Fagherazzi

M Grabow

V Keryvin

A Marchandise

J.-C Grandidier

C Baley

C Le Guennec

O Fagherazzi

Influence of the manufacturing process on the interlaminar tensile strength of thick unidirectional continuous epoxy/carbon fibre composites

Keywords: B. Delamination, E. Automated fibre placement (AFP), E. Prepreg processing, E. Lay-up (manual/automated), through-thickness tensile strength

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Hydrofoils have been revolutionising yacht racing these last ten years (America's Cup; Vendée Globe, sailing race round the world, solo, non-stop and without assistance) as they lift up boats out of the water, permitting to sail at higher speed over the sea.

They are now usually made with continuous fibre reinforced plastics (CFRP; see Fig. 1).

Those of IMOCA class (60 feet monohulls) weigh 250 kg, are 7 m long and 90 mm thick, while those of Ultim class [START_REF] Charrier | Développement de méthodologies dédiées à l'analyse robuste de la tenue de structures composites sous chargements complexes tridimensionnels[END_REF][START_REF] Petersen | Determination of stresses, strains and failure types in multidirectional laminates under pure bending[END_REF](25)(26)(27)(28)(29)(30)(31)(32) m in length multihulls) weigh 450 kg, are 9 m long and 110 mm thick. In terms of dimensioning such appendices, the interlaminar tensile strength (ILTS), also referred to as through-thickness tensile strength, is one key design material parameter, along with the axial compression strength. The loadings-induced stress is either negative if the hydrofoil undergoes the dynamic lift (compressive state) or positive if the reverse case (tensile state) occurs. In the latter case, when the hydrofoil gets opened and the out-of-plane tensile stress reaches the ILTS, the elbow part fails by delamination. This situation is also at stake in aerospace or aeronautics (spars of centre wing boxes, flange-web transitions. . . ).

The measurement of ILTS of layered composite materials has hence been a topic of high interest these last years [START_REF] Xu | Effect of out-of-plane wrinkles in curved multi-directional carbon/epoxy laminates[END_REF][START_REF] Qian | Effect of gap and overlap fiber placement defects on the delamination behavior of L-shaped composite laminates[END_REF][START_REF] Zhou | Deformation and fracture mechanisms of automated fiber placement pre-preg laminates under out-of-plane tensile loading[END_REF] and several experimental methods exist [START_REF] Makeev | Methods for assessment of interlaminar tensile strength of composite materials[END_REF][START_REF] Nikishkov | Structural analysis of composites with porosity defects based on X-ray computed tomography[END_REF].

Jackson and Ifju [START_REF] Jackson | Through-the-Thickness Tensile Strength of Textile Composites[END_REF] proposed a four-points bending (4PB) test configuration with advantages including a pure bending moment in the curved segment of a L shaped beam specimen or a self-aligning test configuration. The 4PB test configuration was later implemented in the standard [START_REF] Astm D | Standard Test Method for Measuring the Curved Beam Strength of a Fiber-Reinforced Polymer-Matrix Composite[END_REF]. It describes the determination of the ILTS of unidirectional L-beam specimens by 4PB using the analytical solutions of Lekhnitskii [START_REF] Lekhnitskii | Anisotropic plates, Gordon & Breach[END_REF] and Kedward et al. [START_REF] Kedward | Flexure of simply curved composite shapes[END_REF].

Few works compared the mechanical response (other than ILTS) of CFRP with respect to the manufacturing process using prepreg semi-products. McManus and Mak [START_REF] Mcmanus | Strain rate and manufacturing technique effects on the damage tolerance of composite laminates[END_REF] investigated the tensile failure and damage tolerance of (un)notched panel specimens made from graphite/epoxy (IM7/977-2, AS4/938) laminates. The specimens were manufactured by manual tape layup and by automated fibre (tow-preg) placement. Lan et al. [START_REF] Lan | Microstructure and tensile properties of carbon-epoxy laminates produced by automated fibre placement: Influence of a caul plate on the effects of gap and overlap embedded defects[END_REF] studied the influence of gaps and overlaps of CFRP specimens made from a Hexcel prepreg material on the microstructure, and on the tensile properties. The reference specimens were fabricated by hand lay-up and by AFP (automated fibre place-ment) technology. Nguyen et al. [START_REF] Nguyen | Effect of Automated Fiber Placement (AFP) Manufacturing Signature on Mechanical Performance[END_REF] investigated the influence of gaps and overlap imperfections implemented in a controlled manner in flat non-unidirectional laminated specimens made from a Toray T800S/3900 prepreg. Panels were manufactured manually or by AFP.

Hydrofoils are usually manufactured by traditional hand lay-up, and more recently by AFP [START_REF] Marchandise | Impact of manufacturing processes on the critical mechanical performances of a hydrofoil[END_REF] 1 . Advantages and drawbacks of both fabrication methods can be found in Kozaczuk [START_REF] Kozaczuk | Automated fiber placement systems overview[END_REF]. Hand lay-up permits the production of complex and long shapes; expensive equipment is usually not required. The quality depends however on the skills of the operator. Ply cut operations prior to hand lay-up and intermediate debulking lead to long production times. Advantages of AFP are the producibility, the part to part repeatability, a low amount of material waste (no ply cut operations prior to layup), and sometimes a short production time. Furthermore, the robot head permits a constant contact pressure, permitting a uniform tacking of the plies, and a constant ply thickness. Limitations can be found in the application of the mould shapes.; for instance, the geometry of the robot head limits female mould radii. Ply edges at the end of a tape path represent a further drawback. Lan [START_REF] Lan | Étude de l'influence des singularités créées par la technique de placement de fibres automatisé sur les performances des matériaux composites[END_REF] emphasises that further advantages of AFP are the realisation of shapes with single or double curvature, or shapes with high variations in the thickness. Potter [START_REF] Potter | Understanding the origins of defects and variability in composites manufacture[END_REF] reviewed sources of variability in the manufacturing of aircraft composite parts and how this leads to the generation of defects, such as voids, fibre misalignment, cure errors. The comparison between two layup processes from prepreg semi-products, namely AFP and hand lay-up, on the out-ofplane tensile behaviour, is an open question that must be addressed.

In some composite laminates, free edges may give rise to stress concentrations due to high interlaminar stresses and reduce ILTS. In other situations, ILTS might be only slightly reduced as reported by Charrier et al. [START_REF] Charrier | Determination of the out-of-plane tensile strength using four-point bending tests on laminated L-angle specimens with different stacking sequences and total thicknesses[END_REF] comparing unidirectional specimens and laminates. The laminates for masts or hydrofoils in racing yachts have usually 80-90 % of unidirectional plies contrary to aeronautics where quasi-isotropic stackings are widely used. Relatively few studies have focused on thick unidirectional specimens, and yet this knowledge is crucial for the nautical sector. Jackson and Martin [START_REF] Jackson | An Interlaminar Tensile Strength Specimen[END_REF] laid-up 1 To our knowledge, the first hydrofoils made by AFP and used on multi-hulls have been manufactured in 2017 (ETF26 class, see Fig. 1) and 2019 (IMOCA class) by Avel Robotics.

unidirectional AS4/3501-06 carbon/epoxy prepreg plies and observed three delaminations but they had several load drops so that they could not capture the first delamination. No indication on the failure locus was given. Ranz et al. [START_REF] Ranz | Experimental research into interlaminar tensile strength of carbon/epoxy laminated curved beams[END_REF] laid-up unidirectional carbon fabrics and used the resin infusion process. They stated that rupture occurred at nearly one-third of thickness from the inner radius but no pictures of fractured specimens were given. Charrier et al. [START_REF] Charrier | Determination of the out-of-plane tensile strength using four-point bending tests on laminated L-angle specimens with different stacking sequences and total thicknesses[END_REF] tested unidirectional T700GC/M21 carbon/epoxy prepreg plies and observed that fracture occurred at between one-fourth and one-third of thickness from the inner radius but again no pictures of fractured specimens were given.

