Enantioselective separation of chiral PCBs by multidimensional gas chromatography techniques. Application to real samples. M.J.

Transcription

1 1 Enantioselective separation of chiral s by multidimensional gas chromatography techniques. Application to real samples L.R. Bordajandi,, L. Ramos, B. Gómara and M.J. González Depart. of I.A. & Environ. Chem.. IQOG (CSIC) Madrid, Spain

2 2 Introduction 209 s 78 s display axial chirality in their non-planar conformations Cl x 0 < x + y < 11 Cl y 19 s present stable enantiomers at room temperature 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 Non racemates Enantioselective transformations

3 3 Introduction Although the enantiomeric enrichment of chiral s 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 s is a rather difficult task

4 4 Introduction Until now, no single enantioselective GC column has been able to separate the 19 chiral s The separation into enantiomers of the 19 chiral congeners has been achieved by the use of capillary columns with different types of chiral stationary phases

5 5 Chiral stationary phases Permethyl β-cd Chirasil-Dex 2,3,6-tri tri-o-methyll β-cd chemicaly bonded to polysiloxane s 84, 91, 95, 132, 135, 136, 149, 174, 176 tert.- buthyldimethylsilyl β-cd BGB-176SE 20% 2,3-di di-o-methyl-6-o-tert.-buthyldimethylsilyl-β-cd s 45, 91, 95, 131, 136, 149, 176 diluted in SE-52 (5 % phenyl methylpolysiloxane) BGB % 2,3,6-tert tert.-butyldimethylsilyl-β-cd diluted in PS086 (15 % phenyl methylpolysiloxane) s 45, 84, 88, 91, 131, 132, 135, 144, 149, 171, 174, 175, 183

6 6 Multidimensional approaches A multidimensional approach is clearly needed when analysing real samples Coelutions problems: With other enantiomers and s With other chemical compounds present in the extract Off line LC /(PYE PYE)-GC Three different multidimensional approaches could be used GC multidimensional Heart-cut MDGC- ECD/MS GC GC-micro micro-ecd

7 7 Heart-cut MDGC Heart-cut MDGC Transference to the 2 D mv A+B+C P C B Min Injector Detector 1 Transfer line Detector 2 C 1 D A B 2 D Deans Valve

8 8 Heart-cut MDGC Selection of capillary columns for the 1 D and 2 D dimension 1 D 2 D (L = 30 m) (L = 30 m) DB-5 Apolar (5% phenyl polysiloxane) Chirasil-Dex s 91, 95, 132, 135, 136, 149, 174, 176 BGB-172 s 84, 135, 171, 183 BGB-176SE s 45, 91, 95, 136

9 9 Heart-cut MDGC mv s in DB-5 ( 1 D) Min mv ,5 33,0 33,5 34,0 Min Transference to Chirasil-Dex ( 2 D ) Min

10 10 Heart-cut MDGC DB-5 Chirasil-Dex s 91, 95, 132, 135, 136, 149, 174 y INJECTIONS Coelutions between enantiomers Chirasil-Dex 132 s 91, 95, 132, 174 s 149, Coelutions with other s Chirasil-Dex 110 Sheep s milk 176 Por lo tanto, para la ,0 77,5 80,0 82,5 85,0 87,5 Min L.R. Bordajandi, PhD Thesis, 2005

11 11 GC x GC micro-ecd (c) Chromatogram in 3D 1 D 2 D Loop modulator Tr in the n 2 D Chromatogram in 2D Tr in 1 D Modulator

12 12 GC x GC micro-ecd Selection of capillary columns for the 1 D and 2 D dimension 1 D 2 D Chirasil-Dex Supelcowax-10 (Polyethylene( glycol) BGB-172 Supelcowax-10 8 chiral s Except chiral s Except 91 BGB-176SE Supelcowax-10 6 chiral s

13 13 GC x GC micro-ecd Chirasil-Dex Supelcowax-10 8 chiral s, except s 6 Retention time in 2 D (s) , Retention time in 1 D (min) , 139

14 14 GC x GC micro-ecd Chirasil-Dex Supelcowax-10 8 chiral s Except s 12 coplanar s 7 indicator s Retention time in 2 D (s) Retention time in 1 D (min) L.R. Bordajandi, PhD Thesis, 2005

15 15 GC x GC micro-ecd Chirasil-Dex Supelcowax-10 Salmon extract 8 s chirals Except Retention time in 2 D (s) , Retention time in 1 D (min)

16 16 GC x GC micro-ecd Chirasil-Dex Supelcowax-10 8 chiral s Except 135 Salmon extract 6 Heart-cut MDGC DB-5 Chirasil-Dex Time of retention in 2 D (s) , Standard Salmon extract Retention time in 1 D (min) L.R. Bordajandi, PhD Thesis, 2005

17 17 MDGC versus GCxGC Heart-cut MDGC DB-5 Chirasil-Dex 4 injections ( 7 hours) 8 chiral s GC GC Chirasil-Dex Supelcowax-10 1 injection ( 3 hours) 6/7 chiral s There is no restrictions in the Length of the column in the 2 nd dimension Very powerful for target compounds e.g. enantiomers of chiral s It is limited to a very short 2 D Very powerful for the resolution of extremely complex mixtures, providing a general overview Provides quantitative analysis Provides qualitative analysis at present

