2442
Anal. Chem. 1904, 56,2442-2447
Isolation of Polychlorinated Dibenzodioxins and Polychlorinated Dibenzofurans from a Complex Organic Mixture by Two-step Liquid Chromatographic Fractionation for Quantitative Analysis H.Y. Tong,D. L. Shore, and F. W. Karasek* Chemistry Department, University of Waterloo, Waterloo, Ontario N2L 3G1,Canada
PCDD and PCDF are present wlth several hundred organic compounds in raw fly ash extract. A first-step normal-phase semlpreparatlve HPLC fractionation with a three-solvent gradient elution program separates PCDD and PCDF into a single fraction wlth about 100 other components. The PCDD and PCDF In this fractlon are further Isolated from the accompanylng compounds by a second-step HPLC fractionation uslng a reverse-phase semlpreparatlve column and a two-solvent gradlenl elution program. The dlstributlon of PCDD and PCDF into five subfractions faclihates Isomer identWlcation and thelr quantitatlon. The separation of PCDD and PCDF by this procedure shows hlgh recovery and good reproduclbillty.
matogram. This serious peak overlapping which is commonly observed in many complex samples makes component indentification very difficult and quantification by GC data impossible. Further separation is needed if a nonselective detector is to be used for specific isomer analysis. In this study, a two-step HPLC procedure is used for the preseparation of fly ash extract. PCDD and PCDF are isolated from more than 600 components by normal-phase followed by reverse-phase HPLC fractionation. Separation is so good that a nonselective detector, such as a flame ionization detector (FID) may be used to quantify most PCDD and PCDF isomers. High recovery and good reproducibility are demonstrated for this technique.
Due to the high toxicity and high stability of polychlorinated dibenzodioxins (PCDD) and polychlorinated dibenzofurans (PCDF), their analysis has received increasing attention (1, 2). Identification and quantification of PCDD and PCDF in a complex mixture are very difficult because of the low levels of dioxins and furans in the presence of a number of other organic components with much higher concentrations. Column chromatography is an established cleanup procedure for the analysis of PCDD and PCDF in a variety of matrices such as soil samples, beef adipose tissue, and wastewater (3-5).As the complexity of the matrix increases, a more sophisticated cleanup procedure is required. Following column chromatography, high-performance liquid chromatography (HPLC) has been used to cleanup some complex samples such as chicken liver and human milk for subsequent dioxin analysis (6, 7). Lamparski et al. reported a multistep separation procedure for the isomer-specific analysis of PCDD in the extract of environmental particulate samples (8). The separation method involves multiple column chromatography followed by reverse-phase and normal-phase isocratic HPLC fractionation. Although this method allows isomer isolation, its complexity makes it very time consuming and the recovery questionable. In most reported cleanup procedures, even after separation, quantification of PCDD and PCDF is usually achieved on selective detectors such as GC/MS with selected ion monitoring (GC/ MS/ SIM). Municipal incinerators burning garbage generate thousands of tons of fly ash throughout the world each year. Approximately 1-2% of the fly ash is not isolated by electrostatic precipitation and escapes with the flue gases (9). Fly ash is a major source of dioxin pollution as well as being one of the most complex environmental samples. A recent study tentatively identified 200 of the more than 600 organic components present in fly ash. A one-step normal-phase HPLC separation was used in the study to exclusively elute all the PCDD and PCDF into a single fraction together with more than 100 other components (IO). Because of the differing numbers and substitution patterns of chlorine atoms, there are 75 PCDD and 135 PCDF isomers. These numerous isomers elute in a narrow temperature range on the gas chro-
Solvents and Standards. The solvents used in this study
EXPERIMENTAL SECTION
0003-2700/84/0356-2442$01.50/0
