Hyphenation of Preparative Liquid Chromatography to Laser-Based Mid-IR Spectroscopy for Monitoring of Proteins in Chromatographic Effluents

Abstract

Mid-infrared spectroscopy provides detailed information about proteins that cannot be obtained with conventional UV detectors. The most powerful IR bands for protein quantification and secondary structure analysis are the amide I (1700-1600 cm-1) and amide II (1600-1500 cm-1) bands. A pronounced challenge in analysis of aqueous protein solutions is the strong absorption of H2O at approx. 1640 cm-1 that overlaps with the amide I band. In conventional Fourier-transform infrared (FTIR) spectroscopy, the optical path-length is restricted to <10 µm to avoid total IR absorption. These small path lengths are unsuitable for flow-through operations due to low robustness. External cavity-quantum cascade lasers (EC-QCLs) offer higher optical powers compared to the thermal sources used in FTIR spectroscopy, leading to increased sensitivity and larger applicable optical path lengths. These advantages open a wide range of possible applications, including near real-time protein monitoring from downstream operations. Here, an EC-QCL based mid-IR spectrometer was coupled to a preparative liquid chromatography (LC) system. Two different model systems, based on ion-exchange chromatography (IEX) and size exclusion chromatography (SEC) were employed to demonstrate the high flexibility of LC-QCL-IR coupling.