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Thin layer chromatography of azoic dyes extracted from cotton fibres
COMMENTARY
Thin layer chromatography of azoic dyes extracted from cotton fibres DAVID K LAING, ANDREW W HARTSHORNE and DANA C BENNETT Central Research and Support Establishment, Home Ofice Forensic Science Service, Aldermaston, Reading, Berkshire, United Kingdom RG7 4PN A thin layer chromatography system has been produced for the examination of azoic dyes extracted from single cotton fibres. Dyes of this class are successfully analysed by using ethyl acetate for initial sharpening of dye spots followed by separation in a mixture of chlorobenzene, 1,2-dichloroethane and acetone (20 :20: 1). Key Wordr: Azoic dyes; Cotton fibres; Thin layer chromatography. Journal of the Forensic Science Society 1990; 30: 309-315 Received 22 March 1990; accepted 21 August 1990
Introduction A scheme for the sequential extraction and classification of dyes from cotton fibres has been presented [I]. The main aim of such schemes is the production of dye solutions suitable for thin layer chromatography (TLC). A subsidiary goal is the identification of the dye class so as to guide the selection of an appropriate TLC elution system. TLC systems have been provided for disperse, acid and basic dyes extracted from polyester, polyamide and acrylic fibres [2], for direct and reactive dyes extracted from cellulosic fibres [3], for acid and metallised acid dyes from wool [4] and for disperse, acid and metallised acid dyes from polypropylene [5]. A system for standardisation of TLC systems using a number of well characterised dyes has also been produced [6]. However, a major omission is that of the azoic dye class, which is commonly found on cotton and viscose fibres. The purpose of this work is to investigate TLC eluent systems suitable for the separation of this dye class.
Experimental Extraction of single cotton fibres for TLC Cotton fibres dyed with the azoic dyes shown in Table 1 were selected from pattern cards produced by Rohner (Rohner Ltd, Pratteln, Switzerland). A single fibre approximately 10 mm long was removed from each sample and placed in a capillary tube together with 5 pl of glacial acetic acid, and the JFSS 1990; 30(5): 309-315
309
TABLE 1 Components of azoic dyes used on cotton and viscose fibres Azoic coupling component
Dye code Yellow A1 A2 A3 A4 A5 A6 A7
Commercial name (Napthanilide)
LRG G LRG L4G G L4G G
CI generic name (ACC)
Azoic diazo component
Commercial name
Diazo Fast Yellow GC Diazo Fast Yellow GC Diazo Fast Orange GGD Diazo Fast Orange RD Diazo Fast Scarlet GG Diazo Fast Red KB Diazo Fast Red Base FG
Red A8 A9 A10 A1 1 A12 A13 A14
Diazo Fast Orange GR Diazo Fast Scarlet GG Diazo Fast Scarlet Fast Red Base SL Diazo Fast Red KB Diazo Fast Red ITR Fast Red Base FG
Orange A15 A16 A17 A18 A19 A20 A2 1 A22
Diazo Fast Yellow GC Diazo Fast Orange GGD Diazo Fast Orange GC Diazo Fast Orange RD Diazo Fast Scarlet GG Diazo Fast Scarlet GG Diazo Fast Red B Fast Red Base SL
Green A23 A24 A25 A26 A27 A28
Diazo Fast Orange GC Diazo Fast Scarlet R Diazo Fast Red B Diazo Fast Corinth V Diazo Fast Blue BB Diazo Fast Blue VFGC
Blue A29 A30 A3 1 A32 A33 A34 A35
Diazo Fast Blue RR Diazo Fast Blue RR Diazo Fast Blue BB Diazo Fast Blue VBN Diazo Fast Blue VRTN Diazo Fast Blue VFGC Diazo Fast Black K
Violet A36 A37 A38 A39 A40
Diazo Fast Yellow GC Diazo Fast Orange GR Diazo Fast Scarlet GG Diazo Fast Red TR Diazo Fast Bordeaux GP
JFSS 1990;
CI generic name ( ADC)
TABLE 1 (continued) Azoic coupling component
Dye code
Commercial name (Napthanilide) LT ITR
Brown A43 A44 A45 A46 Black A47 A48
