兵庫県立健康環境科学研究センター中野　武

低臭素化ダイオキシン類の異性体分析

PCDD/DF 分析法は、毒性係数の示されている 17異性体、 4～ 8塩素化 DD/DF のみを対象

低塩素化 DD/DF の異性体分布は、生成機構や分解過程の解明、毒性評価、起源推定などに非常に重要な情報を提供する

現状

低塩素化ダイオキシン類の異性体分析

●　 SP-2331 ●　 DB-DIOXIN●　 DB5-MS

SP-2331 and DB-DIOXIN

Polarity: 　 DB-DIOXIN 　 < 　 SP-2331 　DB-Dioxin 　 44% methyl, 28% phenyl, 　 20% cyanopropyl　 8% polyoxyethyleneSP-2331

90% 　 biscyanopropyl10% 　 phenylcyanopropyl

Column;

Standard:

a) Authentic standards: 13, 27, 23, 36, 28, 26, 34, 46-DiCDFs

Authentic standards of 136,138,146,147,149,236,238,246,267,346 -TrCDFs

b) Synthesis of PCDD by pyrolysis phenols in presence of KOH at temperature between 250C-300C

c) Synthesis of PCDF by pyrolysis of respective Phenols in pyrex ampoule (370-400C, 15-30min)

d) Synthesis of PCDF by pyrolysis of PCB in pyrex ampoule (400-450C, 15min)

Phenol 2-MCP 3-MCP 4-MCP 23-DCP 24-DCP 25-DCP

Phenol 4-F 1/3-F 2-F 34-F 24-F 14-F

2-MCP 46-F 16/36-F 26-F 346-F 246-F 146-F

3-MCP 17/37/19-F 18/27-F 167/347-F 168/247-F 147/149-F

4-MCP 28-F 267-F 248-F 148-F

PCDF formation from chlorophenols

Synthesis PCDF

Synthesis of single isomers (in one homolog): 2-chloroPhenol : 4,6-DiCDF

Synthesis of mixtures 3,4-dichloroPhenol (result in 1,2- and 2,3-fragment) 3-chloroPhenol (result in 1- and 3-fragment)

O

Cl

ClCl

O

ClCl

Cl

OH

Cl

OH

Cl

Cl

2,3,8-

1,2,8-+

4-MCP

3,4-DCP2,3,8-TrCDF

1,2,8-TrCDF

O

Cl

O

OH

OH

Cl

1,4,7-

1,4,9- Cl

Cl

Cl

Cl

Cl

Cl

Cl

OH

OH

O

Cl

1,4,6- Cl

O

Cl

1,4,8- Cl

Cl

Cl

Cl

Cl

Synthesis of 1,4,X-T3CDF isomers from chlorophenol

Authentic standard (1,4,7- : NMR)

2,5-DCP

4-MCP

3-MCP

2-MCP

1,4,6-TrCDF

1,4,8-TrCDF

1,4,9-TrCDF

1,4,7-TrCDF

NMR 1,4,7-TrCDF

Authentic standard

H9

H6

H8

H2 ; H3

H2

H3

H6

H8

H9

Cl7

Cl1

Cl4

Synthesis PCDD

Synthesis of single isomers 1-MCDD, 2-MCDD, 12-DCDD, 13-DCDD, 23-DCDD, 123-TCDD, 124-TCDD, 236-TCDD, 237-T3CDD

Most cases simple PCDD mixtures with two isomers (smiles rearangement): 2,3+2,3,4 result in 1,2,6- and 1,2,9-T3CDD

Assignment in mixture of two isomers: With the same ring fragment substitution 　the more polar compound elutes later.

