Chemosphere, Vol.28, No.l, pp 89-98, 1994 0045-6535]94 $6.00 + 0.00 Printed in Great Britain Pergamon Press Ltd.

ENANTIOMER SELECTIVE SEPARATION OF TOXAPHENE CONGENERS ISOLATED FROM

SEAL BLUBBER AND OBTAINED BY SYNTHESIS

Roland Kallenborn and Michael Oehme*

Norwegian Institute for Air Research, P.O. Box 64, N-2001 Lillestr~m, Norway

Walter Vetter

Institute of Food and Environment, University of Jena, D-07743 Jena, Germany

Harun Parlar

Department of Analytical Chemistry, University of Kassel, Heinrich-Plett-Str. 40, D-34132 Kassel, Germany

(Received in Germany 12 August 1993; accepted 7 September 1993)

ABSTRACT

Stationary phases based on dimethyl-t-butylsilylated heptakis(2,3,6-tri-O-methyl)-B-cyclodextrin diluted with dif- ferent polysiloxanes were developed for the enantiomer separation of chiral toxaphene congeners by high resolu- tion gas chromatography (HRGC). They allowed to determine the enantiomer ratio of an octa- and nonachloro congener isolated from seal blubber. The found ratios of 1,024 and 1,059 were very close to those of the racema- tes and indicate that enantiomer selective metabolisation is not significant in seals. Furthermore, the enantiomer separation of 11 racemic toxaphene congeners obtained by synthesis was also performed. Two of them had the same retention times as the isolated congeners on 3 or 5 stationary phases, respectively, and similar negative ion chemical ionisation mass spectra. First attempts are made to explain differences in the observed enantiomer separations by the steric structure of the congeners. In addition, general aspects and difficulties of enantiomer selective separations by HRGC are discussed.

INTRODUCTION

Several persistent polychlorinated insecticides are chiral and have been applied as racemates. Examples are com-

pounds of the chlordane group (e.g. cis- and trans-chlordane) [1] and ot-hexachlorocyclohexane (HCH) which is

part of the technical HCH-mixture [2, 3]. Recent progress in the separation of enantiomers by high resolution

gas chromatography (HRGC) made it possible to determine the enantiomer ratio of trace amounts in environ-

mental samples [3]. As a result it could be shown that the original 1:1 enantiomer ratio of the ct-HCH racemate

is altered in higher organisms due to enantiomer selective metabolisation and/or bioaccumulation processes. The

ot(+)-/et(-)-HCH ratio in seal blubber is about 2-3:1 [3, 4]. In brain or muscle tissue from common Eider ducks

89

90

and harbour seals a large excess ofot(+)-HCH is found [4, 5]; et(-)-HCH is obviously less efficiently accumulated

and/or more easily metabolised Corresponding changes of the enantiomer ratios were observed for different

chlordane compounds in marine organisms [ 1].

The determination of enantiomer ratios is important due to the following reasons: First, enantiomers may have

different toxic properties. Second, the less toxic and/or more easily metabolised enantiomer may cause antago-

nistic effects. Third, the change of the enantiomer ratio in biota shows that at least one enantiomer can be meta-

bolised to some degree. This is additional useful information about the biological persistency of the compound.

Bioaccumulation in the food chain is a complex process of uptake, degradation and transfer mechanisms which

normally is enantiomer selective. An enantiomer ratio in biota close to 1 (as found for the applied racemate) indi-

cates a very low degree of biodegradation since it is not very likely that the metabolisation rate of both

enantiomers is similar.

The two most abundant toxaphene congeners in both seal blubber and beluga whale blubber have recently been

isolated [6, 7]. Their structures have been elucidated as 2-exo,3-endo, 5 -exo,6-endo, 8, 8,10,10-octachlorobornane

(abbreviated as TOX8) and 2-exo,3-endo,5-exo,6-endo,8,8,9,10,10-nonachlorobornane (TOX9), respectively,

by different mass spectrometric techniques and by 500 Mhz high resolution nuclear magnetic resonance (NMR)

spectroscopy [7-9]. According to these structure proposals (see Figure I) both compounds are chiral. Therefore,

a separation of the isolated structures into their enantiomers is an additional confirmation that the structur

proposals are correct. Furthermore, the enantiomer ratio of the isolated congeners provides information about

their biodegradation ability in seals.

