Understanding the keywords and background of the absorbance-based methods.
Knowledge Base
Absorbance
Understanding the keywords:
Optical Density (OD) vs. Absorbance
Optical density (OD) and absorbance are terms used in spectrophotometric measurements to understand how light interacts with a solution. Both measure how light passes through a sample and can be calculated using the equation:
Absorbance (A) = Log10(I0/I); I0 = incident light intensity, I is the transmitted light intensity.
While they seem similar, OD and absorbance have some differences:
- Optical density (OD) refers to the extent to which a substance impedes or delays the passage of light. It encompasses both light absorption (where the material absorbs light energy) and light scattering (where light is redirected by the substance). OD is dimensionless and expressed as a ratio.
- Absorbance, on the other hand, precisely quantifies the amount of light absorbed by a substance at a specific wavelength. It solely accounts for light absorption and, like OD, has no units, as it is also expressed as a ratio.
In practice, OD and absorbance are often used interchangeably when light scattering is minimal. However, it's important to note their differences when interpreting data or comparing results.
Transmittance
Absorbance complements transmittance by showing how much light the substance absorbs and retains as energy. Absorption decreases transmission and can be calculated using a logarithmic function of transmittance:
Absorbance (A) = Log10(T)
E.g., If you have a clear glass window, it lets a lot of light through, so it has high transmittance. But if you have a thick foggy window, it doesn't let much light through, so it has low transmittance. So, transmittance tells us how much light can get through a material.
Understanding Spectrophotometry
How does a spectrophotometer measures absorbance:
- The spectrophotometer shines light with different wavelengths.
- Inside the spectrophotometer, there's a special filter called a monochromator. It picks out just one specific wavelength. For example, if we're measuring bacteria at OD600, it only lets through light with a wavelength of 600 nanometers (nm).
- This selected wavelength passes through the sample we're testing.
- On the other side of the sample, there's a detector that measures how much light goes through the sample and how much gets absorbed by it.
- The spectrophotometer shows this as a percentage called transmission (%T). It compares how bright the light was before it went through the sample (I0) to how bright it is after (I).
- Finally, we can calculate absorbance from the transmission percentage.
How to obtain concentration from absorbance:
This can be calculated over the Beer-Lambert Law, which is a fundamental principle that describes the relationship between light absorption and the properties of a substance.
- A = εlc ; A = absorbance (dimensionless), is the molar absorptivity coefficient (M⁻¹cm⁻¹), l is the path length of the light through the solution (cm), c is the concentration of the absorbing sample (M).
Why is transmittance not usually used for concentration determination?
Beer-Lambert's Law links absorbance to concentration in a linear way, making it easier and more accurate to calculate concentration. In contrast, transmittance, which decreases as concentration increases, doesn't show a proportional relationship with concentration. This makes absorbance a preferred choice for concentration calculations in Beer-Lambert's Law
However, there is a threshold for the concentration calculation with the Beer-Lambert’s law. At high concentrations, molecules pack closely, causing stronger interactions and light scattering. Beer's Law predicts a linear relationship between absorbance and concentration, but these interactions disrupt it. This deviation occurs notably beyond a range of about 0.1-0.5 OD units.
Understanding Turbidity vs. Colorimetry
Turbidity measurements and colorimetric measurements serve different purposes in analytical chemistry. Turbidity refers to the cloudiness or haziness of a liquid caused by suspended particles that scatter light, while colorimetric measurements involve the detection and quantification of color changes in a solution.
Turbidity measurements play a crucial role, especially in bacterial, cell culture, and yeast assays, where optical density at 600 nm (OD600) serves as a common indicator of cell density. Turbidity measurements provide a rapid and convenient method to estimate cell concentration by assessing the scattering of light by cells suspended in a solution. OD600 readings are widely utilized to monitor cell growth, evaluate cell viability, and gauge the efficacy of cell culture processes. In addition, this method is commonly employed in environmental monitoring, water quality analysis, and industrial processes where the presence of particulate matter needs to be monitored.
Colorimetric measurements, on the other hand, are also utilized in microbiological assays for various purposes, such as quantifying specific biomolecules (e.g., proteins, nucleic acids) or detecting enzymatic activity. Colorimetric assays offer high sensitivity and specificity, allowing for precise quantification in complex biological samples.
Absorbance-based experimental procedures
Experimental procedures for absorbance-based assays with a microplate reader cover a range of applications in biochemical and cellular analysis:
- Protein assays: Protein quantification assays, such as the Bradford assay or the bicinchoninic acid (BCA) assay, measure the concentration of proteins in a sample based on colorimetric changes resulting from protein-dye interactions. A microplate reader is used to measure the absorbance of the colored product, allowing for precise quantification of protein concentrations.
- Cell viability assays: They assess the viability or metabolic activity of cells in culture. Common assays include the MTT assay or the resazurin assay, where the reduction of a colorless substrate to a colored product by viable cells is measured spectrophotometrically using a microplate reader. Absorbance measurements provide information about cell proliferation, cytotoxicity, or drug efficacy.
- Enzyme-linked immunosorbent assay (ELISA): widely used technique for detecting and quantifying specific antigens or antibodies in biological samples. In ELISA, antigen-antibody interactions are detected using enzyme-linked antibodies and a chromogenic substrate that produces a colored product. The intensity of the color is proportional to the concentration of the target molecule, which can be measured with a microplate reader.
- OD600 measurements: commonly used as a measure of cell density in microbial cultures. It provides a rapid and non-destructive method for monitoring cell growth and assessing cell viability. A microplate reader equipped with an absorbance module allows for precise measurement of OD600 values in microplate wells, enabling high-throughput analysis of microbial growth kinetics.
Overall, absorbance-based assays performed with a microplate reader offer versatile and sensitive methods for quantifying proteins, assessing cell viability, detecting specific molecules, and monitoring microbial growth, making them invaluable tools in biochemical and cellular research.