期刊
ANALYTICAL CHEMISTRY
卷 94, 期 14, 页码 5583-5590出版社
AMER CHEMICAL SOC
DOI: 10.1021/acs.analchem.1c05191
关键词
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资金
- COMET Center CHASE [868615]
- Competence Centers for Excellent Technologies programme by the BMK
- Federal Province of Upper Austria and Vienna
- European Union's Horizon 2020 research and innovation program through NutriShield project [818110]
- Austrian Research Promotion Agency (FFG) [874206]
- Austrian Science Fund FWF [P32644-N]
- Federal Province of Vienna
- Austrian Science Fund (FWF) [P32644] Funding Source: Austrian Science Fund (FWF)
- H2020 Societal Challenges Programme [818110] Funding Source: H2020 Societal Challenges Programme
In this study, an external cavity-quantum cascade laser-based mid-infrared spectrometer was used for in-line monitoring of proteins from preparative ion-exchange chromatography. Advanced background compensation strategies were implemented to obtain high-quality protein spectra, showing the potential to replace laborious off-line methods for protein monitoring.
In this study, an external cavity-quantum cascade laser-based mid-infrared (IR) spectrometer was applied for in-line monitoring of proteins from preparative ion-exchange chromatography. The large optical path length of 25 mu m allowed for robust spectra acquisition in the broad tuning range between 1350 and 1750 cm(-1), covering the most important spectral region for protein secondary structure determination. A significant challenge was caused by the overlapping mid-IR bands of proteins and changes in the background absorption of water due to the NaCl gradient. Implementation of advanced background compensation strategies resulted in high-quality protein spectra in three different model case studies. In Case I, a reference blank run was directly subtracted from a sample run with the same NaCl gradient. Case II and III included sample runs with different gradient profiles than the one from the reference run. Here, a novel compensation approach based on a reference spectra matrix was introduced, where the signal from the conductivity detector was employed for correlating suitable reference spectra for correction of the sample run spectra. With this method, a single blank run was sufficient to correct various gradient profiles. The obtained IR spectra of hemoglobin and beta-lactoglobulin were compared to off-line reference measurements, showing excellent agreement for all case studies. Moreover, the concentration values obtained from the mid-IR spectrometer agreed well with conventional UV detectors and high-performance liquid chromatography off-line measurements. LC-QCL-IR coupling thus holds high potential for replacing laborious and time-consuming off-line methods for protein monitoring in complex downstream processes.
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