Pauline Audigié

Rouaix-Vande Put

André Malié

Carole Thouron

Daniel Monceau

High-temperature cyclic oxidation of Pt-rich γ-γ' bond-coatings. Part II: Effect of Pt and Al on TBC system lifetime

Keywords:

Three kinds of Pt-rich γ-γ' bond-coating were processed with different contents in Pt and Al. The cyclic oxidation tests performed at 1100 °C on TBC systems showed the superiority of the Pt-rich γ-γ' coatings when compared with the β-(Ni,Pt)Al reference system. TBCs with a Pt-only bond-coating provided the highest performance. Whatever the bond-coating, the failure occurred at the TGO/bond-coating interface which appeared to be the weak point of these γ-γ' bond-coating based systems. Al addition during bond-coating fabrication did not improve the durability. A decrease of 2 μm of electroplated Pt thickness led to a higher performance than the reference systems.

Introduction

Thermal Barrier Coating (TBC) failure characterized by macroscopic spallation of the ceramic yttria partially stabilized zirconia (YSZ) can be caused by thermal and/or mechanical stresses [START_REF] Lipkin | Stress development in alumina scales formes upon oxidation of (111) NiAl single crystals[END_REF][START_REF] Tolpygo | Microstructural study of the theta-alpha transformation in alumina scales formed on nickel-aluminides[END_REF][START_REF] Lipkin | Measurement of the stress in oxide scales formed by oxidation of alumina-forming alloys[END_REF], sulphur segregation [START_REF] Funkenbusch | Reactive Element-Sulfur Interaction and oxide scale adherence[END_REF][START_REF] Hou | Oxide Scale adhesion and impurity segregation at the scale/ metal interface[END_REF][START_REF] Haynes | Characterization of commercial EB-PVD TBC systems with CVD (Ni,Pt)Al bond coatings[END_REF][START_REF] Cadoret | Study of the sulfur segregation on Pt-modified Nickel Aluminides and its influence on the TBC spallation mechanism[END_REF], rumpling [START_REF] Zhang | Martensitic transformation in CVD NiAl and (Ni,Pt)Al bond coatings[END_REF][START_REF] Tolpygo | On the rumpling mechanism in nickel-aluminide coatings. Part II: characterization of surface undulations and bond coat swelling[END_REF][START_REF] Balint | Influence of bond coat thickness on the cyclic rumpling of thermally grown oxides[END_REF][START_REF] Tolpygo | Temperature and cycle-time dependence of rumpling in platinum-modified diffusion aluminide coatings[END_REF][START_REF] Tolpygo | Rumpling of CVD (Ni,Pt)Al Diffusion Coatings under Intermediate Temperature Cycling[END_REF] or even void formation after very-long term exposure at high temperature [START_REF] Evans | The influence of oxides on the performance of advanced gas turbines[END_REF]. For this study, the high temperature oxidation behaviour of systems without top coat was firstly investigated while in a second part, the complete TBC systems were studied. In part I of this study [START_REF] Audigié | High-temperature cyclic oxidation behavior of Pt-rich γ-γ' bond-coatings. Part I: oxidation kinetics of coated AM1 systems after very long-term exposure at 1100°C[END_REF], emphasis was placed upon the cyclic oxidation kinetics of superalloy / coating systems after very long-term exposure at 1100 °C. Pt-rich γ-γ' bond-coatings on AM1 doped in hafnium showed superior oxidation resistance than the reference system with a β-(Ni,Pt)Al coating. Among the Pt-rich γ-γ' tested compositions, the best thermal cycling resistance was observed for Pt + Al γ-γ' coatings prepared from a platinum electroplating and a "short-term" aluminizing step, when compared with Pt-only γ-γ' coatings after 6000 cycles of 1 h at 1100 °C. Aluminium addition during fabrication was thus leading to an improvement of the cyclic oxidation performance of superalloy/coating systems.

In order to corroborate such results to the industrial application, cyclic oxidation performance of complete TBC systems with Pt-only γ-γ' and Pt + Al γ-γ' bond-coatings was investigated. Cyclic oxidation tests at 1100 °C were performed to determine the spallation kinetics and the degradation mechanism of TBC systems with a Pt-rich γ-γ' bondcoating. The effect of coating composition was also studied from five compositions of Pt-rich γ-γ' bond-coatings.

Experimental

AM1 first-generation single-crystal superalloy with a low sulphur content (< 0.4 ppm) and a hafnium doping of about 500 ppmw was used as substrate. The base composition of the AM1 alloy was Ni-12Al-9Cr-7Co-2Ti-3Ta-2W-1Mo (at.%), with 0.12S-570Hf-25Zr-23C (ppmw). Superalloys coupons (2 mm thick, 24 mm of diameter) were grit-blasted with α-Al 2 O 3 particles before deposition. Three main kinds of bondcoatings were fabricated and were named P for Pt-only γ-γ', PA for Pt + Al γ-γ' and B for β-(Ni,Pt)Al as described in the Table 1. Pt-only γγ' coatings were prepared by electroplating 5 ± 2 μm of Pt on AM1 superalloy followed by an annealing for 1 h in vacuum at 1100 °C. Four variants of the Pt + Al γ-γ' coatings were prepared from either 5 ± 2 μm or 7 ± 2 μm of electroplated platinum, a heat treatment for 1 h under vacuum at 1100 °C and a "short-term" aluminizing. This latter corresponds to an interrupted high temperature low activity vapor phase aluminizing (APVS®) treatment. This latter is composed of four steps as cited in [START_REF] Benoist | Microstructure of Pt-modified aluminide coatings on Ni-based superalloys[END_REF]:

• Formation of a gaseous phase in the cement allowing aluminium transport,

• Transport of this gaseous phase to the sample surface, • Chemical reduction on the sample surface with aluminium extrac- tion from the gaseous phase,

• Solid diffusion of deposited aluminium in the sample. https://doi.org/10.1016/j.corsci.2019.01.003 ⁎ Corresponding author. E-mail addresses: audigiep@inta.es (P. Audigié), Carole.thouron@ensiacet.fr (C. Thouron).

The usual aluminizing temperature and time of the APVS® are respectively 1100 °C ± 50 °C and between 4 h and 6 h [START_REF] Benoist | Microstructure of Pt-modified aluminide coatings on Ni-based superalloys[END_REF]. In this study, the aluminizing treatment depended on two main factors: the activator quantity which plays a role in the formation of the gaseous phase and the dwell time at high temperature. These parameters were adjusted in order to deposit the equivalent of a layer of either 5 μm or 2 μm of aluminium. A dwell time of 1 h was applied in both cases. Half quantity of activator than usual APVS® was used to deposit 5 μm of Al while no activator was used to deposit 2 μm. For a detailed description of the "short-term" aluminizing, the reader is referred to [START_REF] Audigié | Modélisation de l'interdiffusion et du comportement en oxydation cyclique de superalliages monocristallins à base de nickel revêtus d'une sous-couche γγ' riche en platine[END_REF]. The Pt layer thickness and equivalent Al thickness were calculated from the mass gain after electroplating and aluminizing step by considering that each deposit was fully dense. Coatings were named with their platinum/ aluminium target equivalent thicknesses. Coatings were hence referred to as the 5/2, 5/5, 7/2 and 7/5 compositions. But the actual Pt and Al equivalent thicknesses were different from the targets and are given in Table 1.

