1

EnantioselectiveEnantioselective separation ofseparation ofchiralchiral PCBs by multidimensional PCBs by multidimensional gas chromatography techniques. gas chromatography techniques.

Application to real samplesApplication to real samples

L.R. L.R. BordajandiBordajandi, L. Ramos, B. , L. Ramos, B. GómaraGómara andandM.J. GonzálezM.J. González

DepartDepart. . ofof I.A. & I.A. & EnvironEnviron. . ChemChem. IQOG (CSIC) Madrid, . IQOG (CSIC) Madrid, SpainSpain

2 Introduction

Clx Cly0 < x + y < 11

209 PCBs

19 19 PCBsPCBspresent stable enantiomers

at room temperature

78 78 PCBs PCBs display axial chirality

in their non-planar conformations

PCB 45, 84, 88, 91, 95, 131, 132, 135, 136, 139, 144, 149, 171, 174, 175, 176, 183, 196, 197

Energy of racemization > Free energy in the ecosystems

Racemates

Enantioselective Enantioselective transformationstransformations

Non racemates

3 Introduction

Although the enantiomeric enrichment of chiral PCBs in living organisms has been reported since 1994

Mussels (Hühnerfuss et al., 1994); Sharks (Blanch et al.,1996); Otters ( Ramos et al., 1996); Cetaceans (Vetter and Schurig, 1997; Reich et al., 1998); Predatory birds (Gómara et al., 2006); Human breast milk (Schurig et al., 1995; Bordajandi et al., 2005)

The knowledge about the mechanisms involved in the enantioselective degradation is limited

The analytical separation of chiral PCBs is a rather difficult task

4 Introduction

Until now, no single enantioselective GC column has been able to separate the 19 chiral PCBs

80 100 110 120 130 140 150

PCB 45

PCB 95 PCB 91PCB 136

PCB 149

PCB 176

90

The separation into enantiomers of the 19 chiral congenershas been achieved by the use of capillary columns

with different types of chiral stationary phases

5 Chiral stationary phases

terttert..-- buthyldimethylsilylbuthyldimethylsilyl ββ--CD CD

Chirasil-Dex 2,3,62,3,6--tritri--OO--methyllmethyll ββ--CD CD chemicaly bonded to polysiloxane

BGB-176SE 20% 2,320% 2,3--didi--OO--methylmethyl--66--OO--terttert..--buthyldimethylsilylbuthyldimethylsilyl--ββ--CDCDdiluted in SE-52 (5 % phenyl methylpolysiloxane)

PCBs 45, 91, 95, 131, 136, 149, 176

BGB-172 25% 2,3,625% 2,3,6--terttert..--butbutyyldimetldimethyhylsillsilyyll--ββ--CD CD diluted in PS086 (15 % phenyl methylpolysiloxane)

PCBs 84, 91, 95, 132, 135, 136, 149, 174, 176

PCBs 45, 84, 88, 91, 131, 132, 135, 144, 149, 171, 174, 175, 183

PermethylPermethyl ββ--CD CD

6 Multidimensional approaches

A multidimensional approach is clearly needed A multidimensional approach is clearly needed when analysing real sampleswhen analysing real samples

•With other enantiomers and PCBs•With other chemical compounds present in the extract

CoelutionsCoelutions problems: problems:

Off line LC /(Off line LC /(PYEPYE))--GCGC

Three different Three different multidimensional multidimensional

approaches approaches could be usedcould be used HeartHeart--cutcut MDGCMDGC--

ECDECD/MS/MSGC GC ×× GCGC--micromicro--ECD

GC multidimensionalGC multidimensional

ECD

Heart-cut MDGC7

20 30 40 50 60 70 Min

0

50

100

150

200

mV

Detector 1 Detector 2

1D

2D

Injector

PCB 153+13

2

PCB 105

A+B+C

C

A B

Deans Valve

Transfer line

HeartHeart--cutcutMDGCMDGC

Transferenceto the 2D

8 Heart-cut MDGC

Selection of capillary columns for the Selection of capillary columns for the 11D and D and 22D dimensionD dimension

DB-5 Apolar

11DD 22DD(L = 30 m) (L = 30 m)

Chirasil-DexPCBs 91, 95, 132, 135, 136, 149, 174, 176

BGB-172PCBs 84, 135, 171, 183

BGB-176SEPCBs 45, 91, 95, 136

(5% phenyl polysiloxane)

Heart-cut MDGC9

20 30 40 50 60 70 Min

0

50

100

150

200

mV

32,5 33,0 33,5 34,0 Min

0

50

100

150

200

250

mV

PCB 118

PCB123+149 PCB

118

65 PCBsin DB-5 (1D)

PCB 153+132

PCB 105

Transference to Chirasil-Dex (2D )

