Analyzing Explosive Traces Using 2D-LC

In a recent study led by Bob Pirok and Rick S. van den Hurk of the Van ’t Hoff Institute for Molecular Sciences at the University of Amsterdam a novel heart-cutting two-dimensional liquid chromatography (2D-LC) workflow capable of simultaneously separating and detecting both inorganic and organic explosive residues from a single injection was developed (1). This latest innovation has implications for post-blast forensic analysis.

This new method combines hydrophilic interaction chromatography (HILIC) for separating inorganic anions and cations with reversed-phase liquid chromatography (RPLC) for analyzing organic explosives, using active solvent modulation to transfer and refocus analytes between dimensions (1).

As part of our “From Sample to Verdict” series, we sat down with Rick S. van den Hurk and Bob Pirok of the van ’t Hoff Institute for Molecular Sciences at the University of Amsterdam to ask them about their new method, and how multidimensional liquid chromatography is being used to analyze traces of explosives.

Your study highlights the challenge of detecting both inorganic and organic explosives due to their chemical differences (1). What were some of the key considerations in designing a workflow that could effectively analyze both explosive types within a single injection?

Our Chemometrics and Advanced Separations Team is embedded in the Forensic Analytical Chemistry group of Prof. Arian van Asten, who is professor of on-scene analysis and also affiliated to the Netherlands Forensics Institute (NFI). Our team specializes in advancing fundamental separation science and chemometrics to solve problems such as is the case in forensics.

For this project, the key considerations included the chemical diversity of the analytes, the suitable separation mechanisms to effectively utilize this diversity, and the modulation system to transfer part of the sample from one separation mode to another. The inorganic analytes of interest encompass both positively and negatively charged species, meaning that we have to separate essentially three chemical attributes in a single injection.

Can you explain the advantages of combining HILIC with RPLC in a heart-cutting two-dimensional LC (2D-LC) approach for forensic applications?

Because we have to separate three different chemical attributes in two separation modes, one mode should be capable of separating two attributes. In our case, HILIC was capable of separating both the positively and negatively charged analytes whereas RPLC separates the organic analytes. Because all the organic analytes are not highly polar, they will elute unretained from the HILIC column, a heart-cutting approach is most suitable.

You mentioned using active solvent modulation to transfer and refocus the organic fraction. How did this technique improve the separation or detection process, particularly for complex explosive residues?

The heart-cut fraction we took from the first-dimension HILIC contained a lot of acetonitrile. Meanwhile, some of the organic explosives were still relatively polar and would elute in RPLC already at a low fraction of acetonitrile in the eluent. Active solvent modulation (ASM) can exploit the second-dimension eluent as a means to dilute the heart-cut fraction before it enters the RPLC column. ASM was beneficial for its versatility in selecting the desired dilution factor without requiring an auxiliary pump. With the sufficient dilution obtained from ASM, even the relatively polar organic analytes that may be in explosive residues could be refocused, thus obtaining good separation performance.

Ammonium detection posed a specific challenge in your workflow. Could you elaborate on the derivatization strategy you developed and why ammonium is such a critical ion in explosive residue analysis?

The portfolio of inorganic analytes of interest in explosive casework is broad. Therefore, any sample preparation or mobile phase additives that contain similar ions could interfere with the separation or detection. Among these charged analytes, ammonium is a key indicator for certain types of explosive materials, including, for instance, ammonium nitrate. Because ammonium formate is used in the HILIC mobile phase, we had to perform a derivatization reaction to enable quantification of ammonium originating from the sample. The derivatization strategy was based on earlier works targeting the amine functionality on amino acids. When we tried it for ammonium, it appeared to work. We then implemented minor modifications to minimize the dilution of the original sample during the derivatization workflow to maximize the sensitivity of the complete analytical workflow.

While mass spectrometry (MS) compatibility was built into your mobile phases, you opted to use evaporative light scattering and ultraviolet (UV) detection for this study. What drove that decision, and how do you see mass spectrometry being integrated into future iterations of this method?

