A,B Danielle L Taylor

Cameron J Ferris

Alison R Maniego

B,D Patrice Castignolles
email: p.castignolles@uws.edu.au

Marc In

Het Panhuis

Marianne Gaborieau

Characterization of gellan gum by capillary electrophoresis

Keywords: Gellan gum, capillary electrophoresis, bacterial exopolysaccharides, separation, sonication

come

Table of Contents entry

Gellan Gum is a food thickener and emulsifier, and it has potential in tissue engineering.

Free-solution capillary electrophoresis reveals the presence of oligomers and also separates it according to composition and conformation, even in the presence of aggregates. It provides a tool to assess natural variability, dissolution, ageing and sonication.

Introduction

Gellan gum is a natural exopolysaccharide produced by the bacterium Sphingomonas which is widely distributed from water to terrestrial habitats as well as in plant root systems. The rheological properties of gellan gum make it an ideal multifunctional gelling, stabilizing and suspending agent with uses in food, pharmaceutical, medical and chemical industries. [1][2][3][4][5] With gellan gum's increasingly widespread use its importance has become apparent with its unique properties showing advantages over other naturally occurring polysaccharides. These advantageous properties are its excellent thermal and acid stability, adjustable gel elasticity and rigidity, high transparency and good flavor release. [2] Gellan gum is a linear anionic polysaccharide based on a tetrasaccharide repeat unit composed of two residues of D-glucose, one with up to two acyl substituents (one bearing a vicinal diol), one Dglucuronic acid and one L-rhamnose (Fig. 1). Gellan gum samples differ by the proportion of units with acyl chains attached to the glucose molecule (degree of acylation) and the distribution of these acyl chains along a polysaccharide chain. In other word, gellan gums are copolymers which composition is generally called degree of acylation. The acyl substituents dramatically affect the unique desirable properties, in particularly the rheology of the gels produced. [1,2] It is expected that the distribution of the acyl substituents also has an effect on the properties of gellan gum. [1] Currently there is no definitive method available to produce identical gellan gum samples with the same degree of acyl substituents and equal distribution of molecular weights. [2] Commercial samples are only described as exhibiting a 'high' or 'low' degree of acylation by the supplier (see Table 1). To overcome this natural variability, it is therefore important to determine the degree and distribution of degrees of acylation of a gellan gum. Fig. 1. Molecular structure of fully acetylated gellan gum (a, adapted from [1] ) and fully deacylated gellan gum (b, adapted from [5] ). The monomer unit is constituted from left to right of one D-glucose with two acyl substituents (one acetyl and one glycerate bearing a diol or glycol), one D-glucuronic acid, one D-glucose without substituents and one L-rhamnose. The gellan gums studied in this work are copolymers containing both repeating units (not necessarily forming blocks).

There have been several methods used previously to characterize gellan gum, including rheology, light scattering, nuclear magnetic resonance (NMR) spectroscopy, and dichroism spectroscopy. [3][4][5][6][7] These methods have been used to characterize the conformational transition from a random coil to a double-helix structure (twice higher molecular weight) on cooling. In one instance, the molecular weight of the gellan gum was varied by sonication (the only information given on the sonication was the resulting intrinsic viscosity of the samples). [7] Number-average molecular weights have been measured by osmometry. [8] Weight-average molecular weights have been measured by static light scattering. [5,[7][8] Molecular weight distributions have been determined by size-exclusion chromatography (SEC, also known as GPC or GFC) with state-of-the-art detection. [10][11] Preliminary injections of gellan gums showed that an aqueous LiCl eluent at 60 °C leads to repeatable results. [10] However, further work in other groups relied on different eluents ranging from salt solutions with weaker hydrogen bond breaking properties [11][12] down to pure water [2] . The quality of the separation was not assessed for example in terms of recovery or accuracy of the determined molecular weights in all these latter works despite the use of new eluents and columns. This is of importance since SEC of polysaccharides is commonly plagued by low recovery. [13] Aggregation has been observed in aqueous KCl eluents. [12] Filtration affects the determined molecular weight values in light scattering [14] (or the determination is sometimes impossible due to filters clogging [12] ) and it can also lower the recovery in chromatographic separation of gellan gum. [10] It is thus desirable to apply to gellan gums separation methods that do not require sample filtration. These are methods using no stationary phase: field-flow fractionation [15] or freesolution capillary electrophoresis (detailed later). There is no established method to determine the molecular weight distribution of gellan gums. Furthermore, the influence of a distribution of degrees of acylation has not been considered, although differences in composition could significantly alter the accuracy of the determined molecular weights. [9] The characterization of gellan gum in regard to its average degree of acylation and its distribution of degrees of acylation has not been reported. [1] The aim of this work is to separate the whole gellan gums (without filtering the solution) according to their structure.

Gellan gums are used as sieving media in gel electrophoresis. [16] Characterization of the gellan gums by electrophoresis is a lot scarcer. Free-solution capillary electrophoresis (CE) involves separation in a (fused-silica) capillary filled with a buffer solution (no stationary phase) and placed under high voltages (up to 30 kV). Free-solution CE has been applied once to the determination of enzymatically degraded gellan gums, [17] but not for its separation or structural characterization. Capillary electrophoresis is largely used for separation and characterization of a few natural polymers, DNA and proteins. [18] Applications of CE to polysaccharides are limited. Free-solution CE proved successful to separate chitosan from conjugates [19] or separate oligomers according to their molecular weight [20] . Free-solution CE exhibits a higher resolution than SEC for the separation of oligomers. [21][22][23][24] In the case of polymers, no separation by molecular weight is observed in the cases of single-stranded or double-stranded DNA, poly(styrene sulfonate) and a few other polyelectrolytes. [21,25] This corresponds to the "critical conditions" sought in liquid chromatography at the critical point of adsorption (LC-CC). [26][27][28][29][30][31][32] LC-CC does not separate the compound of interest by its molecular weight. Not separating by the molecular weight of one block in a copolymer, allows determining the molecular weight of the other block. In LC-CC, critical conditions are however tedious to determine and recovery is poor. [31][32] LC-CC has never been successfully applied to polysaccharides. Free-solution CE, or CE in the critical conditions (CE-CC), was on the other hand shown to separate two polysaccharides, pectins [33] and carboxymethyl cellulose, [34] according to their composition, which is named degree of substitution. This separation has a higher selectivity at the lowest charge density, below the Manning's counter-ion condensation. [35][36][37] The aim of this work is to explore the potential of CE-CC for the separation of gellan gums by its composition, namely degree of acylation. In this work, the potential of CE-CC to characterize gellan gums is investigated for the first time examining the repeatability of the sample preparation and separation, and studying the effects of sonication, endotoxin removal, expected degradation stresses (mainly heat) and exceeded expiry dates. These results are compared with ones obtained by rheological analysis.

Results and Discussion

Separation of Gellan Gums by Free-Solution Capillary Electrophoresis.

Sodium borate was used as a buffer like in the literature [17] but with a higher pH (9.2 instead of 8.0) in order to increase the zeta potential of the capillary wall and thus limit potential adsorption of gellan gums to the wall. The separations were performed at 55 °C contrary to 35 °C and 20 °C in the literature [17] . At low temperature, aggregation, through double-helix formation, has been extensively observed by viscosity measurements [38] and light scattering [5] . The separations presented in this work were obtained at 55 °C to obtain random coil conformation and limited aggregation in the capillary. 4 min preconditioning with the buffer was first used as in [17] and led to some separation but with relatively poor resolution and repeatability (data not shown). An additional 1 min flush with NaOH (1 M) was added to the preconditioning (i.e. before each injection) and yielded results with a higher signalto-noise ratio as well as repeatable separations within 10 minutes. Fast separations are important not only in terms of throughput but also to ensure no deacetylation or degradation takes place, which could be caused by long residence to times at high pH and high temperature. The UV spectra yielded by the diode array detector are the same for the different peaks (see Fig. S12). These peaks are thus all consistent with gellan gums and not with some potential impurities such as proteins. The detection might be explained by complexation of the borate ions from the buffer by the glycol moiety present on the acetylated glucose units. Separation with ammonium acetate leads to no peaks detected in equivalent conditions (same pH, data not shown). Complexation of the borate by alcohol groups in neutral monoand oligosaccharides have been shown to be strong enough to create an electrophoretic mobility. [39] A representative example of electropherograms is shown on Fig. 2a and2b, more examples are shown in Supporting information (Fig. S1 to S10). Two main populations with electrophoretic mobilities around respectively 1.5 and 2.8•10 -8 m 2 .V -1 .s -1 are clearly separated. The second peak is broader and exhibits a shoulder showing a likely inhomogeneity in gellan gum structure. These mobilities are lower than those observed for poly(sodium acrylate)s of degree of polymerization above 10 in the same conditions (around 6 m 2 .V -1 .s -1 , see Fig. S13 in supporting information) [23] . This is expected since the charge is due to a carboxylic acid in both cases but the charge density is lower for the gellan gum at our pH (9.2). The first peak has equivalent mobilities to tetrasaccharides in borate buffer (at lower a buffer concentration of 60 mmolL -1 though). [39] It might thus correspond to one gellan gum monomer (which is a tetrasacharide, see Figure 1) or oligomers (see next paragraph). These separations of gellan gums were successfully reproduced with different and longer capillaries, higher voltage and higher injection volume and different operators (Figure 2c). The later parameters (series 2 to 4) resulted in a higher signal-to-noise ratio and also to a higher resolution of the peaks (the latter discussed in the next part).

The separations cannot be compared with literature [17] since the latter does not indicate values of the electrophoretic mobility, only migration times.

Separation of Gellan Gum with Different Degrees of Acylation.

