*Cue the Star Trek music and Captain James T. Kirk voiceover*
Detectors: the eyes of your HPLC. This is the story of charged aerosol detection. Over 15 years: vastly expanding the scope of liquid separations. Allowing liquid chromatographers to see new compounds, boldly showing you what no chemist has seen before.
Before the invention of charged aerosol detection, liquid chromatographers used detectors such as refractive index, low wavelength UV absorbance, and evaporative light scattering for quantitative analyses.
While these HPLC detection methods were useful for analyzing compounds incompatible with higher wavelength absorbance with UV-Vis detectors they were also limited because of low sensitivity and quantitation challenges, which severely impacted method development and prohibited research progression.
Then in the early 2000s, a group of analytical scientists did what scientists do best. They created a more sensitive and universal detection method, which led to the award-winning invention of charged aerosol detector (CAD).
History of the CAD
Used exclusively in liquid-chromatographic separations, charged aerosol detection is a universal technique for quantitative analysis and overcomes the detection sensitivity and analyte versatility issues of other universal approaches.
The first CAD, the Corona® CAD®, was introduced to the scientific community at the Pittsburgh Conference in 2005 and commercially available soon after. The creation of this revolutionary technology resulted from a successful collaboration between TSI and ESA Biosciences Inc (now part of Thermo Fisher Scientific), a chromatography company specializing in high-sensitivity electrochemical detectors.
Fast forward to 2021, CAD is now an integral part of HPLC and UHPLC analysis driven by the sensitive and near-universal analyte response plus standard free quantitation, thanks to over a decade of continued advancements in the technology.
Background to ESA Biosciences
ESA, Inc. was founded in 1968 by a group of graduates from MIT who developed an anodic stripping voltammetry-based instrument to measure minute levels of lead in human blood samples. Lead exposure causes severe changes in brain function and interferes with brain chemistry.
One approach to study changes in brain chemistry is HPLC with electrochemical detection (HPLC-ECD). Unfortunately, commercially available ECDs, at the time, were unreliable and had considerable downtime due to the need for routine maintenance.
ESA, Inc. solved this problem by developing a more robust and reliable instrument using their novel “coulometric” sensor. Research into novel HPLC-based detectors continued, where they later formed ESA Biosciences and introduced the Corona CAD in 2005.
ESA Biosciences, Inc.’s first two CADs were strictly designed for HPLC methods and were compatible with any HPLC system. ESA Biosciences Inc was then acquired by Dionex and CADs were redesigned to be integrated into the Thermo Scientific™ UltiMate™ 3000 HPLC and UHPLC systems. This integration allowed for more ease of use with the seamlessly integrated system with components designed to fit and work together.
Thermo Fisher Scientific went on to develop the Thermo Scientific™ Vanquish™ LC platform.
A Timeline of CAD Evolution
Like all great inventions, design changes over the years improved detector performance and the overall user experience. The stepwise evolution of this technology dramatically transformed the CAD from a research and development tool to an established quality control detector.
[caption id="attachment_23361" align="aligncenter" width="1002"] Table 1. Timeline of CAD evolution.[/caption]
Thermo Fisher Scientific’s contribution to advancing CAD
These major redesigns markedly improved the CAD performance and user-friendliness in a few ways:
Added sensitivity—the change from a crossflow to the FocusJet concentric flow nebulizer markedly reduced the noise level, especially at lower flow rates.
In current CAD models, sub-nanogram levels are now routinely detected with confidence.
More versatility—FocusJet concentric flow nebulizer enabled the use of low flow rates down to 0.01 µL/min making the CAD fully compatible not only with HPLC and UHPLC methods but also with low-flow UHPLC approaches. Early CAD models used a heated nebulizer to prevent evaporative freezing of the nebulizer (e.g., when using THF) but did not control evaporation temperature directly during the droplet drying process. We now know evaporation temperature affects detector response, especially for semi-volatile analytes.
In current CAD models, the evaporation temperature is controllable, and as part of method development, used to improve the analyte response.
Operational convenience—older CAD designs used a “closed system,” requiring the waste levels to be monitored and emptied routinely.
In current CAD models, liquid waste drains freely like all other liquid chromatography detectors. Waste liquid is actively removed from the CAD by a pump, along with active monitoring of the detector status (liquid flow, gas flow, etc.) to ensure optimal performance.