Thus, the topics the present paper would like to address are threefold. (i) Firstly, to our knowledge, no report has been made on the ILTS of AFP specimens, at least in pure bending. (Qian et al. [START_REF] Qian | Effect of gap and overlap fiber placement defects on the delamination behavior of L-shaped composite laminates[END_REF] mimics the AFP-induced defects on hand lay-up specimens but the samples are not made by AFP.) (ii) Secondly, if several studies report the effect of a given manufacturing parameter on ILTS, no work has been dedicated to the comparison of different prepreg lay-up processes on ILTS. (iii) Lastly, the localisation of delamination is sparsely addressed on (thick) unidirectional specimens.

In this study, the influence of the fabrication process of unidirectional CFRP L-beam thick specimens: hand lay-up of classical nautical quality (MAN) vs. AFP under industrial manufacturing conditions. Both types of specimens are made from the same prepreg material. Since both materials will be cured at the same time in an autoclave, it addresses more specifically the question of the influence of the lay-up process on the response of the L-beam specimen. The strength (ILTS) and the localisation of damage will be the indicators of the process quality, and a discussion about the impact of the manufacturing defects and their origin will put these results in perspective.

Materials and experimental methods

Materials

AFP and MAN L-beam specimens were made at Avel Robotics from 42 unidirectional plies, with a fibre weight of 300 g/m 2 , which were composed of carbon fibres with intermediate modulus IM2C from Hexcel and an epoxy matrix Se84LV from Gurit (glass transition T g ∼ 115 • C, cf. datasheet gurit.com). The material for both L-beam types came from a single prepreg roll with a 34.4 % resin weight content. For the AFP production, the material was slitted in eight spools of 6.35±0.125 mm in width tapes (slittapes). The theoretical ply thickness of the L-beams is 299 µm [START_REF] Gay | Composite materials : design and applications[END_REF].

L-beam manufacturing

The fabrication of the MAN L-beam panel consisted in several steps. 42 plies were first cut from the material prepreg roll. After spyking the plies, as this is a common step in the nautical sector to ensure better resin flow during autoclave curing, the plies were laid-up by hand on an aluminium mould in 0 • -direction. Uniform pressure with the thumbs at the corner of the mould and the gentle use of a spatula on the mould sides were exerted on each ply. Intermediate debulking under vacuum during 20 min at less than 1 bar was applied every 3 plies. The total lay-up processing time of the MAN Lbeam panel was 9 h 20 min.

The manufacturing of the AFP L-beam panel was carried out by laying tapes on an aluminium mould with a C1-Coriolis Composites AFP robot in a one-shot sequence.

Several tapes side by side formed thus one ply. The controlled lay-up contact pressure of the robot head of 5 bar, in connection to the local heating provided by the infra-red lamp (∼35-45 • C) which improves tacking, was assumed to be sufficient to ensure correct adhesion between the plies, and thus to avoid intermediate debulking. The total lay up processing time of the AFP L-beam panel was 1 h 05 min for the 42 plies. Thicknesses of both L-beam panels were then measured after the layup, and before curing, with a calliper. The thickness of the AFP panel was quite homogeneous along the span and width of the plate, with a mean thickness of 12.8 mm, resulting in a mean uncured ply thickness of 305 µm. The thickness of the MAN panel had a slightly higher mean thickness of 13 mm resulting in a mean uncured ply thickness of 309 µm. The thickness of the curved part of the MAN panel was generally slightly lower than that of the arms, and was more irregular through the width of the panel. Accurate measurements of the thicknesses across the span and width of the two panels could not be performed as easily on the uncured panels as on the cured ones (see below). Hence the uncured measurements and their analysis are here presented in a synoptical description, which seemed to be consistent with the measurement accuracy.

Both L-beam panels were cured simultaneously in an autoclave using a polycarbonate caul plate. Thermocouples were applied close to the mould and close to the caul plate side. The maximum pressure and temperature were 7 bar and 120 • C, respectively. Monitoring of the one-shot curing cycle had shown that neither the AFP nor the MAN L-beam panel underwent an exothermic reaction. A teflon layer that was applied on the aluminium moulds before laying up the first ply ensured correct demoulding without any significant panel deforming. Lastly, both panels were cut by mitre saw in approximately 25 mm wide L-beam specimens.

The thicknesses were then measured after curing at nine different points along the specimen thickness t using ImageJ2 software [START_REF] Rueden | ImageJ2: ImageJ for the next generation of scientific image data[END_REF], as depicted in Figure 3, andreported in Table 1. The specimens' cross sections were drawn on a sheet, beforehand. The inner radii r i and the angle between the horizontal and the specimen legs φ i (undeformed state) were also measured. The specimen width w, determined at different points along the width, and length L were measured using a calliper. The outer radius r o was calculated from the sum of inner radius r i = 6.4 mm and specimen thickness t (see Fig. 4).

Experimental test procedure

Four-points bending (4PB) tests on seven AFP and seven MAN L-beam specimens were carried out according to the standard [START_REF] Astm D | Standard Test Method for Measuring the Curved Beam Strength of a Fiber-Reinforced Polymer-Matrix Composite[END_REF]. A schematic of the 4PB test setup is given in Figure 4. The diameter D of the loading bars was 20 mm, the upper and lower distances between the loading bar centres l t and l b were 82.7 mm and 91.5 mm, respectively. The loading bars were mounted with bearing bushes. The 4PB tests were carried out using a universal testing machine (Instron 8803, 50 kN load cell) and the applied force, P, was recorded. The vertical displacement speed was set to 0.5 mm/min.

A linear variable differential transducer (type LVDT) was positioned beneath the specimen and the beam's deflection, u, was recorded. Digital image correlation (DIC, Gom Aramis 4M) was used for most of the L-beam specimens. The front side of the L-beam specimens was therefore sprinkled with black and white spray paint. As sudden specimen failure was expected, acoustic emission sensors (Mistras, R15 sensors) were positioned on some L-beams specimens for confirmation. After failure, specimens were cut by water-cooled diamond sawing and cross-sections in the elbow were observed, after standard polishing procedures, by optical microscopy (Olympus BX51).

2.4. Data analyses 2.4.1. Analytical methods ILTS was estimated according to two solutions proposed in the standard [START_REF] Astm D | Standard Test Method for Measuring the Curved Beam Strength of a Fiber-Reinforced Polymer-Matrix Composite[END_REF]. The solution from Lekhnitskii [START_REF] Lekhnitskii | Anisotropic plates, Gordon & Breach[END_REF] was developed for curved mono-layered anisotropic cylinders subjected to an end moment, assuming plane strain conditions. The solution developed by Kedward [9] is based on the strength of materials.

For both solutions, first is computed the Curved Beam Strength (CBS), corresponding to the moment per unit width at failure, generated in the curved section of the L-beam specimen by the force P b = P 2 acting on the lower loading bars, and the distance l 0 along the specimens' legs (see Fig. 4). The lever arm l 0 is given by:

l 0 = (D + t) tan(φ) + d x cos(φ) , (1)
where d x the horizontal distance between the vertical centreline of one upper and one lower loading bar.

