Chemosphere,Vol.23, No.2, pp 193-198, 1991 0045-6535/91 $3.00 + 0.

00
Printed in Great Britain Pergamon Press plc

A S I M P L E METHOD FOR S Y N T H E S I S ~ND ISOIA%TXON OF S I N G L E PCB

CONGENERS SHOWN BY THE EX/%MPLE OF PCB 132, ~ POSSIBLE INTERFERENCE OF
PCB 1 5 3 I%ND PCB 105

Walter Vetter I, Bernd Luckas 2, and Wolfgang Haubold I

1 Universit~t Hohenheim, Institut f~r Chemie,

Garbenstr.30, 7000 Stuttgart 70

Universit~t Hohenheim, Institut fur Lebensmittelchemie,

Pfaffenwaldring 55, 7000 Stuttgart 80

ABSTRACT

PCB 132 was prepared by Cadogan coupling [i] add isolated by semi-preparative
RP-HPLC in purity larger than 98%.
The GC separation of the PCB 153, 132, and 105 on different silica fused
columns (Ultra-2, HP-5, OV-1701, OV-210, OV-225) was discussed.

INTRODUCTION

After the determination of PCBs in environmental samples in 1966 [2], a lot of

investigations have been undertaken for an exact analysis of PCB residues.
Introduction of fused silica capillary columns was an important step in
improvement of the GC separation of technical PCB mixtures and environmental
loads.
Especially the SE-54 column, a slight polar silicone phase, is widely used as
chromatographic column in analysis of organochlorine residues.
However, not all PCB congeners can be separated by application of this GC-
column [i][3].
Investigations using multidimensional GC and mass spectrometry showed that the
main congeners in PCB mixtures of higher chlorine contents, PCB 153, PCB 138,
and PCB i01, which are often used as an integrated part of national
regulations in regards of PCB contamination [4][5], may be interfered by other
PCB congeners on a SE-54 column [6-10].

193
194

Problems may arise since some of the disturbing PCB congeners are not
commercially available. Therefore, a method for preparation of congeners of
analytical interest in high purity must be established.
Most ways to synthesize single PCBs lead to mixtures of two or three PCB
congeners besides other products [ii].
The isolation of PCB congeners of interest, obtained after Cadogan coupling,
was previously done by repeated thin-layer chromatography and recristallation
[i][ii], but in these cases purity larger than 96% became difficult. Using the
described semi-preparative RP-HPLC for the isolation of PCB 132 results in a
purity larger than 99%.

PREPARATION

PCB 132 was formed via Cadogan coupling [i] with small deviations.
2,0g (10mmol) of 2,3,4-trichloroaniline were solved in 13,6g (75mmol) of
1,2,4-trichlorobenzene. After heating (130C), 2,3g (20mmol) of
isopentylnitrite in 15 ml of 1,2,4-trichlorobenzene was added to the solution
during lh.
After stirring (1300C, 2Oh) the crude product was separated by fractionated
destillation at 10 -2 Torr.
An orange viscious liquid was collected at 93-II0OC boiling point (oil
temperature: 250C). The fraction contained the three hexachlorobiphenyls PCB
138, PCB 132, and PCB 130 in almost equal amounts (Fig.l).
The three congeners (dilution 1:20 in acetonitrile) were separated by
isocratic RP-HPLC on a semi-preparative 7-Cl8-column with acetonitrile/water
(86/14 v/v) as the eluent and UV detection (Tab. l).

a) b)

9
Fig. 1 ECD-Chromatogram (HP-5) Fla.2 HPLC-Chromatogram (see Tab.2)
of diluted orange oil of orange oil (dilution 1:20)
(see Tab.2) a) first run; b) second run
 195

The HPLC system can be calibrated with PCB 118, which elutes between PCB 132
and PCB 130/138 [12]. PCB 132 eluted after R t. 11min, while PCB 130 and PCB
138 co-eluted at R t. 13 min (Fig.2a).
The collected PCB 132 containing fractions (120ml) from the semi-preparative
HPLC were extracted four times with 20 ml of n-hexane. The HPLC procedure was
repeated to obtain PCB 132 in high purity (Fig.2b). Finally, we resulted in
140mg of colourless solid PCB 132.
The characterization of the compound was done by 250 MHz-IH-NMR [ppm, D 6-
Acetone: 7.78 (H-6, d, J=8.3Hz), 7.75 (H-5', d, J=8.6Hz), 7.63 (H-4', d,
J=8.6Hz), 7.42 (H-5, d, J=8.3Hz)].

