T.NAKANO and R.WEBER*

Hyogo Pref. Inst. of Public Health & Environ. Sci.

*Inst. of Organic Chem., Univ. of Tuebingen

Analysis of Low Chlorinated PCDD/F -Isomer Specific Analysis of

MCDD/MCDF to T3CDD/T3CDF on DB-Dioxin-Column

Errata

Vol.55, p.123-126

Table 1 and Figure 2 ( 2,3-D 2,8-D )

I am hating computer virus (Klez etc)

DCDD13 28.5127 29.5723 29.5728 29.73

DCDD13 28.5127 29.5728 29.5723 29.73

I found my fixed abstract was not received.

Roland Weber pointed out before, that the 2,7-D is not single authentic standard. (2,7-D contains 2,8-D as impurity, and can not separate)

Introduction Relevance of isomer specific analysis of T4CDD/F-OCDD/F

 Due to the high toxicity of PCDD/F substituted in 2,3,7,8-position, a lot of attention was paid to the isomer specific analysis of tetra- to octachlorinated DD/DF in the last two decades.

Since, exclusively, the congeners substituted in all four 2,3,7,8-position exhibit the dioxin related toxicity, lower chlorinated congeners were not assigned with TEQ values and, therefore, not a lot of attention was paid to them.

Introduction Isomer specific analysis of low chlorinated DD/F 

The analysis of low chlorinated isomers seems interesting from several points of view:

     For the investigation of the mechanisms of PCDD/F formation/degradation, the low chlorinated compounds offer valuable additional information.

For on-line measurement of PCDD/F in waste incineration, some recent research projects focused on estimating TEQ values by measurement of low chlorinated PCDD/F as indicator compound.

To establish this correlation, it seems important to measure not only the total amount of the low chlorinated homologues

but to calculate and correlate specific isomers.

Furthermore, the low chlorinated DD/F may give valuable information for environmental samples. The analysis of isomer distributions in environmental samples is the key to estimating the origin of dioxins and related compounds.

(Also in this field, many data are published for T4CDD/F-OCDD/F, however, insufficient data are available for mono- to tri-chlorinated dioxin/furan.)

GC/MS Analysis.

The analysis was carried out using an HP The analysis was carried out using an HP 5890 II gas chromatograph connected to a 5890 II gas chromatograph connected to a JMS-700 mass spectrometer (JEOL Ltd. JMS-700 mass spectrometer (JEOL Ltd. Japan) (operating at a resolution >10 000).Japan) (operating at a resolution >10 000).

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;

DB-Dioxin (60m, 0.25mm i.d., 0.15um, J&W): Temperature program used for isomer specific separation of the MCDD/F-T3CDD/F on DB-Dioxin column:

50C, 1 min. isothermal; 20C/min. to 150C, 3C /min. to 270C, 14min. isothermal.

Carrier gas flow rate : He 1.0mL/min.

Temperature program

SP-2331: (60m, 0.25mm i.d., 0.25um, SUPELCO): Temperature program used for isomer specific separation of the MCDD/F-T3CDD/F

on SP-2331 column: 120C, 2 min. isothermal; 30C/min. to 200C, 2C/min. to 260C, 6 min. isothermal.

Carrier gas flow rate : He 1.0mL/min.

Temperature program

Standard: a) Authentic standards: commercially available13, 27, 28, 23, 36, 26, 34, 46-DiCDFs (8 isomers / 16 isomers)

Authentic standards of 136,138,146,147,149,234,236,238,246,247,267,346 -TrCDFs(12 isomers / 28 isomers)

b) Synthesis of PCDD by pyrolysis of chlorophenols in presence of KOH (250C-300C, 60min)

c) Synthesis of PCDF by pyrolysis of chlorophenols (370-400C, 15-30min)

