Aerosols of Isocyanates, Amines and Anhydrides

Sampling and Analysis

Jakob Dahlin

Stockholm University

©Jakob Dahlin, Hässleholm 2007 ISBN 978-91-7155-415-4 Printed in Sweden by Universitetsservice, US-AB, Stockholm 2007 Distributor: Department of Analytical Chemistry, Stockholm University Sweden

Abstract

This thesis presents methods for air sampling and determination of isocy-anates, amines, aminoisocyanates and anhydrides. These organic compounds are generated during production or thermal degradation of polymers such as polyurethane (PUR) or epoxy. Isocyanates, amines and anhydrides are air-way irritants known to cause occupational asthma. Some of the isocyanates and diamines are listed as human carcinogens.

Isocyanates and anhydrides are reactive and needs to be immediately de-rivatized during sampling. Methods have been developed for determination of airborne isocyanates and aminoisocyanates using di-n-butylamine (DBA) as the reagent to form stable urea derivatives. Anhydrides were derivatized with DBA to stable amide derivatives. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) enabled instrumental detection limits as low as 10 attomoles. Methods for preparation and characterization of reference so-lutions of technical grade isocyanates, aminoisocyanates and anhydrides are presented. A nitrogen-selective LC-detector enabled the quantification of the DBA-derivatives.

A novel sampler is presented. The sampler consists of a denuder in series with a three-stage cascade impactor and an end filter. The sampler made it possible to reveal the distribution of isocyanates between gas and different particle size fractions. During thermal degradation of PUR, isocyanates were associated to particle size fractions (<1 µm) that may penetrate to the lower airways. The distribution during 8 minutes changes noticeably. Gas phase toluene diisocyanates (TDI) and methylene diphenyl diisocyanate (MDI) become associated to small particles (<1 µm). Many workers are exposed to this kind of aerosol. As a reference method air-sampling was performed us-ing an impinger filled with di-n-butylamine (DBA) in toluene, connected in series with a glass fiber filter. There was a good agreement between the de-nuder impactor sampler and the reference method.

List of Papers

The thesis is based on the papers listed below. References to the papers in the text are assigned with roman numerals.

I Determination of technical grade isocyanates used in the

production of polyurethane plastics. Marand, Å., Dahlin, J., Karlsson, D., Skarping, G., Dalene, M. Journal of Environmental Monitoring, 2004, 6, 606-614

II Determination of Airborne Isocyanates as di-n-butylamine derivatives using liquid chromatography and tandem mass spectrometry. Karlsson, D., Dahlin, J., Marand, Å., Skarping, G., Dalene, M. Analytica Chimica Acta, 2005, 534(2), 263-269

III Size-separated sampling and analysis of isocyanates in work-place aerosols. Part I. Denuder-cascade impactor sampler. Dahlin, J., Spanne, M., Karlsson, D., Dalene, M., Skarping, G. Manuscript

IV Size-separated sampling and analysis of isocyanates in work-place aerosols. Part II. Ageing aerosols from thermal degra-dation of polyurethane. Dahlin, J., Spanne, M., Karlsson, D., Dalene, M., Skarping, G. Manuscript

V Determination of isocyanates, aminoisocyanates and amines formed during thermal degradation of polyurethane. Karlsson, D., Dahlin, J., Skarping, G., Dalene, M. Journal of Environmental Monitoring, 2002, 4, 216-222

VI Determination of airborne anhydrides using LC-MS moni-toring negative ions of di-n-butylamine derivatives. Dahlin, J., Karlsson, D., Skarping, G., Dalene, M. Journal of Environmental Monitoring, 2004, 6, 624-629

In paper I, II and V the respondent contributed to the experimental work, field measurements and the writing of the papers. In paper III the respon-dent was responsible for the majority of the experimental work and the writ-ing of the paper. In paper IV and VI the respondent performed all the ex-perimental work and wrote the papers.

Paper I, V and VI are reproduced by permission of The Royal Society of Chemistry. Paper II is reprinted by permission of Elsevier.

Contents

1 Introduction ..........................................................................................11

2 Aims of the Thesis ...............................................................................13

3 Isocyanates..........................................................................................14 3.1 Introduction.........................................................................................................14

3.1.1 Monoisocyanates.....................................................................................14 3.1.2 Diisocyanates ..........................................................................................15 3.1.3 Isocyanate adducts and Oligomers. ........................................................16

3.2 Health Effects .....................................................................................................17 3.3 Exposure.............................................................................................................18 3.4 Air Sampling .......................................................................................................19

3.4.1 Reagents .................................................................................................19 3.4.2 Sampling Techniques ..............................................................................23

3.4.2.1 Impinger sampling..............................................................................23 3.4.2.2 Filter and solid sorbent sampling .......................................................23 3.4.2.3 Passive sampling ...............................................................................25 3.4.2.4 Fractionated Sampling .......................................................................25

3.4.2.4.1 Denuder...................................................................................26 3.4.2.4.2 Impactor...................................................................................28 3.4.2.4.3 Results from laboratory evaluation..........................................31

3.5 Sample Analysis .................................................................................................33 3.5.1 Colorimetric methods...............................................................................33 3.5.2 Chromatography ......................................................................................34 3.5.3 Detection Methods...................................................................................36

3.5.3.1 Mass Spectrometry ............................................................................37 3.5.3.2 Standard preparation .........................................................................40 3.5.3.3 Internal standard for MS.....................................................................42

4 Amines and Aminoisocyanates............................................................44 4.1 Diamines - properties and uses..........................................................................44 4.2 Exposure and Health Effects..............................................................................45 4.3 Air Sampling .......................................................................................................46 4.4 Amine derivatization ...........................................................................................47

4.4.1 Derivatization with anhydrides.................................................................48 4.4.2 Derivatization with chloroformates ..........................................................48

4.5 Determination of amines and aminoisocyanates ...............................................49 4.5.1 Preparation of standards .........................................................................49 4.5.2 Analysis ...................................................................................................50

5 Organic Acid Anhydrides .....................................................................52 5.1 Chemical propreties............................................................................................52 5.2 Industrial applications .........................................................................................53 5.3 Exposure and health effects...............................................................................53 5.4 Air sampling........................................................................................................54

5.4.1 Filters and solid sorbent sampling...........................................................54 5.4.2 Impinger or bubbler sampling ..................................................................55

5.5 Sample Determination ........................................................................................56

6 Final comments....................................................................................59

7 Conclusions .........................................................................................61

8 Sammanfattning (in Swedish)..............................................................62

9 Acknowledgments................................................................................64

10 References...........................................................................................66

Abbreviations

1-2PP 1-(2-pyridyl)-piperazine 2-MP 1-(2-methoxyphenyl)-piperazine AA Acetic anhydride APCI Atmospheric pressure chemical ionization CLND Chemiluminescent nitrogen detection DBA Di-n-butylamine DEA Di-n-ethylamine DPA Di-n-propylamine EC Electrochemical ECD Electron capture detector ES Electrospray ET Ethyl chloroformate FID Flame ionization detector FL Fluorescence GC Gas chromatography HA cis-Hexahydrophthalic anhydride HAI Hexamethylene aminoisocyanate HDA Hexamethylene diamine HDI Hexamethylene diisocyanate HFBA heptafluorobutyric anhydride HMDI Dicyclohexyl methane diisocyanate HPLC High pressure liquid chromatography HSE Health and safety executive IARC International agency on research of cancer ICA Isocyanic acid ICRP International commission on radiological protec-

tion IPDI Isophoronediisocyanate LC Liquid chromatography MA Maleic anhydride MAI Methylenediphenyl aminoisocyanate MAMA 9-(N-methylaminomethyl)- anthracene MAP 1-(9-anthracenylmethyl)-piperazine MCP Multi channel plate MDA Methylene diphenyl diamine MDI Methylene diphenyl diioscyanate

MIC Methylisocyanate MMNTP 4-methoxy-6-(4-methoxy-1-naphtyl)-1,3,5-

triazine-2-(1-piperazine) MRM Multiple reaction monitoring MS Mass spectrometry NBDPZ 4-nitro-7-piperazino-2,1,3-benzoxadiazole NDI Naphtalene diisocyanate NIOSH National institute of occupational safety and

health Nitro N-4-nitrobenzyl-N-n-propylamine NPD Nitrogen-phosphorous detector OEL Occupational exposure limit OSHA Occupational safety and health administration PA Phthalic anhydride PAC 9-anthracenylmethyl-1-piperazine carboxylate PFBBr Pentafluoro benzylbromide PFPA Pentafluoro propionic anhydride PFU Phenyl-formaldehyde-urea PhI Phenyl isocyanate pMDI Polymeric MDI PUR Polyurethane PVC Polyvinylchloride RH Relative humidity SIR Selective ion recording TA Tetrahydorphthalic anhydride TAI Toluene aminoisocyanate TDA Toluene diamine TDI Toluene diisocyanate TLC Thin layer chromatography TMA Trimellitic anhydride TOF Time of flight TRIG Total reactive isocyanate group TSD Thermionic specific detector TWA Time weighted average UV Ultraviolet

1 Introduction

Exposure to airborne chemicals is very common in several different indus-trial branches. To investigate exposure in different work environments it is necessary to select proper sampling and analytical methods. Authorities in most countries have established limit values for the exposure to different chemicals that results from measurements are compared with.

There are different ways to obtain information about exposure to airborne compounds. For a practicing industrial hygienist and for authorities a total level of some compound with an occupational exposure limit (OEL) value is often sufficient information. If the value obtained is well below the limit, the situation is regarded as acceptable and no measures are needed to improve the situation. On the other hand, if the value obtained is above the OEL, measures must be taken to lower the exposure levels for the workers. How-ever, if not only the OEL is taken into account there are many more aspects to study. In and industrial environment the air is usually not very clean. Dust, particles, different gases and moisture are almost always present. It has been shown that depending on size, particles can reach different parts of the human airways. Some particle size fractions are able to penetrate all the way down to the alveolar region, where the residence time is longer and particle clearance is less efficient. Hazardous compounds may be transported to this region if adsorption of the compounds onto particle surfaces occurs. To ob-tain a more detailed view of an aerosol it is necessary to use methods which not only obtain a total concentration of a certain compound, but also has the ability to separate relevant particle size fractions. If the air level of a com-pound is below the OEL, it may be viewed as acceptable. However, if the compound is associated to small particles that can penetrate deep into the airways, the levels may be too high. This is a very good reason for the de-velopment of samplers that provides results that better reflects the risks. For this purpose there is a need for methods that can show how toxicants are distributed in an aerosol.

The fate of chemicals in the body is an important issue. Some are not very reactive e.g. solvents and may be exhaled after inhalation and only a small amount is absorbed and metabolized. Isocyanates and anhydrides are exam-ples of extremely reactive compounds. If inhaled, there are many compounds in the airways that are highly reactive towards isocyanates and anhydrides. Adducts may be formed with compounds containing e.g. amine groups

11

(-NH2) and hydroxyl groups (-OH). The formation of biological adducts may damage e.g. a protein in an irreversible way.

Isocyanates are chemical compounds used in the manufacture of polyure-thane (PUR) plastics. PUR is a versatile polymeric material. The properties of PUR range from hard and rigid to soft and flexible. In the modern society PUR is used in great amounts for production of paints, soft and rigid foam, elastomers, glues, clothing, shoe soles and several other products. But since the introduction of PUR there have also been published reports about health effects among workers. The health effects arise from exposure to isocy-anates. Isocyanates are highly reactive and in most air-sampling methods used today, amine reagents are used. The amine reagent protects the isocy-anate group, to avoid side reactions with other compounds. Isocyanate sam-pling is a complex field. There are numerous types of isocyanates with dif-ferent properties. Small monoisocyanates are volatile, while diisocyanates and isocyanate adducts are heavier and have lower volatility. Exposure is seldom restricted to just one type of isocyanate. Instead an aerosol consisting of gaseous isocyanates and isocyanates associated to particles is present. Air sampling must be done with methods capable of sampling both gases and particles, without discrimination.

Exposure to reactive monomers may occur during production of poly-mers, but there are also other kinds of exposure. In fact, exposure to thermal degradation products of polymers is more common and hazardous com-pounds may be generated. For PUR, isocyanates are reformed, but diamines and aminoisocyanates that are closely related to diisocyanates, are also gen-erated. Organic acid anhydrides are another type of reactive compounds that can be generated during thermal degradation of epoxy or alkyd resins.

This thesis covers sampling and analysis of reactive compounds that are closely associated to polymers, either used in production or formed during thermal degradation. Sampling methods which are able to separate an aero-sol in different fractions have been developed. Methods that are able to screen for several compounds of different types in the same sample have been developed, to simplify exposure assessment. For sample analysis the instrumental techniques have been improved to obtain lower detection limits and greater selectivity. New methods to bring forward appropriate analytical standards have been developed.

12

2 Aims of the Thesis

• To develop air sampling and analytical methods for complex isocyanates commonly used in the industry.

• To investigate the possibility to use tandem mass spectrometry for analy-

sis of isocyanate-DBA derivatives. • To develop air sampling methods for the assessment of the distribution of

isocyanates in an aerosol and how the aerosol varies with time. • To study reactive aerosols formed during thermal degradation of polyure-

thane regarding amine and aminoisocyanate formation. • To develop methods for the determination of airborne anhydrides simul-

taneously with isocyanates.

13

3 Isocyanates

3.1 Introduction The common feature for all isocyanates is the isocyanate group –NCO. Iso-cyanates are strong electrophils and reacts with compounds containing active hydrogens, such as amines, alcohols, thiols or water. Aromatic isocyanates are usually more reactive than aliphatic isocyanates, due to the electron-withdrawing properties of aromatic rings. The synthesis of isocyanates was discovered in 1848 by Wurtz.1 The most important industrial application for isocyanates, production of polyurethane (PUR) from diisocyanate mono-mers, was invented by Otto Bayer in 1937.1

3.1.1 Monoisocyanates In comparison with diisocyanates the volumes of monoisocyanates used in industrial applications are small. Monoisocyanates are used in the synthesis of pharmaceutical products, for production of pesticides and modification of polymers.1 In the Bhopal accident in 1984 it was methyl isocyanate (MIC) used in the production of an insecticide, carbaryl, that was released and killed at least 3800 people.2 Monoisocyanates have also been identified as thermal degradation products of polyurethane and phenyl-formaldehyde-urea (PFU) resins.3,4 Monoisocyanates studied in this thesis are listed in Table 1.