For comparison purpose, conventional TBC systems with β-(Ni,Pt)Al coatings were fabricated from a 7 ± 2 μm thick platinum layer by electroplating, a diffusion heat treatment of 1 h under vacuum at 1100 °C and a conventional vapor phase aluminizing (APVS). For all systems, a ˜150 μm thick 8 wt.% yttria stabilized zirconia top coat was deposited by electron-beam physical vapor deposition (EBPVD) during two different runs. During the run 1, top coat was deposited on both sides of the coupons while only one side was coated during the run 2. This deposition detail did not affect the results of the study because the observed side for samples from both runs was the chamfered one.

Sample designation and fabrication conditions are detailed in Table 1. One sample of each system was characterized after fabrication; those samples were defined as reference and are indicated with an asterisk in Table 1.

Cyclic oxidation tests with cycles composed of a 1 h dwell at 1100 °C and an air forced 15 min cooling down to room temperature were performed on all systems. Samples were removed periodically from the cyclic oxidation rig to be photographed in order to follow spallation kinetics during thermal cycling. Pictures were used to determine the YSZ unspalled area fraction by image analysis. The lifetime criteria was arbitrarily defined as 75% of unspalled area fraction. Thus, samples were removed from the cyclic oxidation tests and examined postmortem. Cross-sections of TBC systems were characterized by scanning electron microscopy (SEM LEO 435 V P) and energy dispersive spectroscopy (IMIX EDS) from real standards in order to determine which led to failure, i.e. to top coat spallation. Both techniques were also used to characterize the reference samples. As a precision, coating chemical compositions were measured by EDS and correspond to the concentrations averaged on all the coating thickness. Rectangular zones of 215 μm wide and the coating thickness high were drawn to measure the composition. Therefore the height of the analyzed window was adjusted depending on the observed microstructure. It is thus noteworthy that the precision of the measure is theoretically lower when a two-phased volume is analyzed when compared with a single-phase volume but this difference was not quantified.

Results and discussion

As-fabricated coatings

The TBC-PA1 and TBC-PA4 reference systems were characterized after fabrication whereas the TBC-PA11 and TBC-PA15 systems were analyzed after 300 cycles at 1100 °C. Fig. 1 corresponds to the backscattered electron images of the cross-sections of these fourth reference systems and shows the typical microstructure of Pt-rich γ-γ' bondcoatings. Residual alumina particles from the grit-blasting marked the original substrate surface. Above the grit-blasting particles, the coating mainly consisted of a mixture of the γ-Ni and γ'-Ni 3 Al phases enriched in Pt. Below the original surface, the coating consisted of large grains of γ' precipitated in a γ-matrix. By considering the lower limit of the large inward growing Pt-rich γ' grains, the thicknesses of the PA1 and PA4 coatings after fabrication were 26.0 ± 2.0 μm and 23.6 ± 2.0 μm, respectively (to compare, the β-(Ni,Pt)Al coating TBC-B3 had an average thickness of 62.6 ± 3.0 μm). Chemical compositions of each bond-coating were determined by EDS as an average on all the coating thickness and are reported in Table 2. Whatever the coating composition, the platinum and aluminium concentrations were lower than the expected target. Aluminium concentration was comprised between 12 and 17 at.% while the platinum one was 19 at.% at the most after fabrication. In addition, voids were observed at the bond-coating / superalloy interface in all the systems. Neither secondary reaction zones (SRZ) nor topologically-close packed (TCP) phases were observed.

After fabrication, the EBPVD top coat had a good adhesion to the bond-coating due to the formation of a thin layer of Al 2 O 3 during TBC processing.

Additional systems were fabricated in a second processing run in order to obtain coatings with larger platinum and aluminium concentrations for Pt + Al γ-γ' systems. These systems were referenced with the "run 2″ tag. These corresponded to the Pt-only γ-γ' coatings (TBC-P1 to P4), some 5/5 Pt + Al coatings (TBC-PA7 to PA10) and some β-(Ni,Pt)Al coatings (TBC-B3 to B5), the latter being considered as the industrial reference system. The microstructure of these three systems after fabrication was similar to the ones of systems without top coat (Fig. 2) [START_REF] Audigié | High-temperature cyclic oxidation behavior of Pt-rich γ-γ' bond-coatings. Part I: oxidation kinetics of coated AM1 systems after very long-term exposure at 1100°C[END_REF]. The average chemical compositions of the γ-γ' zones for the Pt-only (TBC-P1), Pt + Al γ-γ' (TBC-PA7) coatings and of the β layer for the β-(Ni,Pt)Al coating (TBC-B3) are reported in the Table 2.

Table 2

Chemical compositions in at.% of all the reference bond-coatings, in the as-annealed condition for the TBC-P1, TBC-PA1, TBC-PA4, TBC-PA7 and TBC-B3 systems and after 300 cycles at 1100 °C for the TBC-PA11 and TBC-PA15 systems. Platinum contents of the Pt-rich γ-γ' coatings were close to the ones of systems from the run 1 (16.5 to 19 at%). The aluminium concentration varied between 12.7 at% and 17.7 at% and was poorly correlated with the amount of Al diffused in the coatings during the process (Table 1). We observed that the aluminium concentration of the TBC-PA1 system in the as-fabrication conditions was higher than the one of the TBC-PA4 system although this latter theoretically contained less aluminium according to the aluminium equivalent thickness calculations. Indeed, a 1.5 μm aluminium equivalent thickness and a 13.9 at.% of aluminium were estimated for the TBC-PA1 system while a 2.1 μm thickness and 10.9 at.% were obtained for the TBC-PA4. This difference can be explained on one hand by the thermodynamic effect of platinum on the aluminium activity [START_REF] Gleeson | Effects of Platinum on the Interdiffusion and oxidation Behavior of Ni-Al-Based Alloys[END_REF], promoting the very fast uphill aluminium diffusion from the fabrication step [START_REF] Audigié | Observation and modeling of alpha-NiPtAl and Kirkendall void formations during interdiffusion of a Pt coating with a gamma-(Ni-13Al) alloy at high temperature[END_REF]. Cr and Ti concentrations were similar in all γ-γ' coatings, except for the Pt-only γ-γ' coating which had a slightly higher Cr concentration (6.1 at%) than others.

It appears that the different γ-γ' coatings are differentiated by their quantity of diffused Al, and somehow by the quantity of Pt electrodeposited (Table 1), but this is not clearly visible in the average surface composition of the fabricated coatings.

Spallation kinetics

Fig. 3 exhibits the progression of the top coat spallation for one sample of each system during thermal cycling at 1100 °C. Most of the samples showed localized spallation close to the edges due to a geometrical effect ("the edge effect"). Then spallation progressed towards the disc center, which is clearly visible for TBC-PA10 and TBC-PA18 samples.

As demonstrated by the macroscopic pictures, the lifetime of the systems was increased of about 200 cycles when the platinum equivalent thickness increased from 5.3 μm to 6.9 μm whereas the aluminium level remained close. For a similar platinum content, the TBC system lifetime was extended of more than 400 cycles when the aluminium addition increased from 2.1 μm to 5.2 μm.

In order to better identify the platinum and aluminium effects described above, the YSZ unspalled area fraction was plotted versus the number of cycles at 1100 °C (Figs. 4 and5). The maximum number of cycles undergone by each system was also indicated on the figures.

