Peak separation of all seventeen 2,3,7,8-substituted polychlorinated dibenzo-p-dioxins and dibenzofurans in a single injection using a narrow-bore extended-length DB-17ms chromatography capillary column

ABSTRACT

The chromatographic separation of tetra- to octa-chlorinated dibenzo-p-dioxins and dibenzofurans was evaluated using a DB-17ms column 80 m long, with a 0.18-mm inner diameter (ID) and a 0.18-μm film thickness. This column effectively resolved two 2,3,7,8-substituted congeners, 1,2,3,7,8-pentachlorodibenzo-p-dioxin and 2,3,7,8-tetrachlorodibenzofuran, which had previously been co-eluted with other congeners on a 60-m×0.25-mm ID, 0.25-μm-film-thickness DB-17ms column. All seventeen 2,3,7,8-substituted congeners were separated effectively in a single injection using the narrow-bore extended-length DB-17ms column. To the best of our knowledge, this represents the first report of a column capable of achieving effective separation of all seventeen 2,3,7,8-substituted congeners.

INTRODUCTION

Polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs), collectively referred to as dioxins, are important environmental contaminants. The dioxin family comprises 210 congeners, of which 136 (49 PCDDs and 87 PCDFs) containing four or more chlorine atoms are routinely monitored to determine their environmental levels and industrial emissions. Among these, seventeen 2,3,7,8-chlorine-substituted congeners, characterized by their toxicity equivalency factor values (Van den Berg et al., 2006), must be precisely quantified to determine the toxic equivalency.

Dioxins are usually analyzed using gas chromatography – mass spectrometry (GC-MS) equipped with a capillary column. Although various capillary columns have been evaluated for the separation of dioxin congeners (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; Minomo et al., 2024), no single column has been reported to achieve complete separation of all seventeen 2,3,7,8-substituted congeners. Therefore, multiple measurements using different columns are required to resolve all 17 congeners in accordance with official Japanese analytical methods (Japanese Standards Association, 2020a, 2020b; Ministry of the Environment, Japan, 2022a, 2022b, 2022c). If all 17 congeners could be separated in a single injection, it would reduce the analysis time and cost. We previously conducted a full assignment of the 136 tetra- to octa-chlorinated PCDD/DFs using a DB-17ms capillary column 60 m long, with a 0.25-mm inner diameter (ID) and a 0.25 μm film thickness (hereafter referred to as the normal column). As a result, only two of the 17 congeners, 1,2,3,7,8-pentachlorodibenzo-p-dioxin and 2,3,7,8-tetrachlorodibenzofuran, exhibited co-elution (Minomo et al., 2024). In general, chromatographic separation improves with increased column length and decreased ID (Agilent Technologies, 2016). We therefore further investigated separation using a longer and narrower DB-17ms column.

MATERIALS AND METHODS

REAGENTS

The abbreviations used for the PCDD/DF congeners were as follows: CDD (chlorinated dibenzo-p-dioxin), CDF (chlorinated dibenzofuran), Te (tetra), Pe (penta), Hx (hexa), Hp (hepta), and O (octa).

As previously reported by Minomo et al. (2024), a total of 38 standard solutions prepared from a Comprehensive Polychlorinated Dioxin and Furan Column Defining Kit (Cambridge Isotope Laboratories (CIL), Andover, MA, USA) were used to determine the retention order of all 136 tetra- to octa-substituted CDD/CDFs. These standard solutions contained ¹³C₁₂-1,2,7,8-TeCDF, which was used to establish the relative retention time (RRT) of each peak.

CAPILLARY COLUMN

A DB-17ms chromatographic capillary column (Agilent Technologies, Santa Clara, CA, USA) 80 m long, with a 0.18 mm ID and 0.18 μm film thickness (hereafter referred to as the longer, narrower column) was tested for the separation of PCDD/DFs. As no 80-m column was commercially available, four 20-m columns were connected using Ultra inert universal press fit connectors (Agilent Technologies) to achieve the desired configuration.