The CBS is hence given by

CBS = P b l 0 w = P 2w cos(φ) d x cos(φ) + (D + t) tan(φ) , (2)
In order to obtain a more accurate estimation of the applied moment, the standard [START_REF] Astm D | Standard Test Method for Measuring the Curved Beam Strength of a Fiber-Reinforced Polymer-Matrix Composite[END_REF] recommends to use φ at failure, and not the initial value φ i = 45 • (experimentally measured), as its value changes during loading:

φ = sin -1   -d x (D + t) + d y d 2 x + d 2 y -D 2 -2Dt -t 2 d 2 x + d 2 y   • (3)
Eq. ( 3) is derived by trigonometric functions [START_REF] Astm D | Standard Test Method for Measuring the Curved Beam Strength of a Fiber-Reinforced Polymer-Matrix Composite[END_REF] with

d y = d x tan(φ i ) + D + t cos(φ i ) -∆, (4)
corresponding to the vertical distance between the horizontal centreline of one upper and one lower loading bar, and ∆ representing the vertical stroke displacement during testing.

The Interlaminar tensile strength (ILTS), defined as the maximum radial stress at failure [START_REF] Astm D | Standard Test Method for Measuring the Curved Beam Strength of a Fiber-Reinforced Polymer-Matrix Composite[END_REF], is then determined according to Lekhnitskii [START_REF] Lekhnitskii | Anisotropic plates, Gordon & Breach[END_REF] (noted ILTS L ) through

ILTS L = - CBS r 2 o g 1 - 1 -ρ κ+1 1 -ρ 2κ r max r o κ-1 - 1 -ρ κ-1 1 -ρ 2κ ρ κ+1 r o r max κ+1 (5) with g = 1 -ρ 2 2 - κ κ + 1 (1 -ρ κ+1 ) 2 1 -ρ 2κ + κρ 2 κ -1 (1 -ρ κ-1 ) 2 1 -ρ 2κ , κ = E θ E r , ρ = r i r o , (6)
and

r max = (1 -ρ κ-1 )(κ + 1)(ρr o ) κ+1 (1 -ρ κ+1 )(κ -1)r -(κ-1) o 1 2κ , (7)
where κ represents an anisotropic parameter, r i and r o the inner and outer radii of the curved segment, r max the position of the maximum radial stress, and E r and E θ the elastic moduli in the radial (out-of-plane) and circumferential (fibre) directions, respectively.

Kedward's approximate solution [START_REF] Kedward | Flexure of simply curved composite shapes[END_REF] (noted ILTS K ) is given by

ILTS K = 3 CBS 2 t √ r i r o , (8)
where √ r i r o corresponds, according to Kedward et al. [START_REF] Kedward | Flexure of simply curved composite shapes[END_REF], to the radial location of the maximum radial stress.

These solutions were implemented into Python scripts and validated with respect to literature results [START_REF] Charrier | Determination of the out-of-plane tensile strength using four-point bending tests on laminated L-angle specimens with different stacking sequences and total thicknesses[END_REF].

Finite Element Analysis

Finite element simulations on each AFP and MAN L-beam specimen were performed in order to calculate the ILTS (referred to as ILTS EF ), as well as the stress fields along the specimen thickness and width in the curved segments of the L-beams. The commercial code Abaqus version 6.14 was used.

The 42 unidirectional plies were created with one linear volume element per ply thickness and ten elements through the width. The mechanical behaviour of the plies were taken as transversely isotropic linear elastic. The elastic properties, similar to those used in Keryvin et al. [START_REF] Keryvin | Determination of the longitudinal non linear elastic behaviour and compressive strength of a CFRP ply by bending tests on laminates[END_REF] for the same material, are given in Table 2. As depicted in Figure 5, the kinematics of the upper loading bars was linked to a reference point which could only move in z-direction, the movements in x-and y-direction being blocked.

The lower bars were clamped. Regarding the composite part, the movement of the loading bars has been limited without constraining the L-beam specimen. In the plane of symmetry, a point has been blocked along x. Two points (one on each arm) were blocked along y to establish contact at the start of the simulation to remove rigid body degrees of freedom. The displacement in z-direction was determined by the contact, which was taken as strict (normal component of the reaction force) and frictionless (tangential component of the reaction force). The mesh was refined until differences of less than 1 % on the load-displacement curves were obtained. The loading bars were taken as rigid after finding that taking into account their deformability (steel loading bars) was of very little influence. A numerical model is built for each sample. After completion of the calculation, the ILTS EF (maximum radial stress) was determined at the experimentally recorded load at failure. The numerical model was validated by comparison to literature results [START_REF] Charrier | Determination of the out-of-plane tensile strength using four-point bending tests on laminated L-angle specimens with different stacking sequences and total thicknesses[END_REF], beforehand.

Results

Specimen metrology

Table 1 shows the mean values of the geometrical parameters for AFP and MAN Lbeam specimens. The mean thickness values of both specimens types after autoclave curing differed only by 2.1 % from each other (12.89 mm for AFP and 13.16 mm for MAN specimens). It was found that for MAN L-beam specimens, the difference between the maximum and minimum thicknesses (averaged by batch) along the L-beam is 1.60 mm, approximately twice as high as that of the AFP L-beams (0.87 mm). MAN L-beam specimens were thinner in the curved part, and thicker in the specimens' arms.

The AFP L-beam specimens have shown constant thicknesses along the span of each specimen, with very few thickness differences in the arm and the curved segment of the specimens. Since the differences in geometry were hardly noticeable before curing and since the curing conditions were exactly the same for AFP and MAN, we can conclude that these subtle differences in geometry were exacerbated during curing.

Reproducibility and bending stiffness

Figure 6 shows the force-displacement curves of the 14 samples. An excellent reproducibility of the test realisation was found for both specimen types. Some non-linearity was observed at the beginning of the curves. This was due to the positioning of the LVDT which was not exactly in the symmetry plane at test beginning. The non-linear phase corresponded to a realignment of the measuring system with the specimen that stops after some time. Sample AFP-7 is an exacerbated example of this situation. Its stiffness, after 1 mm, is nonetheless the same as that of the other samples. Instantaneous failure, characterised by a strong force drop, was observed. This was confirmed by the acoustic sensors. Only data prior to load drop were kept. Table 3 shows the results of the AFP and MAN test campaigns in terms of bending stiffness and failure load. The bending stiffness of AFP specimens was 3742 ± 104 N/mm, that of MAN specimens was 3735 ± 104 N/mm, so no differences were noted.

Comparison analytical and numerical estimations of ILTS

Figure 7 shows the evolution of the out-of-plane tensile stress with respect to the radial position along the thickness or to the transverse position along the width of samples as calculated by FEA. The plane strain analytical estimation of Eq. ( 5) is also plotted. Figure 8 exhibits an example of the experimental and numerical force-displacement diagrams of L-beam specimen AFP-6. A very good correlation is found; the numerical model can be therefore used with confidence.

Table 4 shows the values of ILTS L and ILTS K , estimated through Eq. ( 5) and Eq. ( 8), respectively, as well as the FEM calculated ILTS FE . It was found that Kedward et al.

[9] solution is very close to the numerical calculation. In comparison, the solution of Lekhnitskii [START_REF] Lekhnitskii | Anisotropic plates, Gordon & Breach[END_REF] differed from ∼10-15 % from the numerical results. The somehow surprising better estimation of Kedward et al. [START_REF] Kedward | Flexure of simply curved composite shapes[END_REF] solution can be explained by replicating [START_REF] Kedward | Flexure of simply curved composite shapes[END_REF]Fig. 14] and adding the FE simulation results. Figure 9 depicts then the normalised maximum radial stress of the L-beams as a function of the degree of anisotropy κ and of the specimen geometry. Using κ = E θ Er (Eq. ( 6)) and the material properties given in Table 2 (E θ =E 1 , E r =E 2 ), the coefficient of anisotropy is κ = 4.47. As for the geometrical dimensions, is introduced the ratio Rm t = ri+ro 2t ∼ 1 (for this study), with R m representing the mean radius of the curved L-beam segment. Figure 9 thus shows that the ILTS obtained by Kedward et al. [START_REF] Kedward | Flexure of simply curved composite shapes[END_REF] solution is almost equal to that of FEM, and that the solution of Lekhnitskii [START_REF] Lekhnitskii | Anisotropic plates, Gordon & Breach[END_REF] results in clearly smaller ILTS estimations. Whether the solution of Kedward et al. [START_REF] Kedward | Flexure of simply curved composite shapes[END_REF] or Lekhnitskii [START_REF] Lekhnitskii | Anisotropic plates, Gordon & Breach[END_REF] is closer to the numerical calculation depends hence on the elastic properties of the material. This is not mentioned in the standard [START_REF] Astm D | Standard Test Method for Measuring the Curved Beam Strength of a Fiber-Reinforced Polymer-Matrix Composite[END_REF] that states that less than 2% difference is found between the two analytical estimations, for usual CFRP. This is true for low thicknesses but not for high values as in this study.