As already discussed by Buser et al [1], the separation of complex polychlorinated structures such as cis- or

trans-chlordane into their enantiomers is quite demanding. The tailoring of stationary phases which can undergo

optimal steric interactions with given enantiomers is still a highly empirical task. This work discusses the

separation of both isolated toxaphenes by different chiral heptakis(2,3,6-tri-O-methyl)-B-cyclodextrin derivatives

added to polysiloxanes. Furthermore, the results of the enantiomer ratio determination of the isolated TOX8 and

TOX9 congeners are presented.

Recently, Parlar et al. synthesised 11 of those toxaphene congeners which are mainly present in environmental

samples [ 10]. All compounds of which the exact structure has been elucidated, fulfil the criteria for chirality as

described for toxaphenes by Vetter [ 11 ]. The synthetic congeners are racemates. The enantiomer separation abi-

lity of the chosen stationary phases for toxaphene congeners was further tested with a mixture of all 11 synthetic

toxaphenes. In addition, the precision and reproducibility of the enantiomer ratio calculations were determined.

Furthermore, 3 to 5 different stationary phases as well as negative ion chemical ionisation mass spectrometry

were employed to evaluate if the two congeners isolated from seal blubber are among the 11 synthesised com-

pounds.

91

EXPERIMENTAL

Toxaphene congeners 2-exo,3-endo,5-exo,6-endo,8,8,10,10-octachlorobornane (TOX8) and 2-exo,3-endo,5-exo,6-endo,8,8,9,10,10-

nonachlorobomane (TOX9) were isolated from a harbour seal (Phoca vitulina) found dead at the coast close to

Kiel, Germany. The extraction procedure and the structure elucidation have been described in detail in [6, 8, 9].

Solutions of about 30 pg/IJ were prepared in n-hexane (Merck no. 4371, "zur Rtickstandsanalyse").

A mixture of 11 synthesised toxaphene congeners in n-hexane (abbreviated as 11TOX) was obtained from H.

Parlar. Details about the synthesis and structure elucidation are given in [10]. It contained 660 pg/p.l of each of

the following compounds (numbering and structure according to [10]): Octachlorobornane no. 7 (identified as

TOX8), 2,2,5-endo,6-exo,8,9,10-heptachlorobornane (no. 8), octachlorobornene no. 9 (structure still open), 2-

exo,3-endo,5-exo,6-endo,8,8,9,10,10-nonachlorobornane (no. 10, identical with TOX9), nonachlorobornane no.

I1 (structure still open), 2,2,5-endo, 6-exo,8,8,9,10,10-nonachlorobornane (no. 12), 2,2,5,5,8,8,9,10,10-nona-

chlorobornane (no. 13), 2-exo,3-exo,5-endo,6-exo,8,8,9,10,10-nonachlorobornane (no. 14), 2,2,3-exo,5-endo,6-

exo,8,8,9,10,10-decachlorobornane (no. 15), 2,2,5,5,6-exo,8,8,9,10,10-decachlorobornane (no. 16) and 2,2,3-

exo,5,5,8,8,9,10,10-decachlorobornane (no. 17). Further dilutions were made with n-hexane.

7

5

lo(R 1)

CI2HE~ H3

C [ ' ~ ~ . ~ H H / CHC[ 2 ~Ct

TOX8

C[2HC~ H2E[

C t ~ H H" EHEt 2 "EL

TOX9

Figure h Structure of the toxaphene congeners isolated from seal blubber and numbering of the bornane skeleton.

92

Enantiomer selective capillaries Capillary 1: A glass capillary of 12 m length and 0,32 mm i.d. was coated with a 3+1 (w/w) mixture of PS086

and permethylated heptakis(2,3,6-tri-O-methyl)-B-cyclodextrin (PMCD, film thickness: 0,3 ~m). The column

was originally developed for the enantiomer separation of a-HCH. Further details of its preparation are given in

[3].

Capillary 2: The surface of a glass capillary of 20 m length and 0,32 mm i.d. was leached with 20% HCI and

afterwards persilylated according to the procedures described by Blum [12] and Schmid [13]. The surface was

statically coated with a solution of 2,5%0 PS086 and 0,25%0 dimethyl-t-butylsilylated heptakis(2,3,6-tri-O-me-

thyl)-fl-cyclodextrin (DBSCD, prepared according to Blum and Aichholz [14]) in dichloromethane/pentane 1+1

(v/v). Methyltriethoxysilane was added as a crosslinking reagent corresponding to 0.1% of the stationary phase

amount. The resulting film thickness was about 0,15 lam. Further details are given in [3 ].

