development of a procedure for the determination of ... · minutes using a methanol water gradient...

Development of a procedure

for the determination of selected brominated flame retardants (PBB, PBDE) in plastics by HPLC-ICP-MS

Reference materials for industrial development

LGC/GEN/2010/017

September 2010

Development of a procedure for the determination of selected brominated flame retardants (PBB, PBDE) in plastics by HPLC-ICP-MS Reference materials for industrial development

Contact Point:

Nick Boley

Tel: +44 (0)20 8943 7311

Prepared by:

John Entwisle

Approved by:

_____________________________

Date: 02/09/2010

________________________________

http://www.nmschembio.org.uk

The work described in this report was supported by the Department for Innovation, Universities & Skills as part of the National Measurement System Chemical & Biological Metrology Knowledge Base Programme.

Milestone Reference: VAMR7/2b

LGC/GEN/2010/017

© LGC Limited 2010

http://www.nmschembio.org.uk/

Contents 1. Summary page 1

2. Introduction page 2

2.1. Aim of the project page 2

2.2. Properties of PBBs and PBDEs page 2

2.3. Legislation page 2

2.4. Published methadology page 3

3. Sample Preparation page 5

3.1. Extraction background page 5

3.2. Accelerated Solvent Extraction (ASE) pressurised fluid extraction page 6

3.2.1. Preparation of the extraction cell page 7

3.2.2. Selection of extraction solvent page 7

3.2.3. Accelerated solvent extraction (ASE) optimisation experiments for IRMM-310 page 7

3.2.4. Accelerated solvent extraction (ASE) optimisation experiments for ERM-EC591 page 10

4. Investigation into GC/MS quantification of BFRs page 12

5. Determination by HPLC-ICP-MS page 14

5.1. Investigation into the use of ultrasonic nebuliser with desolvation (USN) to reduce

plasma carbon loading page 15

5.2. Investigations into the use of online aqeous dilution to reduce plasma loading page 16

5.3. Investigations into the use of optimal plasma addition gas (oxygen) to prevent carbon

build-up page 16

5.3.1. Optimisation of ICP-MS conditions for bromine detection page 16

5.4. Optimisation of HPLC separation page 17

5.4.1. Pentafluorophenyl propyl stationary phase page 18

5.4.2. Polar-reverse stationary phase page 19

5.4.3. Reverse phase stationary phase page 21

5.4.4. Phenyl-hexyl stationary phase page 21

5.4.5. Investigation into the separation of BFR using π –π interaction column phases page 25

5.5. Selection of an appropriate internal standard page 27

5.6. Initial analysis of IRMM-310 and ERM-EC591 page 28

6. Polybrominated flame retardant standards page 29

6.1. Purity assessment of custom synthesised PBDE-47 page 29

6.2. Assessment of the purity of commercially available 50 µg/mL standards by LC-ICP-MS page 31

7. Validation of the procedure page 32

7.1. Total Br content of ERM-EC591 page 33

7.2. Determination of limit of detection of the LC-ICP-MS system page 34

8. Conclusion page 34

9. Future work page 35

10. Acknowledgements page 36

1. Summary This report details the development of methodology for the accurate quantification of selected polybrominated biphenyl ethers (PBDEs) and polybrominated biphenyls (PBBs) in plastic materials by HPLC-ICP-MS. Chemically, PBDEs are aromatic compounds substituted with up to 10 bromine atoms on a diphenyl ether core. PBBs are the bromine homologues of polychlorinated biphenyls (PCBs). For both substance classes, the numbering of the 209 possible congeners is the same as those for PCBs following IUPAC rules. Accurate quantification of brominated flame retardants (BFRs) in plastics can only be achieved when several significant challenges have been overcome. Among these are the suitable grinding or milling of the plastic, the selective extraction minimising co-extraction of possibly interfering compounds, photolytic degradation in the presence of UV light, thermal instability, especially of BDE-209 during extraction and the chromatographic process, and quantification over a wide concentration range of the individual congeners (1 to 1000 mg/kg). In addition, and more importantly, is the access to traceable calibrants and internal standards of sufficient purity to enable accurate quantification.

The recently produced ERM-EC591 certified matrix reference material, by IRMM (Geel, Belgium) was used to help validate the developed methodology. The CRM was prepared at or about the legislative limit of 0.1% m/m with a mixture of PBDEs and PBBs. The quantification of all possible congeners in the plastic would have taken a significant resource, so the study was restricted to five congeners (PBDE-209, PBDE-206, PBDE-183, PBDE-47, PBB-209) to prove capability as per an international collaborative exercise (CCQM-P14 study). PBDE-206 was chosen as an analyte as it is the thermal debromination product of PBDE-209. Therefore, PBDE-206 can be used as an indicator of unintentional debromination caused during the analytical process. ERM-EC591 was cryogenically ground and extracted under optimal accelerated solvent extraction (ASE) conditions. Investigations into the chromatographic separation by a number of different column stationary phases were performed. Optimum HPLC separation was achieved in sixty minutes using a methanol water gradient applied to a column with a phenyl-hexyl stationary phase. ICP-MS detection was achieved by monitoring both Br ions at m/z 79 and 81. Oxygen was added to the plasma to prevent the formation of elemental carbon derived from the mobile phase causing blockages of the system. A standard bracketing approach was adopted for quantification. Standards were obtained from commercial sources and evaluated for purity prior to use. 9-Bromoantheracene was identified as a suitable internal standard but had to be purified before it could be used. The results for ERM-EC591 agreed well with the certified values and were within the stated uncertainty apart from PBDE-206. The relative standard deviation of six replicates varied from 2.5 to 6.5% for the different congeners. ERM-EC591 is not certified for BDE-206 content because the certification study provided too few data points which were widely scattered (30 ± 11 mg/kg). Using this value a low recovery was obtained (70 ± 3.3 %). However, it is acknowledged that the contributors to the certification study employed GC separation which may have elevated the consensus value. This is due to some degree of thermal debromination occurring of PBDE-209 to from PBDE-206. One participant in the CCQM-P114 study used HPLC-ICP-MS and this value is in very close agreement with the value from this study. It was concluded that the use of HPLC separation performed at ambient temperature ensures minimal congener inter-conversion and therefore provides greater accuracy for the quantification of PBDE-206.

Detection limits of between 2 and 5 µg/g (depending on the congener) in the polymer were achieved which is suitable for measurements close to the regulatory limits (0.1 %). Added confidence was provided by the quantitation of the total Br content of ERM-EC591 by the peak summation technique. The determined total bromine content agreed well with the certified value and was within the uncertainty limits.

The developed procedure provides high accuracy results for BFRs in polymers with minimal thermal debromination. The use of LC-ICP-MS also provides a useful tool for purity and concentration assessments of calibrants.

This work has been presented at Hyphenated Techniques Conference (HTC11) January 2010 in Bruges, Belgium and at the Biannual National Atomic Spectroscopy Seminar (BNASS) July 2010 in Cambridge,UK. An article has been published in the Agilent manufacturers ICP-MS journal in August 2010 and an application note has been drafted. A presentation of the purity evaluation of PBDE-47 is to be made at The Future of Reference Materials conference, Nov 2010 in Geel, Belgium. A power-point presentation has been made at the LGC AT20 internal science meeting in February 2010.

LGC/GEN/2010/017 Page 1

2. Introduction 2.1 Aim of the project

Develop methods for the determination of brominated flame retardants (BFR) in plastics by ICP-MS capable of supporting RoHS directive. The methodology should be capable of accurate quantification of PBBs and PBDEs in materials at the 0.1 % level. Also investigate HPLC-ICP-MS techniques for the assessment of the purity of BFR standards.

2.2 Properties of PBBs and PBDEs

PBBs are manufactured by the bromination of biphenyl and were a widely used commercial flame retardant. They were produced as three primary homologs: hexabromobiphenyl, octabromobiphenyl and decabromobiphenyl, depending on the degree of bromination. FireMaster BP-6 (a yellow-brown powder) is an example of a hexabromobiphenyl product and is mixture of many different congeners with 2,2',4,4',5,5'–hexabromobiphenyl and 2,2',3,4,4',5,5'-heptabromobiphenyl being significant constituents by mass (60-80 % and 12-25 %) respectively. Decabromobiphenyl is the fully brominated biphenyl. These compounds are analogous to PCBs and there are 209 possible congeners depending on the substitutions.

Figure 1: Structure of polybrominated biphenyls (PBB)

BrBr

Commercial PBDEs are manufactured as three mixtures named after the degree of bromination: Penta-mix, Octa-mix and Deca-mix formulations. The industrial production is based on the bromination of diphenyl ether, a process that is terminated at different degrees of bromination. Due to chemical properties, the oxygen atom directs the joining bromine atoms to para and ortho positions. Also steric hindrance of the substituted bromines limits the number of congeners present in PBDE products. The major components in the Penta-mix are tetra and pentaBDEs (PBDE-47 and PBDE-99 being the major congeners). In octa-mix there are hepta and octaBDEs such a BDE-183 and in Deca-mix formulation the major component is the fully brominated, decabromodiphenyl. There are 209 possible congeners, but the number of congeners that are actually in the products is more limited.

Figure 2: Structure of polybrominated biphenyl ethers (PBDE)

O

BrBr

2.3 Legislation

Directive 2003/11/EC places restrictions on the marketing and use of certain dangerous substances and preparations in the EU. The marketing of PentaBDE and OctaBDE was prohibited from August 2004. The Directive on the Restriction of the use of Certain Hazardous Substances in Electrical and


Electronic Equipment 2002/95/EC[1] (commonly referred to as the Restriction of Hazardous Substances Directive or RoHS) was adopted in February 2003 by the European Union1.

The RoHS directive took effect on 1 July 2006, and each member state is required to enforce the directive by passing laws. This directive restricts the use of six hazardous materials in the manufacture of various types of electronic and electrical equipment. It is closely linked with the Waste Electrical and Electronic Equipment Directive (WEEE) 2002/96/EC which sets collection, recycling and recovery targets for electrical goods and is part of a legislative initiative to solve the problem of huge amounts of toxic e-waste2. The restricted compounds include PBBs and PBDEs, and their concentration should not exceed 0.1% w/w. The use of these BFRs was voluntarily phased out in the US. There have also been considerable environmental concerns over these compounds which have led to the inclusion of octa- and penta-bromodiphenyl ethers in the ‘dirty dozen’ list of May 2009. The list is compiled by the United Nations under the Stockholm Convention on Persistent Organic Pollutants3

Also the European Water Framework Directive on drinking water policy Nº (2000/60/CE) states that water should be analysed for the PBDEs (BDE-99, BDE-100, BDE-205, BDE-209) 4.

2.4 Published methodology

For plastics and polymeric materials the methodology drafted by the international electrotechnical commission (IEC) has become widely recognised as the industrial standard. The method is based on extraction using the soxhlet technique followed by GC/MS determination using external calibration or C13 labelled IDMS quantification5.

Significant problems were identified with this approach when the results of an international collaborative study were evaluated6. A reference material was produced for this study (IRMM 310, poly(ethyleneterephthalate) (PET) spiked with PBB and PBDE congeners at the 0.1 % m/m level. A relative between-lab standard deviation ranged from between 22 to 61 % for the congeners. The results highlighted serious deficiencies in the methodology used to determine these compounds in plastic. The report also pointed to the need for certified matrix reference materials and a wider number of certified congener standards solutions.

