How to Identify Peaks in a Chromatogram
Chromatography is one of the core tools in analytical chemistry. It allows scientists to separate and identify complex mixtures with accuracy. However, once you run your sample and see a chromatogram, the real detective work begins—deciding which peak belongs to which analyte. Whether you use TLC, HPLC, or GC, correct peak identification is essential for both qualitative and quantitative analysis.
In this guide, we’ll walk step by step through proven strategies, useful tools, and common mistakes to avoid.
1. Start with Standards
The most reliable way to identify peaks is by comparing your sample chromatogram to known standards.
- Run individual standards: Inject each analyte separately under the same conditions.
- Record retention times (RTs): Write down the RT for each compound.
- Overlay chromatograms: Compare the sample with the standards to match peaks.
💡 Tip: Use a standard mix to save time, but check carefully to avoid co-elution.
2. Use Retention Time as a Key Identifier
Retention time (RT) is the most common metric for peak ID. However, it can shift due to:
- Column age and conditioning
- Mobile phase composition
- Flow rate and temperature changes
- Injection volume differences
To stay consistent:
- Calibrate often with standards.
- Use internal standards to correct shifts.
- Add a neutral marker to confirm system performance.
3. Check Peak Shape and UV/Vis Spectra
If your detector supports it (UV, DAD, or PDA in HPLC), use spectra for confirmation.
- UV/Vis spectra: Match absorbance patterns to known compounds.
- Peak purity analysis: Detect co-elution or impurities.
Poor peak shape can point to problems:
- Fronting peaks – column overload or poor solubility
- Tailing peaks – wrong mobile phase pH or active sites on the column
- Broad peaks – failing column or incompatible mobile phase
4. Use Mass Spectrometry When Possible
In LC-MS or GC-MS, mass spectra provide molecular weight and fragmentation patterns.
- Create Extracted Ion Chromatograms (EICs) for specific m/z values.
- Match spectra to libraries like NIST or Wiley.
- Confirm identity with fragmentation patterns when compounds share a molecular ion.
5. Apply Relative Retention or Rf Values
When working with TLC or when RTs vary slightly:
- Relative Retention Time (RRT):
RRT = T analyte / T reference - Rf values in TLC:
Rf = Distance compound / Distance solvent front
These ratios reduce variability and improve reproducibility.
6. Spike Your Sample
Spiking provides strong confirmation:
- Add a small amount of a known standard.
- If peak height or area increases without shifting RT, the identity is confirmed.
This works well when peaks are close together, when co-elution is suspected, or when spectral data is limited.
Common Pitfalls and How to Avoid Them
| Mistake | Why It Matters | How to Avoid |
|---|---|---|
| Assuming RTs never change | RTs drift due to system shifts | Always run standards under the same conditions |
| Ignoring co-elution | Overlapping peaks mislead ID | Use spectral data or MS for purity checks |
| Using peak area alone | Area ≠ identity | Combine retention time and spectral data |
| Skipping validation | Risk of false positives | Validate methods for specificity, accuracy, and precision |
Tools That Help
- Chromatography software (ChemStation™, Empower™) for integration and matching
- Spectral libraries for UV/Vis or MS data
- Internal standards for normalization and QC
- TLC visualization tools like UV lamps, iodine chambers, or staining reagents
Final Thoughts
Peak identification blends science and skill. It takes experimental rigor, the right tools, and chemical intuition. By combining standards, retention times, spectra, spiking, and validation, you can confidently assign peaks even in complex mixtures.
Whether you are troubleshooting an issue or building a new method, mastering peak identification unlocks the true power of chromatography.
Ray Lombardi, General Manager
With over 43 years’ experience in chromatography, Ray brings a wealth of unprecedented experience in application development, product understanding, and executive management to Sorbent Technologies. Ray is a results-driven professional manager who has held management positions at Thermo Fisher, Agilent Technologies, and Micromeritics Instruments.
Mr. Lombardi’s focus has always centered around the creation of an effective solutions model that is attentive to creating true and tangible value for his customers. Ray has realized that true value is measured only by the success that you bring to your customers in helping them expand their research, improve their products, and increase their innovation.