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Copyright  2007 by Stephen L. Morgan, slide 1 Stephen L. Morgan, Brandi C. Vann, Brittany M. Baguley, and Amy R. Stefan Department of Chemistry & Biochemistry,

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Presentation on theme: "Copyright  2007 by Stephen L. Morgan, slide 1 Stephen L. Morgan, Brandi C. Vann, Brittany M. Baguley, and Amy R. Stefan Department of Chemistry & Biochemistry,"— Presentation transcript:

1 Copyright  2007 by Stephen L. Morgan, slide 1 Stephen L. Morgan, Brandi C. Vann, Brittany M. Baguley, and Amy R. Stefan Department of Chemistry & Biochemistry, The University of South Carolina, Columbia, SC URL: Advances in Discrimination of Dyed Textile Fibers using Capillary Electrophoresis/ Mass Spectrometry Trace Evidence Symposium, Clearwater, FL 16 August 2007

2 Copyright  2007 by Stephen L. Morgan, slide 2  Extraction and subsequent analysis of dye components from fibers offers the possibility for enhanced discrimination of trace fiber evidence. This research addresses development of methods for high-resolution capillary electrophoresis (CE) separation of dyes isolated from limited size samples of textile fibers.  A combinatorial approach to has been employed to optimize the extraction of dyes from nylon, cotton, polyester, and acrylic fibers. The protocols have also been chosen to be compatible (volatile, absence of salts) with subsequent analysis using capillary electrophoresis (CE) or CE/mass spectrometry.  Capillary electrophoresis with both diode array and mass spectrometry (MS) detection has been demonstrated to achieve both discriminating and sensitive analysis of fiber dyes. Introduction

3 Copyright  2007 by Stephen L. Morgan, slide 3 “At present, much of forensic fibre dye casework is based upon comparison rather than identification. This may change, and the emphasis may shift towards comparison and identification, in which case the addition of a mass spectrometric component to the analytical scheme would be desirable. CE appears to offer reduced mechanical complexity and increased resolution of the separated dye components” [Rendle and Wiggins, 1995]. A starting point

4 Copyright  2007 by Stephen L. Morgan, slide 4 Blue acid dye discrimination C.I. acid blue 277 O O NH 2 SO 3 Na HNCH 3 CH 3 SO 2 NHCH 2 CH 2 OH SO - C.I. acid blue 239 H 3 C ONH NH O O H 2 C NH H 2 C C O 3 C.I. acid blue 45 O O OH NH 2 SO 3 - O H NH 2 - O 3 S

5 Copyright  2007 by Stephen L. Morgan, slide 5 Acid blue 45 Time (min) Acid Blue 239 Acid blue 277 neutrals Absorbace (mAU) Electropherogram taken at 600 nm of three blue acid dyes extracted from nylon fibers 15 mM ammonium acetate in 40/60 ACN-H 2 O, pH 9.3 Capillary: 75 μm i.d., 50 cm Separation at 30 kV and 25 °C 3D plot displaying migration time, wavelength and absorbance. Although the absorbance spectra are similar there are clear structural differences based on the migration times of the dyes. Absorbace (mAU) Wavelength (nm) Time (min) Blue dye extracts / similar UV/Vis spectra

6 Copyright  2007 by Stephen L. Morgan, slide 6 Textile fiber dye extraction / CE methods Extracted fiber (+) Reactive (+) Direct (+) Acid (+) Cationic (+) Disperse (+) Vat (-) Na 2 SO °C 30 min Air oxidize 1 hr NaOH 100 °C 30 min Pyridine/H 2 O 100 °C 60 min Pyridine/H 2 O /NH 4 OH 100 °C 60 min Formic acid /H 2 O 100 °C 60 min Chloro- benzene 100 °C 30 min CottonNylonAcrylic Polyester Determine fiber type via PLM or FTIR Original fiber Morgan SL, Nieuwland AA, Baguley BM, Vann BC, Stefan AR, Dockery CR, Hendrix JE, Extraction and capillary electrophoresis for forensic analysis of textile fiber dyes, Submitted to J Forensic Sci 2007.