Despite identical conditions for thermal cycling, reference systems with a β-(Ni,Pt)Al coating showed a lifetime dispersion from 600 to 1350 cycles. The whole top coat was spalled for two samples of the run 1 after 1000 cycles while two others arose from the run 2 reached the lifetime criteria after about 1300 cycles. Local analyses were conducted by Raman spectroscopy on the YSZ spalled flakes. The tetragonal and monoclinic phases were detected on the YSZ of the run 1 whereas only tetragonal phase was identified on the YSZ of the run 2. Thus, the lifetime dispersion was attributed to the presence of the monoclinic phase, which is known to be detrimental for thermal cycling resistance [START_REF] Wood | Protective coatings[END_REF]. For this reason, results from run 1 and run 2 were plotted in separate figures.

Looking at systems from the run 1, the 5/2 systems showed the poorest performance with time to failure of 395 cycles (TBC-PA3) and 565 cycles (TBC-PA2). Then one sample of each bond-coating composition (TBC-PA6, TBC-PA12, TBC-PA16) was removed from the cyclic oxidation tests in order to compare the system microstructures after the same number of cycles (565 cycles). Other samples were tested until the lifetime criterion was reached. Two behaviours of the TBC-PA bondcoating systems stood out by comparison with the reference system. On one hand, for lower initial platinum content than in the reference system and for the lowest initial aluminium content, TBC-PA systems were less performing than the reference systems, as demonstrated by the TBC-PA2 and TBC-PA3 samples containing less than 2 μm equivalent thickness of aluminium. On the other hand, for a similar or even lower initial platinum equivalent thickness (5 μm instead of 7 ± 2 μm) but with a sufficient aluminium addition, TBC cyclic oxidation resistance greatly increased and was superior to the one of the conventional systems. Indeed, most of the samples had preserved more than 95% of the zirconia top coat after more than 1000 cycles at 1100 °C. The TBC-PA18 sample provided the longest life span of 1340 cycles of 1 h at 1100 °C. Considering that AM1 contains a great quantity of titanium, this was clearly promising since the time to failure of TBC systems with the same sample geometry, an EBPVD top coat and either a β-(Ni,Pt)Al or a MCrAlY coating was almost never exceeded 1000 cycles at 1100 °C [START_REF] Vande Put | Effect of Pt addition on the cyclic oxidation resistance of NiCoCrAlYTa base TBC systems[END_REF][START_REF] Wu | On the interfacial degradation mechanisms of thermal barrier coating systems: effects of bond coat composition[END_REF][START_REF] Selezneff | Thermal cycling behavior of EBPVD TBC systems deposited on doped Pt-rich gamma-gamma prime bond coatings made by Spark Plasma Sintering (SPS)[END_REF][START_REF] Smialek | Compiled furnace cyclic lives of EB-PVD thermal barrier coatings[END_REF].

Platinum and aluminium additions during fabrication seem to play a key role in the ceramic spallation and influence the TBC lifetime. Globally, the larger the additions were, the longer lifetime was for the γ-γ' bond coatings.

Considering systems from the run 2, TBCs lifetimes increased and even reached time to failure of 1250 and 1365 cycles at 1100 °C for the TBC-B reference systems containing a β-(Ni,Pt)Al coating. All the TBC-P systems with a Pt-rich γ-γ' bond-coating exhibited a better oxidation resistance than the reference systems, except for the TBC-PA8 sample which suffered from desktop spalling (DTS) [START_REF] Smialek | Moisture-Induced Delayed Alumina Scale Spallation on a Ni(Pt)Al Coating[END_REF]. The longest lifetimes were provided by the TBC-P systems with a Pt-only γ-γ' bond-coating with time to failure from 1455 to 2300 cycles, which is an exceptionally Keeping in mind that all the Pt-rich γ-γ' coatings from the run 2 were fabricated with 5 μm of electroplated platinum instead of 7 ± 2 μm in the β-(Ni,Pt)Al coatings, a reduction of 2 μm of electroplated platinum led to a similar or even higher performance than reference systems. However, the beneficial effect of aluminium evidenced with systems from the run 1 was not observed for systems from run 2. This result was thus surprising because the Pt-only γ-γ' coatings in systems without top coat have previously shown that they were an "amplifier" of the detrimental effect of the titanium and tantalum by supporting the formation of the TiTaO 4 oxide and the oxide spallation [START_REF] Audigié | High-temperature cyclic oxidation behavior of Pt-rich γ-γ' bond-coatings. Part I: oxidation kinetics of coated AM1 systems after very long-term exposure at 1100°C[END_REF]. But, this last observation was done after very-long term exposure (more than 3000 cycles). Consequently, according to the present study, aluminium addition during bond-coating fabrication does not enhance TBC performance. Conversely, this work shows that excellent performances of resistance to thermal cycling of TBC systems are obtained with about 5 μm of electroplating Pt, whatever the Al addition, and that the best behaviour is even obtained without Al addition.

The excellent cyclic oxidation resistance of the TBC-P systems could be explained by the presence of voids at the interdiffusion zone / substrate interface. Voids observed after fabrication may prevent the inward platinum diffusion and/or the outward nickel diffusion, this being responsible for the delay of the YSZ spallation. TBC systems can have an excellent cyclic oxidation resistance but a poor mechanical ----,--------.------.-----"T""----..--------

Microstructure a/ter thennal cycling

Comparison of the microstructures of TBC systems after cyclic oxi dation did not reveal any major difference between ail kinds of coat ings. Fig. 6 shows the end-of-life microstructure of the TBC-PA5 sample after 1170 cycles at 11oo•c. Similarly to ail the systems, the PA bond coating had a duplex structure with a continuous single-phase y layer below the TGO and a two-phase y-y' layer with large Pt-rich y' grains. The thicknesses of these two zones were different between ail systems, those depended on the time to failure but also on the initial platinum and aluminium additions. As an example, the y layer of the TBC-PA5 sample was 9.8 :!: 2.0 µm thick after 1170 cycles while the y-y' layer was 42.8 :!: 7.2 µm thick. Besides, voids were also observed at the bond-coating / superalloy interface but in a smaller proportion when compared with the as-received state.

Chemical compositions of the best performing sample of each system were determined by EDS after thermal cycling (Table 3) Failure occurred at the oxide / bond-coating interface in ail cases. Only AhOJ could be detected in the TGO; precisely alumina was ob served at the base of the YSZ columns with a thickness comprised be tween 5 and 7 µm at the failure for ail the systems. Moreover, no rumpling, no TCP phases and no SRZ were observed whatever the composition of the Pt-rich y-y' coatings. These results demonstrated that the oxidation performance of TBC systems with a Pt-rich y-y' coating is mainly predominated by the TGO / y interface. lmproving chemical bounds between these two layers is necessary to enhance oxidation performance.

Influence of the initial chemical composition

To easily compare the oxidation performance and to better visualize the influence of the bond-coating composition, a Pt-Al performance map was plotted (Fig. 7) where the lifetime was represented versus the platinum and aluminium equivalent thicknesses (see Table 1). In sys tems fabricated by spark plasma sintering, Selezneff et al. [START_REF] Selezneff | Thermal cycling behavior of EBPVD TBC systems deposited on doped Pt-rich gamma-gamma prime bond coatings made by Spark Plasma Sintering (SPS)[END_REF] pre viously reported that it exists a critical aluminium level necessary to sustain the growth of the protective alumina scale and to avoid top coat spallation. However, according to the present study, this schema was not confinned since systems without aluminium addition had the best oxidation behaviour. Nevertheless, the Pt-Al map illustrates that a re duction of 2 µm of platinum led to excellent oxidation performance.

