Skip to main content

Liquid Phase Spectroscopy

In our Liquid Phase Spectroscopy labs we are working with quantum cascade laser (QCL) based infrared (IR) spectroscopy. Select in the sub-navigation the topic of your interest to read more.

QCL-IR Absorption Spectroscopy of Proteins

Motivation

Mid-infrared spectroscopy is a well-established technique for the analysis of polypeptides and proteins. The amide I band (1700-1600 cm-1) originating from C=O stretching coupled to in-phase bending vibration of N-H and the amide II band (1600-1500 cm-1) arising from N-H bending and C-N stretching vibrations are the most useful bands for secondary structure evaluation and quantification of proteins.
Strong absorption of H2O near 1640 cm-1 makes IR studies of proteins in aqueous solutions challenging. For conventional Fourier-transform infrared spectroscopy, very short pathlength of approx. 10 µm are needed to avoid total IR absorption in the spectral region of water. These low path lengths limit the intensities of the IR bands and the signal-to-noise ratio, thus restricting the application to high protein concentrations (approx. 10 mg/mL).
EC-QCLs, which provide spectral power densities several orders of magnitude higher than thermal emitters,  allow the use of 4-5 times higher path length and consequently the detection of proteins at lower concentrations.

Development of Laser-based IR Transmission Setups for Analysis of Proteins

In the last few years, several iterations of EC-QCL based IR transmission setups were developed to analyse the amide I and amide II regions of proteins. The latest iteration provides an improved limit of detection compared to research grade FT-IR instruments at spectra acquisition times lower than 1 min.  

Key Publications:

Book Chapter:

Inline Monitoring of Proteins from Preparative Liquid Chromatography

A commercial EC-QCL based IR Instrument (Chemdetect Analyzer by Daylight Solutions) was employed for inline monitoring of proteins from preparative ion-exchange chromatography (IEX) and size exclusion spectroscopy (SEC).

Key Publications:

Monitoring of perturbation-induced Changes in Protein Secondary Structure

The developed setups were applied to monitor conformational changes in protein secondary structure after different kinds of external perturbation.  

Key Publications:

  • pH titration of β-lactoglobulin monitored by laser-based Mid-IR transmission spectroscopy coupled to chemometric analysis, Spectrochimica Acta A, 2020.
  • External cavity-quantum cascade laser infrared spectroscopy for secondary structure analysis of proteins at low concentrations, Nature Scientific Reports, 2016.
  • EC-QCL mid-IR transmission spectroscopy for monitoring dynamic changes of protein secondary structure in aqueous solution on the example of beta-aggregation in alcohol-denaturated alpha-chymotrypsin, Analytical Bioanalytical Chemistry, 2016.

Quantitation of Individual Proteins in Bovine Milk Samples  

QCL-IR spectroscopy was employed for quantitation of major proteins present in commercial bovine milk. Evaluation of the concentration levels of the temperature sensitive proteins beta-lactoglobulin and alpha-lactalbumin enables the discrimination between different milk types.  

Key Publications:

  • High-throughput quantitation of bovine milk proteins and discrimination of commercial milk types by external cavity-quantum cascade laser spectroscopy and chemometrics, Analyst, 2019.
  • Fast quantification of bovine milk proteins employing external cavity-quantum cascade laser spectroscopy, Food Chemistry, 2018.
  • External cavity-quantum cascade laser (EC-QCL) spectroscopy for protein analysis in bovine milk, Analytica Chimica Acta, 2017.  

Researchers

Alicja Dabrowska
Georg Ramer
Shilpa Vijayakumar

QCL-IR Dispersion Spectroscopy

Acquisition of classical absorption spectra of liquids in the mid-IR range with QCLs is often limited in sensitivity by noise from the laser source. Alternatively, measurement of molecular dispersion (i.e., refractive index) spectra poses an experimental approach that is immune to intensity fluctuations and further offers a direct relationship between the recorded signal and the sample concentration.

In dispersion spectroscopy, the phase shift of the radiation due to passing through a sample is measured. Dispersion and absorption are caused by the same process, thus the same spectral information about the sample can be retrieved by determining either of the two properties.

Very recently, we introduced a Mach-Zehnder interferometer setup to measure the absorption and dispersion spectra of liquid samples.

