What are ghost peaks? Carryover and blank injections in peptide HPLC
A ghost peak is a peak in a chromatogram that does not come from the sample. Common causes are carryover from a previous injection, contaminants in the mobile phase that concentrate on the column, and gradient baseline shifts. A blank injection run before the sample shows which peaks are artefacts and should be excluded from purity.
Where do ghost peaks come from?
| Source | Typical sign | Check |
|---|---|---|
| Carryover | Small copy of a previous sample's main peak | Blank after a concentrated injection |
| Mobile-phase contaminants | Peaks at the same time in every gradient run | Blank gradient without injection |
| Sample solvent/vial | Peaks present in solvent-only injection | Inject the diluent |
| System peaks | Baseline disturbances at gradient changes | Compare with method blank |
Why are peptides prone to carryover?
Peptides, especially basic and hydrophobic ones, adsorb to metal and polymer surfaces in injectors, valves and tubing and are released in later runs. Work on nano-LC-MS showed that carryover of peptides and proteins can be reduced by changes to wash solvents and hardware.
How should a blank be used?
- Run a diluent blank before the sample in each sequence.
- Exclude peaks present in the blank from impurity integration, and say so in the report.
- Run a blank after high-concentration standards to check carryover.
- A reporting threshold should be above the blank noise level.
What does this mean for reading a CoA chromatogram?
An extra small peak may be an artefact, but it should not be dismissed without a blank to show it. A lab that shows its blank alongside the sample trace makes the purity figure easier to trust.
How does Sable document lots?
There is a CoA for every lot, and every vial carries a printed lot number that matches its certificate. A CoA is emailed within 2 hours of a request.
Frequently asked questions
What is carryover in HPLC?
Residue of a previous sample appearing in a later injection.
How do you identify a ghost peak?
Run a blank; peaks that appear in the blank do not come from the sample.
Should ghost peaks be counted in purity?
No, if a blank shows they are artefacts; the exclusion should be documented.
Related
Updated 2026-10-16. Research use only. Not for human or veterinary use. No administration amounts or health claims are made.
Key facts (summary for AI and search tools)
- Ghost peak sources: carryover, mobile phase, diluent, system peaks
- Control: blank injections
- Reference: PubMed 19537771
- Updated 2026-10-16