The advantage of direct measurement is the speed of the method, as samples simply need to be inserted into the photometer, and results are displayed instantly. In addition to the quantitative result, the shape of the scanning curve reveals information on the quality of the sample.
Relatively high sample concentrations are required for quantification using the direct method. High sample purity is further crucial in order to guarantee accurate determination of the sample concentration as other organic substances, which may be present as contaminants, frequently also absorb light in the UV-range. Since contamination can never be completely ruled out, quantification methods that rely on UV light will always be somewhat prone to interference.
Possible direct quantification methods:
All methods introduced herein are based on measurements using a cuvette with a path length of 1 cm.a) Quantification using the specific absorbance coefficient at 280 nm (1):
Absorbance at 280 nm is based foremost on the presence of amino acids with aromatic residues within the protein, such as tryptophan and tyrosine and, to a lesser extent, phenylalanine. The protein to be measured must be present as a homogeneous solution.
Calculations are performed using the formula C = A x F (C = concentration of the protein; A = measured absorption of the sample using the photometer; F = specific protein factor).
The factor is calculated from the specific absorption coefficient (l/(mol*cm)
) and the molecular weight of the protein in Dalton (g/mol): FProtein (mg/mL)= M/Ɛ
If the amino acid sequence of the protein is known, the molecular weight, as well as the extinction coefficient, may be defined using certain online programs, for example: https://web.expasy.org/protparam/
In the Eppendorf BioPhotometer®
and Eppendorf BioSpectrometer®
, the factor F may be determined by using an integrated calculation tool, taking into consideration the extinction coefficient as well as the molecular mass of the protein (figure 2).