MEASUREMENT

PCDD/DFs were analyzed using high-resolution GC-MS: an Agilent 8890A GC system (Agilent Technologies) coupled with a JMS-800D UltraFOCUS MS system (JEOL, Tokyo, Japan). The MS measurement conditions were the same as those previously reported by us (Minomo et al., 2024).

Before measurement of the standard solutions, the GC conditions were optimized using a fly ash sample from a waste incinerator. The final GC conditions were as follows:

• Oven temperature program: 130 °C (2 min), ramping to 210 °C at 5 °C/min, then to 275 °C at 1 °C/min, and finally to 320 °C at 6 °C/min, holding for 27.5 min (total run time: 118 min).

• Injection method: Splitless injection at 280 °C.

• Carrier gas: Helium at a flow rate of 0.9 mL/min. Head pressure at the final oven temperature (320 °C) was 97.5 psi (0.672 MPa).

PEAK ASSIGNMENT

Peaks were identified using the dioxin quantification program DioK ver. 4.02 (JEOL). The retention time (RT) of each peak was determined on the basis of its center of gravity. RRTs were calculated by comparing each peak’s RT with that of ¹³C₁₂-1,2,7,8-TeCDF. To evaluate the reproducibility of chromatographic peak separation, the homolog mixtures were analyzed in triplicate. Peak resolution (R) values for the 2,3,7,8-substituted congeners were calculated in the same way as previously reported by Minomo et al. (2024).

RESULTS AND DISCUSSION

Table 1 presents the retention order of all 136 PCDD/DF congeners, and Fig. 1 illustrates the chromatograms of homolog mixtures. The earliest-eluting congener was identified as 1,3,6,8-TeCDF (RT=54 min, Fig. 1f) and the latest as OCDF (RT=106 min, Fig. 1j). Table 2 presents the average R values for 2,3,7,8-substituted congeners. The chromatographic reproducibility of peak separation was demonstrated to be satisfactory, as indicated by the low relative standard deviation (RSD) values, ranging from 0.5% to 2.0%. The longer, narrower column provided superior separation performance over the normal column. The congeners previously co-eluting on the normal column— specifically 1,2,3,7,8-PeCDD and 2,3,7,8-TeCDF (Minomo et al., 2024)—were effectively separated on the longer, narrower column, with peak resolution values of R=0.6 and R=0.9, respectively (Figs. 1b and 1f). Additionally, the congeners partially separated on the normal column, 2,3,7,8-TeCDD and 1,2,3,4,7,8-HxCDF (Minomo et al., 2024), exhibited improved resolution (R=1.0 for both) on the longer, narrower column (Figs. 1a and 1h). The remaining thirteen 2,3,7,8-substituted congeners demonstrated good separation (R>1.0) on the longer, narrower column, as had occurred with the normal column (Minomo et al., 2024). These findings confirmed that the longer, narrower column achieved effective separation of all seventeen 2,3,7,8-substituted congeners in a single injection.

Table 1 Gas-chromatography elution order of tetra- to octa-CDDs and CDFs on a DB-17ms column with 80-m length, 0.18-mm inner diameter, and 0.18-µm film thickness