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

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

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

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

Synthesis PCDD

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

OH

Cl HO

ClCl

Cl

ClO

O

Cl

Cl

O

O

Cl

Cl

Cl

Cl

+

2,3-CP+2,3,4-DCP 1,2,9-D

1,2,6-D

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

Elution order for D2CDD to T3CDDD2CDD

SP-2331 13 27/28/23 14/17 18 16 12 19

DB-DIOXIN 13 27/28 23 14 17 18 16 12 19

T3CDD

SP-2331 137 138 136 124 139/237 147 123 178 127 128 146 126 129

DB-DIOXIN 137 138 136 124 139 147 237 123 178 146 127 128 126 129

O

O

Cl

Cl

O

O

Cl

ClCl

O

O

Cl

Cl

Cl

1,4,6-D 1,4,7-D 1,4-D O

O

Cl

Cl

O

OCl

Cl

Cl

2,3-D 2,3,7-D

elutes earlier

elutes later

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

Elution order for D2CDF to T3CDFD2CDF

SP-2331 13 17 14 18 16 12 24 37 27 23 36 28 26 19 34 46

DB-DIOXIN 13 17 14 18 24 37 16 12 27 23 28 36 26 34 46 19

T3CDF

SP-2331 137 138 136 168 134 124 147 178 148 123 127 139 146 167 247 128

DB-DIOXIN 137 138 136 168 134 147 124 127 148 123 146 178 247 139 248 167

SP-2331 126 248 246 237 149 234 238 347 236 267 129 346

DB-DIOXIN 126 128 246 237 238 347 236 234 149 267 346 129

elutes earlier

elutes later

Cl

Cl

O O

ClCl

ClOCl

2,4-F 3,7-F 4,6-F

O

Cl

Cl

Cl

O

Cl

O

Cl Cl

1,6-F 1,2-F 1,9-F

1,2,9-F1,3,9-F1,4,9-F

3,4,7-F2,4,8-F2,4,7-F

3,4,6-F

SP-2331 and DB-DIOXIN

Elution order on DB-DIOXIN

1,4- fragment : elutes 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

SP-2331 and DB-DIOXIN

Elution order on DB-DIOXIN

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

1,2-, 1,6-, 1,9- : elutes 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

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 14 15 T3CDF 28 8 10 23 21

Separation of low chlorinated DF

Conclusions

 a) The isomer specific analysis of MCDD/MCDF to T3CDD/T3CDF was established on three standard columns     SP-2331      DB Dioxin     DB 5MS

Conclusions

b) The low chlorinated PCDD/DF can be analysed together with T4CDD/F to OCDD/F ( one GC/MS injection).

c) The DB-Dioxin and the SP-2331 column is effective for an isomer specific analysis of low chlorinated PCDF as it is requested for detailed mechanistic studies.

Acknowledgement

The authors would like to express their thanks to Dr. Brian Gullett and Dr. Dennis Tabor(US-EPA) , Dr. Junji Ogakifor donating some standards,

to Terry Humphries for critical reading of the manuscript.

See you next!

Thank you for your attention.

0

0.1

0.2

0.3

0.4

0.5

0.6

MC

DF

D2C

DF

T3C

DF

T4C

DF

P5C

DF

H6C

DF

H7C

DF

O8C

DF

Homologue distribution of PCDF in Ambient air

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

NMR 1,4,7-TrCDF

Authentic standard

H9

H6

H8

H2 ; H3

H2

H3

H6

H8

H9

Cl7

Cl1

Cl4

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

We can control the ratio of isomers by changing the ratio of chlorophenol.

Case of 3-MCP + 2-MCP

if 2-MCP > > 3-MCP

46- > 16- > 36- > 17- > 37- > 19-F

if 3-MCP >> 2-MCP

17- > 37- > 19- > 16- > 36- > 46-F

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

2,5-dichloroPhenol + 3-chloroPhenol 1,4,9- and 1,4,7- (1,4,9- , 1,4,7- : authentic standard)

3-chloroPhenol and 3,4-dichlorophenol 1,2,7-, 1,2,9-, 1,7,8-, 2,3,7-

Assignment additional synthesis via PCB. 2,3,4' T3CB result in 1,2,7-T3CDF

Roland Weber pointed out before,

that the 2,7-D is not single authentic standard.

(2,7-D contains 2,8-D as impurity, and can not separate)

As for 2,7-D2CDD,

Basically the position is

2,7-D should be presented as an unknown ratio mixture of the two compounds 2,7-/2,8-D .

18.55 18.60 18.65 18.70 18.75 18.800

5000

10000

T im e-->

Abundanc e

Ion 252.00 (251.70 to 252.70): JF 45082B .D

2,7-D C D D

2,7-D C D D

18.55 18.60 18.65 18.70 18.75 18.800200040006000

T im e-->

Abundanc e

Ion 254.00 (253.70 to 254.70): JF 45082B .D

18.55 18.60 18.65 18.70 18.75 18.800

2000400060008000

10000

T im e-->

Abundanc e

Ion 264.00 (263.70 to 264.70): JF 45082B .D

13C 2,3-D C D D

13C 2,7-D C D D

18.55 18.60 18.65 18.70 18.75 18.800

2000

4000

6000

T im e-->

Abundanc e

Ion 266.00 (265.70 to 266.70): JF 45082B .D

2,3-D2,7-/2,8D

2,7-/2,8-D

13C-D2CDD

D2CDD

Dennis Tabor (EPA)

But, I believe the poor peak shape of my standard for 2,3-DCDD has lead to an poor assignment.

