- Email: [email protected]
Synthesis and identification of the 10 hexachlorodibenzo-p-dioxin isomers by high performance liquid and packed column gas chromatography
Chemosphere Vol. i0, pp 3 - 18 ~Per&~amon Press Ltd. 1981. Printed im Great Britain
OO45-6535/81/o10oo3-165o2.oo/o
SYNTHESIS AND IDENTIFICATION OF THE I0 HEXACHLORODIBENZO-P-DIOXIN ISOMERS BY HIGH PERFORMANCE LIQUID AND PACKED COLUMN GAS CHROMATOGRAPHY
L. L. Lamparski and T. J. Nestrick The Dow Chemical Company, Michigan Division Analytical Laboratories,Midland,
MI 48640 USA
INTRODUCTION
Many recent investigations of environmental particulate samples, especially those associated with effluent from combustion processes, have indicated the widespread presence of chlorinated dibenzo-p-dioxins
(CDDs) and chlorinated dibenzofurans (CDFs) (I-8).
two related compound classes are known to be extremely toxic.
Certain members of these
Toxicological investigations
involving CDDs have demonstrated a wide range of biological activities, and also indicate that this activity is highly dependent upon the number and ring position of chlorine atoms present in the molecule (9-14).
All evidence to date regarding the relative toxicity of the 75 possi-
ble CDDs, which range from mono- to octachlorinated species, indicate that peak toxicity is observed for 2,3,7,8-tetrachlorodibenzo-p-dioxin
(2378-TCDD) (9, 15-18).
Although certain
isomers of penta- and hexachlorodibenzo-p-dioxin
(PCDDs and HCDDs) have been found to be less
biologically active than 2378-TCDD (9-10, 19-20), several recent quantitative studies have indicated that such higher chlorinated CDDs are often present in environmental particulates at greater concentrations than TCDDs (1,5,6,8,21).
Because these findings are based upon a limited
number of sample analyses, their consequences with respect to the environment cannot be adequately interpreted.
In anticipation of the possible necessity of accomplishing isomer-specific HCDD analyses on future samples we have synthesized, identified, and most importantly isolated each of the I0 HCDD isomers.
Serving as standards these discrete HCDDs should permit better evaluation of the
isomer specificity of current analytical procedures, allow easier development of truly isomerspecific new procedures, and ultimately lead to a better understanding of the formation, distribution, and transport properties of CDDs in the environment.
Specific
combinations of trichlorophenol
(PCP) were r e a c t e d as t h e i r
(TCP), t e t r a c h l o r o p h e n o l
potassium salts
under controlled-flow
HCDDs.
This synthetic
related
HCDD m i x t u r e s v i a t h e S m i l e s r e a r r a n g e m e n t ( 2 2 - 2 4 ) .
(TeCP), and p e n t a c h l o r o p h e n o l pyrolysis
conditions
t o form
a p p r o a c h h a s b e e n d e s c r i b e d by o t h e r s and i s known t o p r o d u c e i s o m e r i c a l l y
demonstrated the separation
and i d e n t i f i c a t i o n
The work r e p o r t e d by Buser (22)
o f 8 HCDD i s o m e r s v i a c a p i l l a r y
column gas
chromatography-mass
spectrometry (GC-MS).
The remaining 2 HCDD isomers could not be identified
by this procedure nor were any discrete HCDD isomer standards isolated. of the multiple high performance liquid chromatographic
Using a modification
(HPLC) fractionation scheme reported by
us for the separation of the 22 TCDD isomers (25), the I0 HCDD isomers have been separated and isolated in low microgram quantities.
These compounds were characterized by packed column
GC-HS in the selected ion mode (SIH) and were identified as HCDDs via nominal mass and chlorine isotope pattern.
EXPERIMENTAL
Caution.
The described procedures permit the synthesis and isolation of all I0 HCDD isomers,
to include the possibly more toxic homologs 123678-HCDD and 123789-RCDD.
In certain cases the
crude pyrolyzates were found to contain other CDDs including 2378-TCDD.
Persons attempting to
prepare these substances should be experienced in the handling procedures for extremely toxic materials.
Appropriate precautions should be taken so as to minimize the chances of either
personal or environmental exposure to CDDs.
All waste materials, to include equipment washing
solvents, should be carefully packaged and isolated until destroyed by appropriate incineration techniques.
Reagents.
The following chlorophenols were obtained from Aldrich (Milwaukee, WI 53233) in the
best available purity and were used as received:
234-TCP (mp 77-79°C), 236-TCP (99~), 245-TCP
(mp 63-65°C), 246-TCP (98~), 2345-TeCP (98~), 2356-TeCP (mp I14-I16°C), and PCP (Gold Label @ 99+~).
All solvents used were distilled-in-glass quality obtained from Burdick and Jackson
Laboratories
(Huskegon, HI 49442).
Aqueous solutions were prepared using deionized water.
potassium hydroxide was Reagent Grade.
The
Purge gas for the pyrolysis reactor was pre-purified
nitrogen (Hatheson, 99.995~), further purified by passage through a General Electric "Go-Getter" before use.
Silica, 100/200-mesh Bio-Sil A (Bio-Rad Laboratories, Richmond, CA
94804), was used as a sorbent in the pyrolysis reactor.
It was thoroughly cleaned and activated
according to the previously described procedure (25).
HCDD Isomer Syntheses.
(See Table I)
The apparatus and procedure used to synthesize the I0
HCDD isomers is essentially identical to that previously described for TCDD isomers (25).
Hethanolic potassium chlorophenate solutions were prepared by dissolving a total of 0.15
mmol chlorophenol(s) in 1.0 mL of a solution containing 17 mg KOH/I.O mL methanol (0.30 mmol KOH = 2 fold molar excess).
For those cases involving mixed chlorophenates, equimolar portions
of each reactant were combined so as to achieve a total of 0.15 mmol. This solution was then transferred to the glass wool plug positioned in the controlled-flow pyrolysis reactor tube, the methanol solvent purged from the system, and the reaction conducted at 280°C for a period of %15 min.
After cooling the reactor tube to ambient temperature, HCDD products were removed
Table I
P o t a s s i u m C h l o r o p h e n a t e P y r o l y s e s and G e n e r a l i z e d HCDD p r o d u c t s by D i r e c t A d d i t i o n and S m i l e s R e a r r a n g e m e n t R e a c t i o n s .
Pyrolysis
K Chlorophenates
1.
Direct
+
Expected HCDD Products Smiles
~
+
HCDD Isomer SOu~lht
I. 123467-HCDD
H PCP
2.
234-TCP
+
123467-HCDD
--
,~
123467.HCDD
+
II. 123469-HCDD
H PCP
3.
236-TCP
÷
123469.HCDD
A
•
123467.HCDD
÷
III.
123476HCDD
HO PCP
4.
245-TCP
+
123478.HCDD
A
•
12347~HCDD
+
IV. 123468.HCDD
HO PCP
5.
246-TCP
-I-
6
•
2345.TeCP
÷
123468.HCDD
÷
VI. 123789.HCDD
123678.HCDD
.
2356.TeCP
7.
~
HO
2345,TeCP
123468.HCDD
123789.HCDD
÷ 2356.TeCP
-[-
,24669.c00
124679.HCDD
~
•
124689.HCDD
÷
X: 12367~HCDD
HO 2345.TeCP
2356-TeCP
123689.HCDD
123679-HCDD
by i n v e r t i n g t h e t u b e ( e n t r a n c e p o r t up) and e l u t i n g t h e s y s t e m w i t h 50 mL, f o l l o w e d by an additional The t o t a l
25 mL, o f m e t h y l e n e c h l o r i d e i n a f a s h i o n s i m i l a r t o a l i q u i d c h r o m a t o g r a p h i c column. e f f l u e n t was c o l l e c t e d i n a 100-mL b o t t l e
under a stream of s p e c i a l l y p u r i f i e d
nitrogen.
and t h e s o l v e n t removed by e v a p o r a t i o n
The r e s i d u e was r e d i s s o l v e d i n ~I0 mL o f hexane
t o which was t h e n added ~15-20 mL o f aqueous 1 H KOH. A f t e r v i g o r o u s manual s h a k i n g and l a y e r separation,
t h e hexane e x t r a c t was t r a n s f e r r e d
hexane e x t r a c t i o n s
were c o m p l e t e d and t h e e x t r a c t s
liminary qualitative from e a c h p y r o l y s i s
t o a c l e a n 25-mL g l a s s v i a l .
and q u a n t i t a t i v e reaction
hexane and an a l i q u o t
combined.
As a means o f p r o v i d i n g p r e -
i n f o r m a t i o n c o n c e r n i n g HCDD p r o d u c t s , t h e hexane e x t r a c t s
(Table I ) were a d j u s t e d t o a t o t a l
volume o f 20 mL w i t h a d d i t i o n a l
(~I-5~L) removed f o r e x a m i n a t i o n by c a p i l l a r y
with electron capture detection
Two a d d i t i o n a l 5 mL
column gas c h r o m a t o g r a p h y
(HRGC-EC) and packed column gas c h r o m a t o g r a p h y - l o w r e s o l u t i o n
mass spectrometry (GC-LRMS).