The potential of free-solution CEto separate gellan gum is apparent in the comparison of samples with low and high average degrees of acylation shown on Fig. 3. The low-acyl LAGG-Exp has the two relatively broad peaks described above at mobilities 1.45 and 2.8 ×10 -8 m 2 •V -1 •s -1 whereas the high-acyl HAGG has these two peaks but also a broad one at high mobility, 4.95 × 10 -8 m 2 •V -1 •s -1 , and a number of narrow and less repeatable (see supporting information Fig. S2) peaks between the second and the third broad ones. The second broad peaks of both samples differ in shape: LAGG's peak is more symmetrical with a slight tail on the low mobility side, while HAGG's exhibits a slight tail on the high mobility side and a pronounced tail on the low mobility side. The third broad peak has a high mobility value (4.95 × 10 -8 m 2 •V -1 •s -1 ). Various sharp peaks, or spikes, have mobilities between 3 and 4 × 10 -8 m 2 •V -1 •s -1 . These peaks significantly decrease in intensity when the sample is injected 5 days after dissolution rather than a few hours (as shown by comparison of Fig. 3 and4). They disappear completely after sample filtration (Fig. 4). They also disappear with sonication (see last paragraph).

They thus correspond to aggregates and are present mainly, if not only, in the high acyl. This is confirmed by visual observation of the clear low-acyl solution and the cloudy high-acyl gellan gum "solution" (Fig. S11). These aggregates have already been observed to lead to difficulties in light scattering and size-exclusion chromatography. [10,12,14] Capillary electrophoresis is thus complementary to these two methods which are commonly applied to polysaccharides. It could be used to monitor dissolution and is thus complementary with time-resolved solution-state NMR. [40][41] The first peak has mobility equivalent to the tetrasaccharide monomer unit. [39] Oligomers of charged polymers have been observed to have a lower mobility than the polymer for short chains with less than 5-10 monomer units in the case of poly(styrene sulfonate) [21,42] poly(acrylic acid) [23,24] or DNA [25] . The presence of oligomers in gellan gums has never been mentioned in the literature. To confirm the presence of oligomers, we performed a pressure mobilization as performed by Cottet's group for Taylor Dispersion Analysis. [43] They were able to monitor mixtures of monomer and polymer by pressure mobilization due to the large differences in their diffusion coefficient. [44] Pressure mobilization was also applied to our gellan gums (Fig. 5). It is clear that the peak is the sum of narrow and broad peaks corresponding respectively to fast diffusing and slow diffusing species. This confirms the presence of monomer or oligomers. Oligomers have never been reported in Kelcogel gellan gums, but no conditions separating oligomers (such as column with small pore size in SEC) are reported to have been tested. The lowest mobility peak may thus correspond to oligomers. with Gaussian fit (solid lines). Experimental conditions are the same as Series 3 except that mobilization pressure was applied at 50 mbar instead of electric field and injection time was only 5 s.

The relative area of the oligomer peak is compared to the area of the whole electropherogram (Fig. 6).

The ratios of the area of the first peak to the total area are repeatable (see Fig. 6 and supporting information). The first peak is relatively more intense for the low-acyl gellan gum than for the high acyl (compare 3 rd bar to 6 th and 7 th bars in Fig. 6). The low-acyl gellan gums contain more oligomers(s) likely due to some degradation during the deacylation process. The highest mobility peak appears only for the high acyl gellan gums. This peak is of higher intensity when potassium borate is used as buffer instead of sodium borate (Fig. 7b). Injections in potassium borate are repeatable (see Fig. S9 and Fig. S10). The low-acyl gellan gum in potassium borate exhibits slightly different electrophoretic mobilities but no significant change in terms of relative quantities. The change of mobility is expected since the different counter-ion is going to change both electrostatic and hydrodynamic friction (the latter through viscosity change). The same differences are observed with the high-acyl, but the high mobility peak is also more intense. Potassium has been observed to lead to a higher proportion of double-helix conformation. [6] The highest mobility peak, around 5×10 -8 m 2 V -1 s -1 may thus correspond to double-helix conformation. CE has thus potential to study the conformation of gellan gums. Finally the peaks around 3 × 10 -8 m 2 V -1 s -1 may correspond to the gellan gum chains in the expected random coil conformation. This peak is broad for low acyl gellan gums and significantly broader for the high acyl gellan gums. This is attributed to separation by the degree(s) of acylation in CE-CC.

Uncontrolled Degradation of Gellan Gum.

As gellan gum is a natural exopolysaccharide produced by a bacterium and it is expected to be easily degradable in normal environmental conditions, it is usually kept at low temperatures. [1,2] Samples of gellan gum were left on a bench in summer where they experienced temperatures up to 40 ºC for about 14 days. It is expected that under these conditions the gellan gum structure would experience degradation. Fig. 8a depicts a comparison of two LAGG samples, one of which has been exposed to high temperatures. Exposure to 40 °C does not significantly change the mobilities (the second peak's electrophoretic mobility is only 2.8 % lower). The relative intensity of the first peak however significantly increases with exposure to temperature and leads to the higher ratio observed in this work (Fig. 6). Exposure to 'high' temperature thus likely leads to some degradation leading to oligomers formation. Lapse of time is also expected to cause gradual natural degradation of gellan gum. Samples that have exceeded the manufacturer's expiry dates were kept in the same conditions and compared to the current gellan gum samples (Fig. 8b). No significant change is observed in terms of electrophoretic mobility (the largest shift, observed for the second peak, is of 2.1% of the mobility value and may be due to different baselines). No significant increase of the relative area of the first peak is however observed (Fig. 6). This confirms the literature where osmometry showed no change of molecular weight after two years storage of deacetylated gellan gums. [7] .

Endotoxin-free (low-acyl) gellan gum LAEFGG was compared to non-endotoxin free (low-acyl) gellan gum LAGG-Exp in order to determine if the treatment done to remove the endotoxins has any effect on the gellan gum structure (Fig. 8c). LAEFGG's peaks have a similar intensity and electrophoretic mobility compared to LAGG-Exp's corresponding peak. The maximum difference is again observed on the second peak although the LAEFGG's peak has an electrophoretic mobility only 2 % lower: 2.78 compared to 2.84 × 10 -8 m 2 •V -1 •s -1 . A comparison of the relative intensity of the first peak in LAGG-Exp and LAEFGG-Exp (Fig. 6) shows that the treatment done to gellan gum to remove the endotoxins has no effect on its oligomers content or composition that can be observed with CE.

Sonication of Gellan Gum.

A controlled sample treatment technique that has a possible effect on the structure of gellan gum was also examined. Sonication is a useful technique that enables conducting fillers such as carbon nanotubes to be effectively dispersed within a gellan gum solution. [3] However, it has been observed that sonication has a detrimental effect on further processing of gellan gum-carbon nanotubes composites, impeding hydrogel formation [4] and fiber spinning [45] . This has been attributed to a sonication-induced reduction in molecular weight of gellan gum. [4] Fig. 9 displays the apparent viscosity of as-prepared and sonicated solutions of low-acyl and high-acyl gellan gums. An as-prepared solution of HAGG exhibits a relatively high viscosity and shear-thinning behavior, while LAGG-Exp has a lower viscosity which is independent of shear rate across the measured range. This difference is attributed to the higher degree of acylation of HAGG, with more acyl groups promoting stronger hydrogen bonding, which increases viscosity and imparts some pseudo-plasticity. Sonication significantly reduces the viscosity of both LAGG and HAGG, enforcing the conclusion that the process leads to a reduction in molecular weight.

Interestingly, the reduction in viscosity is more marked for HAGG -the viscosity of both sonicated solutions is comparable and the sonicated HAGG is no longer shear-thinning. To investigate this further, the structure of sonicated gellan gum solutions was measured using CE (Fig. 10). The effect of sonication on the gellan gum structure of LAFEGG is illustrated in Fig. 10a. Both peaks of each injection appear very similar. However, the relative intensity of the first peak significantly increases with sonication of sample LAEFGG (Fig. 6), suggesting that sonication leads to some degradation into oligomers in the low-acyl gellan gum. The effect of sonication on the structure of HAGG is shown in Fig. 10b. The spikes consisting in aggregates disappear after sonication. This indicates that the stronger decrease in viscosity for the high acyl than the low acyl is not due so much to a decrease of the molecular weight of gellan gum chains but rather to a decrease in apparent molecular weight, or in other words an aggregate dissolution. The relative intensity of the first peak does not increase after sonication for the high-acyl gellan gum, indicating the absence of significant degradation of the polymer chain themselves into oligomers. The stronger decrease of viscosity in HAGG than in LAGG may be due mainly to dissolution of aggregates of high-acyl gellan gums chains rather than deacylation.

Conclusion

Capillary electrophoresis in the critical conditions (CE-CC) was applied for the first time for the characterization of gellan gum. The method is fast (less than 10 min) and was validated in terms of sample preparation and repeatability. The proof-of-principle was established for the successful separation of gellan gum in regards to its degree of polymerization in the case of oligomers and its degree of acylation for polymers, as well as the conformation (random coil vs double helix). The separations are unaffected by the presence of microgels, as there is no stationary phase or light scattering detection. The microgels can be observed and slow dissolution monitored. Dilute solutions of high-acyl gellan gums contain aggregates that slowly dissolved. Five days are the recommended dissolution time, although it does not provide yet full dissolution, The effect of sonication, endotoxin removal, expected degradation stresses (mainly heat) and exceeded expiry dates on gellan gum were tested. Exposure to high temperatures leads to some degradation of the gellan gum into oligomers, contrary to endotoxin removal treatment. Sonication mainly changes the high-acyl chains through the dissolution of microgels/aggregates. The detection of aggregates initially in high-acyl gellan gum but not in low-acyl ones explains a stronger viscosity decrease for the high-acyl gellan gum.

The CE-CC methods developed in this work proved a valuable tool to determine differences in composition of gellan gums samples. It is complementary to rheology. CE-CC could be applied to a wider variety of gellan gum samples with applications in food or tissue engineering. In particular, it will be valuable for the characterization of the natural variability of gellan gum.

Experimental

Materials.