To improve methodology in this area a CCQM study (P114) was organised (Dec 2008). LGC did not participate in this study as our methodology was yet to be developed. The candidate material ERM-EC591 was used as a test matrix material and two NIST candidate standard solutions (SRM 2257 and 2258) were used for quality control purposes. The study was performed before the certified values were published for these materials. The study produced values which compared very well with the certified values. The relative standard deviation of the means for four congeners determined in ERM-EC591 ranged from 1.9 % to 13.0 %. However for BDE-206 the relative standard deviation of the means was much higher (28.2 %) but this is explained by the fact it is present a comparative low concentration than the other congeners and can be formed from the debromination of BDE-209 by the action of heat or UV light7. 1Directive 2002/95/EC ‘The restriction of the use of certain hazardous substances in electrical and electronic equipment’ (http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2003:037:0019:0023:EN:PDF)) 2Directive 2002/96/EC ‘on waste electrical and electronic equipment (WEEE)’(http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2003:037:0024:0038:EN:PDF 3 The third meeting of the Persistent Organic Pollutants Review Committee (POPRC-3) of the Stockholm Convention on Persistent Organic Pollutants (POPs)) 4 European Water Framework Directive on drinking water policy Nº (2000/60/CE) http://eur-lex.europa.eu/Notice.do?mode=dbl&lang=en&ihmlang=en&lng1=en,es&lng2=da,de,el,en,es,fi,fr,it,nl,pt,sv,&val=256390:cs&page5 Draft International Electrotechnical Commission IEC 62321, Ed1/CDV IEC 111/54/CDV) http://www.iec.ch/cgi-bin/procgi.pl/www/iecwww.p?wwwlang=e&wwwprog=doc-det.p&progdb=db1&wcom=111&wclass=&wdoc=54&wsup6 Anal Bioanal Chem (2008) 390:399-409. 7 R. Zeleny, S. Voorspoels, M. Ricci, R. Becker, C. Jung, W. Bremser, M. Sittidech, N. Panyawathanakit, W. F. Wong, S. M. Choi, K. C. Lo, W. Y. Yeung, D. H. Kim, J. Han, J. Ryu, S. Mingwu, W. Chao, M. M. Schantz, K. A. Lippa, S. Matsuyama.; Anal. Bioanal. Chem., 2010, 369, 1501.


http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2003:037:0019:0023:EN:PDF)

http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2003:037:0019:0023:EN:PDF)

http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2003:037:0024:0038:EN:PDF

http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2003:037:0024:0038:EN:PDF

http://eur-lex.europa.eu/Notice.do?mode=dbl&lang=en&ihmlang=en&lng1=en,es&lng2=da,de,el,en,es,fi,fr,it,nl,pt,sv,&val=256390:cs&page



http://www.iec.ch/cgi-bin/procgi.pl/www/iecwww.p?wwwlang=e&wwwprog=doc-det.p&progdb=db1&wcom=111&wclass=&wdoc=54&wsup

http://www.iec.ch/cgi-bin/procgi.pl/www/iecwww.p?wwwlang=e&wwwprog=doc-det.p&progdb=db1&wcom=111&wclass=&wdoc=54&wsup

A variety of approaches were used by the participants in the CCQM study. Most participants ground the sample pellets before analysis but some did not. A variety of extraction techniques were employed including ASE, soxhlet and soaking. Generally GC-MS systems Table 1 were used to determine the BFRs. However, one participant used GC-ECD and LC-ICP-MS.

Table 1 : Methods used by the participants of CCQM-P114 and columns

Participant Method principle

Column

BAM GC-EI-MS IDMS 15 m x 0.25 mm x 0.1 µm

ZB-5HT, Inferno, Phenomenex

DSS GC-EI-MS

IDMS only for

BDE183 and 206

15 m x 0.25 mm x 0.1 µm

DB-5MS, Agilent

HKGL GC-EI-MS IDMS 15 m x 0.25 mm x 0.1 µm

DB-5MS, J&W

IRMM GC-ECNI-MS

IDMS only for BDE209

15 m x 0.25 mm x 0.1 µm

DB-5, J&W

KRISS GC-EI-MS IDMS 15 m x 0.25 mm x 0.25 µm

Rtx-5MS, Restek

NIM GC-ECD 15 m x 0.25 mm x 0.1 µm

DB-5HT, J&W

NIM LC-ICP-MS 4.6 x 250 mm, 5 µm

TC-C18, Agilent

NIST GC-NCI-MS

IDMS only for PBDE209

15 m x 0.25 mm x 0.25 µm

Rtx-5MS, Restek a

NMIJ GC-EI-MS IDMS 15 m x 0.25 mm x 0.1 µm

ENV-5MS (Kanto Chem. Ltd.) a quantification of BDE47 finally achieved on different column: 15 m x 0.25 mm x 0.15 µm, LC-50, J&K Environmental

IRMM released for sale in May 2009 two matrix CRMs:- ERM-EC590 (polyethylene) and ERM-EC591 (polypropylene). NIST has also released for sale in August 2009 a mixed standard solution of 38 congeners (SRM 2257) and a single congener solution of PBDE-209 (SRM 2258).

LC-MS can also be employed for the determination of BFR but negative ion atmospheric pressure photo-ionisation (NI-APPI) has to be employed to form [M-Br+O]- ions. This technique has been used successfully to determine a number of BDE congeners in house dust at trace level. Unfortunately this technique is not available within the mass spectrometry group at Teddington.¹

¹ Simultaneous Determination by APCI-LC/MS/MS of Hydroxylated and Methoxylated Polybrominated Diphenyl Ethers Found

in Marine Biota by Yoshihisa Kato, Syohei Okada, Kazutaka Atobe, Tetsuya Endo, Futoshi Matsubara, Takayoshi Oguma and

Koichi Haraguchi Anal. Chem., 2009, 81 (14), pp 5942–5948


3. Sample Preparation Initially attempts were made to identify a plastic with relevant levels of BFR from old laboratory equipment. It was envisaged this would provide a cheap material for initial extraction optimisation experiments. However, this proved more problematic than expected. Seven casings of old electrical equipment were sampled. The samples were ground using a domestic coffee grinder with solid carbon dioxide as a refrigerant. Unfortunately none of these materials were found to contain significant levels of PBBs or PBDEs when analysed by HPLC-UV/HPLC-ICP-MS. However one material contained a brominated compound of some type as a response was observed in the HPLC-ICP-MS chromatogram (m/z 79 and 81). However, the peak eluted too early to be either PBDE or PBB congeners. A reference material from IRMM was then identified (IRMM 310) and it was decided to purchase it. ERM-EC591 and 590 were not yet available at this time. IRMM 310 was produced by the gravimetrically controlled additions of BFR to a bulk polymer. The polymer pellets were then extruded a number of times to ensure homogeneity. Two units of IRMM 310 were purchased and stored at -20 °C before use. A domestic coffee grinder was employed to prepare a small quantity of material. Again solid carbon dioxide was used as a refrigerant. This ground material was used for some initial ASE experiments before a more appropriate grinder could be located.

A more suitable cryogenic centrifugal mill was located within LGC and was borrowed. Approx 10 g of IRMM-310 was ground using this cryogenic centrifugal mill (Type 17-140 Glen Creston Ltd, UK). Liquid nitrogen was used as the refrigerant. The integral sieve ensures the material is ground to a uniform particle size of <0.5 mm. The ground material was collected on an aluminium foil sheet and allowed to reach room temperature. During the process the material was protected from excess light exposure. The material obviously picks up lots of condensation so the material was allowed to dry out at room temperature before being transferred to a brown glass vessel for storage (-20 ˚C). The grinder has lots of internal surfaces and required extensive cleaning in order to prevent carry over. Propan-2-ol was used as a rinsing solvent. The contamination problem was evident when a white plastic was ground following a dark green plastic (BCR 601). Visual contamination of the white powder with a few green specks was observed. This highlights the issue of potential cross contamination occurring at this stage of the analysis. Scrupulous attention is needed to ensure grinding equipment is fully cleaned between samples.

It is also important that the particle size of the sample is consistent and controlled as this will have an influence on the sample extraction characteristics. The centrifugal mill ensures this as the material is removed from the grinder as soon as it passes through the sieve and therefore is not re-ground. The mill has an alternative 0.2 mm sieve which can be used. It would be of interest to use this and investigate how particle size affects extraction efficiency.

3.1 Extraction background

Soxhlet extraction is recommended in the IEC draft procedure using either toluene or propanol-2-ol. The sample (0.1 g) is extracted for a total of 2 hours or 20 extraction cycles and the extract made up to 100 mL with solvent.

Participants performing the CCQM-P114 study used a variety of extraction techniques (see Table 2), some more aggressive than others. Some participants performed soxhlet extraction for 21 hours while others did so for only 15 minutes. Ultrasound-assisted extraction was employed by some participants and just soaking in solvent for 50 minutes by others. In addition, a variety of solvents were used, including iso-octane, tetrahydrofuran and hexane but toluene appears to have been the most commonly used.


Table 2 : Method detail employed by the participants of the CCQM-P114 study

Participant Sample pre-treatment Sample intake Extraction Clean-up

BAM Sub-sample ground, cryo-grinding 1 g

Pressurized liquid extraction, toluene,

3 x 20 min -

DSS Whole bottle content, ground

0.1 g for PBDEs 47 and 209 and PBB209;

0.6 g for PBDEs 183 and 206

Soxhlet, n-hexane -

HKGL Whole bottle content, ground 0.1 g Reflux, boiling toluene,

15 min

GPC and neutral alumina column chromatography

IRMM Whole bottle content, ground, cryo-grinding 20 mg

Ultrasound-assisted extraction, isooctane, 75min

Filtration, 0.45 µm

KRISS Sub-sample ground 50 mg Shaking, toluene:tetrahydrofuran

1:1 (v/v), 50 min -

NIM Sub-sample ground 0.2 g Ultrasound-assisted extraction, toluene, 30 min


NIM Sub-sample ground 0.2 g Ultrasound-assisted extraction, toluene, 30 min


NIST Whole bottle content, ground, cryo-grinding 0.5 g Soxhlet, toluene, 21 h

Solvent evaporation, addition of hexane, centrifugation

NMIJ Sub-sample ground 0.95 - 1.37 g Soxhlet, toluene, 10 h -

UV protection applied in all laboratories (alumina foil cover, etc.)

One participant used accelerated solvent extraction (ASE) with toluene and achieved acceptable results so it was decided to initially investigate this technique. Within LGC there is extensive experience of this technique, so minimal development work was required. ASE enables the extraction temperature to be precisely controlled and the extraction process is performed in the dark. Therefore the potential for thermal and photo-degradation is significantly less than for soxhlet extraction. In addition, one of the major advantages of this technique is that the sample cell can be conveniently re-extracted enabling extraction efficiency to be evaluated.

3.2 Accelerated Solvent Extraction (ASE) or pressurised fluid extraction

A proprietary extraction apparatus was used for this work (AS200 Accelerated Solvent Extraction system (ASE), Dionex Ltd.). Briefly, this automated device pumps, under high pressure, an extraction solvent into a pre-heated cell containing the sample. The solvent remains in the heated cell for a specified time, the pressure being maintained by adding solvent or venting excess to a collection vial as required. Finally, the solvent is purged into the collection vial with nitrogen gas. To ensure that a rigorous extraction takes place, the solvent pressurisation step can be repeated for a number of cycles each emptied into the same vial (dependent on the size of the extraction cell used) or the entire process can be repeated for the same cell using several collection vials.


3.2.1 Preparation of the extraction cell

There are a range of cells sizes available and the smallest one, with an internal volume of 1 cm3, which was selected. This minimises the solvent required per cycle thus ensuring a concentrated extract. Care in packing the cell is vital to ensure that channelling does not occur and that particles do not escape to block the transfer lines. The weight of the sample in the cell must be accurately recorded and the sample must be mixed with the dispersal agent before extraction to help prevent channelling of solvent through the cell.