The AFP and MAN L-beam specimens are now compared in terms of their loads at failure ILTS. As seen in Fig. 6, AFP L-beam specimens failed at higher loads (6661 ± 427 N ) than MAN ones (3983 ± 740 N), see Table 3. Table 4 reports the ILTS K mean values of both specimens types. The mean ILTS of the MAN specimens was approximately 40.2 % lower than that of AFP specimens. Moreover, the coefficient of variation (ILTS K ) was three times higher for MAN than for AFP specimens (17 % vs 6 %). Theoretical first failure crack locations were determined analytically using Eq. ( 7) (r max ) from Lekhnitskii [START_REF] Lekhnitskii | Anisotropic plates, Gordon & Breach[END_REF]. The relative quantity, γ = rmax-ri t , was estimated to be 0.34 and 0.32 for AFP and MAN L-beam specimens, respectively. The small geometrical differences (see Table 1), had no influence on this number. The variability on the analytical prediction of the location of the first failure crack was ± 0.02, calculated from the ply (∼ 0.3 mm) and the specimen thicknesses, so that the expected failure location should lie between plies 13 and 16. Theoretical first failure crack locations were also determined with the theoretical failure location ( √ r i r 0 ) from Kedward et al. [START_REF] Kedward | Flexure of simply curved composite shapes[END_REF]. Values of 0.37 ± 0.02 and 0.36 ± 0.02, respectively for AFP and MAN, were found for the relative quantity γ. The first failure crack locations r FE numerically calculated were determined at the locus of the highest out-of-plane stress (see Fig. 7), at γ ∼ 0.32, hence equal to the value found analytically.

Location of the first crack

The number of cracks observed on the edges is around 3 for AFP samples and 1 for MAN samples (see Table 3). The experimental first failure crack location r failure of all AFP and MAN specimens are recapitulated in Table 4, in terms of the relative quantity γ. None of the seven MAN specimens had a γ close to the theoretical values (see Table 4), while five out of seven AFP specimens were in this case. AFP-6, respectively, along with associated side views of the edges. For some MAN samples (as in Fig. 11), some localised porosity can be observed between some plies.

These observations are made for samples where the location of failure is different from the estimated one and for which (for instance MAN-4 Fig. 11) the ILTS values are the lowest. For all MAN samples, the crack is straight through the width, so that its location on the face is the same as inside. Actually, the crack always runs on two plies.

Most of its path is intralaminar; it is interlaminar only when areas of porosity are observed. The position of the crack in the center coincide with that on the edges. In sharp contrast, with AFP (see Fig. 12), the localised areas of porosity, when observed, are much smaller than for MAN samples. The crack is translaminar, running through several plies (up to 6). Actually, the crack probably originating from the inside of the sample (the maximum stress is in the middle plane, see Fig. 7) propagates through the thickness and branches many times, so that many cracks reach the free surfaces. The difference between the location of the initiation crack and the positions of the observed cracks on the edges may differ by up to 3 plies in the different samples (see Fig. 12).

This post-mortem method of defects inspection is not comprehensive. The use of microtomography as in Makeev et al. [START_REF] Makeev | Methods for assessment of interlaminar tensile strength of composite materials[END_REF], Nikishkov et al. [START_REF] Nikishkov | Structural analysis of composites with porosity defects based on X-ray computed tomography[END_REF] is not made. Nevertheless, we highlight here the difference in the type of defects and their location and propagation generated by each lay-up condition rather than trying to describe completely the defect geometry.

Discussion

Influence of manufacturing process on geometry and defects

With regard to the production process of the two specimen types, the following similarities and differences can be noted. MAN and AFP L-beam specimens were made from the same prepreg material under industrial manufacturing conditions. They were laid-up on the same aluminium mould and underwent the same curing cycle. Their dimensions highlight that if the mean behaviour of both processes is identical, their specificities induce geometrical differences in terms of regularity. The robot compacted every ply with a lay-up contact pressure of ∼ 5 bar, and the action of its compaction was always perfectly in line with the normal of the mould surface, resulting in constant ply thicknesses. Such a constant pressure could not be obtained by laying-up plies with thumbs and the spatula as this was the case for the hand lay-up fabrication, even if professionally-made in classical nautical yard conditions. MAN L-beam specimens were only compacted under vacuum every 3 plies. The vacuum compaction acted normally to the surface of the prepreg and not normally to the mould. The ply thickness before each intermediate debulking depended on the manual application and was not uniform all over the part. The developed length of the third ply of each sequence was always longer than the developed length of the first and second plies. The first aspect may be responsible for the apparition of surface irregularities on the prepreg plies: waviness due to air rich zones, plies that were not correctly orientated, misleading the direction of the vacuum compaction. The second aspect results in a difference of fibre tension within each 3 plies sequence, as the first ply is applied on a vacuum compacted surface, and the two subsequent plies on an heterogeneously manually compacted surface.

Similar observations concerning the compaction method and the resulting ILTS were made by Charrier [START_REF] Charrier | Développement de méthodologies dédiées à l'analyse robuste de la tenue de structures composites sous chargements complexes tridimensionnels[END_REF] for L-beam specimens with different ply orientations. The authors studied two different specimen types whose major difference was based on the intermediate debulking. They applied intermediate debulking every fourth to sixth ply on the first L-beam type and every second ply on the second L-beam type. They stated that the ILTS of the second L-beam type was 10 % higher than that of the first one.

The compaction irregularity during layup has three consequences. The first one are small geometrical irregularities that resulted from the manual layup. As intermediate debulking is realised at 0.9 bar, and autoclave curing at 7 bar, the impact of the surface irregularities after layup were exacerbated by curing. In comparison, the panel surface after layup by the robot was sufficiently uniform to ensure a compaction normally to the mould during autoclave curing. The caul-plate stiffness was sufficiently high to correct the irregularities on the AFP panel, but not those of the MAN L-beam one. The second consequence is the result of these geometrical irregularities that happened during curing, and can be interpreted as an uncontrolled resin flow generated by an heterogeneous compaction. Some of the resin in the curved segment of the L-beam panel migrated to the arms of the L-beam, resulting in a fibre volume fraction variation through the span of the panel. The resin content has decreased in the curved part of the L-beam panel where a resin/interface based property is measured. The last consequence comes from the initial reason why intermediate debulking is applied during the prepreg layup: avoiding blocked air that can lead to porosity after autoclave curing. The porosity levels are similar (much less than 1% globally) for both MAN and AFP samples. Yet, larger areas of localised porosities between the plies are found in MAN samples compared to AFP samples. A last difference lies in the fact that, during tape deposition for AFP, the previous layer of tapes is reheated by the robot head to ∼ 25 • C. This lowers the viscosity and increases tacking. This strategy is very efficient for increasing adhesion between layers.

It can thus be concluded, that the AFP manufacturing process has a high influence on various parameters such as the specimen geometry and possible induced defects such as porosities, due to key features of AFP: compaction at every ply, and enhanced tacking between layers.