7 Copyright  2007 by Stephen L. Morgan, slide 7 Nylon and cotton dyes / anionic CE Electropherogram at 214 nm. Peak identification: Peak identification: (1) neutrals; (2) Acid Blue 239; (3) Acid Yellow 156; (4) Acid Blue 324; (5) Acid Red 337; (6) DID 266; (7) Direct Red 84; (8) Direct Orange 39; (9) Direct Yellow 58, (10) Direct Blue 71-1; (11) Direct Blue 71-2; (12) Reactive Blue 250; (13) Reactive Red 198; (14) Reactive Blue 220; (15) Reactive Red 180; (16) Reactive Red 239/241. Conditions: Buffer: 15 mM ammonium acetate in ACN-H 2 O (40:60, v/v), pH 9.3 Injection: 1 psi 5 s Capillary: 50 μm i.d.,50 cm Separation: 30 kV Detection: diode array UV/visible

8 Copyright  2007 by Stephen L. Morgan, slide 8 CE / diode array detection Inlet Outlet

9 Copyright  2007 by Stephen L. Morgan, slide 9 Silanol groups along the wall of the capillary become ionized after the capillary is filled with buffer solution. Positive buffer ions are attracted to the negatively charged wall. Hydrated buffer ions migrate toward the cathode and drag the bulk solution containing cations, neutrals, and anions past the detection window. CE based on electroosmotic flow (EOF) EOF Fused silica capillary + _ CathodeAnode _ _ _ _ _ _ _

10 Copyright  2007 by Stephen L. Morgan, slide 10 Combinatorial optimization experiments 3) Clamp plate, place in oven 2) Deliver solvent combinations to samples 1) Load fibers into 96-well plate Extraction conditions: Ternary solvent mixtures 200 µL solvent in each well 1 mm cm threads Heat to 100  C, 90  C and 60  C Evaporated to dryness Reconstitute in 100 µL water 96-well plate with glass inserts Liquid sampling robot

11 Copyright  2007 by Stephen L. Morgan, slide 11 Nylon / acid dyes Typically, nylon is dyed with acid dyes containing anionic functional groups that produce varying degrees of solubility in water Chemical dye classes include azo (48%), metal-complex azo (31%), and anthraquinone (10%) Dyes are bound to nylon fiber by electrostatic interactions through salt linkages. N H H N O ] n O [ ] n O N H [ Nylon 6,6 Nylon 6 O O OH NH 2 SO 3 - OH NH 2 - O 3 S C.I. Acid Blue 45, Anthraquinone N - O 3 S N OCH 3 H 3 C NN O CH 3 C.I. Acid orange 156, Azo O - N NHA c N S O O NH 2 O - Co 3+ O - N AcHN N S O O H 2 N O - C.I. Acid Blue 171, Metal-Azo

12 Copyright  2007 by Stephen L. Morgan, slide 12 Combinatorial experimental design was applied to the extraction of acid dyes from nylon using water, aqueous ammonia (12 M), and pyridine. Design point % H 2 O% NH 3 % pyridine Mixture designs for three solvents

13 Copyright  2007 by Stephen L. Morgan, slide 13 Anthraquinone Blue B ABB 60 DEG 60 min The design was replicated twice for a total of 20 experiments The amount of dye recovered was measured using a UV/visible microplate reader Pooled relative standard deviations of replicate experiments are %. 96-well plate with glass inserts Nylon / anthraquinone blue B

14 Copyright  2007 by Stephen L. Morgan, slide 14 Acid Blue 171: metal complex Acid orange 156: Azo R 2=.9197 R 2=.9235 A diagonal ridge ranging from equal mixtures of pyridine:water, to pyridine: ammonia is present in the extraction of all acid dye classes Nylon / azo and metal complex azo Stefan AR, Dockery CR, Nieuwland AA.; Roberson SN, Hendrix JE, Morgan SL. Combinatorial optimization for the extraction of anthraquinone, azo, and metal complex acid dyes from nylon fibers for forensic trace analysis. Submitted to J Forensic Sci 2007.

