Peak separation of all 136 tetra- to octa-chlorinated dibenzo-p-dioxins and dibenzofurans on two 50% phenyl-methyl-siloxane-type gas chromatography columns, DB-17ms and VF-17ms

Kotaro MINOMO; Nobutoshi OHTSUKA; Yusuke OCHIAI

doi:10.5985/emcr.20240023

ABSTRACT

The peak separation of all 136 tetra- to octa-chlorinated dibenzo-p-dioxins and dibenzofurans on two types of 50% phenyl methyl siloxane gas chromatography columns, DB-17ms and VF-17ms, is presented. Four congeners of the seventeen 2,3,7,8-congeners, 2,3,7,8-TeCDD, 1,2,3,7,8-PeCDD, 2,3,7,8-TeCDF, and 1,2,3,4,7,8-HxCDF, were not separated clearly on DB-17ms. On VF-17ms, 2,3,7,8-TeCDD, 2,3,7,8-TeCDF, 1,2,3,7,8-PeCDF, and 1,2,3,6,7,8-HxCDF were not clearly separated. The other 2,3,7,8-congeners showed good separation on both columns. Therefore, the concentrations of all seventeen 2,3,7,8-congeners can be obtained by combining the measurement results from DB-17ms or VF-17ms with those from DB-5ms or similar columns.

INTRODUCTION

Polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs), referred to as dioxins, are toxic compounds that are highly persistent in the environment. Up to eight chlorines can be substituted in the basic structure of dioxins; there are 210 dioxin congeners (75 PCDDs and 135 PCDFs), which vary with the number and position of chlorine substitutions. Of these 210 congeners, 136 (49 PCDDs and 87 PCDFs) with four or more chlorine atoms, including seventeen 2,3,7,8-chlorine-substituted congeners, are subject to environmental and industrial emissions monitoring. The toxicity equivalency, which is calculated from the concentrations of the seventeen 2,3,7,8-congeners and the toxicity equivalency factor values assigned to each, is usually used to assess the overall toxicity of dioxins (Van den Berg et al., 2006). The concentrations of the seventeen 2,3,7,8-congeners must be accurately measured to calculate an accurate toxic equivalency. Dioxins are usually quantified by using gas chromatography-mass spectrometry (GC-MS) with a capillary column. Separation of the dioxin congeners on GC is of interest to analysts.

Many researchers have reported the separation of dioxin congeners on various capillary columns (Ryan et al., 1991; Bacher and Ballschmiter, 1992; Fraisse et al., 1994; Abad et al., 1997; Matsumura et al., 2003; Fishman et al., 2004, 2007, 2011; Do et al., 2013; Iwakiri and Enomoto, 2018; Stultz and Dorman, 2020). In particular, reports on the separation of all 136 tetra- to octa-chlorinated congeners (Ryan et al., 1991; Bacher and Ballschmiter, 1992; Matsumura et al., 2003; Fishman et al., 2011; Do et al., 2013) are helpful for accurate peak assignment. However, currently, no capillary column capable of separating all seventeen 2,3,7,8-congeners has been reported. Therefore, multiple measurements on different columns are needed to separate all seventeen. Identifying peak assignments on various capillary columns provides analysts with more options. Some studies have conducted the full assignment of the 136 congeners on “DB-5” (5% phenyl methyl silicone or similar)-type columns (Ryan et al., 1991; Fishman et al., 2011), but there have been few on the “DB-17” (50% phenyl methyl silicone or similar) type (Ryan et al., 1991). Therefore, here, we report the confirmed peak separation of all 136 tetra- to octa-dioxins by using two commercially available “50% phenyl”-type columns, namely DB-17ms and VF-17ms. To our knowledge, full assignment of the 136 dioxin congeners by using these two columns has not been reported before.

MATERIALS AND METHODS

REAGENTS

The abbreviations for the PCDD/PCDF congeners were as follows: CDD=chlorinated dibenzo-p-dioxin; CDF=chlorinated dibenzofuran; Te=tetra; Pe=penta; Hx=hexa; Hp=hepta; O=octa.