Synthesis PCDD

Chromatogram on Columns; Comparison Retention Behavior  Chromatograms - DB-Dioxin   - SP2331 column 

9 10 11 12 13 14 15 16 17 18 19 20 21 22

9 10 11 12 13 14 15 16 17 18 19 20 21 22

9 10 11 12 13 14 15 16 17 18 19 20 21 22

1

4

2

3

MCDF

13 17

1418

1612

3727

36

46

3426

28

24

19

23

137

DiCDF

TrCDF

138

346

129

267

347/

236

137

237/

149

246

248

136 16

813

412

4/14

7

178

148

126

128

247

167

139/

127

146

123

Ambient air SP-2331

DB-DIOXIN

26.5 27.0 27.5 28.0 28.5 29.0 29.5 30.0 30.5 31.0 31.5 32.0

D2CDF

13

17

37

16

12

27

28

36

34

26 46

19

23

24

14

18

32 33 34 35 36 37 38 39

T3CDF

137

138 13

6

167/

248 12

6/1

28/

246

237

238

347/236/234

267

346

129

124

148/

127

178

/14

6/12

3

134/

147

168

247

139 14

9

9 10 11 12 13 14 15 16 17 18 19 20 21 22

9 10 11 12 13 14 15 16 17 18 19 20 21 22

9 10 11 12 13 14 15 16 17 18 19 20 21 22

1

4

2

3

MCDF

13 17

1418

1612

3727

36

46

3426

28

24

19

23

137

DiCDF

TrCDF

138

346

129

267

347/

236

137

237/

149

246

248

136 16

813

412

4/14

7

178

148

126

128

247

167

139/

127

146

123

SP-2331

GC/MS-SIM chromatogram of PCDF

D2CDF

D2CDF

T3CDF

T3CDF

13

137

19

46

13

129137

346

DB-DIOXIN

28.0 28.4 28.8 29.2 29.6 30.0 30.4 30.8 31.2 31.6 32.0

D 2 CDD

13

14

17

1

8

16

12

19

2

7/2

8

23

34.0 34.4 34.8 35.2 35.6 36.0 36.4 36.8 37.2 37.6 38.0 38.4 38.8

T3CDD

129

126

137

138

123/

178

237

136

147

124

139

127

1

28

146

SP-2331

9 10 11 12 13 14 15 16 17 18 19 20 21 22

9 10 11 12 13 14 15 16 17 18 19 20 21 22

9 10 11 12 13 14 15 16 17 18 19 20 21 22

MCDD

DiCDD

TrCDD2

1

13

27/

23/2

814

/17

18

16 1

2

19

137

138

136

124

139/

237

147

129

126

127

17812

3

128 14

6

GC/MS-SIM chromatogram of PCDD

T3CDD

T3CDD

137129

137 129

D2CDD

D2CDD

13

19

1913

Assignment for MCDD/F to T3CDD/F

MCDD TrCDD MCDF DCDF TrCDF TrCDF2 23.76 137 34.18 1 22.57 23 29.87 124 33.80 238 36.331 24.69 138 34.24 3 23.09 28 29.96 127 34.43 347 36.88

DCDD 136 34.61 2 23.56 36 30.16 148 34.43 236 36.8813 28.51 124 35.07 4 24.51 26 30.69 123 34.63 234 36.8827 29.57 139 35.40 DCDF 34 31.09 146 34.65 149 36.9828 29.57 147 35.60 13 26.43 46 31.31 178 34.65 267 37.3723 29.73 237 35.63 17 27.71 19 31.46 247 34.75 346 37.7314 30.05 123 36.19 14 28.48 TrCDF 139 35.08 129 38.4917 30.26 178 36.24 18 28.65 137 31.77 248 35.2218 30.39 146 36.65 24 28.96 138 32.58 167 35.2316 30.85 127 36.86 37 29.08 136 32.87 126 35.4812 31.26 128 37.00 16 29.28 168 33.44 128 35.4819 31.77 126 37.31 12 29.34 134 33.52 246 35.58

