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Isolation and characterisation of polychlorinated biphenyl (PCB) atropisomers
Chmqkre,
PII: SO0456535(96)00131-2
ISOLATION AND CHARACTERISATION
Vol. 32, No. 11, pp. 2133-2140, 1996 Copyright 0 1996 Elsevia Scimce Ltd Printed in Great Britain. All rightsrrsaved 0045-6535/96 $15.W.o0
OF POLYCALORINATED
BH’HENYL (PCB) ATROPISOMERS
Peter Haglund
Institute of Environmental Chemistry, Umel University, S-90187 UmeL, Sweden, (Received in Gem-my 2 lmmry 1996; accepted 14 February 1996)
ABSTRACT
High-performance liquid chromatography on permethylated g-cyclodextrin derivatized silica was used to isolate enantiomers of stable atropisomeric polychlorinated biphenyls. The separations were performed in the reversed phase mode (85 % methanol) at subambient temperature (0 “C). Several injection/ fractionation cycles were performed for each atropisomer. In this way milligram quantities of pure (> 98%) enantiomers could be isolated. These enantiomers were characterised by polarimetry. The specific optical rotation seems to increase as the degree of chlorination decrease, e.g., PCB#196 (octachlorinated) and PCB#84 (pentachlorinated)
exhibit a
rotation of ca. 30 and 240 degree . ml t g-‘. dm”, respectively (h = 436 nm). Furthermore, the (-)-enantiomers always elute prior to the (+)-enantiomers. This implies that PCB atropisomers with similar spatial conformation have similar optical rotation properties.
Copyright
0 I996 Elsevier Science Ltd
Keywords
PCB, atropisomery, liquid chromatography, HPLC, enantioselective separation, polarimetry
INTRODUCTION
Polychlorinated biphenyls (PCBs) in which neither ring is symmetrically substituted may exist as atropisomers. Biphenyls with bulky substituents in the orrho positions sometimes are sufficiently stable to be isolated at room temperature. Three or four chlorine substituents in the ortho positions can sufficiently impede the rotation around the central o-bond such that the enantiomers can be isolated. Nineteen out of the 209 PCB congeners fulfrl the above criteria and are expected to exist as stable atropisomers at physiological temperatures (1). In Figure 1, the structure of a PCB atropisomer is given. 2133
2134
Figure
1:
Optical isomers of 2,2’,3,6-tetrachlorobiphenyl-
a PCB atropisomer
Enantiomeric pairs of PCB atropisomers have been successfully separated by both gas (2-5) and liquid chromatographic methods The high sample capacity of the liquid chromatographic techniques makes it possible to isolated large quantities of pure PCB atropisomers to be used as analytical reference standards, or as test substances in toxicological studies. Puttmann and co-workers used preparative high-performance liquid chromatography (HPLC) on triacetylcellulose to isolate mg quantities of PCB#88, PCB#139, and PCB#197 (IUPAC numbers (6,7) are used throughout the text) (8,9). These substances were carefully characterised (8) and were also subjected to various toxicological tests ( 10) In a recent publication we described how PCB atropisomers can be separated by reversed phase (RP) HPLC on permethylated P-cyclodextrin derivatized silica (PMCD) (11). Using this system, 13 PCB atropisomers could be completely resolved and 6 could be partially separated. Among the fully resolved atropisomers were nine tri- or tetra-ortho substituted PCBs, c.f. Figure 2 The atropisomers of these PCBs have, to our knowledge, never before been isolated in pure form
Figure 2:
84
131
132
135
174
175
176
196
136
Structural formulas of atroptiomeric PCBs which can be fully resolved by BP-HPLC on PMCD
2135 Consequently, the aim of the present study has been to isolate milligram quantities of pure enantiomers of the nine highly stable PCB atropisomers that can be resolved on PMCD. The optical rotation properties of these pure enantiomers should also be studied.
EXPERIMENTAL Chemicals Crystalline material of the individual PCB congeners were obtained from Larodan (Malmti, Sweden). The purity of the compounds was better than 98 %. Methanol of HPLC grade and 99.5 % ethanol of spectroscopic grade were obtained from LabScan (Dublin, Ireland) and Kemetyl (Stockholm, Sweden), respectively. Water was purified using a Milli-Q Plus apparatus (Millipore Corp., Bedford, m
USA).
