Charged Aerosol Detection in Pharmaceutical Analysis

Charged Aerosol Detection (CAD): Principles, Strengths, and Applications

In recent years, the charged aerosol detector (CAD) has emerged as the standard for the quantitative analysis of non-chromophoric substances. It is a universal HPLC detector capable of precisely detecting semi- and non-volatile compounds and is particularly suitable for molecules with no or only a limited number of chromophores.

Pharmaceutical analysis requires instruments with high sensitivity, reliability, and versatility. Many active ingredients, excipients, and impurities have no or only a limited number of chromophoric groups and therefore escape conventional optical detection methods such as UV/VIS or fluorescence. In this context, the CAD has proven to be a pa practical and universal solution to close these gaps and enable the quantitative determination of semi- and non-volatile analytes without derivatization. It is therefore a valuable tool both in the development of new drug products and in quality control in regulated contexts.

1. Principle of operation and operating parameters of the CAD

In CAD, the eluent from the column is nebulized into micrometer-sized droplets. Larger droplets are removed by a high-performance gas stream, while the remaining droplets enter a heated drying tube, where the solvent evaporates. After the solvent evaporates, the non-volatile analyte molecules remain as tiny solid particles, which are charged in the detector with positively charged nitrogen so that their electrical charge can be measured. The measured signal is proportional to the concentration.

Since the ionization and detection process is influenced by particle size, solubility, droplet distribution, and charge transfer, the CAD signal is usually not linear across the entire concentration range. Typically, a saturation curve is observed. The signal can be linearized, for example, by a logarithmic plot.
Figure 1 schematically illustrates-CAD with a reverse gradient pump, which stabilizes the mobile phase composition by reducing noise and fluctuations.

Figure 1: A schematic illustration of an LC-CAD with a secondary pump delivering an inverse gradient (Source 1).

The sensitivity of the detector depends on several factors: the composition of the mobile phase (important for stable nebulization), matrix effects (variable salt content or buffer), and the nebulization and drying conditions (gas flow, temperature). The reverse gradient pump further improves stability and signal-to-noise ratio.
Our model, the Corona VEO RS, operates at up to 2.0 mL/min and is compatible with the most common column formats: from conventional 4.6 mm to modern 2.1 mm UHPLC columns, which are preferred due to their better performance. Typical flow rates range from 0.2–1.5 mL/min (HPLC/UPLC) to 50–200 µL/min (MicroLC), with automatic adjustment of the nebulizer pressure. The evaporation temperature (default 35 °C) is adjustable depending on the mobile phase and concentration. Lower temperatures lead to a more uniform detector response but increase baseline noise due to semi-volatile substances; higher temperatures reduce this noise but may compromise labile analytes.

2. Strengths and Limitations

The CAD offers several advantages that distinguish it from other detectors. It is almost universally applicable, as it reliably detects even non-chromophoric and weakly chromophoric compounds, regardless of the chemical structure of the analyte. Furthermore, the CAD is compatible with gradient elution, allowing its flexible use in modern HPLC methods. Another advantage is the ability to perform quantitative determinations without reference standards, as the signal intensity correlates with the analyte mass. Furthermore, the CAD is robust to the optical properties and refractive indices of the sample, making it a reliable detector in complex matrices.

Nevertheless, certain limitations must be considered when using CAD. The CAD detector requires the use of high-purity volatile additives and solvents in the mobile phase. Non-volatile additives are incompatible. Volatile substances are difficult or impossible to detect because they are lost during the drying process. Sensitivity depends heavily on the composition of the mobile phase, particularly the proportion of organic solvents. Furthermore, CAD does not exhibit perfect linearity over very wide concentration ranges, so logarithmic transformations may be necessary for data processing. The maintenance effort is greater, and the capital investment costs are relatively high.