A Comprehensive Polychlorinated Dioxin and Furan Column Defining Kit (Cambridge Isotope Laboratories (CIL), Andover, MA, USA) was used to determine the retention order of all 136 tetra- to octa-CDDs/CDFs. The standard solution was prepared into 38 mixtures (nos. 1 to 38) containing three to four congeners with no overlapping homologs. ¹³C₁₂-1,2,7,8-TeCDF (Wellington Laboratories, Guelph, Ontario, Canada) was added to the mixtures to allow compensation for shifts in retention time (RT) that may have occurred during the analysis. The concentration of each congener was approximately 5 pg/μL in nonane. In addition, mixed solutions for each homolog (nos. 39 to 42) were prepared. The concentration of each congener was approximately 0.6 pg/μL in nonane. The combinations of congeners in each standard mixture are shown in Table 1.

Table 1 Combination of congeners in the standard mixture

Mixture No.	Congeners
1	1,2,3,4-TeCDF	1,2,3,4,6-PeCDF	1,2,3,4,6,7-HxCDF	1,2,3,4-TeCDD	¹³C₁₂-1,2,7,8-TeCDF
2	1,2,3,6-TeCDF	1,2,3,4,7-PeCDF	1,2,3,4,6,8-HxCDF	1,2,3,6-TeCDD	¹³C₁₂-1,2,7,8-TeCDF
3	1,2,3,7-TeCDF	1,2,3,4,8-PeCDF	1,2,3,4,6,9-HxCDF	1,2,3,7-TeCDD	¹³C₁₂-1,2,7,8-TeCDF
4	1,2,3,8-TeCDF	1,2,3,4,9-PeCDF	1,2,3,4,7,8-HxCDF	1,2,3,8-TeCDD	¹³C₁₂-1,2,7,8-TeCDF
5	1,2,3,9-TeCDF	1,2,3,6,7-PeCDF	1,2,3,4,7,9-HxCDF	1,2,3,9-TeCDD	¹³C₁₂-1,2,7,8-TeCDF
6	1,2,4,6-TeCDF	1,2,3,6,8-PeCDF	1,2,3,4,8,9-HxCDF	1,2,4,6-TeCDD	¹³C₁₂-1,2,7,8-TeCDF
7	1,2,4,7-TeCDF	1,2,3,6,9-PeCDF	1,2,3,6,7,8-HxCDF	1,2,4,7-TeCDD	¹³C₁₂-1,2,7,8-TeCDF
8	1,2,4,8-TeCDF	1,2,3,7,8-PeCDF	1,2,3,6,7,9-HxCDF	1,2,4,8-TeCDD	¹³C₁₂-1,2,7,8-TeCDF
9	1,2,4,9-TeCDF	1,2,3,7,9-PeCDF	1,2,3,6,8,9-HxCDF	1,2,4,9-TeCDD	¹³C₁₂-1,2,7,8-TeCDF
10	1,2,6,7-TeCDF	1,2,3,8,9-PeCDF	1,2,3,7,8,9-HxCDF	1,2,6,7-TeCDD	¹³C₁₂-1,2,7,8-TeCDF
11	1,2,6,8-TeCDF	1,2,4,6,7-PeCDF	1,2,4,6,7,8-HxCDF	1,2,6,8-TeCDD	¹³C₁₂-1,2,7,8-TeCDF
12	1,2,6,9-TeCDF	1,2,4,6,8-PeCDF	1,2,4,6,7,9-HxCDF	1,2,6,9-TeCDD	¹³C₁₂-1,2,7,8-TeCDF
13	1,2,7,8-TeCDF	1,2,4,6,9-PeCDF	1,2,4,6,8,9-HxCDF	1,2,7,8-TeCDD	¹³C₁₂-1,2,7,8-TeCDF