CI generic name ( A C C )
Azoic diazo component
Commercial name
CI generic name ( A D C )
Diazo Fast Violet B Diazo Fast Red RL Diazo Fast Orange CR Diazo Fast Scarlet GG Diazo Fast Red TR Fast Corinth Base LB Diazo Fast Red RL Diazo Fast Red B Diazo Fast Black K Diazo Fast Black M
tube was sealed [I]. The sealed tubes were heated at 100 "C for 20 minutes, and the contents, after cooling, were spotted in small portions onto l00mm X 100 mm Merck DC Alufolien Kieselgel 60F2,, TLC plates (E Merck, Darmstadt, FRG), using a fine capillary and drying between additions with a stream of cold air from a hair dryer. Spots were positioned 10 mm from the lower edge of the plate and at least this distance from the sides. Thin layer chromatography The eluents A to F in Table 2 were investigated for concentration of the dye spots. The edge of the TLC plate bearing the dye spots was dipped into eluent contained in a petri dish which was allowed to run just past the dye spots (typically 10 to 12 mm from the bottom of the plate). The process was repeated as necessary to optimise production of a concentrated line of dye. The eluents G to Z were investigated for the separation of azoic dyes. The eluents were selected from existing systems for other dye classes and pigments. For chromatography, eluent was placed in one compartment of a Camag No25155 twin trough tank (Baird and Tatlock, Ltd, Romford, Essex, UK), the plate placed in the other and the whole allowed to equilibrate for 20 minutes [6]. At the end of this time, the tank was tipped so that eluent flowed into the plate compartment and development was allowed to proceed for 50 to 70 mm up the plate. The plate was then dried and inspected in daylight and with ultraviolet illumination of 254nm wavelength. Rf x 100 values were calculated for each separated component. JFSS 1990; 30(5): 309-315
311
TABLE 2 Eluents investigated for the thin layer chromatography of azoic dyes Eluent
Components
A* B* C* D* E* F* G H I J
Methanol Acetone Ethyl acetate Acetic acid (glacial) Xylene Dimethyl formamide n-Butanol :water : acetic acid (glacial) n-Butanol :water :acetic acid (glacial) Toluene :pyridine n-Hexane :ethyl acetate : acetone Toluene :methanol :acetone Chloroform :acetone Chlorobenzene :1.2-dichloroethane :toluene Chlorobenzene Chlorobenzene :acetone 1,2-Dichloroethane Toluene Acetic acid (glacial) :chlorobenzene Acetic acid (glacial) :toluene n-Butanol :water: acetic acid Chlorobenzene : l,2-dichloroethane : acetone Chlorobenzene : 1,2-dichloroethane :acetone Chlorobenzene :1,2-dichloroethane :acetone Chlorobenzene :1,2-dichloroethane Chlorobenzene :1,2-dichloroethane : acetone Chlorobenzene : l,2-dichloroethane :acetone
K L M N 0 P
Q R S T U V W X
Y Z
Composition by volume
4:4:2 2:1:5 8:2 5:4:1 20:2:1 9: 1 1:l:l 1:l
2: 1 2: 1 1:5:1 1:l:l 9:9:2 9:9:1 1:l 10: 10: 1 20:20:1
* Investigated for pre-development concentration.
Results and discussion Pre-concentration of azoic dyes Neither methanol nor xylene concentrated any of the azoic dyes examined while acetone and glacial acetic acid were effective for only a limited number of samples. Dimethyl formamide was the most successful reagent for this purpose, producing a concentrated line of colourant for all the azoic dyes investigated. However, a yellow discolouration was also produced on the TLC plate which on development moved with the eluent front, carrying the dyes with it. Ethyl acetate was almost as effective, concentrating 48 of the 50 samples examined, with no apparent adverse effects on the plate. In consequence, ethyl acetate was selected for pre-development of TLC plates.