Boric acid and sodium hydroxide pellets (Sigma) were used as received to prepare a 200 mM sodium borate buffer at pH 9.2 (NB200). Boric acid (BDH AnalaR) and potassium hydroxide flakes (Unilab) were also used as received to prepare a 150 mM potassium borate buffer at pH 9.2 (KB150). Ammonium acetate (Scharlau) and a 28% ammonia solution (Fronine Pty LTD) were used as received to prepare a 75 mM ammonium acetate buffer at pH 9.2 (AA75). Gellan gum samples produced by fermentation of a pure culture of Sphingomonas elodea were a gift from CP Kelco. All samples were prepared with gellan gum powder added to hot, approximately 80 ºC, Milli-Q water at 1 g•L -1 , and stirred for 1 h. This yielded a clear solution for low-acyl gellan gum, but a whitish suspension for highacyl gellan gum (see Fig. S11 in supporting information). All low-acyl gellan gum samples where dissolved in Milli-Q water, while the high-acyl samples were dissolved in 1/20 v/v NaOH and NB200 solutions in Milli-Q water, and treated at 60 ºC for 1 h. The samples were not filtered except if stated otherwise. Millipore filters were either 0.45 m (Millex HV, PVDF membrane) or 0.22 m (Millex GP, PES membrane). The high-acyl gellan gum samples used in sonication treatment were dissolved in Milli-Q water (1 g•L -1 and 0.5 g•L -1 ). Due to gellan gum's gelling properties and thermal instability, samples are required to be kept at low temperatures in order to preserve the gellan gum and prevent dissociation. Samples were stored in a fridge unless otherwise specified. The different gellan gum samples are described in Table 1. The low-acyl gellan gum LAGG is (almost) fully deacetylated as shown by the weak acetyl signal at 2 ppm in 1 H NMR [46] (Fig. S14). Unfortunately, NMR and other common analytical techniques are unable to yield information on the glycerate group content. The 1 H NMR spectrum also shows no significant impurity such as residues from bacteria or from the fermentation medium. The capillary electrophoresis experiment was carried out using an Agilent CE 7100 (Agilent Technologies). The capillaries used were 50 µm ID fused silica capillaries (Polymicro, USA). Highsensitivity ("bubble") capillaries were not needed contrary to the literature [17] and only regular capillaries were used. The pretreatment of the capillary included an initial 10 min flush with 1 M NaOH, 5 min flush with 0.1 M NaOH and a final 10 min pretreatment flush with Milli-Q water. This wash was also used after the series in order to clean the capillary. For the latter series of injections extra wash sequences were added after every two or three injections and after every high-acyl gellan gum sample injection. These extra wash sequences were the same as the initial pretreatment flush sequences. The standards used to validate the capillary and act as a reference for the gellan gum sample injections were a model oligoacrylate, AA4, and acetone. [23][24] Both standards and all samples were injected into a 200 mM sodium borate buffer of (NB200). The capillary post-treatment was a flush for 10 min with NaOH and then again with Milli-Q water. Four series of injections were completed, each with different parameters, in sodium borate buffer 200 mM unless otherwise specified. The set conditions for both injection series are illustrated in Table 2. All data was recorded at 200 nm and treated using Origin 7

plotting intensity versus mobility. Electrophoretic mobility was calculated. [23] Sonication of samples was performed using a Branson digital probe sonifier. 10 mL of 0.5 gL -1 samples were subjected to 5 min of pulsed sonication (0.5 seconds on/off) at 30 % amplitude. The apparent viscosity of as-prepared and sonicated samples was measured at 25 °C on an AR-G2 controlled stress rheometer (TA Instuments) using a 60 mm (2°) cone and plate geometry. Shear rate was ramped from 5 to 100 s -1 over a measurement period of 5 min.

Fig. 2 .

 2 Fig. 2. Repeatability tests: (a) electropherograms of the same sample LAEFGG-Exp as a function of

Fig. 3 .

 3 Fig. 3. Electropherograms of low-acyl gellan gum, LAGG-Exp (dotted red line), and high-acyl gellan

Fig. 4 .

 4 Fig. 4. Electropherograms of sample HAGG-Exp after 5 days dissolution (injection series 3): nonfiltered (solid black line), filtered on 0.45 m (dashed red line) or 0.22 m porosity filter (dotted blue

Fig. 5 .

 5 Fig. 5. Pressure mobilization of HAGG (with repeat experiment): experimental signals (dotted lines)

Fig. 6 .

 6 Fig.6. Ratio of the area of the lowest mobility (first) peak to the total area of the electrophoretic

Fig. 7 .

 7 Fig. 7. Electropherograms (series 3) of sample LAGG-Exp (a) and HAGG (b): in potassium borate

Fig. 8 .

 8 Fig. 8. Electropherograms of samples before and after potential degradation (first injection series): (a)

Fig. 9 .

 9 Fig. 9. The apparent viscosity of as-prepared (full symbols) and sonicated (empty symbols) solutions of 0.5 gL -1 of LAGG (circles) and HAGG (triangles) in water.

Fig. 10 .

 10 Fig. 10. Comparison of gellan gum before and after sonication (solid black line and dotted red line,

Table 1 . Gellan gum samples.

 1 LA and HA stand for low and high degrees of acylation, respectively; EF stands for Endotoxin-free.

	Sample	Description	Treatment	Catalog name	Lot number Expiry
	LAGG-Exp	LA	Expired	KELCOGEL-F	7C9228A 26/3/08
	LAGG-Tem	LA	Kept at up to 40 ºC	KELCOGEL-F	7C9228A 26/3/08
	LAEFGG	LA, EF		GELZAN CM	9K6968A 20/11/11
	LAEFGG-Son	LA, EF	Sonicated	GELZAN CM	9K6968A 20/11/11
	LAEFGG-Exp	LA, EF	Expired	GELZAN CM	7K1383A 19/11/08
	HAGG	HA		KELCOGEL-LT100 9K6878A 5/11/11
	HAGG-Son	HA	Sonicated	KELCOGEL-LT100 9K6878A 5/11/11
	HAGG-Exp	HA	Expired	KELCOGEL-LT100	814441A	29/9/09
	Methods.

Table 2 . Experimental conditions for the four injection series

 2

	Parameter	Series 1	Series 2	Series 3	Series 4
	Capillary total length L (m)	0.645	1.20	1.00	1.20
	Capillary detection length l (m)	0.56	1.115	0.915	1.115
	Voltage (kV)	20	25	30	20
	Temperature (ºC)	55	55	55	30
	Injection Pressure (mbar)	30	30	30	30
	Injection Time (s)	10	20	10	20

Acknowledgements

DJT, PC and MG thank the College of Health and Sciences, UWS for a summer studentship and an equipment grant for the capillary electrophoresis. MihP and CJF thank the Australian Research Council (ARC), ARC Future Fellowship (MihP) and University of Wollongong for financial support. CP Kelco are thanked for the gellan gum samples.

Repeatability of Electropherograms of Various Gellan Gum Samples

Appearance of Gellan Gum Samples in Water

UV absorption spectra of the different signals of gellan gums

Fig. S12: UV absorption spectra obtained with the diode array detector of the capillary electrophoresis for the peaks at 1.5 m 2 .V -1 .s -1 (first peak) and 2.8•10 -8 1.5 m 2 .V -1 .s -1 (second peak) for different gellan gums samples.

Mobility of poly(sodium acrylate)

H NMR Spectrum of 'Low-Acyl' Gellan Gum

Relative Intensity of First Peak of Electropherograms


Simon Aubin

Loïc Wingert

Sébastien Gagné

Livain Breau

Jacques Lesage

Comprehensive Methylene Diphenyl Diisocyanate (MDI) Evaluation Method Comparison Using a Laboratory Generation System

Isocyanates are reactive semivolatile contaminants that must be assessed in occupational environments, and specific evaluation methods are required to address the challenges related to isocyanate emission characteristics. Several standard methods exist, but significant differences remain regarding the diversity of industrial isocyanate emissions. This study presents a method to establish a baseline comparison of three sampling principles. A fine aerosol (mass median aerodynamic diameter of 250 nm) of pure methylene diphenyl diisocyanate (MDI) was produced (5-60 µg/m³) using a laboratory generation system (n = 31 generation experiments). Airborne MDI was measured with the following four methods, with an emphasis on the spatial distribution of the collected MDI within the sampler: (1) Swinnex cassette 13 mm, glass fibre filter (GFF), 9-(Nmethylaminomethyl) anthracene (MAMA-Swin); (2) Closed-face cassette (CFC) 37 mm, GFF (end filter and inner walls, MAMA-37; (3) Impinger and backup GGF, 1,2-methoxyphenylpiperazine (MP) (ISO 16702/MDHS 25); and (4) Denuder and GFF (Asset EZ4-NCO), dibutylamine (DBA) (ISO 17334-1). Bland and Altman analyses determined that there were no significant bias between the methods although Asset was not in agreement with MAMA-Swin (95% confidence interval above the ± 20% criteria). Significant correlations (P < 0.05) were observed between airborne MDI concentration levels and their distribution within the Asset (denuder vs end filter) and Impinger (collecting solution vs backup filter) subsections. The presence of impregnated inner walls in the CFC did not increase collection efficiency for the generated MDI aerosol. Non-uniform MAMA impregnation on GFF was demonstrated, whereas the collected MDI was evenly distributed in the air samples. These results provided the basis of comparison for other studies involving more complex isocyanate emissions.