Table 1. Monoisocyanates

Compound Structure Vapour pressure (Pa) CAS-number

ICA 13300 (-19°C) 75-13-8 MIC 46400 (20°C) 624-83-9 EIC 14700 (20°C) 109-90-0

PIC 6900 (20°C) 110-78-1

PhI 250 (20°C) 103-71-9

H NCO

NCO

NCO

NCO

NCO

14

3.1.2 Diisocyanates The majority of isocyanates used industrially are diisocyanates, containing two isocyanate groups. The global market of isocyanates in the year 2000 was about 4.4 million tons. For production of polyurethane a diisocyanate is mixed with a polyol, and the bond formed is called a urethane bond (figure 2).

N

NCO

OR

O N N OR

O N N OR

O N N OR

O N

O O O O

HHHH

OO

H HH

O

H

O

NCO

NCOOCN

NCO

NCOHO

ROH

2,6-TDI 2,4-TDI polyol

Polyurethane Figure 1. Production of PUR, from toluenediisocyanate (TDI) and a polyol.

Several diisocyanates are used in the industry, to achieve different properties of the polyurethane. For production of soft foam, toluene diisocyanate (TDI) is used, but TDI can also be used in lacquers. Rigid foam is produced from methylene diphenyl diisocyanate (MDI). MDI and TDI are also be used in the production of elastomers. MDI-based binder is used for building sand moulds for iron casting. There are also isocyanate glues based on MDI. Coatings are produced from aliphatic isocyanates like hexamethylene diiso-cyanate (HDI), isophorone diisocyanate (IPDI) or dicyclohexylmethane di-isocyanate (HMDI). Coatings need to be UV-resistant, so the aromatic iso-cyanates are not appropriate in this application. Naphthalene diisocyanate (NDI) is another aromatic diisocyanate that is used in the production of hard elastomers used for example for production hard wheels for trucks or roller skates. Polyurethane is one of the most versatile plastic materials known, and the texture of PUR ranges from soft and flexible to hard and rigid.5 Diisocy-anates included in this thesis are summarized in table 2.

Diisocyanates are manufactured by a number of processes, involving dif-ferent starting materials, but the common step for almost all isocyanate pro-duction is a phosgenation reaction, where an amine is reacted with highly toxic phosgene. During the First World War phosgene was used for chemical warfare.6 A lot of research about isocyanate production without phosgene has been performed, but so far no viable processes that completely can re-place phosgenation has been found.7

15

NCOOCN

Table 2. Diisocyanates

Compound Structure Vapor pressure(Pa) CAS-number

1,6-HDI 7 (20°C) 822-06-0

2,6-TDI 2 (20°C) 91-08-7

2,4-TDI 3 (20°C) 584-84-9

IPDI 0.04 (20°C) 4098-71-9

4,4’-MDI 6.65*10-4 (20°C) 101-68-8

NCOOCN

NCOOCN

NCO

NCO

NCO

NCO

*

Some processes for production of polyurethane involves pure diisocyanate monomers, for example production of soft foam where a mixture of 2,4- and 2,6-TDI in the proportions 80:20 is used for the foaming process. Other polyurethane manufacturing involves isocyanate adducts, prepolymeric iso-cyanates or oligomers, containing 3 or more isocyanate groups.

3.1.3 Isocyanate adducts and Oligomers. To obtain different properties for the isocyanate mixture or to lower the volatility, isocyanate adducts or oligomers are often used in the industry. For example is HDI often further reacted during the production, to form biuret, alophanate or isocyanurate adducts. This reduces the volatility of the HDI, and is utilized in the production of polyurethane coatings. During the pro-duction of MDI, a crude mixture of different MDI-isomers (2,2’-, 2,4’- and 4,4’-MDI) and oligomers with 3 or more aromatic rings and isocyanate groups are obtained. For the production of for example rigid foam it is not necessary to separate the isomers, instead the crude mixture of oligomeric isocyanates is used directly. This mixture of MDI, isomers and oligomers are usually referred to as polymeric MDI (pMDI).7 Structures for some isocy-anates found in technical mixtures are summarized in table 3. Isocyanate mixtures are also sometimes prepolymerized, that is mixed with a small amount of polyol, to change volatility, reactivity, storage stability or some other property.

16

Table 3. Isocyanate oligomers and adducts.

rebmun-SAC erutcurtS dnuopmoC

Isocyanurate 3779-63-3

Biuret 4035-89-6

pMDI 9016-87-9

NCO

OCN

OCN

N

HN

NH

O

O

NCO

NCO

OCN

N

N

N

O

O

O

OCN NCONCO

n

3.2 Health Effects A few years after polyurethane production was started industrially, reports were published about respiratory disorders in workers handling the isocy-anates.8 Today, isocyanates are viewed as one of the chemical agents causing the most cases of occupational asthma.9 Between 5-15 % of workers han-dling isocyanates are estimated to develop occupational asthma.10,11 Isocy-anate asthma has been reported for HDI12,13, and HDI adducts14, TDI15,16, TDI prepolymers17 and MDI.18,19 In some cases asthma attacks due to isocy-anates has caused death.20,21 The mechanisms for isocyanate asthma and sensitization have not been completely understood. IgE or IgG antibodys to isocyanates are sometimes found in sensitized patients22, but antibodies are not always present in symptomatic patients, suggesting nonantibody-mediated mechanisms.23 After exposure to isocyanates biomarkers for isocy-anates can be found in blood and urine.24

Besides from asthma, isocyanates also causes other airways disorders like hypersensitivity pneumonitis25,26, rhinitis27,28 and chronic obstructive airway disease.29,30 Isocyanates are also skin irritants, and may cause eczema and contact dermatitis.31,32

Some diisocyanates are suspected carcinogens, and has been studied by the International Agency for Research on Cancer (IARC). 2,4-TDI is classi-

17

fied as a group 2B agent (possibly carcinogenic to humans), while NDI, MDI and pMDI are classified in group 3 (not classifiable as to carcinogenic-ity to humans).33

Since the volumes of monoisocyanates used in the industry are much smaller than for diisocyanates, the number of reports about their health ef-fects is limited. However, much information is available regarding MIC, where victims of the tragic Bhopal disaster have been studied. Several re-views have been published, where the acute symptoms of methyl isocyanate exposure are presented. These include pulmonary edemas, eye and throat injuries, vomiting and nausea, neurological disorders and unconscious-ness.34,35 Animal studies confirm the acute toxic effects of MIC36, but long-term effects showing decreased lung function in exposed workers have been difficult to establish.37 Phenyl isocyanate (PhI), which sometimes has been present as a by-product in polymeric MDI mixtures, has been showed to have sensitizing properties 38, and produce asthma-like symptoms in rats.39

Due to all the negative health effects ascribed to isocyanates, their occu-pational exposure limits (OEL) are very low. In Sweden, the time weighted average (TWA) value, that is the maximum permissible concentration of isocyanates in the air during an eight hour work shift, is 10 ppb for monoiso-cyanates. For diisocyanates the value is 2 ppb.40

3.3 Exposure Exposure to airborne TDI and MDI has been reported during production of soft TDI-based PUR-foam 41,42,43 and rigid MDI-foam.44 Spray painting of polyurethane coating may give rise to high levels of isocyanates, typically HDI-adducts like HDI biuret or isocyanurate that are common components in coatings. Several studies concerning spray painting has been pub-lished.45,46,47,48 Spraying of MDI is also sometimes performed, for example for insulation purposes49 or in more specialized processes like production of “bed liner”, a protective and softening coating on pick-up trucks.50 The iso-cyanate mixture used here, polymeric MDI, is non-volatile but spraying makes airborne exposure possible.

Isocyanate exposure occurs in several industrial branches, not just during manufacture of polyurethane or spray painting. In recent years the exposure associated with thermal degradation of PUR has been thoroughly studied. During flame lamination of TDI-foam with textile the foam is degraded, and TDI is released to the surrounding air.15,51 Other situations where polyure-thane is thermally degraded are for example during welding, cutting or grinding in PUR-coated metal sheets.52,53 Car workshops has been studied to assess this exposure situation.54 MDI glues, or plaster casts used at hospitals has been shown to cause occupational asthma.19,55 Isocyanate exposure can also occur during thermal degradation of other polymers, for example has

18

exposure to isocyanic acid (ICA) and methyl isocyanate (MIC) been ob-served during thermal degradation of phenol-formaldehyde-urea resins.56,57

Another route of exposure to isocyanates is dermal exposure. Reports on contact dermatitis caused by dermal isocyanate exposure have been pub-lished. Glues58, lacquers59, paint or binders60 are example of different prod-ucts that has been showed to cause allergic skin reactions in exposed work-ers.

To study possible exposure situations and evaluate air sampling methods for this thesis a number of different workplace studies have been performed. In paper I spray painting using HDI-based coatings, spray foaming using MDI and casting of PUR plastic details was studied. In paper V different situations where thermal degradation of PUR takes place were studied. Mea-surements were made during welding in coated metal sheets and welding in PUR-insulated heating pipes. The primary goal for the field measurements was not exposure assessment. Instead, field samples were collected to evalu-ate air sampling methods for sampling of different PUR-related compounds.

3.4 Air Sampling Due to the early reports about the isocyanates ability to cause airway disor-der, there were soon developed methods for determination of air levels. In 1957 Marcali presented a procedure for isocyanate determination involving air sampling using a bubbler containing acidic medium for hydrolysis of TDI to toluene diamine (TDA). The formed amines were then diazotized and the air levels were determined photometrically.61 A drawback of the Marcali method is that it not only measures isocyanates, if TDA is present in the air it will also be included in the result.

3.4.1 Reagents The reactivity of isocyanates makes it necessary to protect them already during air sampling. Otherwise, they may be lost in reactions with other compounds present. A number of different derivatizing reagents for isocy-anates have been developed, most of them using the reaction between the isocyanate and an amine to form a urea-derivative. Lower occupational ex-posure limits has been one reason that new reagents are still developed. Re-agents often have some special analytical property highlighted, to make the derivatives suitable for analysis by a certain detector. New reagents have also been developed to obtain faster reaction rates for the derivatization, or increased stability for reagents and derivatives.

The first amine reagent developed was the “nitro” (N-4-nitrobenzyl-N-n-propylamine)-reagent.62 The reagent was not very stable, it had to be stored as the hydrochloride salt, but the formed derivatives were reported stable

19

during analysis. Another amine reagent, 1-(2-pyridyl)piperazine (1-2PP) was introduced in 1979, as having several advantages, for example better stabil-ity and higher molar absorptivity, compared with the nitro reagent.63

To take advantage of fluorescence (FL) detection, a detection method with low detection limits, urea derivatives were formed by reaction of isocy-anates with 9-(N-methylaminomethyl)-anthracene (MAMA).64 When it was introduced it was compared with the nitro-reagent, and a ten to twenty times lower detection limit was claimed with the use of MAMA.

A reagent that still is frequently used is 1-(2-methoxyphenyl)piperazine (2-MP).65 When it was introduced it offered higher selectivity in isocyanate analysis, since it was detected by two detectors, UV and electrochemical. It also had higher collection efficiency for isocyanates when compared with the nitro-reagent and 1-2PP. Another reagent developed for double detection techniques is 3-(2-aminoethyl)indole, also known as tryptamine. Fluores-cence and amperometric detection was used in series for analysis of isocy-anate derivatives.66 In later studies tryptamine has been compared with 2-MP, 1-2PP, and the nitro-reagent. It was concluded that tryptamine and 2-MP had about the same relative reaction rates to isocyanates, while 1-2PP and the nitro reagent reacted much slower with isocyanates.67

1-(9-Anthracenylmethyl)-piperazine (MAP) is a reagent that structurally resembles MAMA as they both contain an anthracene group suitable for UV or FL detection. In the first publication68 presenting the MAP reagent, reac-tion rates were compared with 2-MP, MAMA and tryptamine. The reaction rate for MAP was comparable with 2-MP, and detection properties were in most cases more attractive than for the other reagents in the comparison. MAP has also been used for determination of total reactive isocyanate groups (TRIG). Determination of TRIG is performed by using a reagent that gives equal response for all isocyanate groups, regardless of the structure of the isocyanate. The idea of TRIG measurments is that the concentrations of isocyanate groups can be determined without standards for all analyzed compounds. 2-MP69, MAMA70,71 and tryptamine72 have also been evaluated for measurments of TRIG.

Di-n-butylamine (DBA), the reagent used for air sampling of isocyanates in paper I-VI, was initially used for determination of isocyanate content in technical mixtures used for polyurethane production. A known amount of DBA is added to an isocyanate mixture, and the isocyanate groups are then derivatized and DBA is consumed. The excess DBA is titrated with hydro-chloric acid. The concentration of isocyanate groups in the isocyanate mix-ture can then be calculated. This method was used to determine isocyanate content in technical mixtures in paper I. Air sampling of isocyanates using DBA as a reagent has been used for about a decade.73 A drawback with DBA is that it is an aliphatic molecule, and UV-response for the derivatives is obtained only if it is reacted with aromatic isocyanates. However, mass spec-trometric detection makes it possible to analyze aliphatic isocyanaytes deri-

20

vatized with DBA as well.74 The DBA reagent has other advantages in that it is stable, as are the derivatives formed, due to a high solubility it can be used in high concentrations in toluene for impinger sampling and it has been showed to have high reaction rates when compared with other reagents.75 Excess reagent can easily be removed by evaporation or extraction.

The reagents described so far have been the most studied, but in recent years a few new reagents have been presented, that also should be men-tioned.

