Liquid Chromatography—Liquid Chromatography–Fourier Transform Infrared

Kuligowski, Julia; Quintás, Guillermo; Guárdia, Miguel de la; Lendl, Bernhard

doi:10.1016/b978-0-12-409547-2.14514-7

Cited by 4 publications

(9 citation statements)

References 15 publications

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“…Typical challenges for solvent–evaporation interfaces are, however, the morphology of certain analytes that can change over time and possible spatial inhomogeneity. 9 Moreover, this destructive type of sample preparation prevents fractionation of the effluent after detection.…”

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confidence: 99%

“…The most important mid-IR regions for protein quantification and secondary structure determination are the amide I (1700–1600 cm –1 ) , and amide II (1600–1500 cm –1 ) band. Coupling of IR spectroscopy and LC were successfully demonstrated, , in most cases using organic solvent gradients. However, flow-through mid-IR transmission measurements of proteins in aqueous matrix have several challenges.…”

mentioning

confidence: 99%

“…Here, the eluent is evaporated, while the analyte is (almost) simultaneously deposited onto a suitable substrate prior to FTIR spectra acquisition. Typical challenges for solvent–evaporation interfaces are, however, the morphology of certain analytes that can change over time and possible spatial inhomogeneity . Moreover, this destructive type of sample preparation prevents fractionation of the effluent after detection.…”

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confidence: 99%

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QCL–IR Spectroscopy for In-Line Monitoring of Proteins from Preparative Ion-Exchange Chromatography

et al. 2022

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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.

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QCL–IR Spectroscopy for In-Line Monitoring of Proteins from Preparative Ion-Exchange Chromatography

et al. 2022

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“… 13 Even though these setups enabled protein secondary structure analysis from LC effluents, 14 , 15 solvent evaporation interfaces can bear major challenges such as spatial heterogeneity and changes in analyte morphology over time. 16 Moreover, in preparative LC runs, the effluent is usually fractionated after detection, making a preceding solvent evaporation step inapplicable. More recently, attenuated total reflection-FT-IR spectroscopy 17 was coupled to an LC system for in-line monitoring of proteins.…”

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confidence: 99%

“…For this purpose, complex schemes were developed that evaporate the solvent and deposit the protein almost simultaneously onto a substrate before FT-IR analysis . Even though these setups enabled protein secondary structure analysis from LC effluents, , solvent evaporation interfaces can bear major challenges such as spatial heterogeneity and changes in analyte morphology over time . Moreover, in preparative LC runs, the effluent is usually fractionated after detection, making a preceding solvent evaporation step inapplicable.…”

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Application of Quantum Cascade Laser-Infrared Spectroscopy and Chemometrics for In-Line Discrimination of Coeluting Proteins from Preparative Size Exclusion Chromatography

et al. 2022

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An external-cavity quantum cascade laser (EC-QCL)-based flow-through mid-infrared (IR) spectrometer was placed in line with a preparative size exclusion chromatography system to demonstrate real-time analysis of protein elutions with strongly overlapping chromatographic peaks. Two different case studies involving three and four model proteins were performed under typical lab-scale purification conditions. The large optical path length (25 μm), high signal-to-noise ratios, and wide spectral coverage (1350 to 1750 cm –1 ) of the QCL-IR spectrometer allow for robust spectra acquisition across both the amide I and II bands. Chemometric analysis by self-modeling mixture analysis and multivariate curve resolution enabled accurate quantitation and structural fingerprinting across the protein elution transient. The acquired concentration profiles were found to be in excellent agreement with the off-line high-performance liquid chromatography reference analytics performed on the collected effluent fractions. These results demonstrate that QCL-IR detectors can be used effectively for in-line, real-time analysis of protein elutions, providing critical quality attribute data that are typically only accessible through time-consuming and resource-intensive off-line methods.

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Techniques for the Detection, Quantifications, and Identification of Pharmaceutically Active Compounds and Their Removal Mechanisms

Kamali

Aminabhavi

Costa

et al. 2023

Green Energy and Technology

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Liquid Chromatography—Liquid Chromatography–Fourier Transform Infrared

Cited by 4 publications

References 15 publications

QCL–IR Spectroscopy for In-Line Monitoring of Proteins from Preparative Ion-Exchange Chromatography

QCL–IR Spectroscopy for In-Line Monitoring of Proteins from Preparative Ion-Exchange Chromatography

Application of Quantum Cascade Laser-Infrared Spectroscopy and Chemometrics for In-Line Discrimination of Coeluting Proteins from Preparative Size Exclusion Chromatography

Techniques for the Detection, Quantifications, and Identification of Pharmaceutically Active Compounds and Their Removal Mechanisms

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