Chromatography in Forensic Science

By Stefano Tommasone Ph.D.Reviewed by Lily Ramsey, LLM

Chromatography is a valuable tool frequently used in forensic science. It provides investigators with effective methods for separating and analyzing complex mixtures and helping them understand the substances involved in criminal cases.

Chromatography techniques vary based on the type of separation and detection methods, each offering unique advantages suitable for different purposes. Separation is achieved based on the size or the interaction of analytes with the mobile and solid phases.

Over time, the field has witnessed substantial advancements, significantly improving precision, sensitivity, and application range.

Forensic Science" />Image Credit: Vione/Shutterstock.com

Forensic Chromatography: Advances and Uses

Nowadays, a variety of chromatography techniques are used in forensic applications, including drug analysis, forensic toxicology, and trace evidence examination, thanks to recent innovations that enhance the identification of compounds during forensic research.¹

Planar chromatography, especially thin-layer chromatography (TLC), provides a fast and cost-effective method for identifying ink samples, dyes, and other materials. Despite being a basic method, it is useful in forensic scenarios such as document analysis and forgery.

High-performance thin-layer chromatography-mass spectrometry (HPTLC-MS) has gained attention for its rapid detection of illicit and prescription drugs in biological samples. A validated HPTLC-MS method successfully identified drugs such as citalopram, midazolam, and chlordiazepoxide in forensic toxicology cases.²

Gas chromatography (GC) is one of the most widely used techniques in forensic science, and it is ideal for analyzing volatile compounds. Often coupled with mass spectrometry (GC-MS), it effectively detects drugs, poisons, and flammable liquids involved in arson cases.

The identification of volatile organic compounds (VOCs) linked to postmortem bacterial processes has recently attracted interest in medicolegal death investigations since decomposition odor has been exploited as a forensic trace.

In this regard, two-dimensional gas chromatography (GC×GC) has emerged as a novel tool for the analysis of VOCs produced over time by individual species of bacteria.³

The analysis of thermolabile and non-volatile substances is normally conducted via high-performance liquid chromatography (HPLC). Recently, ultra-high performance liquid chromatography (UHPLC) combined with tandem mass spectrometry (MS/MS) has emerged as a leading method, offering faster analysis times, improved resolution, and enhanced sensitivity.

In drug analysis, forensic experts are increasingly using HPLC and UHPLC to detect and quantify illicit and prescription drugs, thanks to their ability to analyze complex mixtures with high accuracy.

Chromatography techniques are also used in forensic ballistics investigations, offering valuable insights into ammunition brands and firing patterns. Among the various applications, chromatography can be used for the analysis of organic compounds of gunshot residue (OGSR) that originate from the explosives, stabilizers, plasticizers, and other molecules present in gunpowder upon deflagration.⁴

UHPLC-MS/MS was successfully used to identify trace evidence in GSR samples, such as ethyl centralite, diphenylamine and its derivatives, and nitroglycerine. This method’s enhanced sensitivity has improved firearm identification and shooting distance estimations.

Download your PDF copy now!

Case Studies

HPLC analysis of samples of forensic interest enables forensic profiling, which involves the study of the profiles of impurities in drugs like cannabis, cocaine, and opium.

This can be used to support inferences about the similarity between samples and determine their geographical origin. Such information can be used in court for evidential purposes.

Chromatography also allows the identification of drugs of abuse in biological matrices such as hair. For instance, the analysis conducted via UHPLC coupled with high-resolution mass spectrometry (UHPLC-HRMS/MS) positively detected 2C-B (4-bromo-2,5-dimethoxyphenethylamine), mostly known as “pink cocaine,” in the hair of a real forensic case of a polydrug consumer in Spain.⁶

Similarly, UHPLC-triple quadrupole tandem mass spectrometry (UHPLC-QqQ-MS/MS) was used for the detection of modafinil (2-benzhydrylsulfinylacetamide), a psychostimulant used in the treatment of narcolepsy and idiopathic hypersomnia.

The method effectively identified modafinil in evidentiary samples and biological materials. It can be used to evaluate substance use in both legal and illicit contexts, including cases of overdose or misuse. This method also played a key role in post-mortem investigations, detecting modafinil in blood with a concentration of 110 ng/mL.⁷

Top 5 Technologies Reshaping Forensic Science

Challenges and Future Trends

Sample degradation, contamination risks, and data interpretation complexities are some of the challenges that forensic chromatography faces. Achieving fast analytical results on-site with the highest possible accuracy is crucial for investigations.

