UV-Vis spectroscopy in plate reader and cuvette
Coriolis Pharma is specialized in providing contract laboratory services in protein analysis, including a variety of spectroscopic methods.
UV-VIS spectroscopy is a versatile analytical methodology with numerous applications. For proteins, measuring absorbance at 280 nm can be used to directly determine protein concentration according to the Beer-Lambert law:
Absorbance = ɛ x c x l
ɛ = extinction coefficient (ml x mg-1 x cm-1)
c = protein concentration (mg x ml-1)
l = path length (cm)
UV-VIS spectroscopy can also be used to measure protein concentration by using selective colorimetric agents (e.g., BCA assay), for instance, when the extinction coefficient of the protein is not known or when excipients interfere with 280-nm measurements. Other applications of UV-VIS spectroscopy include, amonst others, turbidity measurements (e.g. aggregation index), enzymatic activity measurements, free thiol content determination (Ellman’s reagent), and peroxide concentration measurements.
Coriolis-Pharma has two different spectrophotometers: an Agilent 8453 UV-Vis instrument and a Tecan Safire2 plate reader. Both instruments are capable of full wavelength scans from 190-1100 nm.
The Agilent 8453 can perform spectral reprocessing, such as plotting the second derivative of the full spectrum. This can be useful for obtaining information about protein conformational changes. In addition, the Agilent 8453 uses cuvettes with accurate fixed path lengths (typically 1 mm or 1 cm). This set up allows for the most accurate and precise concentration determinations. The primary limitation of the instrument is that it is a low-throughput system, since only a single sample can be measured at a time.
The Tecan plate reader is a high-throughput system that can measure multiple samples in single set-up (96-well plates). Some accuracy is sacrificed since the path length is not fixed. Path length corrections for each well are used, which makes the loss in accuracy manageable.
Our team of expert scientists can advise which instrument is most appropriate/cost effective for a given application.