Introduction

Isocyanates are highly reactive organic molecules containing the chemical functional group N=C=O. They are mainly employed in the manufacturing of polyurethane materials used in many different products (paint, varnish, insulating foam, mattresses, glue, adhesives, etc.), and workers are potentially exposed to them (1). Isocyanates are respiratory sensitizers and are known to be a cause of occupational asthma (2)[START_REF] Banks | Isocyanate-induced respiratory disease[END_REF][START_REF] Mapp | Polyisocyanates and their prepolymers[END_REF][START_REF] Vandenplas | Occupational asthma and extrinsic alveolitis due to isocyanates: Current status and perspectives[END_REF][START_REF] Malo | Combined alveolitis and asthma due to hexamethylene diisocyanate (HDI), with demonstration of crossed respiratory and immunologic reactivities to diphenylmethane diisocyanate (MDI)[END_REF][START_REF] Vandenplas | Occupational asthma caused by a prepolymer but not the monomer of toluene diisocyanate (TDI)[END_REF]. In order to prevent health problems in workers, over the years, occupational exposure limits (OELs) have been established at levels of a few parts per billion (ppb) [START_REF] Acgih | TLVs and BEIs : based on the documentation of the threshold limit values for chemical substances and physical agents and biological exposure indices[END_REF][START_REF] Ifa | GESTIS database: International limit values for chemical agents (Occupational exposure limits, OELs): Institute for Occupational Safety and Health of the German Social Accident Insurance (IFA)[END_REF]. Air monitoring to evaluate workers' exposure to chemical substances provides occupational hygienists with a base of knowledge to assess the risk to workers' health and the need to control their exposure. Among the numerous chemical substances found in the workplace, it remains challenging to sample "semivolatile" compounds (noting that, to date, the scientific community has not agreed upon a clear and formal definition of the term "semivolatile") due to their dual physical phases [START_REF]Technical Overview of Volatile Organic Compounds: Environmental Protection Agency (EPA)[END_REF][START_REF] Cen | EN 13936 Workplace exposure -Procedures for measuring a chemical agent present as a mixture of airborne particles and vapour -Requirements and test methods[END_REF][START_REF]Workplace Air -Procedures for measuring a semi-volatile chemical agent using pumped samplers -Requirements and test methods[END_REF]. Semivolatile compounds are usually collected by drawing air through a sampler comprised of a filter and a sorbent tube. The main characteristic differentiating isocyanates from other semivolatile substances (such as PAHs and certain pesticides) is their chemical reactivity. This characteristic requires the sampling method to adapt by chemically stabilizing isocyanates into an analyte that can be analyzed in the laboratory [START_REF] Ashley | Selecting isocyanate sampling and analytical methods[END_REF][START_REF] Guglya | Determination of isocyanates in air[END_REF][START_REF] Henneken | Determination of airborne isocyanates[END_REF][START_REF] Streicher | Determination of airborne isocyanate exposure: Considerations in method selection[END_REF]. This process is called derivatization, and its efficiency (which enables optimal measurement of airborne chemical concentration), depends mainly on the ability of the isocyanate function to be in contact with the derivatization reagent added to the liquid or solid support of the sampler [START_REF]-Workplace atmospheres -Guidelines for selecting analytical methods for sampling and analysing isocyanates in air[END_REF].

Numerous methods have been developed over the years, using a secondary amine as the reagent in a collecting solution (impinger) or on an impregnated filter. They have been the subject of many reviews in which diverse aspects have been addressed, such as reaction kinetics, collection efficiency, analytical method optimization and method comparison [START_REF] Rando | Laboratory comparison of sampling methods for reactive isocyanate vapors and aerosols[END_REF][START_REF] Karoly | Stability studies of diphenylmethane diisocyanate (MDI) on glass fiber filters[END_REF][START_REF] Maître | Comparison between Solid Sampler and Impinger for Evaluation of Occupational Exposure to 1,6-Hexamethylene Diisocyanate Polyisocyanates during Spray Painting[END_REF][START_REF] Rudzinski | Sampling and analysis of isocyanates in spray-painting operations[END_REF][START_REF] Kuck | Sampling of diisocyanates (HDI, TDI) in air by derivatisation with secondary amines as reagents[END_REF][START_REF] Tremblay | Investigation of the competitive rate of derivatization of several secondary amines with phenylisocyanate (PHI), hexamethylene-1,6diisocyanate (HDI), 4,4′-methylenebis(phenyi isocyanate) (MDI) and toluene diisocyanate (TDI) in liquid medium[END_REF][START_REF] Gylestam | Dry sampling of gasphase isocyanates and isocyanate aerosols from thermal degradation of polyurethane[END_REF][START_REF] Sennbro | Determination of isocyanates in air using 1-(2-methoxyphenyl)piperazine-impregnated filters: Long-term sampling performance and field comparison with impingers with dibutylamine[END_REF][START_REF] White | MDHS 25 revisited: Part 2, modified sampling and analytical procedures applied to HDI-based isocyanates[END_REF][START_REF] Hext | A comparison of the sampling efficiencies of a range of atmosphere samplers when collecting polymeric diphenylmethane di-isocyanate (MDI) aerosols[END_REF][START_REF] Marand | Solvent-free sampling with di-n-butylamine for monitoring of isocyanates in air[END_REF]. Today, some industrial processes that emit isocyanates require the use of a midget impinger, which is a major concern for industrial hygiene practice because of solvent vapour exposure and the risk of spillage [START_REF] Lesage | Airborne methylene diphenyl diisocyanate (MDI) concentrations associated with the application of polyurethane spray foam in residential construction[END_REF][START_REF] Puscasu | Comparison between the ASSET EZ4 NCO and Impinger Sampling Devices for Aerosol Sampling of 4,4′-Methylene Diphenyl Diisocyanate in Spray Foam Application[END_REF]. Our research team recently carried out studies comparing newer methods to older filter methods [START_REF] Aubin | On site comparison of the OSHA 42, Asset EZ4-NCO, Iso-Chek, DAN and CIP10 methods for measuring toluene diisocyanate (TDI) at a polyurethane foam factory[END_REF][START_REF] Aubin | On-site comparison of the OSHA 47, Asset EZ4-NCO, Iso-Chek, DAN, and CIP10 methods for measuring methylene diphenyl diisocyanate (MDI) at an oriented-strand board (OSB) factory[END_REF]. Although the biases could be explained for some methods, there was a reversal (negative vs positive) between ISO 17734-1 [START_REF]-1 -Workplace atmospheres -Determination of organonitrogen compounds in air uing liquid chromatography and mass spectrometry -Part 1: Isocyanates using dibutylamine derivatives[END_REF] and ISO 14382 [START_REF]1-(2-pyridyl)piperazine-coated glass fibre filters and analysis by high performance liquid chromatography with ultraviolet and fluorescence detectors[END_REF], depending on the type of isocyanate measured.

Although differences between isocyanate concentration measurements can be observed, knowledge is lacking to explain them. It is generally accepted that large fast-curing particles containing isocyanates, such as methylene diphenyl diisocyanate (MDI) polyurethane spray foam aerosols, are efficiently derivatized by their dissolution in the impinger collecting solution, whereas a negative bias has been observed when using impregnated filter methods for such particles [START_REF] Streicher | Determination of airborne isocyanate exposure: Considerations in method selection[END_REF][START_REF]-Workplace atmospheres -Guidelines for selecting analytical methods for sampling and analysing isocyanates in air[END_REF]. This observation can be explained by the fact that coarser particles, or the accumulation of too many over time, significantly decreases isocyanate access to the reagent from the filter. Thus, except for the use of a scanning electron microscope to analyze the surface of a filter [START_REF] Bell | Evaluation of the OSHA 42 and NIOSH 5521 methods in determining the free isocyanate concentration in aerosols and vapor phases during application of two component 1,6-hexamethylene diisocyanate paints[END_REF] and the observation that field extraction enabled higher results [START_REF] Karoly | Stability studies of diphenylmethane diisocyanate (MDI) on glass fiber filters[END_REF], direct investigation into what actually occurs within the sampling device (e.g., spatial distribution within sampler or inner wall deposition), has not been conducted. As a misconception of these differences could lead to underestimating the exposure, it is crucial to increase our knowledge. This article presents a method comparison for the evaluation of MDI using a laboratory generation system that has been recently developed by our research team. The article also highlights how the collected MDI and the reagent are spatially distributed within the sampling devices. Four methods were selected to compare different reagents and sampling principles. A filter method with reagent 9-(N-methylaminomethyl)anthracene (MAMA) was used in a 13 mm Swinnex polypropylene cassette, a sampler type mentioned in a reference method [START_REF] Hsl | Methods for the Determination of Hazardous Substances[END_REF] that is also an intrinsic part of the denuder-filter method described below. This sampler shows no significant collection of particles < 1 µm on its inner walls [START_REF] Hext | A comparison of the sampling efficiencies of a range of atmosphere samplers when collecting polymeric diphenylmethane di-isocyanate (MDI) aerosols[END_REF] and provides a higher density of collected MDI on the filter. The other filter method is an adaptation of ISO 17336 [START_REF]Workplace air quality -Determination of isocyanate in air using a double-filter sampling device and analysis by high pressure liquid chromatography[END_REF] and used MAMA in a 37 mm closed-face cassette (CFC) with its inner wall covered with impregnated filters, which prevents potential inner wall losses [START_REF] Mao | Sampling efficiency of a modified closed-face cassette sampler for airborne toluene diisocyanate determination[END_REF]. A denuder-filter method was used, consisting of the commercially available sampler Asset EZ4-NCO with dibutylamine (DBA) as reagent [START_REF]-1 -Workplace atmospheres -Determination of organonitrogen compounds in air uing liquid chromatography and mass spectrometry -Part 1: Isocyanates using dibutylamine derivatives[END_REF]. An impinger method, with a backup filter inserted in a Swinnex 13 mm cassette, using 1,2methoxyphenylpiperazine (MP) [START_REF] Hsl | Methods for the Determination of Hazardous Substances[END_REF][START_REF]Workplace air quality -Determination of total organic isocyanate groups in air using the 1-(2-methoxyphenyl)piperazine and liquid chromatography[END_REF] was also employed, as impinger methods are still considered to be the only reliable way to efficiently measure isocyanate exposure for processes emitting large fast-curing particles.

The objective of this study is to establish a baseline comparison of the main sampling principles used in airborne isocyanate exposure evaluation. This baseline comparison will be used in further comparison studies to understand how more complex isocyanate emissions behave in different types of samplers.

Materials and Methods

Part 1 -Generation System

The generation system used is described in Aubin et al. 2021(40). Airborne MDI was generated by nebulizing a solution of MDI in hexanes, where the concentration dictated the airborne concentration. Two nominal airborne concentrations were generated: 5-15 µg/m³ and 40-60 µg/m³, referred to as low and high, respectively. These concentrations cover real exposure scenarios in workplace atmosphere since the typical OEL for MDI is 51 µg/m³ (5 ppb).( 9) The generated atmospheres demonstrated a stable concentration of airborne MDI over time, reached after 5 minutes of nebulization, with a Mass Median Aerodynamic Diameter (MMAD) of about 250 nm [START_REF] Aubin | Development and characterization of an adaptable aerosolized methylene diphenyl diisocyanate generation system[END_REF]. Environmental parameters were continuously monitored and fixed at 22-23°C and 28-32% of relative humidity (RH) with an air flow dilution of 20 L/min. A splitter (made in-house in the laboratory) provided eight identical samples from a single intake in the exposure chamber. The samplers were connected to the splitter sampling ports by conductive silicone rubber tubing of minimal length. The system exhibited a continuous flow rate of approximately 21 L/min (nebulizer + air dilution). The system equilibrium (flow rate and internal pressure), to compensate with the air taken for the samples, was reached by adjusting a manual valve connected to the vacuum downstream of the exposure chamber. Generation experiments were performed at periods of approximately 30 minutes and 120 minutes, referred to as short and long, respectively. The short experiments were performed at low and high concentrations, while the long experiments were performed only at low concentrations. The volume of MDI solution was prepared accordingly so that it would suffice for the entire time length of the experiment.