The hexane extracts were then evaporated to dryness under a
stream of specially purified nitrogen and the resulting white, crystalline residues were protected from light and stored dry until further processed.
Capillary Column Gas Chromatosraphy with Electron Capture Detection (HRGC-EC).
Each caustic
extracted pyrolyzate was analyzed qualitatively for the presence of chlorinated species other than HCDDs, and semi-quantitatively for a rough approximation of total HCDDs present, using a Varian 3700 Capillary gas chromatograph equipped with a 6ZNi pulsed electron capture detector. Instrumental calibration was accomplished by injection of an HCDD reference standard containing two different isomers.
An average response factor for HCDDs was computed from this standard
and was subsequently used to determine approximate HCDDs concentration in each pyrolyzate.
The
instrument was equipped with a 0.50 mm i.d. x 30 m-modified surface coated open tubular pyrex glass capillary column.
Chromosorb R-6470-I (1-4 pm) was used as the coated particulate support,
and following a specialized deactivation procedure was mercury-plug dynamic coated using a 15~ (w/v) solution of 60/40 (w/w) OV-17 silicone and Poly S-17g in degassed methylene chloride at a rate of ~2 cm/s.
Prepurified nitrogen was used as the carrier at a linear velocity of 17.5
cm/s, and also supplied as make-up gas to the detector at ~25 cc/min.
A specialized splitless
direct-injection coupling was constructed from pyrex glass tubing and packed with 0.60~ OV-17 silicone + 0.40~ Poly S-179 on 80/lO0-mesh Permabond @ Methyl Silicone (I0 cycle)(available from H~qJ Systems, Inc., Newton, HA 021641.
Operation of the injection port at 270°C permitted
quantitative injection of samples ranging from I to 3.5 pL in iso-octane without column deterioration.
The column was maintained at 280°C isothermal for all analyses and under these
conditions a 2.0 ~L injection of ~70 ppb (ng/mL) 123678-HCDD in isooctane resulted in a k' of 4.7 for this CDD at 30,000 effective theoretical plates. The detector was maintained at 320°C for all analyses.
Preliminary
Gas Chromatography-Low R e s o l u t i o n Mass S p e c t r o m e t r y
HCDDs q u a n t i t a t i o n
by HRGC-EC, t h e c o n c e n t r a t i o n
was a p p r o p r i a t e l y
adjusted
GC-LRHS o p e r a t i n g
i n t h e SIM mode m o n i t o r i n g m/e:
mixed HCDD r e f e r e n c e
for quantitative
of the caustic
HCDDs a n a l y s i s
6-chlorine
isotope pattern
pyrolyzate
388, 390, and 392.
ing conditions
were as f o l l o w s :
+ 0 . 4 0 ~ P o l y S-179 on 8 0 / 1 0 0 - m e s h Permabond O Methyl S i l i c o n e
temperature, jet
x 210 cm s i l y l a t e d
Newton, HA 0 2 1 6 4 ) ; column t e m p e r a t u r e ,
280°C o n - c o l u m n ; c a r r i e r
a t column t e m p e r a t u r e ,
e n e r g y , 70 eV.
glass;
A
separator,
calibra-
by t h e i r
GC-LRMS o p e r a t -
packing, 0.60~ (10 c y c l e )
260°C i s o t h e r m a l ;
g a s , h e l i u m a t 14 c c / m i n . ;
and e l e c t r o n
resolution.
HCDD i s o m e r s were i d e n t i f i e d
OV-I? S i l i c o n e
from HNU S y s t e m s , I n c . ,
aliquot
i s o m e r s was used f o r i n s t r u m e n t a l
m o n i t o r e d a t m/e:
column, 2 mm i . d .
extracted
F o l l o w i n g rough
u s i n g a H e w l e t t - P a c k a r d Model 5992A
388, 390, and 392 a t u n i t
s t a n d a r d c o n t a i n i n g two d i f f e r e n t
t i o n and c o m p u t a t i o n o f an a v e r a g e HCDD r e s p o n s e f a c t o r . characteristic
(GC-LRHS).
(available
injection
port
glass single-stage
Reverse Phase High Performance Liquid Chromatography (RP-HPLC).
The residue from each caustic
extracted pyrolyzate was dissolved in chloroform such that a I0 pL aliquot injected into the RP-HPLC system would contain ~2-4 pg total HCDDs.
This system was composed of a Perkin-Elmer
LC-65T liquid chromatographic column oven and variable wavelength UV detector operating under the following conditions:
column, two 6.2 x 250 mm Zorbax-ODS (Dupont Instruments Div.,
Wilmington, DE 19898) columns in series; isocratic eluent, 92.5/7.5 (v/v) acetonitrile/ water at 2.5 mL/min.; pump, Altex model IIOA; column temperature, 50°C; UV detector, 0.32 aufs at 235 nm; and injector, Rheodyne model 7120 with 50-~L sample loop.
Under these conditions an authen-
tic standard of 2378-TCDD demonstrated a retention time of 15.9 min.
During the course of each
pyrolyzate separation, fractions corresponding to observed peaks eluting between ~18 and 25 min. retention time were collected in 15-mL glass vials equipped with Poly-Seal ® caps containing %2 mL hexane.
HCDDs were recovered by dilution with %8 mL of 2% (w/v) aqueous sodium
bicarbonate and following phase separation the hexane layer was transferred to a clean 4-mL glass ~ial.
Two additional ~I mL hexane extractions were accomplished, the extracts combined,
and the solvent evaporated to dryness under a stream of specially purified nitrogen (RP-HPLC fraction).
Normal Phase Adsorption (Silica) High Performance Liquid Chromatography (Silica-HPLC).
Each
RP-HPLC fraction was redissolved in 2.0 mL of hexane and a 2.0 pL aliquot was examined by GC-LRMS under the previously described conditions to identify fractions containing HCDDs. Selected RP-HPLC fractions were then evaporated to dryness, redissolved in ~80 ~L of hexane, and quantitatively injected into the silica-HPLC system:
Laboratory Data Control model 1204
variable wavelength (IV detector, 0.I aufs at 235 nm; column, two 6.2 x 250 mm Zorbax-Sil (Dupont Instruments Div.) columns in series; isocratic eluent, hexane at 2.0 mL/min.; pump, Altex model IIOA; column temperature, ambient; injector, Rheodyne model 7120 with 100-~L sample loop. Before use these columns were dried according to the procedure of Bredeweg et al. (26).
Fractions
corresponding to each observed peak were collected in clean 4-mL glass vials, and the hexane solvent was then evaporated to dryness under a stream of specially purified nitrogen (Silica-HPLC fraction).
During the course of these fractionations an authentic standard of
2378-TCDD was periodically injected to provide accurate retention time data for assignment of HCDD retention times relative to 2378-TCDD.
Final for
HCDD I s o m e r A n a l y s e s . final
t h e GC-LRMS o p e r a t i n g standard
retention
were calculated.
characteristic
fractions previously
time reference
described
described.
with
excepting
fractions
isotope
the
with
retention
at m/e 398.
Native
times
column temperature
as an internal
for each native
HCDDs w e r e a g a i n
A s e c o n d 2 . 0 ~L a l i q u o t
2378-TCDD i n t o
~2 t o 4 mL o f h e x a n e
1 n g 1ZC-123478-HCDD i n t o
The IZC-123478-HCDD s e r v e d
pattern.
I00 pg native
were dissolved
was c o - i n j e c t e d
from which relative
I t was m o n i t o r e d
6-chlorine
was c o - i n j e c t e d
silica-HPLC
A 2 . 0 pL a l i q u o t
as previously
observed their
Selected
GC-LRMS e x a m i n a t i o n .
HCDD
identified
of the silica-HPLC
t h e HRGC-EC s y s t e m o p e r a t i n g
w h i c h was d e c r e a s e d
by
as
t o 265°C i s o t h e r m a l .
In this case 2378-TCDD functioned as the internal standard retention time reference for computation of HCDD isomer relative retention times.
After characterizing and isolating the I0 discrete
HCDD isomers, these standards were reexamined using the described RP-f[PLC system under conditions normally used for CDDs determination in sample residues (5).
Hence, the eluent was changed to
methanol at 2.0 mL/min., and absolute RP-HPLC retention times were obtained for each HCDD isomer.
CDD Standards.
The authentic standard of 2378-TCDD used in this work was prepared by W. W.
Muelder (Dow Chemical Co) and its properties have been described (25). The 13C-123478-HCDD was synthesized by A. S. Kende (Univ. of Rochester, Rochester, N-Y) and was shown to be 43 at.~ 13C-enriched.
The native HCDD reference standard containing two different isomers was prepared
and described by O. Aniline (23).
RESULTS AND DISCUSSION
Problems associated with macro-synthesis of isomerically pure CDD standards prompted our development of the multiple ¢hromatographies approach for CDDs separation.
