HPLC-CAD Method Development Tips

 
 
 
Overview

 
 
 
Factors to consider for your HPLC-CAD method development

 
 
 
Method setup

Once you’ve properly set up your HPLC-CAD system in the lab, you’re ready to begin method development. Here’s some essential best practices you should follow, recommended by our experts:

1. Have a dedicated HPLC-CAD system, free of use with non-volatile additives, like phosphate buffers or non-volatile ion pairing reagents.
   
   
2. Only use LCMS grade solvents and additives that contain the lowest “residue after evaporation” specification.
   
   
3. Use volatile mobile phase additives only, as non-volatiles are extremely incompatible with the CAD and can adversely affect performance.
   
   
4. Start with a regular gradient and then add an inverse gradient for optimization later, if needed.
   
   
5. For all new methods, use the respective default settings on your CAD as a starting point:
   
   
   • Evaporation temperature at 35 °C
     
     
   • Data collection rate at 10 Hz
     
     
   • Power function value at 1.0
     
     
   • Filter time constant at 5.0 s

 
 
 
Method scouting

Besides screening various column and eluent mixtures to select the best combinations for successful separation, you’ll need to assess if your chemicals and method conditions are compatible with the CAD.

Using a step-by-step systematic approach, you’ll see how each component affects the baseline signal or background current.

Your main goal is to get an idea of what background signal is normal for the method conditions and application.

Here are the general steps to follow for method scouting:

1. Verify your target analytes meet the   CAD volatility requirements

If you do not have the numbers at hand, you can test for analyte volatility as described in our white paper:   A reliable UHPLC/UV/CAD/MS multidetector method for routine quantification and library matching of extractable and leachables in pharmaceutical-grade plastics.

2.  Assess the purity and stability of your nitrogen gas

This step involves running nitrogen gas through your HPLC-CAD system in the absence of a column and mobile phase flow to see if the gas flow and background signal are stable.

3. Verify your HPLC system lines and pump are free of residual salts.*

For this step, you’ll run ultra-pure lab water only, with no additives or column connected, at a flow rate of 1 mL/min and EvapT at 35 °C.

• Your background signal should be no higher than 1 pA.

*We highly suggest having a dedicated HPLC system to use only with your CAD. If this situation is not possible, then you’ll need to flush the instrument to remove residual salts from the lines and pumps, and online degasser (if present) before connecting the CAD.

4. Check the quality of your mobile phase, without and with additives, in the absence of a column with the EvapT at 35 °C

Here, you want to track how the CAD background signal changes as the mobile phase flows, and how you can expect the signal to change with and without mobile phase additives, as well as the influence of the stationary phase.

• Clean solvents with no additives and no column in line should give you a maximum signal of 1 pA.

5. Connect the column to ensure there is no bleed from stationary phase decomposition

Run the mobile phase with any additives through your HPLC-CAD system at your desired method conditions including gradients and column heating to verify the background signal is stable with low current.

• Some columns show significant bleed throughout the operating pH range (e.g. silica-based amino and cyano columns) while others only show significant bleed when operated near the specified operating limits such as pH and temperature (e.g. silica-based C18 columns).
  
  
• Although   column bleed can increase noise and background currents, column performance is often acceptable, and columns may still show fairly stable retention times and peak shapes.

6. Run your analysis

Again, the same rules apply here as for your general method development: stable baseline, resolved peaks and enough equilibration time.

• Check the resolution and, depending on qualification or quantitation focus area, you might proceed with method optimization – like gradient fine-tuning, complementary detectors (in case of overlaying peaks) or complementary gradient (inverse gradient).

 
 
 
Method optimization

Optimizing your HPLC-CAD method involves iterative testing of various method conditions to achieve the best resolution, speed, and reproducibility.

• You can convert non-volatiles to semi-volatiles by   changing the EvapT. Best practice is to increase the EvapT by 5 °C if your model allows, not to exceed the maximum temperature setting on your CAD. Higher temperatures may reduce background noise but will also reduce the signal for semi-volatiles.
  
  
• Apply an   inverse gradient. The inverse gradient composition is the exact opposite of your analytical gradient. Under these conditions, the CAD response among non-volatile analytes is more uniform, which gives you more accurate quantitation with a single calibrant.
  
  • Our inverse gradient wizard allows you to easily generate a complementary gradient by maximizing the organic content to increase the response. Our   white paper highlights a quick overview of the three options available with inverse gradient.
     
• Other possible adjustments include altering the   power function to help you optimize the range over which the CAD response is sufficiently linear for a given method's range of quantitation.
  
  
• In some cases, you can   convert semi-volatile analytes to non-volatile ones with additives. Please contact our technical support team to learn more.

 
 
 
Robustness testing & validation

Robustness testing is done to determine the impact of changing parameters of the separation method and is important for both your   method validation and transfer processes.

• Assess calibration curves and standards at different EvapT settings to look at drift and ranges, and by changing the injection volume.
  
  
• Run your method on different raw materials, columns, bulk and finished good lots to see if small variations cause a large change in data.
  
  
• Test replicates across independent batches to establish your method's reproducibility.

 
 
 
HPLC-CAD method development resources

See our full collection of HPLC-CAD method development and transfer resources.

 
 
 
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