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

Abstract

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.

Extracting Information and Enhancing the Quality of Separation Data: A Review on Chemometrics-Assisted Analysis of Volatile, Soluble and Colloidal Samples

“…One of SEC's most common applications consists of the separation/purification of other proteins and other samples of biological interest such as extracellular vesicles [148,149]. Literature studies exploiting multivariate chemometric approaches to SEC separation of proteins mainly focus on monitoring protein aggregation in different conditions, such as those detailed by Ricker et al [150], on the effect of process passages (such as membrane filtration) [151] and on the discrimination of coeluting species [152]. Extracellular vehicles (EVs) are nano-sized, lipid bilayer-enclosed particles involved in intercellular communication.…”

Mid-Infrared Photothermal Spectroscopy for the Detection of Caffeine in Beverages

“…In the meantime, Fourier-transform infrared (FTIR) spectroscopy has been proposed as an alternative method for the fast and non-destructive determination of caffeine [13,14]. Operating within the mid-IR range (400-4000 cm −1 ), this technique investigates the rovibrational molecular transitions in gases, liquids, and solids, providing molecular-specific information [15][16][17]. Analytical techniques based on mid-IR spectroscopy generally require minimal or no sample preparation, ensuring simplicity.…”