Copyright 2007 by Stephen L. Morgan, slide 1 Stephen L. Morgan, Brandi C. Vann, Brittany M....

Copyright 2007 by Stephen L. Morgan, slide 1

Stephen L. Morgan, Brandi C. Vann, Stephen L. Morgan, Brandi C. Vann, Brittany M. Baguley, and Amy R. Stefan Brittany M. Baguley, and Amy R. Stefan

Department of Chemistry & Biochemistry, The University of South Carolina, Columbia, SC 29208 Email: [email protected]

URL: http:www.chem.sc.edu/faculty/morgan

Advances in Discrimination of Dyed Textile Advances in Discrimination of Dyed Textile Fibers using Capillary Electrophoresis/Fibers using Capillary Electrophoresis/

Mass SpectrometryMass Spectrometry

Trace Evidence Symposium, Clearwater, FL16 August 2007


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.

IntroductionIntroduction


“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 pointA starting point


Blue acid dye discriminationBlue acid dye discrimination

C.I. acid blue 277C.I. acid blue 277

O

O

NH2

SO3Na

HN CH3

CH3

SO2NHCH2CH2OH

SO -

C.I. acid blue 239H3C

O NH

NH

O

O

H2C

NH

H2C

C

O

3

C.I. acid blue 45C.I. acid blue 45

O

O

OH

NH2

SO3-

OH

NH2

-O3S


Acid blue 45

Time (min)

Acid Blue 239

Acid blue 277

neutrals

Ab

sorb

ace

(mA

U)

Electropherogram taken at 600 nm of three blue acid dyes extracted from nylon fibers

15 mM ammonium acetate in 40/60 ACN-H2O, pH 9.3Capillary: 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.

Ab

sorb

ace

(mA

U)

Wavelength (nm) Time (m

in)

Blue dye extracts / similar UV/Vis spectraBlue dye extracts / similar UV/Vis spectra


Textile fiber dye extraction / CE methodsTextile fiber dye extraction / CE methods

Extracted fiber (+)

Reactive

(+)Direct

(+)Acid

(+)Cationic

(+)Disperse

(+)Vat

(-)Na2SO4

100 °C 30 minAir oxidize 1 hr

NaOH100 °C 30 min

Pyridine/H2O100 °C60 min

Pyridine/H2O/NH4OH100 °C60 min

Formic acid/H2O

100 °C60 min

Chloro-benzene100 °C30 min

Cotton Nylon Acrylic Polyester

Determine fiber type via PLM or FTIROriginal

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.


Nylon and cotton dyes / anionic CENylon and cotton dyes / anionic CE

Electropherogram at 214 nm.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:Conditions:Buffer: 15 mM ammonium acetate in

ACN-H2O (40:60, v/v), pH 9.3Injection: 1 psi 5 sCapillary: 50 μm i.d.,50 cmSeparation: 30 kVDetection: diode array UV/visible


CE / diode array detectionCE / diode array detection

Inlet Outlet


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)CE based on electroosmotic flow (EOF)

EOFEOF

Fused silica capillaryFused silica capillary+ _CathodeCathodeAnodeAnode

++

---

--

___ _

+ +

+

+

+

+ ++

+__

++++ ++++ ++++ ++++++++++++

++++++++ ++++ ++++ ++++++++

_


Combinatorial optimization experimentsCombinatorial 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 mixtures200 µL solvent in each well1 mm - 1.0 cm threadsHeat to 100 C, 90 C and 60 C Evaporated to drynessReconstitute in 100 µL water

96-well plate with glass inserts96-well plate with glass inserts

Liquid sampling robotLiquid sampling robot


Nylon / acid dyesNylon / 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 nylonfiber by electrostatic interactions through salt linkages.

NH

HN

O]n

O

[

]nO

NH

[

Nylon 6,6

Nylon 6

O

O

OH

NH2

SO3-

OH

NH 2

-O3S

C.I. Acid Blue 45, AnthraquinoneC.I. Acid Blue 45, Anthraquinone

N

- O3S

NOCH3

H 3CN N

OCH3

C.I. Acid orange 156, AzoC.I. Acid orange 156, Azo

O -

N

N HAc

N

SO

O

N H 2

O -Co3 +

O-

N

AcHN

N

S O

O

H2N

O-

C.I. Acid Blue 171, Metal-AzoC.I. Acid Blue 171, Metal-Azo


Combinatorial experimental design was applied to the extraction of acid dyes from nylon using water, aqueous ammonia (12 M), and pyridine.