129 38.21 27 29.49 147 33.52 237 35.94

The No. of separation peaks

isomer ULTRA-2 DB-5MS SP-2331 DB-DIOXIN

MCDD 2 2 2 2 2 D2CDD 10 5 4 8 9 T3CDD 14 6 5 12 12

Separation of low chlorinated DD

The No. of separation peaks

isomer ULTRA-2 DB-5MS SP-2331 DB-DIOXIN

MCDF 4 4 4 4 4 D2CDF 16 8 10 15 16 T3CDF 28 8 10 23 21

Separation of low chlorinated DF

SP-2331 and DB-DIOXIN

Elution order on DB-DIOXIN

2,4-, 3,7- isomers : earlier

1,2-, 1,6-, 1,9- isomers : later

compare with SP-2331

PCDF

R.T. Behavior : Substitution Fragments

26.5 27.0 27.5 28.0 28.5 29.0 29.5 30.0 30.5 31.0 31.5 32.0

D2CDF

13

17

37

16

12

27

28

36

34

26 4

6 1

9

23

24

14

18

DB-Dioxin

24

3719

12

16

GC/MS-Chromatogram of D2CDF

SP

-233

1

SP

-233

1

32 33 34 35 36 37 38 39

T3CDF 13

7

138 13

6

167

/24

8 126

/128

/24

6 23

7

238

347/236/234

267

346

129

124

148

/12

7 1

78/1

46/1

23

134

/14

7 16

8

247

139 14

9

DB-Dioxin

129

149

139

GC/MS-Chromatogram of T3CDF

SP

-233

1

SP

-233

1

SP-2331 and DB-DIOXIN

Elution order on DB-DIOXIN

1,4- isomers : earlier

compare with SP-2331

PCDD

R.T. Behavior : Substitution Fragments

28.0 28.4 28.8 29.2 29.6 30.0 30.4 30.8 31.2 31.6 32.0

D2 CDD 13

14 17

18

16

12

19

27/2

8 23

DB-Dioxin

14

GC/MS-Chromatogram of D2CDD

SP

-233

1

DB

-Dio

xin

34.0 34.4 34.8 35.2 35.6 36.0 36.4 36.8 37.2 37.6 38.0 38.4 38.8

T3CDD

129

126

137

138 12

3/17

8

237

136

147

124

139

127

128

146

DB-Dioxin

147146

GC/MS-Chromatogram of T3CDD

低臭素化ダイオキシン類の異性体分析

●　 SP-2331 (95%cyanopropyl 5%phenyl-siloxane)

●　 BPX-35 (35%phenylpolysilphenylene-siloxane)

O Si

C H3 6

CN

O Si

C H3 6

CN

C H3 6

CN

Si Si O Si

CH3

O

CH3

CH3

CH3

CH3 x

y

yx

Standards. For the PBDF mixture all three monobromophenol isomers, Phenol and 2, 4-DBP were pyrolysed at 370ºC (15 minutes) in the presence of trace amounts of CuBr2.

Authentic standards 2,7-D2BDF, 2,8-D2BDF, 2,3,8-T3BDF,

合成した臭素化 DF 標品混合物は、

使用した 2-/3-/4- ブロモフェノールの量を反映し、 2->1-,3->4-(4:2:2:1) であった。

DiBrDF の場合、 24- 以外に 28-, 18-, 27- , 17-, 19-, 26-, 37- , 16-, 36- , 46- が含まれ、

TrBrDF は、 248- , 168- , 247- , 246- 、

TeBrDF の場合、 2468- が主要な異性体である。

低臭素化 DD/DF 異性体について、市販標品の RT 、標品混合物中の異性体予測生成比、異性体の溶出順序の規則性から、推定した。

11.5 12.0 12.5 13.0 13.5 14.0 14.5 15.0 15.5 16.0 16.5 Retention Time

0

1587256

M1BDF

16 17 18 19 20 21 22 23 24 25 Retention Time

0

497896

D2BDF

24 26 28 30 32 34 Retention Time

0

102569

T3BDF

46

17

13

18

1637

24

27

36

28

26

247

248

246

346

238

138/136

137 168

1 3

2

4

M1BrDF

D2BrDF

T3BrDF

347/236

237/149

M1BrDF ～ T3BrDF の GC/MS-SIM クロマトグラム　SP2331 ; 60m x 0.32mmid. 0.20um; 120°C (1 min.), 20°C/min. to 200°C, 2°C /min. to 260°C (30min).; He 1.2mL/min.

BPX-35 ; 30m x 0.32mm id. 0.25um; 120°C (2 min.), 20°C/min. to 180°C,

2°C /min. to 220°C, 10°C /min. to 300°C (10min).; He 1.0mL/min.

10 11Retention Time (min)

0

2726152

Inte

nsity

M1BDF / Average

13 14 15Retention Time (min)

503

14887

Inte

nsity

BrClDF / Average

16 17 18 19Retention Time (min)

0

792528

Inte

nsity

D2BDF / Average

1 3

2

4

1346

24

27

28

2617

18

BPX-35 ; 30m x 0.32mm id. 0.25um; 120°C (2 min.), 20°C/min. to 180°C,

2°C /min. to 220°C, 10°C /min. to 300°C (10min).; He 1.0mL/min.