Elution order	TeCDDs		PeCDDs		HxCDDs		HpCDDs		OCDD		TeCDFs		PeCDFs		HxCDFs		HpCDFs		OCDF
	Congener	RRTa	Congener	RRT	Congener	RRT	Congener	RRT	Congener	RRT	Congener	RRT	Congener	RRT	Congener	RRT	Congener	RRT	Congener	RRT
1	1368	0.901	12479	1.135	124679	1.358	1234679	1.499	12346789	1.647	1368	0.860	13468	1.073	123468	1.333	1234678	1.495	12346789	1.683
2	1379	0.922	12468	1.136	124689	1.358	1234678	1.534			1468	0.898	12468	1.077	134678	1.342	1234679	1.513
3	1369	0.953	12368	1.181	123468	1.379					2468	0.906	13678	1.127	124678	1.342	1234689	1.525
4	1378	0.966	12469	1.184	123689	1.395					1378	0.909	12368	1.143	134679	1.358	1234789	1.578
5	1247	0.978	12478	1.195	123679	1.396					1347	0.912	12478	1.144	124679	1.374
6	1248	0.979	12379	1.202	123469	1.406					1247	0.918	13478	1.145	124689	1.389
7	1268	0.999	12369	1.231	123478	1.420					1367	0.926	13467	1.146	123478	1.393
8	1249	0.999	12347	1.240	123678	1.426					1346	0.930	12467	1.156	123467	1.395
9	1246	1.000	12489	1.240	123789	1.437					1379	0.931	13479	1.159	123678	1.399
10	1478	1.002	12467	1.242	123467	1.441					1246	0.940	14678	1.159	123479	1.408
11	1469	1.006	12346	1.256							1348	0.944	12479	1.177	123679	1.423
12	1279	1.017	12378	1.258							1248	0.946	12347	1.182	123469	1.423
13	2378	1.026	12367	1.288							1478	0.954	23468	1.188	234678	1.431
14	1234	1.028	12389	1.306							1268	0.961	13469	1.192	123689	1.433
15	1238	1.029									1467	0.963	12346	1.195	123789	1.469
16	1237	1.032									1237	0.966	12378	1.204	123489	1.478
17	1236	1.036									2368	0.967	12348	1.209
18	1269	1.053									1369	0.977	12469	1.215
19	1239	1.054									1238	0.984	12367	1.226
20	1278	1.063									1678	0.987	12678	1.226
21	1267	1.100									2467	0.992	12379	1.237
22	1289	1.115									1234	0.994	23478	1.266
23											1236	0.995	12369	1.282
24											1278	1.001	23467	1.284
25											1469	1.019	12679	1.287
26											2378	1.023	12489	1.307
27											2347	1.026	12349	1.311
28											1349	1.027	12389	1.352
29											2346	1.033
30											1267	1.035
31											2348	1.036
32											1279	1.040
33											1249	1.047
34											2367	1.058
35											3467	1.070
36											1239	1.087
37											1269	1.101
38											1289	1.157

a)  RRT, relative retention time. The retention time (RT) reference standard was ¹³C₁₂-1,2,7,8-TeCDF. The average RT of ¹³C₁₂-1,2,7,8-TeCDF in 38 standard mixtures was 62.986 min (range: 62.946 to 63.030 min). Bold letters denote 2,3,7,8-PCDD/DFs.

Fig. 1 Chromatograms of tetra- to octa-CDDs and CDFs on a DB-17ms column with 80-m length, 0.18-mm inner diameter, and 0.18-μm film thickness. Peaks in red are 2,3,7,8-chlorine-substituted congeners

Table 2 Average peak resolution and relative standard deviation (RSD) for each 2,3,7,8-congener on the longer, narrower DB-17ms column

Compound	Average peak resolution (N=3)	RSD (%)
2,3,7,8-TeCDD	1.03	0.8
1,2,3,7,8-PeCDD	0.63	2.0
1,2,3,4,7,8-HxCDD	3.04	1.2
1,2,3,6,7,8-HxCDD	3.04	1.2
1,2,3,7,8,9-HxCDD	1.82	1.3
1,2,3,4,6,7,8-HpCDD	15.5	0.7
OCDD	–
2,3,7,8-TeCDF	0.90	0.5
1,2,3,7,8-PeCDF	1.50	1.7
2,3,4,7,8-PeCDF	3.49	1.4
1,2,3,4,7,8-HxCDF	1.02	0.8
1,2,3,6,7,8-HxCDF	1.75	1.1
2,3,4,6,7,8-HxCDF	1.01	0.9
1,2,3,7,8,9-HxCDF	4.52	1.5
1,2,3,4,6,7,8-HpCDF	8.02	0.9
1,2,3,4,7,8,9-HpCDF	21.8	0.8
OCDF	–