Then, in order to correlate the microstructure, the chemical com position and the lifetime of TBCs, SEM observations coupled with EDS analyses were conducted on ail the systems from the run 1. Concentrations of platinum and aluminium after fabrication and thermal cycling were reported in Fig. 8 [START_REF] Audigié | High-temperature cyclic oxidation behavior of Pt-rich γ-γ' bond-coatings. Part I: oxidation kinetics of coated AM1 systems after very long-term exposure at 1100°C[END_REF]). These promising results , in terms of thermal cycling resistance were obtained on a first generation nickel-based superalloy doped in hafnium but containing a large amount of titanium. As the Pt-rich y-y' bond coatings are highly sensitive to the superalloy composition and titanium is known to be detrimental for resistance to cyclic oxidation , the cyclic oxidation resistance of these TBCs could be even improved by using a Ti-free substrate.

Conclusion

The objective of this work • was to study the effect of the bond coating composition on the cyclic oxidation durability of IBC systems with a Pt-rich y-y' bond-coating. Thr ee kind of bond-coatings were processed depending on their platinum and aluminium additions.

The oxidation tests performed on complete TBC s y stems pointed out the superiority of the Pt-rich y-y' coatings when compared with the reference � -(Ni,Pt)Al system. Except for two systems which contained monoclinic phase in the zirconia top coat, all systems had lifetimes superior to 1000 cycles of 1 h at 1100 •c which was very promising when compared with Jiterature data for the sarne superalloy and top coat. In particular, TBCs with a Pt-only y-y' bond-coating provided the highest performance with an exceptional lifetime of up to 2300 cycles For the studied systems of the present work, it was shown that a reduction of 2 μm of electroplated platinum led to higher performance than the reference systems after thermal cycling. Therefore, the reduction of the platinum layer thickness together with the suppression of the aluminizing step allow improved performances at a reduced manufacturing cost.

Whatever the bond-coating, the failure occurred at the TGO / bondcoating interface where the subsurface γ' layer transformed into γ during exposure at high temperature. Improving the adhesion between the oxide and the γ layer acts for the new challenge to enhance oxidation performance of TBCs with a Pt-rich γ-γ' coating.

Fig. 1 .

 1 Fig. 1. Backscattered electron images of metallographic cross-sections of the Pt + Al γ-γ' bond-coatings from the run 1: (a, b) TBC-PA1 and TBC-PA4 in the asannealed condition, (c, d) TBC-PA11 and TBC-PA15 after 300 cycles at 1100 °C.

Fig. 2 .

 2 Fig. 2. Backscattered electron images of the cross-section of the reference bondcoatings from the run 2: (a) TBC-P1, (b) TBC-PA7 and (c) TBC-B3 samples.

Fig. 3 .

 3 Fig. 3. Progression of the top coat spallation for one sample of each system during thermal cycling at 1100 °C.

Fig. 4 .

 4 Fig. 4. Spallation kinetics of systems from run 1 after thermal cycling at 1100 °C.

  Fig. S. Spallation kinetics of sy stems from run 2 after thermal cycling at 1100 •c.

Fig. 6 .

 6 Fig. 6. End-of-life microstructure of the TBC-PAS sampleafter 1170 cycles at 1100 'C (SEM-BSE).

  . The SEM image in the upper right of the figure indicates the zone where concentrations have been measured. Fig. 8 shows that aluminium and platinum concentrations decreased in the coating to finally tend towards the aluminium con centration in the AMl (11.6 at.o/o). This progression was more or Jess rapid depending on the initial platinum and aluminium contents. For instance, the aluminium concentration decreased from 20 at. o/o to 13 at. o/o in 700 cycles for the TBC-PA15 system while it decreased from 14 at. o/o to 12.5 at.o/o in 530 cycles for the TBC-PAl system. Platinum con centrations also decreased more or Jess quickly during thermal cycling

5 Fig. 7 .

 57 Fig. 7. Life span of ail the systems depending on their Pt and Al initial additions (Pt and Al equivalent thicknesses). DTS = Desktop Spalling. Results from Run 2 are underlined.

Fig. 8 .

 8 Fig. 8. Evolution with time of the platinum and aluminium concentrations in the remaining part of the coatings (samples from Run 1).

Table 1

 1 Sample designation and TBC fabrication conditions. In sample ID, P = Pt, PA = Pt + Al and B = β. The asterisk * indicates the sample characterized in the asprepared condition.

	Sample ID	Target coating	Fabrication Conditions			Equivalent Pt (μm)	Equivalent Al (μm)	Run
			Pt	Heat Treatment	Al	Top coat
	TBC-P1 *	Pt-only	5 μm	1h, 1100 °C under vacuum	-	EBPVD	4.8	-	2
	TBC-P2	γ-γ'					4.7	-
	TBC-P3	5/0					4.9	-
	TBC-P4						4.9	-
	TBC-PA1 *	Pt + Al	5 μm		# 1		5.7	1.5	1
		γ-γ'
		5/2
	TBC-PA2						5.6	1.7
	TBC-PA3						5.3	0.8
	TBC-PA4 *	Pt + Al	5 μm		# 2		5.3	2.1	1
		γ-γ'
		5/5
	TBC-PA5						5.3	2.1
	TBC-PA6						5.5	1.5
	TBC-PA7 *	Pt + Al	5 μm		# 3		5.0	7.0	2
		γ-γ'
		5/5
	TBC-PA8						4.6	5.6
	TBC-PA9						4.7	6.4
	TBC-PA10						4.8	5.2
	TBC-PA11 *	Pt + Al	7 μm		# 1		6.9	1.7	1
		γ-γ'
		7/2
	TBC-PA12						6.8	1.7
	TBC-PA13						7.1	2.4
	TBC-PA14						7.3	2.4
	TBC-PA15 *	Pt + Al	7 μm		# 2		7.2	2.9	1
		γ-γ'
		7/5
	TBC-PA16						7.4	2.2
	TBC-PA17						7.0	3.1
	TBC-PA18						6.9	2.2
	TBC-B1	β-(Ni,Pt)Al	7 μm		APVS		7.0		1
	TBC-B2						7.0
	TBC-B3 *	β-(Ni,Pt)Al					6.7	˜22
	TBC-B4						6.6		2
	TBC-B5						6.8

Table 3 Chemical compositions in at.% of the most resistant sample of each system at the failure lime. Sample ID Target Coatlng Ttme at fallure (cycles) Zone

 3

	NI

to finally be close to 4 at.o/o for ail systems when failure occurred. Globally , the failures occurred for all systems for about 4-o ato/o of Pt and 11.7-13.5 at.o/o of Al, i.e. only slightly hlgher of similar than the AMl Al concentration. This value is much higher than the critical Al content (5.9 at.o/o) observed at breakaway for coated AMl without a top coat (first part of this study

Acknowledgements

This study was performed thanks to the financial support of SAFRAN Group. AM1 alloy was furnished by SAFRAN Aircraft Engines. Coatings were done at SAFRAN Aircraft Engines at Châtellerault and Villaroche, France. EBPVD deposits were processed by Ceramic Coating Center, Châtellerault, France.