Part 2 -Air Sampling and Chemical Analysis

-Sampling Methods

The main information about sampling methods and chemical analysis is summarized in Table 1. The MAMA-37 method was based on the approach of Mao et al. 2000(38) using the reagent and analytical procedure described in ISO 17336 [START_REF]Workplace air quality -Determination of isocyanate in air using a double-filter sampling device and analysis by high pressure liquid chromatography[END_REF]. It consisted of a 37 mm CFC with a glass fibre filter (GFF) impregnated with MAMA. Sections of impregnated GFF were added to cover the vertical walls (rim filter) and the inner surface of the inlet section (top filter). The rim filter was 6 mm x 100 mm and the top filter had a diameter of 35 mm with a 6 mm hole punched in the middle. SKC 224-PCXR4 (USA) personal pumps were used to collect samples at 1 L/min (MAMA-Swin, MAMA-37 and Impinger) and the SKC Pocket Pump TOUCH was used for the Asset method at 0.2 L/min. The sampling flow rates were measured with a flowmeter (Mesa Labs Defender, USA) before and after the sampling period to determine whether the flow rate had changed by more than 5%. Except for the Asset, all impregnated filters and subsections described in this study were desorbed immediately after sampling.

To detect unexpected behaviour in the generation system, the Fidas® Frog (Palas, Germany) aerosol optical spectrometer (AOS), which detects particles based on light-scattering principles, was used as a monitoring device (1 min. average measurements). It has the ability to count and size particles with a diameter of 0.150 to 80 µm.

-Sampling Material Preparation Solutions of MAMA (3 mg/L and 10 mg/L) and of MP (30 mg/L), all in toluene, were prepared. Impregnation was done manually by adding the solution to each filter with a volumetric pipette, as described in Table 2. The filters were set on aluminum foil contained in an opaque plexiglass box in which nitrogen was kept at a positive pressure. The filters were left to dry for at least 12 hours. The impregnated filters were inserted immediately into sampling devices or kept away from light at 4°C for a maximum period of 1 month.

Part 3 -Experimental Strategy -Intercomparison of the Derivatizing Reagents

Preliminary experiments were conducted with three sampling methods, each consisting of an impregnated GFF with a different derivatizing reagent inserted into a 37 mm polystyrene CFC (Zefon, USA). The MAMA filters were impregnated following the procedure described above, while the MP filters were impregnated with 500 µL of a solution of MP 10 mg/L in toluene. The DBA filters were prepared according to ISO 17334-1, to obtain the same amount of DBA per surface unit theoretically found in the end filter of the commercially available Asset EZ4-NCO sampler. Six generation experiments were conducted with concentration levels ranging from 3 µg/m³ to 115 µg/m³. The three samplers were used in duplicate in each experiment.

-

Intercomparison of Four Methods

Thirty-one generation experiments were conducted for the comparison study, in which all methods were used in duplicate, with the exception of MAMA-Swin, which had a single replicate due to limitations in sampling port availability on the flow splitter. The number of pairs available for the comparison study of MAMA-Swin vs MAMA-37, vs Asset, and vs Impinger were 61, 48, and 20, respectively.

-

MDI and Reagent Distribution within the Sampling Device

An analysis of the spatial distribution of the reagent and the MDI was conducted to explain potential bias among the methods. Samples collected with the methods described in Table 1 were subdivided into sections prior to their analysis. Table 3 summarizes how the samples were subdivided. Each sub-section was treated and analyzed according to the analytical procedure described in ESI. In order to obtain a more comprehensive comparison, complementary experiments were performed for the MAMA-37 and the Asset methods.

Firstly, the MAMA-37 method was compared to the so-called MAMA-37-single method, in which there is only an end filter (no rim or top filters). These experiments were conducted to evaluate whether or not the use of rim and top filters in MAMA-37 would increase the collecting efficiency, thus causing bias in the results of the comparison with the other methods. Six experiments were done: three at low concentrations and three at high concentrations, where the MAMA-37 was compared to the MAMA-37-single, each in triplicate. The three different sections of MAMA-37 were analyzed separately. Secondly, experiments were conducted to measure the MDI spatial distribution on the 37 mm filter placed in a CFC. The results were compared to the spatial distribution (or mapping) of the derivatizing reagent on the same lot of impregnated filters as the one prepared for MDI evaluation. This experiment, based on the approach of Tucker et al. 2007 [START_REF] Tucker | Investigation of reagent distributions on glass fiber membrane filters used in air sampling[END_REF], consisted of punching 19 circular subsections of 4.8 mm in diameter and analyzing them separately for MAMA or MDI. Mapping of the collected MDI was carried out on samples originating from two generation experiments at high concentration, in triplicate, for 45 minutes.

The analytical procedure for MAMA as the analyte was specifically developed for this experiment and is described in the electronic supplementary information (ESI). The mapping results consisted of amount (in µg) of MAMA or MDI per circular subsection.

Finally, the fractional (per particle size) particle collection efficiency of the denuder section of the Asset sampler was measured on a classical filtration bench test, where the Asset denuder (with the Swinnex cassette removed) replaced the filter holder. The measurements were triplicated.

The Asset denuder was challenged with a sodium chloride (NaCl) test aerosol generated by an atomizer (AGK 2000, Palas Gmbh, Germany) using a 0.5% NaCl solution and air pressure of 2 bars. Upstream of the measurement area, the aerosol was charge neutralized (Aerosol neutralizer 3012, TSI, Shoreview, Minnesota, USA). To derive the fractional collection efficiency, the number particle size distribution was measured upstream and downstream of the tested denuders.

The particle size distributions were measured with a Scanning Mobility Particle Sizer (SMPS 3936, TSI, Shoreview, Minnesota, USA). For the purpose of comparison with other measurements in the project, the equivalent electrical mobility diameters measured by the SMPS were converted into aerodynamic diameters, yielding investigated particle sizes ranging from 100 nm to 865 nm. This particle size range corresponds to mass particle size distribution of the generated airborne MDI (MMAD = 250 nm) [START_REF] Aubin | Development and characterization of an adaptable aerosolized methylene diphenyl diisocyanate generation system[END_REF]. The air flowrate through the denuder was kept at 0.2 L/min. The SMPS flowrate being 0.3 L/min., 0.1 L/min. of clean compressed air was added to the sample flow to compensate for the lower flowrate through the denuder. The compensation flowrate was set and kept constant by a mass flow controller (0-1 L/min., Brooks Instrument SLA5850S, USA).

To reduce uncertainties in measurement due to particle losses, the tubing was set as short as possible. Thus, the upstream/downstream sampling probes were located only a few centimeters in front of/behind the denuder. Moreover, blank tests were performed by measuring the particle collection efficiency of the tubing alone, without the denuder. The denuder results were corrected accordingly.

Part 4 -Data Processing

AOS monitoring data were processed by the Palas software PDAnalyse and exported to Excel for further calculations (average and variability of the particle concentration). MDI concentrations, relative abundance of MDI in samples' subsections, and other sampling parameters were analyzed using STATA version 15.1 (StataCorp LLC, Texas). Bland-Altman (B&A) plots were generated in STATA. B&A plots represent the difference between the responses of an evaluated method and a reference method (bias), as a function of the mean response of both. The comparison method used MAMA-Swin as the basis of comparison for the three other methods. This selection was established based on the ease in sampling device manipulations, its documented absence of inner wall deposition for the particle size involved in this study [START_REF] Hext | A comparison of the sampling efficiencies of a range of atmosphere samplers when collecting polymeric diphenylmethane di-isocyanate (MDI) aerosols[END_REF] and simple laboratory work-up. Added to experimental points, these plots also show the mean bias, its 95% confidence interval and the limits of agreement (±1.96 x standard deviation (s)). B&A analysis has the advantage of showing the agreement (or non-agreement) between methods and its trend and precision over the range of levels measured by the methods [START_REF] Giavarina | Understanding Bland Altman analysis[END_REF]. Prior B&A analysis, data distribution was confirmed as normal for all data sets using Shapiro-Wilk Test. The bias between any given paired measurements (y-axis) was expressed as a fraction of the concentration measured. This fraction was calculated by dividing the difference between the two paired measurements by the mean of these two measurements (x-axis). Bias was considered significant if the line of equality (0% bias) was not within the confidence interval of the mean bias. The agreement between methods was considered acceptable if 95% of the bias values (± 1.96s) were within ± 20%. This criteria was based on the one published by the National Institute for Occupational Safety and Health (NIOSH), ± 25%, and the fact that the methods were tested in a controlled environment. ( 43)

Results and Discussion

-Intercomparison of the Derivatizing Reagents

The comparison using the three reagents with the CFC revealed no significant bias for DBA vs MP, and DBA vs MAMA, with a mean bias and confidence interval (CI) of -1.3% (-8.2, 5.5) and Such a comparison with the same controlled atmosphere used for the formal comparison discussed below was necessary to document any potential differences that might have been observed. For this experiment, the observed bias could originate from the derivatization efficiency and the analytical quantification, since all the other parameters were the same for the three methods. Previous documentation shows that there is no difference in reaction yield or kinetics among the reagents compared in this experiment [START_REF] Kuck | Sampling of diisocyanates (HDI, TDI) in air by derivatisation with secondary amines as reagents[END_REF][START_REF] Tremblay | Investigation of the competitive rate of derivatization of several secondary amines with phenylisocyanate (PHI), hexamethylene-1,6diisocyanate (HDI), 4,4′-methylenebis(phenyi isocyanate) (MDI) and toluene diisocyanate (TDI) in liquid medium[END_REF], therefore the analytical quantification is most likely the principal parameter that may have led to a bias between MAMA and MP. The limits of agreement mentioned above are most likely due to random errors produced along the whole measurement procedure (sampling and analysis). While these limits of agreement can be interpreted as too high, i.e. above ±20%, the B&A plots show that the concentration levels below 10 µg/m³ produced the highest differences between reagents, revealing that the variability becomes relatively less important when the concentration levels increase.