I have 2,7 and 2,3 in my 13C standards and have not gotten the poor peak shape from them.

I believe that the toluene or other solvent must be causing you a problem.

In my standard which do not have toluene 2,7 comes out before 2,3. The first two traces are the 252/254 of the native DCDD. The second two traces are the 264/266 of the 13C DCDD.

The current GC/MS that I am using is Low Res. Therefore the 13C 2,8-DCDF causes me not to be able to see 1,4-DCDD.

Basically the low chlorinated PCDD/PCDF can be analysed together with T4CDDF-OCDD/F.

Behavior on Alumina column.

Low chlorinated isomers may be oxidized during extensive Sulfuric acid treatment.  

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

Elution order for D2CDD to T3CDDD2CDD

SP-2331 13 27/28/23 14/17 18 16 12 19

DB-DIOXIN 13 27/28 23 14 17 18 16 12 19

T3CDD

SP-2331 137 138 136 124 139/237 147 123 178 127 128 146 126 129

DB-DIOXIN 137 138 136 124 139 147 237 123 178 146 127 128 126 129

D2CDD

SP-2331 13 2-3/2-2/23 14/1-3 1-2 1-4 12 1-1

DB-DIOXIN 13 2-3/2-2 23 14 1-3 1-2 1-4 12 1-1

T3CDD

SP-2331 13-3 13-2 13-4 124 13-1 23-3 14-3 123 1-23 12-3 12-2 14-4 12-4 12-1

DB-DIOXIN 13-3 13-2 13-4 124 13-1 14-3 23-3 123 1-23 14-4 12-3 12-2 12-4 12-1

Elution order for D2CDD to T3CDDD2CDD

SP-2331 13 27/28/23 14/17 18 16 12 19

DB-DIOXIN 13 27/28 23 14 17 18 16 12 19

T3CDD

SP-2331 137 138 136 124 139/237 147 123 178 127 128 146 126 129

DB-DIOXIN 137 138 136 124 139 147 237 123 178 146 127 128 126 129

D2CDD

SP-2331 13 27/28/23 14/17 18 16 12 19

13 2-3/2-2/23 14/1-3 1-2 1-4 12 1-1

DB-DIOXIN 13 27/28 23 14 17 18 16 12 19

13 2-3/2-2 23 14 1-3 1-2 1-4 12 1-1

T3CDD

SP-2331 137 138 136 124 139 237 147 123 178 127 128 146 126 129

13-3 13-2 13-4 124 13-1 23-3 14-3 123 1-23 12-3 12-2 14-4 12-4 12-1

DB-DIOXIN 137 138 136 124 139 147 237 123 178 146 127 128 126 129

13-3 13-2 13-4 124 13-1 14-3 23-3 123 1-23 14-4 12-3 12-2 12-4 12-1

Elution order for D2CDF to T3CDFD2CDF

SP-2331 13 17 14 18 16 12 24 37 27 23 36 28 26 19 34 46

DB-DIOXIN 13 17 14 18 24 37 16 12 27 23 28 36 26 34 46 19

T3CDF

SP-2331 137 138 136 168 134 124 147 178 148 123 127 139 146 167 247 128

DB-DIOXIN 137 138 136 168 134 147 124 127 148 123 146 178 247 139 248 167

SP-2331 126 248 246 237 149 234 238 347 236 267 129 346

DB-DIOXIN 126 128 246 237 238 347 236 234 149 267 346 129

D2CDF

SP-2331 13 1-3 14 1-2 1-4 12 24 3-3 2-3 23 3-4 2-2 2-4 1-1 34 4-4

DB-DIOXIN 13 1-3 14 1-2 24 3-3 1-4 12 2-3 23 2-2 3-4 2-4 34 4-4 1-1

T3CDF

SP-2331 13-3 13-2 13-4 1-24 134 124 14-3 1-23 14-2 123 12-3 13-1 14-4 1-34 24-3 12-2

DB-DIOXIN 13-3 13-2 13-4 1-24 134 14-3 124 12-3 14-2 123 14-4 1-23 24-3 13-1 24-2 1-34

SP-2331 12-4 24-2 24-4 23-3 14-1 234 23-2 34-3 23-4 2-34 12-1 34-4

DB-DIOXIN 12-4 12-2 24-4 23-3 23-2 34-3 23-4 234 14-1 2-34 34-4 12-1