Jean-Christophe Garrigues
email: garrigues@chimie.ups-tlse.fr

Marion Cournac

Marlène Oswald

Nicolas Ritter

Muriel Blanzat

Stéphanie Cassel
email: cassel@chimie.ups-tlse.fr

Analysis of complex mixtures of polyglycerol fatty esters using liquid chromatography and high-resolution mass spectrometry: Retention, structural and relative composition study

Keywords: HPLC, HRMS, Formulation, Complex matrices, Polyglycerol esters, Green surfactant

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Polyglycerol is a viscous, non-toxic, water-soluble polyether with a high hydrophilic character and excellent biocompatibility. Polyglycerol esters (PGEs), obtained via esterification of fatty acids on polyglycerols, are odourless, biodegradable non-ionic surfactants. They are mainly used in cosmetics, the food industry and pharmaceuticals and they come from renewable resources [1][START_REF] Hemker | Associative structures of polyglycerol esters in food emulsions[END_REF][START_REF] Oppenlaender | Use of mixtures of polyglycerol fatty esters as emulsifiers in cosmetic and pharmaceutical formulations[END_REF]. These products have excellent skin tolerance, better thermal stability than other conventional non-ionic surfactants, and moderate antimicrobial activity. Furthermore in addition to their surface-active properties, the lipophilic part, consisting of a fatty acid, combined with a hydrophilic part based on glycerol, gives them emollient and moisturizing properties [START_REF] Chailloux | Propriétés amphiphiles des tensioactifs de la famille du monolaurate de polyglycérol et application à la préparation de microémulsions à base d'esters gras. Oléagineux[END_REF]. PGEs are considered as green surfactants with promising physicochemical properties.

Polyglycerol can be synthesised from glycidol [START_REF] Mohammadifar | Green Synthesis of Hyperbranched Polyglycerol at Room Temperature[END_REF] but for safety concern glycerol is preferred.

Glycerol is a safe, renewable material generated as a by-product during the biodiesel process [START_REF] Leoneti | Glycerol as a by-product of biodiesel production in Brazil: Alternatives for the useof unrefined glycerol[END_REF]. The polymerisation of glycerol is ideally conducted at a high temperature in the presence of metal hydroxide [7,[START_REF] Babayan | Preparation and properties of some polyglycerol esters of short and medium chain length fatty acids[END_REF] or more selective catalysts [START_REF] Clacens | Selective etherification of glycerol to polyglycerols over impregnated basic MCM-41 type mesoporous catalysts[END_REF][START_REF] García-Sancho | Etherification of glycerol to polyglycerols over MgAl mixed oxides[END_REF]. As previously mentioned, polyglycerol esters are produced from the esterification of fatty acids, derived from triglycerides of vegetable oils, and polyglycerols. Accordingly, polyglycerol esters are made of a complex mixture of compounds depending on the degree of glycerol polymerisation, on the polyglycerol branching isomers and on the number and type of fatty acids that have reacted per molecule of surfactant. It is important to determine the composition of these complex mixtures both qualitatively and quantitatively. Indeed, the properties of PGEs are connected to their composition [START_REF] Curschellas | Characteristics of polyglycerol ester and its different fractions[END_REF]. Various chromatographic techniques are available for analysing PGEs: thinlayer [START_REF] Michael | Metabolism of polyglycerol and polyglycerol esters[END_REF], GC [START_REF] Nosal | Synthesis and characterization of alkyd resins based on Camelinasativa oil and polyglycerol[END_REF], RP-HPLC [START_REF] Kumar | Analysis of polyglycerols by highperformance liquid chromatography[END_REF] and SFC [START_REF] Macka | Analysis of silanised polyglycerols by supercritical fluid chromatography[END_REF]. Specific methods have also been developed by combining GC and HPLC analyses [START_REF] Meulenaer | Combined liquid and gas chromatographic characterisation of polyglycerol fatty acid esters[END_REF] or using stationary phases of porous graphitic carbon stationary phases [START_REF] Cassel | Liquid chromatography of polyglycerol fatty esters and fatty ethers on porous graphitic carbon and octadecyl silica by using evaporative light scattering detection and mass spectrometry[END_REF]. In this study, the complexity of 4 mixtures of PGEs was analysed by direct infusion in a Q-ToF HRMS system. With these structural data, PGEs retention were studied on five stationary phases (one HPLC and four U-HPLC) in reversed mode, in order to optimise methods adapted to the total range of PGE structures. By integrating molecular modelling data, we connected the PGEs' retention factors to specific mono-, di-and tri-esters. These identified structures were then quantified in relative quantity in the 4 mixtures.

Materials and methods

Synthesis

Chemicals

Products used for synthesis: polyglycerol (Spiga spa, Genova, Italy), stearic acid 98% (Mosselman) and technical oleic acid (Berg & Schmidt).

Procedure for PG distillation

Prior to the synthesis, diglycerol was removed by means of short path molecular distillation (UIC GmbH) to increase the content in polyglycerols with a higher degree of polymerisation.

The short path distillation unit (UIC GmbH) was equipped with temperature regulators (Julabo ME4, HT30M1) and connected to a cold trap filled with liquid nitrogen. The distillation conditions were the following: feeding the polyglycerol-6 into the distillation unit at 80°C and 300mL/h, pressure 0.05 mbar, heating mantle set at 200°C and internal condenser at 90°C.

Analytical methods

Chemicals

HPLC-grade acetonitrile (ACN), methanol (MeOH) and formic acid (HCOOH) were obtained from Sigma-Aldrich Co. (St. Quentin Fallavier, France). Water (H2O) was purified using a milli-Q purification system (Millipore, St. Quentin Yvelines, France).

HRMS

Solutions (1 mg.mL -1 ) of samples S1, S2, S3 and S4 were obtained by dissolution in MeOH with 0.1% HCOOH, sonication at 20°C and filtration through 0.2 µm nylon filters (Waters Corporation, Milford, MA, USA). The samples were injected (5 µL) without using a column on a Xevo G 2 QTof (Waters) system, with elution water 0.1% HCOOH/ACN (90%:10% v:v) and flow 0.5 mL.min -1 , capillary voltage ESI+ (3kV), fragmentor potential (30 V), nitrogen ESI (800L.H -1 ), nitrogen cone (20L.H -1 ) and source temperature (110°C). Masslynx was used to process the results. After summation of 1 min of acquisition, all spectra were analysed and compounds identified by comparison with the theoretical mass with a maximum deviation of 3 ppm.

The deviation is calculated as shown below: ∆ppm = (| theoretical massexperimental mass |) / theoretical mass ×1 000 000 2.2.3 HPLC-MS and U-HPLC-MS HPLC and U-HPLC analyses were performed on an Acquity ® UPLC H-Class system composed of a quaternary solvent manager, a sample FTN manager and an Acquity ® QDA detector (Waters Corporation, Milford, MA, USA). Analyses were monitored by Empower 3 software (Waters Co.). Eluents were filtered through 47 mm, 0.45 µm nylon membrane discs (Waters).

HPLC separations were carried out on Aeris Widepore XB-C8, 3.6µm, 2.1x150 mm (Phenomenex, Torrance, CA, USA) and U-HPLC separations were carried out on 4 columns: Acquity CSH C18 1.7µm 2.1x50mm, Acquity CSH Phenyl-Hexyl 1.7µm 2.1x50 mm, Acquity CSH Fluoro-Phenyl 1.7µm 2.1x50mm from Waters Co and Kinetex F5 1.7µm 2.1x100mm (Phenomenex).