This behaviour can be contrasted with that of other dye classes: acid, disperse, direct and cotton reactive dyes are all concentrated by methanol while basic, vat and sulphur dyes are unaffected [3,6]. The preconcentration step thus provides a further feature for confirming the 312
JFSS 1990; 30(5): 309-315
classification of azoic dyes and discriminating them from the direct and reactive classes also used on cotton fibres.
Thin layer chromatography of azoic dyes Of the eluents investigated (Table 2), dyes moved with the solvent front in eluents G, L, N and T. Eluents H, K and 0 caused tailing of dyes. Chromatography occurred for the remaining eluents examined but overall eluent Z was superior, with eluent I providing a useful alternative. Eluent Z gave a wide range of Rf values and good spot definition for the fifty azoic dyes investigated, the results with this eluent system (chlorobenzene: 1,Zdichloroethane: acetone, 20 :20 : 1, v :v :v) being shown in Table 3. It is interesting to note that only a single component was detected for the majority of the dyes examined. However, when there was a mixture of two azoic coupling components with a single azoic diazo component (A48 and A49, Table I), two spots were produced as expected. The only other examples where multiple spots were produced were dyes A32, A34 and A50. These dyes are distinct in that they alone are formed using CI Azoic Coupling Component 17 [7]. It is thus probable that these two azoic components each contain at least two different chemical compounds.
TABLE 3 Thin layer chromatography of azoic dyes extracted from cotton fibres using chlorobenzene:1,2dichloroethane :acetone (20 :20 :1, v :v :v) as eluent Dye code* A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 All A12 A13 A14 A15 A16 A17 A18 A19 A20 A21 A22 JFSS 1990; 30(5): 309-315
Components
Yellow Yellow Yellow Yellow Yellow Yellow Yellow Pink Oranget Orange? Oranget Oranget PinkJRedt Red7 Redt Oranget OrangeJRedt Oranget Oranget Orange? Oranget Oranget
Rf x 100
TABLE 3 (continued) Dye code*
Components
Rf x lo0
Yellow/Orange GreyIBrown Grey/Purplet Grey/Blue Blue Yellow Violet? Purple? Purple GreyIPinkt Purple? Brown/Pink BrownJPink Purple? Grey/Pink? Purple? Purplet Pink? Purplet Pink? Violett Pink Violet? Pink Brownt Olive? Brown? Brown Purple? Blue Violet Light Blue Dark Blue Blue Blue
* See Table 1 for identification t Fluorescent when illuminated with 254 nm light
Conclusion For azoic dyes extracted from cotton fibres the most favourable eluent system for thin layer chromatography is chlorobenzene: 1,Zdichloroethane : acetone (20 :20 : 1, v :v :v). Pre-development with ethyl acetate results in concentration of dye on the TLC plate and improved separation for the majority of dyes. 314
JFSS 1Y90; 30(5): 309-315
References 1. Laing DK, Dudley RJ, Hartshorne AW, Home JM, Rickard RA and Bennett DC. The extraction and classification of dyes from cotton and viscose fibres. Forensic Science International: in press. 2. Beattie IB, Roberts HL and Dudley RJ. Thin-layer chromatography of dyes extracted from polyester, nylon and poly-acrylonitrile fibres. Forensic Science International 1981; 17: 57-69. 3. Home JM and Dudley RJ. Thin-layer chromatography of dyes extracted from cellulosic fibres. Forensic Science International 1981; 17: 71-78. 4. Macrae R, Dudley RJ and Smalldon KW. The characterization of dyestuffs on wool fibers with special reference to microspectrophotometry. Journal of Forensic Sciences 1979; 24: 117-129. 5. Hartshorne AW and Laing DK. The dye classification and discrimination of coloured polypropylene fibres. Forensic Science International 1984; 25: 133-141. 6 . Laing DK, Boughey L and Hartshorne AW. The standardisation of thin layer chromatographic systems for comparison of fibre dyes. Journal of the Forensic Science Society: 1990; 30: 299-307. 7. The Colour Index, 3rd Edition. Bradford, United Kingdom: Society of Dyers and Colourists, 1987.