In this context multiple
chromatographies refers to the consecutive use of different types of chromatography whose mechanisms for achieving separation involve significantly different molecular properties. definition, applicable forms of chromatography must permit reasonable sample~-~apacity,
By
relatively
constant component retention indices independent of sample matrix, and most importantly permit component recovery as an integral part of the separation process.
Our initial application of
multiple chromatographies permitted the separation, identification, and isolation of the 22 TCDD isomers from limited isomer mixtures synthesized via specific chlorophenate pyrolyses (25).
In this case RP-HPLC fractionation,
followed by normal phase silica-HPLC
refractionation, and finally packed column gas chromatographic separation were used sequentially. Although none of these chromatographies developed more than ~15,000 theoretical plates, their combined use accomplished the separation of all 22 TCDDs where Buser (28) has reported that capillary columns capable of 140,000 theoretical plates have failed.
Additional work in our
laboratory involving the application of these multiple chromatographies to CDDs determination in environmental particulate samples has shown that they permit acquisition of isomer-specific 2378-TCDD quantitative data even in samples intentionally fortified with equivalent amounts of the other 21TCDDs
(5). Hence, we undertook the investigation described in this paper for two
reasons, to provide discrete HCDD isomer standards for analytical purposes, and to demonstrate the applicability of multiple chromatographies for isolating all i0 HCDDs to include the two isomers not currently identified by HRGC means (22).
The information given in Table I indicates that a minimum of six specific chlorophenate pyrolyses can produce the 10 HCDD isomers assuming that direct addition and Smiles rearrange-
ment products are equally favorable routes.
However, in order to maximize the number of known
structural identities one additional pyrolysis (pyrolysis #I.) must be conducted.
Pyrolysis #I
permits synthetic structural identification of 123467-HCDD and 123469-HCDD which are both direct and Smiles rearranged related co-products of pyrolysis #2.
Buser has reported essentially
the same pyrolytic scheme outlined in Table I to produce the 10 HCDD isomers (22). Each of the HCDDs produced in pyrolyses #I through #5 could be discretely identified by Buser using HRGC. Pyrolyses #6 and #7, plus other pyrolyses and variety of reaction condition modifications, were reported to produce only two of the four remaining HCDD isomers when examined by HRGC (22). For this reason we have selected these pyrolyses to demonstrate our approach.
It is currently believed that pyrolytic chlorophenate condensation to CDDs occurs via the mechanisms shown in Figure I for 2356-TeCP and the mixed reaction involving 2345-TeCP and
[
2356'TeCP + 2356.TeCP CI CI
Cl
124679.HCDD
I
I
124689-HCDD I
CI
CI
CI
CI
Cl
Cl
CI
Cl
t
Cl
t
CI
CI
CI
Cl
Cl
Cl
1
123689.HCDD
Cl
Cl
I
CI
CI
Cl
Cl
I
123679.HCDD
CI
CI
c,- y CI 2345"TeCP + 2356.TeCP CI
CI "~~
--c,
"Cl
"0
Fig. I.
"o" y
CI
t
CI t
CI
CI c,
CI
Cl
0 c,
CI
CI c,
CI
0 c,
,
,
c,
c,
c , _Cl_ o c , _ _
Reaction pathways for the condensations of 2356-TeCP (upper) and 2345-TeCP + 2356-TeCP (lower).
Cl
I
IO
When multiple chromatographies are applied to t h e mixed chlorophenate pyrolysis of
2356-TeCP.
2345-TeCP and 2356-TeCP (pyrolysis #7), present in equimolar quantities, both of the expected HCDD isomer products are isolated.
In addition, both HCDD isomers associated with the self-
condensation of 2356-TeCP are observed and can be isolated in significant relative concentration (see Figure i). shown in Figure 2.
The HRGC-EC chromatogram for the caustic extracted crude pyrolyzate is
Preliminary GC-LRMS analysis of this material confirmed that peaks b, c,
and d were HCDDs (see Figure 3). Figure 4.
The RP-HPLC fractionation of this pyrolyzate appears in
Three fractions were collected and each was subjected to GC-LRMS examination to
determine the presence of HCDDs.
As indicated by the mass chromatograms shown in Figure 5,
RP-HPLC fractions #2 and #3 were found to contain the major HCDDs previously observed in the crude pyrolyzate.
These fractions were then refractionated by silica-HPLC.
The silica-HPLC
chromatogram for RP-HPLC fraction #2, (see Figure 6) indicates the presence of two partially resolved species which were appropriately collected in separate fractions (designated RP #2: SIL #1 and RP #2: SIL #2). shown in Figure 7.
Similarly, the silica-HPLC chromatogram for RP-HPLC fraction #3 is
Again two components were observed and collected in separate fractions
(designated RP#3: SIL #1 and RP#3: SIL #2).
When analyzed by GC-LRMS, each
silica-HPLC frac-
tion was found to contain, one major HCDD.
The composite of these mass chromatograms shown in Figure 8 illustrates that the HCDDs present in RP#2: SIL #I and SIL #2 have essentially identical packed column GC retention times.
The
01 HCDDs
B
o I 0
I 5
I 10
I 15
I 20
| 25
I 30
l 35
I 40
I 45
Minutes
F i g . 2.
HRGC-EC chromatogram of t h e c a u s t i c e x t r a c t e d crude p r o d u c t s from p y r o l y s i s #7.
11
HCDDs
m/e 392
B
m/e 390 FS = 96
C
--
-
C
role 388 FS = 50
•
•
•
|
•
•
•
•
2
3
4
5
6
7
8
9
Minutes
Fig. 3.
GC-LRMS chromatogram of the caustic extracted crude products from pyrolysis 417. FS is the full scale factor applied to each ion.
Fi s. 4.
RP-HPLC fractionation of the caustic extracted crude products from pyrolysis #7. major HCDDs
RP Fraction #: 1
2
3
n-
~
_
'
0
~
'
4
(~
. . . . . . . . . . . . . . . . . . . . . . 8 10 12 14 16 18 20 22 24 26 Minutes
28
,
,
,
30
32
34
12
Pyrolysis #7:
RP Fmction#2
m/e 392 FS = 106
Pyrolysis # 7: RP Fraction #3
/ ~
m/e 392 FS = 108
FS = 139
FS = 140
m/e 388
m/e 388
FS = 74
FS = 73
2
3
4
5
6
7
8
9
3
4
5
Minutes Fig. 5.
6
7
8
9
Minut~ GC-LRMS chromatograms of RP-HPLC fractions.
g Fig. 6.
Silica-HPLC refractionation of RP fraction #2 of pyrolysis #7. front side of the first peak.
Sil #2 is the back side of the second peak.
~Z E 0
SI L #1
I
I
I
I
I
0
2
4
6
8
Sil #I is the
I
10 Minutes
SI L #2
I
I
I
l
12
14
16
18
13
A
E c
SI L Fraction #: 0
=
2
R
a> i
=
0
2
,
I
4
,
I
6
,
t
f
8
10
,
I
12
,
,
14
I
I
16
18
Minutes
Fig. 7.
Silica-HPLC refractionation of RP fraction #3 of pyrolysis #7.
RP#2: SIL #1
RP#2: SIL #2
,
'~- FS=55
RP#3: SIL #1
RP#3: SIL #2
~C'123478"HCDD~-'*=] l
I~..~FS=66 ~
S=68
Minutes Fig.
8.
C o m p o s i t i o n mass chromatograms f o r s i l i c a - ] { P L C f r a c t i o n s I o n m/e = 398 i s 13C-123478-HCDD r e t e n t i o n
t i m e marker,
from p y r o l y s i s
#7.
14
same is true for those HCDDs in RP#3: SIL #I and SIL #2.
At this point each of the isolated
HCDDs could be assigned the following chromatographic retention indices:
an absolute RP-HPLC
(CH3CN/H20 eluent) retention time, a silica-HPLC retention time relative to 2378-TCDD, and a packed column GC retention time relative to 13C-123478-HCDD.
When these retention data sets
were collected for all major HCDD products isolated from the seven pyrolysis reactions listed in Table I, exactly 10 discrete sets were found to exist.
By combining synthesis information
derived from the specific chlorophenate precursors and the retention data sets, each of the 10 HCDD isomers could be specified as discrete, isolated species. are presented in Table II.
The final results of this work
As described in the experimental section, additional retention data
for each HCDD isomer were obtained by HRGC-EC at reduced column temperature and by RP-HPLC using an isocratic methanol eluent; these data are also given in Table II.
Table II.
HCDD Isomer Chromatographic Retention Indices
Silica-HPLC
RP-HPLC b
Packed GC
HRGC Rel. RT d
RP-HPLC e
HCDD Isomer
Rel. RT a
(min)
Rel. RT c
(min)
123467-HCDD
1.192
20.05
1.077
2.580
19.47
123469-HCDD
1.081
19.05
0.954
2.284
19.28
123478-HCDD
0.941
22.53
1.006
2.380
21.02
123468-HCDD
0.890
22.55
0.861
2.050
21.87
123678/123789-HCDD
1.060
21.90
1.103
2.587
20.07
123678/123789-HCDD
0.974
22.75
1.016
2.410
20.85
124679/124689-HCDD
0.958
19.87
0.805
1.940
19.62
124679/124689-HCDD
0.972
19.87
0.806
1.940
19.70
123679/123689-HCDD
0.970
21.27
0.903
2.165
20.20
123679/123689-HCDD
1.039
21.27
0.908
2.159
20.28
aRetention time r e l a t i v e to 2378-TCDD, bCH3CN/H20 (92.5/7.5) eluent for absolute retention time, Cpacked column GC retention time relative to 13C-123478-HCDD, d
capillary column GC retention time relative to 2378-TCDD, and emethanol eluent
for absolute retention time.