The cell was partially assembled, a cellulose filter positioned in the cell aperture and the end cap screwed on. The other end of the cell was left open. A portion of the dispersing agent (Ottawa Sand, Fisher Scientific Ltd, Loughborough, Leicestershire, UK) (0.5 g) was added to the cell and the cell was tared on a five figure balance. A small funnel was attached to the cell opening (fashioned by cutting down a 10 mL plastic pipette tip). Using a second balance, 0.11-0.13 g of the sample was weighed in the back end of a glass Pasteur pipette. With the help of the funnel the sample was transferred to the cell. To ensure that the sample was mixed with the dispersed agent, a small drill bit was used to mix the contents. Before the bit was withdrawn from the cell it was tapped to make sure no sand particles were attached. Inevitably some particles of sample do stay attached to the drill bit but these are inconsequential as the sample weight has not been recorded yet. The funnel was removed and the cell was reweighed to record the sample weight. More dispersing agent is then added to the cell until the level is flush with the top of the cell. A second cellulose filter is fitted and the other end cap screwed onto the cell.

The cell was then transferred to the ASE carousel with a 60 mL pre-weighed brown glass collection vessels underneath. The extraction is then performed for example using the conditions detailed in Table 3. After reweighing, a pristine vial cap can be exchanged to prevent evaporation on storage. The sample and extract are fully protected from light throughout the extraction process and during storage before analysis.

3.2.2 Selection of extraction solvent

Ideally it is generally best practice to extract samples with the same solvent composition as the HPLC eluent. This helps prevent the potential problem of co-extractants precipitating out when the solvents mix during the injection process. These precipitates can cause column blockages, increasing back pressure, ultimately resulting in a system failure. However, methanol : water mixtures are far from ideal as an extractant for this type of matrix. It was decided to use toluene as it is known that the most heavily brominated compounds have a good solubility in this solvent and many of the CCQM study participants had used it. Propan-1-ol could also be considered as it would have greater compatibility with the methanol mobile phase and greater UV transparency, but when standards in this solvent were injected on to the system the BFR suffered from peak shape distortion. Ethanol and propan-2-ol might also be good extraction solvents but were not investigated. One problem with using an alcohol is that on exposure to the atmosphere they can quickly absorb moisture. This could cause the hydrophilic, highly brominated flame retardants to potentially drop out of solution as the water content increases. Moisture extracted from the atmosphere was found to be an issue when methanol was left for a few days as a HPLC solvent. The chromatograms were truncated when the system was restarted but original retention times were restored when fresh methanol was used. Toluene extracts have the advantage that they can be stored at low temperature without concerns of precipitation of the higher brominated congeners as they are know to be highly solubility in this solvent. Cold storage minimises potential solvent loss and protects the extracts from stray light.

3.2.3 Accelerated Solvent Extraction (ASE) optimisation experiments for IRMM-310

IRMM-310 was ground using a coffee grinder with solid carbon dioxide as a refrigerant. The extraction cell was packed as described in section 3.2.1 with between 0.11 to 0.13 g of sample.

Table 3 : Accelerated solvent extraction condition of course ground IRMM-310

ASE conditions Toluene Cell pressure (psi) 1500 cycles 1

Cell temperature (ºC) 100 Static time (mins) 20 Preheat time (mins) 1 Flush volume (%) 50

Heat time (mins) 5 Purge time (secs) 60


Four consecutive extraction cycles were performed and collected into separate vials. The static time is quite excessive but was the time quoted by one of the participants in the CCQM P14 report (see Table 2).

An LC-ICP-MS system was used to evaluate these extracts. The system was at a developmental stage providing far from ideal separation. However by summing the peak areas it enable comparisons between the extraction cycles to be made. A Polar reversed-phase column was used under isocratic conditions and is detailed in the chromatographic resolution section 5.4.2 of this report. The material was not sufficiently finely ground by the coffee grinder (<0.5 mm as recommended by IEC) which is reflected by the incomplete extraction, even after 4 cycles.

Figure 3 : Graph of total BFR extracted per extraction cycle of course ground IRMM-310

Sum of peaks Br m/z 79 against extraction cycle

y = 509186x-1.4036

R2 = 0.9746

0100000200000300000400000500000600000

0 1 2 3 4

Extraction cycle

Sum

of p

eak

area

Br

m/z

79

5

The graph in Figure 3 depicts the summed Br peak area in each chromatogram against the extraction cycle. This is a classic example of an extraction profile, demonstrating the exponential decrease in the amount of analyte extracted per cycle. It is clear that efficient extraction was not achieved even after four cycles. Therefore it was concluded that the extraction temperature needs to be increased in order to obtain efficient extraction in a reasonable number of cycles. This experiment also highlights the need to ensure that the sample is finely ground to assist extraction and that the particle size should be consistent thus providing reproducible extraction characteristics.

After this initial extraction, IRMM 310 was ground using a cryogenic centrifugal mill. The material was ground to less than 0.5 mm particle size as stated in the IEC recommendations. It was decided to compare the extraction efficiency at three different temperatures in order to identify the optimum temperature. Three cells were packed as described previously in section 3.2.1 and extracted. One cell was extracted at 140 ˚C, second cell at 150 ˚C and third at 160 ˚C as detailed in Table 4. The extraction cycle time was reduced to a more reasonable 6 minutes.

Table 4 : Accelerated solvent extraction condition of finely ground (0.5 mm) IRMM-310

ASE solvent Toluene Cell pressure (psi) 1500 cycles 5

Cell temperature (˚C) 140, 150, 160 Static time (mins) 6 Preheat time (mins) 1 Flush volume (%) 50

Heat time (mins) 5 Purge time (secs) 60 To provide an assessment of the extraction efficiency the peak area of BDE-209 in the extracts were compared. This is the most problematic BFR in terms of solubility and thermal stability.

Five extraction cycles were performed and collected in separate vessels. It appears that all the PBDE-209 was extracted in the first and second extraction cycle and none in third cycle. The peak area of PBDE-209 in the first extract was expressed as a percentage of the combined peak areas of both the first and second extracts. This value was plotted in the graph below Figure 4 against the extraction temperature.


Figure 4 : Graph of extraction efficiency of PBDE-209 from finely ground (0.5 mm) IRMM-310 at different temperatures

Percentage of total PBDE-209 extracted in first cell extraction

9797.5

9898.5

9999.5100

100.5

135 140 145 150 155 160 165

Cell extraction Temperature C

% o

f PB

DE-

209

in fi

rst

extra

ctio

n

The graph (Figure 4) clearly shows that at higher temperatures the extraction is more efficient. However, at higher temperature there is the increased risk of debromination. Therefore a graph of the ratio of the peak areas of BDE-206 to BDE-209 (extracts one and two combined) was plotted Figure 5.

Figure 5: Graph of the ratio of the PBDE-206 : PBDE-209 extracted from IRMM-310 at different temperatures from finely ground (0.5 mm) IRMM-310

Ratio of BDE-206/BDE-209 peak areas against extraction temperature.

0.02860.02870.02880.0289

0.0290.02910.02920.02930.0294

135 140 145 150 155 160 165

Extraction temperature

Ratio

of B

DE-

206/

BD

E209

pe

ak a

reas

The graph (Figure 5) demonstrates that there is no significant increased formation of BDE-206 from the debromination of PBDE-209 connected to the increase in extraction temperature. In fact the opposite is suggested by the graph, this is an aberration of the data. Unfortunately there was no time available to confirm these findings.

Therefore it was concluded that efficient extraction can be achieved using this technique without excessive debromination occurring and further development should be pursued. The technique will be applicable to other types of plastics.


3.2.4 Accelerated Solvent Extraction (ASE) optimisation experiments for ERM-EC591

As ERM-EC591 is a certified reference material rather than a reference material it was decided to pursue further development work using this material. This material was also used in the CCQM P114 study and participants method details were available. A 10 g portion of ERM-EC591 (polypropylene) was cryogenically ground as described previously and 0.1 - 0.13 g packed into a cell as described previously. Initially the sample was extracted at 150 ˚C with toluene. However, this caused the plastic to melt and it was completely extruded in to the collection vessel. It was concluded that the extraction temperature should be maintained significantly below the melting point of the particular polymer being extracted in order to avoid this problem. The melting points of three common polymers are given in Table 5.

Table 5 : Melting point range of three relevant polymers

Polymer type Melting point (˚C)

Poly(ethyleneterephthalate) (PET) 250-260

Polypropylene 160-170

Polyethylene 120-130

ASE works best when the extraction temperature is above the boiling point of the solvent as it enables pressure to build up in the cell. This enables the solvent to freely permeate the sample. In this case toluene boils at 120 ˚C. However, efficient extraction can be achieved at lower temperatures. It was decided to initially extract at 80 ˚C, 90 ˚C and 100 ˚C and compare the extraction efficiency. Three cells were packed as described previously and extracted using the conditions detailed in Table 6 below.

Table 6 : Accelerated solvent extraction condition of finely ground (0.5 mm) ERM-EC591

ASE solvent Toluene Cell pressure (psi) 1500 cycles 1

Cell temperature (˚C) 80, 90, 100 Static time (mins) 5 Preheat time (mins) 1 Flush volume (%) 50

Heat time (mins) 5 Purge time (secs) 60

In order to obtain more detailed data about the extraction process it was decided to collect five cycles separately at each temperature. It was decided to quantify PBDE-206 and PBDE-209 by external calibration using a single standard. Changes in the relative levels of PBDE-206 should highlight any debromination issues and the fully brominated PBDE-209 is one of the most problematic congeners to extract. It was found that the majority of the BFRs were extracted in the first extraction cycle with the second cycle containing much less. The quantities of PBDE-209 and PBDE-206 extracted in the first two cycles were combined and plotted against the cell extraction temperature see Figure 8 and 9.


Figure 8 : Graph of the amount of PBDE-209 extracted from ERM-EC591 at different temperatures.

Amount of PBDE 209 ug/g extracted against extraction temperature

600

640

680

720

760

800

75 80 85 90 95 100 105

Temperature C

ug/g

ext

ract

ed

Figure 9 : Graph of the amount of PBDE-206 extracted from ERM-EC591 at different temperatures

Amount of PBDE 206 ug/g extracted against extraction temperature

20

22

24

26

28

30

75 80 85 90 95 100 105

Temperature C

ug/g

ext

ract

ed

It is clear from the two graphs above Figure 8 and 9 that extraction efficiency increased with temperature. However, the two graphs do not follow a similar relationship (curve) as they are expected to. Therefore, there must be another factor involved. It was decided to plot the ratio of PBDE-209 to PBDE -206 (debrominated product) Figure 10.


Figure 10 : Graph of the ratio of the PBDE-206 : PBDE-209 extracted from ERM-EC591 at different temperatures.

Ratio of PBDE 206/ PBDE 209 against extraction temperature

0.03

0.032

0.034

0.036

0.038

75 80 85 90 95 100 105

Temperature C

Rat

io o

f tot

al u

g/g

of

PBD

E 20

6: P

BDE

209

From the graph in Figure 10 the ratio of PBDE-206 : PBDE-209 at 80 and 90 ˚C appears to be reasonably consistent, but at 100 ˚C the ratio increases. It was concluded that this is probably caused by a degree of debromination of PBDE-209 and the subsequent formation of PBDE-206. Therefore it can be concluded that an optimal extraction temperature of 90 ˚C should be used for this polymer and a total of 5 cycles should be more than adequate to ensure exhaustive extraction. All further extractions of ERM-EC-591 were performed using these conditions.

Table 7 : Optimal extraction conditions for ERM-EC591 using AS200 ASE (Dionex Ltd, Camberley, UK)

Cell type 1 cm3

Dispersing material Ottawa Sand

Solvent 100% Toluene

Pressure (psi) 1500

Static time (min) 4

Flush 50 %

Number of cycles 5

Temperature (˚C) 90

4. Investigation into GC/MS quantification of BFRs A Thermo Electron Corporation, (Austin, Texas, USA) Finnigan Trace GC ultra with Finnigan Trace DSQ mass spectrometer system was used. It was envisaged that the identity and purity of the HPLC peaks could be determined. Also full ID-GCMS quantification using C13 spikes could be performed to validate the LC-ICP-MS results.