A method for determination of TRIG in air was developed using 9-antrhracenylmethyl-1-piperazinecarboxylate (PAC). Isocyanate groups on all types of isocyanates were reacted to a single analyte. In this way the isocy-anate concentration could be determined without knowledge of the structure or a special standard. However, the method had problems in obtaining blank samples.76 4-nitro-7-piperazino-2,1,3-benzoxadiazole (NBDPZ) is a new reagent that has been tested with several isocyanates.77 Reaction rates have been compared with MAMA, and found to be higher. However, NBDPZ is not very stable in some solvents. Two other new reagents are ferrocenoyl piperazide (Fc-PZ)78 and 4-methoxy-6-(4-methoxy-1-naphtyl)-1,3,5-triazine-2-(1-piperazine) (MMNTP)79, but since the introduction no new reports has been published. For reaction of MMNTP with aliphatic isocyanates a 2 hour “incubation-period” is recommended that seems to make this reagent unsuit-able for air monitoring of isocyanates, where fast reactions are necessary. Structures of different isocyanate reagents are presented in table 4.

Besides from the amine reagents, reports have also been published where isocyanates form urethanes by reaction with ethanol in an impinger or bub-bler. To accelerate the reaction the ethanol was made alkaline by addition of potassium hydroxide.80,81

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Table 4. Isocyanate air sampling reagents

Reagent Structure Reference Reagent Structure Reference

Nitro

NH

NO2

62 1-2PP

NH

N

N

63

MAMA

NH

64 2-MP

HN

N

O

65

Tryptamine

HN

H2N

66 MAP

NH

N

68

DBA NH

73 PAC 76

NBDPZ

NH

N

N

O

N

77 Fc-PZ Fe

O

N

NH

78

MMNTP

O

NN

NO

N

HN

79

O

ON

HN

22

3.4.2 Sampling Techniques Sampling of airborne isocyanates has been performed using both wet (im-pingers or bubblers containing a reagent in some solvent) and dry (filters or different solid sorbents impregnated with reagent) sampling techniques. Dry samplers are generally preferred when personal measurements are per-formed, while impingers have advantages for example regarding the ability to provide reagent to the collected isocyanates. A difficulty is that isocy-anates are present both in the gas phase and associated to particles, which puts extra demands on the sampler.82,83 Since PUR often is manufactured by spraying, it is possible to obtain airborne exposure even for non-volatile isocyanates.

3.4.2.1 Impinger sampling In the first method for airborne isocyanates, the Marcali method, air sam-pling was performed using a bubbler61, and traditionally wet sampling has been the method of choice when new reagents have been introduced. Sam-pling using the nitro reagent62, MAMA64, 1-2PP84, 2-MP65, tryptamine66, DBA73 and MAP85 were all initially presented using impingers. Impingers have also been used for sampling of isocyanates in acid86,87 or alkaline etha-nol.80,81 Impingers are known to have poor sampling efficiency for particles in the sizes between 0.01 and 1.5 µm88,89, but this problem can be solved by placing a filter after the impinger. For 2-MP a reagent impregnated filter has been used after the impinger.90 The DBA method uses an unimpregnated filter after the impinger.53 The high concentration of DBA in the impinger solution and the volatility of the reagent provide efficient derivatization on the filter as well. Impinger-filter sampling using DBA in toluene was used in paper I for samples where the composition of technical isocyanate solutions was compared with the isocyanate composition in air samples. It was also used as a reference method for comparison of total air levels in paper III and IV.

3.4.2.2 Filter and solid sorbent sampling Due to the advantages with dry sampling techniques for personal measure-ments a lot of research has been done to find dry methods that perform as well as impingers. For several reagents impregnated glass fiber filters has been used, but other types of dry samplers, like different solid sorbents, have also been evaluated.

The nitro-reagent has been used in samplers with coated glass powder91, coated glass wool92 and glass fiber filters92,93,94. When comparing with wet sampling methods, air levels were equal93,94 or higher for the dry meth-ods.92,94 MAMA has been used adsorbed on XAD-2. When compared with a bubbler method there was good agreement in air levels for sampling of

23

HDI.95 A special kind of sampler consisting of a MAMA-coated denuder in combination with a MAMA-impregnated filter has also been developed for sampling of HDI and oligomers.96 Impregnated glass fiber filters have been used for dry sampling using 1-2PP in several studies.97,98,99 A method with sampling tubes containing 1-2PP impregnated sorbent has also been pre-sented for sampling of TDI.100 Comparison with an impinger method has been done for glass fiber filters.97 At low humidity the samplers showed comparable results, but when the humidity was almost 100 %, the dry sam-pler failed and much lower levels were obtained for the dry sampler as com-pared to the impinger sampling.

In recent years several studies has been presented using 2-MP impreg-nated glass fiber filters.56,101,102,103,104,105,106 One of the methods use dual fil-ters, to minimize breakthrough.56 In comparisons with impinger methods very different results were obtained. In some studies filters gives the highest air levels101 while equal results for impingers and filters are reported in other studies.102,104,106 There are also examples when impinger sampling has been superior.56,103,105 Besides from the filter methods 2-MP has also been used on extraction columns107, sintered glass108 and a sampler where a PUR sponge is coated with the reagent.109

Tryptamine has also been evaluated for dry sampling using sampling tubes filled with coated XAD-2, but so far the best results for this reagent has been obtained using impinger sampling.110,111 The fairly new reagent MAP has also been tested for dry sampling of HDI and adducts like iso-cyanurate and biuret. It was reported that the MAP-impregnated filter and impinger showed equally good performance.112

Because of the high volatility of DBA it is troublesome to use it in dry samplers alone. The reagent evaporates quickly when air is drawn through the sampler and something that prevents the DBA from evaporating must be present in the sampler. A dry sampler containing DBA in polydimethylsilox-ane for sampling of gaseous TDI was presented a few years ago.113 Polydi-methylsiloxane is also used as a stationary phase for gas chromatography. The sampler consisted of a denuder and a filter in series. The amounts of isocyanates in air were lower than for impinger sampling, when the sampling methods were compared. In later studies the denuder was evaluated for sam-pling of HDI and IPDI114 and MDI115 and reasonable agreement with the impinger method was obtained. Another way to reduce the volatility of DBA is to let it form an ion pair with acetic acid. This principle was used for im-pregnation of a glass fiber-coated denuder in series with a filter.116 This sam-pler has been thoroughly evaluated for several different types of exposure, both laboratory-generated atmospheres and sampling in the field. Compari-sons were made with impinger sampling, and the sampler was found to be a convenient alternative to impinger sampling. The impregnation technique with DBA and acetic acid on glass fiber filters has been used in Paper III and IV for impregnation of the fractionating sampler.

24

3.4.2.3 Passive sampling All air sampling methods presented so far are active sampling methods, i.e. a pump is used to drive the air flow through the sampler. For sampling of ga-seous isocyanates passive sampling methods has also been presented, but the research has been limited, because situations were only gaseous exposure exists are rare. Solid phase micro extraction using DBA as a reagent has been used for sampling of gaseous TDI.117,118 For sampling of MIC diffusive samplers using 2-MP119 and NBDPZ120 has been used for derivatization of isocyanates.

3.4.2.4 Fractionated Sampling The exposure situation for isocyanates is often complex, and isocyanates may, depending on volatility, molecular weight or how they are emitted, be associated with particles or be present as gases. Because of this it is impor-tant to use sampling methods that are able to collect the aerosol in a repre-sentative way. The distribution between gas and particles in the aerosol is also important to consider when lung deposition and the effects on the hu-man airways are studied. Different models for deposition of gas and particles in the human airways have been presented. In one of these models is pre-sented by the International Commission on Radiological Protection (ICRP).121,122 In the ICRP deposition model it can be seen that large particles (>2.5 µm) are mainly deposited in the upper airways due to inertial impac-tion, while smaller particles are able to reach deeper parts (tracheobroncial and alveolar region) of the airways.123 Gaseous substances are deposited in the head and nose region as well, due to diffusion. To better understand where the isocyanates are deposited and obtain a connection between expo-sure types and disease, samplers with the ability to fractionate the aerosol are necessary.

There are a few examples of samplers for isocyanates that can separate gas and particles during sampling. The IsoCheck method uses two filters in series for separation. The first filter is an unimpregnated Teflon-filter that collects particles containing isocyanates. The second filter is a MAMA-impregnated glass fiber filter, that collects gas-phase isocyanate.124 After sampling the front filter is put in a solution containing 2-MP to derivatize the isocyanates associated to particles. However, the use of an unimpregnated Teflon filter probably causes some loss of isocyanates, since the isocyanate groups are not derivatized and therefore unprotected after collection. A vari-ant of the IsoCheck sampler is a triple filter system, introduced in 2003.125 This sampler contains 2 unimpregnated Teflon-filters before a 1-2PP im-pregnated glass fiber filter. The second Teflon filter is used to estimate the gaseous isocyanates collected on the first filter.

A denuder in series with a filter has also been presented as a sampler with ability to separate gas phase from particles. For collection of MDI-aerosols

25

the nitro-reagent was used for coating of denuder and filter.126 For collection of HDI and adducts a denuder-filter system with MAMA-coated parts was used.96 To prevent non respirable particles to enter the samplers, a presepara-tor (a cyclone or an impactor) were used in front of the denuder parts.

Impingers may also be used for fractionated sampling. If the impinger is used with a back-up filter, and the filter is analyzed separately, a fractiona-tion is obtained. Small particles between 0.01 and 1.5 µm are collected on the filter. Large particles and gas phase isocyanates are collected in the im-pinger flask.53 This principle has been used in Paper V.

To obtain a more detailed view of the isocyanate distribution in aerosols a fractionating sampler consisting of a denuder in series with a cascade impac-tor and a glass fiber filter is presented in Paper III. The denuder-impactor separates the aerosol in five different fractions. The sampler was designed for a total flow rate of 5 l min-1. The sampler has been used for sampling of different types of isocyanate aerosols, generated by thermal degradation of polyurethane, evaporation of isocyanates (gas-phase isocyanates) or spraying of polyurethane coating compounds. As a reference sampler for comparison of total air levels, the impinger-filter sampler with DBA in toluene was used.53 The differences in total air levels of isocyanates between the de-nuder/impactor and the reference sampler were small, despite the big differ-ences in the two samplers regarding design and sampling flow. The denuder-impactor sampler is presented in more detail below

3.4.2.4.1 Denuder The first part of the sampler introduced in paper III is a channel-plate de-nuder for collection of gaseous isocyanates (figure 2). The denuder consists of eight parallel glass fiber plates, mounted together in a plastic holder made from polypropylene plastic, and held together with stainless steel bolts. The glass fiber plates mounted in the holder are initially 4 cm wide and 7.2 cm long, but the holder covers about 0.7 cm on each side of a plate, so the effec-tive width facing the air stream is 2.6 cm. The eight denuder plates faces the air stream on both sides, so the total filter surface facing the air stream is 2.6*7.2*8*2 = 299.5 cm2. A thin, but rigid filter material without binder (type MGC, Munktell, Falun, Sweden) was chosen for the denuder plates. Each plate was impregnated with 1.4 M DBA-acetic acid in methanol before the plates were mounted in the denuder.

The efficiency for a denuder can be calculated by using the Gormley-Kennedy equation. The original equations were developed for a cylindrical denuder.

26

26 mm

2 mm

Airflow

Filter-plates

72 m

m

Figure 2. Denuder-part of the combined sampler.

An expression for rectangular denuders have been given by Dasgupta et al. 127 as:

⎟⎟⎠

⎞⎜⎜⎝

⎛−

−= sQLDw

ef ****54.7

91.01 (1)

where f is the efficiency, w is the channel width, D is the diffusion coeffi-cient for the studied compound, L is the length of the channel, Q is the vo-lumetric flow and s is the channel height. Values of the diffusion coefficients for TDI, HDI and IPDI were adopted from Nordqvist.128 The equation is only valid if the flow through the channel is laminar. The flow is considered laminar if the Reynolds’ number is below 2000. The Reynolds’ number for a square channel is calculated as

ηρ**Re dv

= (2)

where v is the linear flow rate, d is the hydraulic diameter ρ is the density of the air and η is the viscosity of the air. For a square channel the hydraulic diameter is calculated as 4*channel area/circumference.129 Design parame-ters for the denuder are presented in table 5

27

Table 5. Design parameters for the denuder

Channel plate denuder

Number of plates Total effective area (cm2)8 300Calculated efficiency (%)a Reynolds’ numberb 4398

a) Calculated accordning to equation 1, for IPDI.b) Calculated accordning to equation 2

To extract the isocyanate-DBA derivatives from the denuder plates after sampling the plates were cut out and placed in test tubes. The plates were extracted by shaking with 1 mM sulfuric acid, methanol and toluene. For efficient extraction it was found that the toluene extraction should be re-peated twice. The toluene was separated and evaporated. The residue was dissolved in acetonitrile for LC-MS analysis.

In paper III the sampling efficiency for gas phase isocyanates was tested by sampling in a test chamber where gaseous isocyanates were generated by evaporation. 2,4- and 2,6-TDI, HDI and IPDI were the isocyanates included in the tests. The breakthrough of gaseous isocyanates was small, and did not exceed 5 % of the total amount collected. This agrees reasonably with the theoretical calculations for the denuder. The calculated efficiency for IPDI (diffusion coefficient: 5.06*10-6 m2 s-1 at 25°C) was about 98 %, using equa-tion 1.

3.4.2.4.2 Impactor A cascade impactor (figure 3) with three impaction stages was connected in series after the denuder part. Cascade impactors are used for fractionation of airborne particles. It is an inertial classifier in the sense that the collection principle is based on the inertia of a particle. An air stream is directed to-wards an impaction plate. Close to the surface of the impaction plate the air stream deviates and flows around the plate. Particles with a high inertia are unable to follow the deviating air stream and are deposited on the plate. In the following impaction stage the diameter of the jet directing the air stream towards the plate is decreased, which increases the velocity of the air passing through the jet. In this way some of the small particles that were able to fol-low the air stream in the first stage now have an inertia that is to big, so these particles are deposited on the following stage instead.