14	1,2,7,9-TeCDF	1,2,4,7,8-PeCDF	1,3,4,6,7,8-HxCDF	1,2,7,9-TeCDD	¹³C₁₂-1,2,7,8-TeCDF
15	1,2,8,9-TeCDF	1,2,4,7,9-PeCDF	1,3,4,6,7,9-HxCDF	1,2,8,9-TeCDD	¹³C₁₂-1,2,7,8-TeCDF
16	1,3,4,6-TeCDF	1,2,4,8,9-PeCDF	2,3,4,6,7,8-HxCDF	1,3,6,8-TeCDD	¹³C₁₂-1,2,7,8-TeCDF
17	1,3,4,7-TeCDF	1,2,6,7,8-PeCDF	1,2,3,4,6-PeCDD	1,3,6,9-TeCDD	¹³C₁₂-1,2,7,8-TeCDF
18	1,3,4,8-TeCDF	1,2,6,7,9-PeCDF	1,2,3,4,7-PeCDD	1,3,7,8-TeCDD	¹³C₁₂-1,2,7,8-TeCDF
19	1,3,4,9-TeCDF	1,3,4,6,7-PeCDF	1,2,3,6,7-PeCDD	1,3,7,9-TeCDD	¹³C₁₂-1,2,7,8-TeCDF
20	1,3,6,7-TeCDF	1,3,4,6,8-PeCDF	1,2,3,6,8-PeCDD	1,4,6,9-TeCDD	¹³C₁₂-1,2,7,8-TeCDF
21	1,3,6,8-TeCDF	1,3,4,6,9-PeCDF	1,2,3,6,9-PeCDD	1,4,7,8-TeCDD	¹³C₁₂-1,2,7,8-TeCDF
22	1,3,6,9-TeCDF	1,3,4,7,8-PeCDF	1,2,3,7,8-PeCDD	2,3,7,8-TeCDD	¹³C₁₂-1,2,7,8-TeCDF
23	1,3,7,8-TeCDF	1,3,4,7,9-PeCDF	1,2,3,7,9-PeCDD		¹³C₁₂-1,2,7,8-TeCDF
24	1,3,7,9-TeCDF	1,3,6,7,8-PeCDF	1,2,3,8,9-PeCDD		¹³C₁₂-1,2,7,8-TeCDF
25	1,4,6,7-TeCDF	1,4,6,7,8-PeCDF	1,2,4,6,7-PeCDD		¹³C₁₂-1,2,7,8-TeCDF
26	1,4,6,8-TeCDF	2,3,4,6,7-PeCDF	1,2,4,6,8-PeCDD		¹³C₁₂-1,2,7,8-TeCDF
27	1,4,6,9-TeCDF	2,3,4,6,8-PeCDF	1,2,4,6,9-PeCDD		¹³C₁₂-1,2,7,8-TeCDF
28	1,4,7,8-TeCDF	2,3,4,7,8-PeCDF	1,2,4,7,8-PeCDD		¹³C₁₂-1,2,7,8-TeCDF
29	1,6,7,8-TeCDF	1,2,3,4,6,7-HxCDD	1,2,4,7,9-PeCDD		¹³C₁₂-1,2,7,8-TeCDF
30	2,3,4,6-TeCDF	1,2,3,4,6,8-HxCDD	1,2,4,8,9-PeCDD		¹³C₁₂-1,2,7,8-TeCDF
31	2,3,4,7-TeCDF	1,2,3,4,6,9-HxCDD	1,2,3,4,6,7,8-HpCDF		¹³C₁₂-1,2,7,8-TeCDF
32	2,3,4,8-TeCDF	1,2,3,4,7,8-HxCDD	1,2,3,4,6,7,9-HpCDF		¹³C₁₂-1,2,7,8-TeCDF
33	2,3,6,7-TeCDF	1,2,3,6,7,8-HxCDD	1,2,3,4,6,8,9-HpCDF		¹³C₁₂-1,2,7,8-TeCDF
34	2,3,6,8-TeCDF	1,2,3,6,7,9-HxCDD	1,2,3,4,7,8,9-HpCDF		¹³C₁₂-1,2,7,8-TeCDF
35	2,3,7,8-TeCDF	1,2,3,6,8,9-HxCDD	OCDF		¹³C₁₂-1,2,7,8-TeCDF
36	2,4,6,7-TeCDF	1,2,3,7,8,9-HxCDD	1,2,3,4,6,7,8-HpCDD		¹³C₁₂-1,2,7,8-TeCDF
37	2,4,6,8-TeCDF	1,2,4,6,7,9-HxCDD	1,2,3,4,6,7,9-HpCDD		¹³C₁₂-1,2,7,8-TeCDF
38	3,4,6,7-TeCDF	1,2,4,6,8,9-HxCDD	OCDD		¹³C₁₂-1,2,7,8-TeCDF
39	TeCDFs,
40	PeCDFs	HxCDDs
41	HxCDFs	PeCDDs	Hp–OCDFs	Hp–OCDDs
42	TeCDDs