were “distilled in glass”, UV grade from Caledon Laboratories, Ltd. (Georgetown, Ontario, Canada). The standards of 1,2,3,4tetrachlorodibenzo-p-dioxin (1234-TCDD), 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin (123478-H6CDD), 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin (1234678-H7CDD),octachlorodibenzo-pdioxin (OCDD),and octachlorodibenzofuran (OCDF) were purchased from Ultra Scientific,Inc., (Hope, RI). The standards of 1,2,3,4,7-pentachlorodibenzo-p-dioxin(12347-P5CDD) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (2378-TCDD)were obtained from Cambridge Isotope Laboratories Inc. (Woburn, MA) and Foxboro/Analabs (North Haven, CT), respectively. After being cleaned by ultrasonic agitation in detergent, rinsed with deionized water, and dried at 250 “C for 3 h, all glassware waa rinsed three times with methylene chloride and then benzene immediately before its use. Sample Collection and Extraction. The Ontario fly ash sample was collected from the electrostatic precipitator of a municipal incinerator in Toronto, Ontario, Canada. The sample was stored away from sources of light. The 435-g fly ash sample was Soxhlet-extracted with benzene. The extraction and following concentration procedure are described previously (IO). The raw extract of fly ash sample was finally concentrated to 1.8 mL and stored in the freezer at -15 OC before the first-step HPLC fractionation. First-Step High-Performance Liquid Chromatographic Separation. The instrument used for the first-step HPLC
separation was a Spectra-Physics SP-8000 HPLC equipped with SP-8400 UV/vis detector and SP-4100integrator. The monitoring wavelength was 254 nm. A 10-pm, semipreparative Spherisorb silica column (250 X 9.4 mm, Tetrochem, Toronto, Canada) was employed with a 140-pL sample loop. The gradient elution program consisted of 100% n-C8H14for 20 min, programmed to 100% CHzC1, over 30 min and held at 100% CHzClzfor 20 min, programmed to 100% CH3CN over 10 min and held at 100% CH3CN for 1 min, and programmed back to 100% CHzClz in 5 min and finally to 100% hexane in another 5 min. During this gradient program five separate fractions were collected at elution times of 0 to start of first peak, 20, 50, 70, and 91 min. The flow rate was 5 mL/min. To avoid column overload and obtain enough sample material for the second-step HPLC separation, the raw extract was separately loaded on the HPLC four times and the corresponding fractions were combined, The combined fraction 2 was concen0 1984 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 56, NO. 13, NOVEMBER 1984
trated to 800 pL by the procedure previously described (IO). This fraction was stored in the freezer at -15 "C prior to the second-step HPLC separation. The SP-8000 HPLC was carefully cleaned by running large volumes of CBH14,CH2C12,and CH3CN through the system before loading the sample. Second-Step High-Performance Liquid Chromatographic Separation, The second-step HPLC separation was achieved on a Varian 5000 HPLC with a Vista 402 data system. This instrument was also equipped with a built-in UV detector at a fixed wavelength of 254 nm, a Fluorichrom detector allowing excitation over the 280-700 nm range, and an automated Rheodyne injector with 100-pL sample loop. A semipreparative reverse-phase column, MicroPak MCH-10 (300 X 8 mm, Varian Associates Inc., Walnut Creek, CA), was used. A gradient elution program was developed. It consisted of 100% CHSCN for 3 min, linear programmed to 90% CH3CNand 10% CH2C12over 10 min and held for 5 min, and then linearly programmed back to 100% CH3CN in 2 min and held at 100% CH3CN for another 10 min. During this gradient program, six subfractions were collected at elution times of 4 to 10.75,13.5,15,16,20, and 27 minutes. The flow rate was 2 mL/min. A 70-pL portion of fraction 2 obtained from the fmt-step HPLC separation was loaded for second-step HPLC separation and six subfractions were collected separately. Each subfraction was concentrated to a tiny volume and finally adjusted to 50 pL with benzene. These subfractions were subjected to GC/MS and GC analyses. The second-step HPLC separation of fraction 2 was performed, under exactly the same conditions, in duplicate. Run 1and run 2 were used to test the reproducibility of the second-step HPLC separation. The Varian 5000 HPLC was carefully cleaned up running CH3CN and CHzClzthrough the system before loading the sample. A 500-mL benzene