HPLC-CAD Response Factors

Overview

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.

The CAD shows uniform response (<5% RSD variation) among all non-volatile analytes (0.5 μg; flow injection analysis). More information can be found in Technical Note TN72806.

HPLC-CAD learning center

Related resources

Summary of factors impacting the CAD 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

Analyte volatility

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:

Boiling point > 400 °C
Molecular weight > 350 g/mol and enthalpy of vaporization > 64 kJ/mol

The general rule of analyte volatility with respect to CAD compatibility is:

Non-volatile analytes are highly compatible and give a similar uniform response.
Semi-volatile analytes tend to show a non-uniform response, which depends upon the analyte volatility, EvapT and ability to form salts with mobile phase additives.
Volatile analytes show no response.

CAD response by flow injection after correction

Click to enlarge
Uniform response of Charged Aerosol Detection for a range of chemically diverse test substances using flow injection analysis (0.5 μg into a mobile phase stream of water/acetonitrile 20/80 (v/v)). The variability of response was < 6%.

Mobile phase composition

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:

Mobile phase quality and age
Mobile phase additives and impurities
Glassware and pH adjustments
Gradient elution

We discuss each of these factors in detail and the effect on the CAD response, along with best practices for preparing your mobile phase.

Mobile phase preparation

Click to enlarge
HPLC-CAD gradient analysis of a non-volatile analyte (2840 ng on column (o.c.)) A: blue trace using mobile phase with contaminated additive, B: black trace using good quality phase prepared with fresh additive). Indicators of poor mobile phase quality like baseline artifacts and accumulated contaminants are shown in the figure. Poor response is an obstacle to allow the detection of even relatively high levels of analytes, which is no problem by using the appropriate solvent quality.

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:

Only use solvents labeled as LC/MS grade or better that contain the lowest “residue after evaporation” specification.
For all commercial solvents, record lot number and solvent quality. Document the residue after evaporation for a given lot of solvent. Using a permanent marker, log date received and opened to avoid using contaminated or old solvents inadvertently.
If a different grade, supplier, lot etc. of the solvent is used, consider testing the solvent against a solvent of known quality.
Do not store mobile phases for later use as impurities and contaminants can increase over time.

Aqueous solvents

A common cause for high background currents in the CAD response can come from impurities in the water used for mobile phase preparation.

Use Type 1 ultrapure water as defined by ASTM D1193 with key specifications of 18.2 MΩ-cm or greater resistivity at 25 °C and maxima of 50 ppb total organic carbon, 1 ppb sodium, 1 ppb chlorides, and 3 ppb total silica when possible.
If you cannot obtain Type 1 ultrapure water then we suggest using a high-grade of bottled water with low levels of impurities is recommended, such as UHPLC-MS grade water. Keep in mind using bottled water will usually produce higher background currents and noise levels than fresh ultrapure water.
Another approach is not to use pure water but rather water with a low percentage of the organic solvent like methanol or acetonitrile but make sure to adjust the gradient profile to account for this change.

Organic solvents

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.

Some grades of organic solvents like THF are often stabilized with additives like butylated hydroxytoluene.
Other “high purity” solvent grades are usually tailored for a specific purpose (electrochemical or spectrophotometric grade) and optimized to contain the least possible amount of a certain class of impurities (redox active or chromophoric contaminants) but may contain significant impurities of other classes.
Not all vendors have the same “residue after evaporation” specification for a given grade of solvent. Choosing solvent grades with the lowest “residue after evaporation” specification can help mitigate all of these issues and keep the CAD at optimal performance.

Click to enlarge
CAD baseline behavior for four different commercial sources of methanol. Black, blue, and pink traces – LC/MS grade; brown – UHPLC grade. Two filter constants are shown: 7 (0–3.5 min) and 0 (3.5–6.0 min). For details refer to Technical Note 140: Optimizing and monitoring solvent quality for UV-Vis absorption, fluorescence and charged aerosol detectors.

Solvent aging

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:

Establish shelf lifetimes and refrain from using solvents exposed to air for a prolonged time.
Choose the volume of your reservoir bottles according to solvent use to ensure the shortest shelf time possible.
Use fresh ultrapure deionized water to prepare aqueous mobile phases and prepare daily to maintain quality.
Prepare organic mobile phases at least on a weekly basis to preserve quality.

Click to enlarge
Effects of solvent storage on CAD baseline behavior. Black – fresh bottle of LC/MS grade acetonitrile (ACN) (lot A) used directly; pink – same bottle of ACN lot A but used following repetitive opening and closing of solvent bottle; blue – different lot.

Glassware

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.