COLUMNS

Two chromatographic columns (length: 60 m; id: 0.25 mm; film thickness: 0.25 μm), namely DB-17ms (Agilent Technologies, Santa Clara, CA, USA) and VF-17ms (Agilent Technologies), were tested for the separation of PCDDs/PCDFs. Both columns are 50%-phenyl-type columns, but the substitution position of the phenyl group in the liquid phase differs, with silphenylene–dimethylsiloxane for DB-17ms and diphenylsiloxane–dimethylsiloxane for VF-17ms (Fig. 1).

Fig. 1 Structure of liquid phase for (a) DB-17ms and (b) VF-17ms. Chemical formulas are referenced from Agilent Technologies (2016)

MEASUREMENT

PCDDs/PCDFs in the standard solutions were measured by using high-resolution GC-MS (Agilent 7890A GC (Agilent Technologies)+JMS-800D (JEOL, Tokyo, Japan)). MS was performed under positive electron ionization (resolution: >10,000), and data were acquired in selected ion monitoring mode. The same GC temperature program was used for the two columns: 130°C (2 min) to 210°C (15°C/min) to 310°C (3°C/min), and finally to 320°C (5°C/min, 17 min) (total: 59.7 min). Injection was performed in splitless mode at a temperature of 280°C. Helium was used as the carrier gas for the column, and the flow rate was 1.5 mL/min. The monitoring ion channels (m/z) were 321.8936 for TeCDDs; 355.8546 for PeCDDs; 389.8156 for HxCDDs; 423.7767 for HxCDDs; 459.7348 for OCDD; 305.8987 for TeCDFs; 339.8597 for PeCDFs; 373.8207 for HxCDFs; 407.7818 for HpCDFs; 443.7398 for OCDF; and 317.9389 for ¹³C₁₂-1278-TeCDF. The data acquisition time of each ion channel was 27 ms and the cycle time was 500 ms. Increasing the number of monitoring channels reduces the data acquisition time, resulting in reduced sensitivity. To confirm peak separation more clearly, the number of monitoring channels per homolog was set to one, and the number of data points in the chromatogram was increased. Although analytical manuals in Japan require the monitoring of two or more homolog channels for the determination of dioxins (JSA, 2020a, 2020b; MOE, 2022a, 2022b, 2022c), one monitoring channel was considered sufficient because each congener of the standard material used was prepared with high purity.

Peak assignment was performed by using the dioxin quantitative program DioK ver. 4.02 (JEOL). The time of each peak (the center of gravity) was treated as the RT. Relative retention times were calculated for each peak relative to the RT of ¹³C₁₂-1,2,7,8-TeCDF.

RESULTS AND DISCUSSION

Table 2 shows the retention order of all 136 PCDDs/PCDFs congeners on the two columns. Additionally, the retention order is visually displayed in Figs. 2, 3, 4, 5, 6, 7, 8.

Table 2 Gas chromatography elution order of tetra- to hexa-CDDs and CDFs on two 50%-phenyl-type capillary columns

