New CE methods for the determination of inorganic ions and carbohydrates in real matrices

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1 New CE methods for the determination of inorganic ions and carbohydrates in real matrices C. Sarazin a,b, N. Delaunay a, C. Costanza b, V. Eudes b, P. Gareil a a Laboratory of Physicochemistry of Electrolytes, Colloids and Analytical Sciences, Chimie ParisTech, Paris, France b Central Laboratory of Prefecture de Police, Paris, France

home-made formulations high number of targeted inorganic and organic

2 Analysis of post-explosion residues identification / quantitation of explosives used by terrorists: military, industrial, pyrotechnics or home-made formulations high number of targeted inorganic and organic compounds in a large concentration range great variety of matrices

Explosive formulations Military and industrials primary: metallic salts (PbN 3 ),

nitrocellulose, nitroglycerol Smoke-producing, colored light-emitting, sound-emitting

) elements (C, S) carbohydrates (lactose, ) Home-made formulations mixtures containing

acid + aluminum foil mixtures peroxide-based mixtures (hydrogen peroxide + acetone /

3 Explosive formulations Military and industrials primary: metallic salts (PbN 3 ), organics secondary: nitroaromatics, nitrocellulose, nitroglycerin Propelling powders nitrocellulose, nitroglycerol Smoke-producing, colored light-emitting, sound-emitting effects anions (ClO 3-, ClO 4-, NO 3-, SO 4 2- ) cations (Mg 2+, K +, Ba 2+, Sr 2+, Na + ) elements (C, S) carbohydrates (lactose, ) Home-made formulations mixtures containing oxidative salts (ammonium nitrate + fuel oil, chlorate / perchlorate + carbohydrate ) acid + aluminum foil mixtures peroxide-based mixtures (hydrogen peroxide + acetone / hexamethylene tetramine) IC, GC-MS, TLC, for forensic confirmation: complementary analytical techniques + CE

4 Aims and Scope CE development of 2 original methods for inorganic anions and cations with LOD ~ 1 ppm, complementary to IC 11 Anions: Cl -, NO 2-, NO 3-, S 2 O 2-3, ClO 3-, ClO 4-, SO 2-4, SCN -, HPO 2-4, HCO 3-, N formate (internal standard) 8 Cations: Na +, K +, NH 4+, MeNH 3+, Sr 2+, Ca 2+, Ba 2+, Mg 2+ + Li + (internal standard) an original method for the simultaneous analysis of anions and cations in acidic/aluminum mixtures an original method for carbohydrates (sucrose, lactose, glucose, fructose) validation application for routine analysis with real matrices

5 Anion analysis initial conditions developed by Hutchinson et al*: EOF reversed by Polybrene 40 mm Tris, ph mm Na 2 CrO 4 & 10 mm CrO 3 (anionic chromophore) Absorbance (mau) ClO 3- + ClO 4- + N SCN Time (min) improvement in resolution ionic strength capillary length solvent content voltage temperature *Hutchsinson et al., Anal. Chem., 79 (2007)

6 Anion analysis: typical electrophoregram targeted anions potential interferents 1 Iodide 2 Bromide 3 Chloride 4 Nitrite 5 Nitrate 6 Thiosulfate 7 Perchlorate 8 Thiocyanate 9 Azide 10 Chlorate 11 Sulfate 12 Cyanate 13 Fluoride 14 Formate (IS) 15 Chlorite 16 Acetate 17 Carbonate 18 Phosphate 19 Oxalate Bare fused-capillary 50 µm x 96 cm (detection, 87.5 cm) modified by 0.1% (w/w) polybrene. BGE: 25 mm CrO 3, 25 mm Na 2 CrO 4, 100 mm Tris, ph 8.2, 6 % EtOH. Temperature, 15 C. Applied voltage, - 30 kv. Electrokinetic injection, -2 kv, 50 s. Anions at 20 mg L -1 injected in water/bge (9/1, v/v). Indirect UV absorbance at 254 nm. Sarazin et al., J. Chromatogr. A, 1217 (2010)

