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Capillary Electrophoresis: A Second Look at Peptide Purity
If two impurities have the same hydrophobicity as the main peptide, an HPLC column will not separate them, however good the column is. A different technique that sorts molecules by a different property can. That is the idea behind orthogonal methods, and capillary electrophoresis (CE) is one of the best.
Key takeaways
- CE separates molecules by their charge-to-size ratio in an electric field.
- It resolves deamidation and other charge-changing impurities well.
- It needs tiny sample volumes and gives very sharp peaks.
- Two methods that separate by different principles are more convincing than one.
How it works
A hair-thin fused silica capillary is filled with a buffer, and a few kilovolts of voltage are applied between its ends. A tiny plug of sample is injected at one end. Charged molecules migrate at different speeds depending on their charge and size, and a detector near the other end records them as peaks. In the common form, capillary zone electrophoresis, the buffer pH is set so that peptides carry a defined charge, often an acidic phosphate buffer around pH 2.5.
The silica wall also carries charge, which creates a bulk flow of liquid called electroosmotic flow that drags everything along. Analysts control it with buffer pH and coatings.
Why it sees different things than HPLC
- HPLC sorts by hydrophobicity. CE sorts by charge and size.
- A deamidated peptide gains a negative charge, which shifts it clearly in CE but only slightly in HPLC. See deamidation.
- Truncated or extended peptides change both charge and size and appear at distinct positions.
- Peptides that differ only in a buried hydrophobic residue may co-elute in HPLC yet separate in CE if the charge differs.
Strengths and limits
- Efficiency. Hundreds of thousands of theoretical plates give very sharp peaks.
- Tiny samples. Injection volumes are in nanoliters.
- Low solvent use. Mostly aqueous buffers.
- Limits. UV path length is only the capillary diameter, so sensitivity is lower than HPLC, and results are more sensitive to buffer and capillary condition. It is also less widely run.
Using both methods together
The reasoning is similar to using two independent witnesses. If both HPLC and CE show a single dominant peak with the same estimated purity, the case for a clean sample is much stronger than with either alone. If they disagree, the disagreement is itself informative: an impurity that one method hides is showing up in the other. Add mass spectrometry for identity, and you have three different views of the same sample (see HPLC and mass spectrometry).
What to look for on a report
- Whether the supplier states a second, orthogonal purity method.
- The buffer and voltage conditions, if CE is reported.
- Whether the purity values from the two methods are comparable.
The bottom line
One chromatogram is a single viewpoint. Orthogonal methods such as CE are how analytical chemists check that nothing is hiding behind the main peak. The basic report layout is covered in how to read a peptide COA.
Related reading
- How to Read an HPLC Chromatogram: Peaks, Shoulders and Baselines
- Deamidation in Peptides: Why Asparagine-Glycine Is a Weak Spot
- How to Read a Certificate of Analysis (COA) for Research Peptides
- Browse the full Research Library
For laboratory research use only. Not for human or veterinary use, and not intended to diagnose, treat, cure or prevent any condition. This article is educational and is not medical advice.