Homolog	DB-17ms capillary column						VF-17ms capillary column
Homolog	Elution order	Congener	RRT^a	Elution order	Congener	RRT	Elution order	Congener	RRT	Elution order	Congener	RRT
TeCDD	1	1368	0.913	12	1279	1.013	1	1368	0.912	12	1279	1.013
	2	1379	0.931	13	2378	1.020	2	1379	0.930	13	2378	1.016
	3	1369	0.958	14	1234	1.023	3	1369	0.959	14	1238	1.022
	4	1378	0.969	15	1238	1.023	4	1378	0.966	15	1237	1.023
	5	1247	0.979	16	1237	1.026	5	1247	0.979	16	1234	1.026
	6	1248	0.979	17	1236	1.029	6	1248	0.979	17	1236	1.029
	7	1268	0.996	18	1269	1.043	7	1268	0.995	18	1269	1.044
	8	1249	0.997	19	1239	1.045	8	1478	0.998	19	1239	1.046
	9	1246	0.998	20	1278	1.052	9	1249	1.000	20	1278	1.050
	10	1478	0.999	21	1267	1.082	10	1246	1.001	21	1267	1.081
	11	1469	1.003	22	1289	1.095	11	1469	1.007	22	1289	1.096
PeCDD	1	12479	1.110	8	12489	1.198	1	12479	1.106	8	12347	1.193
	2	12468	1.111	9	12347	1.198	2	12468	1.106	9	12489	1.193
	3	12368	1.148	10	12467	1.199	3	12368	1.141	10	12467	1.194
	4	12469	1.152	11	12346	1.212	4	12469	1.150	11	12378	1.204
	5	12478	1.160	12	12378	1.213	5	12478	1.152	12	12346	1.211
	6	12379	1.167	13	12367	1.237	6	12379	1.160	13	12367	1.229
	7	12369	1.190	14	12389	1.253	7	12369	1.186	14	12389	1.247
HxCDD	1	124679	1.298	6	123469	1.359	1	124679	1.291	6	123469	1.355
	2	124689	1.298	7	123478	1.379	2	124689	1.291	7	123478	1.367
	3	123468	1.323	8	123678	1.388	3	123468	1.314	8	123678	1.375
	4	123689	1.344	9	123789	1.407	4	123689	1.333	9	123789	1.394
	5	123679	1.344	10	123467	1.412	5	123679	1.334	10	123467	1.404
HpCDD	1	1234679	1.502	2	1234678	1.553	1	1234679	1.492	2	1234678	1.538
OCDD			1.701						1.686
TeCDF	1	1368	0.879	20	1678	0.989	1	1368	0.879	20	2467	0.991
	2	1468	0.911	21	2467	0.992	2	1468	0.913	21	1678	0.992
	3	2468	0.918	22	1236	0.995	3	2468	0.918	22	1236	0.997
	4	1378	0.922	23	1234	0.996	4	1378	0.921	23	1234	0.999
	5	1347	0.924	24	1278	1.001	5	1347	0.924	24	1278	1.001
	6	1247	0.929	25	1469	1.016	6	1247	0.929	25	2378	1.017
	7	1367	0.937	26	2378	1.019	7	1367	0.937	26	1469	1.018
	8	1346	0.940	27	2347	1.021	8	1379	0.940	27	2347	1.020
	9	1379	0.941	28	1349	1.023	9	1346	0.942	28	1349	1.025
	10	1246	0.948	29	2346	1.027	10	1246	0.950	29	2346	1.029
	11	1348	0.951	30	2348	1.029	11	1348	0.951	30	2348	1.029
	12	1248	0.953	31	1267	1.030	12	1248	0.953	31	1267	1.029
	13	1478	0.960	32	1279	1.034	13	1478	0.961	32	1279	1.034
	14	1268	0.966	33	1249	1.040	14	1268	0.965	33	1249	1.041
	15	1467	0.968	34	2367	1.049	15	2368	0.970	34	2367	1.047
	16	2368	0.971	35	3467	1.059	16	1467	0.970	35	3467	1.059
	17	1237	0.972	36	1239	1.074	17	1237	0.970	36	1239	1.076
	18	1369	0.980	37	1269	1.085	18	1369	0.981	37	1269	1.085
	19	1238	0.986	38	1289	1.133	19	1238	0.987	38	1289	1.133
PeCDF	1	13468	1.060	15	12346	1.163	1	13468	1.056	15	12346	1.162
	2	12468	1.063	16	12378	1.170	2	12468	1.058	16	12378	1.163
	3	13678	1.106	17	12348	1.174	3	13678	1.101	17	12348	1.170
	4	12368	1.118	18	12469	1.178	4	12368	1.113	18	12469	1.175
	5	12478	1.119	19	12367	1.188	5	12478	1.114	19	12678	1.182
	6	13478	1.120	20	12678	1.188	6	13478	1.115	20	12367	1.182
	7	13467	1.121	21	12379	1.198	7	13467	1.118	21	12379	1.191
	8	12467	1.129	22	23478	1.220	8	12467	1.125	22	23478	1.212
	9	13479	1.132	23	12369	1.234	9	13479	1.126	23	23467	1.228
	10	14678	1.132	24	23467	1.235	10	14678	1.129	24	12369	1.230
	11	12479	1.147	25	12679	1.238	11	12479	1.141	25	12679	1.232
	12	12347	1.152	26	12489	1.255	12	12347	1.148	26	12489	1.249
	13	23468	1.155	27	12349	1.259	13	23468	1.148	27	12349	1.257
	14	13469	1.159	28	12389	1.297	14	13469	1.157	28	12389	1.291
HxCDF	1	123468	1.275	9	123678	1.351	1	123468	1.266	9	123678	1.340
	2	134678	1.283	10	123479	1.364	2	134678	1.274	10	123479	1.354
	3	124678	1.284	11	123679	1.386	3	124678	1.275	11	123679	1.374
	4	134679	1.301	12	123469	1.386	4	134679	1.292	12	123469	1.380
	5	124679	1.319	13	234678	1.397	5	124679	1.309	13	234678	1.385
	6	124689	1.338	14	123689	1.400	6	124689	1.327	14	123689	1.389
	7	123478	1.343	15	123789	1.458	7	123478	1.332	15	123789	1.446
	8	123467	1.346	16	123489	1.472	8	123467	1.338	16	123489	1.463
HpCDF	1	1234678	1.496	3	1234689	1.540	1	1234678	1.482	3	1234689	1.528
	2	1234679	1.523	4	1234789	1.610	2	1234679	1.511	4	1234789	1.595
OCDF			1.747						1.729