HPLC separation The RP-HPLC were performed on a HP1050 liquid chromatographic system (Hewlett-Packard, Waldbronn, Germany) consisting of a quaternary pump, a helium degassing unit, an autosampler, and a variable wavelength UV-detector (set at h = 210 nm). A Hewlett-Packard ChemStation PC software was used for instrument control, data collection, and data analysis. Enantiomer separation was obtained by chromatography on two serially connected 4.6 x 250 mm Nucleodex P-PM columns (Macherey-Nagel, Diiren, Germany). These columns are filled with 5 pm Nucleosil 100 silica, which has been surface modified with covalently bond PMCD groups. A column heater/chiller, Model 7950, from Jones Chromatography (Hengoed, United Kingdom) was used to regulate the column temperature. The atropisomer separations were performed at optimum chromatographic conditions, that is: 85 % methanol at a flow rate of 0.40 ml / min and a column temperature of 0 “C (11). Aliquots (20 to 100 ~1) of saturated methanol solutions (1 to 5 PCB mg / ml) were injected and appropriate fractions were collected by a fraction collector. The injection/ fractionation process was repeated until mg quantities of each PCB enantiomer had been isolated. Corresponding fractions were pooled, and a small aliquot was subjected to HPLC analysis (using the same HPLC equipment and conditions) to check the purity of the isolated enantiomers After completion of the fractionation process the solvent was evaporated by rotary evaporation under reduced pressure and argon blow-down. The amount of atropisomeric PCBs isolated was determined gravimetrically.
Measurement of optical rotation The isolated atropisomers were dissolved in 1 ml of spectrometric grade ethanol (1 to 2 mg / ml) and the optical rotation of plane polarised light was measured using a Perkin-Elmer 141 electronic polarimeter (ijberlingen, Germany). The rotation was measured at three wavelengths, viz 578, 546, and 436 nm, and the specific optical rotation values were calculated.
2136
RESULTS AND DISCUSSION
HPLC separation
All atropisomeric PCBs studied was successfully separated Due to the relatively high peak resolution (R > 2 for all enantiomeric pairs) relatively large amounts of PCBs could be separated. In Figure 3 the effect of various column loads on resolution is illustrated. Normally, as much as 200 to 500 ug of material could be separated using these analytical sized (4.6 mm ID) PMCD columns In some cases, however, the limited solubility of the PCBs in methanol posed a problem For PCB# 196 only 1 mg / ml could be dissolved even if heat and ultrasonic treatment were applied Consequently, approximately 50 injections had to be done before all of the PCB#196 solution (ca. 5 mg) had been fractionated To increase the speed of the fractionation, the time between the injections was reduced to less than the retention time of the PCB atropisomers. Thus, the material from several injections was simultaneously separated on the chromatographic columns. The injection frequency was adjusted so that the time between two consecutive enantiomeric pan clusters became approximately five minutes, c.f Figure 4. In this way, the capacity of the system was increased to about 1 mg per hour. An alternative approach would be to use a preparative or semipreparative cohunn that has larger sample capacity, but no such column was available at our laboratory since the cost of these columns is very high. One problem that appears then the HPLC columns is overloaded is that the peak resolution deteriorates and the enantiomers start to co-elute. To maintain enantiomer purity a shaving technique was
used
The major
part of the first eluting enantiomer was collected in a first fraction (F I), the effluent was then directed to waste (w) during one minute, and finally the second eluting enantiomer was collected in a second fraction (F2), c f Figure 4. This procedure resulted in an enantiomer purity of > 98 %
2137
IllAlJ 00 60
40
20 0
loo0 ma&o400200.. 0 -
1200 -
800 -
400-
oa 20
Figure 3:
i‘li‘::
30
40
50 min
Chromatograms illustrating the effect of various sample load on the separation of the PCB atropisomers. The injected amounts are (from top to bottom): 5, 100, and 500 pg. The instrumentation and experimental conditions are given in the Experimental Section.
2138
i\ \
1 /F
30 _~_____
50
70
90
min
Chromatograms from the fractionation of an atropisomeric PCB (PCB#84) on PMCD. F1, F2,
Figure 4:
and W denote the first, second, and waste fractions, respectively. The instrumentation and experimental conditions are given in the Experimental Section.