-

Intercomparison of Four Methods

The particle concentration monitoring demonstrated that the MDI concentration was stable throughout the generation experiments, with RSDs ranging from 3 to 33% with 94% of RSDs below 20%. Intra-method precision was evaluated by calculating the difference between replicates. Average, minimum, and maximum differences for the methods were as follows: MAMA-37: 2.5% (min. 0.1% max. 7.4%); Asset: 3.6% (min. 0.1% max. 13%) and Impinger: 2.5% (min. 0.3% max. 8.1%).

Figure 1 presents B&A plots for the three pairs of method combinations, including all experiment results. The three methods did not show a significant bias overall, because their confidence interval of the mean bias includes the zero bias value. MAMA-37 and Impinger exhibited limits of agreement, approximately ±17%, within the acceptable range defined a priori for this study (±20%). Asset exhibited limits of agreement of (-40, 32)%, which is above the acceptable range. The lack of agreement of Asset vs MAMA-Swin seems explained by the fact that Asset introduce a higher variability in the measurements, which is supported by the the variability observed within its duplicates mentioned above. B&A plots were applied to subgroups of data and no significant effect on bias was observed for the sampling time, the concentration levels or the collected amount of MDI.

The agreement between the tested methods in such a controlled environment was somewhat anticipated due to the chemical and physical forms of the airborne MDI. Firstly, MDI was present in its simplest form, i.e., monomeric, without any copolymer that could have promoted its reaction in other chemical species, thus compromising MDI derivatization. Secondly, the particulate phase, composed of particles below 500 nm, does not imply a challenge in terms of collecting or derivatizing efficiency for the air sampling principles used in this study.

Impinger and MAMA-37 are indeed in agreement to MAMA-Swin when measuring the MDI in the described generated atmosphere. Although MP introduced a small but significant bias compared to MAMA in the reagent intercomparison, the combination of reagent + sampling method and the increase of the number of experiments over the concentration level range made it insignificant. This increase in number of experiments would also be an explanation for the decrease of the limits of agreement observed in the method intercomparisons (Impinger (MP) vs MAMA-Swin and MAMA-37 vs MAMA-Swin). The source of the variability causing the nonagreement of Asset could be the denuder (this only difference compared to MAMA-Swin in terms of sampling, see figure 4) and its separate laboratory work up procedure on two subsections. However, this would only explain a small portion of the variability since the MDI analyzed in Asset denuders accounts for less than 10% (see below). Therefore, the observed variability would most likely be a combination of different other sources.

Figure 1. Bland-Altman plots comparing MAMA-37, Impinger or Asset methods to MAMA-Swin. The relative bias (y axis) consists of the difference between the two paired measurements divided by the mean of these two measurements. The MDI concentration (x axis) consists of the mean of the two paired measurements.

-MDI Partition within the Sampling Device

MDI partition within the MAMA-Swin cassette

Rinsing of the inlet part of the MAMA-Swin cassette was stopped after the first series of experiments since it did not show any detection of MDI even at the highest load of collected MDI. This further confirms the appropriate choice of the MAMA-Swin as the basis of comparison.

MDI partition within the Impinger sampling train

Figure 2A shows the partition of MDI in the impinger + cassette sampling device for the generation tests performed during the intercomparison study (n = 20). On average, the proportions of MDI collected with the impinger method were 14% and 86% in the impinger and the backup filter, respectively. The partition between impinger and filter is comparable to Hext et al. 2003 (27) for which 60% to 65% of the MDI aerosol (< 1 µm) was found on the backup filter of the sampling device. Regression analysis showed a correlation (R² 0.62, P<0.05) between MDI apportionment in the impinger or the backup filter and MDI concentration but did not show any correlation with sampling duration. A decreasing trend of the proportion of MDI found in the impinger was observed along with the increase of MDI air concentration levels. The proportion of MDI in the impinger decreased from 16% to 12% when its airborne concentration was increased from 13 µg/m³ to 57 µg/m³ (figure 2B). This could be explained by the fact that a higher MDI concentration promotes the formation of larger particles, which are less prone to evaporation. Thus, the distribution difference seems most likely due to a decrease of the relative abundance of vapour phase MDI, since the increase in MDI particle size is not great enough to be more efficiently collected by the impinger. The presence of larger particles corroborated by the mass particle-size distribution (measured with an electrical low-pressure impactor) of the generated aerosol determined in our previous study [START_REF] Aubin | Development and characterization of an adaptable aerosolized methylene diphenyl diisocyanate generation system[END_REF] confirms this hypothesis (see the two comparative particlesize distribution histograms in ESI) with MMAD changing from 250 nm to 350 nm at low and high concentrations, respectively. Other studies demonstrated that the impinger is efficient at collecting vapour and large particles but not efficient for particles below 2 µm, hence the addition of a backup filter downstream of the impinger [START_REF] Hext | A comparison of the sampling efficiencies of a range of atmosphere samplers when collecting polymeric diphenylmethane di-isocyanate (MDI) aerosols[END_REF][START_REF] Spanne | Collection efficiency for submicron particles of a commonly used impinger[END_REF]. It should be noted that the impinger solution does not act as a delimitation between vapour and particle phase MDI, as the solution collection efficiency is not zero for the MDI mass particle-size distribution (MPSD) generated in this study. Experiments conducted by drawing NaCl particles solely through the denuder section made it possible to evaluate the proportion of particles of given sizes that are collected by the denuder at the sampling flow rate at which the Asset sampler is used. Figure 3 presents the fraction of the particles retained by the denuder in function of their diameter at an airflow rate of 0.2 L/min. These results highlight the fact that the Asset "denuding" step does not only apply to vapour phase contaminants but also to a portion of the particles. While the denuder principle has been demonstrated to be efficient in the past as a method to determine the partition between vapour and particle phases (45), the denuder section of the Asset sampler collected a significant amount, from 15 to 25% of < 865 nm particles when challenged with the NaCl aerosols. While it was beyond the scope of the study to investigate the Asset denuding capabilities, the results obtained enable a better understanding of the partition between the denuder and end filter (see below). The robustness of these measurements is supported by the low collection efficiency of the tubing without the denuder, i.e., 1.4% on average, with a standard deviation of 1.3%. Thus, uncertainties about the denuder results due to correction on the basis of blank test data are strongly reduced. [START_REF] Aubin | Development and characterization of an adaptable aerosolized methylene diphenyl diisocyanate generation system[END_REF], where the partition between the denuder and end filter was 8:92, on average. Correlation tests demonstrated that concentration levels had a significant (R² 0.68, P<0.05), but small, effect on the distribution of MDI in the denuder section (D1+D2). Similar to what was observed in the impinger and its backup filter, a decreasing trend in the proportion of MDI found in the denuder was observed along with the increase of airborne MDI concentration. Correlation tests did not demonstrate any significant effect of sampling duration or weight of MDI collected on the proportion found in the four subsections of the Asset sampler. The effect of the concentration is illustrated in figure 5.B, which presents the proportion of MDI retrieved in the denuder (D1 + D2) for two subsets of data for which the MDI concentration levels are different, while sampling duration is the same. The same explanation described above for the MDI distribution in the impinger and backup filter applies, i.e., the increase of MDI particle size diminishes its evaporation and therefore its relative abundance in the denuder section.

Two other subsets of data enabled the comparison of MDI proportions found in sections D1 and D2 simply by visualizing the results above the LOQ. Figure 5.C and 5.D compare the MDI proportion found in D1 and D2 when the weight of collected MDI was approximately the same because of the longer sampling duration for the lower MDI concentration. The D1/D2 ratio, approximately equal to 2 for both levels, shows that the longitudinal distribution of MDI is not affected by the increase of MDI levels and its associated change in particle size distribution. This can be explained by the denuder collection efficiency measurements. Indeed, in both cases (low and high concentrations), the mass particle size distribution (MPSD) lies mainly in the range of 100 nm to 900 nm, (MMAD = 250 nm). In this range, Figure 3 shows a very flat profile of particle losses, i.e., between 15% and 25%, approximately, and between 15% and 18%, approximately, from 200 nm to 800 nm. Thus, even if the MPSD shifts slightly toward larger particles, the percentage of particles lost per denuder unit of length remains unchanged.

The connection between denuder and the end filter sections, which consist of a disposable 3-mL syringe and a Swinnex cassette, respectively, involves a luer lock, which forces the air to flow from an 8-mm to a 1.6 mm diameter cross-section (Figure 4B). The turbulence created by such an abrupt change could lead particles to impact and stick to the sampler inner walls, and the rinsate sub-section (Table 3) addressed this potential occurrence. The MDI proportion found in the rinsate was minimal (95% of the samples below 2%, 52% of the samples were below the LOQ), most likely due to the small size of the MDI aerosol.

As established by Gylestam et al. 2014 (24), these results highlight the fact that the partition observed (approximately 8:92 for the denuder and the end filter, respectively) does not express a clear separation between the vapour and particle phases. Therefore, given the results observed in the denuder collection efficiency experiments (see above) and the MPSD of the MDI aerosol generated (MMAD = 250 nm) (40), a very low proportion of vapour phase MDI is anticipated, most likely lower than 8%.

Comparison between MAMA-37 and MAMA-37-Single

A B&A analysis, mean bias of 1.4%, CI (-0.7, 3.6) and limits of agreement of (-6.9, 9.8)%, demonstrated that the total MDI measured by both sampling devices was not significantly different over the MDI concentration range evaluated (10 to 60 µg/m³) (see ESI). This observation is made despite the fact that MAMA-37 collected, on average, 7% of the MDI on the CFC inner walls (n = 18). MDI would therefore be collected accidentally by the impregnated media added inside the sampler, since MDI is collected in any case by the filter when the inner walls are not impregnated.