PCDD/DFs in fly ash samples from waste incinerators were quantified by using the longer, narrower column. Representative chromatograms (Fig. 2) demonstrate good resolution of the 2,3,7,8-substituted congeners. The analytical results obtained via the longer, narrower column were subsequently compared with those acquired through established standard methodologies (Fig. 3). There was strong concordance between the two approaches, with relative concentration ratios ranging from 0.92 to 1.10. The results indicated that the longer, narrower column achieved effective separation of 2,3,7,8-substituted congeners within actual sample matrices.

Fig. 2 Chromatograms of 2,3,7,8-substituted tetra- to hexa-CDDs and CDFs in fly ash samples from a waste incinerator, as analyzed by using the longer and narrower column. Peaks in red are 2,3,7,8-chlorine-substituted congeners

Fig. 3 Ratio of 2,3,7,8-substituted congener concentration in fly ash samples measured on the longer, narrower column to those measured by the standard method (N=5). The columns used in the standard method: DB-17ms (60 m long, with a 0.25 mm ID and a 0.25 μm film thickness, applied for PeCDFs and 2,3,4,6,7,8-/1,2,3,7,8,9-HxCDFs) and DB-5ms (60 m long, with a 0.25 mm ID and a 0.25 μm film thickness, applied for the other congeners)

CONCLUSIONS

The seventeen 2,3,7,8-chlorinated congeners were separated effectively in a single injection using a DB-17ms capillary column 80 m long, with a 0.18 mm ID and 0.18 μm film thickness. To the best of our knowledge, this is the first report of a column capable of effectively separating all seventeen 2,3,7,8-substituted congeners. Although the conditions established here may not be highly practical owing to the extended analysis time of 118 min, more efficient and practical separation could potentially be achieved by modifying the column size, temperature program, or other parameters. Even with columns for which the elution order has been previously reported, adjustments in column size may lead to improved separation efficiency. Our findings provide valuable insights that could help in the development of new column designs optimized for dioxin analysis.

ACKNOWLEDGMENTS

This research was performed by the Environment Research and Technology Development Fund (JPMEERF20255004) of the Environmental Restoration and Conservation Agency under the auspices of the Ministry of the Environment of Japan.