Key Publications:

  • Mid-IR dispersion spectroscopy – A new avenue for liquid phase analysis, Spectrochimica Acta Part A, 2023
  • The next generation of mid-IR laser-based refractive index (dispersion) spectroscopy of liquid-phase analytes, SPIE Proceedings, 2022.
  • Mid-IR refractive index sensor for detecting proteins employing an external cavity quantum cascade laser-based Mach-Zehnder interferometer, Optics Express, 2020.
  • Beyond Beer’s law – why the index of refraction depends (almost) linearly on concentration, ChemPhysChem, 2020.
  • External Cavity Quantum Cascade Laser-Based Mid-Infrared Dispersion Spectroscopy for Qualitative and Quantitative Analysis of Liquid-Phase Samples, Applied Spectroscopy, 2019.

Researchers

Alicja Dabrowska

QCL-IR Spectroscopy for Downstream Process Monitoring

The aim of this project is to advance laser-based IR spectroscopy methods for analysis of proteins as well as to apply and establish IR spectroscopy as a monitoring and quality control tool for studying downstream bioprocesses.

In the first step, this project focusses on harnessing the unique properties of external cavity – quantum cascade laser (EC-QCL) light sources in order to advance the field of IR spectroscopy, which has long been coined by the characteristics of thermal light sources and measurement approaches based on direct absorption spectroscopy. 

Subsequently, enabled by this progress in instrument design and performance, the QCL-IR spectroscopy is employed to investigate individual bioprocess steps in the recombinant production of a model protein. In downstream bioprocessing, low levels of protein concentration have been prohibitive so far for employing IR spectroscopy for process monitoring. QCL-IR spectroscopy will be introduced as tool for structure-based product analytics at significant points along downstream bioprocessing (IB analytics, refolding kinetics, downstream chain analytics and heme incorporation kinetics) and utilized for characterization and quality control of the targeted unit operations. Changes in structure and activity of the intermediate products and the final protein are correlated and effects of systematically varied process parameters will be investigated.

Key Publications:


FWF-Project: Advancing QCL-IR Spectroscopy of Proteins for Downstream Process Monitoring, No. P32644-N

Researchers

Georg Ramer

QCL based Vibrational Circular Dichroism (VCD)

Motivation

Chirality in the chemical world refers to the inability to superimpose one molecule on its mirror image. It is a prevalent concept in biological systems, being present in most biopolymers like proteins and DNA, and intrinsically linked to the function of those chiral biological molecules. As a consequence, the accurate assessment of the chiral identity of any given drug candidate is an important analytical goal, as the different forms of chiral molecules differ significantly in their pharmacokinetics or even toxicity levels.

Vibrational Circular Dichroism (VCD) relies on the difference of the absorbance for left- and right-handed circular polarised light in the mid-infrared (MIR). It combines the broad applicability of MIR spectroscopy with a sensitivity to the chirality of the analyte. However, it is plagued by low intensity and typical VCD spectra can take up to several hours of measurement time.

We have recently succeeded in constructing a MIR-laser based VCD instrument, leveraging the high power and inherent polarisation of quantum cascade lasers (QCL). By employing a balanced detection system, we are able to significantly reduce the noise introduced by the laser and reduce the measurement time for high quality VCD spectra to a few minutes. We are confident that the higher time resolution offered by QCL-VCD can shine light on the chiral changes in dynamic systems like catalysis or protein folding in a way not possible before. 

Key Publications:

  • Lendl, B.; Hermann, D.-R.; Ramer, G.; Zeiler, M. Enantiomeric Excess Determination by Quantum Cascade Laser Vibrational Circular Dichroism: A Chemometric Approach. In Photonic Instrumentation Engineering X; Soskind, Y., Busse, L. E., Eds.; SPIE: San Francisco, United States, 2023; p 52, doi.org/10.1117/12.2650526.
  • Hermann, D. R.; Ramer, G.; Kitzler-Zeiler, M.; Lendl, B. Quantum Cascade Laser-Based Vibrational Circular Dichroism Augmented by a Balanced Detection Scheme. Anal. Chem. 2022, 94 (29), 10384–10390,doi.org/10.1021/acs.analchem.2c01269.

          Researchers

          Daniel-Ralph Hermann