JFSS 1990; 30(5): 309-315
Thin layer chromatography of azoic dyes extracted from cotton fibres DAVID K LAING, ANDREW W HARTSHORNE and DANA C BENNETT Central Research and Support Establishment, Home Ofice Forensic Science Service, Aldermaston, Reading, Berkshire, United Kingdom RG7 4PN A thin layer chromatography system has been produced for the examination of azoic dyes extracted from single cotton fibres. Dyes of this class are successfully analysed by using ethyl acetate for initial sharpening of dye spots followed by separation in a mixture of chlorobenzene, 1,2-dichloroethane and acetone (20 :20: 1). Key Wordr: Azoic dyes; Cotton fibres; Thin layer chromatography. Journal of the Forensic Science Society 1990; 30: 309-315 Received 22 March 1990; accepted 21 August 1990
Introduction A scheme for the sequential extraction and classification of dyes from cotton fibres has been presented [I]. The main aim of such schemes is the production of dye solutions suitable for thin layer chromatography (TLC). A subsidiary goal is the identification of the dye class so as to guide the selection of an appropriate TLC elution system. TLC systems have been provided for disperse, acid and basic dyes extracted from polyester, polyamide and acrylic fibres [2], for direct and reactive dyes extracted from cellulosic fibres [3], for acid and metallised acid dyes from wool [4] and for disperse, acid and metallised acid dyes from polypropylene [5]. A system for standardisation of TLC systems using a number of well characterised dyes has also been produced [6]. However, a major omission is that of the azoic dye class, which is commonly found on cotton and viscose fibres. The purpose of this work is to investigate TLC eluent systems suitable for the separation of this dye class.
Experimental Extraction of single cotton fibres for TLC Cotton fibres dyed with the azoic dyes shown in Table 1 were selected from pattern cards produced by Rohner (Rohner Ltd, Pratteln, Switzerland). A single fibre approximately 10 mm long was removed from each sample and placed in a capillary tube together with 5 pl of glacial acetic acid, and the JFSS 1990; 30(5): 309-315
309
TABLE 1 Components of azoic dyes used on cotton and viscose fibres Azoic coupling component
Dye code Yellow A1 A2 A3 A4 A5 A6 A7
Commercial name (Napthanilide)
LRG G LRG L4G G L4G G
CI generic name (ACC)
Azoic diazo component
Commercial name
Diazo Fast Yellow GC Diazo Fast Yellow GC Diazo Fast Orange GGD Diazo Fast Orange RD Diazo Fast Scarlet GG Diazo Fast Red KB Diazo Fast Red Base FG
Red A8 A9 A10 A1 1 A12 A13 A14
Diazo Fast Orange GR Diazo Fast Scarlet GG Diazo Fast Scarlet Fast Red Base SL Diazo Fast Red KB Diazo Fast Red ITR Fast Red Base FG
Orange A15 A16 A17 A18 A19 A20 A2 1 A22
Diazo Fast Yellow GC Diazo Fast Orange GGD Diazo Fast Orange GC Diazo Fast Orange RD Diazo Fast Scarlet GG Diazo Fast Scarlet GG Diazo Fast Red B Fast Red Base SL
Green A23 A24 A25 A26 A27 A28
Diazo Fast Orange GC Diazo Fast Scarlet R Diazo Fast Red B Diazo Fast Corinth V Diazo Fast Blue BB Diazo Fast Blue VFGC
Blue A29 A30 A3 1 A32 A33 A34 A35
Diazo Fast Blue RR Diazo Fast Blue RR Diazo Fast Blue BB Diazo Fast Blue VBN Diazo Fast Blue VRTN Diazo Fast Blue VFGC Diazo Fast Black K
Violet A36 A37 A38 A39 A40
Diazo Fast Yellow GC Diazo Fast Orange GR Diazo Fast Scarlet GG Diazo Fast Red TR Diazo Fast Bordeaux GP
JFSS 1990;
CI generic name ( ADC)
TABLE 1 (continued) Azoic coupling component
Dye code
Commercial name (Napthanilide) LT ITR
Brown A43 A44 A45 A46 Black A47 A48
CI generic name ( A C C )
Azoic diazo component
