Charged Aerosol Detection is sensitive and offers near-universal detection independent of the analyte structure. Because of these unique properties liquid chromatographers across all industries frequently use the Charged Aerosol Detector (CAD) over other universal detectors like low-wavelength UV, evaporative light scattering (ELSD) and refractive index (RID).
As with every LC detector, there are key pointers you should know and address to optimize the performance of your CAD. Having a basic understanding of how the CAD works and what factors can negatively impact performance is critical for generating high-quality, reliable data and preventing damage to your detector.
There are multiple aspects to consider when optimizing the performance of your Charged Aerosol Detector and more importantly, conserving the uniform analyte response. Things such as analyte volatility, mobile phase quality and composition, as well as salt formation have the largest effect on uniform response.
|Factor||Consideration||Impact on the CAD response|
|Eluent stream||Any introduction of semi- or non-volatile components in mobile phase||Increases background signal, noise and drift. Non-volatile additives may cause serious damage to the CAD|
|Use highest quality additives with lowest residue of evaporation||LCMS grade additives are preferred to reduce the baseline noise|
|Salt formation between ionic analytes and charged additives||Depletes uniform analyte response and may cause serious damage to the detector|
|Changing solvent composition with gradient elution||Gives a reproducible drift. Depletes uniform analyte response. Sudden changes in solvent composition, such as a step gradient, creates artifact peaks|
|Mobile phase quality and purity||LCMS grade solvents are preferred. Aged solvents increase background and noise|
|Nebulization||Analyte volatility||Impacts uniform analyte response|
|Gas flow rate and stability||Any changes in nitrogen flow rate can increase the baseline signal. Nitrogen is preferred over compressed air for safety reasons and decreased baseline noise|
|Aerosol stability and efficiency||Buildup of salts at the nebulizer tip can impact droplet formation|
|Evaporation temperature (EvapT)||Drying efficiency of the solvent versus the analyte||Increasing EvapT can improve background signal and response. Decreasing EvapT can increase the detection range of semi-volatiles|
|Other factors||Contamination with non-volatiles from other LC system components||Increases background, noise and drift. Depletes uniform analyte response|
|Residual soaps and detergents in lab glassware||Produces baseline artifacts and ghost peaks|
|pH adjustments with pH electrodes||Gives baseline artifacts and ghost peaks|
|Leaching of chemicals from vials and caps||Generates baseline artifacts and ghost peaks|
|Standard quality and purity||Adversely affects purity measurement|
|Column bleed/decomposition||Increases noise and background currents|
Because the response of the CAD for a given analyte is primarily influenced by the volatility of a compound during aerosol evaporation, the ability to predict whether a compound is a good candidate for Charged Aerosol Detection begins with volatility. This primary consideration should be a starting point for developing your HPLC-CAD methods.
Compounds compatible with the CAD should exhibit AT LEAST ONE of the following physical properties:
The general rule of analyte volatility with respect to CAD compatibility is:
An important pre-requisite for any mobile phase used with the CAD is the solvent must be volatile and not contain any non-volatile components.
Several factors associated with the mobile phase composition can influence the performance, and more importantly, the response uniformity including:
We discuss each of these factors in detail and the effect on the CAD response, along with best practices for preparing your mobile phase.
The solvent purity requirements for the CAD and MS are very similar. When you use mobile phases of poor quality, the CAD may show high noise, poor response, baseline drift and gradient-related artifact peak.
A few best practices you can follow to ensure your mobile phase is fresh and pure are:
A common cause for high background currents in the CAD response can come from impurities in the water used for mobile phase preparation.
Most organic HPLC solvents have boiling points lower than water, so most solvents meet the requirement for mobile phase volatility. But many solvent grades contain significant levels of non-volatile impurities that can adversely affect CAD performance and you should avoid use.
Aging of the mobile phase can also affect CAD performance by increasing the background current and noise. To avoid adverse detector response from aged mobile phases so you should:
Your laboratory glassware is a common source of non-volatile contaminants and includes chemicals that leach from the glass over time, left-over residue from prior use, or from cleaning detergents.
