New spectrophotometers and cuvettes have been designed to allow the measurement of absorbance values from samples using microliter volume sizes. These measurements are done using short pathlengths to decrease the sample volumes required. The major applications for these spectrophotometers and cuvettes are samples that are difficult to obtain in large amounts, such as proteins and nucleic acids that absorb in the UV range. Existing UV-absorbance standards have been designed for longer pathlength measurements. SRM 2082 was developed to validate the pathlength calibration of these cuvettes and instruments using materials with absorbance spectra that are similar to the most commonly used samples. SRM 2082 consists of three components, a blank buffer solution, a solution of the amino acid tryptophan in the buffer, and a solution of the nucleobase uracil in the buffer. The tryptophan solution has an absorbance spectrum (peak at 280 nm) similar to proteins and the uracil has an absorbance spectrum (peak at 260 nm) similar to nucleic acids. The absorbance of these solutions were determined using a series of cuvettes with calibrated pathlengths from 0.1 mm to 2 mm. The effects of temperature and spectral bandwidth variations on the absorbance values of SRM 2082 were also investigated.
Journal of Research (NIST JRES) -
absorbance, standard, pathlength, certified reference material, UV visible, protein, nucleic acid, spectrophotometer