For HPLC analysis, a gradient mode was used with all time conditions : (% vol.) t0min: 40% H20, 50% ACN, 10% ACN + 1% HCOOH; t10min: 90% ACN, 10% ACN + 1% HCOOH; Flow 1 mL.min -1 ; the run time was 10 minutes and the column temperature was 25°C. For U-HPLC analysis, all-time conditions were (% vol.) t0min: 50% H20, 40% ACN, 10% ACN + 1% HCOOH; t5min: 90% ACN, 10% ACN + 1% HCOOH; Flow 0.5 mL.min -1 ; the run time was 5 minutes and the column temperature was 35°C. MS analyses were performed with an electrospray ionisation source (ESI) in positive mode with a sampling frequency of 8.8 Hz. The capillary and fragmentor potentials were set respectively to 0.8 kV and 15 V. The QDA analyser worked in full scan mode, and the mass range was set at m/z 145-1250. To identify and analyse each PGE, full-scan survey spectra were used to generate extracted ion chromatograms (XICs) using the [M+Na] + or [M+H] + mass value adjusted at 2 decimals, from theoretical or experimental masses (Table S1 to S4 in supplementary data). For the semi-quantitative analysis of each PGE, the peaks obtained for each XIC were integrated and the relative abundance was calculated by the ratio of each PGE's peak area on the sum of the integrated peaks areas.

Molecular modelling

All PGE structures were optimised using semi-empirical AM1 quantum mechanical calculations in the MOPAC application, using Chem 3D Pro version 14.0 software (CambridgeSoft) with an RMS gradient of 0.100 to minimise energy. The optimised structures were used to calculate the octanol/water partition coefficient (log P) and Connolly molecular area (CMA) with Chem 3D Pro 14.0 software (CambridgeSoft).

Results and discussion

Modelling of structures

Prior to the experiments, modelling was performed for all the structures that could possibly be present. This comprised free polyglycerols (PG), with a degree ranging from 1 to 12, and including possible loss of water molecules, whether because of intrinsic cyclic structures or dehydration processes in the mass spectrometer. Then the same kind of work was done for fatty esters, combining every single free PG with up to 12 fatty chains. Since the analysis of PGEs mixtures by HRMS, HPLC and U-HPLC-MS did not resolve all probable isomers and branched PGs, modelling was limited to linear PGs with 1, 2 or 3 chains of esterified fatty acids in terminal positions for mono and di-esters. For tri-esters models, the 3 rd fatty chain is positioned on the central OH function of the PGs. For PGs and PGEs with water molecule loss, the models incorporate 1 or 2 water loss.

The molecules will be named as follows:

-PGn = free polyglycerol with a polymerisation degree n For the study, we had samples of the mixtures of the free PGs used in the esterification reaction, one centred around PG3 and two around PG6 (either distilled or not).

We had 4 samples of PGEs obtained with different fatty acid mixtures, whose initial reactive compositions are given in Then, for every possible molecule, the molecular formula was determined, and the exact mass at the fourth decimal level was calculated for both hydrogen and sodium adducts (which may form during the synthesis process or in the ESI source).

Possible molecules are modelled and three-dimensional structures minimised, for calculation of the physicochemical parameters log P and Connolly molecular area. These physicochemical parameters will provide information in the structural study on possible formulae, linking the HRMS and experimental retention data obtained using the two chromatographic methods.

HRMS analyses

Once this preliminary work was done, all 4 samples were analysed using high resolution mass spectrometry in order to identify all the molecules present in each sample. Figure 1 shows the ESI+ TOF mass spectrum obtained with sample S3. In all acidified and dissolved methanol samples, [M+H] + and [M+Na] + adducts were detected in ESI positive mode. This can be explained by the ability of the polyglycerol polar-head group to behave as a relatively good chelators for sodium ions, depending on the PG structure. The analysis of these complex HRMS spectra consisted of elemental analysis of the PGEs corresponding to the molecular formulae of the modelled PGEs for signals greater than 10% of the relative intensity.

For S1, (Table S1, supplementary data) obtained from distilled PG6 and C18 fatty acid, esters with a polar head comprising between 3 and 8 glycerol units combined with one or two fatty chains were mainly detected, as expected as this is consistent with the starting materials used.

In this sample, 27 PGEs were found with a relative intensity above 10% for 143 modelled, corresponding to 19% identification score.

For S2, (Table S2, supplementary data) which resulted from the reaction between distilled PG6 and a mixture of fatty acids mainly composed of oleic acid, here again esters with a polar head comprising between 3 and 8 glycerol units esterified by one to up to 3 fatty chains were detected. For compounds that have two or more chains, one cannot distinguish between a combination of 2 C18:1 and 1 C18 + 1 C18:2, for they have the exact same formulas and therefore theoretical mass. Nonetheless, as there is nearly 80% of oleic acid and only less than 2% stearic acid in the starting mixture of fatty acids used to prepare S2, it is statistically most likely that it is the combination of 2 C18:1 fatty chains. In this sample, 33 PGEs were found with a relative intensity above 10% for 327 modelled, corresponding to 10% identification score. S3, supplementary data was obtained from the reaction between non distilled PG6 and a 1:1 mixture of palmitic and stearic acid. As expected, many more compounds can be detected here as diglycerol and other fairly volatile compounds (cyclic ones) were not removed by the distillation step, as in S1 and S2. PG2 to PG7 combined with up to 3 chains can therefore be detected in S3. In sample S3, 59 PGEs were found with a relative intensity above 10% for 150 modelled, corresponding to 39% identification score.

S3 (Table

Finally, S4 (Table S4, supplementary data was obtained by the combination of PG3 with the same fatty acid mixture as for S2, mainly composed of oleic acid. As expected, PG2 to PG5 bearing up to 3 chains can be found in the mixture, with the same uncertainty for compounds having 2 or more chains, whether 2C18:1 or 1C18 + 1C18:2. Same conclusion applies here. .

In sample S4, 32 PGEs were found with a relative intensity above 10% for 327 modelled, corresponding to 10% identification score.

In every sample mixture, non-reacted free PGs can be detected, depending on the starting free PGs mixture, along with the esters.

In all mixtures, no PG with a degree exceeding 12 was found, even at very low level.

No structure with more than 2 water loss was detected.

HPLC-MS

In order to check that the analytical methods ensure the separation and identification of the various classes of PGEs, we chose test compounds, for which the probable developed formulas are given in Figure 2 and optimised structures after AM1 minimisation are given in Figure S1 (supplementary data). The esterification reaction involves the terminal hydroxyl of each PG, as they are more reactive than the other OH groups [START_REF] Marques-Alvarez | Solid catalysts for the synthesis of fatty esters of glycerol, polyglycerols and sorbitol from renewable resources[END_REF]. The terminal positions are therefore chosen in all models to represent the monoesters and di-esters. The test compounds were selected to simplify method development and to verify that they could be separated on the columns used in this study.

These 4 test compounds were identified by HRMS in sample S3 (table S4, supplementary data).

We chose esterified PGEs from PG4, 2 monoesters with a single C16 and a single C18 chain and 2 di-esters with 2 C16 and 2C18 chains. Because the exact structures and positions of the fatty acids are unknown, the formulae given in Figure 2 are only probable structures, in reference to the more reactive terminal hydroxyl groups. We used these four test compounds because they show strong variations in their hydrophobic (log P) and steric (Connolly molecular area) values (Table 3) and enable us to assess the selectivity of the stationary phase and the conditions of gradient on these model PGEs with varied steric and hydrophobicity footprints.