In this study, our main focus was to demonstrate the feasibility of separating as many explosive trace analytes of interest as possible. For ease of method development, using evaporative light scattering detection (ELSD) for the inorganic analytes and UV for the organic analytes was preferred. However, the sensitivity of these detectors is worse than what MS could offer. In its current form, the sensitivity for most analytes is insufficient to be used in forensic explosive casework because these traces are typically retrieved in low sample amounts. The use of MS in future iterations may resolve this limitation. However, because of the heart-cutting nature of the method, two MS instruments are ideally used, one for each separation.

From a forensic casework perspective, how do you see this single-injection, multi-class detection method impacting routine analysis workflows in forensic laboratories dealing with post-explosion scenes?

The current workflow for unknown samples from a post-explosion crime scene involves extensive (manual) sample preparation to separate the three different classes of analytes. Then, three separate analyses are performed on the respective sample fractions. Once fully developed and validated, our single-injection approach intends to minimize sample loss during the sample preparation process as the entire sample is subjected to the derivatization workflow with minor dilution. Instead of three separate analysis, one analytical workflow in which all analytes of interest are separated, may speed up the process and reduce the required manual labor for explosive-residue screening.

How has 2D-LC evolved as a technique in analyzing explosives over the past decade or so?

2D-LC has hardly been explored for analyzing explosives so far, as most forensic laboratories do not have access to 2D-LC instrumentation. However, as a technique, 2D-LC has seen many technical developments over the past decade. One of the major improvements to the applicability of 2D-LC has been the development of modulation strategies to overcome solvent mismatch between the two separation modes and to maintain sensitivity. As a result, 2D-LC as a technique has seen growing interest from non-academic laboratories and the scope for applications has significantly broadened. The analysis of explosives is merely a new area in which 2D-LC can now offer interesting separation performance, which previously remained largely unexplored.

How does your 2025 study advance or expand the work conducted in your 2022 study (2)?

In the 2022 study, we focused on a specific type of explosive material called smokeless powders. These are typically used as propellant in ammunition and rockets. Smokeless powders comprise for a significant fraction of nitrocellulose, a polymeric explosive compound. The characterization of nitrocellulose has not yet been exploited for forensic differentiation between types and brands of commercial smokeless powder products. This method required the analyst to know that they are dealing with a smokeless powder before doing the analysis, for instance a shooting incident where ammunition was recovered. In the 2025 study, we aimed to develop universal 2D-LC method that is capable of identifying as many different types of explosive materials as possible, as opposed to only one type. As such, its more universally applicable to completely unknown samples of explosive traces recovered from a crime scene to aid in identifying the class of energetic material.

What are the current ongoing challenges that chromatographers face in forensic analysis, and what steps are underway to resolve these issues?

Many of the chromatographic methods used in forensic laboratories are not regularly updated because of the lengthy validation process required for the results to be used in a criminal investigation. To streamline method development and possibly the validation procedure, the use of artificial intelligence may be of use. Similarly, with the rise of more complex explosives and accelerants and also complex illicit drug isomers, the conventional methods may not be sufficient to separate new analytes of interest.

In addition, with state-of-the-art separation technology, many of the existing methods could be performed faster. High-resolution systems, such as 2D chromatography, can be further explored for their separation power and also to circumvent sample preparation steps and ultimately reduce labor. They may also be used to investigate new means of chemical discrimination of sample types, such as what we demonstrated in our 2022 study for nitrocellulose-based explosive materials.

References

1. Van den Hurk, R. S.; Belina, E.; Verduin, J.; et al. Simultaneous Analysis of Organic and Inorganic Explosive Traces by Online Two-dimensional Liquid Chromatography. J. Chromatogr. A. 2025, 1758, 466187. DOI: 10.1016/j.chroma.2025.466187
2. Van den Hurk, Abdulhussain, N.; van Beurden, A. S. A.; et al. Characterization and Comparison of Smokeless Powders by On-line Two-dimensional Liquid Chromatography. J. Chromatogr. A. 2022, 1672, 463072. DOI: 10.1016/j.chroma.2022.463072