3. Comparison with other detector types

Classic HPLC detectors include the refractive index detector (RID), the evaporative light scattering detector (ELSD) and the Nitrogen Chemiluminescence Detector (CLND). Each of these detectors has specific limitations: The ELSD exhibits limited reproducibility and a nonlinear response; the RID, while easy to handle, has low sensitivity and is not compatible with gradient elution; and the CLND thus is susceptible to interference and has limited specificity. It only detects nitrogen-containing analytes and the mobile phases as well as buffers should not contain any additional nitrogen sources. CLND operation requires technical effort, in particular regular maintenance as well as a reliable gas supply and periodic calibrations.

Most HPLC detectors are based on the detection of chromophores, with the UV/VIS detector being the most commonly used. Its disadvantage is that only molecules with suitable chromophores can be reliably detected. The CAD is independent of this limitation and reliably detects non-chromophoric or weakly chromophoric substances.

Compared to ELSD, RID, and fluorescence detectors, the CAD offers several advantages: It exhibits a more uniform and linear response than the ELSD, is more sensitive and flexible than the RID, and covers a significantly broader substance spectrum than fluorescence detectors, which are only selective for fluorescent or labeled molecules. Its sensitivity exceeds that of the RID and is comparable to that of the UV/VIS detector but remains inferior to that of the fluorescence detector.

The universality of the CAD is based on the fact that it detects virtually any substance that can be converted into particles. Compounds with a boiling point above 400 °C, an enthalpy of vaporization above 65 kJ/mol and a molecular weight above 350 g/mol are generally considered non-volatile and are accordingly reliably detected.
However, the analysis of complex samples often requires the combination of multiple detectors. While UV/VIS detectors enable the precise quantification of chromophoric molecules, the CAD is ideally suited as a secondary detector to cover the broadest possible substance spectrum.

4. What is CAD used for?

The CAD is used particularly when UV/VIS detectors are unsuitable. It is often used in combination with chromatographic techniques such as SEC-CAD and HILIC-CAD in pharmaceutical analysis. SEC-CAD enables the characterization of complex biomolecules such as polymers and proteins, thus monitoring their purity, size, and the presence of aggregates. These are important quality attributes of pharmaceutical products. HILIC-CAD, on the other hand, enables the sensitive detection of small polar molecules – carbohydrates, oligosaccharides, glycans, and amino acids – without derivatization and is therefore useful for assessing the composition and safety of therapeutic proteins and excipients. In both modes, CAD allows the detection of chromophore-free substances, thus enabling monitoring of impurities, degradation products, and associated ions of active substances and excipients (e.g.: Chloride, Natrium, Potassium, Acetate).

An important application area is the analysis of polysorbates and polyethylene glycols (PEGs), common excipients in biopharmaceuticals: CAD enables their quantification and monitoring of degradation products such as free fatty acids or short-chain fragments. It is also used in lipidomics to characterize phospholipids, free fatty acids, and glycolipids, providing important information for formulation development and stability studies. Another important field is the analysis of carbohydrates and glycans in therapeutic proteins and monoclonal antibodies: CAD enables the profiling of these molecules without derivatization, which shortens analysis times and supports quality control. Finally, CAD is suitable for the direct quantification of amino acids, which are essential for the correct composition and stability of therapeutic proteins and enables the precise monitoring of both free amino acids and degradation products.

Figure 2: Application of the CAD in the analysis of solutions containing various amino acids to evaluate separation and detection performance.

CAD is also used for the detection of many active pharmaceutical ingredients (APIs), process contaminants, and natural products with poor UV properties. It is utilized in stability and degradation studies and for the quantification of substances that are difficult to detect using conventional methods.
In conclusion, CAD represents a valuable and widely applicable detection technology in pharmacy and biotechnology thanks to its high sensitivity and the ability to detect almost all non - or semi-volatile components.

Are you interested?
Please do not hesitate to contact us. The experts from Laboratory Services will be happy to provide you with further information at any time. CAD is one of many devices that are part of our technical equipment. Click here for the PDF Equipment Laboratory Services.

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Literature sources

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