Introduction
Capillary conditioning is one of the most important maintenance procedures in Capillary Electrophoresis (CE). Proper conditioning prepares the inner capillary surface for analysis by removing contaminants, establishing a consistent surface charge, and ensuring stable electroosmotic flow (EOF).
Whether using a new capillary or a capillary that has been previously used, routine conditioning can help improve reproducibility and reduce run-to-run variability.
Why Capillary Conditioning Is Important
The inner surface of a fused silica capillary can be affected by:
- Manufacturing residues
- Buffer deposits
- Sample matrix components
- Environmental contamination
- Adsorbed analytes
Without proper conditioning, these materials may contribute to:
- Migration time shifts
- Peak shape distortion
- Poor reproducibility
- Reduced efficiency
- Unstable electroosmotic flow
Routine conditioning helps maintain consistent separation performance.
Conditioning New Capillaries
New capillaries should always be conditioned before their first use.
The initial conditioning process helps:
- Remove residual manufacturing materials
- Hydrate the capillary surface
- Establish consistent operating conditions
- Improve reproducibility from the first analysis
Failure to properly condition a new capillary may result in inconsistent performance during early runs.
Recommended Conditioning Procedure
Step 1: Filter All Solutions
Before conditioning or operation, filter all solutions through a:
- 0.45 µm syringe filter, or
- 0.20 µm syringe filter
Filtering helps prevent particulates from entering the capillary and causing restrictions or performance issues.
Step 2: Flush with Sodium Hydroxide
Condition the capillary by flushing with: 0.1 N Sodium Hydroxide (NaOH) using the CE instrument.
The sodium hydroxide treatment helps:
- Clean the capillary surface
- Remove contaminants
- Activate the silica surface
- Promote consistent electroosmotic flow
Step 3: Rinse with High-Purity Water
Following NaOH treatment, flush the capillary with: CE-grade water or HPCE-grade water for approximately: 5 minutes. This rinse removes residual sodium hydroxide and prepares the capillary for buffer equilibration.
Step 4: Equilibrate with Run Buffer
As the final conditioning step, flush the capillary with the intended run buffer for: 10 minutes This allows the capillary surface and separation buffer to reach equilibrium before sample injection.
Daily Conditioning Recommendations
This conditioning procedure is not limited to new capillaries.
For best performance, the same conditioning sequence should be performed:
- Before daily use
- Before beginning a new analysis sequence
- After extended storage
- Whenever reproducibility issues are observed
Routine conditioning can significantly improve long-term method consistency.
Benefits of Routine Conditioning
Proper conditioning can help achieve:
- Stable electroosmotic flow
- Reproducible migration times
- Improved peak shape
- Reduced analyte adsorption
- Better method robustness
- More reliable quantitative results
These benefits are particularly important in regulated environments and validated analytical methods.
Additional Good Practices
To maximize capillary performance:
- Use only high-purity reagents and buffers.
- Filter all solutions before use.
- Replace contaminated buffers regularly.
- Avoid introducing particulates into the capillary.
- Follow manufacturer-recommended storage procedures.
- Perform routine conditioning before analytical sequences.
These practices help extend capillary life and maintain consistent analytical results.
Conclusion
Capillary conditioning is a critical step in achieving reliable Capillary Electrophoresis performance. By flushing with 0.1 N sodium hydroxide, rinsing with high-purity water, and equilibrating with the run buffer, analysts can establish consistent capillary surface conditions and improve reproducibility. Regular conditioning of both new and previously used capillaries helps ensure stable electroosmotic flow, consistent migration times, and dependable analytical results.