Absorbance in Solutions:

UV-Vis Absorbance of Solutions

USB4000-UV-VIS General-purpose spectrometer is preconfigured for 200-850 nm and has a 25 µm slit and order-sorting filter
DH2000-BAL Balanced deuterium tungsten halogen light source provides illumination from 215-2000 nm
CUV-UV This sturdy cuvette holder accepts 1 cm pathlength cuvettes
CV-Q-10 A quartz cuvette is recommended for UV applications in particular
QP450-2-XSR Two 450 µm extreme solarization-resistant optical fibers will transmit and receive light in this setup
Absorbance Standards (optional) NIST-traceable photometric absorbance standards for 200-450 nm (STAN-ABS-UV) and 400-900 nm (STAN-ABS-VIS) ranges
OceanView Spectroscopy software

What light source should I use for illumination?
What is the best sampling optic for my measurement?
What spectrometer should I use for detection?
Are there any integrated systems I can use?
Why do I need a reference measurement?
Do I need an absorbance standard?
Why is stray light important?
How do I use Beer's Law to calculate concentration or extinction coefficient?
Why isn't my calibration curve linear?
What's the difference between relative and absolute absorbance?
How do I take the best dark measurement?
How do I take the best reference measurement?
How do I make repeatable measurements?
How do I measure very low concentrations or low absorbance?
How do I measure very high concentrations or high absorbance?
Is there an easy way to make kinetics measurements in the software?
Should I correct for electrical dark?
Should I use the non-linearity correction?
Education Kits for Science

Education Kits for Science

Spectroscopy Experiment Kit for Teaching STEM Labs
Flame Spectrometer

Flame Spectrometer

High Thermal Stability, Interchangeable Slits
FLAME-CHEM Spectrometer Systems

FLAME-CHEM Spectrometer Systems

Compact Spectrophotometers for Educators and Lab Pros
JAZ Spectrometer

JAZ Spectrometer

Handheld Spectrometer for UV-Vis Measurements
SpecLine – Analysis Software

SpecLine – Analysis Software

Software for Compound Identification
FLAME-BUNDLE-ABS

FLAME-BUNDLE-ABS

Application-Ready System for Absorbance
USB2000+ (Custom)

USB2000+ (Custom)

Custom Configured Spectrometer for Setup Flexibility
STS Developers Kit

STS Developers Kit

Connect, Code, Create with New Sensing Tools
QE Pro (Custom)

QE Pro (Custom)

High-sensitivity Spectrometer for Low Light Level Applications
Maya2000 Pro (Custom)

Maya2000 Pro (Custom)

High Sensitivity Spectrometer
NIRQuest512

NIRQuest512

Small-footprint Spectrometer for Near-Infrared Measurements
STS-UV

STS-UV

UV Spectral Analysis in a Tiny Footprint
USB4000 (Custom)

USB4000 (Custom)

Custom Configured for Maximum Flexibility
Maya LSL Spectrometer

Maya LSL Spectrometer

Low Stray Light with High Sensitivity
NIRQuest512-2.5

NIRQuest512-2.5

Small-footprint Spectrometer for Near-Infrared Measurements
HR4000 (Custom)

HR4000 (Custom)

High Resolution Spectrometer for Maximum Flexibility
Maya2000 Pro-NIR

Maya2000 Pro-NIR

High-sensitivity Spectrometer for Raman and NIR Applications
STS-VIS

STS-VIS

Vis Spectral Analysis in a Tiny Footprint
QE Pro-ABS

QE Pro-ABS

High-sensitivity Spectrometer for Absorbance
STS-NIR

STS-NIR

NIR Spectral Analysis in a Tiny Footprint
USB2000+UV-VIS

USB2000+UV-VIS

Application-ready Spectrometer for the UV-VIS
EMBED Spectrometer

EMBED Spectrometer

Robust, Stable Spectrometer for OEM Applications
NIRQuest512-1.9

NIRQuest512-1.9

Small-footprint Spectrometer for Near-Infrared Measurements
USB2000+XR1

USB2000+XR1

Extended Range Spectrometer for UV-NIR applications
NIRQuest512-2.2

NIRQuest512-2.2

Small-footprint Spectrometer for Near-Infrared Measurements
USB-TC Temperature Controller

USB-TC Temperature Controller

Increase Thermal Wavelength Stability
USB2000+UV-VIS-ES

USB2000+UV-VIS-ES

Application-ready Spectrometer for the UV-VIS with Enhanced Sensitivity
NIRQuest (Custom)

NIRQuest (Custom)

Custom configured for Near-Infrared Measurements
USB2000+VIS-NIR

USB2000+VIS-NIR

Application-ready Spectrometer for the Visible and near-IR