28

Airflow

Figure 3. Cross-cut view of impactor stages and collection principle. Particle-sizes are not to scale.

The design of an impactor is based on numerical solutions of the Navier-Stokes equations which were presented in 1974 by Marple and Liu.130 Im-paction stages are usually described with the cut-off diameter, d50, which is the aerodynamic diameter of particles that are collected by the stage with an efficiency of 50 %. By calculation of the dimensionless Stokes’ number, the cut off diameter for an impaction stage can be obtained. For the type of im-pactor used in this work, that is with a single orifice and a circular jet d50 can be calculated using the formula131

5050 ***

**9 StkUC

Wdcpρ

η= (3)

In this equation η is the viscosity of the medium surrounding the particle, W is the nozzle width, ρp is the particle density and U is the velocity through the nozzle. Cc is the Cunningham slip correction factor that becomes more important with decreasing particle size. When the particle diameter comes close to the mean free path of the individual molecules, the surrounding gas no longer acts as a continuous media, but as individual gas molecules exert-ing forces on the particle. The Cunningham slip correction factor compen-

29

sates for this. For particles larger than 1 µm the Cc has a value close to 1. Stokes’ number is a dimensionless parameter and represents the ratio be-tween a particles stopping distance to a characteristic diameter. Stk50 is the Stokes’ number for 50% collection of the particles with the specified diame-ter d50. For a circular jet impactor the Stk50 is 0.22.

To obtain a sharp cut-off curve for an impactor, the Reynolds’ number should be between 500 and 3000 calculated as:

ηρ**Re Dv

= (4)

In equation 4 the linear air velocity is represented by v, D is the diameter of the circular jet, ρ is the density of the air and η is the viscosity of the air.

0

20

40

60

80

100

particle size (µm)

colle

ctio

n ef

ficie

ncy

(%)

impactor 2impactor 1

0.1 1.0 10

Figure 4. Collection efficiency for the two top impaction stages. The efficiency was determined by measuring the penetration of a polydisperse glass aerosol.

The three impaction stages used for the measurements had cut-off values (d50) of 2.5, 1.0 and 0.5 µm. Design parameters for the impactor stages are presented in table 6. The collection efficiency for the top impactor stages was checked by measuring the penetration of a glass aerosol by connecting the stages to an aerodynamic particle sizer (figure 4). For stage 1 and 2, the cut-off diameters were close to the calculated values (paper III).

Due to the reactive nature of the isocyanates the derivatizing reagent must be applied to the impaction plates where the particles are deposited. As sub-strates for the impactors thin DBA-acetic acid impregnated glass fiber plates

30

were used in paper III and IV. To avoid filtration and retention of small particles on the top plates, a concentrated and highly viscous solution of DBA and acetic acid was used.

Table 6. Design parameters for the impactor stages.

Impactor Stage 2Stage 1 Stage 3

1.02.7 0.5Nozzle width (mm)2.353.55 1.90Jet to plate distance (mm)1.571.32 1.85S/Wa

46532585 6980Reynolds’ numberb

4714.6 106Linear velocity (m s-1)1.082.5 0.58Calculated d50 (µm)c

a) S is the distance between the nozzle exit and the substrate, W is the nozzle width.b) Calculated according to equation 4c) Calculated according to equation 3

For collection of the smallest particles that passes the last impaction stage a 25 mm glass fibre filter was mounted after the last impaction stage. The end filter was impregnated with a solution of DBA-acetic acid in methanol.

After sampling, the impactor substrates and the end filter were extracted in test tubes containing 1 mM sulfuric acid and toluene. The samples were shaken and placed in an ultrasonic bath before the toluene phase was sepa-rated and evaporated. After evaporation the samples were dissolved in ace-tonitrile before the LC-MS analysis.

3.4.2.4.3 Results from laboratory evaluation In paper III measurements with the denuder-impactor sampler are pre-sented. Besides from sampling of gas phase isocyanates, thermal degradation products of polyurethane and spraying of polyurethane coating compounds were studied. Sample generation was performed in a 0.3 m3 sampling cham-ber, built from stainless steel and glass. The humidity in the chamber was controlled by mixing dry and humidified air. To avoid isocyanate aerosol to escape outside the chamber, the pressure in the chamber was below the am-bient pressure (controlled by the exhaust ventilation of the chamber).

The results from the measurements with the denuder-impactor showed that different isocyanate distributions were obtained depending on how the isocyanates were generated. When comparing the total air-levels measured with the denuder-impactor with air-levels measured simultaneously using impinger-filter, good agreement between the samplers was obtained.

31

10

20

30

40

50

60

70

80

90

100

Sampler part

Per

cent

of t

otal

am

ount

in s

ampl

erICA MIC HDI2,4-TDI MDI

0Impactor 1d50= 2.5 μm

Impactor 2d50= 1.0 μm

Impactor 3d50= 0.5 μm

End Filter Denuder

Figure 5. Relative distribution of isocyanates in the denuder-impactor. Sample col-lected in the sampling chamber during thermal degradation of PUR.

The isocyanate distribution for samples collected during thermal degradation of PUR showed that gas-phase isocyanates and isocyanates associated to small particles dominated in the aerosol (figure 5).

biuretisocyanuratediisocyanurate

Impactor 1d50= 2.5 μm

Impactor 2d50= 1.0 μm

Impactor 3d50= 0.5 μm

End Filter Denuder

10

20

30

40

50

60

70

80

0Per

cent

of t

otal

am

ount

in s

ampl

er

Sampler part Figure 6. Relative distribution of isocyanates in the denuder-impactor sampler. Sample collected in the sampling chamber during spraying of PUR-coating.

32

Non-volatile MDI was exclusively found on the impaction stages, probably due to fast condensation.

Samples collected during spraying of isocyanate coating showed that iso-cyanates for this type of generation were associated with the larger particles, and the top stage with a d50 of 2.5 µm collected the majority of the isocy-anates (figure 6).

In paper IV, ageing isocyanate aerosols were studied. After isocyanate generation, three consecutive denuder-impactor measurements were made. The analysis of these samples revealed that the isocyanate distribution in the aerosol was time dependent. Aromatic isocyanates were initially associated with the gas phase or small particles, but after a few minutes the distribution of isocyanate was changed, and the aromatic isocyanates became associated with particles. The monoisocyantes and HDI remained in the gas phase for the whole sampling period.

The results obtained with the denuder-impactor have shown that this sampler has great potential for investigation of aerosols containing isocy-anate. However, some points should be highlighted regarding the evaluation. For example, this type of sampler has not been designed for personal meas-urements. Instead the sampler was constructed as a tool with ability to study how different isocyanate aerosol behaves. So far, the denuder-impactor has only been evaluated in chamber measurments. To fully characterize the sam-pler, field measurments are also necessary. The sampler should also be evaluated for other types of aerosol associated with different work opera-tions like casting of PUR or spray foaming.

3.5 Sample Analysis

3.5.1 Colorimetric methods The low occupational exposure limits for isocyanates and complex composi-tion in air samples has contributed to the development of sensitive and selec-tive analytical methods. The first air sampling methods for isocyanates relied on spectrophotometric detection of isocyanates. Before analysis the isocy-anates are hydrolyzed to amines, that in turn are diazotized, and the colored complexes are analyzed.61 This way of analysis also analyses the corre-sponding amines and aminoisocyanates if they are present in the air, so a sum of these compounds is obtained. To measure the isocyanates alone, an improvement of the Marcali-method was introduced in 1970. Sampling was performed using two different sampling solutions, one containing hydrochlo-ric acid for hydrolysis of isocyanates. The other solution contained an amine, for reaction with the isocyanates. After sampling both solutions were diazo-

33

tized, and the difference in response between the samples represented the response for the isocyanates.132

3.5.2 Chromatography When derivatizing agents for isocyanates were introduced, it became neces-sary to develop methods that could separate the formed derivatives from the excess reagent. Separation of the derivatives also provides a higher selectiv-ity, which is necessary if several isocyanates are present in the air sample. For the first derivatizing reagent, the nitro reagent, thin layer chromatogra-phy (TLC) was used for separation.62 TLC has also been used for analysis of isocyanates derivatized using 1-2PP.84

The dominating technique for separation of isocyanate derivatives is liq-uid chromatography. A LC method for analysis of the nitro reagent was de-volped in 1976133, and for methods using other reagents as 1-2PP63, MA-MA64, 2-MP65, tryptamine66, MAP68 and DBA73, HPLC separation was used for sample analysis when the reagents were introduced.

Chromatographic analysis in paper I-VI has been performed using mi-cro-LC, that is with columns with small internal diameters and low flow-rates between 70-100 µl min-1. Micro-LC has also been used for determina-tion of MAMA-derivatives of isocyanates.134 Compared with conventional LC there is several advantages, as for example a low consumption of mobile phases and less maintenance of the instrument. Low flow rates also allow small column particles to be used without getting a high column back pres-sure. This increases the resolution and selectivity of the chromatography. All columns used have been 50 mm long reversed-phase columns with octade-cylsilica (C18) particles in sizes between 2.5-3.5 µm.

To improve the chromatography, sample injection has been performed us-ing on-column concentration in paper I-VI. This means that a small sample plug, usually about 2.5-5 µl, surrounded on both sides with a weak eluent (usually 95/5 water/acetonitrile) is injected to the column. When this pack-age reaches the column the analytes in the sample are retarded on top of the column, and focused in a narrow band. The analytes are released when the gradient starts, but the peaks eluted are much sharper than for conventional injection methods, where the sample is injected directly into the mobile phase. This is illustrated in figure 7 where the same amount of isocyanate-DBA derivatives has been injected.

34

0.78

1.96 2.53 3.20 4.42

6.28

6.55

6.93

7.748.34

9.11

0 1 2 3 4 5 6 7 8 9 100

100

Time (min)

Rel

ativ

e ab

unda

nce

(%)

0

1000

100

50

50

50

1.56

1.95 2.46

3.07 4.26 6.19

6.46

6.86

7.72 8.32

9.07

9.11

8.347.74

6.91

6.52

6.274.39

3.18

2.572.01

1.58

A

B

C

Figure 7. The effect of on column concentration. Chromatogram A: sample dis-solved in acetonitrile, injection without focusing. Chromatogram B: sample dis-solved in mobile phase. Chromatogram C: concentrated sample dissolved in acetoni-trile, sample surrounded by 95/5 water/acetonitrile in the injection loop.

The sample was dissolved in 20 µl acetonitrile, 20 µl mobile phase (60/40 water/acetonitrile) and 2.5 µl acetonitrile surrounded by 17.5 µl 95/5 water acetonitrile. Pure acetonitrile is a good solvent for the derivatives, but the peaks are severely broadened and distorted. For the sample dissolved in mo-bile phase it is only the early eluting peaks that are broadened, but there could be solubility problems for some derivatives when the concentration is increased. The concentrated sample surrounded by weak mobile phase shows excellent peak-shape for all the derivatives, and there are no problems with the solubility of the derivatives. If the sample was dissolved in water the effect would have been the same as for on column focusing, but due to the low solubility of the derivatives in water this is not appropriate.

35

3.5.3 Detection Methods For the early chromatographic methods for determination of urea derivatives of isocyanates ultraviolet (UV) detection has been one of the most popular detection techniques. Analysis of monomeric isocyanates and prepolymers or adducts using LC-UV has been presented for both the nitro re-agent46,133,135,136 and 1-2PP.63,137,138 UV detection has also been used for MAMA64, 2-MP65, MAP68 and DBA.73 All the reagents except for DBA con-tain aromatic rings, so absorbance in the UV-region is obtained for aromatic as well as aliphatic isocyanates. Analysis of isocyanates using UV and DBA can only be performed for aromatic isocyanates.

To obtain lower detection limits and higher selectivity for isocyanates in complex air samples, several other detection methods has been used for analysis. Sometimes dual detection systems are used to confirm analytical peaks as isocyanates.

Fluorescence (FL) detection has been used for MAMA64, tryptamine66 and MAP.68 MAMA and MAP both contain an anthracene group, suitable for fluorescence detection. Tryptamine may also be detected using fluorescence, but the response is lower than for MAP and MAMA.68 Compared with UV detection, lower detection limits are obtained using FL-detection.

The advantage of using two detectors in series is increased selectivity since the ratios between the detector responses must be the same for samples and standards. This makes it possible to exclude peaks that are not isocy-anates. TRIG-measurements are usually performed using dual detectors. When 2-MP was introduced as a reagent, UV detection in series with elec-trochemical (EC) detection65 was used for sample analysis. The detection technique has later been adapted for sampling of prepolymers and ad-ducts.69,139 Prepolymer identification was originally based on the response ratio between the EC and UV detector, and quantification was done by com-paring the EC-response to the response of a monomer. This way of isocy-anate identification and quantification makes standards containing anything else but monomers unnecessary, which simplifies the analysis. Later re-search has showed that some isocyanates may be quantified wrong140 or not identified as isocyanates141 using the response ratio method for 2-MP.

MAP was introduced as a better alternative to determination of unknown isocyanates using a monomer for determination of isocyanate concentra-tion.68 UV detection in combination with EC has been used, as well as UV in combination with FL-detection.68,85,142 The FL-detector was introduced as a more robust alternative than an EC-detector.142

For determination of TRIG, MAMA has also been evaluated using dual detection with UV and FL. However, the ratio between the different detec-tors was not used for identification, instead the ratio between absorption at two different wavelengths was used for identification, since this ratio was found to be fairly constant for different isocyanates.70,71 Tryptamine has also

36

been used for determination of TRIG, by using a combination of a FL and an amperometric detector.72

3.5.3.1 Mass Spectrometry Mass spectrometry (MS) is a sensitive and selective detection technique and very low detection limits can be obtained. This makes MS a very attractive technique for analysis of isocyanates in air, where OEL values are low. However it is only in the last ten years that MS has been used as a routine detection technique for isocyanates. Partly this depends on that it initially was an expensive technique, not affordable for many laboratories. Old mass spectrometers were also difficult to use, the response could be unstable, and the interfaces that were possible to use in combination with LC were poorly developed. Nowadays, it is possible to build inexpensive and more robust mass spectrometers, and with the electrospray (ES) interface coupling to LC is straightforward. DBA, which has been used as a reagent for isocyanates in paper I-VI, was initially analyzed using LC-UV73, but LC-MS methods for determination of aromatic143,144 and aliphatic4,74 isocyanates were soon intro-duced. In paper I LC-MS was used for determination of isocyanates used in technical grade qualities and for analysis of air samples.