The system was set up following a Thermo Scientific application note1. The application note details a detection capability of 2 pg/µL (equivalent to 2 ng/mL) for each individual congener. The most important issue with GC is ensuring an efficient transfer of analyte on to the column with minimal thermal break down. To ensure this a pseudo on-column liner system was adopted with a 0.53 mm 2 m deactivated retention gap (Phenomenex Inc, Torrance, California, USA) connected to the column, a Zebron HT-5, 15 m x 0.25 mm I.D. x 0.25 µm film thickness (Phenomenex Inc, Torrance, California, USA).

The system was initially used to confirm the extraction optimisation experiments and single ion monitoring windows were established. No obvious thermal breakdown of BDE-209 was observed using this system. The peak shape of BDE-209 was Gaussian and not distorted. Full spectra chromatograms of the five compounds were established and the single ion monitoring (SIM) programmed into a method file.

Table 8 : Oven temperature program

Rate (˚C/min)

Temp (˚C) Hold time (min)

Initial 90 0.5 Ramp 1 60 255 2 Ramp 2 45 300 17

The early part of the chromatogram is quite crowded with many peaks and BDE-47 peak suffered from an unresolved shouldering peak in the SIM chromatograms. Extensive optimisation experiments were tried but full baseline resolution was difficult to achieve. Less abundant ions from BDE-47 were monitored in an effort to identify an interference ‘free’ ion but unresolved peaks were still observed.

Table 9 : Retention times of the five BFRs of interest

Compound Retention time (window) minutes

BDE-47 3.62 (2-4.5)

BDE-183 5.37 (4.5-8.8)

BDE-206 11.36 (8.8-12)

BB-209 12.73 (12-15)

BDE-209 17.57 (15-20)

To achieve results with a suitably low uncertainty by GC-IDMS it is important to obtain low relative standard deviations for the ratio. In order to achieve this the dwell time is critical and needs to be increased with the increasing width of later eluting peaks. Early eluting peaks will be sharp and short in duration and therefore require a fast dwell time. Late eluting peaks require an increase in the dwell time in order to achieve low standard deviation of the ratio. This is an important factor that needs to be taken into account of when performing this type of analysis. 1 Quadrupole GC/MS Analysis of Polybrominated Diphenyl Ethers (PBDE) in environmental samples by Thermo Scientific Application no 10047 http://www.sorvall.com/eThermo/CMA/PDFs/Articles/articlesFile_25566.pdf


http://www.sorvall.com/eThermo/CMA/PDFs/Articles/articlesFile_25566.pdf

Table 10 : Mass spectrometer settings and appropriate m/z ions

Compound Dwell time Major ion C13 label ion Confirmation ion

Confirmation ion

BDE-47 10 325.89 337.8 485.79 403.9

BDE-183 30 561.72 573.72 721.62 627.5

BDE-206 100 719.61 731.61 879.46 785.3

BB-209 100 943.42 955.42 783.63 959.19

BDE-209 200 799.39 811.39 Not required Not required

It was concluded that it would take a considerable amount of recourse to fully optimise the system. Unfortunately the system was required for other project work, therefore it was decided to abandon this approach and concentrate on the HPLC-ICP-MS procedure as initial results using this technique appeared promising. GC-MS is not a novel way to quantify BFRs as the technique has been well researched and documented in recent years. In addition, many National Measurement Institutes (NMIs) have applied traceable metrology using this technique, as has been demonstrated in the CCQM P114 study. Therefore GC-MS development was curtailed as the research would not be innovative and therefore of little merit.

If a certification study is to be performed in the future, it is recommended that GC-MS is employed as a confirmatory technique. Also HPLC fractions can be collected and injected onto the GC-MS system to confirm the purity of HPLC peaks.

5. Determination by HPLC-ICP-MS There are many challenging aspects to the detection and quantification of Br species by quadrupole ICP-MS. Historically, detection of bromine by ICP-MS has been limited due to its high first ionization potential (11.84 eV) leading to poor ionization in the plasma (Approx 5 %).

Table 11 : Isotopic masses and abundance of bromine and potential ICP-MS interferences

Mass Number Natural Abundance Interference

79

81

50.7%

49.3%

38Ar40ArH+

CuO+

ArK+ 40Ar40ArH+

CuO+

ArK+

ZnO+

The argon-argon hydrides are the most significant interferences. In addition, 40Ar-40Ar + dimer are very large and may affect 79Br as they are adjacent masses. The eluent from the HPLC will not contain significant quantities of Cu, K, or Zn and so these interferences are unlikely. Also the fact that a continuous signal is being monitored ensures that even if some argon hydride is formed it is of little significance.

The effect of carbon addition to the plasma on the ionisation and, therefore, the sensitivity of Br detection was unknown, and so was initially investigated. A 50 mg/L potassium bromide solution was introduced into the plasma and a source of carbon introduced into the plasma. It was found that the addition of methane to the plasma caused a small but significant drop of the signal at m/z 79 and 81. Therefore the amount of carbon/organic solvent entering the plasma should be minimised to ensure


that the sensitivity is not suppressed. The participant in the CCQM-P114 study who used LC-ICP-MS employed a standard dimension column, i.e. 250 mm in length and 4.6 mm internal diameter. Flow rates from this column are generally in the range of 0.5-1 mL/min. This flow would provide significant difficulties due to the quantity of carbon loading on to the plasma. One of the easiest ways to reduce carbon loading on the plasma is to employ narrow bore columns. These HPLC columns require reduced flow rates (0.1-0.3 mL/min) thus reducing the plasma load but provide similar resolving capacity as they contain smaller particle size packing material. In addition the amount of solvent vapour entering the plasma from the spray chamber can be reduced by maintaining the spray chamber at -5 °C.

There are also alternative ways in which the amount of solvent entering the plasma can be reduced and these were investigated.

5.1 Investigation into the use of ultrasonic nebuliser with de-solvation (USN) to reduce plasma carbon loading

The aim of this experiment was to determine if BFRs are suited to this type of nebulisation and if sensitive detection can be achieved. The system was set up and the carrier gas from the instrument connected to the USN. Solutions were introduced to the USN system using a peri-pump. The system works by impinging the flow of solution onto an ultrasonic transducer plate. The resultant cloud is passed over a de-solvation membrane where the majority of water vapour and the more volatile components are removed from the gas stream. The gas stream is then directed to the plasma. The effect is to improve sample introduction efficiency to the plasma by up to an order of magnitude whilst reducing the formation of hydride and oxide interferences that derive from the aqueous media.

The CETAC U6000AT+ (M60103) system was set up using the parameter detailed below and the sweep gas from the unit was connected to the plasma transfer line of the instrument.

Table 12 : Optimal sensitivity setting for bromide ions for CETAC U6000AT +(M60103) ultra sonic nebuliser.

Parameter Setting

Heater ˚C 120

Cooler ˚C -8

Heater ˚C 130

Sweep gas 2.00

A 50 mg/L elemental bromine standard in 70 % aqueous methanol was prepared and introduced to the system at a flow rate of 0.7 mL/min. It was discovered that an extremely long wash out time were required to reduce the bromine signal. This caused significant difficulty when tuning the system as it took so long for the sensitivity to be affected after one of the parameters was altered. It was found that a 5 % ammonia solution made an excellent wash solution. The ammonia reacts with the elemental bromine to form ionic ammonium bromide which has a much lower vapour pressure than elemental bromine. A solution of 50 mg/L ammonium bromide in 70 % aqueous methanol was prepared and used to tune the system instead. This improved the sensitivity of the system and the washout time was reduced. The system was quite noisy, as expected, with an RSD of 15 % compared to normal nebuliser of <2 %. This would cause significant noise in the chromatographic baseline and reduce the detection limit of the system. Unfortunately the USN requires a significant flow of liquid uptake (i.e. 1 mL/min) in order to obtain stable signals.

The eluent of the chromatographic system (Polar RP, 80 % aqueous ethanol isocratic) was connected to the USN and an injection of 50 mg/L ammonium bromide injected onto the HPLC-ICP system. An un-retained peak was observed at m/z 81 and 79 which corresponds to ionic bromide. However when a 50 mg/L standard of Penta-BDE was injected onto the system no peaks were observed. It was noted that significant proportion of the organic modifier was not removed by the de-solvation unit and carbon build up occurred on the cones of the ICP-MS.


It can be concluded that the BFRs probably adhered to one of the internal surfaces in the system and did not elute from the system. Using a USN complicates the system and it is appears that BFRs do not pass through the USN system therefore it was decided not to pursue this further.

5.2 Investigations into the use of online aqueous dilution to reduce plasma carbon loading

Online dilution is a technique that can be used to reduce the carbon loading on the plasma. The column eluent is diluted with water before entering the nebuliser. This reduces the evaporation of the solvent and therefore the carbon loading on the plasma. The system was set up as previously and a second Shimadzu LC 3A HPLC pump was added and its flow of water matched with that from the analytical system (0.15 mL/min). The eluents were combined at a T-piece and the combined flow directed to the ICP-MS nebuliser. The dilution caused a very dramatic loss of sensitivity, which was much greater that expected from dilution alone. Also, the baseline of the system was very noisy which significantly increased detection limits. The peaks also tailed badly; this was probably caused by solubility problems due to the increased aqueous content of the mixed eluent. It was particularly a problem with the heavily brominated flame retardants which may be partially precipitating out when the eluents were combined. Therefore this approach was abandoned.

5.3 Investigations into the use of optional plasma addition gas (oxygen) to prevent carbon build-up

To overcome the build up of elemental carbon that is derived from the organic component from the mobile phase, oxygen can be added to the plasma gases. The oxygen reacts with the carbon in the plasma to form carbon monoxide and carbon dioxide which are removed in the gas stream. Bromine is not known to readily form oxides in the gas phase. Adding oxygen to the plasma reduces the available energy for ionization and therefore can suppress sensitivity. However it is an effective way to prevent elemental carbon blocking the cones and thus stopping ions getting to the mass spectrometer. It also needs to be considered that oxygen atoms are highly reactive species and will cause heavy oxidation of nickel cones. Therefore, it is recommended that both the sampler and skimmer cones are replaced with those made from platinum which can resist the chemical attack. The system was set up and the minimum amount of pure oxygen (5 % optional gas valve setting ≈ 100 mL/min) added to the plasma gas. A tune solution (50 mg/L ammonium bromide in 80 % aqueous methanol) was introduced into the spray chamber and the system optimised to obtain optimal sensitivity for 79 and 81 m/z. When the system was optimised the eluent from the HPLC (0.2 mL/min) was connected to the nebuliser and (Polar RP column, isocratic 80 % aqueous ethanol). A 50 mg/L standard solution of PBDE 209 was injected onto the system and a peak was observed at a retention time of 28 minutes. The system appears to be working and shows potential for further investigations.

5.3.1 Optimisation of ICP-MS conditions for bromine detection

In order to fine tune the system and achieve the best sensitivity, a more relevant tune solution was prepared. A 20 mg/L solution of hexabromobiphenyl technical mix in 90 % aqueous methanol was prepared. Unfortunately it was found that this solution deteriorated relatively quickly and needed to be prepared on a daily basis from a methanol stock solution. When an aged solution was used severe spiking of the signal occurred when tuning the ICP-MS.

The gas flow for the PFA nebuliser was found to be a critical parameter in obtaining optimal sensitivity. The PFA nebuliser was found to work with greater efficiency when the carrier gas was reduced. With aqueous solutions, a carrier gas flow rate of 0.9 mL/min has been found to be optimal but with 90 % aqueous methanol it was reduced to 0.67 mL/min to achieve optimal sensitivity.