Advances in artificial intelligence (AI) models aim to mitigate some of these issues by streamlining chromatographic data analysis. AI-driven techniques can improve result accuracy and reduce false-positive or false-negative outcomes.

AI can be integrated into existing testing and analysis processes to make the whole procedure rapid and more accurate. Data fusion (DF) techniques, by combining information from multiple sensors, can significantly enhance the accuracy of forensic investigations, reducing errors and increasing reliability.⁸

There is also growing interest in portable instruments for forensic chromatography, especially for the analysis of drugs of abuse.

Portable GC-MS has been used in illicit drug identification to detect highly potent synthetic opioids, including the differentiation of fentanyl analogs, which is not always possible in the field with more commonly used methods such as fentanyl test strips.⁹

Conclusion

Chromatography plays a crucial role in forensic science, identifying drugs and toxins and tracing evidence. Recent advancements, especially in HPLC and UHPLC-MS/MS techniques and AI-driven data analysis, have significantly improved applications in forensic investigations.

Interest in portable, miniaturized systems that facilitate rapid on-site analysis is growing. Such systems will accelerate evidence collection and improve investigative timelines.

Further advancements in AI methods will continue to enhance the precision and reliability of forensic chromatography, ensuring more accurate results and timely insights in criminal cases.

References

1. Young, G. M. & Lurie, I. S. (2022). Recent forensic applications of enhanced chromatographic separation methods. Journal of Separation Science, 45, 369-381.https://doi.org/10.1002/jssc.202100513. Available: https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/abs/10.1002/jssc.202100513
2. Choudhary, P., Verma, K. L. & Kalra, D. (2022). Validated simultaneous high-performance thin-layer chromatography‒mass spectrometry method for analysis of citalopram prochlorperazine, midazolam, and chlorodiazepoxide in urine for forensic analysis. 35, 363-73.10.1007/s00764-022-00191-3.
3. Furuta, K., Byrne, J., Luat, K., Cheung, C., Carter, D. O., Tipton, L. & Perrault Uptmor, K. A. (2024). Volatile organic compounds produced during postmortem processes can be linked via chromatographic profiles to individual postmortem bacterial species. Journal of Chromatography A, 1728, 465017.https://doi.org/10.1016/j.chroma.2024.465017. Available: https://www.sciencedirect.com/science/article/pii/S0021967324003911
4. Serol, M., Ahmad, S. M., Quintas, A. & Família, C. (2023). Chemical Analysis of Gunpowder and Gunshot Residues. Molecules, 28.10.3390/molecules28145550.
5. Tittarelli, R., Dagoli, S., Cecchi, R., Marsella, L. T. & Romolo, F. S. (2024). 75 years of forensic profiling: A critical review. Heliyon, 10, e39490.10.1016/j.heliyon.2024.e39490.
6. Matey, J. M., López-Fernández, A., García-Ruiz, C., Montalvo, G., Zapata, F. & Martínez, M. A. (2021). Identification of 2C-B in Hair by UHPLC-HRMS/MS. A Real Forensic Case. Toxics, 9.10.3390/toxics9070170.
7. Nowak, K., Chłopaś-Konowałek, A., Szpot, P. & Zawadzki, M. (2025). The Issue of "Smart Drugs" on the Example of Modafinil: Toxicological Analysis of Evidences and Biological Samples. J Xenobiot, 15.10.3390/jox15010015.
8. Felizzato, G., Iacobellis, G., Liberatore, N., Mengali, S., Sabo, M., Scandurra, P., Viola, R. & Romolo, F. S. (2025). Machine Learning-Driven Data Fusion of Chromatograms, Plasmagrams, and IR Spectra of Chemical Compounds of Forensic Interest. ACS Omega, 10, 7048-7057.10.1021/acsomega.4c10107.
9. Gozdzialski, L., Aasen, J., Larnder, A., Ramsay, M., Borden, S. A., Saatchi, A., Gill, C. G., Wallace, B. & Hore, D. K. (2021). Portable gas chromatography–mass spectrometry in drug checking: Detection of carfentanil and etizolam in expected opioid samples. International Journal of Drug Policy, 97, 103409.https://doi.org/10.1016/j.drugpo.2021.103409. Available: https://www.sciencedirect.com/science/article/pii/S0955395921003145

Further Reading

• All Forensics Content
• Collecting Evidence at a Crime Scene
• Using Forensics to Tackle Child Exploitation
• Forensic Investigations of Natural Disasters
• Forensic Identification in Mass Disasters