84 85 86 87 88 89 90 91Min

0

10

20

30

40

50

PCB 123

PCB 118

PCB 149

PCB 153PCB 105PCB

132

10 Heart-cut MDGC

DBDB--5 5 –– ChirasilChirasil--DexDex PCBs 91, 95, 132, 135, 136, 149, 174 y 176

PCBsPCBs 91, 95, 132, 174 91, 95, 132, 174 PCBsPCBs 149, 176149, 176PCB 135 PCB 135 PCB 136PCB 136

4 4 INJECTIONSINJECTIONS

Por lo tanto, para la

75,0 77,5 80,0 82,5 85,0 87,5 Min

Sheep´s milk

PCB 136

PCB 110

Coelutions with other PCBs Chirasil-Dex

Coelutions between enantiomers

100 105 110 115

PCB 176

Chirasil-Dex

PCB 135

PCB 132

L.R. Bordajandi, PhD Thesis, 2005

GC x GC micro-ECD11

1D 2D

(c)

Chromatogramin 3D

Tr in 1D

Tr in the n 2D

Chromatogramin 2DLoop modulator

Modulator

12 GC x GC micro-ECD

Selection of capillary columns for the Selection of capillary columns for the 11D and D and 22D dimensionD dimension

11DD 22DD

ChirasilChirasil--DexDex ×× SupelcowaxSupelcowax--10 (10 (Polyethylene glycol)Polyethylene glycol)

BGBBGB--172 172 ×× SupelcowaxSupelcowax--1010

BGBBGB--176SE 176SE ×× SupelcowaxSupelcowax--1010

8 chiral PCBs Except PCB 135

8 chiral PCBs Except PCB 91

6 chiral PCBs

GC x GC micro-ECD13

ChirasilChirasil--DexDex ×× SupelcowaxSupelcowax--1010

8 chiral PCBs, except PCB 135

65 PCBs

0

6

1

2

3

4

5

50 60 70 80 90 120110100 130 150140 160

Retention time in 1D (min)

Ret

entio

ntim

e in

2 D (s

)

9591

136

176

174132

149

84

135, 139

PCB 135, PCB 135, 139139

L.R. Bordajandi, PhD Thesis, 2005

GC x GC micro-ECD1414

ChirasilChirasil--DexDex ×× SupelcowaxSupelcowax--1010

8 chiral PCBs Except PCB 135

7 indicator PCBs

12 coplanar PCBs65 PCBs

0

6

1

2

3

4

5

50 60 70 80 90 120110100 130 150140 160Retention time in 1D (min)

Ret

entio

ntim

e in

2 D (s

)

77

126

169

123

118 157156

105

167

11481

189

5228

153

138

180

101

118

L.R. Bordajandi, PhD Thesis, 2005

15 GC x GC micro-ECD

ChirasilChirasil--DexDex ×× SupelcowaxSupelcowax--1010

8 PCBs chirals Except PCB 135

SalmonSalmon extractextract

Retention time in 1D (min)

Ret

entio

ntim

e in

2 D (s

)

0

6

1

2

3

4

5

50 60 70 80 90 120110100 130 150140 160

9591

136 176

174132

149

84

135,139

PCB 135PCB 135

L.R. Bordajandi, PhD Thesis, 2005

16 GC x GC micro-ECD

ChirasilChirasil--DexDex ×× SupelcowaxSupelcowax--1010

8 chiral PCBs Except PCB 135

SalmonSalmon extractextract

Retention time in 1D (min)

Tim

e of

rete

ntio

nin

2 D (s

)

0

6

1

2

3

4

5

50 60 70 80 90 120110100 130 150140 160

9591

136 176

174132

149

84

135,139

Standard

135 149

Salmonextract

136

HeartHeart--cutcut MDGCMDGCDBDB--5 5 —— ChirasilChirasil--DexDex

L.R. Bordajandi, PhD Thesis, 2005

17 MDGC versus GCxGC

8 8 chiral chiral PCBs PCBs

DBDB--5 5 –– ChirasilChirasil--DexDex

HeartHeart--cutcut MDGCMDGC

4 injections (∼ 7 hours)ChirasilChirasil--DexDex × SupelcowaxSupelcowax--1010

GC GC ×× GCGC

6/7 6/7 chiral chiral PCBsPCBs1 injection (∼ 3 hours)

There is no restrictions in the Length of the column in the2nd dimension

It is limited to a very short 2D

Very powerful for target compoundse.g. enantiomers of chiral PCBs

Very powerful for the resolution of extremely complex mixtures, providing a general overview