solvent was run through the Soxhlet extraction and concentration procedure, and then it was used as the extraction blank (IO). Injections of pure benzene were run through the first-step and second-step HPLC separation procedure. The collection of fraction 2 from the first-step HPLC separation and that of subfractions from the second-step HPLC separation were used as the blank of HPLC separation. These blanks were analyzed by GC and GC/MS to test for impurities present in the methods. Gas Chromatographic/Mass Spectrometric Analysis. GC/MS analysis was done on a Hewlett-Packard HP5987A GC/MS system with HPl00O data system and an HP7914 Winchester disk drive. In this study, an ionization voltage of 70 eV and a source temperature of 200 OC were used. An open split interface heated to 310 OC interfaced an HP5880A GC to the mass spectrometer. Both linear scanning (50-500 amu) and selected ion monitoring were used. The data acquisition and storage system allows mass chromatograms to be reconstructed at any mass in the scan range. In addition, the mass spectra of any peak on total ion current (TIC) trace may be obtained during or after a sample run. Different modes of background substraction at TIC peaks are available for obtaining clean spectra. The library search system includes probability based matching (PBM) based on 70 000 reference spectra as well as a self-training interpretive retrieval system (STIRS). In the selected ion monitoring mode, five groups of 20 selected ions may be monitored at different times. In this study, a 30 m X 0.32 mm i.d. DB-5 fused silica capillary column (J & W Scientific Inc., Rancho Cordova, CA) was used. The GC conditions were cool on-column injector less than 50 "C and column temperature programmed from 80 OC to 300 OC at a rate of 4 OC/min with initial 1min and final 5 min isothermal periods. The helium carrier gas flow rate was 3 mL/min at room temperature. High-%solution Gas Chromatographic Analysis. GC analysis was performed on a Hewlett-Packard HP5880A GC equipped with FID and electron capture detector (ECD). A microcomputer data system with a cartridge tape allows storage of chromatographic information for future calculations. A cool on-column injector and a 30 m X 0.32 mm i.d. DB-5 column were used. The chromatographic conditions were the same as those in the GC/MS analysis except the rate of temperature programming was 3 "C/min.
1
2443
S o x h l e t E x t r a c t l o n o f F l y a s h Sample
I HPLC F r a c t i o n a t i o n p h a i e column, 3 8oivant gradient elution 1
I at-atop
( normal
I Fraction 2 (
PAH. PCDD a n d PCDF )
2nd-atop
HPLC F r a c t l o n a t i o n
( r o v 8 r a s phage column,
2 8oIvont g r a d i e n t e l u t i o n 1
I Subfraction 2 t o 6 (
J
a
GC/MS A n a l y a l a
PCDD a n d PCDF
)
\
m GC/FID A n a l y a l a
Flgure 1. Scheme of two-step HPLC fractionation and subsequent analysis of PCDD and PCDF in a fly ash sample from a municipal
Incinerator. Identification and Quantification of PCDD and PCDF in Fly Ash Sample. Since PCDD and PCDF have very characteristic mass spectra,their identification in the sample was based on the mass spectra. The position of PCDD and PCDF on the total ion current trace (TIC) was further confirmed by the mass chromatograms of M+, (M - 2)+,and (M + 2)' for each PCDD or PCDF. However, the identification of PCDD and PCDF in this study was not completely isomer specific due to the lack of standards for all the individual isomers. Both GC/MS/SIM and GC/FID techniques were used for quantitation of some PCDD and PCDF in the Ontario fly ash sample. The results in this study are also not isomer specific owing to unavilability of all individual isomer standards. Since only the 1234-TCDD standard was available, the area response of ita M+ ion was used to obtain a quantitative value for the sum of all TCDD isomers detected by M+ ions in the GC/MS/SIM analyses. This same technique applied to quantitation of P,CDD, H6CDD, H,CDD, OCDD, and OCDF. A similar method was used in the quantification of PCDD and PCDF by GC/FID. Namely, the sum of peak areas of identified PCDD with the same number of chlorine atoms was compared to the corresponding PCDD isomer standard. PCDF were quantified with the same PCDD standards except for OCDF where its standard was available.