^a RRT, relative retention time. The RT reference standard was ¹³C₁₂-1,2,7,8-TeCDF. The RT of ¹³C₁₂-1,2,7,8-TeCDF in standard mixtures nos. 1 to 38 averaged 26.972 min (range: 26.948 to 26.997 min) on DB-17ms and 27.161 min (range: 27.135 to 27.188 min) on VF-17ms. Bold letters denote 2,3,7,8-congeners.

Fig. 2 Chromatograms of TeCDD congeners (standard solution no. 42) on DB-17ms (a) and VF-17ms (b). The peak in red is 2,3,7,8-TeCDD

Fig. 3 Chromatograms of PeCDD congeners (standard solution no. 41) on DB-17ms (a) and VF-17ms (b). The peak in red is 1,2,3,7,8-PeCDD

Fig. 4 Chromatograms of HxCDD congeners (standard solution no. 40) on DB-17ms (a) and VF-17ms (b). The peaks in red are 2,3,7,8-chlorine-substituted congeners

Fig. 5 Chromatograms of TeCDF congeners (standard solution no. 39) on DB-17ms (a) and VF-17ms (b). The peak in red is 2,3,7,8-TeCDF

Fig. 6 Chromatograms of PeCDF congeners (standard solution no. 40) on DB-17ms (a) and VF-17ms (b). The peaks in red are 2,3,7,8-chlorine-substituted congeners

Fig. 7 Chromatograms of HxCDF congeners (standard solution no. 41) on DB-17ms (a) and VF-17ms (b). The peaks in red are 2,3,7,8-chlorine-substituted congeners

Fig. 8 Chromatograms of hepta- and octa-CDD/CDF congeners (standard solution no. 41) on DB-17ms (a) and VF-17ms (b). The peaks in red are 2,3,7,8-chlorine-substituted congeners

For the 2,3,7,8-congeners, peak resolutions (R) were calculated by using Eq. 1.

R= t 2 - t 1 1 2 ( w 1 + w 2 )

(1)

where t₁: RT of first peak; t₂: RT of second peak; w₁: peak width at base of the first peak; and w₂: peak width at base of the second peak.

TETRA- TO HEXA-CDDs

We constructed chromatograms of the TeCDD congeners (Fig. 2). On DB-17ms, 2,3,7,8-TeCDD was partially separated from the closest congeners (1,2,3,4-/1,2,3,8-TeCDD) (Fig. 2a), with a peak resolution of R=0.6. VF-17ms partially separated 2,3,7,8-TeCDD from the closest congener, 1,2,7,9-TeCDD (Fig. 2b), with R=0.9.

We also constructed chromatograms of the PeCDD congeners (Fig. 3). 1,2,3,7,8-PeCDD was co-eluted with 1,2,3,4,6-PeCDD on DB-17ms (Fig. 3a). In contrast, there was baseline separation of 1,2,3,7,8-PeCDD from the nearest congener, 1,2,3,4,6-PeCDD, on VF-17ms (Fig. 3b), with R=1.9.