Provides quantitative analysis Provides qualitative analysis at present

18 Chiral PCBs in real samples

EnantiomericEnantiomeric enrichment ofenrichment of chiralchiralPCBs in real samplesPCBs in real samples

19 Chiral PCBs in real samples

Enantiomeric Enantiomeric enrichmentenrichment ofof chiral chiral PCBsPCBs in real in real samplessamples

HeartHeart--cutcut MDGCMDGCGCxGCGCxGC--mmicroicro--ECDECD

84, 91, 95, 132, 84, 91, 95, 132, 135, 136, 149, 171, 135, 136, 149, 171,

174, 176, 181174, 176, 181

20042004SpainSpainDairy productsDairy products(milk, cheese, (milk, cheese, yoghurt)yoghurt)

HeartHeart--cutcut MDGCMDGCGCxGCGCxGC-- mmicroicro--ECDECD

84, 91, 95, 132, 84, 91, 95, 132, 135, 136, 149, 171, 135, 136, 149, 171,

174, 176, 181174, 176, 181

20052005SpainSpainHuman breast milkHuman breast milk

HearHear--cutcut MDGCMDGC//ECDECD84, 91,95,132, 135, 84, 91,95,132, 135, 136, 149, 174, 176136, 149, 174, 176

20022002DNPDNP, Spain, Spain3 predatory bird3 predatory birdspecies (eggs)species (eggs)

LC(LC(PYEPYE))--GC/GC/ECDECDHeartHeart--cutcut MDGCMDGC/MS/MS

84, 91,95,132, 135, 84, 91,95,132, 135, 136, 149, 174, 176136, 149, 174, 176

19991999Mediterranean SeaMediterranean Sea5 cetacean species5 cetacean species(liver and fat)(liver and fat)

LC(LC(PYEPYE))--GC/GC/ECDECD84, 91,95,132, 135, 84, 91,95,132, 135, 136, 149, 174, 176136, 149, 174, 176

19961996DNPDNP, Spain, SpainOttersOtters(liver and fat)(liver and fat)

HeartHeart--cutcut MDGCMDGC/MS/MS95, 132, 14995, 132, 14919199966Atlantic oceanAtlantic oceanSharks Sharks (liver)(liver)

MultidimensionalMultidimensionalTechnique Technique

PCBsPCBsYear of Year of publpubl..

OriginOriginSpeciesSpecies

20 Chiral PCBs in real samples

ENANTIOMERICENANTIOMERIC FRACTION (FRACTION (EFEF))Range between 0 and 1Range between 0 and 1

EFEF ≈≈ 0.5 0.5 racemic racemic

EFEF > 0.5 > 0.5 excess 1excess 1stst enantienantioomermer

EFEF < 0.5 < 0.5 excess 2excess 2ndnd enantienantioomermer

EFEF = = Area 1Area 1stst enantienantioomermer

ÁreaÁrea 22ndnd

enantienantioomermerArea 1Area 1stst

enantienantioomermer +

2 nd

Only EF values out of the range of EF ± SD of PCB standards were considered as non-racemic

