The use of a UV-Vis spectrometer for determining the concentration of proteins in a solution is one of the fundamental methods in bioanalytics. All previous methods have utilized the Beer-Lambert law to determine concentration based on measured absorbance at a single wavelength and a constant optical path length. Due to the limitations of the Beer-Lambert law, solutions need to be diluted. Dilution can have a significant impact on the optical properties of samples, the accuracy of acquired results, and is time-consuming. Therefore, we have developed a new method based on the modified Beer-Lambert law, in which we vary the optical path length - i.e., the optical path is variable. To precisely vary the optical path, we use the SoloVPE system. As part of this master's thesis, we compare measurements where the optical path is varied, with existing systems where the optical path is constant. The results obtained using the variable optical path are compared with results obtained from classical UV-Vis methods, high-throughput UV methods, and HPLC methods. The comparability of the accuracy of the measured results was verified on antibodies, denatured proteins, structural proteins, enzyme reaction products, and Polysorbate 80. We also verified the accuracy of the measurements at different wavelengths. The measurement results showed reproducibility (RSD ≤ 2% between measurements of the same samples). The relative difference against the results obtained with existing UV-Vis methods was generally ≤ 3%. At high concentrations, the new system was shown to be more accurate (deviation from the theoretical value ≤ 5%, with existing systems ≤ 20%). The system proved to be comparable to existing systems for determining protein concentration in a solution. Only in the determination of the concentration of enzyme reaction products were the results non-reproducible. Therefore, it would be sensible to conduct additional research and determine the causes of the non-reproducibility.
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