It was found that solvent held in glass bottles quickly became contaminated with Br and noisy baselines were observed. HPLC solvents were prepared and stored in high density polyethylene bottles (HDPE) to avoid inorganic Br contamination from glass.


Table 13 : Agilent 7500CE optimised operating conditions

Agilent 7500CE ICP-MS instrument conditions

narrowTorch

-5Spray chamber temperature ºC

1Rotary pump

5% Oxygen (100mL/min)

Optional plasma gas flow

0.19Makeup gas L/min

0.66Carrier gas L/min

Pt sampler Pt skimmer

Cones

1540 WPlasma Power

PFA Agilent Nebuliser

60Total acquisition time min

1.5Integration time per point (sec)

79 Br81 Br

MassesAcquisition parameters

5.4 Optimisation of HPLC separation Optimal HPLC separation of the congeners of interest will be constrained due to the use of ICP-MS detection. ICP-MS detectors have certain limitations. Isocratic conditions are compatibility with ICP-MS as the organic load is constant and the plasma can be tuned for this steady state. However it is highly unlikely that an isocratic separation will be optimal as many structurally similar compounds require resolution. Most probably a gradient elution will be required. There is a limit to the maximum differential between the starting composition and the final composition of a gradient which an ICP-MS can accommodate. This is limited by the maximum reflected power that the system allows before it switches off the plasma and goes to standby mode. Reflected power heats up the torch and coil and damages the instrument. The cut-off value is in the order of 25 W and therefore limits the use of extreme gradients. Also, the stability of the system has to be considered. If reflected power spikes over 25 W occur, then again the system switches off the plasma and goes to standby mode.

Therefore initially UV detection (215 nm) was used to detect the flame retardants enabling cost effective development of the HPLC separations. It was also decided to use less costly technical mix standards to gauge the degree of congener separation that might be achieved for a given set of HPLC conditions. When separation conditions are established ICP-MS detection can be substituted.

Generally in the literature C18 type stationary phases have been used to separate BFRs. However significant gradients were employed with large solvent composition differential. These are incompatible with ICP-MS detection.

Therefore it was decided to initially investigate columns that provided π-π interactions as opposed to purely hydrophilic interaction. It is known that BDE-209 is highly hydrophobic and will require significant levels of organic modifier for it to elute from such a column. It is envisaged that interactions between an aromatic stationary phases and the two aromatic rings of the congeners should hopefully resolve some of these very structurally similar compounds. This has been observed for the separation of aromatic explosives using a phenyl-hexyl stationary phase. Generally it is hoped that the gradient modifier differential between start and end should be less than for a C18 column.

Initially significant problems with the HPLC auto-injector were encountered. The needle blocked and high back pressures caused the LC to shut down. Also the injection seat blocked and needed


changing. The solution to these problems was to use a syringe rinse of toluene with every injection and to limit the number of injections per vial septum. This appeared to resolve the problem and injector blockages were not a significant issue after these changes were adopted. It is thought that a punctured septum allows toluene into the body of the septum which softens it. The needle can then readily core the septum on subsequent injections. The core particle is then transferred by the needle resulting in blockages.

Another significant consideration is the compatibility of the extraction solvent with the mobile phase of the chromatographic system. The optimum situation is for the extract to have the same solvent composition as the initial mobile phase of the gradient to avoid excessive perturbation of the chromatographic system. In addition, the risk of co-extractant precipitating out on injection and causing column blockages needs to be reduced. For this application to ensure PBDE-209 solubility high levels of organic modifier are required in the extractant and therefore high levels of organic modifier are required in the initial mobile phase. In other words, because highly brominated PBDEs have a very poor solubility in polar solvents they need to be enriched in an organic modifier (typically >85 %) to ensure solubility. This constraint serves to limit the development of an efficient gradient elution system using hydrophilic interaction alone. These parameters have to be considered when developing a chromatographic system for BFRs.

5.4.1 Pentafluorophenyl Propyl stationary phase

The first column to be investigated was an Allure PFP Propyl 5 µm 150 x 2.1 mm from Thames Restek Corporation (Windsor, UK). This phase type was chosen due to the similarly of the phase to the analytes. The phase consists of pentafluorophenylpropyl groups. It was decided to use propan-2-ol as the organic modifier as it can be used as an extraction solvent for PBDEs from plastics and therefore it is expected that no compatibility problems on injection would occur. It also has a lower volatility compared to methanol or ethanol, which should reduce the carbon loading of the plasma when connected up. The column eluent was monitored at 215 nm using a DAD detector.

Table 14 : HPLC gradient condition (propan-2-ol) using Allure PFP column

Time min (Flow rate 0.17 mL/min)

% Propan-2-ol in mobile phase

0 65

1 65

20 99

31 99

32 65

51 65

It was found that PBDE-209 eluted faster than was expecting for these conditions as a peak at 15.2 minutes was observed. However significant back pressure were observed which was close to the column manufactures recommended maximum. This could cause significant problems for future analysis so this separation is far from ideal and was not pursued.

It was decided to try the column with a lower viscosity solvent (ethanol) under the same gradient conditions.


Table 15 : HPLC gradient condition (ethanol) using for Allure PFP column

Time min

(Flow rate 0.17 mL/min)

% Ethanol in mobile phase

0 65

4 65

25 90

31 90

32 65

51 65

A set of technical standards were prepared in methanol and 20 µl of each injected onto the system. The technical materials are a mixture of congeners and so each give a series of peaks. The retention times of the peaks are tabulated below

Table 16 : Retention times of observed peaks from the BFR technical products using Polar-RP column Deca- PBDE (min) Octa-PBDE (min) Penta- PBDE (min) Hexa-PBB (min)

28.5 20.1 18.4 15.2

22.5 19.7 18.4

23.9 20.6 19.8

25.9 23.7 21.2

23.5

25.9

Bold= major peak

As expected, PBDE-209 elutes significantly later at 28.5 minutes instead of 15 minutes, as previously, due to the more polar nature of the ethanol solvent. Although significant separation of the components has been achieved there is a lot of overlap. It also appeared that these technical mixes contain fewer congeners than might be expected.

5.4.2 Polar-reverse stationary phase

Another column phase type, a Polar-RP 80H 4 µm 150x2 mm (Phenomenex, UK) was tried using the gradient in the table below. This phase is ether-linked phenyl with polar end capping, and provides polar and aromatic reversed phase selectivity. The DAD system (215 nm) was again used as the detection system.

Table 17 : HPLC gradient condition (propan-2-ol) using Polar-RP column


% Propan-2-ol in mobile phase

0 35

4 35

25 80

31 80

32 35

51 35


Table 18 : Retention times of observed peaks from the BFR technical products using Polar-RP column

Deca-PBDE (min) Octa-PBDE (min) Penta-PBDE (min) Hexa-PBB (min)

33.0 14.8 10.9 14..2

18.1 13.8 15..3

20.7 17.7 18.2

21..9 21.7

22..9 25.5

26..2

Bold= major peak PBDE-209 elutes at 33 mins with this system and the separation of the component peaks is better than the PFP column. However, there is considerable overlap of the major brominated compounds. The peaks are broad and the fact that the major components of the Octa-PBDE and Hexa-PBB overlap at 18 mins means that this system is far from ideal. Increasing the retention times would increase the resolution but as the Hexa-PBB and Octa-PBDE, peaks overlap to such a large extent, it is doubtful that this stationary phase will resolve them. However, it was decided to use ethanol to try and achieve better resolution.

Table 19 : HPLC gradient condition (ethanol) using Polar-RP column

Time min

(Flow rate 0.17mL/min)


0 35

4 35

25 80

31 80

32 35

51 35

Table 20 : Retention times of observed peaks from the BFR technical products using Polar-RP column


35.9 22.4 17.8 21.6

25.5 21.1 23.1

28.1 22.3 25.6

29.1 24.7 27.2

32.6 28.6

35.9 31.7

Bold= major peak

There was increased resolution of the peaks of interest but the major peaks of Octa-PBDE and Hexa-PBB co-elute so another phase should be studied.


This column was connected to the ICP-MS system and eluted 80 % aqueous ethanol at a flow rate of 0.2 mL/min under isocratic conditions. This system was used to analyse some extracts to determine ASE extraction efficiency.

5.4.3 Reverse stationary phase C18

Therefore the more traditional C18 phase was investigated to try to achieve better separation. A Phenomenex Luna C18 column, 150 mm x 2.1 mm id, 3 µm was investigated using the following conditions. Table 21 : HPLC gradient condition (ethanol) using Reverse phase C18 column



0 75

4 75

25 90

31 90

32 75

51 75

Table 22 : Retention times of observed peaks from the BFR technical products using C18 column


34.6 22.9 14.9 16.6

25.7 16.4 18.3

26.3 19.9 20.4

28.0 22.1 21.3

28.9 23.4 23.4

30.8 26.1 26.3

31.4 28.6

29.7

30.7

35.4

Bold= major peak

Again there is increased resolution of the peaks of interest but the major peaks of Octa-PBDE and Hexa-PBB co-elute. There is also the possible overlap of one Hexa-PBB peak with Deca PBDE-209. As this is a component of significant interest this separation is not ideal. A column with a C8 phase was also tried, but similar separation was achieved and the resolution of the major component of Octa-PBDE and Hexa-PBB could not be improved.

5.4.4 Phenyl-Hexyl stationary phase

A Luna 3µm Phenyl-Hexyl 50 mm x 2.0 mm id column in series with a Luna Cyano column with the same dimensions, eluted with an ethanol gradient, was tried. This system was connected to the ICP-MS. Luna Phenyl-Hexyl is a reproducible, extremely stable phenyl stationary phase. Most phenyl phases use a short propyl (3 carbon) linker, which limits phase stability. The Phenyl-Hexyl bonded phase employs a phenyl ring with a hexyl (6 carbon) linker and is densely bonded to Luna silica surface. Dense bonding and the hexyl linker reduce bonded phase hydrolysis and increases chemical stability.


Table 23 : HPLC gradient condition (ethanol) using Luna phenyl-hexyl column

Time min

(Flow rate 0.15 mL/min)


0 73

2 73

33 84

35 84

36 73

50 73

Table 24 : Retention times of observed peaks from the BFR technical products using Phenyl-hexyl column

Deca-PBDE (min)

Octa-BDE(min) Penta-PBDE (min) Hexa-PBB ( min) Deca-PBDE (min)

42.5 14.3 8.1 13.5 24.0

18.0 9.9 14.6

22.5 12.1 15.0

25.1 12.9 15.8

27.5 14.1 19.2

31.0 15.5 22.0

33.4 17.5 24.6

42.5 31.0

Bold= major peak This system gave a separation interval of more than one minute between the major component of Hexa-PBB (19.2 mins) and Octa-PBDE (18.0 mins). In addition, the majority of the peaks were well resolved. Therefore this system has provided the best separation so far and was further intensively investigated.

The phenyl-hexyl column was then used without the cyano column, as it appeared the cyano column was not affecting the chromatography. The phenyl-hexyl stationary phase should provide significant interaction π-π with the aromatic rings of the BFRs. In order to maximise this interaction, the column manufacturer’s instructions recommend methanol is used as the sole organic modifier. The system was set using isocratic conditions of 87 % aqueous methanol and an extract of ERM-EC591 was injected onto the system. The peaks of interest were identified by injecting the individual congeners. Excellent separation of the congeners of interest was achieved. However, two of the congeners appeared to have small co-eluting peaks: PBDE-183 and PBB-209 (see Figure 9). Therefore significantly more resolving power is required to enable accurate quantitation of these two congeners. The arrows on Figure 14 chromatogram highlight the unresolved peaks on BDE-183 and BB-209 peaks. This isocratic system appears to be sufficiently fit-for-purpose for more routine-type analysis as the major congeners are well resolved.