Quality control

1st

21 Aquatic ecosystems

0

0,5

1

PCB 95 PCB 132 PCB 149

SHARK SPECIESSHARK SPECIES

EF < 0.5PCB 132EF ≈≈ 0.5PCBs 95, 149

G.P.Blanch, R. Serrano, A. Glausch, V.Schurig and M.J. González. J Sep Sci, 1996

5 CETACEAN SPECIES5 CETACEAN SPECIES

0

0,5

1

PCB84

PCB91

PCB95

PCB132

PCB135

PCB136

PCB149

PCB174

PCB176

EF

EF < 0.5PCBs 84, 91, 132, 135 , 149

EF ≈≈ 0.5PCBs 95, 136, 174, 176

S. Reich, B. Jiménez, L. Marsilli, L.M. Hernández, V. Schurig and M.J. González. Env Sci Technol, 1999

22 Terrestrial ecosystems

0,00

0,50

1,00

PCB45

PCB84

PCB91

PCB95

PCB132

PCB135

PCB136

PCB149

PCB174

PCB176

EF

3 PREDATORY BIRD SPECIES3 PREDATORY BIRD SPECIES

EF > 0.5 PCB 45, 84, 95, 132EF < 0.5 PCB 135, 174, 176EF ≈≈ 0.5 PCB 91, 149

B. Gómara and M.J. González. Chemosphere, 2006

0,00

0,50

1,00

PCB84

PCB91

PCB95

PCB132

PCB135

PCB136

PCB149

PCB174

PCB176

EF

OTTER SPECIES

EF < 0.5 PCB 132, 135, 174, 175

EF ≈≈ 0.5 PCB 84, 91, 95, 136, 149

L. Ramos, B. Jiménez, M. Fernández, L. Hernández and M.J. González. Organohalogen Comp 26, 1996

23 Human breast milk

Enantiomeric Enantiomeric enrichment

PCBs 91, 95 y 149 EF EF ≈≈ 0.5 racemic mixture0.5 racemic mixtureEF ≈ EF ≈ ≠≠ 0.5 depending of the sample0.5 depending of the samplePCBs 84 y 174EF < 0.5 EF < 0.5 exceesexcees ofof the the 22ndnd enantenant..PCBs 132, 135, 171, 176 y 183

enrichment

PCB 171 and 183 high enantiomeric enrichment of the 2PCB 171 and 183 high enantiomeric enrichment of the 2ndnd enantiomerenantiomer

100 110 120 130 Min

Standard

PCB 183 PCB 171

Breast milk

L.R. Bordajandi and M.J. González. Organohalogen Comp 67, 2005

24 Enantioselectve degradation

EnantioselectiveEnantioselective transformation oftransformation of chiralchiral PCBs in PCBs in living organismsliving organisms

The species could influence theThe species could influence the enantioselectiveenantioselectivedegradation ofdegradation of chiralchiral PCBsPCBs

The mechanisms involved are still unknownThe mechanisms involved are still unknown

PCBs could already arrive to the living organisms with PCBs could already arrive to the living organisms with a nona non--racemicracemic composition, ifcomposition, if enantioselectiveenantioselectivetransformations have previously occurredtransformations have previously occurred

25 Milk and dairy products

Dairy products:

Milk , cheese Milk , cheese && yogurtyogurt from three species: cow, goat from three species: cow, goat & & sheepsheep

PCBs 135, 136, 176 EF < 0.5 EF < 0.5 exceesexcees ofof thethe 22ndnd enantiomerenantiomer

PCBs 84, 91, 95, 132, 149, 174 EF = 0.5 EF = 0.5 racemicracemic mixture mixture

Enantiomeric Enantiomeric enrichmentenrichment

PCBsPCBs 171 171 andand 183 183 HighHigh deviationdeviation fromfrom thethe racemicracemic compositioncomposition in in somesome samples samples

DifferencesDifferences amongamong speciesspecies andand dairydairy productsproducts

L.R. Bordajandi, L. Ramos and M.J. González. Organohalogen Comp, 66, 2004

Milk and dairy products26

PCB 183PCB 183

0,00

0,50

1,00

0,00

0,50

1,00

0,00

0,50

1,00

EF

EF

EF

Cow

Goat

Sheep

milk cheese yoghurt

Milk and dairy products27

PCB 183PCB 183

0,00

0,50

1,00

0,00

0,50

1,00

0,00

0,50

1,00

EF

EF

EF

Cow

Goat

Sheep

milk cheese yoghurt

28 EnantioselectiveEnantioselective transformation oftransformation of chiralchiral PCBsPCBs

TheThe enantiomericenantiomeric enrichmentenrichmentofof PCBs 171 and 183 foundPCBs 171 and 183 foundin humans could be due to in humans could be due to the uptake of nonthe uptake of non--racemicracemic

PCBs through the dietPCBs through the diet

Conclusions29

Conclusions1. Heart-cut MDGC and GC x GC are suitable to separate PCB

enantiomers in real samples.

2. The differences found between species in the enantiomeric enrichment of chiral PCBs could be due to both,

The species capacity to degraded PCBs

The non-racemic PCB uptake through the diet

More research and data are needed to drawmore reliable conclusions

30 AcknowledgementsAcknowledgements

DoñanaDoñana National Park (National Park (CSICCSIC), La Laguna University (Spain) and ), La Laguna University (Spain) and Centro Centro StudiStudi CetaceiCetacei (Italy)(Italy)for sampling collectionfor sampling collection

CSICCSIC, CM,, CM, INIAINIA,, CICYTCICYT (Spanish institutions)(Spanish institutions)for financial supportfor financial support

Prof. V. Prof. V. SchürigSchürig ((TübingenTübingen University,Germany)University,Germany)for kindly providing for kindly providing chirasilchirasil--DexDex columnscolumns

My working group atMy working group at CSICCSIC and other collaborators and other collaborators L.R. L.R. Bordajandi Bordajandi L. RamosL. RamosB. B. Gómara Gómara B. B. JiménezJiménezR. SerranoR. Serrano O. RamosO. RamosG. BlanchG. Blanch S. ReichS. Reich