These hydrophobic and steric parameters are used for modelling the reversed-phase retention mechanisms [START_REF] Lafossas | Analysis of the retention of tetracyclines on reversed-phase columns: Chemometrics, design of experiments and quantitative structureproperty relationship (QSPR) study for interpretation and optimization[END_REF] Wide-pore core-shell columns showed good efficiency and peak capacity for the separation of macromolecules [START_REF] Fekete | Evaluation of a new wide pore core-shell material (AerisTM WIDEPORE) and comparison with other existing stationary phases for the analysis of intact proteins[END_REF]. The Aeris™ WIDEPORE XB-C8 3.6 µm silica particles, surrounded by a 0.2 µm porous layer, showed good selectivity in the separation of the 4 test PGEs. The semiquantitative analysis was performed from XIC chromatograms for all PGEs theoretically present in each sample. Tables 4 to 6 present the results for samples S1 to S4 for relative abundances above 1%.

For S1 (Table S5, supplementary data), the major species were PG4, PG5 and PG6 esters containing two C18 chains, accounting for 7.3 to 8.1% (relative abundance) of the mixture.

There was also 5.9% PG3+2 C18. Di-esters accounted for 35.7% of the S1 sample. The majority-identified monoesters were PG3 and PG4 with one C18 chain, at 6.5 and 4.6%

respectively. Other classes of monoesters were found with different PGs with loss of a water molecule: PG3 [1], PG4 [1], PG5 [1] and PG6 [1] associated with one C18 chain. Monoesters accounted for 42.5% of the S1 sample. The remaining 21.8% were made up of 70 free PGs, mono-and di-ester minority species, all identified with this analytical method. All free PGs were found at retention times between 0.20 and 0.35 minutes. Monoesters are found between 0.59 and 1.96 min and di-esters separated between 7.3 and 9.3 min. PG8, PG6, PG5 and PG3 esters with one C18 chain are found with a loss of one water molecule with higher retention times.

For PG8 [1], PG6 [1], PG5 [1] and PG3 [1] esters with one C18 chain, this water loss occurred prior to separation during the esterification reaction. For PG7 [1], PG4 [1], and PG3 [1] ] esters with one C18 chain, This water loss can also occur in the ESI source and the water-loss PGEs are then identified at the same retention time as linear PGEs.

For S2 (Table S6, supplementary data), a very complex mixture obtained from distilled PG6, the analysis showed that the major species were PG3, PG4, PG5 and PG6 esters bearing two alkyl chains (C18, C16, C18:1, C18:2), accounting for 13.5 to 6.9% (relative abundance) of the mixture. The simple quadrupole system resolution does not discriminate isobaric compounds.

The PGE identified with an experimental mass of 791.78 can be attributed to either PG3+1C18+1C18:2, PG3+2C18:1 or PG4+2C16. As the C18:1 fatty acid accounts for nearly 80% of the fatty acid mixture used for the preparation of sample 2, we can only suggest that PG3+2C18:1 may be the major compound having this mass. Di-esters account for 50.1% of sample S2. The majority-identified monoesters were PG3, PG4 and PG4 [1] with one C18:1 chain, at 4.6, 4.1 and 3.0% respectively. Monoesters account for 26.9% of the S2 sample. The remaining 23% made up of 70 free PGs, mono and di-ester minority species. Most of the monoesters and di-esters with one or two identified water loss are found at the same retention time as linear PGEs, this mechanism is linked to the ESI source.

For S3 (Table S7, supplementary data), a complex mixture of non-distilled PG6 with C18 and C16, the analysis showed that the major species were PG4 esters containing one C18 and one C16 alkyl chains and PG3 [1] monoester with one C18 chain, accounting for 6.6 and 6.1%

(relative abundance) of the mixture, respectively. Di-esters account for 46.9 % of the S3 sample.

The majority-identified monoesters comprised all the PGs from PG2 to PG8 [1] with one C18

or one C16 chain. Monoesters accounted for 25.7% of sample S3. The remaining 27.4% were made up of 65 free PGs, mono and di-ester minority species. PG3, PG4, and PG5 esters with one C16 or one C18 chain are found with a loss of one water molecule with higher retention times.This water loss occurred prior to separation during the esterification reaction.

The S4 sample analysis (Table S8

U-HPLC-MS analyses

The first work was to select the suitable column for separating the PGEs according to the chosen gradient. We followed the 4 typical PGEs ( Connoly molecular areas (from 617 to 993), (Table 2). The Acquity fluoro-phenyl column was compatible with the monoester PG6+1C18:1, as analysed with the U-HPLC-MS method. This signal was not detected in the S3 sample. The comparison of the retention time and the molecular calculated parameters log P and Connolly molecular area, can be used to discriminate probable PGEs structure for isobaric ions.

HRMS results confirm the interpretation given with the comparison of the retention time with the calculated values for log P and the Connolly molecular area. In sample S2, the elemental analysis confirmed the probable formulae of PG6+1C18:1 (Table S2, supplementary data), and for S3 the identification of the di-ester PG3 [1]+1C18+1C16 (Table S3, supplementary data).

The development of the two HPLC-MS and U-HPLC-MS methods is complementary in analysing these complex matrices. Some di-esters of the S3 sample, such as PG3 [1]+1C18+1C16, are identified by the U-HPLC MS method (m/z: 749.80, Table S11, supplementary data) and not detected by the HPLC-MS method. These very hydrophobic diesters (log P: 20.9) have a high steric effect with the stationary phase (Connolly molecular area 876.3 Ų) and are therefore not correctly eluted by the HPLC-MS method, whereas this PGE is identified with HRMS (Table S3, m/z: 749.5917, supplementary data). The simple HRMS analysis of the samples, without chromatographic separation, cannot identify some of the less apolar monoesters, such as PG6+1C18:1. This PGE was identified well in sample S2

using HRMS (Table S2, m/z: 749.4655) and was also identified with the HPLC-MS and U-HPLC-MS methods, with a relative abundance ranging from 1.3% to 1.4% (Table S6 and S10, m/z 749.68 and 749.59, supplementary data). PG6+1C18:1 was not identified with HRMS for sample S4 (Table S4, supplementary data), whereas it was found using HPLC-MS and U-HPLC-MS, with a relative abundance of 1.3% to 1.4% with the two methods, for the same sample (Table S8 and S12, m/z: 749.69 and m/z 749.62, supplementary data). It appears that the development of the two chromatographic methods brings complementary resolution to HRMS for the study of these complex matrices.

Both the HPLC and U-HPLC methods can discriminate cyclic PG, generated by ESI reaction or during the polymerisation process, which are found in various PGE structures. For sample 1, PG4+2C18 was the most abundant PGE, identified at an rt of 8.30 min with HPLC (Table S5).

An intramolecular condensation was found with the identification of PG4 [1] + 2C18 at an rt of 9.33 min, related to an increase in hydrophobic and steric interactions with the stationary phase.