7 Anion analysis: improvement in LODs IC: LODs of ca 1 ppm for all anions (certified method ISO/CEI 17025) Injection Anions LODs (ppm) Cl - NO - 2 NO - 3 S 2 O 2-3 ClO - 4 SCN - N - 3 ClO 3- SO 2-4 HPO 2-4 Hydrodynamic, classical (50 mbar, 5 s, 0.25 % V cap ) Hydrodynamic, FASS* (50 mbar, 60 s, 3 % V cap ) / 5 / Electrokinetic, FESI** (- 2 kv, 50 s) final LODs < 1 ppm FESI: controlled and constant sample conductivity *FASS: Field Amplified Sample Stacking **FESI: Field Enhanced Sample Injection Sarazin et al., J. Chromatogr. A, 1217 (2010)

5 % RSD (t Mi /t formate ) 0.4-0.9 % RSD (A i ) 2.6-4.8 % RSD (A i /A formate ) 0.6-2.

8 Anion analysis: validation (1/2) LODs (calculated for S/N = 3) from 0.3 to 0.6 mg L -1 intermediate precision (3 repeated analyses over 3 consecutive days) RSD (t Mi ) % RSD (t Mi /t formate ) % RSD (A i ) % RSD (A i /A formate ) % internal standard internal standard Lab requirement < 5 % linearity range from 2 to 20 mg L -1 Sarazin et al., J. Chromatogr. A, 1217 (2010)

9 Anion analysis: validation (2/2) matrix effects tested matrices: cotton, plastic, cloth, paper, soil, metal, glass calibration curves for standards in water and in matrix extracts normalized area case of NO paper water cloth concentration (ppm) statistical comparison dispersions Snedecor s test slopes and intercepts Student s test bias < 20 % for all tested matrices negligible matrix effects Sarazin et al., J. Chromatogr. A, 1217 (2010)

10 Cation analysis targeted cations: lack of UV absorbance choice of a cationic chromophore imidazole, pyridine: carcinogenic, mutagenic, reprotoxic creatinine, morpholine, ephedrine: too low electrophoretic mobilities guanidine Absorbance (AU) steepness of the UV spectrum ~ 190 nm Wavelength (nm) Sarazin et al., Electrophoresis, 32 (2011)

11 Cation analysis targeted cations: lack of UV absorbance choice of a cationic chromophore imidazole, pyridine: carcinogenic, mutagenic, reprotoxic creatinine, morpholine, ephedrine: too low electrophoretic mobilities guanidine Absorbance (mau) [guanidinium] (mm) steepness of the UV spectrum ~ 190 nm low linearity range: 0-18 mm guanidinium concentration fixed at 15 mm Sarazin et al., Electrophoresis, 32 (2011)

12 Cation analysis: analysis time SMIL approach: Successive Multiple Ionic-polymer Layer faster separation more stable and ph-independent cathodic EOF st layer: flush with 1 % (w/w) Polybrene in water 2 nd layer: flush with 0.1 % (w/w) polyvinylsulfate in water Sarazin et al., Electrophoresis, 32 (2011)

13 Cation analysis: typical electrophoregram Absorbance (mau) Cs + Ca 2+ MeNH + 3 NH + K + 4 Na + Mg 2+ Fe 2+, Co 2+, Mn 2+, Ni 2+ Sr 2+ Li + (IS) Zn 2+ Cu 2+ Ba 2+ targeted cations potential interferents Pb Time (min) Bare fused-capillary 75 µm x 80 cm (detection, 70 cm) modified (i) 1% (w/w) Polybrene and (ii) 0.01% (w/w) PVS. BGE: 15 mm guanidine acetate adjusted at ph 4.0 with acetic acid, 3 mm 18-C-6. T, 20 C. Applied voltage, + 30 kv. Indirect UV detection, 190 nm. Hydrodynamic injection (50 mbar, 4 s ). Cations at 15 mg L -1 (except Li + (2 mg L -1 ) and Pb 2+ (50 mg L -1 )) in water. Sarazin et al., Electrophoresis, 32 (2011)

14 Cation analysis: validation LODs (calculated for S/N = 3) from 0.6 to 1.1 mg L -1 intermediate precision (3 repeated analyses over 3 consecutive days) RSD (t Mi ) % RSD (t Mi /t Li+ ) % RSD (A i / t Mi ) % Lab requirement < 5 % linearity range from 3 to 30 mg L -1 matrix effects tested: cotton, cloth, soil bias < 20 % Sarazin et al., Electrophoresis, 32 (2011)