REFERENCES

•  Abad,  E.,  Caixach,  J.,  Rivera,  J., 1997. Application of DB-5ms gas chromatography column for the complete assignment of 2, 3, 7, 8-substituted polychlorodibenzo-p-dioxins and polychlorodibenzofurans in samples from municipal waste incinerator emissions. J. Chromatogr. A 786, 125–134. doi: 10.1016/S0021-9673(97)00538-4.
• Agilent Technologies, 2016. The essential chromatography & spectroscopy catalog 2016/17 GC and GC/MS. https://hpst.cz/sites/default/files/download/2024/08/5991-5213EN_GC_Catalog_Columns.pdf (accessed 1 May 2025)
•  Bacher,  R.,  Ballschmiter,  K., 1992. Separation of polychlorodibenzo-p-dioxins and-dibenzofurans on the new polar stationary phase DB DIOXIN. Chromatographia 34, 137–142. doi: 10.1007/BF02276126.
•  Do,  L.,  Liljelind,  P.,  Zhang,  J.,  Haglund,  P., 2013. Comprehensive profiling of 136 tetra-to octa-polychlorinated dibenzo-p-dioxins and dibenzofurans using ionic liquid columns and column combinations. J. Chromatogr. A 1311, 157–169. doi: 10.1016/j.chroma.2013.08.070.
•  Fishman,  V. N.,  Martin,  G. D.,  Lamparski,  L. L., 2004. Comparison of Series 5 gas chromatography column performances from a variety of manufacturers for separation of chlorinated dibenzo-p-dioxins and dibenzofurans using high-resolution mass spectrometry. J. Chromatogr. A 1057, 151–161. doi: 10.1016/j.chroma.2004.09.034.
•  Fishman,  V. N.,  Martin,  G. D.,  Lamparski,  L. L., 2007. Comparison of a variety of gas chromatographic columns with different polarities for the separation of chlorinated dibenzo-p-dioxins and dibenzofurans by high-resolution mass spectrometry. J. Chromatogr. A 1139, 285–300. doi: 10.1016/j.chroma.2006.11.025.
•  Fishman,  V. N.,  Martin,  G. D.,  Wilken,  M., 2011. Retention time profiling of all 136 tetra-through octa-chlorinated dibenzo-p-dioxins and dibenzofurans on a variety of Si-Arylene gas chromatographic stationary phases. Chemosphere 84, 913–922. doi: 10.1016/j.chemosphere.2011.06.012.
•  Fraisse,  D.,  Paisse,  O.,  Hong,  L. N.,  Gonnord,  M. F., 1994. Improvements in GC/MS strategies and methodologies for PCDD and PCDF analysis: I. Evaluation of the non-polar DB-5ms column. Fresenius’ J. Anal. Chem. 348, 154–158. doi: 10.1007/BF00321621.
•  Iwakiri,  R.,  Enomoto,  T., 2018. Elution order of PCDD/DFs and DL-PCBs on commercially available capillary columns. Abstracts for the 27th Symposium on Environmental Chemistry, P-011 (in Japanese).
• Japanese Standards Association, 2020a. Method for determination of tetra- through octa-chlorodibenzo-p-dioxins, tetra- through octa-chlorodibenzofurans and dioxin-like polychlorobiphenyls in stationary source emissions. JIS K 0311, Tokyo, Japan.
• Japanese Standards Association, 2020b. Method for determination of tetra- through octa-chlorodibenzo-p-dioxins, tetra- through octa-chlorodibenzofurans and dioxin-like polychlorobiphenyls in industrial water and waste water. JIS K 0312, Tokyo, Japan.
•  Matsumura,  T.,  Masuzaki,  Y.,  Seki,  Y.,  Ito,  H.,  Morita,  M., 2003. Development of new capillary columns for dioxin analysis. Organohalogen Compd. 60, 375–378.
• Ministry of the Environment, Japan, 2022a. Manual on determination of dioxins in ambient air (in Japanese).
• Ministry of the Environment, Japan, 2022b. Manual on determination of dioxins in sediment (in Japanese).
• Ministry of the Environment, Japan, 2022c. Manual on determination of dioxins in soil (in Japanese).
•  Minomo,  K.,  Ohtsuka,  N.,  Ochiai,  Y., 2024. 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. Environ. Monit. Contam. Res. 4, 117–125. doi: 10.5985/emcr.20240023.
•  Ryan,  J. J.,  Conacher,  H. B.,  Panopio,  L. G.,  Lau,  B. P. Y.,  Hardy,  J. A.,  Masuda,  Y., 1991. Gas chromatographic separations of all 136 tetra-to octapolychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans on nine different stationary phases. J. Chromatogr. A 541, 131–183. doi: 10.1016/S0021-9673(01)95990-4.
•  Stultz,  C.,  Dorman,  F., 2020. The Rtx-Dioxin2 and Rxi-17SilMS as alternative gas chromatographic confirmation columns for dioxin analysis. J. Chromatogr. A 1625, 461263. doi: 10.1016/j.chroma.2020.461263.
•  Van den Berg,  M.,  Birnbaum,  L. S.,  Denison,  M.,  De Vito,  M.,  Farland,  W.,  Feeley,  M.,  Peterson,  R. E., 2006. The 2005 World Health Organization reevaluation of human and mammalian toxic equivalency factors for dioxins and dioxin-like compounds. Toxicol. Sci. 93, 223–241. doi: 10.1093/toxsci/kfl055.