Capillary 3: Furthermore, a fused silica capillary of 20 m length and 0,25 mm i.d. was statically coated without

further pretreatment with a solution of 2,5%00V-1701 OH (Ohio Valley, Marietta, OH, USA) and 0,875%0 di-

methyl-t-butylsilylated heptakis(2,3,6-tri-O-methyl)-B-cyclodextrin corresponding to 35% (w/w) of the statio-

nary phase. The solvent was dichloromethane/pentane 1+1 (v/v). The calculated film thickness was 0,21 ~m.

Other employed capillaries In addition, the presence of TOX8 and TOX9 in the mixture of synthetic congeners was verified with the follo-

wing fused silica capillaries.

Capillary 4:25 m length x 0,2 mm i.d., coated with 0,11 I.tm 95%-methyl-5%-phenyl polysiloxane (Ultra 2, Hew-

lett-Packard).

Capillary 5:30 m length x 0,25 mm i.d., coated with 0.1 ~tm 90% biscyanopropyl + 10% phenyl cyanopropyl

polysiloxane (RTx2330 , Restek Corp., Bellefonte, PA, USA).

Instrumentation Gas chromatography combined with electron capture detection was carried out on a Carlo Erba 5300 Mega

series gas chromatograph equipped with an HT40 electron capture detector (ECD) and an EC 400 control unit.

Nitrogen at a flow rate of 35 ml/min was used as moderation gas for the ECD. Splitless injections of 1 p.l were

carried out at an injector temperature of 250°C and a detector temperature of 300°C. The chromatograms were

recorded by a Hewlett-Packard 3365 ChemStation. Compound identification was carried out on a Hewlett-

Packard (HP) 5987 mass spectrometer with an HP 5890 series II gas chromatograph and a HP 5988 GC/MS

interface. Negative ion chemical ionisation (NICI) was carried out at an ion source temperature of 200°C and an

ion source pressure of 0.5 bar using methane as reagent gas. The ion source was tuned for optimum performance

with PFTBA at m/z 312, 414 and 464. Mass spectra were recorded within a mass range of m/z 34-500 (scan

speed, 600 ainu/s). Helium was used as carrier gas at an flow rate of 35-45 cm/s for all separations.

Separation conditions Capillary 1 (PS086/PMCD): Splitless injection at 60°C, splitless/isothermal period 2 rain, 20°C/min to 150°C,

150-240°C at 3°C/rain, isothermal at 240°C.

93

Capillary 2 (PS086/DBSCD): Splitless injection at 120°C, splitless~isothermal period 2 min, 5°C/min to 170°C,

20 min isothermal, 170-200°C at 3°C/min, 20 min isothermal, 200-220°C/min at 10°C/min, isothermal at 220°C

for 20 min.

Capillary 3 (OV-1701/DBSCD): Splitless injection at 60°C, splitless/isothermal period 2 min, 25°C/min to

200°C, 50 min isothermal, 200-210°C at 3°C/min, 10 min isothermal, 210-220°C/min at 10°C/min, isothermal at

220°C for 20 min.

Capillary 4 (Ultra 2): Splitless injection at 90°C, splitless/isothermal period for 2 min, then 30°C/min to 150°C,

and 4°C/min to 260°C, isothermal at 260°C for 10 min.

Capillary_ 5 (RT.x2330): Splitless injection at 90°C, splitless/isothermal period for 2 min, then 30°C/min to 160°C,

and 3°C/min to 260°C, isothermal at 260°C for 10 min.

RESULTS AND DISCUSSION

Enantiomer separation by HRGC is still based on an empirical selection of the stationary phase. At present it is

not possible to predict the exact enantiomer selective behaviour of a stationary phase. According to our

experience the following factors are important when derivatised cyclodextrins are added to ordinary stationary

phases to achieve enantiomer selectivity.

The steric structure of such cyclodextrins does not only interact with enantiomers but causes also changes of the

polarity of the stationary phase. This might lead to co-elution of compounds which were well separated on the

not modified stationary phase. Furthermore, the interactions with the enantiomers are weak. A careful adaptation

of the sterie structure of the chiral modificator to that of the enantiomers is necessary to obtain as high Van der

Waal interactions as possible.

Since the strength of such forces decreases significantly with increasing temperature, enantiomer separations

have to be carried out at the lowest possible capillary temperature. Consequently, temperature programmes

applied for difficult enantiomer separations should start at low temperatures using slow programme rates. Fur-

thermore, the employment of longer isothermal periods in the beginning of the programme might be favourable.