15
As described, pyrolysis #7 (2345-TeCD + 2356-TeCP) produced four discrete major HCDD products. Based on synthetic data it is possible that six major HCDD products could be formed; the selfcondensation products from either 2345-TeCP (HCDDs V and VI) or 2356-TeCP (HCDDs VIII), plus the expected mixed reaction products (HCDDs IX and X).
VII and
Since the HCDDs (V and VI)
resulting from the self-condensation of 2345-TeCP (pyrolysis #5) have RP-HPLC retention times of 21.90 min. and 22.75 min, Figure 4 shows that these HCDDs are not major products of pyrolysis #7 (note HRGC-EC and packed column GC-LRHS retention data are confirmatory).
However, the
self-condensation products of 2356-TeCP (pyrolysis #6) co-elute at a retention time of 19.87 min by RP-HPLC as shown in Figure 9.
Therefore the HCDDs present in RP#2 of pyrolysis #7
should be identical to those from pyrolysis #6.
Subsequent silica-HPLC refractionation of the
HCDDs from pyrolysis #6 yielded the chromatogram shown in Figure I0, which when compared to Figure 6 confirms the identity of these HCDDs as 124679-HCDD (VII) and 124689-HCDD (VIII). Hence, the HCDDs isolated during silica-HPLC refractionation of pyrolysis #7 RP#3 (see Figure 7) are the mixed reaction products 123689-HCDD (IX) and 123679-HCDD (X). This conclusion is further confirmed by the remainder of the retention indices data given in Table II.
CONCLUSIONS
Capillary column gas chromatography (HRGC) is one of the most efficient separation techniques currently in practical use for organic compounds. Application of HRGC to the difficult area of isomerically related CDD separations has been amply reported by Buser et al. (4,8,18,22,24,27).
A
E r" U~
~q
HCDD products (2 isomers) o
no HCDDs
I
I
!
I
|
|
I
I
f
0
2
4
6
8
10
12
14
16
.
18
I
|
f
|
20
22
24
26
,
|
28
,
]
30
,
|
,
32
Minutes
Fig. 9.
RP-HPLC chromatogram of the s e l f - c o n d e n s a t i o n products of 2356-TeCP ( p y r o l y s i s #6).
16
Although HRGC is a powerful single-approach procedure for separating complicated mixtures, it inherently suffers two problems when applied to CDDs determination. column capacity.
The first of these is
Because capillary columns require very small amounts of material to be separ-
ated, their practical use for isolating and collecting species for examination by alternate procedures is poor.
Secondly, considering the 22 TCDD and I0 HCDD isomers, to our knowledge
there have been no reports demonstrating complete isomer specificity for these compounds from either synthetically prepared mixtures or from environmental residues which employ only HRGC systems.
Our response to these problems has been to trade a single high efficiency-low capacity
chromatographic column for three consecutive low efficiency-high capacity chromatographies. Although the HRGC approach offers simple and speedy results, its realm of application is consider ably restricted when compared to multiple chromatographies.
Nowhere are these restrictions
more evident than in the field of isomer-specific CDD determinations, especially TCDDs and HCDDs which are currently topics of environmental concern.
The application of multiple chromatographies is not a new analytical technique.
However, with
ever improving instrumentation and automation, especially in the field of HPLC, it is an old area which should be reexamined.
Although the RP-HPLC, silica-HPLC, and packed column-GC
systems described in this report are not highly efficient, we have demonstrated that they can be used in conjuction to produce extreme selectivity.
As previously published, we have shown
that this system can separate and identify all 22 TCDD isomers (25). Essentially the same system can also provide such specificity in a wide variety of environmental samples at ppt concentrations (5).
In addition, multiple chromatographies was shown to be capable of resolv-
ing the I0 discrete HCDD isomers.
As a final means of demonstrating its CDD isomer separation
capabilities we have included the silica-HPLC chromatogram showing the resolution of 2,8-dichlorodibenzo-p-dloxln (28-DCDD) from 27-DCDD in a mixture confirmed by infrared spectroscopy to be 95~ 27-DCDD (see Figure 11, silica-HPLC conditions same as text).
REFERENCES
i.
Dow Chemical Company, "Trace Chemistries of Fire," Nov. 1978.
2.
K. Olie, P. L. Vermeulen, O. Hutzinger, Chemosphere, 6, 455 (1977).
3.
H . R . Buser, H. P. Bosshardt, C. Rappe, Chemosphere, 7, 165 (1978).
4.
C. Rappe, H. R. Buser, H. P. Bosshardt, "Polychlorinated Dibenzo-p -dioxins (PCDDs) and Dibenzofurans (PCDFs):
Occurrence, Formation, and Analysis
of Environmentally Hazardous Compounds," Presented at CIPAC-Symposium, Baltimore, Md., June 5-6, 1979. 5.
L . L . Lamparski, T. J. Nestrick, Anal.Chem., in press.
6.
R . E . Clement, G. A. Eicemen, F. W. Karasek, W. D. Bowers, H. L. Parsons, J~.of Chromatogr~ 189, 53 (1980).
7.
A. Eiceman, R. E. Clement, F. W. Karasek, Anal. Cbem:, 51, 2343 (1979).
17
A
E
eU~ ¢O ¢q
124679.HCDD
U3 O
and 1 2 4 6 8 ~ H C D D
C O
>
\ i 0
i 2
I 4
,
I 6
,
I 8
,
I 10
I 12
,
I 14
,
I 16
,
I 18
Minutes
Fig. I0.
Silica-HPLC refractionation of RP-HCDD fraction from pyrolysis #6.
c 03 ¢N
27-DCDD
d tO e~
2378-TCDD
!
I
I
I
!
I
!
!
I
I
!
0
2
4
6
8
10
12
14
16
18
20
Minutes Fig.
11.
SiIica-HPLC fractionation
o f 9 5 : 5 m i x t u r e o f 2 7 - and 28-DCDD.
18
8.
H.R.
9.
E . E . McConnell, J. A. Moore, J. K. Haseman, M. W. Harris, Toxicol. Appl.
Buser, H. P. Bosshardt, Mitt. Gebiete Lebensm. Hyg., 69, 191 (1978).
Pharmacol., 3_~7, 146 (1976). I0.
A. Poland, E. Glover, A. S. Kende, J_.c.Biol. Chem,, 251, 4936 (1976).
11.
G. R. Higginbotham, A. Huang, D. Firestone, J. Verrett, J. Ress, A. D. Campbell, Nature
12.
B. A. Schwetz, J. M. Norris, G. L. Sparsehu, V. K. Rowe, P. J. Gehring, J. L.
(London), 220~ 702 (1968). Emerson, C. G. Gerbig, Environ. Health Perspect., 5, 87 (1973). 13.
R. L. Johnson, P. J. Gehring, R. J. Kociba, B. A. Schwetz, Environ. Health Perspect., 5, 171 (1973).
14.
J. S. Cantrell, N. C. Webb, A.J. Mabis, Acta Crystallogr. Sect. B, 25, 150 (1969).
15.
A. P. Gray, S. P. Cepa, I. J. Solomon, O. Aniline, J_c.Org. Chem., 4_!, 2435 (1976).
16.
A. S. Kende, J. J. Wade, D. Ridge, A. Poland, J. Org. Chem., 39,
17.
B. A. Schwetz, J. M. Norris, G. L. Sparschu, V. K. Rowe, P. J. Gehring,
931 (1974). J. L. Emerson, C. G. Gerbig, Adv. Chem. Ser., 120, 55 (1973). 18.
H. R. Buser, Thesis, University of Ume~, Sweden, 1978.
19.
J. A. Moore, NIEHS Report, March I, 1977.
20.
D. Firestone, Ecol. Bull. (Stockholm), 2~7, 39 (1978).
21.
O. Hutzinger, University of Amsterdam, The Netherlands, Personal
22.
H. R. Buser, J. of Chromatogr., 114, 95 (1975).
23.
O. Aniline, Adv. Chem. Ser., 12__O0,126 (1973).
24.
C. Rappe, S. Marklund, H. R. Buser, H. P. Bosshardt, Chemosphere, Z,
25.
T. J. N e s t r i c k , L. L. Lamparski, R. H. S t e h l , Anal. Chem., 51, 2273 (1979).
26.
R. Bredeweg, L. Rothman, C. D. P f e i f f e r , Anal. Chem., 51, 2061 (1979).
27.
C. Rappe, H. R. Buser, H. P. Bosshardt, Chemosphere , 7, 431 (1978).
28.