15 Copyright  2007 by Stephen L. Morgan, slide 15 Hydrogen bonding provides substantivity of direct dyes to cotton Reactive dyes are covalently attached to hydroxyl groups C.I. Reactive Red 1 covalently bonded to cellulose Rendle, D.F.; Crabtree, S.R.; Wiggins, K.G.; Salter, M.T. "Cellulase Digestion of Cotton Dyed with Reactive Dyes and Analysis of the Products by Thin-Layer Chromatography," J. Soc. Dye. Colour 1994, 110, Using a combinatorial approach, best extraction of direct dyes from cotton was achieved at 50:50 water:pyridine Reactive dyes are extracted from cotton using 1.5% NaOH Dye extracts from mixture combinatorial design Cotton / direct and reactive dyes

16 Copyright  2007 by Stephen L. Morgan, slide mM ammonium acetate in 40/60 ACN-H 2 O, pH 9.3 Capillary: 50 μm i.d., 50 cm (82 cm for CE-MS) Separation at 30 kV and 25 °C using 214 nm anionic dyes Peak identification: (1) neutrals; (2) Reactive Blue 21; (3) Reactive Yellow 160; (4) Reactive Orange 72; (5) Reactive Blue 19; (6) Reactive Yellow 176; (7) Reactive Violet 5; (8,9) Reactive Black 5 and Reactive Blue 250; (10) Reactive Red 198; (11) Reactive Blue 220; (12) Reactive Red 180; (13) Reactive Red 239/241. O O NH 2 NH SO 3 - SO 2 CH 2 CH 2 OSO 3 - Reactive blue 19 Cotton / direct and reactive dyes / CE

17 Copyright  2007 by Stephen L. Morgan, slide 17 Cotton fiber /reactive dye extracts / CE Electropherogram at 214 nm of a reactive dye (C.I. reactive violet 5, marked by an arrow) extract after SPE clean up. Electropherogram at 214 nm of a reactive dye (C.I. reactive violet 5, marked by an arrow) extracted from cotton using NaOH C.I. reactive violet 5 Dockery CR, Stefan AR, Nieuwland AA, Roberson SN, Baguley BM, Hendrix JE, Morgan SL. Automated extraction of direct, reactive, and vat dyes from cellulosic fibers for forensic trace analysis by capillary electrophoresis. Submitted to J Forensic Sci 2007.

18 Copyright  2007 by Stephen L. Morgan, slide 18 Cotton / vat dye extracts / CE Buffer: 15 mM ammonium acetate in acetonitrile-water (40:60, v/v), pH reducing agent (sodium dithionite) NaS 2 O 4 OH HO C.I. vat orange 9 DAD electropherograms at 280 nm

19 Copyright  2007 by Stephen L. Morgan, slide 19 Wavelength (nm) Absorbance (mAU) UV/Vis spectrum of vat dye standard UV/Vis spectrum of vat dye extract Wavelength (nm) Absorbance (mAU) Spectra of vat dye standard and extract are the same No change in dye constitution due to extraction conditions Vat dye / UV/visible confirmation Dockery CR, Stefan AR, Nieuwland AA, Roberson SN, Baguley BM, Hendrix JE, Morgan SL. Automated extraction of direct, reactive, and vat dyes from cellulosic fibers for forensic trace analysis by capillary electrophoresis. Submitted to J Forensic Sci 2007.