Measurement
of optical
rotation
The results of the optical rotation measurements are shown in Table I. The specific optical rotations of corresponding fractions are of the same magnitude, but of opposite signs, indicating that the optical purity is equal for both of the fractions. These results support the outcome of the HPLC purity check
h
84
84
131
131
132
132
135
135
136
136
174
174
175
175
176
176
196
196
El
E2
El
E2
El
EZ
El
EZ
El
E2
El
E2
El
EZ
El
EZ
El
E2
578nm
-115
125
-52
60
-51
54
-21
17
-35
37
-15
14
-17
16
-33
31
-22
21
546 nm
-135
138
-63
63
-62
61
-22
18
-41
43
-21
20
-20
20
-39
35
-24
26
436nm
-242 241 -110 111 -111 110
-42
31
-70
73
-30
31
-34
34
-66
63
-29
30
Table I:
Specific optical rotation (degree 1ml * g -’. dm -‘) of the pure PCB atropisomers. El and E2 means first and second eluting enantiomer, respectively.
2139 The optical rotation seems to increase as the degree of chlorination decreases, e.g., the specific optical rotation for the octachlorinated PCB#196 and the pentachlorinated
PCB#84 is ca. 30 and 240 degree
??
ml 1 g -’1
dm -l, respectively, at h = 436 nm. Another interesting observation is that the (-)-enantiomer always elute prior to the (+)-enantiomer of the same congener. This might indicate that PCB atropisomers with similar spatial conformation also have the similar optical rotation properties. The specific optical rotation data reported here is of the same magnitude as the optical rotation data reported by Ptittmann et. al. (8) Their optical rotation data for PCB#88, PCB#139, and PCB#197 ranged between ca. 30 and 180 degree
??
ml I g -’ dm -’(h = 436 nm). ??
CONCLUSIONS
The PMCD columns proved very usetil as fractionation tools for atropisomeric PCBs. This is mainly due to the high peak resolution (R > 2) for the congeners studied. Multiple injection/ separation/ fractionation cycles are however necessary when mg quantities have to be isolated Then several separations are performed simultaneously the capacity of the system increased to 1 mg per hour. The optical rotation measurements indicate a relationship between the retention order of the enantiomers and the direction in which plane polarised light is rotated. Thus, it is probably possible to predict the optical rotation properties on the basis of retention data on atropisomeric PCBs. Since it is known that the optical rotation properties depend on the conformation of the enantiomers it is plausible that the absolute spatial conformation of the PCB enantiomers can be predicted as soon as some reference materials of known absolute conformation becomes available. The isolated atropisomers have a high (> 98%) enantiomeric purity. The compounds will therefore be very useful as analytical reference standards as well as test substances for toxicological studies aiming at enantioselective properties.
REFERENCES
1
K.L.E. Kaiser, Environ. Polut. 7, 93 (1974).
2
V. Schurig and A. Glausch, Naturwissenschaften
3
W.A. Kiinig, B. Gehrcke, T. Runge and C. Wolf, J. High Res. Chromatography
4
A. Glausch, G.J. Nicholson, M. Fluck and V. Schurig, J, High Res. Chromatography
5
I.H. Hardt, C. Wolf, B. Gehrcke, D H. Hochmuth, B. Pfaffenberger,
80,468 (1993). 16, 376
(1993).
17, 347 (1994)
H. Hiihnentiss and W.A. Kbnig,
J. High Res. Chromatogr. 17, 859 (1994). 6
K. Ballschmitter and M. zell, Frezenius Z. Anal. Chem 302, 20 (1980).
7
E. Schulte and R. Malisch, Frezenius Z. Anal. Chem 3 14, 545 (1983).
8
M. Piittmsnn, F. Oesch and L.W. Robertson, Chemosphere
15, 2061 (1986).