H. R. Buser, C. Rappe, Chemosphere, 7, 199 (1978).
Communication, 1978.
269 (1978).
(Received i n The N e t h e r l a n d e 31 October 1980)
OO45-6535/81/o10oo3-165o2.oo/o
SYNTHESIS AND IDENTIFICATION OF THE I0 HEXACHLORODIBENZO-P-DIOXIN ISOMERS BY HIGH PERFORMANCE LIQUID AND PACKED COLUMN GAS CHROMATOGRAPHY
L. L. Lamparski and T. J. Nestrick The Dow Chemical Company, Michigan Division Analytical Laboratories,Midland,
MI 48640 USA
INTRODUCTION
Many recent investigations of environmental particulate samples, especially those associated with effluent from combustion processes, have indicated the widespread presence of chlorinated dibenzo-p-dioxins
(CDDs) and chlorinated dibenzofurans (CDFs) (I-8).
two related compound classes are known to be extremely toxic.
Certain members of these
Toxicological investigations
involving CDDs have demonstrated a wide range of biological activities, and also indicate that this activity is highly dependent upon the number and ring position of chlorine atoms present in the molecule (9-14).
All evidence to date regarding the relative toxicity of the 75 possi-
ble CDDs, which range from mono- to octachlorinated species, indicate that peak toxicity is observed for 2,3,7,8-tetrachlorodibenzo-p-dioxin
(2378-TCDD) (9, 15-18).
Although certain
isomers of penta- and hexachlorodibenzo-p-dioxin
(PCDDs and HCDDs) have been found to be less
biologically active than 2378-TCDD (9-10, 19-20), several recent quantitative studies have indicated that such higher chlorinated CDDs are often present in environmental particulates at greater concentrations than TCDDs (1,5,6,8,21).
Because these findings are based upon a limited
number of sample analyses, their consequences with respect to the environment cannot be adequately interpreted.
In anticipation of the possible necessity of accomplishing isomer-specific HCDD analyses on future samples we have synthesized, identified, and most importantly isolated each of the I0 HCDD isomers.
Serving as standards these discrete HCDDs should permit better evaluation of the
isomer specificity of current analytical procedures, allow easier development of truly isomerspecific new procedures, and ultimately lead to a better understanding of the formation, distribution, and transport properties of CDDs in the environment.
Specific
combinations of trichlorophenol
(PCP) were r e a c t e d as t h e i r
(TCP), t e t r a c h l o r o p h e n o l
potassium salts
under controlled-flow
HCDDs.
This synthetic
related
HCDD m i x t u r e s v i a t h e S m i l e s r e a r r a n g e m e n t ( 2 2 - 2 4 ) .
(TeCP), and p e n t a c h l o r o p h e n o l pyrolysis
conditions
t o form
a p p r o a c h h a s b e e n d e s c r i b e d by o t h e r s and i s known t o p r o d u c e i s o m e r i c a l l y
demonstrated the separation
and i d e n t i f i c a t i o n
The work r e p o r t e d by Buser (22)
o f 8 HCDD i s o m e r s v i a c a p i l l a r y
column gas
chromatography-mass
spectrometry (GC-MS).
The remaining 2 HCDD isomers could not be identified
by this procedure nor were any discrete HCDD isomer standards isolated. of the multiple high performance liquid chromatographic
Using a modification
(HPLC) fractionation scheme reported by
us for the separation of the 22 TCDD isomers (25), the I0 HCDD isomers have been separated and isolated in low microgram quantities.
These compounds were characterized by packed column
GC-HS in the selected ion mode (SIH) and were identified as HCDDs via nominal mass and chlorine isotope pattern.
EXPERIMENTAL
Caution.
The described procedures permit the synthesis and isolation of all I0 HCDD isomers,
to include the possibly more toxic homologs 123678-HCDD and 123789-RCDD.
In certain cases the
crude pyrolyzates were found to contain other CDDs including 2378-TCDD.
Persons attempting to
prepare these substances should be experienced in the handling procedures for extremely toxic materials.
Appropriate precautions should be taken so as to minimize the chances of either
personal or environmental exposure to CDDs.
All waste materials, to include equipment washing
solvents, should be carefully packaged and isolated until destroyed by appropriate incineration techniques.
Reagents.
The following chlorophenols were obtained from Aldrich (Milwaukee, WI 53233) in the
best available purity and were used as received:
234-TCP (mp 77-79°C), 236-TCP (99~), 245-TCP
(mp 63-65°C), 246-TCP (98~), 2345-TeCP (98~), 2356-TeCP (mp I14-I16°C), and PCP (Gold Label @ 99+~).
All solvents used were distilled-in-glass quality obtained from Burdick and Jackson
Laboratories
(Huskegon, HI 49442).
Aqueous solutions were prepared using deionized water.
potassium hydroxide was Reagent Grade.
The
Purge gas for the pyrolysis reactor was pre-purified
nitrogen (Hatheson, 99.995~), further purified by passage through a General Electric "Go-Getter" before use.
Silica, 100/200-mesh Bio-Sil A (Bio-Rad Laboratories, Richmond, CA
94804), was used as a sorbent in the pyrolysis reactor.
It was thoroughly cleaned and activated
according to the previously described procedure (25).
HCDD Isomer Syntheses.
(See Table I)
The apparatus and procedure used to synthesize the I0
HCDD isomers is essentially identical to that previously described for TCDD isomers (25).
Hethanolic potassium chlorophenate solutions were prepared by dissolving a total of 0.15
mmol chlorophenol(s) in 1.0 mL of a solution containing 17 mg KOH/I.O mL methanol (0.30 mmol KOH = 2 fold molar excess).
For those cases involving mixed chlorophenates, equimolar portions
of each reactant were combined so as to achieve a total of 0.15 mmol. This solution was then transferred to the glass wool plug positioned in the controlled-flow pyrolysis reactor tube, the methanol solvent purged from the system, and the reaction conducted at 280°C for a period of %15 min.
After cooling the reactor tube to ambient temperature, HCDD products were removed
Table I
P o t a s s i u m C h l o r o p h e n a t e P y r o l y s e s and G e n e r a l i z e d HCDD p r o d u c t s by D i r e c t A d d i t i o n and S m i l e s R e a r r a n g e m e n t R e a c t i o n s .
Pyrolysis
K Chlorophenates
1.
Direct
+
Expected HCDD Products Smiles
~
+
HCDD Isomer SOu~lht
I. 123467-HCDD
H PCP
2.
234-TCP
+
123467-HCDD
--
,~
123467.HCDD
+
II. 123469-HCDD
H PCP
3.
236-TCP
÷
123469.HCDD
A
•
123467.HCDD
÷
III.
123476HCDD
HO PCP
4.
245-TCP
+
123478.HCDD
A
•
12347~HCDD
+
IV. 123468.HCDD
HO PCP
5.
246-TCP
-I-
6
•
2345.TeCP
÷
123468.HCDD
÷
VI. 123789.HCDD
123678.HCDD
.
2356.TeCP
7.
~
HO
2345,TeCP
123468.HCDD
123789.HCDD
÷ 2356.TeCP
-[-
,24669.c00
124679.HCDD
~
•
124689.HCDD
÷
X: 12367~HCDD
HO 2345.TeCP
2356-TeCP
123689.HCDD
123679-HCDD
by i n v e r t i n g t h e t u b e ( e n t r a n c e p o r t up) and e l u t i n g t h e s y s t e m w i t h 50 mL, f o l l o w e d by an additional The t o t a l
25 mL, o f m e t h y l e n e c h l o r i d e i n a f a s h i o n s i m i l a r t o a l i q u i d c h r o m a t o g r a p h i c column. e f f l u e n t was c o l l e c t e d i n a 100-mL b o t t l e
under a stream of s p e c i a l l y p u r i f i e d
nitrogen.
and t h e s o l v e n t removed by e v a p o r a t i o n
The r e s i d u e was r e d i s s o l v e d i n ~I0 mL o f hexane
t o which was t h e n added ~15-20 mL o f aqueous 1 H KOH. A f t e r v i g o r o u s manual s h a k i n g and l a y e r separation,
t h e hexane e x t r a c t was t r a n s f e r r e d
hexane e x t r a c t i o n s
were c o m p l e t e d and t h e e x t r a c t s
liminary qualitative from e a c h p y r o l y s i s
t o a c l e a n 25-mL g l a s s v i a l .
and q u a n t i t a t i v e reaction
hexane and an a l i q u o t
combined.
As a means o f p r o v i d i n g p r e -
i n f o r m a t i o n c o n c e r n i n g HCDD p r o d u c t s , t h e hexane e x t r a c t s
(Table I ) were a d j u s t e d t o a t o t a l
volume o f 20 mL w i t h a d d i t i o n a l
(~I-5~L) removed f o r e x a m i n a t i o n by c a p i l l a r y
with electron capture detection
Two a d d i t i o n a l 5 mL
column gas c h r o m a t o g r a p h y
(HRGC-EC) and packed column gas c h r o m a t o g r a p h y - l o w r e s o l u t i o n
mass spectrometry (GC-LRMS).