Analysis of acid-aluminum mixture plastic bottle with HCl chemical reaction or HNO 3 acid + balls = important of aluminum foils gazeous release explosion = acidic projection post-blast residue

15 Analysis of acid-aluminum mixture plastic bottle with HCl chemical reaction or HNO 3 acid + balls = important of aluminum foils gazeous release explosion = acidic projection post-blast residue samples: acidic yellow liquid + metal foil pellets need for simultaneous determination of Cl -, NO 3- and Al(III) electrolyte containing 2,6-pyridine dicarboxylic acid (PDC) anionic chromophore for indirect UV detection of anions anionic Al(III) chelate with absorbance in UV Sarazin et al., J. Sep. Sci., 33 (2010)

16 Acid-aluminum mixtures: typical electrophoregram Absorbance (mua) NO 3 - Al(III)-PDC chelate system peak Cl - EOF Temps Time (min) Bare fused silica capillary, 50 µm id x 60 cm (detection, 50 cm) coated with Polybrene (2.5 g L -1 in water). Electrolyte: 20 mm PDC adjusted at ph 4.5. Applied voltage, - 30 kv. T, 20 C. UV detection, 214 nm. Hydrodynamic injection, 50 mbar, 15 s. Sample: 20 ppm Al(III), 40 ppm anions in water simulation of the speciation diagram of aluminum with PDC Sarazin et al., J. Sep. Sci., 33 (2010)

17 Acid-aluminum mixtures: validation Selectivity tested with Zn(II), Cu(II), Fe(II), Ni(II), Co(II), Fe(III), Ca(II), Sr(II), Ba(II), Mg(II), S 2 O 2-3, SO 2-4, Cl -, NO 3-, ClO 3-, ClO 4-, HPO 2-4, SCN -, HCO - 3 LODs (calculated for S/N = 3) 0.3 mg L -1 for Al(III) and 1.2 mg L -1 for NO 3- and Cl - intermediate precision (3 repeated analyses over 3 consecutive days) RSD (t Mi ) % Lab requirement RSD (t Mi /t EOF ) % < 5 % RSD (A i / t Mi ) % linearity range up to 30 mg L -1 matrix effects tested with yellow acid viscous liquid, metallic pellet extracts no effect Sarazin et al., J. Sep. Sci., 33 (2010)

18 Analysis of carbohydrates difficult to analyze «neutral» carbohydrates in CE no easily ionizable functions (pka ) hydrophilic compounds: no interaction with micelles lack of chromophore function for direct UV detection analytical approaches derivatization complexation high ph and indirect UV high ph and direct UV: enediolate form at 270 nm* * Rovio et al., Electrophoresis, 28 (2007)

Carbohydrate analysis: method development 130 mm NaOH + 36 mm Na 2 HPO 4 (Rovio) vs 130 mm NaOH NaOH Man Suc Lac Glu Fru NaOH + Na 2 HPO 4 vs NaOH LOD ~ LOD t anal > t anal N ~ N NaOH + phosphate Rs

19 Carbohydrate analysis: method development 130 mm NaOH + 36 mm Na 2 HPO 4 (Rovio) vs 130 mm NaOH NaOH Man Suc Lac Glu Fru NaOH + Na 2 HPO 4 vs NaOH LOD ~ LOD t anal > t anal N ~ N NaOH + phosphate Rs ~ Rs Glu Suc Lac Fru Man Conditioning: Polybrene 0.1% (m/m) Electrolyte: 130 mm NaOH NaOH EOF inversion Capillary 50 µm x 60 cm (det. UV at 50 cm). V, +16kV. T, 20 C. Injection, 50 mbar, 4 s. Carbohydrates at 50 ppm in water. Beckman Coulter CE-DAD system. capillary 50 µm x 60 cm (det. UV at 50 cm) modified by Polybrene 0.1% (m/m). Electrolyte: 130 mm NaOH, ph T, 20 C. λ analyse = 270 nm. V, - 16 kv. Hydrodynamic injection, 50 mbar, 4 s. Carbohydrates and IS at 50 mg L -1 in water. separation in 12 min direct UV detection photo-oxidation Sarazin et al., Anal. Chem., 83 (2011)