Influence of manufacturing process on failure location and ILTS

For AFP specimens, ILTS values are rather homogeneous and the failure locations are those expected by analytical or numerical estimations. There is no weak area such as large zones of localised porosity, which is rather diffuse and located between the tapes.

The crack path is translaminar, running through different plies and exhibiting many branches. In contrast, the MAN manufacturing process of L-beam specimens with such short radiuses is very intolerant to the smallest layup defects that will vastly affect the dimensional stability of even simple parts. ILTS values are ∼ 40 % lower than the AFP ones and more scattered (17 vs 6 %). This scattering is related to samples for which the ILTS value is low, the failure location different from the estimated one and for which relatively large zones of localised porosity are observed, and from which, most of the time, the crack originates. When ILTS is high, no such zones are observed, so that no "defect" (weak area) is at stake. For all MAN samples, the crack path is straight through the thickness and intralaminar. Still, the maximum value for MAN samples is lower than the minimum value of AFP samples. The enhanced bonding between plies during AFP processing is likely to be responsible for this.

Petersen et al. [START_REF] Petersen | Determination of stresses, strains and failure types in multidirectional laminates under pure bending[END_REF] carried out ILTS measurements following the standard used in this paper on thin (∼ 3 mm) or thick (∼ 8 mm) UD (M21/T700GC). They observed an increase in scatter with higher thickness in addition to a reduction of ILTS: 97 and 80 MPa for thin and thick samples, respectively. They also related a failure locus γ of 0.42 and 0.36 for thin and thick UDs respectively. In comparison to Lekhnitskii's estimation (our calculations), γ is found to be 0.42 and 0.34 for thin and thick UDs respectively. The authors related the scatter with a possible scale effect linked to a larger amount of voids. Charrier et al. [START_REF] Charrier | Determination of the out-of-plane tensile strength using four-point bending tests on laminated L-angle specimens with different stacking sequences and total thicknesses[END_REF] carried out the same measurements on the same material for thicknesses between ∼ 4 and 12 mm and observed no drastic reduction in ILTS: 47 and 40 MPa for thin and thick samples, respectively. However no information on the precise failure location was given apart from between one fourth and one half of the thickness. Makeev et al. [START_REF] Makeev | Methods for assessment of interlaminar tensile strength of composite materials[END_REF] carried out ILTS measurements following the standard used in this paper on 6.3 mm in thickness UD (8552/IM7). They observed a large scatter in ILTS and in the failure locations (γ ∈ [0.24; 0.59]; the theoretical value being 0.37). They linked this variability to the presence of porosity. Indeed, by systematic use of computed tomography (CT) they associated the initiation of the crack to the presence of porosity. They also stated that this standard is not adequate for measuring ILTS and recommended the use of the short beam test. Manufacturing quality thick laminates by hand lay-up is a challenge. The use of ILTS from bending tests on curved beams seems to us more appropriate in a design sequence on curved composite parts such as hydrofoils since it is more representative (partially) of the process quality then by using flat parts, for which the "true" ILTS can be measured.

None of our MAN L-beam specimens did break mechanically, i.e. at the radial position where the out-of-plane tensile stress is the highest, but due to defects and heterogeneities in the material, since porosities are observed. This in complete accordance with the latter studies [START_REF] Makeev | Methods for assessment of interlaminar tensile strength of composite materials[END_REF][START_REF] Charrier | Determination of the out-of-plane tensile strength using four-point bending tests on laminated L-angle specimens with different stacking sequences and total thicknesses[END_REF][START_REF] Petersen | Determination of stresses, strains and failure types in multidirectional laminates under pure bending[END_REF]. The correspondance between experimental and theoretical failure locations in Petersen et al. [START_REF] Petersen | Determination of stresses, strains and failure types in multidirectional laminates under pure bending[END_REF] as well as the much higher ILTS values with respect to those from Charrier et al. [START_REF] Charrier | Determination of the out-of-plane tensile strength using four-point bending tests on laminated L-angle specimens with different stacking sequences and total thicknesses[END_REF] on the same material, in connection with Makeev et al. [START_REF] Makeev | Methods for assessment of interlaminar tensile strength of composite materials[END_REF] observations of porosities, suggests that when there are some differences between estimated and measured failure locations, some defects (in this case porosities) are at stake and responsible for ILTS reduction.

In contrast, five out of seven AFP L-beam specimens broke at a location very close to estimations. For these AFP L-beam specimens it can thus be assumed that defects within the material were sufficiently small to exclude premature specimen failure during loading. The mechanical limit of those specimens could therefore be reached. It can hence be concluded that AFP L-beam specimens are more resistant than MAN ones of this study.

In order to estimate more accurately ILTS, the locations of the first failure cracks (on the edges), r failure , were entered into the FE model, and ILTS FE (r failure ) were calculated. The results are given in Table 4 and summarised in Table 3. Hydrofoils used in racing yachts are thick (up to 120 mm) and have a double curvature, which is less favourable for MAN manufacturing as compared to AFP, and is likely to promote the creation of defects such as porosities. In terms of applications of this study to hydrofoils, let us note that the internal radius of the samples are smaller that those the hydrofoils elbows. The smaller the radius the higher the swelling so that this work maximises this possibility.

Fig- ure 2

 2 depicts the manufacturing process of the L-beam panel.

Figure 10 shows

 10 Figure 10 shows an example of how instrumentation permits to correlate the time and location of the onset of the first failure crack on the edges of a specimen. The sudden drop in force is associated to the occurence of a strain discontinuity (DIC) and a violent acoustic event in terms of energy, localisation and intensity (AE).

Figures 11 and 12

 12 Figures 11 and 12 are post mortem observations by optical microscopy of the crosssections of L-beams in the elbow where the cracks are located, for samples MAN-4 and

  The resulting mean values (67.0±6.6 MPa for AFP and 35.6±5.[START_REF] Makeev | Methods for assessment of interlaminar tensile strength of composite materials[END_REF] MPa for MAN L-beam specimens) show that the difference in ILTS between AFP and MAN specimens increases to 46.8 %.5. ConclusionUnidirectional thick (13 mm) L-beam specimens were fabricated by hand lay-up (MAN) using classical nautical manufacturing conditions and by AFP technology from the same CFRP prepreg material and tested in four-points bending. MAN and AFP specimens were compared in terms of test reproducibility, specimen metrology and material bending stiffness, first failure crack locations, failure loads and Interlaminar TensileStrengths (ILTS).Specimens should theoretically fail at a given estimated specific location from the inner radius. All AFP specimens failed close to the estimated location. Their mechanical limit had therefore been reached. By contrast, none of MAN specimens broke at the estimated location. Defects induced by the manufacturing process are localised porosities.The 47 % lower ILTS values for MAN specimens, their scatter three times higher, as well as these failure locations different from the estimations, indicate that the MAN manufacturing process is more difficult to carry out for thick curved composite parts other than simple plates without inducing defects. Key features of AFP (staggering, compaction every ply, enhanced tacking by local heating) make it possible for composite structures to reach high ILTS values in a reproducible way.

Fig. 1 .

 1 Fig. 1. Hydrofoils manufactured by AFP in January 2017: (left) during sailing with ETF26 class catamaran and (right) after manufacturing (Avel Robotics).

Fig. 2 .Fig. 3 .

 23 Fig. 2. AFP L-beam specimen production. the arrow indicates the direction of the fibres.

Fig. 4 .

 4 Fig. 4. 4PB test setup for ILTS determination.

Fig. 5 .Fig. 6 .Fig. 7 .

 567 Fig. 5. Numerical model of the 4PB on L-beam with boundary conditions.

Fig. 8 .Fig. 9 .ForceFig. 10 .Fig. 11 .Fig. 12 .