Figure 9 : Chromatogram of an extract of ERM EC591 highlighting unresolved peaks

Isocratic HPLC condition of Toluene extract of ERM EC591

02000400060008000

100001200014000

0 10 20 30 40 5

Time in minutes

Cou

nts

per s

econ

d B

r79+

81

0

To investigate if co-eluting compounds (especially toluene) were suppressing the bromine signal, the mobile phase was spiked with a small quantity of isotopically-labelled inorganic bromide (Br81). A toluene extract of ERM-EC591 was injected onto the system and both Br81 and Br79 were monitored (Figure10). The upper (pink) signal is Br81 and the lower (blue) is Br79 signal. It can be concluded that there is very little suppression or enhancement of the signal from the solvent or co-extractants.

Figure 10 : Chromatogram showing the elevated baseline of Br81

0100002000030000400005000060000700008000090000

100000

0 10 20 30 40

Intensity cps for Br

Time (min)

Therefore, it was decided to purchase a longer phenyl-hexyl column and also employ a slight gradient to reduce the retention time of the last component. A Luna phenyl-hexyl 5µm 150 mm x 2.1 mm id column was used. The slightly longer column was used to improve the resolution between PBB-209 and a very minor peak that eluted just prior to it. This system was tried to also check whether resolution of a potential internal standard (PBDE209 with two Br atoms replaced with F in the molecule) from a minor component in the extract could be achieved. Unfortunately, this resolution was not achieved and this internal standard was rejected.


Table 25 : Optimised chromatographic conditions

Column (40 ºC) Luna Phenyl-Hexyl Phenomenex 3µm (2mm i.d., 100 mm length)

Mobile phase

gradient

Time min methanol% water%

0 82 18

17 85 15

26 93 7

48 93 7

50 82 18

60 82 18

Flow rate program (mL/min) 0.18

Injection volume (µl) (10 ºC) 1

(needle rinse used)

To increase the sensitivity of the system, larger injection volumes were investigated. However, peak resolution was lost for the minor components and the peak shape deteriorated. Single congener standards in pure nonane were injected onto the system, but resulted in distorted peak shapes; all standards were subsequently diluted in toluene before injecting on the system.

Figure 11 : Bromogram of an ASE toluene extract of ERM EC-591

05000

10000

1500020000

2500030000

35000

40000

0 10 20 30 40 50

Time / minutes

79B

r + 81

Br i

nten

sity

/ cp

s

PBDE 47

Internal standard

PBDE183

PBB209

PBDE206

PBDE209

05000

10000

1500020000

2500030000

35000

40000

0 10 20 30 40 50

Time / minutes

79B

r + 81

Br i

nten

sity

/ cp

s

PBDE 47

Internal standard

PBDE183

PBB209

PBDE206

PBDE209

Baseline resolution of all the congeners of interest has been achieved (Figure 11). The established system enables the characterisation of the method to be performed in terms of sensitivity and recovery from ERM-EC591.

Some further work into the separation was performed as a column manufacturer supplied them under a free trial scheme.


5.4.5 Investigation into the separation of BFR using π –π interaction column phases

COSMOSIL PYE column (Nacalai Tesque, Japan) was initially identified as a potential candidate for this project. Using normal phase a separation of a selection of PBDE congeners has been published1.

This column was not purchased due its high price ($1,450), and it was uncertain that it would achieve a similar separation if reversed phase was used. Normal phase chromatography cannot be used as it would load too much carbon on to the ICP-MS plasma. The column manufacturer loaned two columns from its range (PYE and NAP). Both provide greater π –π interactions with the analytes. The PYE column has pyrene groups on the stationary phase the other NAP has naphthalene groups. Using the established HPLC gradient and ICP-MS conditions, the columns were evaluated by injecting a mixed solution of 38 lower brominated congeners (SRM 2257). Both columns showed better separation of many of the lower brominated congeners than the phenyl-hexyl column. This resolving power will be of particular use for the quantitation (or purity analysis) of some of the lower brominated congeners. Unfortunately, the ICP-MS did not perform as well as normal in terms of sensitivity and a more complete comparison could not be carried out. Full evaluation of the separation potential of these columns remains to be determined.

Figure 12 : Chromatogram of SRM 2257 on a Cosmosil 5PYE column

Gradient elution of SRM2257 on Cosmosil 5PYE column 150 X 2.0 mm

70000

120000

170000

220000

270000

0 10 20 30 40 50

Time min

Cou

nts

per s

econ

d m

/z

81,7

9

The BFRs appear to have greater retention on this column than the phenyl-hexyl system and can be used for the quantification of early eluting BFRs.

Figure 13 : Chromatogram of SRM 2257 on a Cosmosil π NAP column

Gradient elution of SRM 2257 on Cosmosil π NAP

column 150 x2.0 mm

1900029000

39000490005900069000

0 10 20 30 40 50

Time in min

Cou

nts

per s

econ

d m

/z 7

9 +8

1

The separation on the NAP column appears to be similar to the phenyl-hexyl system but the peaks are not as well shaped. This could be due to the ICP-MS not operating at optimum performance, or the column may have needed further conditioning.

1 Kannan Narayanan et al (Bulletin of the Korean Chemical Society ISSN 0253-2964 2005 vol 26 number 4 page 529-536


Figure 14 : Chromatogram of SRM 2257 on a Luna Phenyl hexyl column

Gradient elution of SRM 2257 on Luna 3u Phenyl-

Hexyl column 100 x 2.0 mm

3800

5800

7800

9800

11800

0 10 20 30 40 50

Time in min

Cou

nts

per s

econ

d m

/z

81+7

9

Using the phenyl-hexyl system the resolution of the 38 peaks is very good considering it is carried out by HPLC. Further improvements could be made to optimise the separation if it required for environmental samples.

Figure 15 : Chromatogram of commercial Penta PBDE on a Luna Phenyl hexyl column

PentaPBDE Technical mix (20ug/mL)

0

2000

4000

6000

8000

10000

12000

0 10 20 30 40 50 6

Time min

cps

79Br

plu

s 81

B

0

r

Figure 16 : Chromatogram of commercial Octa-PBDE on a Luna Phenyl hexyl column

OctaPBDE technical mix (50ug/mL)

05000

10000150002000025000300003500040000

0 10 20 30 40 50 6

Time min

cps

79B

r and

81Br

0

The chromatograms in Figures 15 and 16 demonstrate that a clear distinction can be made between Octa and Penta PBDE technical mixtures.


5.5 Selection of an appropriate internal standard

The requirements of an internal standard for this analysis are quite exacting. The compound must contain at least one bromine atom and it also has to elute in a part of the LC-ICP-MS chromatogram that is ‘free’ from PBB or PBDE congeners.

Several brominated compounds were investigated : dibromocyclohexane, hexabromobenzene and pentabromophenol. These compounds had little retention on the column and eluted close to the void volume. Potentially many unretained compounds elute in the void volume, which could possibly affect the ICP-MS response, and so they are far from ideal. The mobile phase contains a relatively high level of organic modifier and therefore it is expected that only very hydrophobic compounds might be expected to be retained.

For GC analysis a pseudo BDE-209 is used as an internal standard. It has two fluorine atoms substituted for two bromine atoms (4’,6-Difluoro-2,2’,3,3’,4,5,5’,6’-octabromodiphenyl ether) or (F-PBDE 201) (Chiron AS, Norway). This compound was injected on to the system but was retained less well than expected and partially co-eluted with one of the extracted BFRs in the ERM-EC591 extract. Another similar compound with one Br atom substituted with F is available, but this compound was not tried.

It was decided to investigate compounds with significant aromatic character that would have more affinity with the column. 9-bromoanthracene was purchase from Sigma and a solution injected onto the system. The retention was again much less than expected and the compound eluted in 6 minutes. This retention time is significantly greater than the void volume (2.5 mins) and therefore is an ideal candidate as this part of the chromatogram is ‘free’ from PBB and PBDE congener peaks. However this material was found to be not sufficiently pure and another minor bromine containing compound eluted at the same time as PBDE-47.

Therefore it was decided to purify the 9-bromoanthracene by semi-prep HPLC using a semi-prep phenyl-hexyl column. A small quantity of material was purified using this column. A 1200 µg/g solution was prepared from the solid in methanol and five injections were carried out. The 9-bromoanthracene fractions were collected as they eluted from the HPLC system into a brown glass vial. The collected fractions were combined and evaporated to dryness under a stream of nitrogen at room temperature. The solid was re-dissolved in toluene to provide a solution containing approximately 40 µg/g of purified 9-bromoanthracene.

Table 26 : Chromatographic condition for the purification of 9-bromoanthracene

Column Phenomenex Luna phenyl-hexyl 250 mm x 10 mm 5µm

Mobile phase 100 % methanol

Flow rate 2.2 mL/min

Column temperature 40 ˚C

Injection volume 100 µl

Retention times min 9-bromoantracene (9.6)

Unknown impurity (12)


5.6 Initial analysis of IRMM-310 (PET) and ERM-EC591

An HPLC-ICP-MS system considered to be fit-for-purpose had been established (see Table 25) and it was therefore decided to try to quantify all the congeners of interest. A single point calibration standard was prepared at approximately the same concentration as the sample.

Table 27 : Recovery of BFR from IRMM-310 extracted at two different temperatures by ASE

Identification Recovery % Recovery % Recovery % µg/g Recovery % PBDE-47 PBDE-183 PBB-209 PBDE-206 PBDE-209

140 1a 94.7 69.5 120.0 3.9 199.4 150 2a 105.1 75.5 135.5 4.4 257.2

Two extractions were carried out at two different temperatures of IRMM-310 reference material detailed in section 3.2.3, Table 4. Unit 140 1a was extracted at 140 ˚C and Unit 150 2a at 150 ˚C. It appears that there is a significantly better extraction at 150 ˚C but this needs to be confirmed as this was a fairly basic investigation, without the use of an internal standard. Reasonably good recoveries were obtained demonstrating the feasibility of the approach. However, the recovery of PBDE-209 of over 200 % is of concern. PBDE-209 is the most important congener and so further investigations were required. To investigate the repeatability of the system 140 1a was injected onto the system a number of times and the data is tabulated below.

Table 28 : Repeatability exercise of HPLC-ICP-MS system by injecting extract 140 1a

PBDE-47 PBDE-183 PBB-209 PBDE-206 PBDE-209 Retention

time (Mins) 10.36 24.15 32.89 39.23 47.81

2487401 1300551 10787377 279954 8760655 2443107 1299215 10655945 277127 8805571 2511097 1276382 10833986 269028 9133406 2463208 1279021 10817748 271791 8932088

Mean 2476203 1288792 10773764 274475 8907930 Stdev 29479.9 12863.3 80886.6 4964.1 166923.5 CV % 1.2 1.0 0.8 1.8 1.9

The coefficient of variation for the various congeners varies from 0.8 to 1.9, which demonstrates that the system is stable and reproducible. It can be concluded that the variability of the recovery between the extracts 140 1a and 150 2a is not due to the HPLC-ICP-MS system fluctuations but to the extraction process.

To build upon this initial work, it was decided to analyse the certified reference material ERM-EC591. Three replicates were extracted under the optimum extraction conditions detailed in Table 7.

Table 29 : Recovery of BFR from ERM-EC591 using optimised ASE conditions

Replicate PBDE-47 PBDE-183 PBB-209 PBDE-206* PBDE-209 1 90.1 107.3 98.5 100.7 190.0 2 90.0 96.4 108.4 118.3 203.4 3 91.5 102.4 109.2 119.7 208.2

Mean 90.5 102.0 105.4 112.9 200.5 stdev 0.9 5.4 6.0 10.6 9.5 CV % 0.9 5.3 5.7 9.4 4.7

* There is no certified value but from the certification study a consensus value of 30.4 ±11 mg/kg (n=4) was used as the reference value.