In a mass spectrometer ions are separated by their mass to charge ratio (m/z). Mass spectrometric detectors using two different types of MS instru-ments, quadropole and time of flight (TOF), has been used in this thesis.

For routine analysis of air samples different types of quadropole instru-ments have been used. In the single quadropole used in paper I, V and VI charged ions from the electrospray ion source are focused by different elec-tronic lenses and introduced between four rods. The rods work as electrodes, and dc and ac voltages are applied to the rods. The mass spectrum is scanned by increasing the ac and dc voltages, while the ratio between the voltages is kept constant. Depending on the voltage applied, ions with a unique value of m/z can pass through the quadropole in stable trajectories. The other ions are lost by collision with the quadropole rods. Scanning of a spectrum is ob-tained by increasing the voltage gradually. Selective ion recording (SIR) can be performed by selecting a few number of voltage settings, that only allows ions with a specified m/z to pass through the quadropole. By selecting only a few ions for analysis lower detection limits are obtained, because the analy-sis time for these ions is increased.

A triple quadropole instrument, which was used in paper II-IV, is con-structed in the same way as a single quadropole, but three mass analyzers are combined in series. The second quadropole is used as a collision cell for fragmentation of ions from the first quadropole. The ions are collided with a collision gas, usually argon, and the fragments obtained are analyzed by the third quadropole. For routine analysis multiple reaction monitoring (MRM) was used. In MRM ions with one m/z are selected by the first quadropole, these ions are collided with argon, and a second fragment ion with another

37

m/z is selected by the third quadropole. The selectivity in determination is improved compared with a single quadropole used in the SIR-mode. A triple quadropole can be used in several different modes, for example in daughter scanning mode ions with one m/z are selected by the first quadropole, the ions are collided, and third quadropole is scanned to obtain a spectra of the collision fragments. In parent-scanning mode the first quadropole is scanned and the third is held static. In this way all ions releasing a selected fragment in the collision cell are detected. For detection of ions in quadropole instru-ments electron multipliers are used.

In paper I a time of flight (TOF) MS was used for identification of isocy-anate derivatives. In the TOF-MS all ions entering the mass analyzer are accelerated with an electrical pulse of several kilovolts. The ions are acceler-ated into a field free drift tube, all with the same kinetic energy. Depending on the mass of the ions they will travel through the tube with different veloc-ity. Light ions pass faster through the tube than heavy ones. At the end of the tube the ions are detected by a multi channel plate (MCP) detector. The MCP detector can register the response from several ions at the same time, in contrast to the electron multiplier, that only can detect one ion at the time. Depending on the time it takes for an ion to travel through the drift tube, the m/z of the ion can be calculated. Exact masses of ions at the ppm level can be obtained with TOF-MS, but this type of instrument needs to be calibrated more often than quadropoles. Because they are less robust than quadropoles, they are usually not suited for routine measurements of several hundred samples.

All the MS-instruments used in paper I-VI were equipped with electros-pray (ES) interfaces. In this type of interface the effluent from the column is sprayed through a capillary, by assistance of a coaxial nitrogen gas-flow. Between the capillary tip and the MS inlet a voltage of several kilovolts is applied. The sprayed droplets become charged, and the solvent is evaporated from the droplets by assistance of a heated nitrogen gas flow and the electric field. There are different theories about how the charged ions escape from the droplets. In one theory ions are released from the small droplets when the concentration of charges becomes too high on the droplet surface. In the other theory solvent evaporation continues until all solvent is evaporated and only the ions are left. The ions enter the MS by assistance of electrical fields, and a negative pressure gradient. Electrospray is a “soft” ionization method, and little fragmentation of the ions is obtained. By varying voltage settings in the entrance to the mass analyzer, for example sample cone voltage, the degree of fragmentation can be increased. By using this method it is possible to perform some fragment analysis in TOF and single quadropole instru-ments as well. The ES interface can operate both in the positive and negative ionization mode.

The fragmentation pattern for isocyanate-DBA derivatives usually con-tains a few typical ions when positive ionization is used. The molecular ion

38

[M+H]+ is usually abundant, as well as the adduct ions with sodium [M+Na]+. Sometimes the potassium [M+K]+ adduct is present as well. Ions where one DBA molecule has been lost are present in the spectra as [M+H-129]+. Ions from the reagent are also present as m/z = 130, [DBA+H]+ and m/z = 156, [DBACO]+.

When the single quadropole was used for isocyanate analysis in paper I and V the instrumental detection limits for the monoisocyanates was be-tween 20 and 70 fmol. For the diisocyanates the same values were about 10 times lower, between 3 and 5 fmol. In paper II-IV a triple quadropole in-strument was used instead. This lead to improved detection limits, between 0.5-5 fmol for monoisocyanates, and 0.01-0.06 fmol for the diisocyanates. The lowest detection limits were obtained using MRM, with analysis of the reaction [M+H]+ [DBA+H]+.

Except for isocyanate-DBA derivatives, LC-MS has also been used for determination of isocyanates derivatised with 2-MP. In the first publication about MS determination of 2-MP derivatives particle beam ionization was used for the MS analysis145, but in later methods electrospray ionization has been used.104 MS/MS analysis using either a triple quadropole104 or ion trap MS146 has also been presented. To lower detection limits a method for 2-MP using coordination ion spray (adduct formation with lithium ions) has been presented.147 In paper II this was tested for DBA-derivatives and lithium acetate was added to the LC mobile phases for adduct formation (figure 8). The molecular ion disappeared from the spectra, and was replaced by [M+Li]+, [M+H-129]+ was replaced by [M+Li-129]+ and [DBA+H]+ was replaced by [DBA+Li]+. When MS/MS analysis was preformed, and the [M+Li]+ ion was chosen in the first quadropole for fragmentation, the prod-uct spectra was dominated by [M+Li-129]+. If the same procedure is carried out without lithium in the mobile phase, [DBA+H]+ is the dominating ion in the product spectra. The fragmentation pattern was clearly different for the lithium adducts compared with the fragmentation of protonated molecular ions, but when the detection levels were compared, analysis of protonated molecular ions was still superior compared with the analysis of lithium ad-ducts.

39

130

304 433

100

0

100

0

136328

310

439

400m/z300200

Rel

ativ

e ab

unda

nce

(%)

A

B

Figure 8. Addition of lithium to the mobile phase alters the fragmentation pattern for the isocyanate-DBA derivatives (spectra A), compared to regular reversed phase chromatography (spectra B). All ions formed for 2,4-TDI-DBA were lithium ad-ducts, when lithium acetate was added to the mobile phase.

For DBA-derivatives, GC-MS methods have also been used for determina-tion of monoisocyanates.4 Mass spectrometric detection has also been used for isocyanates derivatized with the NBDPZ-reagent. Atmospheric pressure chemical ionization in combination with MS/MS analysis works well for methyl isocyanate120 and different mono- and diisocyanate derivatives.148 Another recently presented isocyanate reagent, ferrocenoyl piperazide has also been analysed using LC-MS/MS with atmospheric pressure chemical ionization (APCI).78

3.5.3.2 Standard preparation A demand for all analytical methods including mass spectrometry is stan-dards for all compounds that shall be analyzed. Response factors for MS are very different even for molecules with similar structures. For some isocy-anate monomers it is possible to obtain pure substances that can be used as standards after derivatization, but for the different compounds present in e.g. polymeric isocyanate mixtures and thermal decomposition products of PUR are not available as purified chemicals.

A method for preparation of reference solutions of different isocyanates derivatives has been used in papers I, II and V. By using characterized reference solutions, the problem with synthesis and isolation of separate compounds present in technical mixtures is circumvented. For preparation of

40

reference solutions, a nitrogen selective LC-detector, chemiluminescent ni-trogen detector (CLND) has been used. The CLND has an equimolar re-sponse for nitrogen-containing organic compounds, which makes it possible to use any nitrogen containing compound as external standard.149 In the de-tector the effluent from the LC-column is mixed with oxygen and argon and nebulized into a pyrolytic tube placed in an oven heated to 1050°C. Organic compounds are oxidized to carbon dioxide, water and different other oxides. Nitrogen bonded to organic molecules is converted to nitrogen oxide (NO). The pyrolysis products from the oven are splitted and one part is passed to a membrane dryer, where water is removed. The other part goes to the exhaust ventilation. The dried oxides are lead to a chamber where they are reacted with ozone. The NO reacts with ozone to nitrogen dioxide in the excited state (NO2*). The NO2* relaxes to its ground state by emission of a photon.

F

E

D

C

B

A

G

m/z = 1144.9

m/z = 912.2

m/z = 1358.1

m/z = 892.7

m/z = 427.4

TIC

LC-CLND0 5 10 15 20 25

Time (min) Figure 9. LC-CLND chromatogram (A) and total ion current chromatogram (B) for a technical isocyanate mixture, Desmodur N 3300 derivatized with DBA. Chroma-tograms C-G represents extracted mass chromatograms for HDI-DBA, isocyanurate-DBA, diisocyanurate-DBA, triisocyanurate-DBA and tetraisocyanaurate-DBA.

41

The photons emitted by NO2* are detected by a photomultiplier tube. Re-sponse for an organic compound is thus obtained only if the compound con-tains nitrogen. Mobile phases used for CLND-analysis must be free from nitrogen, which makes it impossible to use acetonitrile. Instead methanol and water are used for preparation of mobile phases. As a standard for determi-nation of the nitrogen content in the analytical peaks, caffeine dissolved in water was used. Caffeine standards are easily prepared by weighing. The standard solutions are stable for long time, and caffeine is an inexpensive chemical. Caffeine is also rather easy to handle as it has a high melting point and it is not very hygroscopic.

For the preparation of standards from technical mixtures, small amounts of different technical mixtures were dissolved in toluene containing DBA in excess. The solutions were stirred and agitated in an ultrasonic bath. For completion of the reaction between DBA and the isocyanate groups the solu-tions were left standing for a few hours, before they were evaporated to dry-ness in a vacuum centrifuge. The residues were dissolved in methanol, and the samples were analyzed by LC-CLND (figure 9).

No structural information is obtained from CLND-measurements. This makes it necessary to use mass spectrometry for identification of the differ-ent compounds. To simplify the mass spectrometric analysis and identifica-tion, the same chromatographic parameters were used as for the CLND-analysis. For identification a time of flight mass spectrometer was used. Deuterium-labeled DBA was also used for the identification of different isocyanate-DBA derivatives. By comparing spectra from isocyanates deri-vatized with the deuterium-labeled DBA and derivatives with non-labeled DBA the number of isocyanate groups for different ions can be calculated. These methods have been used in paper I for identification of prepolymeric isocyanates and isocyanate adducts.

When the concentrations of all isocyanate species in a reference solution were determined, the solution was diluted in methanol to a concentration that is easy to use for preparation of standards for the quantitative MS analysis. The standard concentration used for most of the reference solutions was usually set to 1 µg ml-1 for the most abundant compound in the solution.

3.5.3.3 Internal standard for MS For determination of isocyanates with LC-MS it is necessary to use internal standards. An ideal internal standard for mass spectrometry has a similar structure as the analyte and a retention time in the LC separation that is close to the analyte. Isotope-labeled analogues of the analytes are often used as internal standards. In paper V deuterium-labeled isocyanate-DBA deriva-tives, prepared from deuterated amines were used as internal standards. The internal standards were prepared by the thermal decomposition of deuterium-labeled urethane derivatives of deuterium labeled amines. When these types of derivatives are heated they are thermally degraded to deuterium labeled

42

isocyanates. The isocyanates were collected in an impinger containing tolu-ene-DBA, and deuterium-labeled isocyanate-DBA derivatives were ob-tained. The synthesis has been described by Karlsson et al.4,74 Internal stan-dards with deuterium were available for MIC, HDI, 2,6- and 2,4-TDI and MDI. These internal standards were also used for quantification of other isocyanate-DBA derivatives, even though they are not ideal internal stan-dards. In papers I and II deuterium-labeled isocyanate-DBA derivatives prepared by reaction of isocyanates with deuterium labeled DBA were used instead. The advantage of using the later internal standards is that internal standards can be easily prepared for all the isocyanates analyzed. These in-ternal standards are prepared in the same way as described for standards obtained from technical isocyanate mixtures by dissolution of the appropri-ate isocyanate in a solution containing deuterium-labeled DBA. For prepara-tion of the DBA-derivatives of isocyanic acid (ICA) and MIC a somewhat different method was used. Isocyanic acid is not commercially available. It is possible to purchase MIC, but due to its high volatility and its toxic effects it is not desirable to handle this compound in the laboratory. Instead ICA and MIC were generated by thermal degradation of urea for ICA and 1,3-dimethylurea for MIC. The isocyanates are emitted during the thermal deg-radation, and these are collected in toluene containing deuterium-labeled DBA. This method was also used for synthesis of standards for of ICA and MIC. 57

Two different types of deuterium-labeled DBA were evaluated for syn-thesis of internal standards, one containing nine deuterium atoms and one with eighteen deuterium atoms. The more deuterium that is incorporated in the molecule, the more different it becomes compared with the non-labeled derivative. The different types of DBA and internal standards synthesized from amines were compared in paper II. The retention time differences are increased with the number of deuterium in the molecules. A diisocyanate derivative with two DBA containing 18 deuterium atoms each elutes almost 20 seconds earlier than the analyte. This affects the analytical precision and the spread of the residuals are much greater for this calibration than for iso-cyanate-DBA derivatives containing nine deuterium atoms. The lowest spread of the residuals is obtained for the DBA-derivatives obtained from DBA-derivatives of the deuterium-labeled amines. These derivatives also had retention times almost the same as the non-labeled derivatives. How-ever, regarding the possibility to have internal standards for all analyzed compounds, and the easier way of preparation of internal standard deriva-tives, the nona-deuterium labeled DBA was chosen for analysis. This type of internal standard was also used for determination of air samples in paper III and IV.