Tab. 1 HPLC - system Tab.2 GC - system

HPLC system: GC system:

pump: Bischoff Analysentechnik, oven: Hewlett Packard 5890

Model Nr. 2200 injection: 220C (splitless)
detector: UV, Jasco Uvidec-100-1I detection: 300C (ECD)
injector: rheodyne 7125 loop carrier gas: ECD-N 2
with 200 ~i sample
Temperature Drouram for GC-Dhases:
HPLC conditions for PCB separation:

column: 250/%/10-nucleosil 7-C18 Fig. 1,3,4 180C, 4min; 6C/min;

Macherey-Nagel, No.715002 210C, 4min; 4"C/min;
length: 25cm, diam.: 10mm 260C, 25min.
(semi-preparative column)
Fig. 5 100C, 2min; 6C/min;
eluent: CH3CN/H20 (86/14 v/v) 240C, 25min.

flow: 5,0 ml/min Fig. 6 180C, 4min; 6C/min;

210C, 4min; 4"C/min;
detector: UV, 254 nm 240C, 25min.

DISCUSSION

Duinker et al. reported, that PCB 153 may be interfered by PCB 132 and PCB 105
on a middle wide 25m SE-54 column with an isocratic temperature program
[63[7].
Using a 50m SE-54 column and multistep temperature program (Tab.2), the
separation of PCB 132 from PCB 153 becomes less problematic [10][13].
Additionally, Ballschmiter et al. reported differences in separation with the
same silicon phase fabricated by different manufacturers [14]. This may also
be originated in differences of the coating of the phases, since some columns
which are widely known under the same name differ in the percentual
constitution (see Tab.3).
196

Using a 50m HP-5 c o l u m n w i t h ID 0,32mm and N 2 as the c a r r i e r gas, we did not

succeed in s e p a r a t i o n of PCB 132 and 105 (Fig.3a), w h i l e by u s e of a 50m
Ultra-2 c o l u m n w i t h ID 0 , 2 m m and He as c a r r i e r gas, it was p o s s i b l e to p a r t l y
r e s o l v e PCB 132 and 105 (Fig.3b). Therefore, we p r o p o s e to use the s e p a r a t i o n
of PCB 105 and PCB 132 to c o n t r o l the e f f i c i e n c y of the c h r o m a t o g r a p h i c system
applied for PCB d e t e r m i n a t i o n .
Recently, n o n - o r t h o and m o n o - o r t h o c o n g e n e r s w e r e found to be the m o s t toxic
PCB c o m p o n e n t s in t e c h n i c a l PCB m i x t u r e s and PCB 105 (a m o n o - o r t h o PCB
congener) b e c a m e m o r e i n t e r e s t i n g b e c a u s e of its e x p e c t e d toxicity.

a) HP-5 b) U l t r a - 2

~.~

Fig.3 E C D - C h r o m a t o g r a m on 50m SE-54 Fig.4 E C D - C h r o m a t o g r a m on O V - 1 7 0 1

a) ID: 0,32mm, film: 0 , 5 2 ~ m (HP) 50m, ID: 0,32mm, Film: O , 5 1 ~ m
b) ID: 0,2mm, film: 0 , 3 3 ~ m (HP) (Macherey-Nagel) (see Tab. 2)

L_ J

Fiq.5 E C D - c h r o m a t o g r a m on O V - 2 1 0 Fiq.6 E C D - C h r o m a t o g r a m on O V - 2 2 5
30m, ID: 0,32mm, film: 0,25~m 50m, ID: 0,32mm, film: 0,25~m
(Pierce) (see Tab.2) (Macherey-Nagel) (see Tab.2)
 197

However, it must be proven that neither PCB 105 nor PCB 153 co-elute with PCB
132.
Duinker et al. separated the congeners by multidimensional GC with a SE-54
column and an OV-210 phase (see Fig.5) as the second column [6].
We were able to resolve all three components with an OV-1701 phase (Fig.4),
but using this phase PCB 105 may be interfered by PCB 141 [13]. Therefore, we
propose the application of an OV-225 phase (Fig.6), according to Larsen and
Riego [10], which expected that problems in separation of PCB congeners on a
SE-54 column should be solved by using more polar phases.

Tab.3 Phases, coatinas and the effiencv in separation of PCB 153. 132. 105

phase coated with comparable phases Efficency in

(besides methyl) separation

SE-54" 5% phenyl CP-Sil-8, DB-5, HP-5 153, 105/132

Ultra-2, SE-52, OV-73

OV-1701** 7% phenyl, i% vinyl, CP-SiI-19, DB-1701, 153, 132,

7% cyanopropyl BP-10, SFB-7, 105(141)