When adjusting the pH of your mobile phase solution, keep in mind that pH electrodes are stored in concentrated solutions of potassium chloride. Residual drops on the electrode surface can introduce a measurable amount of this non-volatile salt to the mobile phase and may adversely affect the CAD performance by increasing the background noise.
Mobile phase additives often include buffers, pH modifiers, and ion-pairing reagents. If your HPLC-CAD method requires mobile phase additives, you need to consider:
|Non-volatile||Highly compatible||Not compatible||Not compatible|
Non-volatile mobile phase additives are extremely incompatible with the CAD and can adversely affect performance.
The organic content of the mobile phase entering the detector influences the nebulization process and affects the uniformity of detector response during gradient elution.
As a result the CAD response is dependent on mobile phase composition and changes in the mobile composition may impact the uniform response and subsequent quantification capabilities.
One straightforward way you can overcome the impact of gradient elution on the CAD response is by applying an inverse gradient workflow with our Inverse Gradient configuration. This setup uses a second pump to generate a second inverse gradient, which restores the uniform analyte response and enables standard-free quantitation.
Learn more about using Inverse Gradient in our Technical Note 73449: Why use charged aerosol detection with inverse gradient?
You can generally expect to see an increase in background current and noise with mobile phase additives and this effect increases with additive concentration and decreases with additive volatility. If your method requires additives, we recommend you use volatile and semi-volatile additives at the lowest level necessary. But there are also some situations in which salt formation can increase the CAD response.
Here’s what you should know about salt formations:
1. Unwanted salt formation decreases detector performance in certain cases
2. Intentional salt formation can actually broaden the range of analytes detected by the CAD
Read more about the effect of salt formation on the CAD response in our Technical Note 73914: Charged aerosol detection - factors affecting uniform analyte response.
*The issue with basic mobile phases, especially ammonia-based ones, is that carbonate forms over time from the air above the mobile phase. Carbonate is non-volatile and leads to poor detector performance. Freshly made mobile phases are typically okay but deteriorate with age. Increasing the evaporation temperature can markedly reduce issues and allows the use of basic phases with the CAD. But make sure your column is stable with basic phases otherwise column bleed may be problematic.
Changes in EvapT can improve detector performance (higher EvapT decreases noise) and shift the response of the semi-volatile to behave more like a non-volatile and thereby improves response (lower EvapT).
You can also alter the EvapT to see which compounds behave as semi-volatiles when doing impurity and mass balance studies and deciding when to use the standard free quantitation approach with a surrogate standard.
The quality of standards for a particular chromatographic method can influence the accuracy of your results and affect the noise and background current of the CAD. The purity level for commercial standards is often determined by a single analytical technique that may miss the contribution of some impurities.
Standards may absorb moisture from the atmosphere (hygroscopic) or absorb moisture from the atmosphere and dissolve to form a liquid (deliquescent). Both are problematic when preparing standards for calibration studies. Other compounds may be unstable with exposure to the atmosphere or degrade when dissolved in a solvent. To address these issues:
Current versions of the CAD are compatible with flow rates up to 2.0 mL/min and all commonly available analytical column formats. You should choose stable columns free from bleed that are compatible with the method temperature and pH requirements, near aqueous conditions. Column bleed can introduce semi-volatile and non-volatile impurities and may give higher noise and background current.
Vials consist of two parts – the body and the cap with septum – and each may be a source of contamination that produces baseline artifacts and ghost peaks.
Before selecting an autosampler vial you should check if the material type negatively impacts analytical performance. Fill vials with mobile phase and each of the solutions used for sample and standard preparation. Cap, vortex, and leave for about 72 hours. Vortex each day to make sure the vial cap is exposed to the solution. Analyze the contents of each vial and check if:
If there are issues, you will need to test other vials for compatibility.
If no issues, then your vials and caps are safe to use.
Do you have questions? Want to learn more about Charged Aerosol Detection? Speak to a solutions specialist today.