18 18 Chiral s in real samples Enantiomeric enrichment of chiral s in real samples

19 19 Chiral s in real samples Enantiomeric enrichment of chiral s in real samples Species Origin Year of publ. s Multidimensional Technique Sharks (liver) Atlantic ocean , 132, 149 Heart-cut MDGC/MS Otters (liver and fat) DNP,, Spain , 91,95,132, 135, 136, 149, 174, 176 LC(PYE PYE)-GC/ GC/ECD 5 cetacean species (liver and fat) Mediterranean Sea , 91,95,132, 135, 136, 149, 174, 176 LC(PYE PYE)-GC/ GC/ECD Heart-cut MDGC/MS 3 predatory bird species (eggs) DNP,, Spain , 91,95,132, 135, 136, 149, 174, 176 Hear-cut MDGC/ECD Dairy products (milk, cheese, yoghurt) Spain , 91, 95, 132, 135, 136, 149, 171, 174, 176, 181 Heart-cut MDGC GCxGC-micro icro-ecd Human breast milk Spain , 91, 95, 132, 135, 136, 149, 171, 174, 176, 181 Heart-cut MDGC GCxGC- micro-ecd

20 20 Chiral s in real samples 1 st 2 nd ENANTIOMERIC FRACTION (EF( EF) Range between 0 and 1 EF = Area 1 st enantiomer Area 1 st enantiomer + Área 2 nd enantiomer Quality control EF 0.5 racemic EF > 0.5 excess 1 st enantiomer EF < 0.5 excess 2 nd enantiomer Only EF values out of the range of EF ± SD of standards were considered as non-racemic

21 21 Aquatic ecosystems 1 SHARK SPECIES 0, EF < EF 0.5 s 95, 149 G.P.Blanch, R. Serrano, A. Glausch, V.Schurig and M.J. González. J Sep Sci, CETACEAN SPECIES EF 1 0,5 EF < 0.5 s 84, 91, 132, 135, EF 0.5 s 95, 136, 174, 176 S. Reich, B. Jiménez, L. Marsilli, L.M. Hernández, V. Schurig and M.J. González. Env Sci Technol, 1999

22 22 Terrestrial ecosystems 1,00 EF 0,50 3 PREDATORY BIRD SPECIES EF > , 84, 95, 132 0, EF < , 174, 176 EF , 149 B. Gómara and M.J. González. Chemosphere, ,00 EF 0,50 OTTER SPECIES EF < , 135, 174, 175 0, EF , 91, 95, 136, 149 L. Ramos, B. Jiménez, M. Fernández, L. Hernández and M.J. González. Organohalogen Comp 26, 1996

23 23 Human breast milk Enantiomeric enrichment s 91, 95 y 149 s 84 y 174 s 132, 135, 171, 176 y 183 EF 0.5 racemic mixture EF 0.5 depending of the sample EF < 0.5 excees of the 2 nd enant. 171 and 183 high enantiomeric enrichment of the 2 nd enantiomer Standard Breast milk Min L.R. Bordajandi and M.J. González. Organohalogen Comp 67, 2005

24 24 Enantioselectve degradation Enantioselective transformation of chiral s in living organisms The species could influence the enantioselective degradation of chiral s The mechanisms involved are still unknown s could already arrive to the living organisms with a non-racemic composition, if enantioselective transformations have previously occurred

25 25 Milk and dairy products Dairy products: Milk, cheese & yogurt from three species: cow, goat & sheep Enantiomeric enrichment s 84, 91, 95, 132, 149, 174 EF = 0.5 racemic mixture s 135, 136, 176 EF < 0.5 excees of the 2 nd enantiomer s 171 and 183 High deviation from the racemic composition in some samples Differences among species and dairy products L.R. Bordajandi, L. Ramos and M.J. González. Organohalogen Comp, 66, 2004

26 26 Milk and dairy products 183 1,00 EF 0,50 Cow 0,00 1,00 EF 0,50 Goat 0,00 1,00 EF 0,50 Sheep 0,00 milk cheese yoghurt

27 27 Milk and dairy products 183 1,00 EF 0,50 Cow 0,00 1,00 EF 0,50 Goat 0,00 1,00 EF 0,50 Sheep 0,00 milk cheese yoghurt

28 28 Enantioselective transformation of chiral s The enantiomeric enrichment of s 171 and 183 found in humans could be due to the uptake of non-racemic s through the diet

29 29 Conclusions Conclusions 1. Heart-cut MDGC and GC x GC are suitable to separate enantiomers in real samples. 2. The differences found between species in the enantiomeric enrichment of chiral s could be due to both, The species capacity to degraded s The non-racemic uptake through the diet More research and data are needed to draw more reliable conclusions

30 30 Acknowledgements Doñana National Park (CSIC( CSIC), La Laguna University (Spain) and Centro Studi Cetacei (Italy) for sampling collection CSIC,, CM, INIA, CICYT (Spanish institutions) for financial support Prof. V. Schürig (Tübingen University,Germany) for kindly providing chirasil-dex columns My working group at CSIC and other collaborators L.R. Bordajandi L. Ramos B. Gómara B. Jiménez R. Serrano O. Ramos G. Blanch S. Reich