This may result in long retention times. Therefore, capillaries with thin films should be used to diminish the capa-

city factors. However, this is contradictory to the experience that a reduction of the film thickness often leads to

a deterioration of the enantiomer separation ability. Furthermore, most enantiomer separations require a relative-

ly high plate number which limits the possibility to reduce retention times by employing shorter capillaries.

The stationary phase originally developed for the separation ofct(+)-HCH and ct(-)-HCH (PS086 + 30% perme-

thylated heptakis(2,3,6-tri-O-methyl)-13-cyclodextrin) did not show any enantiomer selectivity for the TOX8 or

TOX9 congener. This confirms that steric effects are important. The partly chlorinated bridge makes toxaphenes

more voluminous than HCH (see also Figure 1). The capillary coated with PS086 + 10% dimethyl-t-butylsilyla-

ted heptakis(2,3,6-tri-O-methyl)-13-cyclodextrin (DBSCD) was originally developed for the separation of cis- and

trans-chlordane enantiomers. These chlordanes have about the same steric size as toxaphenes due to the chlori-

nated carbon bridge atom and the additional ring system. Figure 2 shows that this stationary phase also is able to

separate TOX8 (R-value 0,89) and TOX9 (R-value 1,46) into enantiomers. A very careful optimisation of the

94

temperature programme was necessary applying a start temperature below 100°C and a long isothermal period at

200°C (see also comments before). For TOX9 an even better separation was obtained with the more polar

stationary phase OV-1701 to which 35 % of the chiral modificator DBSCD was added. The obtained resolution

R for TOX8 and TOX9 were 0,85 and 1,93, respectively.

Figure 2:

A T O X 8

/ 30 31

TOX9

46 48 50

T O X 8

37 38 39

t [m in i ' ' t [ rain] 32 33

T O X 9

t [ rain] t [m in i 54 55 56 57

ECD gas chromatogram of the enantiomer selective separation of the isolated TOX8 and TOX9

congeners on (A) PS086/DBSCD and (B) OV-1701/DBSCD.

Enantiomer ratios of TOX8 and TOX9 were determined by 10 successive injections on OV-1701 + 35%

DBSCD (see Table I). Taken the measuring error into account (about 2%) the enantiomer ratio of 1,024 found

for the TOX8 congener cannot be considered different from 1. For TOX9 the deviation of 0,059 from the

racemate ratio of 1 is larger than the standard deviation of the measurements (+ 0,033) indicating that a slight

shift might have occurred. However, the change is not statistically significant at a 95% confidence level. For

other chiral polychlorinated compounds such as ct-HCH and cis/trans-chlordane enantiomer ratios were found in

seal blubber which were significantly different from the racemate. The TOX8 and TOX9 congeners behave

obviously different. The nearly unchanged 1:1 ratio indicates that enantiomer selective biodegradation processes

are very slow or even missing in seals. This might also explain their high accumulation in seal blubber compared

to other polychlorinated insecticides.

95

Table I: Enantiomer ratio (signal l/signal 2) of TOX8 and TOX9 isolated from seal blubber and synthesised

TOX8. Results of successive injections on OV-1701/DBSCD are shown using electron capture detection.

Injection no. TOX8 TOX9

isolated synthesised

1 1,06 1,05 1,03

2 1,05 1,01 1,03

3 1,01 1,00 1,03

4 1,03 1,01 1,02

5 1,02 1,00 1,07

6 1,02 1,02 1,10

7 1,00 1,01 1,04

8 0,99 1,11

9 1,05 1,07

10 1,01 1,09

Average 1,024 1,010 1,059

s 0,023 0,017 0,033

Systematic deviations from the 1:1 ratio were occasionally observed for racemic ~x-HCH when detection me-

thods based on electron capture processes were used (electron capture detector or negative ion chemical ioni-

sation mass spectrometry). Then, the area of the first enantiomer signal was always larger than the second one.

An explanation can be a disturbance of the detector response due to the following reason: After detection of the

first enantiomer, the original electron population might not be completely re-established before the second enan-

tiomer elutes leading to a smaller signal area. A reason can be surface adsorption of some traces of the first elu-

ring enantiomer, since these deviations are often concentration dependent and are reduced at lower concentra-

tions. Furthermore, they are more pronounced after injections of real samples containing some matrix residues

[15]. Therefore, it is very important to verify enantiomer ratio determinations in biological samples by control

measurements with the racemate at the same concentration level.