We then created chromatograms of the HxCDD congeners (Fig. 4). The elution order of HxCDDs on DB-17ms and VF-17ms was the same. All 2,3,7,8-chlorine-substituted congeners were separated. DB-17ms gave R values of 1.8 for 1,2,3,4,7,8-HxCDD, 1.8 for 1,2,3,6,7,8-HxCDD, and 1.4 for 1,2,3,7,8,9-HxCDD (Fig. 4a). VF-17ms separated the 2,3,7,8-congeners more clearly, with R=2.0 for 1,2,3,4,7,8-HxCDD, 2.0 for 1,2,3,6,7,8-HxCDD, and 2.2 for 1,2,3,7,8,9-HxCDD (Fig. 4b).

TETRA- TO HEXA-CDFs

Chromatograms of the TeCDF congeners were constructed (Fig. 5). The elution orders of 2,3,7,8-TeCDF and the nearby congeners differed on the two columns tested. The order on DB-17ms was 1,4,6,9-TeCDF, 2,3,7,8-TeCDF, and 2,3,4,7-TeCDF, and that on VF-17ms was 2,3,7,8-TeCDF, 1,4,6,9-TeCDF, and 2,3,4,7-TeCDF. On DB-17ms, 2,3,7,8-TeCDF was slightly separated from its closest congener, 2,3,4,7-TeCDF (Fig. 5a), with R=0.3. In contrast, 2,3,7,8-TeCDF was co-eluted with 1,4,6,9-TeCDF and 2,3,4,7-TeCDF on VF-17ms (Fig. 5b).

We then created chromatograms of the PeCDF congeners (Fig. 6). The two columns demonstrated good separation of 2,3,4,7,8-PeCDF on DB-17ms (Fig. 6a), with R=3.7, and on VF-17ms (Fig. 6b), with R=3.3. In addition, 1,2,3,7,8-PeCDF was separated on DB-17ms, with a peak resolution of R=1.0, from the closest congener, 1,2,3,4,8-PeCDF. In contrast, 1,2,3,7,8-PeCDF was co-eluted with 1,2,3,4,6-PeCDF on VF-17ms.

Next, we constructed chromatograms of the HxCDF congeners (Fig. 7). DB-17ms separated 1,2,3,6,7,8-HxCDF, 2,3,4,6,7,8-HxCDF, and 1,2,3,7,8,9-HxCDF from the other congeners (R=1.1, 1.0, and 3.2, respectively), and it partially separated 1,2,3,4,7,8-HxCDF (R=0.7). VF-17ms separated 1,2,3,4,7,8-HxCDF, 2,3,4,6,7,8-HxCDF, and 1,2,3,7,8,9-HxCDF from the other congeners (R=1.1, 1.1, and 3.2, respectively); however, 1,2,3,6,7,8-HxCDF was co-eluted with 1,2,3,4,6,7-HxCDF.

HEPTA- AND OCTA-CDDs/CDFs

We also constructed chromatograms of the hepta- and octa-CDD/CDF congeners (Fig. 8a, b). The hepta-CDD/CDF congeners were separated from each other, and the elution orders were the same on the two columns we tested, as on the many columns previously reported (Ryan et al., 1991; Bacher and Ballschmiter, 1992; Matsumura et al., 2003; Fishman et al., 2011). The elution order of the hepta- and octa-CDD congeners was 1,2,3,4,6,7,9-HpCDD and 1,2,3,4,6,7,8-HpCDD, followed by OCDD, whereas that of the hepta- and octa-CDF congeners was 1,2,3,4,6,7,8-HpCDF, 1,2,3,4,6,7,9-HpCDF, 1,2,3,4,6,8,9-HpCDF, and 1,2,3,4,7,8,9-HpCDF, followed by OCDF.