20 Copyright  2007 by Stephen L. Morgan, slide 20 Acrylic / basic (cationic) dyes Acrylic is composed of 85% repeating acrylonitrile units and 15% monomers and is typically dyed with basic (cationic) dyes Dyes are bound to acrylic fiber through salt linkages provided by initiator and terminator fragments (sulfonate or sulfate acid groups) Optimum extraction conditions were found at 50:50 formic acid:water x y n * H 2 C H C H 2 C C* CN R R' N N C H 3 CH 3 N N NN CH 3 H 2 C C.I. Basic Red 46 Acrylic

21 Copyright  2007 by Stephen L. Morgan, slide 21 Electropherogram at 214 nm. Peak identification: (1) Basic Red 22, (2) Basic Yellow 21, (3) Basic Blue 159, (4) Basic Red 14, (5) Basic Blue 41, (6) Basic Blue 45, (7) Basic Red 18. Conditions:  Buffer: 45 mM ammonium acetate in acetonitrile-water (60:40, v/v), pH 4.7  Capillary: 50 µm i.d., 50 cm  Injection: 1 psi, 5 s  Separation: 20 kV, 25  C  Detection: diode array UV/vis 214 nm Acrylic / basic cationic dyes / CE Peak 1: C.I. basic red 22

22 Copyright  2007 by Stephen L. Morgan, slide 22 Polyester does not contain ionic or covalent dye sites and is typically dyed with water insoluble disperse dyes. The literature (e.g., Laing, et al.) suggests that disperse dyes can be extracted from polyester with chlorobenzene and heat for 30 min. We agree. Disperse dye extracts O O O O [ ]n]n Electron microscope photo of polyester fibers O 2 NNN OCH 3 N N HO C.I. Disperse Orange 29 Polyester / disperse dyes

23 Copyright  2007 by Stephen L. Morgan, slide 23 Absorbance (mAU) C.I. Disperse Yellow 114 NACE conditions for disperse dyes: Buffer: 80 mM ammonium acetate in acetonitrile-methanol (75:25, v/v), pH 9 Separation: 20 kV Peak identification: (1) Disperse Red 343, (2) Disperse Blue 73, (3) Disperse Yellow 114, (4) Disperse Orange 29 Polyester / disperse dyes / CE Electropherogram at 214 nm of Disperse Orange 29 extracted from polyester

24 Copyright  2007 by Stephen L. Morgan, slide 24 Electropherogram at 400 nm of C.I. Acid orange 156 standard Absorbance (mAU) Time (min) Wavelength (nm) Acid dye (standard) / CE

25 Copyright  2007 by Stephen L. Morgan, slide 25 Time (min) Electropherogram at 400 nm of C.I. Acid orange 156 extracted from 10 cm nylon fiber Acid dye nylon extract (10 cm) / CE Wavelength (nm) Absorbance (mAU)

26 Copyright  2007 by Stephen L. Morgan, slide 26 Acid dye nylon extract (5 cm) / CE Electropherogram at 400 nm of C.I. Acid orange 156 extracted from 5 cm nylon fiber Time (min) Wavelength (nm) Absorbance (mAU)

27 Copyright  2007 by Stephen L. Morgan, slide 27 Acid dye nylon extract (2.5 cm) / CE Electropherogram at 400 nm of C.I. Acid orange 156 extracted from 2.5 cm nylon fiber Time (min) Wavelength (nm) Absorbance (mAU)

28 Copyright  2007 by Stephen L. Morgan, slide 28 Acid dye nylon extract (1 cm) / CE Electropherogram at 400 nm of C.I. Acid orange 156 extracted from 1 cm nylon fiber Time (min) Wavelength (nm) Absorbance (mAU)