2140
9
M. Piittmann, A. Mannschreck, F. Oesch and L.W. Robertson, Biochem. Pharmacol. 38, 1345 (1989)
10
A. Msnnschreck, N. Pustet, L W Robertson, F. Oesch and M. Piittmann, Liebigs Ann. Chem. 2101
11
P. Haghmd, J. Chromatogr., In Press
(1985)
PII: SO0456535(96)00131-2
ISOLATION AND CHARACTERISATION
Vol. 32, No. 11, pp. 2133-2140, 1996 Copyright 0 1996 Elsevia Scimce Ltd Printed in Great Britain. All rightsrrsaved 0045-6535/96 $15.W.o0
OF POLYCALORINATED
BH’HENYL (PCB) ATROPISOMERS
Peter Haglund
Institute of Environmental Chemistry, Umel University, S-90187 UmeL, Sweden, (Received in Gem-my 2 lmmry 1996; accepted 14 February 1996)
ABSTRACT
High-performance liquid chromatography on permethylated g-cyclodextrin derivatized silica was used to isolate enantiomers of stable atropisomeric polychlorinated biphenyls. The separations were performed in the reversed phase mode (85 % methanol) at subambient temperature (0 “C). Several injection/ fractionation cycles were performed for each atropisomer. In this way milligram quantities of pure (> 98%) enantiomers could be isolated. These enantiomers were characterised by polarimetry. The specific optical rotation seems to increase as the degree of chlorination decrease, e.g., PCB#196 (octachlorinated) and PCB#84 (pentachlorinated)
exhibit a
rotation of ca. 30 and 240 degree . ml t g-‘. dm”, respectively (h = 436 nm). Furthermore, the (-)-enantiomers always elute prior to the (+)-enantiomers. This implies that PCB atropisomers with similar spatial conformation have similar optical rotation properties.
Copyright
0 I996 Elsevier Science Ltd
Keywords
PCB, atropisomery, liquid chromatography, HPLC, enantioselective separation, polarimetry
INTRODUCTION
Polychlorinated biphenyls (PCBs) in which neither ring is symmetrically substituted may exist as atropisomers. Biphenyls with bulky substituents in the orrho positions sometimes are sufficiently stable to be isolated at room temperature. Three or four chlorine substituents in the ortho positions can sufficiently impede the rotation around the central o-bond such that the enantiomers can be isolated. Nineteen out of the 209 PCB congeners fulfrl the above criteria and are expected to exist as stable atropisomers at physiological temperatures (1). In Figure 1, the structure of a PCB atropisomer is given. 2133
2134
Figure
1:
Optical isomers of 2,2’,3,6-tetrachlorobiphenyl-
a PCB atropisomer
Enantiomeric pairs of PCB atropisomers have been successfully separated by both gas (2-5) and liquid chromatographic methods The high sample capacity of the liquid chromatographic techniques makes it possible to isolated large quantities of pure PCB atropisomers to be used as analytical reference standards, or as test substances in toxicological studies. Puttmann and co-workers used preparative high-performance liquid chromatography (HPLC) on triacetylcellulose to isolate mg quantities of PCB#88, PCB#139, and PCB#197 (IUPAC numbers (6,7) are used throughout the text) (8,9). These substances were carefully characterised (8) and were also subjected to various toxicological tests ( 10) In a recent publication we described how PCB atropisomers can be separated by reversed phase (RP) HPLC on permethylated P-cyclodextrin derivatized silica (PMCD) (11). Using this system, 13 PCB atropisomers could be completely resolved and 6 could be partially separated. Among the fully resolved atropisomers were nine tri- or tetra-ortho substituted PCBs, c.f. Figure 2 The atropisomers of these PCBs have, to our knowledge, never before been isolated in pure form
Figure 2:
84
131
132
135
174
175
176
196
136
Structural formulas of atroptiomeric PCBs which can be fully resolved by BP-HPLC on PMCD
2135 Consequently, the aim of the present study has been to isolate milligram quantities of pure enantiomers of the nine highly stable PCB atropisomers that can be resolved on PMCD. The optical rotation properties of these pure enantiomers should also be studied.
EXPERIMENTAL Chemicals Crystalline material of the individual PCB congeners were obtained from Larodan (Malmti, Sweden). The purity of the compounds was better than 98 %. Methanol of HPLC grade and 99.5 % ethanol of spectroscopic grade were obtained from LabScan (Dublin, Ireland) and Kemetyl (Stockholm, Sweden), respectively. Water was purified using a Milli-Q Plus apparatus (Millipore Corp., Bedford, m
USA).