43

4 Amines and Aminoisocyanates

There are some diamines that are closely related to the diisocyanates dis-cussed in chapter 3, the diamines used for production of diisocyanates, hex-amethylenediamine (HDA), toluenediamine (TDA) and methylenedi-phenyldiamine (MDA). Exposure to diamines and also aminoisocyanates has been demonstrated during thermal degradation of PUR.81,150 An aminoisocy-anate is a diisocyanate where one of the isocyante groups has been converted to an amine group. Aminoisocyanates can not be synthesized as pure sub-stances because they would polymerize with themselves, but they exist as airborne intermediates in the smoke during thermal degradation of polyure-thane plastics.

4.1 Diamines - properties and uses Except for being used in the production of isocyanates, where diamines are phosgenated in the last reaction step, diamines are used in the production of other type of polymers.

Table 7. Diamines

Compound Structure Vapor pressure(Pa) CAS-number

1,6-HDA H2NNH2 25 (20°C) 124-09-4

2,6-TDANH2H2N 2130 (150°C) 823-40-5

2,4-TDA

NH2

NH2

100 (106°C) 95-80-7

4,4’-MDANH2H2N

2.7*10-5 (25°C) 101-77-9

Hexamethylene diamine (HDA) is used in the production of nylon, used for production of textile fibers. Nylon polymers are polyamides, formed by reac-

44

tion of diamines with diacids. For production of Nylon 6/6, HDA is reacted with adipic acid.151

Methylenediphenyl diamine (MDA) is used as a curing agent in some types of epoxy plastics. In contrast to anhydride cured epoxy, it is not neces-sary to heat diamine-cured epoxy for the curing reaction to take place.151,152

No examples for the use of toluenediamines (2,4- and 2,6-TDA) outside the isocyanate and polyurethanes industry have been found for this review. However, in the production of soft foam from TDI, the reaction of TDI with water, where TDI is converted to TDA, is often used. TDI and TDA react and form polyurea. Carbon dioxide is released during the conversion of iso-cyanates, and used as a blowing agent.153 Another isomer, 2,5-TDA, is used in some types of hair-dye.154

Aminoisocyanates only exists as reactive intermediates during thermal degradation of PUR. The structures for the aminoisocyanates studied are presented in table 6.

Table 8. Aminoisocyanates

Compound Structure Vapor pressure(Pa) CAS-number

HAI NH2OCN 95566-41-9

2,6-TAINH2OCN

- 22683-71-2

2,4-TAI (1)

NCO

NH2

- 99626-87-6

2,4-TAI (2) NH2

NCO

- 99626-88-7

MAINH2OCN

- 148913-78-4

4.2 Exposure and Health Effects Except for MDA, there are rather few reports on occupational exposure to diamines used as pure chemicals. MDA is used for epoxy curing, and both long term exposure155 and acute toxic effects156 have been studied.

Exposure to diamines also occurs in the PUR-industry. During thermal degradation of PUR, diamines are formed, as well as aminoisocyanates. For example, flame lamination of soft foam with textile generates 2,4-, and 2,6-

45

TDA as well as the corresponding tolueneaminoisocyanates (TAI).157 Dur-ing welding in PUR-coated metal HDA can be detected, as well as hexame-thylene aminoisocyanate (HAI).81 In paper V it was shown that MDA and methylenediphenyl aminoisocyanate (MAI) was formed during welding in PUR-insulated district heating pipes. The pipes were insulated with rigid PUR foam based on MDI. Exposure to amines and aminoisocyanates was also demonstrated during welding in coated metal.

Human exposure not related to industrial activity has also been consid-ered for MDA and TDA. MDA has been shown to be released from medical products made from MDI-based polyurethane.158,159 Several studies have investigated exposure to TDA from PUR-covered breast implants. The PUR material surrounding the implants has been shown to be non stable and eas-ily degraded, with the formation of TDA.160,161

Two of the diamines studied, 2,4-TDA and MDA, have been classified by IARC as group 2B agents (possibly carcinogenic to humans).33 Reports about acute toxicity for MDA have described liver injuries and jaundice for the exposed individuals. The reports concerns both suspected dermal expo-sure156 as well as accidental ingestion162,163 causing these symptoms. For HDA, the knowledge about effect on humans is limited, but several studies on animals have shown that HDA is an irritant to the upper respiratory tract as well as the eyes and the skin. Similar effects have been found on exposed humans due to airway exposure.164 The situation about known effects on humans for TDA is somewhat similar as for HDA, in that most of the infor-mation comes from animal studies. In a criteria document, the toluenedia-mines are described as irritants to eyes, skin and the upper respiratory tract. The 2,4-, 2,5- 2,6- isomers are all mutagenic, but only 2,4-TDA is carcino-genic in animals. Some investigations have found TDA as a reproductive toxin affecting spermatogenesis, but the available data was limited.165

In Sweden no OEL values are given for the diamines discussed here. However, 2,4-TDA and MDA may only be handled by authorized laborato-ries, due to the fact that these diamines are suspected carcinogens. 40

4.3 Air Sampling Air sampling methods devoted only for sampling of aromatic amines have been developed, but there are also methods that are developed for simultane-ous sampling of amines and isocyanates, as they often occur together in the industry.

For collection of airborne amines several methods using acid-impregnated glass fiber filters have been presented. Usually the filters are treated with sulfuric acid, and during sampling the amines are stabilized as sulfates. For desorption from the filters after sampling, alkaline solutions are used. It is possible to analyze free amines by chromatography, but derivatization before

46

the analysis is common. Amine derivatization will be described in more de-tail later in the text.

The American organizations National Institute of Occupational Safety and Health (NIOSH) and Occupational Safety and Health Administration (OSHA) have presented filter methods for MDA. In the OSHA method166 the amines are derivatized before analysis, while the NIOSH method167 analyses free amines. The OSHA method have been developed and adapted for analy-sis of toluenediamines as well.168 Another MDA method using acidic glass fiber filters, where the amines were derivatized before analysis has also been published.169

Methods using acidic filters are not suitable for determination of amines in the presence of isocyanates, since isocyanates will be converted to amines in contact with acids. In methods that can measure amines and isocyanates in the same sample the isocyanates are derivatized upon sampling, and the amines are either derivatized after sampling or analysed as such. In the early methods where both types of compounds can be determined, alkaline ethanol in impinger bottles was often used for sampling. Isocyanates react with the ethanol forming urethanes, and are in this way protected from hydrolysis to corresponding amines or further reaction with the sampled amines. This technique has been used for sampling of PUR degradation products from coatings81 and soft foam during flame lamination.150 Derivatisation was per-formed before the analysis. A method where the amines are left underivat-ized has also been used for sampling during flame lamination81, and for sampling of MDA in the presence of MDI.80

Isocyanates react faster with amines than with ethanol and most isocy-anate derivatization reagents used today contain an amine group for protec-tion of the isocyanates. In wet sampling methods the amine reagent is dis-solved in some organic solvent, for example toluene. Amines and aminoiso-cyanates present in the sampled air are also collected in the reagent solution. This collection principle has been used in the NIOSH 2-MP method. 170 Af-ter sampling the impinger solution is analyzed for the presence of both iso-cyanates and amines.

Collection of amines and aminoisocyanates in the isocyanate sampling so-lution has also been used in paper V. DBA dissolved in toluene was used for protection of the isocyanate groups and by derivatisation after sampling it was possible to analyze airborne amines and aminoisocyanates as well. Air sampling was performed using an impinger connected in series with a 13 mm glass fiber filter, with a pore-size of 0.3 µm.

4.4 Amine derivatization For analysis of amines with chromatographic methods it is common to deri-vatize the amines before the analysis. Amine groups are active, and amines

47

may if they are not derivatized become adsorbed by hydrogen bonding to different surfaces in the analytical instrument. Derivatization protects the amine group from oxidation. Derivatization has also often been used to ob-tain lower detection levels and better selectivity in the analytical procedure.

4.4.1 Derivatization with anhydrides Acid anhydrides react with amines, and different anhydrides have been used for derivatization of amines. In the NIOSH air sampling method for sam-pling of amines in the presence of isocyanates, acetic anhydride is added for derivatization of amines. The acetic anhydride also consumes the excess isocyanate reagent, 2-MP, which facilitates the chromatographic analysis. The acetic anhydride reacts slowly with toluene diamines present in the sample, and heating is necessary for completion of the reaction. Acetic an-hydride derivatization of MDA (and m-phenylenediamine) has also been used in a filter method. Filters were extracted with an aqueous base (phos-phate buffer) mixed with acetonitrile, and acetic anhydride was then added to this solution.169

Fluorinated acid anhydrides are very common for derivatization of amines, not just for air sampling, but also for analysis of amines in biological samples171 and in PUR-foam.172 The fluorinated acid anhydrides react fast with amines in organic solvent, and high recoveries are obtained for aro-matic amines.173 The acid formed by the reagent is extracted from the or-ganic phase with a buffer prior to analysis.

Heptaflurobutyric anhydride (HFBA) is used in two NIOSH methods for filter sampling of toluenediamines168 and MDA.166 The filters are shaken with alkaline solution and toluene. The amines are extracted to the toluene. The toluene is separated and the HFBA reagent is added to for derivatization of the amines.

HFBA have also been used for amine-derivatization when amines and isocyanates have been collected in alkaline ethanol. After sampling the sam-pling solution was evaporated to dryness. Phosphate buffer and toluene was added, together with HFBA.81 HDA and HAI were determined in the toluene phase. A similar derivatiztion method has also been used for derivatization of toluenediamines, but instead of HFBA pentafluropropionic anhydride (PFPA) was used.150

4.4.2 Derivatization with chloroformates Another reagent, ethyl chloroformate (ET), have been used for amine deri-vatization in the DBA method for isocyanates.143 The amines are reacted to urethanes in this type of derivatization. The reaction of amines with ET takes place in a two-phase reaction, with an aqueous and an organic phase. The water solution should be alkaline to force the amines into the organic solvent

48

where they react with the chloroformate. After the reaction the organic layer containing amine-ET and isocyanate-DBA derivatives is separated and eva-porated. The residue is dissolved in a solvent appropriate for the following analysis. Chloroformate reagents have been widely used for derivatization of amines preceding GC-analysis of amines.174,175 The reaction is quickly com-pleted without heating, and by selecting an appropriate buffer the selectivity of the reaction can be very high. In paper V ET was used for derivatization of amines and aminoisocyanates before analysis.

4.5 Determination of amines and aminoisocyanates

4.5.1 Preparation of standards Standards for diamine analysis are easily prepared as the diamines are com-mercially available. Aminoisocyanate standards for analysis are not com-mercially available, but different methods have been used for synthesis of derivatives. In two previous methods where aminoisocyanates were ana-lyzed, diamines were used as starting material for synthesis of derivatives. In the first method, where aminoisocyanates were analyzed with underivatized amine groups, a deficit of ethyl chloroformate was used for derivatization of diamins.157 The second method also included the reaction with ethyl chloro-formate in deficit, and the unreacted amine-groups were then derivatized with HFBA, to obtain the aminoisocyanate derivaties.81 In previous methods where TAI was derivatized with DBA and ET, standards for determination of aminoisocyanates were obtained by collecting thermal degradation of TDI-foam in toluene-DBA, and derivatize the amine-groups with ET after sampling.143 Aminoisocyanate derivatives can also be prepared from the corresponding diisocyanate in a two-step reaction. In paper V a method for the synthesis of aminoisocyanates derivatized with DBA and ET in reference solutions is presented. Isocyanates were dissolved in an organic solvent, usually isooctane, and reacted in two steps, with ethanol and DBA. The reac-tion of an isocyanate group with ethanol results in the same urethane deriva-tive as is obtained when an amine group is reacted with ET. The amount of reagent added in the first step was just sufficient to derivatize one isocyanate group on each diisocyanate. After completion of the first reaction the second reagent was added in excess, to derivatize the remaining isocyanate groups. For synthesis of HAI, MAI and 2,6-TAI, DBA was added in the first step, then ethanol was added. The amount of derivatives in the reference solution was determined using LC-CLND as described in chapter 3 for preparation of isocyanate-DBA reference solutions. Identification of the derivatives was

49

performed using TOF-MS. From 2,4-TDI it is possible to obtain two types of aminoisocyanate derivatives, with the amine group either on position 2 or position four. One of the isocyanate groups on 2,4-TDI, probably the group in position four, reacts faster with the added reagent than the other group. By varying which reagent is added first, both types of derivatives can be ob-tained. Small amounts of the diamine-ET and diisocyanate-DBA derivatives were also found in the prepared reference solutions (figure 10).

0 5 10 15

A

A

B CD E

E

C

D

Time (min) Figure 10. LC-CLND chromatograms from the synthesis of 2,4-TAI-ET-DBA. Peak A is the chromatographic front, B 2,4-TDA-ET, C 2,4-TAI-ET-DBA isomer 1, D 2,4-TAI-ET-DBA isomer 2, E 2,4-TDI-DBA. To the mixture in the lower chroma-togram ethanol was added before DBA, while DBA was added first in the top chro-matogram.

When solutions where the aminoisocyanate derivatives were the dominating compounds were obtained, diamine-ET and diisocyanate-DBA derivatives were added, so all of the compounds in the reference solutions had the same concentration, to facilitate when the solutions were used as analytical stan-dards.