Commercial name
CI generic name ( A D C )
Diazo Fast Violet B Diazo Fast Red RL Diazo Fast Orange CR Diazo Fast Scarlet GG Diazo Fast Red TR Fast Corinth Base LB Diazo Fast Red RL Diazo Fast Red B Diazo Fast Black K Diazo Fast Black M
tube was sealed [I]. The sealed tubes were heated at 100 "C for 20 minutes, and the contents, after cooling, were spotted in small portions onto l00mm X 100 mm Merck DC Alufolien Kieselgel 60F2,, TLC plates (E Merck, Darmstadt, FRG), using a fine capillary and drying between additions with a stream of cold air from a hair dryer. Spots were positioned 10 mm from the lower edge of the plate and at least this distance from the sides. Thin layer chromatography The eluents A to F in Table 2 were investigated for concentration of the dye spots. The edge of the TLC plate bearing the dye spots was dipped into eluent contained in a petri dish which was allowed to run just past the dye spots (typically 10 to 12 mm from the bottom of the plate). The process was repeated as necessary to optimise production of a concentrated line of dye. The eluents G to Z were investigated for the separation of azoic dyes. The eluents were selected from existing systems for other dye classes and pigments. For chromatography, eluent was placed in one compartment of a Camag No25155 twin trough tank (Baird and Tatlock, Ltd, Romford, Essex, UK), the plate placed in the other and the whole allowed to equilibrate for 20 minutes [6]. At the end of this time, the tank was tipped so that eluent flowed into the plate compartment and development was allowed to proceed for 50 to 70 mm up the plate. The plate was then dried and inspected in daylight and with ultraviolet illumination of 254nm wavelength. Rf x 100 values were calculated for each separated component. JFSS 1990; 30(5): 309-315
311
TABLE 2 Eluents investigated for the thin layer chromatography of azoic dyes Eluent
Components
A* B* C* D* E* F* G H I J
Methanol Acetone Ethyl acetate Acetic acid (glacial) Xylene Dimethyl formamide n-Butanol :water : acetic acid (glacial) n-Butanol :water :acetic acid (glacial) Toluene :pyridine n-Hexane :ethyl acetate : acetone Toluene :methanol :acetone Chloroform :acetone Chlorobenzene :1.2-dichloroethane :toluene Chlorobenzene Chlorobenzene :acetone 1,2-Dichloroethane Toluene Acetic acid (glacial) :chlorobenzene Acetic acid (glacial) :toluene n-Butanol :water: acetic acid Chlorobenzene : l,2-dichloroethane : acetone Chlorobenzene : 1,2-dichloroethane :acetone Chlorobenzene :1,2-dichloroethane :acetone Chlorobenzene :1,2-dichloroethane Chlorobenzene :1,2-dichloroethane : acetone Chlorobenzene : l,2-dichloroethane :acetone
K L M N 0 P
Q R S T U V W X
Y Z
Composition by volume
4:4:2 2:1:5 8:2 5:4:1 20:2:1 9: 1 1:l:l 1:l
2: 1 2: 1 1:5:1 1:l:l 9:9:2 9:9:1 1:l 10: 10: 1 20:20:1
* Investigated for pre-development concentration.
Results and discussion Pre-concentration of azoic dyes Neither methanol nor xylene concentrated any of the azoic dyes examined while acetone and glacial acetic acid were effective for only a limited number of samples. Dimethyl formamide was the most successful reagent for this purpose, producing a concentrated line of colourant for all the azoic dyes investigated. However, a yellow discolouration was also produced on the TLC plate which on development moved with the eluent front, carrying the dyes with it. Ethyl acetate was almost as effective, concentrating 48 of the 50 samples examined, with no apparent adverse effects on the plate. In consequence, ethyl acetate was selected for pre-development of TLC plates.