The hexane extracts were then evaporated to dryness under a
stream of specially purified nitrogen and the resulting white, crystalline residues were protected from light and stored dry until further processed.
Capillary Column Gas Chromatosraphy with Electron Capture Detection (HRGC-EC).
Each caustic
extracted pyrolyzate was analyzed qualitatively for the presence of chlorinated species other than HCDDs, and semi-quantitatively for a rough approximation of total HCDDs present, using a Varian 3700 Capillary gas chromatograph equipped with a 6ZNi pulsed electron capture detector. Instrumental calibration was accomplished by injection of an HCDD reference standard containing two different isomers.
An average response factor for HCDDs was computed from this standard
and was subsequently used to determine approximate HCDDs concentration in each pyrolyzate.
The
instrument was equipped with a 0.50 mm i.d. x 30 m-modified surface coated open tubular pyrex glass capillary column.
Chromosorb R-6470-I (1-4 pm) was used as the coated particulate support,
and following a specialized deactivation procedure was mercury-plug dynamic coated using a 15~ (w/v) solution of 60/40 (w/w) OV-17 silicone and Poly S-17g in degassed methylene chloride at a rate of ~2 cm/s.
Prepurified nitrogen was used as the carrier at a linear velocity of 17.5
cm/s, and also supplied as make-up gas to the detector at ~25 cc/min.
A specialized splitless
direct-injection coupling was constructed from pyrex glass tubing and packed with 0.60~ OV-17 silicone + 0.40~ Poly S-179 on 80/lO0-mesh Permabond @ Methyl Silicone (I0 cycle)(available from H~qJ Systems, Inc., Newton, HA 021641.
Operation of the injection port at 270°C permitted
quantitative injection of samples ranging from I to 3.5 pL in iso-octane without column deterioration.
The column was maintained at 280°C isothermal for all analyses and under these
conditions a 2.0 ~L injection of ~70 ppb (ng/mL) 123678-HCDD in isooctane resulted in a k' of 4.7 for this CDD at 30,000 effective theoretical plates. The detector was maintained at 320°C for all analyses.
Preliminary
Gas Chromatography-Low R e s o l u t i o n Mass S p e c t r o m e t r y
HCDDs q u a n t i t a t i o n
by HRGC-EC, t h e c o n c e n t r a t i o n
was a p p r o p r i a t e l y
adjusted
GC-LRHS o p e r a t i n g
i n t h e SIM mode m o n i t o r i n g m/e:
mixed HCDD r e f e r e n c e
for quantitative
of the caustic
HCDDs a n a l y s i s
6-chlorine
isotope pattern
pyrolyzate
388, 390, and 392.
ing conditions
were as f o l l o w s :
+ 0 . 4 0 ~ P o l y S-179 on 8 0 / 1 0 0 - m e s h Permabond O Methyl S i l i c o n e
temperature, jet
x 210 cm s i l y l a t e d
Newton, HA 0 2 1 6 4 ) ; column t e m p e r a t u r e ,
280°C o n - c o l u m n ; c a r r i e r
a t column t e m p e r a t u r e ,
e n e r g y , 70 eV.
glass;
A
separator,
calibra-
by t h e i r
GC-LRMS o p e r a t -
packing, 0.60~ (10 c y c l e )
260°C i s o t h e r m a l ;
g a s , h e l i u m a t 14 c c / m i n . ;
and e l e c t r o n
resolution.
HCDD i s o m e r s were i d e n t i f i e d
OV-I? S i l i c o n e
from HNU S y s t e m s , I n c . ,
aliquot
i s o m e r s was used f o r i n s t r u m e n t a l
m o n i t o r e d a t m/e:
column, 2 mm i . d .
extracted
F o l l o w i n g rough
u s i n g a H e w l e t t - P a c k a r d Model 5992A
388, 390, and 392 a t u n i t
s t a n d a r d c o n t a i n i n g two d i f f e r e n t
t i o n and c o m p u t a t i o n o f an a v e r a g e HCDD r e s p o n s e f a c t o r . characteristic
(GC-LRHS).
(available
injection
port
glass single-stage
Reverse Phase High Performance Liquid Chromatography (RP-HPLC).
The residue from each caustic
extracted pyrolyzate was dissolved in chloroform such that a I0 pL aliquot injected into the RP-HPLC system would contain ~2-4 pg total HCDDs.
This system was composed of a Perkin-Elmer
LC-65T liquid chromatographic column oven and variable wavelength UV detector operating under the following conditions:
column, two 6.2 x 250 mm Zorbax-ODS (Dupont Instruments Div.,
Wilmington, DE 19898) columns in series; isocratic eluent, 92.5/7.5 (v/v) acetonitrile/ water at 2.5 mL/min.; pump, Altex model IIOA; column temperature, 50°C; UV detector, 0.32 aufs at 235 nm; and injector, Rheodyne model 7120 with 50-~L sample loop.
Under these conditions an authen-
tic standard of 2378-TCDD demonstrated a retention time of 15.9 min.
During the course of each
pyrolyzate separation, fractions corresponding to observed peaks eluting between ~18 and 25 min. retention time were collected in 15-mL glass vials equipped with Poly-Seal ® caps containing %2 mL hexane.
HCDDs were recovered by dilution with %8 mL of 2% (w/v) aqueous sodium
bicarbonate and following phase separation the hexane layer was transferred to a clean 4-mL glass ~ial.
Two additional ~I mL hexane extractions were accomplished, the extracts combined,
and the solvent evaporated to dryness under a stream of specially purified nitrogen (RP-HPLC fraction).
Normal Phase Adsorption (Silica) High Performance Liquid Chromatography (Silica-HPLC).
Each
RP-HPLC fraction was redissolved in 2.0 mL of hexane and a 2.0 pL aliquot was examined by GC-LRMS under the previously described conditions to identify fractions containing HCDDs. Selected RP-HPLC fractions were then evaporated to dryness, redissolved in ~80 ~L of hexane, and quantitatively injected into the silica-HPLC system:
Laboratory Data Control model 1204
variable wavelength (IV detector, 0.I aufs at 235 nm; column, two 6.2 x 250 mm Zorbax-Sil (Dupont Instruments Div.) columns in series; isocratic eluent, hexane at 2.0 mL/min.; pump, Altex model IIOA; column temperature, ambient; injector, Rheodyne model 7120 with 100-~L sample loop. Before use these columns were dried according to the procedure of Bredeweg et al. (26).
Fractions
corresponding to each observed peak were collected in clean 4-mL glass vials, and the hexane solvent was then evaporated to dryness under a stream of specially purified nitrogen (Silica-HPLC fraction).
During the course of these fractionations an authentic standard of
2378-TCDD was periodically injected to provide accurate retention time data for assignment of HCDD retention times relative to 2378-TCDD.
Final for
HCDD I s o m e r A n a l y s e s . final
t h e GC-LRMS o p e r a t i n g standard
retention
were calculated.
characteristic
fractions previously
time reference
described
described.
with
excepting
fractions
isotope
the
with
retention
at m/e 398.
Native
times
column temperature
as an internal
for each native
HCDDs w e r e a g a i n
A s e c o n d 2 . 0 ~L a l i q u o t
2378-TCDD i n t o
~2 t o 4 mL o f h e x a n e
1 n g 1ZC-123478-HCDD i n t o
The IZC-123478-HCDD s e r v e d
pattern.
I00 pg native
were dissolved
was c o - i n j e c t e d
from which relative
I t was m o n i t o r e d
6-chlorine
was c o - i n j e c t e d
silica-HPLC
A 2 . 0 pL a l i q u o t
as previously
observed their
Selected
GC-LRMS e x a m i n a t i o n .
HCDD
identified
of the silica-HPLC
t h e HRGC-EC s y s t e m o p e r a t i n g
w h i c h was d e c r e a s e d
by
as
t o 265°C i s o t h e r m a l .
In this case 2378-TCDD functioned as the internal standard retention time reference for computation of HCDD isomer relative retention times.
After characterizing and isolating the I0 discrete
HCDD isomers, these standards were reexamined using the described RP-f[PLC system under conditions normally used for CDDs determination in sample residues (5).
Hence, the eluent was changed to
methanol at 2.0 mL/min., and absolute RP-HPLC retention times were obtained for each HCDD isomer.
CDD Standards.
The authentic standard of 2378-TCDD used in this work was prepared by W. W.
Muelder (Dow Chemical Co) and its properties have been described (25). The 13C-123478-HCDD was synthesized by A. S. Kende (Univ. of Rochester, Rochester, N-Y) and was shown to be 43 at.~ 13C-enriched.
The native HCDD reference standard containing two different isomers was prepared
and described by O. Aniline (23).
RESULTS AND DISCUSSION
Problems associated with macro-synthesis of isomerically pure CDD standards prompted our development of the multiple ¢hromatographies approach for CDDs separation.
In this context multiple
chromatographies refers to the consecutive use of different types of chromatography whose mechanisms for achieving separation involve significantly different molecular properties. definition, applicable forms of chromatography must permit reasonable sample~-~apacity,
By
relatively
constant component retention indices independent of sample matrix, and most importantly permit component recovery as an integral part of the separation process.
