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.

中文翻译：

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量子级联激光-红外光谱和化学计量学在制备型尺寸排阻色谱中在线鉴别共流出蛋白质中的应用

将基于外腔量子级联激光器 (EC-QCL) 的流通式中红外 (IR) 光谱仪与制备型尺寸排阻色谱系统相一致，以展示对具有强烈重叠色谱峰的蛋白质洗脱物的实时分析。在典型的实验室规模纯化条件下进行了涉及三种和四种模型蛋白的两个不同案例研究。大光路长度 (25 μm)、高信噪比和宽光谱覆盖范围（1350 至 1750 cm ^–1) 的 QCL-IR 光谱仪允许在酰胺 I 和 II 波段进行稳健的光谱采集。通过自建模混合物分析和多变量曲线分辨率进行的化学计量分析能够在蛋白质洗脱瞬态中进行准确的定量和结构指纹识别。发现获得的浓度曲线与对收集的流出物馏分进行的离线高效液相色谱参考分析非常一致。这些结果表明，QCL-IR 检测器可以有效地用于蛋白质洗脱的在线实时分析，提供通常只能通过耗时且资源密集的离线方法访问的关键质量属性数据。