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. 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:.
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.
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. Protein concentration can be measured directly, via absorbance at 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:. The regulations for food and drug in the United States, described in the Title 21 of the Code of Federal Regulations, are critical in ensuring safe and ethical drug administration. View Blog. Absorbance microplate readers are widely used in research, drug discovery, bioassay validation, quality control, and manufacturing processes in the pharmaceutical, biotech, food and bev…. Download eBook.
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. Read Application Note. 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.
Nucleic acid and protein quantitation are essential measurements upstream of many sophisticated assays in genetics and molecular biology. Various methods have been developed to…. 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…. 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…. Absorbance microplate readers are widely used in basic research, drug discovery, bioassay validation, quality control, and manufacturing processes in pharmaceutical, biotech, food and b….
Learn how absorbance microplate reader provides a colorimetric readouts for cell viability or proliferation. 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.
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…. 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…. In spectrophotometers, samples are read through cuvettes or tubes with a horizontal cross…. Endpoint readers are prolific in the laboratory since absorbance has become the detection of choice for many applications.
A frequent contaminant, endotoxin, can cause fever,…. In biology and chemistry, the principle of absorbance is used to quantify absorbing molecules in solution. Many biomolecules are absorbing at specific wavelengths themselves. 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. Further below, many examples for absorbance measurements are given, from measuring protein concentration to cell viability. Traditionally, absorbance measurements were performed in a cuvette: A solution with an analyte of known absorbance characteristics is placed into a cuvette.
An absorbance reader then determines the absorbance by sending light with known intensity through the sample and detecting the intensity behind the sample. Light that did not make it through to the detector was either absorbed or scattered. The scattered part is determined separately by measuring appropriate blanks and is subtracted from this value to obtain pure absorbance of the substance of interest.
The portion of light that is able to pass the sample is also called transmission and is mainly given as percentage Fig. The more analyte is found in solution, the more light is absorbed by it and the lower is the transmission. The absorbance, however, is the part of the light that was taken up by the analyte. It is the absolute value of the logarithm to the power of 10 of the transmission 1.
Here are the mathematical equations and some numbers to explain what is described by transmission and absorbance:. After performing an absorbance measurement the result is a value given in either transmission or optical density. However, the goal of the measurement is the quantification of a substance in solution, the obvious question is how to convert the signal into the concentration value. Generally, there are two ways: by employing the Beer-Lambert law or by measuring a standard curve in parallel to samples of unknown concentrations.
The Beer-Lambert law describes the relation of absorbance, path length and concentration of an absorbing substance:. It says absorbance is linear to the concentration multiplied by the path length and extinction coefficient 2.
The path length refers to the length of sample the light has to go through. For instance, in a cuvette the path is standardized to 1 cm. The extinction coefficient is a constant specific for an absorbing substance and a specific wavelength, typically the absorbance maximum of the substance. It provides information on how strongly the specific substance absorbs light at the specific wavelength. As an example, the mass extinction coefficient for bovine serum albumin BSA is 0.
The Beer-Lambert law is very helpful as it allows quantification of absorbing substances without the need to add any other reagents. However, it is limited as outlined below. For instance, colorimetric protein quantification assays such as the Bradford assay depend on a substance that increases absorbance in presence of proteins. The increase is measured in a standard curve with known protein concentrations as well as in samples, so that their concentration can be calculated.
Different light sources can be used for absorbance measurements. They differ in the spectral range they cover, in their intensity and in the stability of the light they emit. Tungsten Halogen lamps cover the range from nm to more than nm and are often used due to their cost-effectiveness. Xenon flashlamps, however, cover the spectral region from nm to nm and cover the UV-range of the spectrum.
This makes the detection and quantification of nucleic acids nm and proteins nm possible. BMG LABTECH microplate readers are equipped with a xenon flashlamp for absorbance measurements and hence provide maximum flexibility to measure any wavelength between and nm, or all the whole spectrum of wavelengths. The liquids of interest need to be transferred either to a cuvette or to a microplate in order to measure their absorbance.
The material of cuvette and microplate is always clear to ensure maximum light transmission, as absorbance of the solution and not of the material is of interest for the researcher. Still, various materials are common and differ from one to another. Polystyrene is most often used and may be altered to provide specific binding characteristics, either for cell culture or ELISA applications. However, they are non-transmissive in the UV-range which requires the use of special plates of cyclic olefin copolymer to measure absorbance below nm.
A second aspect to consider is the sample volume that is needed for reliable absorbance measurements. Standard cuvettes require approximately 4.
The cuvette measurement is horizontal: light is directed from one side into the sample and on the opposite side the transmitted light is detected. Therefore, the geometry of cuvettes provides a standardized path length of 1 cm. This is one of the major differences to microplate measurements as they run vertically top to bottom or bottom to top.
The FS5 Fluorescence Spectrometer enables transmission and absorbance measurements of liquid samples in the wavelength range nm nm and optical densities of OD 0. Download here: Transmission and Absorbance of Liquid Samples. If you have enjoyed reading this Technical Note, and want to be the first to see the latest news, applications, and product information from Edinburgh Instruments, sign-up to our infrequent newsletter via the red sign-up button below, and follow us on social media.
For more information the LP or to enquire about our other high-end instrumentation, why not contact a member of our sales team at sales edinst. Instrument and Procedure for Absorbance Measurements The FS5 Fluorescence Spectrometer is equipped with a silicon detector that allows for transmission and absorbance measurements. FS5 Spectrofluorometer from Edinburgh Instruments. Results Figure 1 shows the transmission spectra of fluorescein samples at different concentrations measured in an FS5 fluorescence spectrometer.
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