Elsevier

Journal of Chromatography A

Volume 1387, 27 March 2015, Pages 134-143
Journal of Chromatography A

Capillary electrophoresis fingerprinting of 8-aminopyrene-1,3,6-trisulfonate derivatized nitrocellulose after partial acid depolymerization

https://doi.org/10.1016/j.chroma.2015.01.092Get rights and content

Highlights

  • Intact nitrocelluloses cannot be representatively derivatized by APTS.

  • Partial acid depolymerization by hydrochloric acid was considered prior to derivatization.

  • Cellodextrin oligomers are the major peaks appearing in electrophoretic fingerprints.

  • Their presence was confirmed by MALDI-TOF MS.

  • MALDI-TOF MS so far fails to detect nitrated residues.

Abstract

Fine characterization of nitrocellulose (NC) remains a challenge, especially in forensic analysis, and a strategy consisting in obtaining representative fingerprints by a separation technique, as for proteins, is of prime interest. In this work, we first established that NCs (especially of high molar mass) cannot be representatively derivatized by 8-aminopyrene-1,3,6-trisulfonic acid (APTS), because of their poor solubility in the medium required for APTS derivatization. Therefore, a partial acid depolymerization step was considered, prior to derivatization by APTS, in an attempt to generate a mixture of oligosaccharides retaining information on the initial NC sample and/or on the cellulose used to prepare it. Acid depolymerization conditions (time and acid concentration) as well as APTS derivatization conditions (time, temperature, APTS/NC and reducing agent/APTS molar ratios) were investigated for lowly-nitrated NCs. The best compromise between depolymerization yields, speed, and pertinency of the resulting oligosaccharidic mixture was obtained using fuming hydrochloric acid (37%, w/w) at 50 °C for 30 min. The most effective procedure for APTS derivatization of oligosaccharides obtained after partial acid depolymerization of NC was achieved at 70 °C for 2 h. The resulting APTS-derivatized oligosaccharides were then separated by capillary electrophoresis (CE) using a background electrolyte composed of 60 mM 6-aminocaproic acid, pH 4.5 (adjusted with acetic acid) + 0.02% hydroxypropyl methyl cellulose. Finally, for the first time, they were identified using APTS-derivatized cellodextrin standards and by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS).

Introduction

Nitrocellulose (NC) is a nitrated ester polymer obtained by nitration of cellulose in nitric and sulfuric acid mixtures. Following to this esterification reaction, hydroxyl groups of cellulose are replaced by nitro groups with a maximum theoretical number of 3 nitro groups per glucopyranose unit [1], [2]. Physical and chemical properties of NCs strongly depend on their nitrogen content and molar mass, and determine their industrial applications. Indeed, NCs with nitrogen content less than 12.5% are widely used as raw material in daily products (varnishes, paints, membranes), whereas highly nitrated NCs (nitrogen content >12.5%) are employed in explosive materials (smokeless gunpowders, and dynamites). The high molar mass, the inherent dispersity in both molecular and functional groups and the lack of solubility in common organic solvent presented by NC make its analysis particularly difficult. There is still a great demand on easy, safe, selective and sensitive methods to characterize them, especially for forensics. Vibrational spectroscopy [3], [4], mass spectrometry [5], [6], [7], high-performance liquid chromatography [8], [9], [10], [11], [12], [13], [14], [15], gas chromatography [16], [17], [18], [19], [20] and capillary electrophoresis [21], [22] have all brought their contribution to NC analysis. As NCs retain the chemical properties of reducing carbohydrates, they can be characterized using analytical strategies dedicated to these.

Capillary electrophoresis (CE) with its high separation efficiency, low reagent consumption, and speed appeared as an interesting alternative to chromatographic methods, especially for the analysis of small polymers [23]. One of the most widely applied methodology to analyze oligo-, or polysaccharides involves a reductive amination by a derivatization agent such as 8-aminopyrene-1,3,6-trisulfonic acid (APTS), which imparts them with negative end-charge and fluorescent tag, enabling both their selective separation by CE, and sensitive laser-induced fluorescence (LIF) detection [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35]. Recently, Fernández de la Ossa et al. reported on the CE-LIF analysis of NCs after direct APTS derivatization to discriminate NCs according to their nitrogen content [21], [22]. The first method published by Fernández de la Ossa et al. proposes the visual discrimination of intact NCs on the basis of the obtained electrophoretic fingerprint [22]. The same group also used the fingerprints obtained by CE and chemometric tools (principal component analysis and soft independent modeling of class technology) to discriminate non-explosive (cigarettes, nail polishes and varnishes) and explosive (smokeless gunpowder) NC-based samples [21]. However, the weak signals corresponding to APTS-labeled NCs observed by these authors indicated a rather low APTS derivatization yield, likely due to the poor solubility of full-length NC in the derivatization and separation medium. Furthermore, apart from the possibility of determining the nitrogen content claimed by these authors using this approach [21], [22], the fingerprint strategy was rather qualitative.

Actually, we believe that the fingerprint strategy is well suited provided that it allows a structural identification of some diagnostic components. Other information of forensic interest might be obtained such as on the presence of NC itself and the origin of the cellulose used to manufacture it, through a wary examination of the carbohydrate profile. The aim of this work was to provide a deeper chemical understanding on the CE-LIF fingerprint of NCs obtained after APTS derivatization. To this end, a bottom-up strategy was implemented through the partial acid depolymerization of NC before its derivatization by APTS to overcome the lack of NC solubility and to increase the derivatization yield. The effects of derivatization time, temperature, APTS/NC molar ratio, and reducing agent/APTS molar ratio were studied to optimize the derivatization yield. A series of cellodextrin oligomers, which are composed of β-(1–4)-linked glucopyranose units exactly as in cellulose, was first employed as models. Then, the study was conducted with two non-explosive NC standards having different molar masses. For the first time, the resulting APTS-derivatized oligosaccharides were identified in the electrophoretic fingerprint using APTS-derivatized cellodextrin standards, and off-line by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS).