HPLC separation The RP-HPLC were performed on a HP1050 liquid chromatographic system (Hewlett-Packard, Waldbronn, Germany) consisting of a quaternary pump, a helium degassing unit, an autosampler, and a variable wavelength UV-detector (set at h = 210 nm). A Hewlett-Packard ChemStation PC software was used for instrument control, data collection, and data analysis. Enantiomer separation was obtained by chromatography on two serially connected 4.6 x 250 mm Nucleodex P-PM columns (Macherey-Nagel, Diiren, Germany). These columns are filled with 5 pm Nucleosil 100 silica, which has been surface modified with covalently bond PMCD groups. A column heater/chiller, Model 7950, from Jones Chromatography (Hengoed, United Kingdom) was used to regulate the column temperature. The atropisomer separations were performed at optimum chromatographic conditions, that is: 85 % methanol at a flow rate of 0.40 ml / min and a column temperature of 0 “C (11). Aliquots (20 to 100 ~1) of saturated methanol solutions (1 to 5 PCB mg / ml) were injected and appropriate fractions were collected by a fraction collector. The injection/ fractionation process was repeated until mg quantities of each PCB enantiomer had been isolated. Corresponding fractions were pooled, and a small aliquot was subjected to HPLC analysis (using the same HPLC equipment and conditions) to check the purity of the isolated enantiomers After completion of the fractionation process the solvent was evaporated by rotary evaporation under reduced pressure and argon blow-down. The amount of atropisomeric PCBs isolated was determined gravimetrically.
Measurement of optical rotation The isolated atropisomers were dissolved in 1 ml of spectrometric grade ethanol (1 to 2 mg / ml) and the optical rotation of plane polarised light was measured using a Perkin-Elmer 141 electronic polarimeter (ijberlingen, Germany). The rotation was measured at three wavelengths, viz 578, 546, and 436 nm, and the specific optical rotation values were calculated.
2136
RESULTS AND DISCUSSION
HPLC separation
All atropisomeric PCBs studied was successfully separated Due to the relatively high peak resolution (R > 2 for all enantiomeric pairs) relatively large amounts of PCBs could be separated. In Figure 3 the effect of various column loads on resolution is illustrated. Normally, as much as 200 to 500 ug of material could be separated using these analytical sized (4.6 mm ID) PMCD columns In some cases, however, the limited solubility of the PCBs in methanol posed a problem For PCB# 196 only 1 mg / ml could be dissolved even if heat and ultrasonic treatment were applied Consequently, approximately 50 injections had to be done before all of the PCB#196 solution (ca. 5 mg) had been fractionated To increase the speed of the fractionation, the time between the injections was reduced to less than the retention time of the PCB atropisomers. Thus, the material from several injections was simultaneously separated on the chromatographic columns. The injection frequency was adjusted so that the time between two consecutive enantiomeric pan clusters became approximately five minutes, c.f Figure 4. In this way, the capacity of the system was increased to about 1 mg per hour. An alternative approach would be to use a preparative or semipreparative cohunn that has larger sample capacity, but no such column was available at our laboratory since the cost of these columns is very high. One problem that appears then the HPLC columns is overloaded is that the peak resolution deteriorates and the enantiomers start to co-elute. To maintain enantiomer purity a shaving technique was
used
The major
part of the first eluting enantiomer was collected in a first fraction (F I), the effluent was then directed to waste (w) during one minute, and finally the second eluting enantiomer was collected in a second fraction (F2), c f Figure 4. This procedure resulted in an enantiomer purity of > 98 %
2137
IllAlJ 00 60
40
20 0
loo0 ma&o400200.. 0 -
1200 -
800 -
400-
oa 20
Figure 3:
i‘li‘::
30
40
50 min
Chromatograms illustrating the effect of various sample load on the separation of the PCB atropisomers. The injected amounts are (from top to bottom): 5, 100, and 500 pg. The instrumentation and experimental conditions are given in the Experimental Section.
2138
i\ \
1 /F
30 _~_____
50
70
90
min
Chromatograms from the fractionation of an atropisomeric PCB (PCB#84) on PMCD. F1, F2,
Figure 4:
and W denote the first, second, and waste fractions, respectively. The instrumentation and experimental conditions are given in the Experimental Section.