29 Copyright  2007 by Stephen L. Morgan, slide 29 Petrick, et al. analyzed dyes extracted from acrylic and polyester fibers followed by HPLC/DAD/MS 1:1 formic acid: water extraction of basic dyes Compared extraction efficiency of 4:3 mixtures of pyridine and water to 4:3 mixtures of acetonitrile and water for disperse dyes Analyzed dyes from 5 cm single acrylic fiber and polyester fibers Huang, et al. analyzed acid, basic, and disperse dye pairs by LC/MS Extracted unknown dyes from 10 different red cotton fibers Unknown fiber length Fibers distinguished based on extraction protocol (SWGMAT), ionization in the mass spectrometer, chromatographic behavior, and mass spectra Huang, M.; Russo, R.; Fookes, B.; Sigman, M. “Analysis of Fiber Dyes by Liquid Chromatography Mass Spectrometry (LC-MS) with Electrospray Ionization: Discrimination Between Dyes with Indistinguishable UV-Visible Absorption Spectra” J. For. Sci (50) 3. Petrick, L.; Wilson, T.; Fawcett, R. “High-Performance Liquid Chromatography-Ultraviolet-Visible Spectroscopy-Electrospray Ionization Mass Spectrometry Method for Acrylic and Polyester Forensic Fiber Dye Analysis,” J. For. Sci (51), Recent literature ( )

30 Copyright  2007 by Stephen L. Morgan, slide 30 CE with UV/visible / MS detection  High sensitivity  Reliability  Ease of use  minimized daily maintenance EOF

31 Copyright  2007 by Stephen L. Morgan, slide 31 Co-axial Sheath flow interface. (1) capillary adjustment; (2) capillary from CE; (3) extension capillary; (4) microvolume tee; (5) stainless steel capillary, end of extension capillary, and nebulizing tip; (6) nebulizer gas (N 2 ); (7) make-up flow. [Waters/Micromass] CE-DAD/MS sheath flow interface External xenon lamp seen in background. (1) inlet buffer block; (2) deuterium lamp aperture (now redundant); (3) DAD; (4) coolant T-piece; (5) fiber optic from xenon lamp; (6) fiber optic to DAD; (7) standard Beckman aperture with rubber donut ring; (8) capillary; (9) CE-MS interface. The xenon lamp and power supply can be seen in the background

32 Copyright  2007 by Stephen L. Morgan, slide 32 Basic dyes / CE/MS with positive ESI/MS m/z 273 m/z 317 m/z 434 m/z 347 m/z 344 m/z 371 m/z 428 Conditions: Sheath flow rate: 1.7 μL/min Nebulization gas: 8 psi ESI voltage: 3718 V Cone voltage: 17 V Estimated LOD’s for basic dyes vary from 0.2 to 0.4 µg/mL. For a 5 µL injection volume, this represents ~2 pg.

33 Copyright  2007 by Stephen L. Morgan, slide 33 2 mm acrylic / basic dye extract / CE/MS Peak identification: (1), (2), (3). Extraction conditions: 2 mm tri-dyed acrylic fiber 10 µL water:formic acid (1:1, v/v) added Heated at 100  C for 1 h Evaporated to dryness Reconstituted in 5 µL water CE conditions: 45 mM ammonium acetate in acetonitrile-water (60:40, v/v), pH 4.7 Injection: 20 kv 10 sec MS Conditions: Sheath flow rate: 1.7 μL/min Nebulization gas: 8 psi ESI voltage: 3718 V Cone voltage: 17 V Basic Violet 16 Basic Blue 159 Basic Yellow 28

34 Copyright  2007 by Stephen L. Morgan, slide m/z 427 m/z 659 m/z 424 Acid dye (anionic) / CE-DAD-MS with ESI (-) Dye 1 Dye 2 Dye 3 Sample: 0.1 mg/mL acid dye mixture CE conditions: 30 kV with 3 psi (separation), 2psi – 10 sec injection, 50 µm i.d. Buffer conditions: 15 mM Ammonium Acetate with 40 % Acetonitrile pH 9.3 MS conditions: V (cap), 50 V (cone), 10 psi (gas), 4 µL/min 50:50 IPA/ H 2 O, 1 % TEA N CE/DAD at 319 nm CE/MS UV/visible spectra