 89101112 Fig. 8. Confrontation of experimental/numerical force-displacement diagram of L-beam specimen AFP-06.

Table 1

 1 Metrology mean values of AFP and MAN specimens. w, t, r i , ro, L, Φ i are respectively, the width, thickness, internal and outer radii, arm length and initial half-angle of L-beam. w mean t mean t max,mean r i,mean r o,mean L mean Φ i,mean

	Mean values	[mm] [mm]	-t min,mean [mm]	[mm]	[mm]	[mm]	[ • ]
	AFP	24.68 12.89	0.87	5.99	18.9	110	45
	MAN	25.10 13.16	1.60	6.02	19.2	110	45
	Difference [%]	1.7	2.1		0.5	1.7	0	0

Table 2

 2 Elastic properties of SE84LV/IM2C ply (transverse isotropy)

	E 1	E 2	ν 12 ν 23	G 12	G 23
	[MPa] [MPa] [-]	[-] [MPa] [MPa]
	160000	8000	0.3 0.3	4000	3077

Table 3

 3 Summary of results between AFP and MAN L-beam specimens tested in 4PB.

		AFP	Difference	MAN
	Mean stiffness [N/mm]	3743 ± 104	0.3 %	3735 ± 104
	Mean load at failure [N]	6661 ± 427	40.2 %	3983 ± 740
	Location of first failure crack	5/7 at r max	-	0/7 at r max
	Number of edge cracks	1-6 (mean 3)	-	1
	ILTS FE (r max ) [MPa]	76.7±5.5	43.0 %	43.7±8.0
	ILTS FE (r failure ) [MPa]	67.0±6.6	46.8 %	35.6±5.4

Table 4

 4 First failure cracks locations (r failure , edges). Confrontation of analytical (superscripts K Eq. (8) and L Eq. (5)) and numerical (FE, for rmax, Eq. (7) and (r failure )) ILTS of AFP and MAN L-beam specimens. Numbers in bold indicate that r failure is close to rmax.

	Sample	First failure crack γ = r failure -r i t [-]	ILTS K [MPa]	ILTS L [MPa]	ILTS FE (rmax) ILTS FE (r failure ) [MPa] [MPa]
		1	0.57	79.1	68.9	81.7	58.4
	AFP lay-up	2 3 4 5	0.33 0.36 0.17 0.33	73.4 73.4 66.8 68.2	65.5 65.8 59.4 61.0	77.1 77.0 67.6 67.7	70.0 69.4 57.2 65.2
		6	0.33	79.9	71.1	82.8	75.2
		7	0.36	75.4	67.1	80.9	73.8
	Mean value	73.7±4.6 65.5±3.8	76.7±5.5	67.0±6.6
		1	0.38	57.1	50.8	58.3	42.3
	MAN lay-up	2 3 4 5	0.38 0.50 0.55 0.43	49.0 47.3 41.3 32.0	43.3 41.9 36.9 28.4	47.2 47.2 41.0 31.3	42.0 37.6 30.9 26.7
		6	0.43	44.5	39.4	43.6	37.4
		7	0.19	37.3	32.9	36.9	32.5
	Mean value	44.1±7.6 39.1±6.8	43.7±8.0	35.6±5.4
	Difference AFP -MAN	40.2 %	40.3 %	43.0 %	46.8 %

Acknowledgments

We would like to thank A. Launay and A. Le Palabe (IRDL) for stimulating discussion and to K. Henry (IRMA) and R. Le Borgne (Avel Robotics) for experimental assistance. Région Bretagne and CD56 (project DURAFOIL) are acknowledged for the PhD. grant of M. Grabow.

Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:


Jérémy Molineau

Maria Hideux

Philippe Hennig

Sophie Bertin

Fabien Mauge

Eric Lesellier

Caroline West
email: caroline.west@univ-orleans.fr

Analysis of short-chain bioactive peptides by unified chromatography-electrospray ionization mass spectrometry. Part II. Comparison to reversed-phase ultra-high performance liquid chromatography

Keywords: Peptides, Reversed-phase liquid chromatography, Supercritical fluid chromatography, Unified Chromatography

Introduction

The use of biomolecules as active pharmaceutical ingredients (API) has strongly intensified since the beginning of this century. Among them, peptides are amino-acid oligomers and polymers, with a wide diversity of molecular weights and polarities.

Peptides have been used as APIs since the introduction of insulin for diabetes treatment in the 1920s' [START_REF] Home | Insulin therapy development beyond 100 years[END_REF]. Compared to the small synthetic chemical molecules mostly used in occidental medicine, the low toxicity usually associated with peptide drugs is attractive to favor patient's safety [START_REF] Fazlullah | Toxicity of Biologically Active Peptides and Future Safety Aspects: An Update[END_REF].

Historical perspectives on therapeutic peptides were clearly exposed in previous reviews [START_REF] Uhlig | The emergence of peptides in the pharmaceutical business: From exploration to exploitation[END_REF][START_REF] Lau | Therapeutic peptides: Historical perspectives, current development trends, and future directions[END_REF]. In addition, pharmaceutical peptides of biological or synthetic origin under 5 000 Da and commercialized in northern countries (Europe, Japan and North America) for therapeutic and diagnostic applications are all freely listed in the PepTherDia database (http://peptherdia.herokuapp.com/) [START_REF] D'aloisio | PepTherDia: database and structural composition analysis of approved peptide therapeutics and diagnostics[END_REF]. This database currently comprises around 100 compounds information such as physicochemical and pharmacokinetic properties. Examining this database, it appears that short-chain peptides (between 300 Da and 2000 Da) stand for 75% of commercialized pharmaceutical peptides. Among them, about a third (a quarter of the total) has a molecular weight below 1000 Da. Until the 1990s, most of the therapeutic peptides entering in clinical study were short-chain peptides [START_REF] Lau | Therapeutic peptides: Historical perspectives, current development trends, and future directions[END_REF]. Then, as the synthesis and manufacturing technologies improved [START_REF] Bray | Large-scale manufacture of peptide therapeutics by chemical synthesis[END_REF][START_REF] Thayer | Making peptides at large scale[END_REF], peptides with higher molecular weights were more often described. Short-chain peptides however continued to be developed in a steady fashion.