This behaviour can be contrasted with that of other dye classes: acid, disperse, direct and cotton reactive dyes are all concentrated by methanol while basic, vat and sulphur dyes are unaffected [3,6]. The preconcentration step thus provides a further feature for confirming the 312
JFSS 1990; 30(5): 309-315
classification of azoic dyes and discriminating them from the direct and reactive classes also used on cotton fibres.
Thin layer chromatography of azoic dyes Of the eluents investigated (Table 2), dyes moved with the solvent front in eluents G, L, N and T. Eluents H, K and 0 caused tailing of dyes. Chromatography occurred for the remaining eluents examined but overall eluent Z was superior, with eluent I providing a useful alternative. Eluent Z gave a wide range of Rf values and good spot definition for the fifty azoic dyes investigated, the results with this eluent system (chlorobenzene: 1,Zdichloroethane: acetone, 20 :20 : 1, v :v :v) being shown in Table 3. It is interesting to note that only a single component was detected for the majority of the dyes examined. However, when there was a mixture of two azoic coupling components with a single azoic diazo component (A48 and A49, Table I), two spots were produced as expected. The only other examples where multiple spots were produced were dyes A32, A34 and A50. These dyes are distinct in that they alone are formed using CI Azoic Coupling Component 17 [7]. It is thus probable that these two azoic components each contain at least two different chemical compounds.
TABLE 3 Thin layer chromatography of azoic dyes extracted from cotton fibres using chlorobenzene:1,2dichloroethane :acetone (20 :20 :1, v :v :v) as eluent Dye code* A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 All A12 A13 A14 A15 A16 A17 A18 A19 A20 A21 A22 JFSS 1990; 30(5): 309-315
Components
Yellow Yellow Yellow Yellow Yellow Yellow Yellow Pink Oranget Orange? Oranget Oranget PinkJRedt Red7 Redt Oranget OrangeJRedt Oranget Oranget Orange? Oranget Oranget
Rf x 100
TABLE 3 (continued) Dye code*
Components
Rf x lo0
Yellow/Orange GreyIBrown Grey/Purplet Grey/Blue Blue Yellow Violet? Purple? Purple GreyIPinkt Purple? Brown/Pink BrownJPink Purple? Grey/Pink? Purple? Purplet Pink? Purplet Pink? Violett Pink Violet? Pink Brownt Olive? Brown? Brown Purple? Blue Violet Light Blue Dark Blue Blue Blue
* See Table 1 for identification t Fluorescent when illuminated with 254 nm light
Conclusion For azoic dyes extracted from cotton fibres the most favourable eluent system for thin layer chromatography is chlorobenzene: 1,Zdichloroethane : acetone (20 :20 : 1, v :v :v). Pre-development with ethyl acetate results in concentration of dye on the TLC plate and improved separation for the majority of dyes. 314
JFSS 1Y90; 30(5): 309-315
References 1. Laing DK, Dudley RJ, Hartshorne AW, Home JM, Rickard RA and Bennett DC. The extraction and classification of dyes from cotton and viscose fibres. Forensic Science International: in press. 2. Beattie IB, Roberts HL and Dudley RJ. Thin-layer chromatography of dyes extracted from polyester, nylon and poly-acrylonitrile fibres. Forensic Science International 1981; 17: 57-69. 3. Home JM and Dudley RJ. Thin-layer chromatography of dyes extracted from cellulosic fibres. Forensic Science International 1981; 17: 71-78. 4. Macrae R, Dudley RJ and Smalldon KW. The characterization of dyestuffs on wool fibers with special reference to microspectrophotometry. Journal of Forensic Sciences 1979; 24: 117-129. 5. Hartshorne AW and Laing DK. The dye classification and discrimination of coloured polypropylene fibres. Forensic Science International 1984; 25: 133-141. 6 . Laing DK, Boughey L and Hartshorne AW. The standardisation of thin layer chromatographic systems for comparison of fibre dyes. Journal of the Forensic Science Society: 1990; 30: 299-307. 7. The Colour Index, 3rd Edition. Bradford, United Kingdom: Society of Dyers and Colourists, 1987.
JFSS 1990; 30(5): 309-315