Measurement
of optical
rotation
The results of the optical rotation measurements are shown in Table I. The specific optical rotations of corresponding fractions are of the same magnitude, but of opposite signs, indicating that the optical purity is equal for both of the fractions. These results support the outcome of the HPLC purity check
h
84
84
131
131
132
132
135
135
136
136
174
174
175
175
176
176
196
196
El
E2
El
E2
El
EZ
El
EZ
El
E2
El
E2
El
EZ
El
EZ
El
E2
578nm
-115
125
-52
60
-51
54
-21
17
-35
37
-15
14
-17
16
-33
31
-22
21
546 nm
-135
138
-63
63
-62
61
-22
18
-41
43
-21
20
-20
20
-39
35
-24
26
436nm
-242 241 -110 111 -111 110
-42
31
-70
73
-30
31
-34
34
-66
63
-29
30
Table I:
Specific optical rotation (degree 1ml * g -’. dm -‘) of the pure PCB atropisomers. El and E2 means first and second eluting enantiomer, respectively.
2139 The optical rotation seems to increase as the degree of chlorination decreases, e.g., the specific optical rotation for the octachlorinated PCB#196 and the pentachlorinated
PCB#84 is ca. 30 and 240 degree
??
ml 1 g -’1
dm -l, respectively, at h = 436 nm. Another interesting observation is that the (-)-enantiomer always elute prior to the (+)-enantiomer of the same congener. This might indicate that PCB atropisomers with similar spatial conformation also have the similar optical rotation properties. The specific optical rotation data reported here is of the same magnitude as the optical rotation data reported by Ptittmann et. al. (8) Their optical rotation data for PCB#88, PCB#139, and PCB#197 ranged between ca. 30 and 180 degree
??
ml I g -’ dm -’(h = 436 nm). ??
CONCLUSIONS
The PMCD columns proved very usetil as fractionation tools for atropisomeric PCBs. This is mainly due to the high peak resolution (R > 2) for the congeners studied. Multiple injection/ separation/ fractionation cycles are however necessary when mg quantities have to be isolated Then several separations are performed simultaneously the capacity of the system increased to 1 mg per hour. The optical rotation measurements indicate a relationship between the retention order of the enantiomers and the direction in which plane polarised light is rotated. Thus, it is probably possible to predict the optical rotation properties on the basis of retention data on atropisomeric PCBs. Since it is known that the optical rotation properties depend on the conformation of the enantiomers it is plausible that the absolute spatial conformation of the PCB enantiomers can be predicted as soon as some reference materials of known absolute conformation becomes available. The isolated atropisomers have a high (> 98%) enantiomeric purity. The compounds will therefore be very useful as analytical reference standards as well as test substances for toxicological studies aiming at enantioselective properties.
REFERENCES
1
K.L.E. Kaiser, Environ. Polut. 7, 93 (1974).
2
V. Schurig and A. Glausch, Naturwissenschaften
3
W.A. Kiinig, B. Gehrcke, T. Runge and C. Wolf, J. High Res. Chromatography
4
A. Glausch, G.J. Nicholson, M. Fluck and V. Schurig, J, High Res. Chromatography
5
I.H. Hardt, C. Wolf, B. Gehrcke, D H. Hochmuth, B. Pfaffenberger,
80,468 (1993). 16, 376
(1993).
17, 347 (1994)
H. Hiihnentiss and W.A. Kbnig,
J. High Res. Chromatogr. 17, 859 (1994). 6
K. Ballschmitter and M. zell, Frezenius Z. Anal. Chem 302, 20 (1980).
7
E. Schulte and R. Malisch, Frezenius Z. Anal. Chem 3 14, 545 (1983).
8
M. Piittmsnn, F. Oesch and L.W. Robertson, Chemosphere
15, 2061 (1986).
2140
9
M. Piittmann, A. Mannschreck, F. Oesch and L.W. Robertson, Biochem. Pharmacol. 38, 1345 (1989)
10
A. Msnnschreck, N. Pustet, L W Robertson, F. Oesch and M. Piittmann, Liebigs Ann. Chem. 2101
11
P. Haghmd, J. Chromatogr., In Press
(1985)