A mixed standard was used to construct three point calibration graphs for the individual congeners, apart from BDE-209 where a single matched standard was used for quantification. All the calibration graphs were linear and had regression coefficients greater than 0.998. 9-bromoanthracene solution (40 μg/g) was added by weight in a 1:1 ratio to the extracts to act as an internal standard.

The recoveries for PBDE-47, 183 and PBB-209 were within ±10 % of the certified values and are within the range of uncertainty. The recovery for PBDE-206 was of the correct order of magnitude, but it is difficult to prove accuracy as the reference value is not a certified value. The reference value comes from the certification study and is a consensus value of only four determinations 30.4 ± 11 mg/kg and is subject to a significant level of uncertainty.

For the important congener PBDE-209 a recovery of more than 200 % was again obtained, even though this is a completely different reference material.

It was suspected that the PBDE-209 standard was approximately 50 % of its expected concentration and this was confirmed by comparing this unit with NIST SRM 2258. Another unit from the same batch was purchased to confirm this finding so that this evidence could be presented to the supplier. However this second unit’s concentration appeared to be correct so it was concluded that something could have occurred to the first unit during storage.

The recoveries of all the BFRs in these three replicates are in close agreement and the precision appears to be relatively good so it can be concluded that most parameters in the procedure are under control.

6. Polybrominated flame retardant standards Neat solid congeners are available from a very limited number of suppliers, and are expensive and generally synthesised on request. Chiron AS (Trondheim, Norway) was contacted through Greyhound Ltd, their UK distributor, and they provide the prices and delivery times for some mg quantities.

6.1 Purity assessment of custom synthesised PBDE-47

To demonstrate LGC capability for purity evaluations a quantity of PBDE-47 was purchased. PBDE-47 was chosen as it was among the less expensive BFRs and it is a congener of significant toxicity and environmental concern.

The LGC Purity Team applied a well established characterisation approach to PBDE-47. The approach has been successfully applied to many pure reference materials in the past. PBDE-47 is thought to be formed using the synthetic route shown in Figure 17. The evidence for this route being used is that iodine was identified as a significant contributor to the inorganic residue.

Figure 17 : Abbreviated reaction equation for the synthesis of PBDE-471

Six different techniques were applied in this exercise and the results are tabulated below together. A full detailed report has been complied 2 but a summary table of results is below in Table 30. 1 Synthesis and characterisation of highly polybrominated diphenyl ether PhD thesis by Daniel Teclechiel at the Department of Environmental Chemistry Stockholm University 2008 http://www.mk.su.se/page.php?pid=156

2 LGC report numbers : AT20/RMCS/2009/BDE_47_Residual_Solvents_AT20_PCS_2276_HS_GC_MS_ZB624, AT20/RMCS_2009/ BDE_ 47_Purity_AT20_PCS_2276_GC_FID_ZB_5HT AT20/RMCS/2009/BDE_47_Residual_Solvents_AT20_PCS_2276_HS_GC_MS_ZB624 AT20/RMCS/2009/ BDE_ 47_Purity_AT20_PCS_2276_HPLC_C18

OH

Br

Br

I

Cl Br

BrBr

Br

Br

Br

O

Br

Br

Br

BrBr2,Iron NaOH, H2O


http://www.mk.su.se/page.php?pid=156

Table 30 : Purity assessment results for PBDE-47 solid.

Analytical results % m/m u (% m/m)

HPLC-PDA 99.22 0.007

GC-FID 99.31 0.003

DSC 99.38 0.014

Purity organic 99.30 0.048

Moisture 0.01 0.008

Inorganic residue 0.02 0.004

Residual solvent 0.01 0.005

Homogeneity - 0.006

Purity total 99.2 0.148

A complementary purity evaluation was carried out by HPLC-ICP-MS. A 180 µg/g solution of PBDE-47 in toluene was injected onto the system as previously described. Peak normalisation was used to evaluate the purity. An example chromatogram is provided below were five bromine containing impurities were resolved.

Figure 18 : HPLC-ICP-MS Chromatogram of toluene solution of PBDE-47 (180 µg/g)

Note : Efforts to identify the impurities was not made.

Table 31 : Results of the complementary purity evaluation by HPLC-ICP-MS.

Injection no. % m/m 1 99.35 2 99.30 3 99.37 4 99.38 5 99.76 6 99.82 7 99.80

mean 99.54 Standard deviation 0.24


PBDE-47 was characterised for purity using a well established approach at this laboratory. Its purity was calculated to be 99.2 ± 0.3 mass % at the 95 % confidence interval (k = 2). The HPLC-ICP-MS value of 99.5 ± 0.2 % (n=7) is in good agreement. The procedure has significant potential for efficient purity assessment of BFRs. The potential to perform simultaneous PDA-UV detection and also the detection of other elements, such as iodine, could provide significant added value.

Additionally, efforts were made to confirm the identity and purity of this solid PBDE-47 material. A 25 µg/g solution of PBDE-47 in toluene was prepared and injected onto the system three times alternately with a 25 µg/g commercial solution of PBDE-47 (Cambridge Isotopes Ltd). The retention times were used to confirm the material was BDE-47 and the concentration was also calculated using the commercial standard. Using the commercial standard as a calibrant, a recovery of 99.7 % was obtained. This value is very close to 100 % so provides additional confirmation of the purity of BDE-47.

6.2 Assessment of the purity of commercially available 50 µg/mL standards by LC-ICP-MS

Most of the 209 possible congeners are now available as single standard solutions from commercial suppliers. Generally the most concentrated solution available of a single congener is 50 µg/mL. The solvent is generally n-nonane but other solvents are used e.g. isooctane or toluene. Also complex mixtures of congeners are available at lower concentrations.

Single congener standards 50 µg/mL (≈ £100 / 1 mL) were purchased for the method development work. 50 µg/mL solutions of PBDEs 47, 183, 206 and 209 in n-nonane were obtained from Cambridge Isotopes laboratories (via LGC Standards, UK) The standard compounds had certified purity and concentration claiming traceability to NIST. However, no values were provided on the certificates for the solvent density. It was decided to evaluate these solutions in terms of congener purity using the HPLC-ICP-MS system. In order to asses the purity of these standards they were injected without dilution onto the system in triplicate. Assuming compound independent (bromine) response potentially the purity, in terms of bromine, can be determined.

Table 32 : Purity of individual congener standard by HPLC-ICP-MS

Component Purity %

N=3

Possible identity of impurity

BDE-47 100 nd

BDE-183 100 nd

BB-209 99.74

(±0.01)

Not identified

BDE-206 95.97

(±0.03)

Not identified

BDE-209 98.82

(±0.06)

BDE-206 (retention time)

The congeners were found to be relatively pure with purities generally greater than 95 %. However, it was found that the 50 µg/mL PBDE-209 standard contained a small quantity of PBDE-206. The most efficient and convenient way to determine the congeners in the plastic extracts is to prepare a mixed standard of the congeners of interest. However, the small amount of BDE-206 in the PBDE-209 standard will cause significant quantification problems for PBDE-206. This is because the PBDE-209 is at a relatively high concentration while BDE-206 is approximately 25 times lower. The


other standard solutions did not contain significant quantities of other congeners that could be readily identified.

Using HPLC-ICP-MS to access purity could provide a means to demonstrate congener purity and concentration at LGC. If the LGC characterised PBDE-47, or other certified bromine-containing compound, was added quantitatively to the standard congener solution by mass, the bromine peak from the certified material will be of known concentration and, using this, the amount of Br in the other peak can be calculated and related to the congener concentration using the known molecular weight. However, this experiment was not performed due to time constraints, but has the potential to be commercialised. The fact that HPLC causes negligible de-bromination offers a significant advantage in terms of metrology

7. Validation of the procedure To validate the procedure ERM-EC591 was analysed. The cryogenically ground (<0.5 mm) material (0.11 g) was packed into six replicate accelerated solvent extraction cells and extracted under optimal conditions Table 7. Toluene was added gravimetrically to the extracts in order that all extracts had theoretically the same concentration as the most dilute. Using this estimated concentration, gravimetric standards were prepared, one just above, and one just below (bracketing) the theoretical concentration.

The preparation of the bracketing standards from the individual congener solutions was planned in detail as the 50 µg/mL (≈69 µg/g) solutions are expensive and only 1 mL units are available. The dilution schemes are detailed below. The bracketing approach was adopted as it was considered to be the most accurate for this application. With chromatographic runs of approximately an hour, instrumental drift is a major consideration and its affects should be minimised by this technique. The mixed standards were prepared at twice the expected concentration and then diluted 1:1 gravimetrically with the internal standard. The sample extracts were also diluted 1:1 gravimetrically with the internal standard.

Figure 19 : Representing dilution scheme of mixed congener standards

Dilution scheme for individual congener standards (69ug/g) for bracketing standards

69ug/g individual std diluted 0.11g → …… g toluene

BDE47 BDE183 BDE206 BB209

Mixed low level std

4µg/g 1.2µg/g 40µg/g

0.11g of each

LOW level standard

12µg/g

0.11g and0.11g of internal std

Low level injection standardBDE47 2.7 µg/gBDE183 0.98 µg/gBB209 8.38 µg/gBDE206 0.30 µg/g

BDE47 BDE183 BDE206 BB209

Mixed high level std

6µg/g 2µg/g 43µg/g

0.11g of each

High level standard

15µg/g

0.11g and 0.11g of internal std

High level injection standardBDE47 2.8µg/gBDE183 1.0µg/gBB209 8.5µg/gBDE206 0.35µg/g

69ug/g individual std diluted 0.11g → …… g toluene

The purity of the individual congener standard BDE-209 was found to contain a small but significant amount of PBDE-206. This prevented BDE-209 from being added in the mixed standard as it would


have artificially enhanced the BDE-206 concentration causing quantification problems. A separate dilution scheme for PBDE-209 is detailed below.

Figure 20 : Representation of dilution scheme of PBDE-209 stock standard

Preparation of PBDE 209 single standard solutions and sample extract from bracketed determination

Single congener std diluted 0.11g → 0.96 g toluene

PBDE209 69ug/g

Low level std

LOW level standard


Low level injection standardBDE209 8.5 µg/g

PBDE209 69ug/g

High level std

HIGH level standard


High level injection standardBDE209 9.3 µg/g

Single congener std diluted 0.11g → 0.89 g toluene

0.11-0.13g sample ≈ 9g solvent

Toluene added so that all

the extracts have the same

expected concentration

Extraction of sample


Extract injection solution

Using this approach described previously, the excellent recoveries (detailed in Table 33) were obtained. ERM-EC591 is not certified for BDE-206 content as the certification study provided too few data points and they were widely scattered (30.4 ± 11.1 mg/kg). However, it is acknowledged that this value is probably artificially high as most participants used gas chromatography. BDE-206 is the thermal degradation product of BDE-209. This explains the apparent low recovery for BDE-206. One participant in the CCQM P114 study also used LC-ICP-MS which avoids the issue of thermal degradation and obtained a value of 21 ± 2 mg/kg. This value is in excellent agreement with the value obtained using the approach detailed in this report of 21 ± 1.4 mg/kg.