CAD versus 0ther detection methods
CAD vs. ELSD
Both CAD and Evaporative Light Scattering Detectors (ELSD) are evaporative aerosol detectors able to detect non-volatile and many semi-volatile compounds. But how the particles are detected differs between the two technologies. This difference can significantly impact detector performance.
In ELSD, aerosol detection depends on the light-scattering properties of the analyte and not on the charge of the particle. For CAD, the aerosol is charged, and the size of the charge depends on the particle size. The total charge is measured as a current, independent of optical properties.
CAD response is not affected by the analyte’s optical properties (e.g., refractive index, absorbance, and fluorescence).
A single nebulizer works for all flow rate ranges.
CAD vs. LW UV
UV-Vis detectors require the analyte to possess a chromophore in order to be detected. But not all compounds have a sufficiently strong chromophore. For these compounds low wavelength ultra-violet (LW UV) detection is used but, as so many compounds are now detected, selectivity issues can arise when distinguishing between target versus unknown peaks. Furthermore, this approach lacks sensitivity and can only be used with certain solvents.
For instance, you cannot easily detect a peak for an unknown if the compound absorbs in the cut-off wavelength of your eluents, like acetonitrile at 190 nm.
With CAD you can easily detect your target compounds and impurities regardless of solvent absorbance characteristics leading to higher detection sensitivity with better baselines.
CAD vs. RID
Refractive index detectors (RIDs) are universal but have shortcomings like incompatibility with gradient elution and an extensive startup time of easily several hours.
CAD on the other hand works with both isocratic and gradient separations, is more sensitive and requires less time for equilibration.
CAD vs. MS
Although mass spectrometry (MS) detectors are powerful the technology only works if your compounds form gas-phase ions. The response might suffer from ion suppression, therefore it is difficult to quantify without isotope standards.
[caption id="attachment_23350" align="alignright" width="960"] Figure 1: Literature references for CAD Detection sorted by area.[/caption]
Scientists working in diverse areas like pharmaceutical/biopharmaceutical, food/nutrition, natural products/botanicals, fundamental research, and environmental/industrial utilize CAD. Almost two decades later, there are now over 860 CAD-based publications covering many applications and markets.
The vast applications relate to the technology features. Analyte response is uniform, standard free quantitation is possible, which is critical when pure standards are not available for situations like drug impurity measurement or the analysis of complex matrices such as botanicals.
Additionally, the CAD does not require the presence of a chromophore. This feature expands the range of substances measured following HPLC/UHPLC separation including, carbohydrates, inorganic and organic ions, lipids, surfactants and excipients.
Lastly, CAD is used in numerous standardized methods, including those from the United States Pharmacopeia, European Pharmacopeia, and International Organization for Standardization.
Ways to maximize your CAD analysis
The superior analytical power of CAD lies in the ability to combine with other detectors like UV and MS in an “omni” platform to more fully interrogate the sample (e.g., Figure 2).
[caption id="attachment_23356" align="aligncenter" width="960"] Figure 2. Results of a cell culture bag extract demonstrating the “omni” platform. The numbers refer to the standard names listed in the table. The table shows the analysis of 18 common extractables/leachables and the response on CAD, UV, and MS.[/caption]
This triple detection method extends the scope of compounds measured in a sample, enabling you to use CAD to quantify non-volatiles and semi-volatiles without chromophores, UV for volatiles and non-volatiles chromophores, and MS to confirm analyte identity.
Both CAD and MS are destructive detectors, so they must be placed after the UV flow cell, connected in parallel using a T-piece. Sometimes a DAD replaces the UV detector, as the 3D-field gives in-depth analytical information because the use of a single wavelength to measure multiple analytes in a sample can be problematic due to differences in relative response between the analytes.
For instance, what happens if you get a peak from an unknown – does this compound have a chromophore like your target molecule?
For a small unknown peak is the response due to a low abundance of the compound, or a high abundance of a compound that does not absorb at that wavelength?
With CAD you work will not endure these limitations.
Explore new frontiers with your own Thermo Scientific™ Vanquish™ HPLC and UHPLC System with charged aerosol detection
Curiosity and a desire to make the world a better place lie at the heart of every scientist. The driving force behind many breakthroughs is the ability to think and solve problems, like when the founders at ESA invented the Corona CAD to help people suffering from lead toxicity.