Use glassware dedicated only for use with the CAD, including mobile phase reservoirs, beakers, flasks, and graduated cylinders.
The CAD is an excellent tool for analyzing detergents and surfactants, so we recommended detergent-free cleaning agent, washing your CAD-dedicated glassware manually, or taking special care to rinse your glassware after washing AND prior to use.
Always rinse your glassware with a suitable solvent, followed by triple-rinsing with high-purity deionized water, followed by rinsing with the mobile phase solvent or weaker eluting mobile phase solvent when a mobile phase contains a solvent mixture.
Allow the glassware to air dry after cleaning (refrain from using paper towels) and cover any open glassware not in use to prevent the entry of dust.

pH adjustments

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.

Thoroughly rinse the electrode surface with ultrapure water before use.
Avoid immersing the pH electrode into the mobile phase, use small aliquots to measure the pH and discard after the pH adjustment is complete.

Mobile phase additives

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:

Concentration of semi-volatile and nonvolatile additives in the mobile phase. We recommended you restrict the concentration of additives to the minimum required to achieve the desired separation.
Level of impurities in the mobile phase additives. Higher levels of impurities typically give higher background currents, higher baseline noise, and more pronounced baseline drift.
Some additives are hygroscopic or react with air. Your best option is to purchase single vials use containers and only use freshly opened vials.
Ability of additives to form salts with ionic analytes during nebulization. This is a balance between (i) trying to form a salt between the additive and the ion of interest to increase response for semi-volatile and (ii) avoiding salt formation between the additive and charged contaminants in the phase that will lead to more noise and decreased performance.

Relationship between chemical volatility and CAD compatibility

	Analyte	Mobile phase	Additives
Non-volatile	Highly compatible	Not compatible	Not compatible
Semi-volatile	Compatible	Compatible	Compatible
Volatile	Not compatible	Compatible	Compatible

Non-volatile mobile phase additives are extremely incompatible with the CAD and can adversely affect performance.

Short-term exposure to non-volatile additives gives increased noise, decreased response and marked baseline drift.
Long-term exposure, especially at high levels, may require your CAD to undergo major service or repairs.

Gradient elution

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?

Click to enlarge
Chromatograms and calibration curves of four pharmaceuticals without (A) and with (B) inverse gradient compensation. Similar analyte response curves for all induvial analytes are only obtained when using inverse gradient compensation, which allows you to quantify with only a surrogate standard.

Salt formation

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.

Click to enlarge
Observed noise at different concentrations of a volatile additive. The CAD was used at default settings with 1 mL/min isocratic flow of ammonium formate dissolved in 80/20 (v/v) water/methanol.

Here’s what you should know about salt formations:

1. Unwanted salt formation decreases detector performance in certain cases

When salts derive from ionized contaminants in the mobile phase
If salts form between an ionized contaminant in the mobile phase and ionized additives
When you add both anionic and cationic ion pairing agents to the mobile phase

2. Intentional salt formation can actually broaden the range of analytes detected by the CAD

Formation of a salt between an ion pairing agent and an oppositely charged semi-volatile analyte can force the analyte to behave more like a non-volatile species. This is important when using standard free quantitation

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.

Click to enlarge
Utilization of salt formation to enhance signal intensity for the semi-volatile compound oxalic acid (0.5 µg each). By building a non-volatile complex the detected peak area is multiplied for the former semi-volatile, hard to detect oxalic acid.

Volatile additives and typical concentrations compatible with the CAD

Acidic additive	mM	mg/mL	pKa	pH
Acetic acid	17.4	1.04	4.8	3.27
Formic acid	26.3	1.09	3.8	2.7
Trifluoroacetic acid	13	1.48	0.0	1.9
Buffer	mM	mg/mL	pKa	Buffer range
Ammonium acetate	10	0.77	4.8	3.8-5.8
Ammonium format	10	0.63	3.8	2.8-4.8
*Ammonium carbonate	10	0.96	10.3 9.3 7.8	7-11

*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.

Other factors

EvapT

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.

Standards

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.

Check the label or refer to the Certificate of Analysis to learn more about analytical approaches used for purity determinations.

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:

Purchase small quantities of standards preferably in single use sealed vials or use pre-weighed sealed ampules for standard preparation.
Store standards over an active desiccant and replace when necessary.
Record in a logbook the date you purchased the standard, when opened and after each use.
Set appropriate expiration date and replace when expired.

Columns/bleed

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.

Some columns are notoriously unstable like silica, and silica-based amino and cyano; avoid these modes when possible.
Use the lowest column temperature that provides good resolution to help prevent column bleed.

Vials and caps

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:

New interfering peaks appear in the chromatogram that affect analyte peaks of interest.
Changes in the solvent front impact early eluting peaks.
Large perturbations in the baseline chromatogram.

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.

Click to enlarge
Comparison of three different vials. A significant amount of ionic leachables is seen in a non-passivated vial (red line). Another vial type shows a high amount of unidentified leachable(blue line) eluting in the void volume. A third vial did not show any significant amount of leachables and was acceptable for use with this method (purple line).