Editorial Feature

Sensitivity Beyond the ATR Limit from Inline Mid-IR Liquid Analyzers

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This article is focused on the mid-infrared. This is roughly 3,000 nanometers to 14,000 nanometers spectral range, or about 3,300 wavenumbers down to about 700 wavenumbers. Compared to the visible, this is about 10 times the wavelength range.

We’re interested in Mid-IR frequencies because the fundamental vibrational absorbance features of most organic molecules exist within this spectral range. That enables the user to be very selective and highly sensitive. If you compare Mid-IR to the near infrared, with near infrared largely containing overtones of the vibrational modes in the mid-infrared, the relevant strength is several orders of magnitude stronger in the Mid-IR. Furthermore, using simple transmission-based techniques, simple application of Beer's Law can enable easy quantitation of molecular concentrations. It's a label-free technique, meaning you don't have to tag molecules or add dyes to liquid samples.

It's also well known that Mid-IR is highly valuable for looking at protein based biological therapeutics, as it's sensitive to key attributes of monoclonal antibodies, antibody drug conjugates, fusion proteins, vaccines, and virus like particles. There are also modifications that can be performed on the proteins themselves, including the conjugation of polysaccharides, glycans, and other post-translational modifications, and conformational changes. One of the things Daylight Solutions has learned from interactions with many customers is that there is a growing need for new analytical capabilities to address the growing challenges in this space.

Why isn't Mid-IR More Commonly Used as a Part of the Workflow for High Sensitivity, Small-Volume Liquid Analysis?

The FTIR, which has been the workhorse infrared instrument for decades, has several key limitations. One, the incandescent Globar source lacks an intensity that would enable longer path lengths greater than about 10 microns in aqueous solutions. Secondly, that incandescent beam is very difficult to couple into small volumes without losing a significant amount of photon energy. Furthermore, you typically must couple to cryogenically cool the detectors, which have very limited linear dynamic range.

Why are we Visiting the Mid-Infrared Based Techniques?

The answer is that in the last decade, the quantum cascade laser has really become a mature, robust, reliable laser source for performing very challenging Mid-IR based spectroscopy analysis. The quantum cascade laser spectral brightness provides over four orders of magnitude higher spectral brightness, at any given wavelength range when compared to a globar source and even a synchrotron source, which can be found in a handful of large multi-million-dollar institutions around the world.

With a quantum cascade laser, you're able to punch through much thicker columns of liquid that might have strongly absorbing media such as water. The longer path lengths enable higher sensitivity and the ability to sample more of your sample volume and have a truer representation of the sample. In FTIR, you typically must use attenuated total reflection-based techniques, which limits the interaction volume to very close to the surface.

When studying biologics, with very short path-length cells or looking at surface characteristics, you can have confounding effects due to the stickiness of the material, and its adherence to the surface. By performing a transmission-based measurement, these effects can be alleviated.

What Types of Products do Daylight Solutions Offer, and what Areas Would They Typically be Used in?

Daylight Solutions is the most trusted provider of quantum cascade laser systems. They have a varied portfolio with a wide range of quantum cascade laser sources that are part of their scientific product line. Each quantum cascade laser that they routinely sell into the scientific and commercial markets can be applied to the ChemDetect liquid analyzer that is discussed in this article. Depending on the analyte of interest, the ChemDetect can be optimized to focus on a subset of analytes of interest, to maximize signal to noise ratio and sensitivity for an application.

In addition to the quantum cascade laser, Daylight Solutions Core Technologies also include advanced Mid-IR imaging optics, individual sensor detectors, and large-area focal array technologies. These Core Technologies form the building blocks of developing higher level integrated systems. Their laser subsystems serve as end user instruments for scientific, commercial and military applications.