MDI partition in sampling device of MAMA-37

Figure 6 shows the distribution of MDI in the MAMA-37 sampling device for the generation tests performed during the intercomparison study (n = 61). On average, the proportions of MDI collected were 93%, 5% and 2% in end, rim and top filters, respectively. Regression analysis showed no correlation between MDI apportionment inside the CFC and concentration, sampling duration or mass of MDI collected. These results, combined with those of the comparison between MAMA-37 and MAMA-37-Single, demonstrate that the generated MDI is not likely to be deposited in a significant proportion on the internal walls of the CFC. Indeed, some of the aerosol or traces of vapour seem to diffuse once they enter the cassette but they would be collected in any case by the end filter even if the inner walls had no impregnated media. This conclusion differs from that reported in a study evaluating toluene diisocyanate (TDI) with the same sampler configuration, in which one-third of the TDI was found in the top and rim filters [START_REF] Mao | Sampling efficiency of a modified closed-face cassette sampler for airborne toluene diisocyanate determination[END_REF]. This difference can mostly likely be explained by the vapour pressure difference between TDI and MDI (2.3x10 -2 mm Hg and 5.0x10 -6 mm Hg, respectively). The higher relative abundance of vapour phase TDI makes it more likely to diffuse once it enters the sampler inlet and therefore to be collected by the filters covering the cassette's inner walls. The change in MDI concentration levels, and its associated effect on the relative abundance of vapour phase MDI (see sections on Impinger and Asset), was most likely not important enough to induce a substantial change in MDI spatial distribution within the CFC. Many publications over the last decade have pointed out the significance of exposure measurement underestimation caused by analyte deposition on sampler inner walls [START_REF] Ashley | Analytical performance issues Closed-Face Filter Cassette (CFC) sampling-guidance on procedures for inclusion of material adhering to internal sampler surfaces[END_REF][START_REF] Hendricks | On wiping the interior Walls of 37-mm closed-face cassettes: An OSHA perspective[END_REF][START_REF] Harper | Analytical Performance Criteria[END_REF][START_REF] Chapter | Factors affecting aerosol sampling NIOSH Manual of Analytical Methods (NMAM) 5e édition Washington DC: NIOSH-CDC[END_REF]. It may have such an effect on the result that at least one standard specifies that any analyte found on sampler inner surfaces must be accounted for in the exposure measurement [START_REF]D8358-21 Standard Guide for Assessment and Inclusion of Wall Deposits in the Analysis of Single-Stage Samplers for Airborne Particulate Matter[END_REF]. Our study demonstrated that a fine aerosol of pure MDI, a reactive chemical requiring in situ derivatization, is not subject to underestimation due to inner wall deposition in a polystyrene CFC. A and B show one out of four replicates obtained from two different lots of filters, but they are representative of all the replicates obtained. Figure 7.A illustrates how the impregnated MAMA was distributed on the 37 mm filter. Preliminary tests demonstrated that the amount of MAMA or MDI was not related to the weight of the discs sampled. Therefore, the weight of subsections was not recorded. Figure 7.B illustrates how MDI is distributed after 45 min. of air sampling at 57 µg/m³, resulting in approximately 2.6 µg of MDI collected on the filter. This amount is of the same order of magnitude as the amount collected in the experiments at high concentrations or long sampling durations described above. Figure 7.C shows the sampling pattern made to map the MAMA or MDI on filters.

C

The MAMA was not evenly distributed on filters, with twice as much accumulating on one side of the filter compared to the other and even to the centre area of the filter. This non-uniformity can be explained by factors such as the solvent and the procedure used to impregnate the filter [START_REF] Tucker | Investigation of reagent distributions on glass fiber membrane filters used in air sampling[END_REF].

The actual MAMA distribution seemed most likely influenced by the impregnation procedure in which the filters were not perfectly leveled, leading to higher levels of solution flowing toward a given portion of the filter, especially when wetted filters adhered to the surface by capillarity. Once dried, yellowish filter coloration was also visible toward the edge of filters, a known occurrence caused by faster evaporation of solvent along the edges, pulling the solution containing the reagent towards the edges by capillarity [START_REF] Tucker | Investigation of reagent distributions on glass fiber membrane filters used in air sampling[END_REF].

The spatial distribution of MDI on filters from air sampling was almost uniform, with a maximum amount in the centre and edge areas showing only a maximum decrease of 19%. For several reasons, this was expected due to the fine MDI aerosol. Firstly, the largest particles are below 1000 nm and are therefore not efficiently captured by inertial impaction after flowing through the inlet of the cassette (no accumulation at the centre). Secondly, the filter in the cassette acts as a flow straightener, enabling a more uniform flow pattern across the filter surface. Finally, for the smallest particles below about 200 nm, the diffusion promotes a homogenization of the particle concentration upstream of the filter once the particles enter the cassette. Even though the distribution of MAMA on the filters shows a much lower surface concentration in certain areas, it is still sufficient to collect and derivatize the MDI as it was generated in this study. This observation cannot be generalized to all isocyanate emission scenarios and raises concerns that aerosols producing a non-uniform concentration on a filter may not be efficiently derivatized if some areas of the filter have a low amount of derivatizing reagent, as this will lead to underestimating the measurement, and consequently underestimating workers' exposure.

Conclusion

Experiments demonstrated that the derivatizing reagents (MAMA, MP, and DBA), when used in the same sampling device, produced no or very little bias in the airborne MDI measurements. Biases between the MAMA-Swin and Impinger (mean 0.3% CI (-3.6, 4.3)), Asset (-4.1% (-9.4, 1.2)) and MAMA-37 (-0.7% CI (-2.9, 1.4)) were not significant over a concentration range of 5 µg/m³ to 60 µg/m³. In terms of agreement, Impinger and MAMA-37 showed limits of agreement of ± 17% when compared with MAMA-Swin, which was within the acceptable range of ±20% defined for the purpose of this study. The limits of agreement for Asset vs MAMA-Swin was (-40, 31)% and therefore above the acceptable range. The authors attributes this non-agreement to Asset measurement higher variability compared to the other three methods. These conclusions are valid for the generated MDI aerosol, which had a MMAD of 250 nm.

The MDI was found at 86% in the backup filter of the impinger. A significant relationship was observed between the proportion of MDI in the filter and MDI concentration level, most likely explained by an increase in the size of MDI particles observed at higher concentrations, therefore leading to a smaller proportion of vapor phase MDI.

On average, 8% of the MDI was found on the denuder section of the Asset sampler. Denuder sections were found to collect 15% to 25% of NaCl particles < 865 nm when challenged on the classic filtration bench test. Therefore, the proportion of MDI collected by the denuder section is most likely due to fine MDI particles and, to a lesser extent, MDI in the vapour phase. The same explanation given above for the Impinger method applies to the significant correlation between MDI concentration and its distribution within the sampler. The MDI proportion found in the denuder section decreased when the airborne MDI concentration increased.

Although up to 7% of the MDI was found on rim and top filters, experiments demonstrated that covering the CFC inner walls with MAMA impregnated filter did not change the overall collection efficiency for the generated airborne MDI. Our study demonstrated that a fine aerosol of pure MDI, a reactive chemical requiring in situ derivatization, is not subject to underestimation originating from inner wall deposition in a polystyrene CFC at a concentration above the most common exposure limit value of 51 µg/m³ for a duration of 30 minutes.

The comparison of the MAMA and MDI spatial distributions on a 37-mm filter enabled the observation of a potential MAMA deficit in specific filter areas. Whereas airborne MDI collected on the filter was uniform, the relative amount of MAMA was twice as much on one side of the filter compared to the other. Although this non-uniformity did not have an effect on the MDI measurements, these results demonstrate that any aerosol producing a non-uniform accumulation on a filter may not be efficiently derivatized if some areas of the filter have a low amount of derivatizing reagent, as this will lead to underestimating the measurement, and consequently underestimating workers' exposure.

Method comparison results combined with additional data from parameters such as particle-size distribution, vapour-particle partition and spatial distributions of collected MDI within a sampling device are useful for understanding potential biases between evaluation methods. This study used an aerosol consisting of fine particles of pure MDI, which sets the basis of comparison for other studies involving more complex isocyanate emissions.

Electronic Supplementary Information (ESI)

Methods

-Chemicals

The MDI (98%); N-methyl-amino-methyl-9 anthracene (MAMA) (98%); dibutylamine; DBA (> 99%); acetone (HPLC grade), and acetic anhydride, AA, (98%) were purchased from Sigma-Aldrich (USA) and were used without further purification. MDI-DBA and the MDI-DBA-d9 standard kits were supplied by Supelco (USA). MAMA-MDI was prepared by Nuchem Therapeutics (Canada). Acetonitrile (ACN), water (H2O), formic acid (FA) (optima grade), and phosphoric acid (H3PO4) (> 99%) were obtained from Fisher Scientific (Canada). Toluene (> 99%) was purchased from EMD Millipore (USA) and dimethylformamide from the J.T. Baker Company (Fisher Scientific, Canada). The glacial acetic and sulfuric (96%) acids, dimethylformamide, triethylamine (HPLC grade), DMF (HPLC grade), and methanol, MeOH (HPLC grade) were obtained from J.T. Baker (Fisher Scientific, Canada).

-Instrumentation

Lab preparation involved the use of an Eberbach shaker (Michigan, USA), a Branson B-52 ultrasonic bath (Danbury, USA), and a Sorvall ST 40R centrifuge (Thermo Scientific, Canada). A Zymark Turbovap LV-ZW700 evaporator was used (Biotage, USA). The two MAMA methods samples were analyzed using an ultra-high-performance liquid chromatographic (UPLC)photodiode array (PDA) system consisting of an LC1290 from Agilent (USA) with a Zorbax Bonus RP 3.0 X 100 mm 1,8 μm (Agilent, USA). Impinger method samples were analyzed using the same instrumentation with a Zorbax Bonus RP 4.6 X 150 mm, 3.5μm (Agilent, USA).