RESULTS AND DISCUSSION Figure 1 shows the scheme of this two-step HPLC fractionation procedure and the subsequent analysis of PCDD and PCDF by GC/MS and GC/FID. The first-step HPLC fractionation was designed to classify the numerous components in the raw fly ash extract according to their polarities. A normal-phase, semipreparative HPLC column and gradient elution with n-C6HI4, CH2ClZ,and CH&N were used and five fractions were Collected. The major compounds found in each fraction and number of GC peaks observed on GC/FID chromatograms of each fraction are listed in Table I. Among the several hundred components observed, more than 200 have been tentatively identified in our earlier study (10). In the first-step HPLC separation, PCDD and PCDF exclusively elute in fraction 2 together with
2444
ANALYTICAL CHEMISTRY, VOL. 56, NO. 13, NOVEMBER 1984
Table I. Distribution of Organic Compounds Found among Fractions of First-Step HPLC Separation of an Ontario Fly Ash Extract
I
710 TRACE
no. of
fraction
components obsd
1 2
205 157
3
120
4
64
5
82
major compds found
aliphatic hydrocarbon PAH with less than three rings, PCB, PCDD, PCDF PAH with more than three rings, oxygenated PAH (oxy-PAH),chlorinated oxy-PAH, nitrogen-containing PAH oxy-PAH (small amount), phthalate ester polar compounds
ECO TRACE
80
I00
30c
200
TEHP
Figure a. Gas chromatogram of subfraction 1 of second-step HPLC
Separation: FID trace (upper) and ECD trace (lower). Chromato-
/
eo.
. I0
rnn
graphic conditions were as follows: 30 m X 0.32 mm 1.d. DB5 fused silica column; temperature at 80 O C for 1 min, programmed to 300 O C at 3 'C/min. SF2
FL TRACT
C
4
c- P I
I
IO _c
~t
fa
6
i w c
-
1 0 " " " " 26 ' .-c- F(
"
FB
cum)
OQLCGTDY
Figure 2. Scheme of second-step HPLC separation: gradient elution program (upper trace): fluorescence trace (middle trace) and UV trace
4
SF4
(lower) of fractlon 2 obtained from first-step HPLC separation: and subfraction collection interval. HPLC conditions are described in the Experimental Sectlon. more than 100 other components. The chromatogram of fraction 2 is still too complicated for detailed quantitative analysis. More complete isolation of PCDD and PCDF from other organic compounds found in fraction 2 was achieved by a second-step HPLC separation. A reverse-phase semipreparative HPLC column and gradient elution program with CH&N and CHzClz was used. In this study, fraction 2 refers to fraction 2 of the first-step HPLC separation while fractions of the second-step HPLC separation program are referred to as subfractions (SF). Figure 2 shows the second-step HPLC separation of fraction 2 of the Ontario fly ash extract. The six subfractions collected are designated SF1 to SF6. The major compounds found in fraction 2 of the raw extract of Ontario fly ash are polycyclic aromatic hydrocarbone (PAH), PCDD, and PCDF (Table I). A small amount of polychlorinated biphenyl (PCB), tetrachlorobiphenyl, nonachlorobiphenyl, and decachlorobiphenyl, were present in the same fraction of the sample (IO),but these did not interfere with the analysis of TCDD to OCDD or the corresponding PCDF isomers. Figure 3 can be used to contrast the GC/FID and GC/ECD trace of SF1. The two major peaks seen on the ECD trace were identified by their mass spectra as chlorinated benzenes. No PCDD and PCDF eluted in this subfraction. The major components eluting in SF1 are PAH. The second-step HPLC separation isolated PCDD and PCDF from most other organic compounds. The distribution pattern of PCDD and PCDF among SF2 to SF6 is shown in Figure 4. In this figure, PCDD and PCDF are marked differently. The GC retention time of specific PCDD and PCDF is noted where isomeric standards were available. Because
"TF l SF6
1
OCDF
u 200
230
260
290 TEMPPC)
Figure 4. Gas chromatograms showing the distribution of PCDD and PCDF among subfraction 2 to 6 of second-step HPLC separation. Chromatographic conditions are ghren in Figure 3. FID detector. PCDD is denoted as "0"and PCDF is denoted as "X". Subtraction (SF) GC retention times were (1) 1234-TCDD isomer, (2) 2378-TCDD Isomer, (3) 12347-P5CDD isomer, (4) 123478-H6CDD, (5) 1234578-H7CDD isomer, and (6) 1234678-H7CDD Isomer.