4.5.2 Analysis Amines derivatized with different fluorinated anhydrides have often been analyzed using gas chromatography. The derivatives formed are suitable for analysis with for example electron capture detector166,168 (ECD) which is very sensitive for compounds containing halogens, or thermionic specific detector81,150 (TSD), which is sensitive towards nitrogen containing com-pounds. Anhydride-amine derivatives of this type has often been analyzed using GC, but it is also possible to use LC-methods for separation of the derivatives.172

LC methods for underivatized amines use buffered mobile phases for the separation. To obtain retention on a reversed-phase column it is necessary to

50

keep the amines deprotonized. Methods where free amines are analyzed by LC are all developed for aromatic amines, so UV-detection have been cho-sen for detection.80,81,167 Electrochemical detection was also used in two of the methods.81,167

One advantage with derivatization of amines before LC analysis is that active groups are made less active. This minimizes the possibilities for ad-sorption in the analytical system. Alternatively, that retention is lost due to charging of the amine group. The use of buffered mobile phases can also be avoided and this is an advantage for some types of detectors, for example mass spectrometers. LC-UV analysis have been used for amines derivatized with acetic anhydride.169,170 For analysis of amines and aminoisocyanates derivatized with ET LC-MS has been used for analysis.143 The urea bond present in the aminoisocyanate derivatives is prone to degradation during GC-analysis, so this type of derivatives is better suited for LC analysis at ambient temperature.

In paper V LC-MS was used for analysis of amines and aminoisocy-anates in air samples. For amines and aminoisocyanates deuterated amine-ET derivatives were used as internal standards. Air samples were also simul-taneously analyzed for isocyanates. The fragmentation patterns of amine derivatives contained molecular ions, [M+H]+, sodium adducts, [M+Na]+, and the fragments [M-46]+ and [M-92]+, that represents neutral loss of one or two ethanol. The fragmentation pattern for aminoisocyanate derivatives was recognized from both amine derivatives and isocyanate derivatives, that is except for molecular ions [M+H]+ and sodium adducts, [M+Na]+, [M-46]+ [M-129]+, [DBA+H]+ and [DBACO]+ can be found in the spectra. The in-strumental detection limits for the aminoisocyanates was as good as for iso-cyanate-DBA-derivatives analyzed by the single quadropole, about 2-4 fmol. The detection limits for amine derivatives was about ten times higher.

The amine-ET derivatives have retention times close to the monoisocy-anates, and aminoisocyanates derivatized with ET and DBA elutes in the time window between their corresponding diamines and diisocyanates on reversed-phase LC. Thus air samples could be analyzed for the presence of amines and aminoisocyanates with the same analysis time as for isocyanates only. However, to achieve separation between the two different isomers of 2,4-TAI, some modifications of the mobile phase were made. Instead of using 95/5/0.05 (% v/v/v) acetonitrile/H2O/formic acid as the strong eluent, the acetonitrile was replaced by a mixture of 75/25 acetonitrile/methanol (% v/v).

The excess DBA, present in the air samples also reacts with ET in the work-up procedure. The DBA-ET derivative is also eluted during the chro-matographic analysis as a broad overloaded peak, but fortunately this deriva-tive elutes between the last 2,4-TAI-ET-DBA and MAI-ET-DBA, and the determination of these peaks is not disturbed.

51

5 Organic Acid Anhydrides

5.1 Chemical properties Acid anhydrides are formed by reaction of two carboxylic groups, with eli-mination of water. Anhydrides can be formed by reaction between two acid molecules, for example acetic anhydride formed by reaction of two mole-cules acetic acid, or when two carboxylic groups in a diacid react with each other, for example maleic acid forms maleic anhydride. In the production of polymers only anhydrides from diacids are used, because they are difunc-tional and can be used for crosslinking.152 Anhydrides are reactive towards water, amines and alcohols. They are also strong oxidizers.176 The anhy-drides studied in paper VI, maleic anhydride (MA), phthalic anhydride (PA), tetrahydrophthalic anhydride (TA) and cis-hexahydrophthalic anhy-dride (HA) are all solids at room temperature. Structures are given in table 9.

Table 9. Organic acid anhydrides.

Compound Structure Vapor pressure(Pa) CAS-number

MA O

O

O

21.3 (20°C) 108-31-6

PA O

O

O

1.3 (20°C) 85-44-9

TA O

O

O

1.3 (20°C) 85-43-8

HA O

O

O

H

H

1 (20°C) 13149-00-3

52

5.2 Industrial applications Organic acid anhydrides are important industrial chemicals, and used in many chemical processes. In production of polymers, anhydrides are used as hardeners and curing agents for epoxy, alkyd or polyester resins. Compared to amines, which also can be used as curing agents for epoxy resins, anhy-drides need heat for the curing reaction.152 This makes them more expensive to use in production, but it is easier to control the properties of the formed resin.177 Besides from production of resins anhydrides are found in several different applications. Phthalic anhydride is also used for production of plas-ticizers, dyes, pharmaceuticals and pesticides.178,179 Maleic anhydride is pre-dominantly used for production of polyester resins. It is also used in the syn-thesis of other chemicals as fumaric acid and hexahydrophthalic anhydride. In the production of the sweetener aspartame maleic anhydride serves as a raw material.180 Tetrahydrophthalic anhydride and hexahydrophthalic anhy-dride are used in different anhydride-epoxy resins.181 Hexahydrophthalic anhydride is also used in the production of insecticides and rust preserva-tives.179 Other anhydrides used in the industry are for example trimellitic anhydride, tetrachlorophthalic anhydride and methyltetrahydrophthalic an-hydride.177,179

5.3 Exposure and health effects Several reports about exposure to acid anhydrides have been published. The effects of anhydrides on workers has often been studied during production of different resins, where for example PA182,183, MA182,184, HA185 or other anhy-drides are used in the production. Anhydrides are often used as solids in the production of resins. Dust containing anhydrides lead to exposure via the airways or skin.

Besides from production exposure to anhydrides can occur during appli-cation of powder paints186, or during thermal degradation of paints for exam-ple during welding or flame cutting of painted steel.187,188,189 In paper VI generation of different anhydrides during heating of different coatings was demonstrated.

Because of their reactivity and oxidizing properties organic acid anhy-drides are irritants to human airways, eyes, skin and mucous membranes. Besides from being direct toxins, exposure to acid anhydrides may also cause hypersensitivity that for example leads to the development of occupa-tional asthma.177 This has been shown for example for exposure to PA183 and HA.185 Rhinitis and chronic bronchitis are other symptoms associated with workplace exposure. Trimellitic anhydride causes influenza-like symptoms, or may lead to pulmonary hemorrhage.177 Trimellitic or phthalic anhydride

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may also cause allergic alveolitis.186 Anhydrides are also suspected to cause contact dermatitis190 and urticaria.184

Methods have been developed for biological monitoring of organic acid anhydrides. In the human body the anhydrides are hydrolysed to their corre-sponding diacids, and these can be found in urine or blood as biomarkers of anhydride exposure. Methods for determination of phthalic acid191 and hexa-hydrophthalic acid192 in urine and hexahydrophthalic acid and methylhexa-hydrophthalic acid in plasma193 have been presented. Antibodies (IgE and IgG) are also sometimes found in workers exposed to anhydrides. Presence of antibodies shall only be regarded as biomarkers of exposure, since the connection between antibodies and disease is weak.179

In Sweden the TWA values for PA is 2 mg m-3. For MA it has been set to 1.2 mg m-3. No limit values are given for TA and HA, but because they are known to cause hypersensitivity, they may only be handled by authorized laboratories.40

5.4 Air sampling A lot of different air sampling methods for determination of airborne anhy-drides have been developed since the early eighties. If the development of sampling devices and collection methods is compared with the development of isocyanates sampling it can be noted that there are few similarities be-tween the anhydride methods. For isocyanate methods developed at the same time almost all methods contain an amine reagent for the isocyanate deri-vatization. The anhydride methods presented and reviewed here uses deri-vatization during sampling, collection of anhydrides as such and hydrolysis of the anhydrides during sampling. Anhydrides are reactive compounds, but they are not as reactive as isocyanates, and methods where derivatization occurs during the collection are less common. Due to higher exposure limits the development of new air sampling methods for anhydrides has not been as extensive as for isocyanates.

5.4.1 Filters and solid sorbent sampling Glass fiber filters have been a common choice for collection of airborne anhydrides. If the filters used are unimpregnated, the extraction usually takes place in aqueous solutions to hydrolyze the anhydrides to corresponding acids. Glass fiber filter methods that involves hydrolysis before analysis have been presented for MA194, PA195, HA196 and TMA.197 In the method for HA, the acid formed during hydrolysis was derivatized with pentafluorben-zylbromide (PFBBr) before analysis.

OSHA has published several methods for different airborne anhydrides using impregnated glass fiber filters for collection and derivatization of the

54

anhydrides upon collection. One method uses filters impregnated with 1-2PP for collection of acetic anhydride (AA).198 1-2PP is a reagent that has also been used for sampling and derivatization of isocyanates.63 The other reagent used for filter sampling with impregnated filters is 3,4-dimethoxybenzylamine, also known as veratrylamine. Four almost identical methods have been presented for sampling of MA199, PA200, TMA201 and AA.202 When the methods for TMA and AA was published it had been found that addition of di-n-octylphthalate to the filters improved the contact be-tween the reactants.201

In an earlier method published by OSHA, MA was collected using a solid sorbent, tenax, coated with p-anisidine for derivatization of the anhydride.203 Tenax as an adsorbent for anhydrides has also been used in a method devel-oped by Health and Safety Executive (HSE) in Great Britain. The method has been evaluated for sampling of PA, TMA and tetrachlorophthalic anhy-dride. After sampling the anhydrides are extracted and hydrolyzed in aque-ous solutions.204 Sampling on tenax has also been performed for MA, in a method where the anhydride is determined as such, i.e. without prior deri-vatization or hydrolysis.205 In some methods a glass fiber filter has been placed in front of the tenax-tube to prevent large particles from enter-ing.206,207 In the PA-method206 the anhydrides are analyzed as such, while the corresponding acid is esterfied with methanol in the presence of boron tri-flouride (BF3) in the TMA method.207

XAD-2 is another type of sorbent that has been used for anhydride sam-pling. Methods has been developed for HA196,208, TA196 and methyltetrahy-drophthtalic anhydride.209 In all the methods the anhydrides are analyzed without modification.

Sampling of TMA on PVC-coopolymer filters has been published by Pa-lassis and co-workers210 and the National Institute for Occupational Safety and Health (NIOSH).211 Both the methods use esterfication of the acid of TMA, with methanol/BF3.

A somewhat unusual sampling device, a spiral glass trap has been devel-oped by Pfäffli. The spiral glass trap has been used for determination of PA212 and chlorendic anhydride.213 The sampler has been built for fume sampling, and to be able to separate gases and particles. A filter in front of the spiral glass trap collects particles, gaseous anhydrides are collected on the walls of the spiral glass trap and anhydrides not collected in the two first stages are collected on a tenax-tube that is used as a back up. After sampling the different parts of the sampler are extracted, and anhydrides are deter-mined as such.

5.4.2 Impinger or bubbler sampling In all the impinger and bubbler methods found in the literature, some aque-ous solution is used in the impinger, and the anhydrides collected are imme-

55

diately hydrolyzed. NIOSH has a method for MA, where anhydrides are collected in distilled water214, A similar method for MA was published a few years earlier by Geyer and Saunders194, where the anhydrides were collected in dilute phosphoric acid. For collection of HA208 and TA196 two methods using 0.1 M NaOH in bubblers has been presented by Jönsson and co-workers.

In paper VI it was investigated if it was possible to use an isocyanate me-thod53,73 using impinger sampling for screening of organic acid anhydrides as well. Anhydrides are reactive towards amine reagents, and there are possi-bilities for isocyanates and anhydrides to occur in the same exposure situa-tions for example during thermal degradation of polymeric coatings, that often consists of several different layers. It was found that with some modi-fications of the isocyanate method anhydrides and isocyanates could be col-lected simultaneously. One modification of the existing method was that instead of pure toluene as a solvent for the DBA reagent a mixture of toluene and acetonitrile was used instead. This increased the recovery of TA and HA. In pure toluene the recovery of these anhydrides was just about 20 %.

5.5 Sample Determination For the determination of anhydrides several different methods have been used. The for anhydrides corresponding acids that have UV response, LC with UV detection has been performed.194,195,197,204,214,215 Also the methods where derivatizing reagents has been used for anhydride sampling, LC-UV has been the method of choice.199,200,201,202,203

GC has also been used for sample analysis. Various detection principles have been used, such as flame ionization detection (FID)196,208,209,210,211, elec-tron capture detector (ECD)205,206,207,212,213, MS196,213 or nitrogen phosphorous detector (NPD).198

In paper VI organic acid anhydrides derivatised with DBA were deter-mined by LC-MS. A quadropole MS was used, with electrospray ionization. The ability of anhydrides to react with DBA was discovered when scanning of isocyanate air-samples was performed. Early eluting peaks were sus-pected as anhydrides, and it was confirmed by reacting pure anhydrides with DBA in solution and comparing the mass spectra. Anhydrides were also derivatized with di-n-propylamine (DPA) and di-n-ethylamine (DEA). The same fragmentation pattern as for the DBA derivatives were obtained. Ini-tially positive electrospray ionization was used. However, when quantifica-tion of anhydrides should be performed, it was found that calibration graphs were non-linear. When the ionization was switched to the negative mode, linear calibration graphs and a gain in selectivity were obtained. In the posi-tive mode protonated molecular ions and sodium and potassium adducts were seen. Some of the anhydride derivatives also showed more complex

56

adducts, and an ion at m/z = M+64 is probably [M+Na+CH3CN]+, that is an adduct with sodium and acetonitrile. Increasing the cone voltage lead to more fragmentation, and [M+H-18]+ (loss of water) and [M+H-129]+ was seen, as well as the [DBA+H]+ ion. The two last ions are also very common in the fragmentation patterns of isocyanate-DBA derivatives. Switching to negative ionization, the spectra was dominated by [M-H]-, the molecular ion without one proton at low cone voltages. At higher cone voltages fragmenta-tion was increased and ions like [M-H-44]- (loss of carbon dioxide) and [M-H-129]- (loss of DBA) were seen in the spectra.