Our initial application of
multiple chromatographies permitted the separation, identification, and isolation of the 22 TCDD isomers from limited isomer mixtures synthesized via specific chlorophenate pyrolyses (25).
In this case RP-HPLC fractionation,
followed by normal phase silica-HPLC
refractionation, and finally packed column gas chromatographic separation were used sequentially. Although none of these chromatographies developed more than ~15,000 theoretical plates, their combined use accomplished the separation of all 22 TCDDs where Buser (28) has reported that capillary columns capable of 140,000 theoretical plates have failed.
Additional work in our
laboratory involving the application of these multiple chromatographies to CDDs determination in environmental particulate samples has shown that they permit acquisition of isomer-specific 2378-TCDD quantitative data even in samples intentionally fortified with equivalent amounts of the other 21TCDDs
(5). Hence, we undertook the investigation described in this paper for two
reasons, to provide discrete HCDD isomer standards for analytical purposes, and to demonstrate the applicability of multiple chromatographies for isolating all i0 HCDDs to include the two isomers not currently identified by HRGC means (22).
The information given in Table I indicates that a minimum of six specific chlorophenate pyrolyses can produce the 10 HCDD isomers assuming that direct addition and Smiles rearrange-
ment products are equally favorable routes.
However, in order to maximize the number of known
structural identities one additional pyrolysis (pyrolysis #I.) must be conducted.
Pyrolysis #I
permits synthetic structural identification of 123467-HCDD and 123469-HCDD which are both direct and Smiles rearranged related co-products of pyrolysis #2.
Buser has reported essentially
the same pyrolytic scheme outlined in Table I to produce the 10 HCDD isomers (22). Each of the HCDDs produced in pyrolyses #I through #5 could be discretely identified by Buser using HRGC. Pyrolyses #6 and #7, plus other pyrolyses and variety of reaction condition modifications, were reported to produce only two of the four remaining HCDD isomers when examined by HRGC (22). For this reason we have selected these pyrolyses to demonstrate our approach.
It is currently believed that pyrolytic chlorophenate condensation to CDDs occurs via the mechanisms shown in Figure I for 2356-TeCP and the mixed reaction involving 2345-TeCP and
[
2356'TeCP + 2356.TeCP CI CI
Cl
124679.HCDD
I
I
124689-HCDD I
CI
CI
CI
CI
Cl
Cl
CI
Cl
t
Cl
t
CI
CI
CI
Cl
Cl
Cl
1
123689.HCDD
Cl
Cl
I
CI
CI
Cl
Cl
I
123679.HCDD
CI
CI
c,- y CI 2345"TeCP + 2356.TeCP CI
CI "~~
--c,
"Cl
"0
Fig. I.
"o" y
CI
t
CI t
CI
CI c,
CI
Cl
0 c,
CI
CI c,
CI
0 c,
,
,
c,
c,
c , _Cl_ o c , _ _
Reaction pathways for the condensations of 2356-TeCP (upper) and 2345-TeCP + 2356-TeCP (lower).
Cl
I
IO
When multiple chromatographies are applied to t h e mixed chlorophenate pyrolysis of
2356-TeCP.
2345-TeCP and 2356-TeCP (pyrolysis #7), present in equimolar quantities, both of the expected HCDD isomer products are isolated.
In addition, both HCDD isomers associated with the self-
condensation of 2356-TeCP are observed and can be isolated in significant relative concentration (see Figure i). shown in Figure 2.
The HRGC-EC chromatogram for the caustic extracted crude pyrolyzate is
Preliminary GC-LRMS analysis of this material confirmed that peaks b, c,
and d were HCDDs (see Figure 3). Figure 4.
The RP-HPLC fractionation of this pyrolyzate appears in
Three fractions were collected and each was subjected to GC-LRMS examination to
determine the presence of HCDDs.
As indicated by the mass chromatograms shown in Figure 5,
RP-HPLC fractions #2 and #3 were found to contain the major HCDDs previously observed in the crude pyrolyzate.
These fractions were then refractionated by silica-HPLC.
The silica-HPLC
chromatogram for RP-HPLC fraction #2, (see Figure 6) indicates the presence of two partially resolved species which were appropriately collected in separate fractions (designated RP #2: SIL #1 and RP #2: SIL #2). shown in Figure 7.
Similarly, the silica-HPLC chromatogram for RP-HPLC fraction #3 is
Again two components were observed and collected in separate fractions
(designated RP#3: SIL #1 and RP#3: SIL #2).
When analyzed by GC-LRMS, each
silica-HPLC frac-
tion was found to contain, one major HCDD.
The composite of these mass chromatograms shown in Figure 8 illustrates that the HCDDs present in RP#2: SIL #I and SIL #2 have essentially identical packed column GC retention times.
The
01 HCDDs
B
o I 0
I 5
I 10
I 15
I 20
| 25
I 30
l 35
I 40
I 45
Minutes
F i g . 2.
HRGC-EC chromatogram of t h e c a u s t i c e x t r a c t e d crude p r o d u c t s from p y r o l y s i s #7.
11
HCDDs
m/e 392
B
m/e 390 FS = 96
C
--
-
C
role 388 FS = 50
•
•
•
|
•
•
•
•
2
3
4
5
6
7
8
9
Minutes
Fig. 3.
GC-LRMS chromatogram of the caustic extracted crude products from pyrolysis 417. FS is the full scale factor applied to each ion.
Fi s. 4.
RP-HPLC fractionation of the caustic extracted crude products from pyrolysis #7. major HCDDs
RP Fraction #: 1
2
3
n-
~
_
'
0
~
'
4
(~
. . . . . . . . . . . . . . . . . . . . . . 8 10 12 14 16 18 20 22 24 26 Minutes
28
,
,
,
30
32
34
12
Pyrolysis #7:
RP Fmction#2
m/e 392 FS = 106
Pyrolysis # 7: RP Fraction #3
/ ~
m/e 392 FS = 108
FS = 139
FS = 140
m/e 388
m/e 388
FS = 74
FS = 73
2
3
4
5
6
7
8
9
3
4
5
Minutes Fig. 5.
6
7
8
9
Minut~ GC-LRMS chromatograms of RP-HPLC fractions.
g Fig. 6.
Silica-HPLC refractionation of RP fraction #2 of pyrolysis #7. front side of the first peak.
Sil #2 is the back side of the second peak.
~Z E 0
SI L #1
I
I
I
I
I
0
2
4
6
8
Sil #I is the
I
10 Minutes
SI L #2
I
I
I
l
12
14
16
18
13
A
E c
SI L Fraction #: 0
=
2
R
a> i
=
0
2
,
I
4
,
I
6
,
t
f
8
10
,
I
12
,
,
14
I
I
16
18
Minutes
Fig. 7.
Silica-HPLC refractionation of RP fraction #3 of pyrolysis #7.
RP#2: SIL #1
RP#2: SIL #2
,
'~- FS=55
RP#3: SIL #1
RP#3: SIL #2
~C'123478"HCDD~-'*=] l
I~..~FS=66 ~
S=68
Minutes Fig.
8.
C o m p o s i t i o n mass chromatograms f o r s i l i c a - ] { P L C f r a c t i o n s I o n m/e = 398 i s 13C-123478-HCDD r e t e n t i o n
t i m e marker,
from p y r o l y s i s
#7.
14
same is true for those HCDDs in RP#3: SIL #I and SIL #2.
At this point each of the isolated
HCDDs could be assigned the following chromatographic retention indices:
an absolute RP-HPLC
(CH3CN/H20 eluent) retention time, a silica-HPLC retention time relative to 2378-TCDD, and a packed column GC retention time relative to 13C-123478-HCDD.
When these retention data sets
were collected for all major HCDD products isolated from the seven pyrolysis reactions listed in Table I, exactly 10 discrete sets were found to exist.
By combining synthesis information
derived from the specific chlorophenate precursors and the retention data sets, each of the 10 HCDD isomers could be specified as discrete, isolated species. are presented in Table II.
The final results of this work
As described in the experimental section, additional retention data
for each HCDD isomer were obtained by HRGC-EC at reduced column temperature and by RP-HPLC using an isocratic methanol eluent; these data are also given in Table II.
Table II.
HCDD Isomer Chromatographic Retention Indices
Silica-HPLC
RP-HPLC b
Packed GC
HRGC Rel. RT d
RP-HPLC e
HCDD Isomer
Rel. RT a
(min)
Rel. RT c
(min)
123467-HCDD
1.192
20.05
1.077
2.580
19.47
123469-HCDD
1.081
19.05
0.954
2.284
19.28
123478-HCDD
0.941
22.53
1.006
2.380
21.02
123468-HCDD
0.890
22.55
0.861
2.050
21.87
123678/123789-HCDD
1.060
21.90
1.103
2.587
20.07
123678/123789-HCDD
0.974
22.75
1.016
2.410
20.85
124679/124689-HCDD
0.958
19.87
0.805
1.940
19.62
124679/124689-HCDD
0.972
19.87
0.806
1.940
19.70
123679/123689-HCDD
0.970
21.27
0.903
2.165
20.20
123679/123689-HCDD
1.039
21.27
0.908
2.159
20.28
aRetention time r e l a t i v e to 2378-TCDD, bCH3CN/H20 (92.5/7.5) eluent for absolute retention time, Cpacked column GC retention time relative to 13C-123478-HCDD, d
capillary column GC retention time relative to 2378-TCDD, and emethanol eluent
for absolute retention time.