of the possibility of coeluting isomers, all isomeric standards me needed for positive peak identification of most PCDD and PCDF isomers. Since there are only two H7CDD isomers possible, both peaks may be assigned with the use of one H7CDD isomer standard. OCDD and OCDF peaks can also be postively identified. Compound identification is illustrated with TCDD and TCDF in Figure 5. The mass chromatograms of m / z 321.9 (TCDD), m / z 305.9 (TCDF), and the total ion
ANALYTICAL CHEMISTRY, VOL. 50, NO. 13, NOVEMBER 1984
2445
a 0 C D D\,OC
30.0
32.0
31.0
33.0
3S.0
34.0
‘“‘1 b
36.0
I
300
M/Z
Ir
305.9
TIC
x
(MIN)
Flgure 5. TCDD and TCDF identiflcatlon: ma58 chromatogram of m l r 321.9 (TCDD), m l r 305.9 (TCDF) and total ion current trace (TIC). Chromatographic conditions were as follows: 30 m X 0.32 mm i.d. DE5 fused silica column; temperature at 80 OC for 1 mln, programmed to 300 OC at 4 OVmin. PCDD is denoted as “0” and PCDF 1s denoted as “X”.
/I
3, 40
, ,
!I,
!,,
.I,I, , l,ll/l.
, ,
BO
1,
120
,
,
,
,
//I, ,
,
I LID
I,
,
,
,
,,
200
,
,
,
,
, ,
,
l!
1.0
Flgure 7. Gas chromatogram of OCDD and OCDF In fraction 2 of Ontario fly ash sample: (a) on a 30 m X 0.32 mm i.d. DB-5 column, (b) on a 60 m X 0.32 mm i.d. DB-5 column. GC conditions were as follows: temperature at 80 OC for 1 min, programmed to 300 OC at 3 ‘Clmln; FID detector.
P
M/Z
339.9
I/
Ill
I ,
DF
, ,
,,
, ,
,,
Zen
,
,
,
Ill,
,
,
, ,
,
,
,_I
320
Flgure 8. Mass spectra of TCDD (a) and TCDF (b) obtained from SF2 of the Ontario fly ash extract.
current trace (TIC) are shown for the corresponding region. Compound identification was also confirmed by mass chromatograms of (M - 2)+ and (M + 2)+ ions, as well as by carefully examining their full mass spectra shown in Figure 6. PCDD and PCDF with the same number of chlorine atoms exhibit very similar retention behavior on many GC columns. When many PCDD and PCDF isomers are present, such as in complex environmental samples, they often elute in clusters within a small temperature range on the chromatogram. This serious overlapping of components makes isomer identification difficult and quantitation with nonselective detectors such as FID impossible. The second-step HPLC separation reduces the complexity of the sample by fractionating the numerous PCDD and PCDF isomers into different subfractions. TCDD and TCDF separation can be used to illustrate this. The overlapping of components has been greatly reduced by removing some of the TCDF isomers into SF3 (Figure 4). The separation of other PCDD and PCDF isomers is much better, HOCDD, HBCDF, H,CDD, H,CDF, and OCDD and OCDF haye been completed separated. In most cases, specific isomer identification is possible if isomeric standards are available. Figure 7 illustrates the usefulness of this technique for the analysis of OCDD and OCDF. These two compounds have the same retention time on a 30-m DB-5 capillary column and are only slightly separated on a 60-m capillary column.
a400 D n ~ sF5
Flgure 8. Mass chromatogram of m l z 339.9 (P,CDD) of subfraction 2 to 5. Chromatographic conditions are given In Figure 5.