Design point

% H2O % NH3 % pyridine

1 100 0 0

2 0 100 0

3 0 0 100

4 0 50 50

5 50 0 50

6 50 50 0

7 66 16 16

8 16 66 16

9 16 16 66

10 33 33 33

Mixture designs for three solventsMixture designs for three solvents


Anthraquinone Blue BAnthraquinone 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 4.0-7.0%.

96-well plate with glass inserts96-well plate with glass inserts

Nylon / anthraquinone blueNylon / anthraquinone blue BB


Acid Blue 171: metal complexAcid Blue 171: metal complexAcid orange 156: AzoAcid orange 156: Azo R2=.9197R2=.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 azoNylon / 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.


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 ReactiveDyes and Analysis of the Products by Thin-Layer Chromatography," J. Soc. Dye. Colour 1994, 110, 338-341.

Using a combinatorial approach, best extraction of direct dyes from cottonwas 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 dyesCotton / direct and reactive dyes


15 mM ammonium acetate in 40/60 ACN-H2O, pH 9.3Capillary: 50 μm i.d., 50 cm (82 cm for CE-MS)Separation at 30 kV and 25 °C using 214 nm

anionicanionic dyes 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

NH2

NH

SO3-

SO2CH2CH2OSO3-

Reactive blue 19Reactive blue 19

Cotton / direct and reactive dyes / CECotton / direct and reactive dyes / CE


Cotton fiber /reactive dye extracts / CECotton 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

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.


Cotton / vat dye extracts / CECotton / vat dye extracts / CE

Buffer: 15 mM ammonium acetate in acetonitrile-water (40:60, v/v), pH 9.3 + reducing agent (sodium dithionite)

NaS2O4

OH

HO

C.I. vat orange 9

DAD electropherograms at 280 nmDAD electropherograms at 280 nm


Wavelength (nm)

Ab

sorb

ance

(m

AU

)

UV/Vis spectrum of vat dye standard

UV/Vis spectrum of vat dye extract

Wavelength (nm)A

bso

rban

ce (

mA

U)

Spectra of vat dye standard and extract are the same No change in dye constitution due to extraction conditions

Vat dye / UV/visible confirmationVat 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.


Acrylic / basic (cationic) dyesAcrylic / 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

*H2C

HC

H2C C *

CN

R

R'

NNCH3

CH3N

N

N N

CH3

H2C

C.I. Basic Red 46

Acrylic


Electropherogram at 214 nm.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: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 / CEAcrylic / basic cationic dyes / CE

NCH3

N

N

N

S

CH3

Peak 1: C.I. basic red 22Peak 1: C.I. basic red 22


Polyester does not contain ionic or covalentdye 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

Electron microscope photo of polyester fibers

O2N N N

OCH3

N

N

HO

C.I. Disperse Orange 29

Polyester / disperse dyesPolyester / disperse dyes


Ab

so

rba

nc

e (

mA

U) 70

60

50

40

30

20

10

0

1

2 3 4

C.I. Disperse Yellow 114

NACE conditions for disperse dyes:Buffer: 80 mM ammonium acetate in

acetonitrile-methanol (75:25, v/v), pH 9Separation: 20 kVPeak identification: (1) Disperse Red 343,

(2) Disperse Blue 73, (3) Disperse Yellow 114, (4) Disperse Orange 29

Polyester / disperse dyes / CEPolyester / disperse dyes / CE

Electropherogram at 214 nm of Disperse Orange 29 extracted from polyester


Electropherogram at 400 nm of C.I. Acid orange 156 standard

Abs

orba

nce

(mA

U)

Time (min)

Wavelength (nm)

Acid dye (standard) / CEAcid dye (standard) / CE


Time (min)

Electropherogram at 400 nm of C.I. Acid orange 156 extracted from 10 cm nylon fiber

Acid dye nylon extract (10 cm) / CEAcid dye nylon extract (10 cm) / CE

Wavelength (nm)

Abs

orba

nce

(mA

U)




Time (min)

Wavelength (nm)