DIFFERENCE BETWEEN DB-17 AND DB-17ms

The full assignment of all 136 PCDD/DFs on DB-17, a column analogous to DB-17ms, has been reported by Ryan et al. (1991). DB-17ms is a version of DB-17 designed to MS specifications (Agilent Technologies, 2016). A brief comparison of the two columns is provided here for the reader’s convenience. The 2,3,7,8-congeners that DB-17ms, but not DB-17, can separate are 2,3,7,8-TeCDD, 1,2,3,7,8-PeCDF, and 1,2,3,6,7,8-HxCDF. Conversely, the 2,3,7,8-congeners that DB-17, but not DB-17ms, can separate are 1,2,3,7,8-PeCDD and 2,3,7,8-TeCDF. The remaining 12 congeners can be separated on both columns.

COMBINATION WITH OTHER COLUMNS FOR COMPLETE SEPARATION OF THE SEVENTEEN 2,3,7,8-CONGENERS

The separation of 2,3,7,8-chlorine substituted congeners on the two columns is summarized in Table 3. Six 2,3,7,8-congeners were partially separated or co-eluted on the two columns: 2,3,7,8-TeCDD (DB-17ms and VF-17ms), 1,2,3,7,8-PeCDD (DB-17ms), 2,3,7,8-TeCDF (DB-17ms and VF-17ms), 1,2,3,7,8-PeCDF (VF-17ms), 1,2,3,4,7,8-HxCDF (DB-17ms), and 1,2,3,6,7,8-HxCDF (VF-17ms). Therefore, the two columns tested can be combined with columns capable of separating these six congeners to separate all of the 2,3,7,8-congeners. According to the literature, DB-5ms, VF-5ms, VF-Xms (Fishman et al., 2011), and BPX-DXN (Matsumura et al., 2003) demonstrate good separation of these six congeners. Therefore, their combination (DB-17ms or VF-17ms×DB-5ms, VF-5ms, VF-Xms, or BPX-DXN) should provide complete separation of all seventeen 2,3,7,8-congeners.

Table 3 Separation of 2,3,7,8-chlorine-substituted PCDD/DFs on the two columns

Compound	DB-17ms	VF-17ms
2,3,7,8-TeCDD	+−	+−
1,2,3,7,8-PeCDD	−−	++
1,2,3,4,7,8-HxCDD	++	++
1,2,3,6,7,8-HxCDD	++	++
1,2,3,7,8,9-HxCDD	++	++
1,2,3,4,6,7,8-HpCDD	++	++
OCDD	++	++
2,3,7,8-TeCDF	−−	−−
1,2,3,7,8-PeCDF	++	−−
2,3,4,7,8-PeCDF	++	++
1,2,3,4,7,8-HxCDF	+−	++
1,2,3,6,7,8-HxCDF	++	−−
2,3,4,6,7,8-HxCDF	++	++
1,2,3,7,8,9-HxCDF	++	++
1,2,3,4,6,7,8-HpCDF	++	++
1,2,3,4,7,8,9-HpCDF	++	++
OCDF	++	++

++ Peak well separated, R≥1.0; +− peak partially separated, 0.6≤R<1.0; −− peak co-eluted, R<0.6

CONCLUSIONS

Here, we presented the complete assignment of all 136 tetra- to octa-CDDs/CDFs separated on two gas chromatography columns, DB-17ms and VF-17ms. Of the seventeen 2,3,7,8-congeners, 2,3,7,8-TeCDD, 1,2,3,7,8-PeCDD, 2,3,7,8-TeCDF, and 1,2,3,4,7,8-HxCDF were partially separated or co-eluted on DB-17ms, and 2,3,7,8-TeCDD, 2,3,7,8-TeCDF, 1,2,3,7,8-PeCDF, and 1,2,3,6,7,8-HxCDF were partially separated or co-eluted on VF-17ms. The other 2,3,7,8-congeners showed good separation on both columns. The concentrations of all seventeen 2,3,7,8-congeners can be obtained by combining the measurement results from DB-17ms or VF-17ms with those from DB-5ms or similar columns.

Currently, the combination of BPX-DXN and RH12ms is the mainstream method for dioxin analysis in Japan (Iwakiri and Enomoto, 2018). Although this combination can separate almost all 2,3,7,8-congeners, the separation of 2,3,4,6,7,8-HxCDF is insufficient (Matsumura et al., 2003). By applying the combination of DB-17ms or VF-17ms with DB-5ms or similar columns, as mentioned above, sufficient separation of all 2,3,7,8-congeners—including 2,3,4,6,7,8-HxCDF—is possible.

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