Reversed-phase liquid chromatography (RPLC) is the most employed method for peptide analysis [START_REF] Yang | Analysis of peptides and protein digests by reversed phase high performance liquid chromatographyelectrospray ionisation mass spectrometry using neutral pH elution conditions[END_REF][START_REF] Peterson | Analysis of RP-HPLC loading conditions for maximizing peptide identifications in shotgun proteomics[END_REF][START_REF] Mant | HPLC Analysis and Purification of Peptides[END_REF][START_REF] Boysen | HPLC of Peptides and Proteins[END_REF] because of its robustness, high-resolution power, short analysis times and compatibility to mass spectrometry (MS). Different authors described RPLC as a flexible and versatile technique, which could also explain its attractiveness for peptides investigations [START_REF] Boysen | HPLC of Peptides and Proteins[END_REF][START_REF] Fekete | New trends in reversed-phase liquid chromatographic separations of therapeutic peptides and proteins: Theory and applications[END_REF]. However, RPLC-UV-MS is not always enough to assess peptide structure and purity as co-elutions may occur, especially with isobaric compounds are isomeric species are frequent in such mixtures. Complementary techniques are always desirable to obtain a more complete picture of a sample [START_REF] Agrawal | Orthogonal Separations in Reversed-Phase Chromatography[END_REF]. Besides, solubilization of the peptides in adequate dilution solvent and carryover effects have been reported as significant issues in RPLC [START_REF] Issaq | Separation, detection and quantitation of peptides by liquid chromatography and capillary electrochromatography[END_REF][START_REF] Maes | Strategies to reduce aspecific adsorption of peptides and proteins in liquid chromatography-mass spectrometry based bioanalyses: An overview[END_REF]. In addition, retention in RPLC is mostly governed by hydrophobic interactions between the analyte and aqueous mobile phase, and by dispersive interactions between hydrophobic residues and hydrophobic stationary phase ligands. As a result, the more polar peptides (typically encountered in short-chain peptides) are not always adequately retained in RPLC chromatographic systems. Polar peptides should be best analyzed with hydrophilic interaction liquid chromatography (HILIC), which is frequently used as an orthogonal method for peptide purification and identification [START_REF] Simon | Evaluation of hydrophilic interaction chromatography (HILIC) versus C18 reversed-phase chromatography for targeted quantification of peptides by mass spectrometry[END_REF][START_REF] Le Maux | Improved short peptide identification using HILIC-MS/MS: Retention time prediction model based on the impact of amino acid position in the peptide sequence[END_REF][START_REF] Peng | Peptidomic analyses: The progress in enrichment and identification of endogenous peptides[END_REF]. The solvent-rich mobile phases employed in HILIC are particularly advantageous for sensitive electrospray ionization (ESI) MS detection [START_REF] Simon | Evaluation of hydrophilic interaction chromatography (HILIC) versus C18 reversed-phase chromatography for targeted quantification of peptides by mass spectrometry[END_REF][START_REF] Periat | Systematic comparison of sensitivity between hydrophilic interaction liquid chromatography and reversed phase liquid chromatography coupled with mass spectrometry[END_REF]. However, in our experience, HILIC methods often lack the sufficient robustness to be suitable for a generic method in the R&D laboratory. Capillary electrophoresis is also a favorite method to analyze therapeutic biomolecules [START_REF] Han | Intact mass analysis of monoclonal antibodies by capillary electrophoresis-Mass spectrometry[END_REF][START_REF] Kaur | Capillary electrophoresis and the biopharmaceutical industry: Therapeutic protein analysis and characterization[END_REF]. However, when purification is desirable, transfer to preparative scale is impossible.

Looking for complementary methods that could cover a wide polarity range for short-chain and long-chain peptides, and could possibly be transferred to preparative scale when needed, Unified Chromatography (UC) appears as an interesting option. Indeed, UC mobile phase gradients may start with a non-polar composition comprising large proportions of pressurized carbon dioxide (usual working range of supercritical fluid chromatography -SFC), and end with a rather polar composition containing large proportions of liquid solvents (usual working range of enhanced fluidity liquid chromatography -EFLC), possibly ending in pure-liquid solvent conditions (LC). This promises the possible analysis of non-polar and polar analytes in a single run [START_REF] Taguchi | Simultaneous analysis for water-and fatsoluble vitamins by a novel single chromatography technique unifying supercritical fluid chromatography and liquid chromatography[END_REF], or makes it possible to propose a single and generic method to analyze both non-polar and polar peptides. Although carbon-dioxide mobile phases are usually perceived as being incapable to elute polar analytes, SFC, EFLC and UC have all been demonstrated to be useful for this purpose [START_REF] West | How Good is SFC for Polar Analytes?[END_REF][START_REF] Molineau | Analysis of flavonoids with unified chromatography-electrospray ionization mass spectrometry-method development and application to compounds of pharmaceutical and cosmetic interest[END_REF][START_REF] Raimbault | Analysis of free amino acids with unified chromatography-mass spectrometry-application to food supplements[END_REF][START_REF] Bennett | Enhanced fluidity liquid chromatography of inulin fructans using ternary solvent strength and selectivity gradients[END_REF][START_REF] Losacco | Metamorphosis of supercritical fluid chromatography: A viable tool for the analysis of polar compounds?[END_REF]. Some examples of peptides and proteins separations by SFC, EFLC or UC appeared in literature recently [START_REF] Ventura | Advantageous use of SFC for separation of crude therapeutic peptides and peptide libraries[END_REF][START_REF] Losacco | Expanding the range of sub/supercritical fluid chromatography: advantageous use of methanesulfonic acid in water-rich modifiers for peptide analysis[END_REF][START_REF] Bennett | Protein separations using enhanced-fluidity liquid chromatography[END_REF].

However, most of the peptides appearing in previous works had molecular weights above 1000 Da, which set different issues in terms of solubility and multiple conformations than short-chain peptides [START_REF] Aguilar | High-resolution reversed-phase high-performance liquid chromatography of peptides and proteins[END_REF]. Further information on the topic of biomolecules analysis with SFC, EFLC and UC can be found in a recent review [START_REF] Molineau | Chromatographic analysis of biomolecules with pressurized carbon dioxide mobile phases -A review[END_REF].

In the first part of this series of papers [START_REF] Molineau | Analysis of short-chain bioactive peptides by unified chromatographyelectrospray ionization mass spectrometry. Part I. Method development[END_REF], a UC method with UV and ESI-MS detection was developed for the purpose of determining the purity of peptide drug candidates, with molecular weights below 1000 Da. The method was developed on a modern instrument included reduced dead volumes, and with a stationary phase based on superficially porous particles offering improved efficiencies, thus the method could be called "ultra-high-performance UC". In this second part, a thorough comparison between this UC-MS method and a reference, previously optimized, reversed-phase ultra-high-performance liquid chromatography -mass spectrometry (RP-UHPLC-MS) method is proposed. Previously, we had conducted a similar comparison between ultra-high-performance SFC-MS and RP-UHPLC-MS for small chemical drugs [START_REF] Lemasson | Comparison of ultra-high performance methods in liquid and supercritical fluid chromatography coupled to electrospray ionization -mass spectrometry for impurity profiling of drug candidates[END_REF] that demonstrated a high level of orthogonality between the two methods. It was therefore of interest to examine the complementarity and compare method performance in more details on these short-chain peptides. For this purpose, 43 peptides comprising 10 linear tripeptides and 33 cyclic pentapeptides were analyzed with both methods. The comparison was focused on performance for the intended application to purity analysis, thereby examined the impurities detection and profile, overall purity comparison, and quality attributes like the symmetry of the main peak and observed peak capacity. Some attention was paid to the separation of isomeric species, which are often encountered in synthetic mixtures of peptides.

Material and methods

Chemicals and solvents

From the 76 peptides used to develop the UC-MS method in the first part of this series [START_REF] Molineau | Analysis of short-chain bioactive peptides by unified chromatographyelectrospray ionization mass spectrometry. Part I. Method development[END_REF], 43 peptides were available in sufficient quantity to ensure reliable method comparison. They comprised 10 linear tripeptides and 33 cyclic pentapeptides. These compounds have confidential structures not exposed in this paper. They are however described by their significant features in Table S1. It can be observed in Figure S1 (supplementary information) that adequate diversity was retained with this shorter selection, as the molecular weights ranged from 392 to 736 g/mol, calculated log P values ranged from -6.9 to 2.9 and topological polar surface area (TPSA) ranged from 130 to 292. These features were determined with Vortex v2019.04.82972.15-s (Dotmatics Limited). The scattering of points according to these significant features can be compared to that of the larger set of peptides used to develop the UC-MS method.

The peptides were provided in dimethylsulfoxide (DMSO) solutions at 10 mM. Some of them having basic residues were present as trifluoroacetic acid salts, as indicated in Table S1. The DMSO solutions were further diluted at 1 mM with (i) methanol (MeOH) for UC analysis and (ii) water/acetonitrile/trifluoroacetic acid 98/2/0.1 (v/v/v) for LC analysis.

For UC analysis, gradient-grade MeOH was purchased from VWR (Fontenaysous-bois, France) and CO2 with a purity of 99,995% was provided by Air Liquide (Bagneux, France). Water was provided by Purelab flex from Veolia (Wissous, France). Ammonium hydroxide solution was obtained from Sigma-Aldrich (Merck, France). For RPLC analysis, HPLC-grade acetonitrile (ACN) was purchased from Merck (VWR international SAS, France), water was obtained from a Milli-Q Purification System from Millipore (Millipore SAS, France) and trifluoroacetic acid (TFA) for spectroscopy was obtained from Merck (VWR International SAS, France).