26 27 28 29

Retention Time (min)

0

312320

Inte

nsity

T3BDF / Average

31 32 33

Retention Time (min)

0

87023

Inte

nsity

T4BDF / Average

Standards. The PBDD standards were synthesized by pyrolysis of bromophenols in Pyrex ampoules.

The PBDD was synthesized by condensation of 2-monobromo phenol, 2, 4- and 2, 6- dibromophenol (DBP) and 2, 4, 6-tribromophenol (TBP) at 350ºC (30 min).

Authentic standards:2,7/2,8-D2BDD, 2,3,7-T3BDD.

合成した臭素化 DD 標品混合物は、

DiBrDD は、 13- 、 27/28- 、

TrBrDD は、 137-, 138- 、 136-, 139-

TeBrDD は、 1368-, 1379- が含まれていた。

11 12 13 14 15 16 Retention Time

0

3095833

M1BDD

16 17 18 19 20 21 22 23 24 Retention Time

0

760425

D2BDD

25 26 27 28 29 30 31 32 Retention Time

0

398216

T3BDD

138124

237129

136

137

2

1

13

19

12

27/28/23

18

16

14/17

M1BrDD

D2BrDD

T3BrDD

　 M1BrDD ～ T3BrDD の GC/MS-SIM クロマトグラムSP2331 ; 60m x 0.32mmid. 0.20um; 120°C (1 min.), 20°C/min. to 200°C, 2°C /min. to 260°C (30min).; He 1.2mL/min.

BPX-35 ; 30m x 0.32mm id. 0.25um; 120°C (2 min.), 20°C/min. to 180°C, 2°C /min. to 220°C, 10°C /min. to 300°C (10min).; He 1.0mL/min.

10.4 10.8

Retention Time (min)

0

4457267

Inte

nsity

M1BDD / Average

14 15 16

Retention Time (min)

524

9893

Inte

nsity

BrClDD / Average

17 18 19

Retention Time (min)

0

2389477

Inte

nsity

D2BDD / Average

2

1

13

27/28

BPX-35 ; 30m x 0.32mm id. 0.25um; 120°C (2 min.), 20°C/min. to 180°C,

2°C /min. to 220°C, 10°C /min. to 300°C (10min).; He 1.0mL/min.

27 28 29

Retention Time (min)

0

1307231

Inte

nsity

T3BDD / Average

31.0 31.5 32.0 32.5

Retention Time (min)

0

191064

Inte

nsity

T4BDD / Average

1368

1379

PCB 濃度が高い場合、DiBrDF(323.8785, 325.8765) は

PeCB(323.8834,325.8805) の妨害を受ける。

排ガス試料

臭素系 DD/DF はBrDF>>BrClDF ＞ BrCl2DF 、BrDD>>BrClDD ＞ BrCl2DD

16 20 24

Retention Time (min)

0

649323

16 20 24

Retention Time (min)

0

568627

D2BrDF

D2BrDF

STD mix

Sample

27

28

#118

#105#114

28

27

GC/MS-SIM クロマトグラム (D2BrDF) 　　　　　SP2331 ; 60m x 0.32mmid. 0.20um; 120°C (1 min.), 20°C/min. to 200°C, 2°C /min. to 260°C (30min).; He 1.2mL/min.

PCB 濃度が高い場合、 DiBrDF(323.8785, 325.8765) は PeCB(323.8834,325.8805) の妨害を受ける。

14 16 18 20

Retention Time (min)

0

171522

14 16 18 20

Retention Time (min)

0

86858

BrClDF

BrClDF

Sample-B

Sample-A

GC/MS-SIM クロマトグラム (BrClDF)SP2331 ; 60m x 0.32mmid. 0.20um; 120°C (1 min.), 20°C/min. to 200°C, 2°C /min. to 260°C (30min).; He 1.2mL/min.

臭素系 DD/DF は BrDF>>BrClDF ＞ BrCl2DF 、 BrDD>>BrClDD ＞ BrCl2DD