Recently, Parlar and co-workers synthesised 11 toxaphene congeners including those which are most abundant in

marine organisms. Two of these 11 compounds had an identical NICI mass spectrum and within + 0,01 min the

same retention time as TOX8 on 5 (capillaries 1-5) and as TOX9 on 3 (capillaries 1, 4, 5) stationary phases of

different polarity. Therefore, it can be assumed that they are identical with the isolated congeners. TOX8 was

identified as no.7 (structure not identified in [10]) and TOX9 as no. 10 (same structure independently elucidated

in [10]) in the 11TOX mixture. Figure 3 shows the separation of this standard containing all 11 congeners on

OV-1701 + 35% DBSCD. The enantiomer ratio was 1,010 for the compound identified as TOX8 in the synthetic

mixture. The slight deviation of 1% from the 1:1 ratio is within the measuring error. This confirms that the deter-

mined enantiomer ratio of the isolated TOX8 is very close to 1 and that the observed increase to 1,02 cannot be

considered as significant.

96

Unfortunately, TOX9 co-elutes on both enantiomer selective stationary phases with another congener in the

synthetic standard mixture 11TOX. Enantiomer selective separations are more exposed to such interferences

due to the following reason. A complete enantiomer separation (R-values > 1,4) replaces single signals by pairs of

signals with retention time differences in the order of 1-2 min. For compounds with comparable retention time

differences this might cause co-elution of their enantiomers. As can be seen from Figure 3, this problem is al-

ready quite serious for the not very complex mixture 11TOX where two co-elutions could be observed. Also co-

elution of cis- and trans-Chlordane enantiomers has been reported [1]. An even larger number of interferences

between separated pairs of enantiomers might be possible for real sample extracts containing more congeners.

Then further fractionation steps will be necessary.

o0 :K 0

J

Ob x

+

L_.___J

20 30

E d I=

0 I--

40 t [rain] 50

Z"

d

0 d ' - c X v 0 0

x ©

6o 7o 8o 9o loo t [min]

Figure 3: Enantiomer selective separation of a mixture of the 11 synthetic congeners on OV-1701/DBSCD

using electron capture detection. Selected congeners and coeluting enantiomers (Int.) are marked.

97

Figure 3 shows further that the steric structure of the enantiomers has a strong influence on the achievable

separation. All nona- and decachlorocongeners with identified structures in the 11TOX mixture have a

8,8,9,10,10-chlorine substitution of the bridge and the methyl group at the bridge head. Of the 3 decachloro

compounds only one (2,2,3-exo,5-endo,6-exo,8,8,9,10,10-decachlorobornane) is completely separated into

enantiomers. The two others (2,2,5,5,6-exo,8,8,9,10,10-decachlorobornane and 2,2,3-exo,5,5,8,8,9,10,10-deca-

chlorobornane) are not or only slightly separated. Therefore, it can be assumed that the distribution of the

chlorine atoms in the ring is an important factor. There are some indications that the presence of the following

substructures is necessary for an enantiomer separation on the applied stationary phases:

1. Chlorine atoms must be present at the carbon atoms 2, 3, 5 and 6 of the ring.

2. Chlorine atoms must be present alternating in both exo- and endo positions.

Work is in progress to study the relation between structure and enantiomer separation in more detail using

selected toxaphene congeners and stationary phases.

CONCLUSIONS

Chiral toxaphene congeners can be separated into enantiomers by adding derivatised 13-cyclodextrins to common

stationary phases. The obtained separation is dependent on both the steric structure of the applied chiral modifi-

cator and the toxaphene congener. The applied stationary phases allowed to separate the two most abundant

congeners present in seal blubber into enantiomers. This confirms further that the elucidated chiral structures (see

Figure 1) are correct.

The determined enantiomer ratios of both the isolated octa- and nonachloro congener (1,024 and 1,059) were

not different from 1 at a 95% confidence level. The 1:1 ratio of the racemates in the applied technical toxaphene

is obviously not significantly changed in seals neither by bioaccumulation nor by metabilisation. The 1:1 enan-

tiomer ratio is a further proof of the high persistency of these toxaphene congeners in marine mammals.

The retention times of both TOX8 and TOX9 on 3-5 stationary phases of different polarities as well as their

NICI mass spectra were similar to those of two congeners recently synthesised by Parlar and co-workers.

ACKNOWLEDGEMENT

R. Kallenborn thanks for a DEM1NEX research fellowship. This work was part of the institute programme

"Organochlorines in the Arctic" which is financially supported by the Norwegian Ministry of Environment. The

help of Dr. M. D. M011er, Swiss Federal Research Station in preparing the enantioselective capillaries is greatly

acknowledged.

98

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[15] Oehme, M., not published results.