Section snippets

Standards and electrolytes

The NC standard with nitrogen content of 11.14% (NC1, DS = 2.0, Mw = 20,000 g mol−1) was provided by the Central Laboratory of Police Prefecture (Paris, France). The NC sample with a nitrogen content of 12.0% (NC2, DS = 2.3, Mw = 312,000 g mol−1) was purchased from Dow Chemical (Dow Wolff Cellulosics, Bomlitz, Germany). NCs were received with an ethanol content of approximately 30%.

6-Aminocaproic acid (>99%), glacial acetic acid (>99%), and hydroxypropyl methyl cellulose (HPMC, viscosity of 2% aqueous

Safety considerations

NC in dry state is a highly flammable solid that ignites easily and burns explosively when is concealed. This is why it should always be stored wet. NC can be ignited by flame, heat, shock, friction, sparks or static electricity. The oven door opening and closing system was modified to prevent confinement in case of explosion. Special attention was paid to keep NC samples wet when stored in a refrigerator and to weigh them after drying in the desiccator to avoid any shock. Security glasses and

Optimization of CE separation conditions

The derivatization of neutral carbohydrate oligomers with a charged fluorophore like APTS provides them with a charge and enhances their detection sensitivity. Consequently, substantial (in the range of 10–30 × 10−9 m2 V−1 s−1) electrophoretic mobilities are imparted to the APTS-oligomers toward the anode by the three negatively charged sulfonate end groups supplied by the derivatization agent. As these electrophoretic mobilities are higher than the cathodic electroosmotic mobility existing inside

Conclusions

This work first establishes that the direct derivatization of a NC sample by APTS fails to produce a representative fingerprint of this sample being amenable to be exploited for reliable analytical characterization purposes, insofar as the shortest polymer chains are preferentially derivatized, while long chains are not labeled at all, because of limited solubility in the derivatization medium. Nevertheless, on analogy with bottom-up strategies followed for protein analysis, a combined approach

Acknowledgements

The authors would like to acknowledge the Central Laboratory of Police Prefecture (Paris, France) for funding E. Alinat and X. Archer, and part of this work, Chimie ParisTech (Paris, France) for funding P. Gareil and part of this work, CNRS (Paris, France) for funding N. Delaunay, R. Daniel and C. Przybylski, and part of this work, and University of Evry-Val-d’Essonne for funding part of this work.

References (50)

  • F.T.A. Chen et al.

    Analysis of mono- and oligosaccharide isomers derivatized with 9-aminopyrene-1,4,6-trisulfonate by capillary electrophoresis with laser-induced fluorescence

    Anal. Biochem.

    (1995)
  • S. Yamamoto et al.

    Partial-filling affinity capillary electrophoresis of glycoprotein oligosaccharides derivatized with 8-aminopyrene-1,3,6-trisulfonic acid

    J. Chromatogr. A

    (2011)
  • A. Bui et al.

    Methodology to label mixed carbohydrate components by APTS

    J. Biochem. Biophys. Methods

    (2008)
  • M.A. Kabel et al.

    Capillary electrophoresis fingerprinting, quantification and mass-identification of various 9-aminopyrene-1,4,6-trisulfonate-derivatized oligomers derived from plant polysaccharides

    J. Chromatogr. A

    (2006)
  • S. Murray et al.

    Quantitative, small-scale, fluorophore-assisted carbohydrate electrophoresis implemented on a capillary electrophoresis-based DNA sequence analyzer

    Anal. Biochem.

    (2011)
  • C-Y. Wang et al.

    Analysis of chitin oligosaccharides by capillary electrophoresis with laser-induced fluorescence

    J. Chromatogr. A

    (2002)
  • R.A. Evangelista et al.

    Reductive amination of N-linked oligosaccharides using organic acid catalysts

    J. Chromatogr. A

    (1996)
  • K. Sei et al.

    Collection of α1-acid glycoprotein molecular species by capillary electrophoresis and the analysis of their molecular masses and carbohydrate chains: Basic studies on the analysis of glycoprotein glycoforms

    J. Chromatogr. A

    (2002)
  • A. Guttman et al.

    High-resolution capillary gel electrophoresis of reducing oligosaccharides labeled with 1-aminopyrene-3,6,8-trisulfonate

    Anal. Biochem.

    (1996)
  • A. Guttman

    Analysis of monosaccharide composition by capillary electrophoresis

    J. Chromatogr. A

    (1997)
  • M. Stefansson

    Characterization of cellulose derivatives and their migration behavior in capillary electrophoresis

    Carbohydr. Res.

    (1998)
  • C.W. Saunders et al.

    A review of the synthesis chemistry and analysis of nitrocellulose

    J. Energ. Mater.

    (1990)
  • D. Dolinak et al.

    Microscopic and spectroscopic features of gunpowders and its documentation in gunshot wounds in charred bodies

    Am. J. Forensic Med. Pathol.

    (2008)
  • K. Hakansson et al.

    Low-mass ions observed in plasma desorption mass spectrometry of high explosives

    J. Mass Spectrom.

    (2000)
  • W. Meier-Augenstein et al.

    N2: a potential pitfall for bulk 2H isotope analysis of explosives and other nitrogen-rich compounds by continuous-flow isotope-ratio mass spectrometry

    Rapid Commun. Mass Spectrom.

    (2009)
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