However, the second-step HPLC separates OCDD from OCDF into SF5 and SF6, respectively. Some positional isomers of PCDF and PCDD are separated into different subfractions. Figure 8 shows the distribution of P&DF isomers among subfraction 2 to 5. Although some of the same isomers may elute in SF2 and SF3, generally, different positional isomers elute into different subfractions. This facilitates the identification of specific isomers. Since the collection intervals cannot be optimized for all PCDD and PCDF isomers simultaneously, it is difficult to isolate each positional isomer into one subfraction. Some isomers may have eluted while the subfractions were being changed and therefore were found in both subfractions. This, however, does not seriously affect their quantification. On the other hand, compounds which have the same retention time in different subfractions may not necessarily be the same isomers. The distribution of PCDD and PCDF among the different subfractions is summarized in Table 11. From these data as
2446
ANALYTICAL CHEMISTRY, VOL. 56, NO. 13, NOVEMBER 1984
Table 11. Distribution of PCDD and PCDF Detected among Subfractions of Second-Step HPLC Fractionationo
SF2 SF3 SF4 SF5 SF6
TCDD
TCDF
P&DD
P&DF
H&DD
D (100)
D (56) D (44)
D (51) D (49)
D (19) D (10) D (13) D (58)
D (59) D (41)
H&DF
H7CDD
D (10) D (90)
D (27) D (73)
H&DF
OCDD
D (13) D (87)
D (100)
OCDF
D (100)
Parentheses indicate percentage of PCDD (or PCDF) with same number of chlorines found in subfraction.
a
Table 111. Concentrations of PCDD and PCDF in an Ontario Fly Ash Sample as Determined by GC/FID and GC/MS/SIM
compd
concn, ng/g of fly ash, determined by GC/FID (% re1 std dev)"
TCDD TCDF P&DD P&DF H&DD H&DF HTCDD H7CDF OCDD OCDF
436 (2.5) 294 (6.1) 504 (2.5) 508 (13.8) 668 (8.9) 420 (7.3) 622 (4.5) 428 (10.6) 533 (1.7) 101 (9.0)
concn, ng/g of fly ash, determined by GC/MS/SIM (% re1 std dev)'
impurity
b
344 (2.2) 454 (3.3)
I
588 (4.1) 634 (1.2) 563 (12.8) 130 (3.2)
'Based on two injections. well as from the information presented in Figure 4 a few generalization about this reverse-phase HPLC column can be made: (1) PCDD and PCDF elute in order of increasing chlorine atom substitution; (2) PCDD elutes earlier than PCDF with the same number of chlorine atoms; and (3) PSCDF isomers spread into more subfractions than any other PCDD and PCDF isomers. In most cases the positional isomers of PCDF, which elute later on the reverse-phase HPLC column, elute earlier on the DB-5 GC column. Generally, this also applies to PCDD positional isomers. Most likely, this observation is related to the chlorine substitution symmetry of different positional isomers. Good isolation of PCDD and PCDF from almost all other organic components and effective reduction in complexity of GC chromatograms in the PCDD and PCDF region greatly facilitate their quantification With a nonselective detedor such as FID. Table I11 lists the quantitative results of PCDD and PCDF found in the Ontario fly ash sample by GC/FID. The method for their quantification is described in the Experimental Section. If individual isomer standards are available, it would be possible to quantify specific PCDD and PCDF isomers. Some overlapping GC peaks of PCDD and PCDF can be seen in Figure 4. The peak ratio obtained from GC/MS/SIM is used to divide the peak area for quantification by GC/FID (Figure 5). This method was used to divide a few peak areas between TCDD and TCDF and between P&DD and PSCDF. Other PCDD and PCDF were well separated and no difficulty was encountered in their quantification by GC/FID. On the basis of the observation of response behavior of a number of organic compounds on a flame ionization detector, it was assumed that PCDD and PCDF which have the same number of chlorine atoms will have quite similar FID response factors (11). Therefore, other PCDF in the sample were quantified by using the FID response factors obtained from the PCDD standard having the same number of chlorine atoms. The determined FID response factor of OCDD (5.0 area counta/ng) and OCDF (5.1 area counts/ng) partly supports this assumption.
40
50
60
70
MIN
Flgure 0. Gas chromatograms showlng the reproducibility In both elution pattern and recovery of the second-step HPLC separation: (a) subfraction 3 of run 1, (b) subfraction 3 of run 2. Both samples were treated In the same manner with the same volume belng Injected. Chromatographic condltlons are glven in Figure 2.