Abs

orba

nce

(mA

U)


Acid dye nylon extract (2.5 cm) / CEAcid 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)

Abs

orba

nce

(mA

U)




Time (min)

Wavelength (nm)

Abs

orba

nce

(mA

U)


Petrick, et al. analyzed dyes extracted from acrylic and polyester fibers followed by HPLC/DAD/MS

1:1 formic acid: water extraction of basic dyesCompared extraction efficiency of 4:3 mixtures of pyridine and water to 4:3

mixtures of acetonitrile and water for disperse dyesAnalyzed dyes from 5 cm single acrylic fiber and polyester fibers

Huang, et al. analyzed acid, basic, and disperse dye pairs by LC/MSExtracted 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. 2005 (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. 2006 (51), 771-779.

Recent literature (2005-2006)Recent literature (2005-2006)


CE with UV/visible / MS detectionCE with UV/visible / MS detection

High sensitivity Reliability Ease of use minimized daily

maintenance

EOF


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 (N2); (7)

make-up flow. [Waters/Micromass]

CE-DAD/MS sheath flow interfaceCE-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


Basic dyes / CE/MS with positive ESI/MSBasic 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.


2 mm acrylic / basic dye extract / CE/MS2 mm acrylic / basic dye extract / CE/MS3

2

1

Peak identification: (1), (2), (3).

Extraction conditions:2 mm tri-dyed acrylic fiber10 µL water:formic acid

(1:1, v/v) addedHeated at 100 C for 1 hEvaporated to drynessReconstituted 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


1

2

3m/z 427

m/z 659

m/z 424

Acid dye (anionic) / CE-DAD-MS with ESI (-)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: -3300 V (cap), 50 V (cone), 10 psi (gas), 4 µL/min 50:50 IPA/ H2O, 1 % TEA

N

12

3

CE/DAD at 319 nm

CE/MS

UV/visible spectra


CE-DAD/MS of extracted nylon fiberCE-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: -3300 V (cap), 50 V (cone), 10 psi (gas), 4 µL/min 50:50 IPA/ H2O, 1 % TEA

1

2

m/z 427

m/z 659

m/z 424

m/z 676

3

nylon

N

1 23

CE/DAD at 319 nm

CE/MS

UV/visible spectra


Dye identification/characterizationDye identification/characterization

Interpretation of fragmentation pattern. All fragments are singly charged species.

Ion

ab

un

dan

ce

MS/MS spectrum of C.I. Basic Blue 3 obtained using positive ion electrospray ionization (ESI+)

Ion mass (m/z)

O

N

NN

O

N

NHN

m/z 296

O

N

NN

m/z 280

O

N

NNH

O

N

NN

m/z 252

m/z 236

m/z 324324

280

236


CE/DAD/MS analysis: nylon fiber extract with CE/DAD/MS analysis: nylon fiber extract with 3 acid dyes after laundering with Tide3 acid dyes after laundering with Tide®®

UN

U

U

FB

U

2

unknown

unknown

Dye 3

Dye 2

Dye 1

m/z 438

m/z 394

m/z 427.7

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/DADat 319 nm


CE/DAD/MS analysis: nylon fiber extract with CE/DAD/MS analysis: nylon fiber extract with 3 acid dyes after 12 mo. accelerated weathering3 acid dyes after 12 mo. accelerated weathering

N

1 23

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


Nylon polymer components in extractsNylon polymer components in extracts

*N

N

O

O

H

H

NN

O

O

H

H

NN

*O

O

H

H

m/z 1014 (4.5-Nylon 6,6 units)

m/z 901 (4-Nylon 6,6 units)


m/z 676 (3-Nylon 6,6 units)


NN

O

O

H

H

**

C12H22N2O2••

Exact Mass: 226.17N

N*

O

O

H

H

**

*

Adipic acidC6H14N2

••

Exact Mass: 114.12

Hexamethylene DiamineC6H8O2

••

Exact Mass: 112.05

The 0.5 unit on these masses is due to adipic acid on the end of the polymer chain

RIC

Minutes

CE/MS


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.

ConclusionsConclusions


AcknowledgementsAcknowledgements

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.

Copyright 2007 by Stephen L. Morgan, slide 1 Stephen L. Morgan, Brandi C. Vann, Brittany M....

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