Since 2014, Daylight Solutions have taken these Core Technologies and laser sub-systems, and integrated them into advanced end user Life Sciences instruments, beginning with the Spero microscope. This article will discuss the ChemDetect liquid analyzer. These Life Sciences instruments have been used to enable a wide range of new applications including polymer science, label-free digital pathology in clinically relevant timescales, molecular binding assays at a single protein level, API analysis in tablets, forensics of packaging defects, and microfluidic imaging. Here, liquid chromatography applications will be examined.

What are the Specifics of the ChemDetect Liquid Analyzer?

The ChemDetect liquid analyzer is a new instrument based on Daylight Solution’s quantum cascade laser technology. What's special about it is that it's taking a tried and true technique, direct infrared spectroscopy, and using this new technology to get to sensitivity levels and timescales previously inaccessible with previous generation technology.

The ChemDetect Liquid Analyzer is a quantitative Mid-IR liquid analyzer, with sensitivities down to the 10 microgram per milliliter level. It has a small flow cell volume, down to one microliter; and fast spectral sweeps up to 10 spectra per second. It also has a very large linear dynamic range which allows measurement of high sensitivities with precision over multiple decades of concentration. Furthermore, because of the use of the quantum cascade laser and advanced sensor technology, no cryogenic cooling is required, and can therefore use a small footprint.

The removable and autoclavable microliter transmission flow cell works very easily. It mates with typical off the shelf chromatography fittings and tubing. It can be taken apart to adjust path length and optimized for an application of interest.

What are the Differences Between Attenuated Total Reflection (ATR) Versus Transmission Based Techniques and which one has Daylight Solutions Chosen?

There are some differences between ATR versus transmission-based techniques, with Daylight Solutions choosing the transmission architecture. In ATR, light is incident on the surface of a lower-index medium at a critical angle, such that you have an evanescent wave coupling into the first few microns of the cell. For a typical transmission style, the light passes simply through the entire volume of the flow cell from top to bottom. In the ATR technique, the interaction length is limited to the surface of the cell, typically limited to the first few microns of a cell.

Furthermore, that interaction length is dependent on wavelength, angle of incidence, as well as the energies of refraction of the incident material as well, as the liquid material itself. Versus the transmission cell, the path length is fixed by the mechanical structure of the device, and by design. That allows the path length to be dialed in, to optimize the signal to noise ratio for an analyte of interest. Furthermore, because it's a simple application of Beer's Law, quantitation becomes simple and straightforward.

What is the Layout of the ChemDetect Liquid Analyzer?

From top to bottom it contains onboard CPU and control electronics which drive the high fidelity through the quantum cascade laser, and an internet communication port which has established digital communication between the instrument and a remote PC. The ChemDetect liquid analyzer contains a single high-fidelity tunable quantum cascade laser, as well as two balanced thermoelectrically cooled detectors, which enable high precision transmission-based measurements.

The transmission flow cell resides in the sample compartment. It can be removed and cleaned periodically as necessary. The flow cell also has an adjustable path length component based on a precision gasket, which will be discussed in more detail below. Looking after the ChemDetect liquid analyzer is very simple and it requires only meeting up with standard chromatography tubing and fittings.

The flow cell is sandwiched by two mid-infrared transparent windows. The fluid flows from top to bottom and is schematic, with the path length set by a precision spacer that is calibrated at the factory. Then the Mid-IR light is focused through the flow cell and is attenuated by the analyte as it goes through, using Beer's Law.

Based on the known path length and the amount of attenuated light going through the cell, the concentration of the analyte of interest can be easily measured.

Graphs produced by the ChemDetect liquid analyzer are basically a spectra versus time. That spectra can be converted to concentration versus time, through a simple chemometric model, that's either downloaded onto the instrument, or offloaded onto a data management server. Spectra can be collected at up to 10 hertz. That is 10 complete high signal-to-noise ratio spectrum per second at two-wave interfacing. Furthermore, that data is easily exportable in either a raw spectral format or converted to a concentration by chemometric model.

How does the ChemDetect Operate?