OV-210 50% trifluoropropyl QF-I, SP-2401, 153, 132, 105

RSL-400, sufficient

0V-225 25% phenyl, CP-Sil-43, DB-225, 153, 132, 105

25% cyanopropyl BP-15, XE-60, CS-5 very good

* sometimes reported as 94% methyl, 5% phenyl, 1% vinyl

** sometimes reported as 86% methyl, 7% phenyl, 7% cyanopropyl

In conclusion, we propose the application of an efficient SE-54 column as

standard column to determine PCB 153 without interferences from PCB 105 and
PCB 132. The OV-1701 phase is suitable as a second column besides the SE-54
column in PCB analyses with regard to food control regulations [16].
The application of the more polar columns OV-210 and OV-225 makes it possible
to separate PCB 105 and PCB 132 (Fig. 5 & 6).
Especially the OV-225 column can be an interesting addition to apolar phases,
since the elution sequence on this phase is very different from an apolar
phase (e.g. 8-HCH appears behind p,p'-DDE; p,p'-DDD appears behind PCB 138 in
the chromatograms). The best resolution of the three investigated PCB
congeners 153, 132, and 105 was obtained on this phase.
198

REFERENCES

[1] Mullin, D.M, Pochini, C.M., McCrindle, S., Romkes, M., Safe, S.H., Safe,
L.M. (1984) High-resolution PCB analysis: Synthesis and Chromatographic
Properties of All 209 PCB Congeners. Environ. Sci.Technol. 1 8 : 4 6 8 - 4 7 6

[2] Jensen, S. (1966) Report of a new chemical hazard. New Sci. 32: 621.

[3] Safe, S., Safe, L., Mullin, M. (1985) Polychlorinated Biphenyls:

Congener-Specific Analysis of a commercial Mixture and a Human Milk
Extract. J.Agric. Food Chem. 3 3 : 2 4 - 2 9

[4] Schadstoffh~chstmengen-VO, BGBI.I vom 23.M~rz 1988, Anlage 9 : 3

[S] Nederlandse Staatscourant 6 December 1984, 239

[6] Duinker, J.C., Schulz, D.E., Petrick, G. (1988) Multidimensional Gas

Chromatography with Electron Capture Detection for the Determination
of Toxic Congerners in Polychlorinated Biphenyl Mixtures.
Anal. Chem. 6 0 : 4 7 8 - 8 2

[7] Schulz, D.E., Petrick, G., Duinker, J.C. (1989) Complete Characterization
of Polychlorinated Biphenyl Congeners in Commercial Aroclor and Clophen
Mixtures by Multidimensional Gas Chromatography-Electron Capture
Detection. Environ. Sci.Technol. 2 3 : 8 5 2 - 8 5 9

[8] De Boer, J., Dao, Q.T. (1991) Interferences in the Determination of

2,4,5,2',5'-Pentachlorobiphenyl (CB101) in Environmental and Technical
Samples. Intern.J.Environ.Anal. Chem. 4 3 : 2 4 5 - 2 5 1

[9] Roos, A.H., Kienhuis, P.G.M., Traag, W.A., Tuinstra, L.G.M.Th. (1989)
Problems encountered in the Determinatio~ of 2,3,4,2',4',5' Hexachloro-
biphenyl (CB 138) in Environmental Samples.
Intern.J.Environ.Anal. Chem. 3 6 : 1 5 5 - 1 6 1

[i0] Larsen, B., Riego, J. (1990) Interference from 2,3,5,6-3',4'-Hexachloro-

biphenyl (CB 163) in the Determination of 2,3,4-2',4',5'-Hexachlorobi-
phenyl (CB 138) in Environmental and Technical Samples.
Intern.J.Environ.Anal.Chem. 40:59-68

[11] Hutzinger, 0., Safe, S., Zitko, V. (1971) Polychlorinated Biphenyls:

Synthesis of Some Individual Chlorobiphenyls.
Bull. Environ.Contam.Toxicol. 6:209-219

[12] Brodsky, J., Ballschmiter, K. (1989) Trennung polychlorierter Biphenyle

(PCB) dutch RP-FlUssig-Chromatographie. Fres. Z.Anal.Chem. 3 3 5 : 8 1 7 - 2 5

[13] De Boer, J., Dao, Q.T. (1989) The Analysis of Individual Chlorobiphenyl
Congeners in Fish Extracts on 0.15 mm i.d. Capillary Columns.
HRC & CC 1 2 : 7 5 5 - 7 5 9

[14] Ballschmiter, K., Sch~fer, W., and Buchert, H. (1987) Isomer-specific

identification of PCB congeners in technical mixtures and environmental
samples by HRGC-ECD and HRGC-MSD. Fres. Z.Anal.Chem. 3 2 6 : 2 5 3 - 2 5 7

[15] Safe, S., Yao, C., Davis, P. (1990) Development of Toxic Equivalency
F a ~ o r s for Polychlorinated Biphenyls (PCBs) Dioxin-90, September 10 th-
14 , Bayreuth (F.R.G.); Volume 2 : 5 5 - 5 9

[16] Beck, H., Mathar, W. (1985) Analysenverfahren zur Bestimmung yon ausge-
w~hlten Einzelkomponenten in Lebensmitteln. Bundesgesundheitsbl. 2 8 : 1 - 1 2

(Received in Germany 4 June 1991; accepted i July 1991)