Institute of Chemical Technologies and Analytics, Technische Universität Wien, Getreidemarkt 9, 1060 Vienna, Austria.
Institute of Chemical, Environmental and Bioscience Engineering, Technische Universität Wien, Getreidemarkt 9, 1060 Vienna, Austria.
Anal Chem. 2022 Apr 12;94(14):5583-5590. doi: 10.1021/acs.analchem.1c05191. Epub 2022 Mar 30.
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, 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.
在这项研究中，我们应用基于外腔量子级联激光的中红外（IR）光谱仪对制备型离子交换层析过程中的蛋白质进行在线监测。25μm 的大光程允许在 1350 至 1750cm 的宽调谐范围内采集稳健的光谱，涵盖了用于确定蛋白质二级结构的最重要的光谱区域。蛋白质的中红外吸收带重叠以及由于 NaCl 梯度导致的水的背景吸收变化带来了重大挑战。通过实施先进的背景补偿策略，我们在三个不同的模型案例研究中获得了高质量的蛋白质光谱。在案例 I 中，直接从具有相同 NaCl 梯度的样品运行中减去参考空白运行。在案例 II 和 III 中，样品运行的梯度轮廓与参考运行的不同。在这里，我们引入了一种基于参考光谱矩阵的新补偿方法，其中使用电导率检测器的信号来关联合适的参考光谱，以校正样品运行光谱。使用这种方法，只需进行一次空白运行即可校正各种梯度轮廓。血红蛋白和β-乳球蛋白的获得的 IR 光谱与离线参考测量进行了比较，所有案例研究均显示出极好的一致性。此外，从中红外光谱仪获得的浓度值与传统的 UV 检测器和高效液相色谱离线测量值吻合良好。因此，LC-QCL-IR 耦合有望替代繁琐且耗时的蛋白质离线监测方法，用于复杂下游工艺中的蛋白质监测。