4.17

226.2

20 4 6Time (min)

226.2

182.2180.1

3002001000

50

100

Rel

ativ

e ab

unda

nce

(%)

m/z

A

4.84

276.2300200100

0

50

100

176.1232.2

276.2

344.2

Rel

ativ

e ab

unda

nce

(%)

m/z

B

5.03

280.23002001000

50

100

151.1 236.2

280.2

348.2

Rel

ativ

e ab

unda

nce

(%)

m/z

C

5.51

282.2300200100

0

50

100

153.1

282.2

350.2

Rel

ativ

e ab

unda

nce

(%)

m/z

D

Figure 11. Chromatograms and negative ion spectra for the studied anhydride-DBA derivatives. A: Maleic anhydride-DBA, B: Phthalic anhydride-DBA, C: Tetrahy-drophthalic anhydride-DBA, D: cis-hexahydrophthalic anhydride-DBA.

57

The instrumental detection limits for the anhydride-DBA derivatives in the negative ionization mode ranged from 10-30 fmol. For SIR determination acquisition using two functions simultaneously was performed. One function was set for acquisition of the negative anhydride-DBA ions, while the other function was used for acquisition of positive ions of monoisocyanate-DBA derivatives. As internal standard during the MS analysis amines derivatized with trifluoro acetic anhydride and PFPA were used. These internal stan-dards are not ideal, in that the resemblance with the analyzed derivatives are small, but the derivatives used had similar retention times and a good re-sponse in the negative ionization mode.

For the chromatography of the anhydride-DBA derivatives it was found that it was necessary to use formic acid in the mobile phase, to keep the de-rivatives uncharged. Exclusion of formic acid removes the proton on the carboxyl group, resulting in decreased retention times, and the anhydride derivatives were eluted with the chromatographic front.

Standards of anhydrides-DBA derivatives were obtained by derivatization of anhydrides with DBA in solutions. The concentration of the derivatives in solution was determined using LC-CLND, as previously described for the synthesis of isocyanate standard solutions. In paper VI a number of differ-ent paint samples were heated, and the thermal degradation products were analyzed for anhydrides. Phthalic anhydride was the dominating anhydride, present in most of the paint samples, but in some paint samples it was also possible to detect maleic and hexahydrophthalic anhydride.

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6 Final comments

The sampling of airborne chemicals is justified for many reasons and several interest groups are involved. Workers have a justified interest to be informed about possible health risk at workplaces. Workers want to be ensured that the work is safe. They very often demand independent measurements and opin-ions. Sometimes they want data and information regarding different kinds of legal actions when e.g. workers are believed to have been sick due to the work. The companies (employers) have a great interest to make sure that workers do not become ill due to the work, in addition to the concern of workers health. It is expensive to replace sick workers and the production will be affected. It is also of importance for the employer to demonstrate that all is done to make the workplace safe. Many things in working life are regu-lated by different kinds of laws. The authorities have a direct responsibility to enforce current legislation. This results in practice in inspections of work places and e.g. demands of measurements of airborne chemicals.

However, exposure assessment by air sampling is a very demanding pro-cedure. One random sample does not mean much. Several samples are nec-essary to obtain a representative overview. A complete study involves many samples, takes long time and is very costly. If the exposure is complex, a lot of efforts need to be done and a battery of different methods must be applied.

Nowadays, most professional work in the industry is performed in a way that meets demands from ISO standards. Subcontractors also have a demand to fulfill the same requirements. Contract laboratories and industrial hygiene consultants are such subcontractors. This results in standardized protocols and procedures. It has many advantages but there is a conflict with the scien-tific community. Scientists are dedicated to find out new things and to de-velop new methods and procedures. Results are in most cases presented in peer reviewed papers, but this is not always sufficient as scientific papers are read only by a limited group of people. The time to implement new scientific achievements into national or international standards is very long. In prac-tice, many present standards are obsolete.

Today, there are many powerful analytical instruments commercially available. The modern instruments are much better than the ones that were available a few years ago. In the pharmaceutical industry such instrumenta-tion are routinely used. In the field of work environment chemistry there are many examples of laboratories with obsolete equipment. The equipment

59

used in this thesis is up to date and the author has experienced the necessity to have these kinds of equipment available to solve the aims of this thesis.

The aims of this thesis have essentially been fulfilled. Many new scien-tific questions were found during the work. The field of thermal decomposi-tion of plastics is complex. The focus has been on isocyanates, amines and anhydrides, but there are many more compounds to take into account regard-ing health risks. There is an immediate need for future studies as many workers are exposed and may be of great risk.

There are unsolved problems regarding air sampling of reactive com-pounds. The denuder-impactor sampler has so far mostly been applied to sampling of monomeric isocyanates. But isocyanate aerosols also consist of more compounds such as reaction products between isocyanates and polyols. Is it possible to also sample isocyanates that have partly reacted with poly-ols?

Reversed-phase chromatography has been shown to have limitations re-garding larger isocyanate compounds. Should other chromatographic meth-ods as gel permeation chromatography, where other solvents can be used, be considered as an alternative?

Further, sampling of amines and aminoisocyanates using dry sampling is still an unsolved problem. There is almost no knowledge on how amines and aminoisocyanates are distributed in reactive aerosols. In addition, sampling of anhydrides has not yet been studied with the new sampler.

Even though “wet” sampling methods as the impinger-filter may be cum-bersome to handle, they should still be regarded as very important for the development of new methods and reagents. It is the simplest and most effec-tive way to bring reactants and reagents together.

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7 Conclusions

In this thesis methods for air sampling and determination of isocyanates, amines, aminoisocyanates and anhydrides generated during production or thermal degradation of polymers such as polyurethane (PUR) or epoxy have been developed. The methods are based on derivatization of reactive groups with di-n-butylamine (DBA). Isocyanates and aminoisocyanates form stabile urea derivatives with DBA. Anhydrides form amide derivatives with DBA. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) enabled a reduction of instrumental detection limits 100 times, to as low as 10 atto-moles for isocyanate-DBA derivatives. Methods for preparation and charac-terization of reference solutions of technical grade isocyanates, aminoisocy-anates and anhydrides have been developed. A nitrogen-selective LC-detector enabled the quantification of the DBA-derivatives. By using deute-rium labeled DBA it was possible to synthesize internal standards for all studied isocyanates.

A novel sampler is developed. The sampler consists of a denuder in series with a three-stage cascade impactor and an end filter. The sampler made it possible to reveal the distribution of isocyanates between gas and different particle size fractions. As a reference method, simultaneous sampling was performed using an impinger filled with DBA in toluene, connected in series with a glass fiber filter. There was a good agreement between the denuder impactor sampler and the reference method. It was found that during thermal degradation of PUR, isocyanates were associated to particle size fractions (<1 µm) that may penetrate to the lower airways. The distribution of isocy-anates in aerosols during 8 minutes changes noticeably. Gas phase TDI and MDI became associated to small particles (<1 µm). This type of sampler can be an effective tool to study how different isocyanates can reach different parts of the human airways.

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8 Sammanfattning (in Swedish)

Denna avhandling behandlar provtagning av luftburna organiska ämnen som uppkommer vid framställning eller bearbetning av olika plastmaterial. Tyngdpunkten ligger på luftprovtagning av isocyanater, vilket är en av be-ståndsdelarna i polyuretanplast. Polyuretan är en av våra mest använda plas-ter, och olika typer av polyuretan används för produktion av t ex skumgum-mi, lacker, lim, fogskum och isolering. Isocyanater bildas vid termisk ned-brytning av polyuretan. Utöver isocyanater bildas också diaminer och ami-noisocyanater. En aminoisocyanat är en molekyl som innehåller en amingrupp och en isocyanatgrupp. Aminoisocyanater kan inte renframstäl-las, de skulle omedelbart reagera med varandra, men de kan existera i den aerosol som uppkommer vid termisk nedbrytning av polyuretan. Organiska syraanhydrider kan bildas vid termisk nedbrytning av epoxiplast, där dessa ämnen ingår som härdare.

Isocyanater är reaktiva ämnen som kan irritera luftvägarna och orsaka astma. Flera aromatiska aminer och isocyanater är klassade som möjligt cancerframkallande. Anhydrider är irriterande och kända allergener. Särskil-da tillstånd krävs för hantering av flera typer av isocyanater, aminer och anhydrider. Små partiklar (0.1-5 µm) har egenskapen att kunna nå långt ner i andningsvägarna och orsaka ohälsa. Det är därför viktigt att kemiskt kunna studera dessa små partiklars sammansättning, men metodik har saknats.

I arbetslivet exponeras många för termiska nedbrytningsprodukter av po-lymerer. Exponering förekommer inte bara i plastindustrin, utan även inom många andra områden. Heta arbeten där termiska nedbrytningsprodukter bildas förekommer i bilverkstäder, byggarbetsplatser och vid borttagning av färg. Även andra plastmaterial kan avge isocyanater vid uppvärmning såsom bakelit och fenol/formaldehyd/urea-harts som finns i mineralull. Även i hemmiljö kan man bli exponerad om man upphettar plast eller färg. Isocya-naterna förekommer både i gas- och partikelform.

Isocyanater är reaktiva ämnen. Vid luftprovtagning måste därför isocya-natgrupperna skyddas, för att de inte skall reagera med andra ämnen annars underskattas halten. I avhandlingen görs detta genom att isocyanaterna rea-gerar med en amin, di-n-butylamin (DBA) redan vid insamlingen. DBA rea-gerar också med organiska syraanhydrider, och denna princip har utnyttjats för luftprovtagning av dessa ämnen.

Eftersom isocyanater är mycket giftiga har de mycket låga gränsvärden. Därför har analysmetoder med extremt låga detektionsgränser utvecklats.

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Vätskekromatografi (LC) med masspektrometrisk (MS) detektering kan nu bestämma isocyanater i attomolmängder med en hög selektivitet. Detta är nödvändigt när man ska analysera flera ämnen samtidigt i ett och samma luftprov. Den utvecklade analystekniken har använts för analys av flera un-dersökta plasttyper. För en säker bestämning har stor vikt har lagts på att ta fram nödvändiga standardlösningar. LC-MS-analys kräver att standarder finns för samtliga bestämda ämnen. För detta ändamål har en LC kvävese-lektiv detektor, chemiluminescent nitrogen detector (CLND) använts.

För luftprovtagning av isocyanater har en ny typ av provtagare utvecklats. Provtagaren fraktionerar en aerosol i en gasfraktion och fyra olika partikel-fraktioner. Dessa fraktioner analyseras var för sig. Gas samlas i en denuder, partiklar på 3 impaktorsteg och mycket små partiklar på ett filter. I denudern samlas gasformiga isocyanater in med hjälp av diffusion. Isocyanatpartiklar-na impakterar beroende på storleken på de olika impaktorplattorna, där de fångas upp. Denuder-delen, impaktorplattor och slutfiltret impregnerades med DBA.

Den utvecklade provtagaren jämfördes med en etablerad provtagningsme-tod och en god överensstämmelse erhölls. Beroende på hur isocyanataeroso-len genererades bildades en varierande gas/partikel fördelning med en stor andel små partiklar. För en åldrande aerosol ändrades fördelningen av isocy-anater mellan gas och partiklar så att andelen små partiklar ökade.

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9 Acknowledgements

I want to express my sincere gratitude to the following people that everyone in some way has contributed to this thesis: My supervisor, Professor Gunnar Skarping, for your support, your great knowledge in chemistry and your special ability to always find solutions to my analytical problems. Without your skills and interest, this work would still be an unfinished project. My co-supervisor, Associate professor Marianne Dalene, for support, re-views and comments on manuscripts, interesting projects and for finding laboratory assistance to help me complete my studies. Co-author Dr Daniel Karlsson for valuable discussions and advice when-ever I needed, for teaching me how chromatography and mass spectrometry works in practice, for your excellent supervision during my diploma-work and at last for being a good friend. Co-author Dr Mårten Spanne for your never ending enthusiasm, your in-credible knowledge about aerosols, for always having the time to perform yet another necessary experiment in the laboratory (making me work even in the weekends!) and for being a good friend. Co-author Dr Åsa Marand for help and support when I started my PhD studies, good collaboration and friendship, despite our different opinions about roadkill elks. Mikael Adamsson for helping me with all strange orders of chemicals, glassware and everything else I needed. And for being a good friend, with whom I can discuss all work-related problems (and other things as well) on our almost daily afternoon train-trips home. Monica Hassgård for excellent laboratory assistance, taking care of thou-sands of samples. Your help has been incredibly valuable. Viveka Bursius for assistance in the laboratory, and for always being such a nice and happy person, no matter how early in the morning I meet you.

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Ralf “Allt går” Gunnarsson for manufacture of impactors, denuders and several other devices used for air sampling. Your skills in construction of equipment are truly impressive. Johannes Hall for assistance with all kinds of computer-problems. Jakob Riddar Johnson, Daniel Gylestam, Marcus Gustavsson, the “next generation” of PhD students, good luck in your future studies. Dr Sven Andersson and Professor Peter Michelsen, for your sometimes strange sense of humor, creating an amusing atmosphere in the lunch-room. Professor Paul Scheepers, Vivian van Hooren, Anjoeka Pronk and Jan Hendrix, for truly interesting workplace-studies in Belgium and the Nether-lands during 2002. My old-time friends, Olof, Nisse, Peter and Magnus. I now that I am a real worst case regarding phone-calls and meetings, but it is really nice to have friends like you, who always are supportive, ready to party, and when we finally find time to get together it is always a pleasure! My parents Stina and Michaël and my sister Johanna for support and al-ways being there for me when I need your help. My wonderful girlfriend Hanna, no one mentioned here is more important than you. You are the one, who knows how to say the right things to make me happy after miserable days at work, you are truly supportive and the best friend there is. Together with you, I know that I am always happy. I love you so much!

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