20 Carbohydrate analysis: separation optimization IS fructose, glucose, lactose, sucrose + 5 carbohydrate interferents (xylose, ribose, maltose, mannose and galactose) Time (min) Bare fused-silica capillary 50 µm id x 60 cm (det. at 50 cm) modified with Polybrene 0.1% (m/m). Electrolyte, 130 mm NaOH, ph T, 20 C. λ det = 270 nm. V, - 16 kv. Hydrodynamic injection, 50 mbar, 4 s (0.5 % Vcap). Carbohydrates and IS at 50 mg L -1 in water. IS = naphthalenesulfonic acid Sarazin et al., J. Sep. Sci., 35 (2012) Factors NaOH concentration in the electrolyte separation temperature electrolyte conductivity

21 Carbohydrate analysis: typical electrophoregram targeted carbohydrates potential interferents Rs = 1.34 (IS) naphthalenesulfonic acid (1) ribose (2) xylose (3) fructose (4) mannose (5) glucose (6) galactose (7) maltose (8) lactose (9) sucrose Bare fused-silica capillary 50 µm id x 60 cm (det. at 50 cm) modified with Polybrene (1 g/l in water). Electrolyte: 98 mm NaOH (ph 13,0), 120 mm NaCl. T, 26.5 C. Voltage, -14 kv. Hydrodynamic injection, 5 s, 50 mbar. Direct UV detection at 270 nm. Concentration, 0.05 mm each in water. Sarazin et al., J. Sep. Sci., 35 (2012)

22 Carbohydrate analysis: validation intermediate precision (5 repeated analyses on 3 different days) LOD (S/N = 3) RSD (t Mi /t NSA ) % RSD (normalized corrected areas) % 5 µm for lactose and sucrose, 7 µm for glucose, and 10 µm for fructose linearity range µm for glucose and fructose µm for sucrose and lactose matrix effects tested matrices: soil, cloth, red wine, sucrose solution significant effects for extracts having high ionic or carbohydrate concentrations but the resulting bias did not exceed 10 % < laboratory specifications Sarazin et al., Talanta, 99 (2012)

23 Applications: context Simulation of a suicide attack in a bus mixture of NH 4 NO 3 & icing sugar residues collected on cotton swabs, extracted by water and analyzed by IC & CE

24 Applications: CE analysis of anions 1 Cl - 3 NO 3-8 SO HCO 3-10 HPO 4 2-

25 Applications anions: CE vs IC Extract 1 C (mg L -1 ) Cl - NO 3 - SO 4 2- HCO 3 - HPO 4 2- IC 28 ± ± ± 4.2 NR 11 ± 2.2 CE 24 ± ± ± ± ± 1.3 Extract 2 IC 7.2 ± ± ± 1.4 NR ND CE 8.1 ± ± ± ± 3.1 ND NR, not researched; ND, not detected Good correlation between CE and IC NO 3- : main anion present in the residue extracts

26 Applications: CE analysis of cations 1 NH K + 4 Ca 2+ 5 Na + 6 Mg 2+ IS Li +

27 Applications cations: CE vs IC Extract 1 (dilution 1/10) Extract 2 C (mg L -1 ) NH 4 + K + Ca 2+ Na + Mg 2+ IC 54.5 ± ± ± ± ± 0.7 CE 57.3 ± ± ± ± ± 0.5 IC 45.6 ± ± ± ± 1.2 < LOQ CE 44.9 ± ± ± ± 1.4 < LOQ NR, not researched; ND, not detected Good correlation between CE and IC NH 4+ : main cation present in the residue extracts

28 Applications: CE analysis of carbohydrates Extract 1: no carbohydrate detected 1 fructose 2 glucose 4 sucrose quantitation Sucrose 77 mg L -1 Glucose 31 mg L -1 Fructose 28 mg L -1 established use of NH 4 NO 3 & sucrose in the explosive charge

Conclusion new CE methods are available for inorganic anions inorganic cations neutral carbohydrates analysis of explosive residues these methods were validated for real

29 Conclusion new CE methods are available for inorganic anions inorganic cations neutral carbohydrates analysis of explosive residues these methods were validated for real samples and matrix effects were negligible good correlations between IC and CE results were obtained CE shows great interest as a method confirmation for justice inquiries

30 Acknowledgements Prof. Pierre Gareil Dr. Jérôme Vial Prof. J.-M. Mallet Dr. Cédric Sarazin Christine Costanza Véronique Eudes All of you for your attention

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