Quantitation of TCDD, PsCDD, H,CDD, H7CDD, OCDD, and OCDF in the fly ash sample was also done by GC/ MS/SIM for comparison with those results obtained from GC/FID. This method is also described in the Experimental Section. Since characteristic ions of PCDD and PCDF were monitored, the possible peak contamination problem in the GC/FID analysis is not encountered in GC/MS/SIM analysis. Quantitative results of PCDD and PCDF in the Ontario fly ash by GC/MS/SIM are listed in Table 111. Although peak contamination was encountered in the quantification of TCDD, TCDF, P,CDD, and P&DF by GC/FID, the results obtained from the GC/FID and GC/MS/SIM analyses are still reasonably consistent. The reproducibility of the second-step HPLC is illustrated in Figure 9. The gas chromatograms of subfraction 3 obtained from the two separate HPLC runs (run 1 and run 2) are contrasted. The subfraction of each run was treated in the same manner and the same volume was injected on the GC. As illustrated, the distribution pattern and peak area of PCDD and PCDF are identical in both runs except for a larger amount of an unidentified impurty found in run 2. The average recovery of the first-step HPLC using TCDD, H6CDD, H7CDD, and OCDD standards was 105% (IO). The average recovery of the second-sbp HPLC was calculated from four HPLC fractionations. The recovery of TCDD and OCDD standards was determined to be 90% with a relative standard deviation of 5%. No significant impurities were found in the blanks of the Soxhlet extraction and in fraction 2 of the first-step HPLC separation (IO). An unidentified impurity shown in Figure 9 was found in the second-step HPLC separation. However, this impurity does not interfere with the analysis of PCDD and PCDF.
CONCLUSION The two-step semipreparative HPLC separation consisting of a normal-phase followed by a reverse-phase column and solvent gradient elution can be used to effectively isolate PCDD and PCDF from several hundred organic components
2447
Anal. Chem. 1084, 56,2447-2451
existing in raw fly ash extract. The distribution of numerous PCDD and PCDF isomers among several subfractions greatly reduces the peak overlapping in the gas chromatogram. Consequently this facilitates isomer identification, and permita their quantification with nonselective detection such as FID. High recovery and good reproducibility can be obtained in this simple separation procedure. Registry NO.1234-TCDD)30746-5&8; 2378-TCDD)1746-01-6; 12347-PSCDD, 39227-61-7; 123478-H&DD, 39227-28-6; 1234678-H&DD, 35822-46-9; OCDD, 3268-87-9; OCDF, 3900102-0; TCDF, 30402-14-3;PsCDF, 30402-15-4;HBCDF,55684-94-1; HVCDF, 38998-75-3. LITERATURE CITED (1) Esposito, M. P.; Tlernan, T. 0.; Dryden, F. E. “Dioxlns”; U.S. Envlronmental Agency Report, EPA-600/2-80-197, Nov 1980.
(2) Hutzinger, O., Frei, R. W.. Merian, E., Pocchiari, F., Eds. ”Chlorinated Dioxins and Related Compound”; Paragon Press: Toronto, 1982. (3) Camoni, I.; Di Muccio, A.; Pontecorvo, D.;Vergori, L. J. Chromatogr. i m , 153,233-238. (4) Gross, M. L.; Tung Sun; Lyon, P. A,; Wojlnski, S. F.; Hllker, D. E.; Dupuy, A. E., Jr.; Heath, R. G. Anal. Chem. 1881, 53, 1902-1906. (5) Nowicki, H. G.; Kieda. C. A,; Current, V.; Schaefers, T. H. HRC CC, J . High Resoiut Chromatogr . Chromatogr Commun . 198 1 4 178-179. (6) Ryan, J. J.; Pilon, J. C. J. Chromatogr. 1980, 197, 171-180. (7) Langhorst, M. L.; Shadoff, L. A. Anal. Chem. 1980, 52, 2037-2044. (8) Lamparskl, L. L.; Nestrlck, T. J. Anal. Chem. 1980, 52, 2045-2054. (9) Karasek, F. W.; Onuska, F. I . Anal. Chem. 1982, 54, 309A-315A. (10) Tong, H. Y.; Shore, D. L.; Karasek, F. W. J. Chromatogr. 1984, 285, 423-441. (11) Tong, H. Y.; Karasek, F. W. Anal. Chem. 1984, 56,0000.
