DWD-1 UV detector (high-performance dual beam dual wavelength) with built-in data processing

Wavelength range: 254nm, 280nm (simultaneous detection), and select two wavelengths between the 254nm, 280nm-400nm spectral lines for simultaneous detection.

*DWD-1 Instrument Features

Protein 280nm/254nm dual wavelength measurement:

Aromatic amino acids such as tyrosine and tryptophan containing conjugated double bonds in protein molecules. They have the property of absorbing ultraviolet light, with the absorption peak at 280nm wavelength, and the optical density value of the absorption peak within this wavelength is proportional to its concentration. Therefore, it can be used as a basis for qualitative and quantitative determination of proteins. Therefore, the ultraviolet absorption method at 280nm is usually used for continuous monitoring of protein concentration in chromatography systems. However, due to the different contents of tyrosine and tryptophan in various proteins, accurate quantification requires the use of pure samples of the protein to be tested as standards for comparison, or the extinction coefficient of the protein already known as a reference. In addition, many non protein substances also have a certain absorption capacity at a wavelength of 280nm, which may cause interference. Among them, the impact of nucleic acids (purine and pyrimidine bases) is particularly severe. The absorption at 280nm is 10 times stronger than that of proteins (per gram), but the absorption of nucleic acids is stronger at 254nm, with a peak around 254nm. The extinction coefficient at 254nm for nucleic acid is twice that at 280nm,

Proteins, on the other hand, have a higher UV absorption value at 280nm than at 254nm.

usually：

The light absorption ratio of pure protein: A280/A254>1.8

Light absorption ratio of pure nucleic acid: A280/A254<0.5

Therefore, when nucleic acids are present simultaneously in protein solutions (as is the case in most biological systems), both OD254nm and OD280nm must be measured simultaneously. Then, based on the ratio of absorbance of two wavelengths, the true protein content is calculated by correcting it through empirical formulas to eliminate the influence of nucleic acids.

Protein concentration=1.45 × A280-0.74 × A254 (mg/ml)

This empirical formula is established based on data obtained from a mixture of proteins (yeast enolase) and nucleic acids (yeast nucleic acid) at known concentrations and ratios.