The software used to operate this system and analyze the data was OpenLab CDS, also from Agilent. Asset samples were analyzed using an ultra-high-performance liquid chromatographicmass spectrometry (UPLC-MS/MS) system consisting of a Waters Acquity UPLC coupled with a Waters Xevo TQ triple quadrupole MS (USA) equipped with an electrospray source and an autosampler having a partial loop and a needle overfill feature (10 µL). The column was an Acquity UPLC BEH C18, 1.7 µm, 2.1 mm x 100 mm, from Waters (Santry, Ireland). The software used to operate the system and analyze the data was Masslynx, V4.1, from Waters (USA).

Analysis -MDI-MAMA Methods

Calibration standards with MAMA-MDI derivative were prepared in DMF to cover a concentration range of 0.015 to 0.700 µg of MDI per 2 mL. The mobile phase consisted of trimethylamine (TEA) buffer (30 mL TEA + 940 mL H2O, pH 3 with H3PO4) (eluant A) and ACN (eluant B), respectively. Each sample was desorbed in 2 mL of desorption solution (60 mL TEA buffer + 140 mL ACN and 400 mL DMF). The LC method used an isocratic elution program, 25% A and 75% B, at 1 mL/min, 8 µL sample injection and column kept at 30°C. PDA detection was performed at  = 254 nm. All calibration standards and samples were filtered on 0.22 µm (polytetrafluoroethylene) prior their transfer in 2 mL vials. Run time for each analysis was 8 minutes.

-MAMA (as analyte) method Calibration standards were prepared by dissolving MAMA in ACN in order to obtain concentrations from 2.5 µg/mL to 20 µg/mL. Samples were extracted in 1 mL of ACN and vortexed for a few seconds. The same LC method than the one for MAMA-MDI was applied with the exception of the injection volume of 4 µL and a run time of 4 minutes. All calibration standards and samples were filtered on 0.22 µm prior their transfer in 2 mL vials.

-Impinger method Calibration standards were prepared by mixing, in glass tubes, MDI solutions of various concentration in toluene with MP in toluene at 0,1 mg/mL. The resulting MDI-MP derivative standard solutions, of concentration range 0.05 µg/mL to 0.96 µg/mL, were obtained by evaporating the toluene followed by a re-dissolution in 1 mL of acetic anhydride 0.5% in ACN. The mobile phase consisted of sodium acetate buffer 0.1% in water at pH 6 (acetic acid) (eluant A) and ACN (eluant B), respectively. The LC method used an isocratic elution program, 38% A and 62% B, at 1 mL/min, 20 µL sample injection and column kept at 30°C. PDA detection was performed at  = 250 nm. Run time for each analysis was 8 minutes. Filter samples, already immersed in toluene, and impinger samples were quantitatively transferred in glass tubes and the toluene were evaporated. The samples were then dissolved in 1 mL of acetic anhydride 0.5% in ACN. All calibration standards and samples were filtered on 0.22 µm prior their transfer in 2 mL vials.

-Asset Method Asset standards and samples were prepared according to Halpenny et al. (1) The prepared solutions were injected (10 µL) into the UPLC-MS via its autosampler, set at 15°C. The mobile phase was composed of ACN + 0.1% FA (eluant A) and water + 0.1% FA (eluant B). The run started with a gradient of 70% eluant A (0.5 min.), ramped to 90% eluant A (2.5 min.), held at 90% eluant A (2 min.), ramped to 100% eluant A (3 min.), and finally equilibrated at 70% eluant A (2 min.). The flow rate in the column was 0.6 mL/min. and the temperature was maintained at 50°C. The Xevo TQ was used in positive mode with the capillary voltage set at 2 kV and the source temperature at 150°C. The desolvation temperature and flow were 500°C and 1,000 L/h, respectively, while the collision gas flow was set at 0.15 mL/min. The [M+H]+ for MDI-DBA is m/z 509.3 and that of the internal standard, MDI-DBA-d18, is m/z 527.4. The data were acquired in multiple reaction monitoring (MRM) mode (Table S1). Manual adjustments were made on the integrations to ensure that the entire peak was covered before the data were recorded. The results were used to create a regression calibration curve with linear fit.

Figure S1. B&A plots of intercomparison of the derivatizing reagents, The relative bias (y axis) consists of the difference between the two paired measurements divided by the mean of these two measurements. The MDI concentration (x axis) consists of the mean of the two paired measurements.

Figure 2 .

 2 Figure 2. (A) MDI distribution within the Impinger method sampling train; (B) Change of MDI proportion in the impinger over the airborne MDI concentration NaCl particle collection in the Asset sampler (denuder section)

Figure 3 .

 3 Figure 3. NaCl particle losses, in percentage of particle number concentration, flowing through the denuder section of the Asset sampler, as a function of the particle size (circles = mean (triplicates), error bars = standard deviation).

Figure 4 .Figure 5 .

 45 Figure 4. Asset sampler diagram (A) inlet top view; (B) side view MDI partition within the Asset sampler Figure 5.A presents the relative amounts of MDI found in the four different sections of the Asset sampler. These results confirm the trend observed in Aubin et al. 2021[START_REF] Aubin | Development and characterization of an adaptable aerosolized methylene diphenyl diisocyanate generation system[END_REF], where the partition between the denuder and end filter was 8:92, on average. Correlation tests demonstrated that concentration levels had a significant (R² 0.68, P<0.05), but small, effect on the distribution of MDI in the denuder section (D1+D2). Similar to what was observed in the impinger and its backup filter, a decreasing trend in the proportion of MDI found in the denuder was observed along with the increase of airborne MDI concentration. Correlation tests did not demonstrate any significant effect of sampling duration or weight of MDI collected on the proportion found in the four subsections of the Asset sampler. The effect of the concentration is illustrated in figure5.B, which presents the proportion of MDI retrieved in the denuder (D1 + D2) for two subsets of data for which the MDI concentration levels are different, while sampling duration is the same. The same explanation described above for the MDI distribution in the impinger and backup filter applies, i.e., the increase of MDI particle size diminishes its evaporation and therefore its relative abundance in the denuder section.

Figure 5 .

 5 Figure 5. MDI distribution found within (A) the four sub-sections of the Asset sampler; (B) Denuder over the concentration; (C) Denuder subsections at low concentration; (D) Denuder sub-sections at high concentration (n = 60 for each graph)

Figure 6 .

 6 Figure 6. MDI distribution within MAMA-37 (n = 61)

Figure 7 .

 7 Figure 7. (A) Distribution of impregnated MAMA on 37 mm GFF; (B) Distribution of collected airborne MDI on impregnated MAMA GFF; (C) Sampling pattern. Percentages (A and B) describe the MAMA or MDI distribution (normalized vs the highest value (100%) of mass per circular subsection)

Table 1 .

 1 Summary of sampling and analysis methods for MDI

	Method	Sampling	Reagent(s)	Analysis	LOQ A	Other details
	designation
		GF Denuder, GF
	ISO 17734-1 (Asset)	13 mm (Asset L/min EZ4-NCO), 0.2	Dibutylamine (DBA)	LC-MS	0.42 µg/m³ 0.0025 µg	No field extraction
		Impinger +
	MDHS 25/4 ISO 16702 (Impinger)	backup GF in Swinnex 13 mm, 1 L/min	1,2-methoxyphenylpiperazine (MP)	LC-UV	0.004 µg 0.13 µg/m³	Field extraction in toluene (backup filter)
		GF 37 mm + GF
	MAMA-37	rim and top filters, closed-face cassette, 1 L/min	9-(N-methylaminomethyl)anthracene (MAMA)	LC-UV	0.014 µg 0.47 µg/m³	Field extractionin DMF/ACN/buffer
	MAMA-Swin	GF 13 mm, Swinnex, 1 L/min	9-(N-methylaminomethyl)anthracene (MAMA)	LC-UV	0.014 µg 0.47 µg/m³	Field extraction in DMF/ACN/buffer
	GF: glass fibre filter; LC: liquid chromatography; UV: ultraviolet detection; MS: mass spectrometry; LOQ: limit of quantification,
	expressed in MDI monomer A : LOQ per sample and in air for a sampling time of 30 min.

Table 2 .

 2 Parameters used for GFF impregnation

Type of filters Dimensions (mm) Method Reagent and Concentration (mg/L) Volume transferred (µL)

	Filtering membrane	37	MAMA-37	MAMA 3	500
	Top filter (inner wall)	35	MAMA-37	MAMA 3	425
	Rim filter (inner wall)	6 x 100	MAMA-37	MAMA 3	400
	Filtering membrane	13	MAMA-Swin	MAMA 10	2 x 50
	Filtering membrane	13	Impinger	MP 30	2 x 50
	-Laboratory Analysis
	Details on chemical, instrumentation and sample treatment are described in the electronic
	supplementary information (ESI).

Table 3 .

 3 Description of sample collector sub-sections analyzed separately

	Method designation	Sub-section description
		1) First half of denuder (D1)
	Asset	2) Second half of denuder (D2) 3) End filter (F)
		4) Rinsing of empty D2 + inlet part of cassette (Rinsate)
	Impinger	1) Impinger solution 2) Backup filter
		1) 37 mm end filter
	MAMA-37	2) Rim filter
		3) Top filter
	MAMA-Swin	1) 13 mm end filter 2) Rinsing of inlet part of cassette

Acknowledgements

The authors would like to thank Camille Nicolas-Henry and Asma Daoud for their support in laboratory generation and analyses. We also thank the IRSST for its financial and technical support.

-Quality Control

For each method, all analytical sequences incorporated quality control samples (QCS). For each sequence, the calibration standards were analyzed, followed by a reagent blank (RB), a QC at a value representing 50% of the dynamic range (QC50%) and then followed by the samples. Every 10 samples, the QC50% was analyzed to verify the calibration of the analytical run. At the end of the sequence, the RB, and QC50% were analyzed again, followed by the calibration standards. Each result was reported considering the fact that the QCS were in the acceptable range, which were ± 20% for the QC50. Figure S3. B&A plot of the comparison MAMA-37 vs MAMA-37-single. The relative bias (y axis) consists of the difference between the two paired measurements divided by the mean of these two measurements. The MDI concentration (x axis) consists of the mean of the two paired measurements.