.
.
I
I
RECEIVED for review March 7,1984. Accepted June 4,1984. Financial support for the Natural Sciences and Engineering Research Council of Canada is gratefully acknowledged.
Use of Multivariate Curve Resolution and a High-speed Diode Array Ultraviolet Detector in Size-Exclusion Chromatography of Lignin-Based Copolymers John C. Nicholson, John J. Meister, Damodar R. Patil, a n d L a r r y R. Field* Southern Methodist University, Department of Chemistry, Dallas, Texas 75275
A copolymer of ilgnln containing a bound chromophore adduct is analyzed by a new technlque that uses the combined methods of slre-exclusion chromatography and multivariate curve resoiutlon. Even though the UV absorptlon spectra of lignin and the bound chromophore differ only slightly, this technique Is able to display graphically the spectral contribution of the chromophore adduct throughout the sire-distributlon chromatogram of the polymer. I n addition, quantitatlve information can be obtained for the contribution of the mass of the adduct to the polymer as a whole or to a partlcuiar molecular sire fraction.
Size-exclusion chromatography (SEC) has become a popular technique for determining the molecular size distribution of polymers. Even though narrow molecular weight standards may not exist for the assessment of weight-average or number-average molecular weight, this technique can still be quite useful in following the course of a particular polymer reaction. Moreover, the use of a series of selective detectors at the end of a SEC column may enable one to deduce important information regarding the distribution of a particular functional group or groups within a specific polymer. Numerous specific detectors have been used for this purpose (e.g., infrared, ultraviolet, and fluorescence) (I) with varying degrees of success. In general these detectors work particularly well for both qualitatively and quantitatively monitoring some spectroscopically responsive portion of the polymer as it elutes from a size-exclusion column. In actual practice a nonspecific refractive index detector is usually inserted in series with one or several of these specific detectors to provide a quantitative point of reference. In cases where the spectral properties of a functional group of interest are not substantially different from that of the parent polymer itself, it is a difficult problem to distinguish 0003-2700/84/0356-2447$01.50/0
these two by ordinary spectral techniques. For example, in this work Kraft lignii,a polymer extracted from woody plants, was grafted with a 2-propenamide monomer containing a 4-methoxyphenyl group. The product of the grafting reaction contains lignin grafted with 1-amidoethylene as well as pure poly(1-amidoethylene) homopolymer that is not connected to any lignin. In this case both graft polymer and ungrafted homopolymer have very similar UV absorption spectra. Thus, there is no unique wavelength to monitor one and not the other during a chromatographic analysis. The UV spectra were, however, found to be sufficiently different to allow the application of a new technique combining multivariate curve resolution mathematics and size-exclusion chromatography to be very successful in spectroscopically resolving the contribution of the grafted agent to the total UV absorption spectra. Thus, by using this procedure it is possible to track the amount of grafted agent added to the polymer as well as its distribution throughout the size-exclusion chromatogram of the polymer even though the spectra of both are quite similar. EXPERIMENTAL SECTION Synopsis of Curve Resolution Software. The multivariate curve resolution software (MCR-2) used in this work was developed by Infometrix Inc., of Seattle, WA, and was designed to be used with the Hewlett-Packard HP1040A high-speed spectrophotometric detector. The multivariate methematics embodied in the MCR-2 software provides the user with a complete solution to a two-component chromatographic resolution problem. The method makes no limiting assumptions and requires only a marix of spectral absorbances, measured over a period of time. In this work the MCR-2 uses d of the spectral absorbance data for six data input channels over a range of time defined by the user as the peak width. If the peak being analyzed contains only one component, all spectra will be nearly the same, and their differences will be below a preset threshold (1%in this case). The program then reports 0 1984 American Chemical Society