Table 33 : Validation data:- Recovery of selected BFR from ERM-EC591

PBDE-47 PBDE-183 PBB-209 PBDE-206a PBDE-209

Certified value (g/kg) 0.245 0.087 0.748 0.030 0.78

Certified uncertainty (g/kg) 0.023 0.008 0.08 0.011 0.09

Mean obtained values (g/kg) 0.259 0.081 0.725 0.021 0.793

Standard deviation, n = 6 (g/kg) 0.006 0.006 0.034 0.001 0.021

Mean recovery (%) 105.7 93.1 96.9 70.0 101.6

Standard deviation (%) 2.4 6.8 4.5 3.3 2.7 aNot a certified value in EC591 uncertainty but stdev (n = 4).

BDE-209 was determined separately because the stock standard from CIL was found to contain trace amounts of BDE-206. A recovery of 102 ± 2.7 % was obtained. To confirm the recovery the extracts were re-analysed against NIST SRM 2258 and similar recovery of 103 ± 4.3 % was obtained. All the certified compounds gave recoveries which agreed well with the certified values and are well within the certified uncertainty of ±10 %.

7.1 Total Br content of ERM-EC591

Using the existing data it was clear that the total bromine content of ERM-EC591 could easily be calculated. PBDE-47 was used as the calibrant as its purity had been determined traceably, but


potentially any of the congeners could have been used. Alternatively, a well-characterised Br containing compound could be used that was retained on the column at an appropriate retention time. The molecular weight to Bromine conversion factor was calculated and used to convert the standard compound concentration to Br concentrations. All the peaks in the extract chromatograms were integrated and the total peak area summed. Using the peak areas for the PBDE-47 peak in the bracketing standards the Br content of each extract chromatogram was calculated. This value was then related back to the total Br content of the weight of sample. Therefore by utilising the peak area summation technique over the whole chromatogram, the total Br content of the extract can be easily calculated. An average recovery of 102 ± 2.5 % (RSD n=6) for total Br from the certified value (2.08 g/kg with an expanded uncertainty (k=2) of 0.07 g/kg) (± 3.4 %) was achieved.

This approach utilises the extensive linear range capability and compound independent response of the ICP-MS technique.

7.2 Determination of limit of detection of the LC-ICP-MS system

The system was set up and tuned as previously described. A mixed standard of all five congeners of interest was prepared. This was used to prepare 4 standards in the range of the expected limit of detection. The low level of BDE-206 in the BDE-209 solution will be insignificant at these dilute levels. Calibration graphs were constructed for all the components using peak height rather than peak area against concentration. For each blank chromatogram the data was converted to an excel table. A standard deviation was calculated for each blank over the expected elution time of each peak. These standard deviations were then multiplied by a factor of three and using the slope of the calibration lines for each compound converted to µg/g. A factor of 10 was used to calculate the reporting limit of each component. An average of the six injections is quoted.

Table 34 : Detection limit of LC-ICP-MS system

ng/g µg/g 9 g

solvent Sample /0.11 g

BDE-47 25 0.025 0.227 2.1 BDE-183 22 0.022 0.202 1.8 BB-209 18 0.018 0.159 1.4

BDE-206 24 0.024 0.219 2.0 BDE-209 55 0.055 0.491 4.5

Table 35 : Reporting limit of LC-ICP-MS system

ng/g µg/g 9 g

solvent Sample /0.11 g

BDE-47 84 0.084 0.755 6.9 BDE-183 75 0.075 0.673 6.1 BB-209 59 0.059 0.529 4.8

BDE-206 81 0.081 0.729 6.6 BDE-209 182 0.182 1.635 14.9

8. Conclusion Accurate quantification of PBDEs and PBBs in plastic has been achieved by overcoming some significant analytical challenges. This is demonstrated by the recoveries obtained for ERM-EC591.

The milling of the plastic pellets by cryogenically cooled centrifugal mill minimises thermal degradation from friction heating. The centrifugal mill also ensures that the material is ground to a uniform particle size (0.5 mm) which provides consistent extraction properties. Photolytic degradation and migration of BFR out of the surface of particles is minimised by storing the sample at -20 ˚C in brown glass vessels.

ASE provides a convenient and efficient extraction system that protects BFRs from photolytic degradation during the extraction process. Optimisation of the extraction temperature for different


polymers can be performed with relative ease. Efficient extraction can be achieved by the ability of the ASE system to perform numerous extraction cycles on the same sample cell. This also minimises the time the BFRs are subjected to elevated temperatures and therefore minimises the possibility of thermal degradation. Exhaustive extraction of the cell can be used to confirm efficient extraction has been achieved.

Photolytic degradability and solvent loss were minimised by storing the extracts in brown glass vessels at 4 ˚C.

During the separation thermal degradation was minimised by performing the chromatographic separation at 40 ˚C rather than 250 ˚C plus for GC separations. The developed HPLC conditions provide adequate separation of the congeners of interest. The separation cannot be expected to resolve all the possible congeners, however of the 38 congeners in the NIST SRM 2757, 29 peak tops can be observed. This is very good separation for an HPLC system. With further optimisation and the possible use of (or combination with) other π-π interaction columns, better resolution could be achieved if required.

The ICP-MS detector has a very wide linear range and so quantification is possible over a wide range of concentrations (1 and 1000 mg/kg) with relative ease. ICP-MS provides very specific detection of Br and is not affected by signal suppression of co-eluting compounds. This enables peak summation to be performed enabling the accurate quantification of total Br to be performed.

The use of the internal standard 9-Bromoanthracene added by weight corrects for injection volume variability and solvent evaporation. The use of bracketed standards prepared by weight/weight reduces errors cause by instrument drift and provides greater traceability.

The major issues associated with the determination of BFRs in plastics have been reconciled. However, there are some significant issues associated with the commercial standards which are of concern.

The calibrants were sources from commercial suppliers and generally have found to be suitable apart from one standard unit which was 50 % in error. However, certified values are quoted in m/vol which is relatively straightforward to convert to m/m if it is in a pure solvent. Wellington Labs supplied a standard solution in mixed solvent 10 % toluene : nonane. This adds a complication to the conversion to m/m. Commercial suppliers should be advised to include a density figure for the solvent on their certificates at specific temperatures (e.g.18, 20 and 25 ˚C) together with an expansion coefficient figure.

Another important finding was that the single congener standard of PBDE-209 contained a small but significant amount of PBDE-206. Generally it is more efficient to produce mixed working standards for quantitation. The level of PBDE-206 would cause significant underestimate of this minor congener in the sample. Therefore a single component standard was prepared for the determination of PBDE-206. Checking the purity of the solution standards is therefore necessary.

Added validity to the procedure was provided by the determination of the total Br content of ERM-EC591. Using PBDE-47 (purity traceable to LGC) as a calibrant and utilising the peak area summation technique over the whole chromatogram, the total Br content of the extract was calculated. A recovery of 102 ± 2.5 % (RSD n=6) was achieved.

Over all the results form this study are very encouraging as all the recoveries for ERM-EC591 appear to agree well with the certified value and PBDE-206 agrees with the most creditable value for this material. The limits of detection are suitable for assessment of materials for compliance with the regulations.

9. Future work The cost of operating an LC-ICP-MS is quite considerable, especially as this particular separation takes over 60 minutes per sample. Therefore there has to be some significant advantage in using this technique. The procedure obviously has advantages when high accuracy analysis is required. However, for more routine applications the run time could be shortened with some sacrifice of congener separation, but full advantage could be taken of the ICP’s species un-specific response. A single congener standard solution has been used to quantify the total BFR content to good affect thus simplifying quantification considerably¹.

¹Shao, Mingwu; Wei Cho; Jia, Yongjuan et al; Analytical Chemistry 2010 ACS ASAP


These steps should make LC-ICP-MS determination more economically viable.

GC-MS analysis is less expensive to operate than LC-ICP-MS and also provides fragment ions which can be used to confirm compound identity. It needs to be considered that LC-ICP-MS technique does not provide structural identity which is generally required for legal purposes. Identification is derived only from retention time standard comparisons. However, GC-MS quantification is complex as each congener has to be injected onto the system and the concentration calculated and summed. Further work into establishing more cost effective LC-ICP-MS analysis would be of benefit for routine analysis.

9-Bromoanthracene has proved to be very useful as an internal standard for this analysis. There may be potential for LGC to produce purified material for sale. If 9-bromoanthracene is thermally stable compound it might have wider applicability in GC-MS analysis.

The purity and concentration of individual congener standards is an important area which could yield benefits if investigated further. The LC-ICP-MS system provides an excellent approach to accurate quantification of any BFR single standard by relating its concentration to a certified bromine containing compound that gives a resolved peak in the chromatogram, e.g. BDE-47. Purity in terms of congener content is also assessed simultaneously by the system. This analysis requires only a few µL of 50 µg/g standard solutions. This could be provided as a commercial service to the producers of standards, i.e. Cambridge Isotopes Ltd or Wellington Labs, but would require further validation. This technique of purity assessment has clear advantages when applied to C13 labelled materials which are extremely expensive as only a small amount of the material is required.

Isotopically labelled C13 BFRs are available; however, compounds labelled with isotopic bromine are currently not available. Therefore BDE-209 would make an excellent candidate for Br labelled synthesis. The combination of C13 and Br labelled compounds would provide an excellent analytical tool for the investigating of the environmental fate of theses compounds. Also it would enable accurate quantification of low levels of BDE-209 in very complex environmental matrix types using SSIDMS.

For trace analysis in water or other environmental matrices, greater sensitivity will be required and further work into improving the systems sensitivity will need to be performed:

1) Investigate the use of the improved model Agilent 7700 ICP-MS as LOD for Br of 17ng/mL has been reported¹

2) Investigate the use of the Apex nebuliser as means to increase sensitivity of the system.

3) An online UV digester (approx 1 metre coil wrapped around the UV source) could be placed after the column as this has been described in a HPLC conductivity paper before the ICP-MS². This would decompose the BFR releasing bromine atoms. If the mobile phase is made slightly acidic, hydrogen bromide or molecular Br2 will be formed both of which are significantly more volatile than the intact BFRs. This will increase the transfer of Br to the plasma and result in much greater sensitivity of the system. However, they may also cause increased tailing of peaks as it could take time for them to be flushed from the spray chamber.

4) Use of other extraction solvents that have greater compatible with the HPLC system, e.g. ethanol or propan-2-ol should be investigated. The use of more volatile solvents other than toluene opens up the opportunity to concentrate extracts before analysis. An estimate of the detection limit that can be achieved using DAD detection should be performed.

The EU is currently negotiating about extinction to include those compounds mentioned in the RoHS recast and those substances identified under REACH as being of very high concern. Potentially they include hexabromocyclododecane (HBCDD), diethylhexyl phthalate (DEHP), butylbenzylphthalate (BBP) and dibutyl phthalate (DBP)³. Beryllium has also been mentioned as a possible addition to the list.

¹ Katarzyna Bierla; Anne Riu et al J. Anal. Spectrum, 2010, 25, 889-892

² Uchiyama, Karzuhisa et al; Bunseki Kagaku., 2010, 59(3), 207-212 Japanese)

³A draft proposal for the recast of the RoHS Directive was recently published by the European Parliament (EP). http://www.element-14.com/community/docs/DOC-20143


10. Acknowledgements I wish to acknowledge the help of

Mr. Paul Norris (LGC Ltd) whose experience in the use of ASE was of great assistance to this research project.

Reinhard Zeleny, (IRMM) (CCQM P114 study organiser) for materials which allowed the project to proceed.

Chris Hopley (LGC Ltd) for his assistance with GC-MS set up and analysis.

Steve Wilbur (Agilent) for advice on ICP-MS optimisation.

Ian Axford and Andy Earls (LGC Ltd) for some background information on brominated flame retardants.

Frank Torma and Thierry LeGoff (LGC Ltd) for the purity evaluation of PBDE-47.

LGC Runcorn for the loan of the cryogenic grinder.

Nick Boley and Victor Martinez (LGC Ltd) for help in compiling the report.

Chris Smith (NMO) RoHS UK compliance enforcer.


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