The PG3 + 1C18 monoester was identified at an rt of 1.32, and the corresponding cyclic PG3 [1] + 1 C18 was found at the same rt of 1.32, related to ESI-induced dehydration and at rt 1.96, also related to an increase in hydrophobic and steric interactions with the stationary phase (Table S5). The same analysis can be achieved for all samples, for a precise characterisation of these specific classes of cyclic PGEs, in accordance with the previous work of Chaimbault et al. [START_REF] Chaimbault | Direct Analysis of Industrial Oligoglycerols by Liquid Chromatography with Evaporative Light-Scattering Detection and Mass Spectrometry[END_REF] and Crowther et al. [START_REF] Crowther | Electrospray mass spectrometry for characterizing polyglycerols and the effects of adduct ion and cone voltage[END_REF]. The detailed PGEs analysis with one or two water loss is identical to the analysis performed with the U-HPLC method.

Conclusions

The study of the composition of polyglycerol ester mixtures remains a challenge, even after decades of studies, as they display numerous structures that have very close features.

Thanks to the modelling of all possible PG and PGE structures up to 12 PG units and High Resolution Mass Spectrometry (HRMS), we were able to identify the compounds present in the

-

  PG[x] = PG with a loss of x water molecules (maximum 2) -PGn+mCx = PGE with PGn and m linear chains of x carbon atoms, for a set of four possible fatty acids (C16, C18, C18:1 and C18:2) -PGn+mCx/pCy = PGE with PGn and m linear chains of x carbon atoms and p linear chains of y carbon atoms, for a set of four possible fatty acids (C16, C18, C18:1 and C18:2) -[M+H] + = ionic structure of a PGE with a hydrogen adduct -[M+Na] + = ionic structure of a PGE with a sodium adduct

The

  XIC chromatogram of sample S3 (Figure 3) shows the separation of the 4 PGEs from the least hydrophobic (A: log P 4.8) to the most hydrophobic (D: log P 15.2) in less than 10 minutes, with a simple aqueous gradient using ACN as an organic modifier. This XIC chromatogram was obtained by summation of m/z: 557.36, 563.41, 813.60 and 851.65. Each separated chromatograms obtained with the 4 m/z values is given in Figure S2 (supplementary data).

Figure 2 )

 2 , by injecting sample S3 for the 4 study columns with a XIC chromatogram extraction for each compound. With the Acquity CSH C18 column, only PG4[1]+1C16 and PG4[1]+1C18 were identified. With the CSH Phenyl-Hexyl and Kinetex F5 columns, the 2 test monoesters PG4[1]+1C16, PG4[1]+1C18 and one di-ester PG4+2C16 were identified. The XIC chromatogram of sample S3 (Figure 4) shows the separation of the 4 test PGEs on the Acquity fluoro-phenyl column in less than 5 minutes, with a simple aqueous gradient using ACN as an organic modifier. The column selectivity of the Acquity fluoro-phenyl column was adapted to the separation of the test PGEs in a wide range of log Ps (from 4.8 to 15.2) and

4

  different samples provided by the Gattefossé company, and to construct a structural database with HRMS data. HPLC-MS and U-HPLC-MS analyses using a QDA detector (Waters), enabled us to perform PGE separations of PGEs in 10 minutes (HPLC) and 5 minutes (U-HPLC) gradients. The detection produced extracted ion chromatograms (XICs), based on all the possible adducts identified in the HRMS analysis. Using 2 different gradient times and comparing the retention factors and the log P of the different species, we were able to distinguish between different possible isobaric ions and therefore to make a reliable relative quantification of all the PGEs identified in order to compare different synthesised samples with each other.

Table 1

 1

		below.
	Sample	Free PGs	Fatty acids
	S1	Distilled PG6	C18 (98.8%)
	S2	Distilled PG6	C18:1 (79.4%)

Table 1 .

 1 Samples S1 to S4 initial reactive composition.

Table 2

 2 summarises the rough composition of each sample.

	Sample Free PGs	PG esters
	S1	PG3 to PG8 PG3 to PG8 with up to 2 C18 chains
	S2	PG3 to PG6 PG3 to PG7 with up to 3 chains / mixed grafting
			(C16/C18/C18:1/C18:2)
	S3	PG2 to PG8 PG2 to PG7 with up to 3 chains / mixed grafting (C16/C18)
	S4	PG3 to PG4 PG3 to PG5 with up to 3 chains / mixed grafting
			(C16/C18/C18:1/C18:2)

Table 2 .

 2 Samples 1 to 4 composition.

  and are therefore relevant indicators of the structural variability of interactions.

	Table 3. Log P and Connolly molecular area calculated for PG4[1]+1C16, PG4[1]+1C18,
	PG4+2C16, PG4[1]+2C18.
	Compound	Compound	log P	Connolly Molecular Area (Ų)
		code
	PG4[1]+1C16	A	4.8	617.9
	PG4[1]+1C18	B	5.9	654.9
	PG4+2C16	C	12.1	932.7
	PG4[1]+2C18	D	15.2	993.0

  Monoesters accounted for 21.2% of the S4 sample. The remaining 27.4% were made up of 60 free PGs, mono and di-ester minority species. As observed for sample S2, most of the monoesters and di-esters with one or two identified water loss are found in S4 at the same retention time as linear PGEs, this mechanism is linked to the ESI source.

	, supplementary data) showed results close to S2. The major
	species were PG3, PG4 and PG5 di-esters containing C18, C18:1 and C18:2 alkyl chains,
	accounting for 19.3 and 11.5% (relative abundance) of the mixture. Di-esters account for 55.9%
	of the S4 sample. The majority-identified monoesters comprised PG3 and PG4 with one C18:1
	chain.

Acknowledgements

The results of this study were obtained by the "Polyglycerol Ester Characterisation" research project funded by the Gattefossé company.

selected for all identification and semi-quantitative analysis of the PGEs samples, with the U-HPLC method. These analytical conditions gave an equivalent resolution compared to the HPLC method (Figure 3) while dividing the analysis time by 2. This XIC chromatogram was obtained by summation of m/z: 557.36, 563.41, 813.60 and 851.65. Each separated chromatograms obtained with the 4 m/z values is given in Figure S3 (supplementary data).

The semi-quantitative analysis was performed from XIC chromatograms for all PGEs theoretically present in each sample. Tables S9 to S12 summarise the results for samples S1 to S4 for relative abundances above 1%. For sample S1 (Table S9, supplementary data), the U-HPLC method provided identification of 37 compounds with more than 1% relative abundance, which is 24 additional compounds compared to the HPLC method. It is possible with the U-HPLC to identify 6 free PGs (PG3 to PG7) at an rt close to 0.31 min, accounting for between 1.0 and 1.5% of all PGEs and PGs detected. The major species were PG3 and PG4 esters containing two C18 chains, accounting for 6.1 and 5.81% (relative abundance) of the mixture.

All the most abundant di-esters identified by HPLC, from PG3 to PG6, detected and quantified with similar yields. With U-HPLC, di-esters accounted for 36% (35.7% with HPLC), monoesters accounted for 37.1% (42.5% with HPLC) and free PGs were quantified at 6.9% (ND with HPLC) of the S1 sample. The remaining 20% are made up of free PGs, mono and di-ester minority species. For sample S2 (Table S10, supplementary data), the U-HPLC method provided identification of 33 compounds with more than 1% of relative abundance, which is 7 additional compounds compared to the HPLC method. With the U-HPLC analysis, the majority identified species were PG3, PG4, PG5 and PG6 esters containing two alkyl chains (C18, C16, C18:1, C18:2), accounting for between 7.9 and 3.3% (relative abundance) of the mixture. All the most abundant di-esters identified by HPLC, from PG3 to PG6, were detected and quantified with similar yields except for the compound with an experimental mass of 791.71 attributed to