15
As described, pyrolysis #7 (2345-TeCD + 2356-TeCP) produced four discrete major HCDD products. Based on synthetic data it is possible that six major HCDD products could be formed; the selfcondensation products from either 2345-TeCP (HCDDs V and VI) or 2356-TeCP (HCDDs VIII), plus the expected mixed reaction products (HCDDs IX and X).
VII and
Since the HCDDs (V and VI)
resulting from the self-condensation of 2345-TeCP (pyrolysis #5) have RP-HPLC retention times of 21.90 min. and 22.75 min, Figure 4 shows that these HCDDs are not major products of pyrolysis #7 (note HRGC-EC and packed column GC-LRHS retention data are confirmatory).
However, the
self-condensation products of 2356-TeCP (pyrolysis #6) co-elute at a retention time of 19.87 min by RP-HPLC as shown in Figure 9.
Therefore the HCDDs present in RP#2 of pyrolysis #7
should be identical to those from pyrolysis #6.
Subsequent silica-HPLC refractionation of the
HCDDs from pyrolysis #6 yielded the chromatogram shown in Figure I0, which when compared to Figure 6 confirms the identity of these HCDDs as 124679-HCDD (VII) and 124689-HCDD (VIII). Hence, the HCDDs isolated during silica-HPLC refractionation of pyrolysis #7 RP#3 (see Figure 7) are the mixed reaction products 123689-HCDD (IX) and 123679-HCDD (X). This conclusion is further confirmed by the remainder of the retention indices data given in Table II.
CONCLUSIONS
Capillary column gas chromatography (HRGC) is one of the most efficient separation techniques currently in practical use for organic compounds. Application of HRGC to the difficult area of isomerically related CDD separations has been amply reported by Buser et al. (4,8,18,22,24,27).
A
E r" U~
~q
HCDD products (2 isomers) o
no HCDDs
I
I
!
I
|
|
I
I
f
0
2
4
6
8
10
12
14
16
.
18
I
|
f
|
20
22
24
26
,
|
28
,
]
30
,
|
,
32
Minutes
Fig. 9.
RP-HPLC chromatogram of the s e l f - c o n d e n s a t i o n products of 2356-TeCP ( p y r o l y s i s #6).
16
Although HRGC is a powerful single-approach procedure for separating complicated mixtures, it inherently suffers two problems when applied to CDDs determination. column capacity.
The first of these is
Because capillary columns require very small amounts of material to be separ-
ated, their practical use for isolating and collecting species for examination by alternate procedures is poor.
Secondly, considering the 22 TCDD and I0 HCDD isomers, to our knowledge
there have been no reports demonstrating complete isomer specificity for these compounds from either synthetically prepared mixtures or from environmental residues which employ only HRGC systems.
Our response to these problems has been to trade a single high efficiency-low capacity
chromatographic column for three consecutive low efficiency-high capacity chromatographies. Although the HRGC approach offers simple and speedy results, its realm of application is consider ably restricted when compared to multiple chromatographies.
Nowhere are these restrictions
more evident than in the field of isomer-specific CDD determinations, especially TCDDs and HCDDs which are currently topics of environmental concern.
The application of multiple chromatographies is not a new analytical technique.
However, with
ever improving instrumentation and automation, especially in the field of HPLC, it is an old area which should be reexamined.
Although the RP-HPLC, silica-HPLC, and packed column-GC
systems described in this report are not highly efficient, we have demonstrated that they can be used in conjuction to produce extreme selectivity.
As previously published, we have shown
that this system can separate and identify all 22 TCDD isomers (25). Essentially the same system can also provide such specificity in a wide variety of environmental samples at ppt concentrations (5).
In addition, multiple chromatographies was shown to be capable of resolv-
ing the I0 discrete HCDD isomers.
As a final means of demonstrating its CDD isomer separation
capabilities we have included the silica-HPLC chromatogram showing the resolution of 2,8-dichlorodibenzo-p-dloxln (28-DCDD) from 27-DCDD in a mixture confirmed by infrared spectroscopy to be 95~ 27-DCDD (see Figure 11, silica-HPLC conditions same as text).
REFERENCES
i.
Dow Chemical Company, "Trace Chemistries of Fire," Nov. 1978.
2.
K. Olie, P. L. Vermeulen, O. Hutzinger, Chemosphere, 6, 455 (1977).
3.
H . R . Buser, H. P. Bosshardt, C. Rappe, Chemosphere, 7, 165 (1978).
4.
C. Rappe, H. R. Buser, H. P. Bosshardt, "Polychlorinated Dibenzo-p -dioxins (PCDDs) and Dibenzofurans (PCDFs):
Occurrence, Formation, and Analysis
of Environmentally Hazardous Compounds," Presented at CIPAC-Symposium, Baltimore, Md., June 5-6, 1979. 5.
L . L . Lamparski, T. J. Nestrick, Anal.Chem., in press.
6.
R . E . Clement, G. A. Eicemen, F. W. Karasek, W. D. Bowers, H. L. Parsons, J~.of Chromatogr~ 189, 53 (1980).
7.
A. Eiceman, R. E. Clement, F. W. Karasek, Anal. Cbem:, 51, 2343 (1979).
17
A
E
eU~ ¢O ¢q
124679.HCDD
U3 O
and 1 2 4 6 8 ~ H C D D
C O
>
\ i 0
i 2
I 4
,
I 6
,
I 8
,
I 10
I 12
,
I 14
,
I 16
,
I 18
Minutes
Fig. I0.
Silica-HPLC refractionation of RP-HCDD fraction from pyrolysis #6.
c 03 ¢N
27-DCDD
d tO e~
2378-TCDD
!
I
I
I
!
I
!
!
I
I
!
0
2
4
6
8
10
12
14
16
18
20
Minutes Fig.
11.
SiIica-HPLC fractionation
o f 9 5 : 5 m i x t u r e o f 2 7 - and 28-DCDD.
18
8.
H.R.
9.
E . E . McConnell, J. A. Moore, J. K. Haseman, M. W. Harris, Toxicol. Appl.
Buser, H. P. Bosshardt, Mitt. Gebiete Lebensm. Hyg., 69, 191 (1978).
Pharmacol., 3_~7, 146 (1976). I0.
A. Poland, E. Glover, A. S. Kende, J_.c.Biol. Chem,, 251, 4936 (1976).
11.
G. R. Higginbotham, A. Huang, D. Firestone, J. Verrett, J. Ress, A. D. Campbell, Nature
12.
B. A. Schwetz, J. M. Norris, G. L. Sparsehu, V. K. Rowe, P. J. Gehring, J. L.
(London), 220~ 702 (1968). Emerson, C. G. Gerbig, Environ. Health Perspect., 5, 87 (1973). 13.
R. L. Johnson, P. J. Gehring, R. J. Kociba, B. A. Schwetz, Environ. Health Perspect., 5, 171 (1973).
14.
J. S. Cantrell, N. C. Webb, A.J. Mabis, Acta Crystallogr. Sect. B, 25, 150 (1969).
15.
A. P. Gray, S. P. Cepa, I. J. Solomon, O. Aniline, J_c.Org. Chem., 4_!, 2435 (1976).
16.
A. S. Kende, J. J. Wade, D. Ridge, A. Poland, J. Org. Chem., 39,
17.
B. A. Schwetz, J. M. Norris, G. L. Sparschu, V. K. Rowe, P. J. Gehring,
931 (1974). J. L. Emerson, C. G. Gerbig, Adv. Chem. Ser., 120, 55 (1973). 18.
H. R. Buser, Thesis, University of Ume~, Sweden, 1978.
19.
J. A. Moore, NIEHS Report, March I, 1977.
20.
D. Firestone, Ecol. Bull. (Stockholm), 2~7, 39 (1978).
21.
O. Hutzinger, University of Amsterdam, The Netherlands, Personal
22.
H. R. Buser, J. of Chromatogr., 114, 95 (1975).
23.
O. Aniline, Adv. Chem. Ser., 12__O0,126 (1973).
24.
C. Rappe, S. Marklund, H. R. Buser, H. P. Bosshardt, Chemosphere, Z,
25.
T. J. N e s t r i c k , L. L. Lamparski, R. H. S t e h l , Anal. Chem., 51, 2273 (1979).
26.
R. Bredeweg, L. Rothman, C. D. P f e i f f e r , Anal. Chem., 51, 2061 (1979).
27.
C. Rappe, H. R. Buser, H. P. Bosshardt, Chemosphere , 7, 431 (1978).
28.
H. R. Buser, C. Rappe, Chemosphere, 7, 199 (1978).
Communication, 1978.
269 (1978).
(Received i n The N e t h e r l a n d e 31 October 1980)