35 Copyright  2007 by Stephen L. Morgan, slide 35 CE-DAD/MS of extracted nylon fiber Dye 1 Dye 2 Dye 3 Sample: Extract from nylon 6,6 fiber with 3 acid dyes CE conditions: 30 kV with 3 psi (separation), 2psi – 20 sec injection, 50 µm i.d. Buffer conditions: 15 mM Ammonium Acetate with 40 % Acetonitrile pH 9.3 MS conditions: V (cap), 50 V (cone), 10 psi (gas), 4 µL/min 50:50 IPA/ H 2 O, 1 % TEA 1 2 m/z 427 m/z 659 m/z 424 m/z nylon N CE/DAD at 319 nm CE/MS UV/visible spectra

36 Copyright  2007 by Stephen L. Morgan, slide 36 Dye identification/characterization Interpretation of fragmentation pattern. All fragments are singly charged species. Ion abundance MS/MS spectrum of C.I. Basic Blue 3 obtained using positive ion electrospray ionization (ESI+) Ion mass (m/z)

37 Copyright  2007 by Stephen L. Morgan, slide 37 CE/DAD/MS analysis: nylon fiber extract with 3 acid dyes after laundering with Tide ® U N U U FB U 2 unknown Dye 3 Dye 2 Dye 1 m/z 438 m/z 394 m/z m/z 659 m/z 424  Washing adds 4 unknown CE/DAD/MS peaks  Dyes 1 and 3 not visible in CE-DAD at 319 nm due to loss after laundering  Dyes 1 and 3 visible in MS signal, but signal significantly noisier and peaks broader FB peak from sample Tide Standard Wavelength, nm UV/visible spectra RIC Minutes CE/MS CE/DAD at 319 nm

38 Copyright  2007 by Stephen L. Morgan, slide 38 CE/DAD/MS analysis: nylon fiber extract with 3 acid dyes after 12 mo. accelerated weathering N Dye 1 Dye 2 Dye 3  Weathering is affecting all three dyes in a similar manner  Few degradation products seen  Nylon polymer present in MS signal due to extraction process or what? Dye 3 Dye 2 Dye 1 Nylon m/z 427 m/z 659 m/z 424 m/z 676 CE/DAD at 319 nm RIC Minutes UV/visible spectra Wavelength, nm CE/MS

39 Copyright  2007 by Stephen L. Morgan, slide 39 Nylon polymer components in extracts m/z 1014 (4.5-Nylon 6,6 units) m/z 901 (4-Nylon 6,6 units) m/z 788 (3.5-Nylon 6,6 units) m/z 676 (3-Nylon 6,6 units) m/z 563 (2.5-Nylon 6,6 units) C 12 H 22 N 2 O 2 Exact Mass: The 0.5 unit on these masses is due to adipic acid on the end of the polymer chain RIC Minutes CE/MS

40 Copyright  2007 by Stephen L. Morgan, slide 40  Although microextraction/CE/MS is destructive to the sample, only an extremely small sample is required (~1-2 mm of a single 15  diameter fiber). Automated micro-extractions and CE offer the forensic analyst reproducible analyses (% RSDs ranging from 5-25%) with limits of detection in the picogram range.  CE methods compatible with MS have been developed. The sheath- flow CE/MS interface is suited to routine dye analysis, exhibits stability and ease of use, and requires little maintenance.  CE/DAD-MS methods for cationic textile dyes have been optimized. CE/DAD-MS methods for anionic dyes have been developed and applied to extracted samples.  CE/DAD and CE/MS provide qualitative and semi-quantitative “fingerprint” for dyes extracted from evidence fibers. Discrimination may be enhanced through matching CE migration times, molecular weight, and structural fragmentation. Conclusions

41 Copyright  2007 by Stephen L. Morgan, slide 41 Acknowledgements This research was supported under a contract award from the Counterterrorism and Forensic Science Research Unit of the Federal Bureau of Investigation’s Laboratory Division. Points of view in this document are those of the authors and do not necessarily represent the official position of the Federal Bureau of Investigation.


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