<div class="csl-bib-body">
<div class="csl-entry">Akhgar, C. K., Ebner, J., Spadiut, O., Schwaighofer, A., & Lendl, B. (2022). QCL-IR Spectroscopy for In-Line Monitoring of Proteins from Preparative Ion-Exchange Chromatography. <i>Analytical Chemistry</i>. https://doi.org/10.1021/acs.analchem.1c05191</div>
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dc.identifier.issn
0003-2700
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dc.identifier.uri
http://hdl.handle.net/20.500.12708/19832
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dc.description.abstract
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 μm 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 β-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.
en
dc.description.sponsorship
Fonds zur Förderung der wissenschaftlichen Forschung (FWF)
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dc.description.sponsorship
FFG - Österr. Forschungsförderungs- gesellschaft mbH
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dc.description.sponsorship
European Commission
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dc.description.sponsorship
FFG - Österr. Forschungsförderungs- gesellschaft mbH
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dc.language.iso
en
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dc.publisher
AMER CHEMICAL SOC
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dc.relation.ispartof
Analytical Chemistry
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dc.rights.uri
http://creativecommons.org/licenses/by/4.0/
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dc.subject
infrared spectroscopy
en
dc.subject
Quantum cascade lasers
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dc.subject
Ion-exchange chromatography
en
dc.title
QCL-IR Spectroscopy for In-Line Monitoring of Proteins from Preparative Ion-Exchange Chromatography