This section will discuss three specific examples that highlight some of the key features and feasibility of the instrument. The first being civil quantitation of target molecules in aqueous solutions. Secondly, looking at single analyte quantization in complex mixtures such as growth media. The final example is the detection of individual spectral components in a liquid chromatography separation.

The first example is quantitation of individual analytes in aqueous solution, specifically glucose and ammonia in water solutions. In this example three other typical analytes that might be found in a bioprocess were also included in the solution: glutamine, glutamate and lactate. The glucose and ammonia both show very high degrees of linearity in this case, over multiple decades of concentration, and RMS concentration errors on the order of 10 micrograms per milliliter.

Will that performance still hold even in complex media? In the second example the ChemDetect analyzer is used for quantitation of a single analyte, in this case glutamine, in a complex medium, Hams' F-12. Here there is still excellent linearity over multiple decades; and again, sensitivities are in the same order as discussed in the previous example, on the order of a few tens of micrograms per milliliter.

ChemDetect analyzer from Daylight Solutions

ChemDetect analyzer from Daylight Solutions

The final example is using the ChemDetect liquid analyzer in combination with a standalone HPLC system. In this case an Agilent 1260 series system is used, with two ChemDetect systems in series.

As that occurs, the solution flows past the flow cell with different characteristics at different times. By having spectroscopy in real-time, it’s possible to add an additional dimension and degree of information that wouldn’t be available with standard in-line HPLC analyzers.

With two different ChemDetects looking at two different spectrum ranges, with the first ChemDetect looking at 1,450 to 1,800 wavenumbers (the amide MI one and amide MI two bands) and the second ChemDetect measuring from 1,000 to 1,250 wavenumbers (mostly the carbohydrate bands). There is very little signal associated with the amide MI one and amide MI two bands, for this example. As an extra sample there isn’t much signal in the amide MI region. But there is strong response in the carbohydrate region. As a function of time, that elution rises and then falls as the material flows past the flow cell and down to the waste bin.

It's important to point out here that looking at the total integrated mid-infrared signal, the temporal spread in the elution is on par with what you might expect from a typical UV signal coming off that same HPLC system. That's a real testament to the small flow cell volume that Daylight Solutions can achieve, based on the ability to focus the light down to small areas.

The final one is an interesting example. Again, an HPLC system is used, but in this case looking at 20 kilodalton dextran dissolved in water, flowing through a size exclusion chromatography column. The expectation is that this sample is homogeneous, and that the spectra should reflect that nature. Instead, what is found is at the end of the elution, there is a substantial change in the spectra of the sample coming off the column, particularly in the shorter wavenumber region.

The ChemDetect uncovers previously hidden heterogeneity of the sample, and allows further investigation, possibly with additional techniques downstream. The highlight is that the UV response to this polysaccharide was almost nonexistent. But the mid-infrared has a very strong response. So, for sugars, polysaccharides, the mid-infrared has a very strong advantage, not only in simulated activity, but also providing additional information that would be previously hidden to the end user.

Find out more about sensitivity beyond the ATR limit from inline Mid-IR liquid analyzers in this webinar

Summary

Several innovations in the photonics technology sector have come together to bring back this classic mid-infrared spectroscopy technique. Daylight Solutions are doing so in a way that allows them to apply traditional mid IR direct spectroscopy to challenging biological analysis. DRS Daylight Solutions has a track record of transitioning lifesaving technologies into very highly demanding, highly regulated markets.

They’ve done so in the defense industry, taking their lasers, ruggedizing them, and putting them into very demanding applications with very severe regulations. This is something they're committed to do with the Life Sciences sector as well.

They remain committed to bringing innovative solutions to the Life Sciences space, working with individual partners on taking this technology into the R&D centers, and transitioning them into GMP compliant process areas.

This information has been sourced, reviewed and adapted from materials provided by Daylight Solutions Inc.

For more information on this source, please visit Daylight Solutions Inc.

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