UC-MS Instrument

The ultra-high performance supercritical fluid chromatography system was a Waters Corporation (Guyancourt, France) ACQUITY Ultra Performance Convergence Chromatography TM (UPC2 ® ) instrument, equipped with a photodiode-array (ACQUITY PDA) detector and an ACQUITY QDa ® single-quadrupole mass detector with electrospray ionization source. Instrument details were provided in the first part of this work [START_REF] Molineau | Analysis of short-chain bioactive peptides by unified chromatographyelectrospray ionization mass spectrometry. Part I. Method development[END_REF]. Empower ® 3 was used for system control, data acquisition and integration of peaks for symmetry and peak capacity measurements.

UC-MS method

The UC-MS method development was previously described [START_REF] Molineau | Analysis of short-chain bioactive peptides by unified chromatographyelectrospray ionization mass spectrometry. Part I. Method development[END_REF]. The stationary phase was Ascentis Express OH5 (150 x 4.6 mm; 2.7 µm) from Sigma Aldrich. The mobile phase was a CO2-cosolvent gradient, with a cosolvent composed of MeOH containing 2% H2O (v/v) and 20 mM NH4OH. Two different methods were used. The first one, referred to as "generic gradient", is the gradient optimized in the first part of this series. The co-solvent gradient starts at 5% and reaches 80% in 10 min, together with a reverse flow-rate gradient from 3.0 to 1.5 ml/min (see details in Table 1a). Total run duration is 15 min. A second gradient, referred to as "focus gradient", was developed in the course of this study, to enhance the resolution of impurities eluting close to the target peptide. The co-solvent gradient is adjusted individually for each target peptide based on its elution composition Ce, corresponding to the proportion of co-solvent necessary to elute the analyte with the generic gradient. A slow gradient is then conducted with a co-solvent proportion varying from Ce -5% to Ce + 5% in 10 min. Before and after this slow gradient, a fast gradient is applied, so the total analysis duration is 15 min. Because the Ce values were all rather large, a constant flow-rate of 1.5 ml/min was used in this case to avoid reaching the pressure limit of the pumping system (414 bar). Details are provided in Table 1b.

For both gradient methods, the back pressure was set at 120 bar, the oven temperature was set at 60°C and the sample compartment temperature at 10°C. UV detection wavelength was set at 210 nm with resolution 1.2 nm. ESI-MS parameters were set as follows: both positive and negative ionization single-ion-recordings (SIR) were observed, together with positive ionization for total ion chromatograms (TIC). The needle temperature was set at 600°C and the cone voltage at 10 V. For positive TIC and SIR acquisition, the capillary voltage was set at 0.3 kV, while it was set at -0.8 kV for negative SIR. Injection volume was 5 µl for every blank and peptide analysis.

When the proportion of co-solvent is low (at the beginning of UC gradient), it is useful to introduce an additional flow of liquid prior to entering the MS. It is however unnecessary when the proportion of co-solvent reaches high values (at the end of UC gradient). Consequently, the MS make-up flow-rate for "large gradient" also applied a reverse gradient from 0.4 to 0 ml/min in 3 min, as previously optimized. However, no make-up fluid was necessary in the case of focus gradients, because all peptides analyzed with a focus gradient were eluted with a large proportion of co-solvent. The composition of make-up fluid was identical to that of the chromatographic co-solvent (MeOH comprising 2% H2O and 20 mM NH4OH).

UHPLC-MS Instrument

The ultra-high-performance liquid chromatography system was an ACQUITY UPLC® I-Class from Waters Corporation. It was equipped with a binary solvent delivery pump compatible with the conditions of the present method (mobile phase flow rate of 0.7 mL/min and pressures up to 528 bar), an autosampler with flowthrough-needle injection system, two 2-position column ovens compatible with 150 mm length columns and a photodiode-array (ACQUITY PDA) detector. The main flow stream was splitted by a CORONA VEO RS splitter (Thermo Scientific) placed before the MS entrance. With this system, the split ratio is about 1/10: only 1/10 of the column flow goes to the MS. ACQUITY TQMQ XEVO® triple-quadrupole mass detector (Waters Corporation) with electrospray ionization source was used for mass spectrometry identification. MassLynx® software (V4.1) was used for system control and data acquisition and Empower®3 for integration of peaks for column efficiency measurements. Waters Data Converter (V2.1) was used to convert data from MassLynx to Empower.

RP-UHPLC-MS method

The RP-UHPLC-MS method serving here as reference has been routinely used at Servier Research Laboratories since 2016 as a generic method to analyze peptides of varied sizes and polarities. The stationary phase was ACQUITY CSH C18 (100 × 2.1 mm, 1.7 µm) from Waters. Mobile phase A was H2O / ACN / TFA 100/1/0.1 (v/v/v) and mobile phase B was ACN / H2O / TFA 100/1/0.1 (v/v/v). Similarly to the UC methods, two different gradient programs were used: a large gradient referred to as "RPLC generic gradient" and a slower gradient focused on the target peptide, individually adapted to each peptide, referred to as "RPLC focus gradient". In the generic gradient, the B solvent proportion was increased from 0 to 60% in 11 min (see details in Table 1c). The total analysis time was 14.3 min. In focus gradient, the B solvent proportion was slowly increased from Ce -3% to Ce +3. Details are provided in Table 1d. Flow-rate was set at 0.7 ml/min in both methods with an oven temperature settled at 70°C and sample temperature at 20°C. Injection volume was held at 1 µl for every blank and peptide analysis. The lower injected volume was necessary to maintain comparable ratios of injected volume to column volume between the UC and RPLC methods (0.3 vs. 0.4%).

UV detection wavelength was set at 210 nm with resolution 1.2 nm. For MS detection, mass to charge ratio between 100 and 1000 Da were recorded with a scan time of 0.2 s. For positive TIC and SIR, acquisition, the MS parameters were set as follows: capillary voltage at 3.8 kV, cone voltage at 30 V, extractor 3 V, source temperature 150°C, desolvation temperature 600°C, cone gas flow 0 L/h, desolvation gas flow 1000 L/h, collision gas flow 0.14 mL/min.

Design of experiment for UC robustness assessment

To assess the method robustness, a Design of Experiments (DoE) with Doehlert matrix was carried out. The DoE was prepared and evaluated with Ellistat software version 6.5 2021/01 (Ellistat, Chavanod, France). Three parameters were considered: back-pressure (ranging from 111.3 to 128.7 bar), column oven temperature (ranging from 51.8 to 68.2°C) and the final co-solvent concentration in gradient elution (ranging from 70 to 90%). The full matrix is presented in supplementary information (Figure S3).

Results & Discussion

Orthogonality of the two methods

Before comparing the performance of the methods, it is useful to examine their orthogonality. Because the UC method was developed on a polar stationary phase (polyhydroxylated ligand), it was expected that elution order would be most different from the elution order obtained in RPLC on an octadecylsiloxane stationary phase. For this purpose, we followed the method proposed by D'Attoma et al. [START_REF] D'attoma | On-line comprehensive two-dimensional separations of charged compounds using reversed-phase high performance liquid chromatography and hydrophilic interaction chromatography. Part I: Orthogonality and practical peak capacity considerations[END_REF] and plotted the peptides eluted with both methods in terms of the mobile phase composition (percentage of organic solvent) at the moment of elution. Considering the regression line and the 95% confidence limits, a polygon is drawn to provide a view of the distribution of target peaks in the retention space. The elution composition was computed according to the method proposed by Fekete et al. [START_REF] Fekete | Method development for the separation of monoclonal antibody charge variants in cation exchange chromatography, Part I: Salt gradient approach[END_REF] using Eq. ( 1):

= + ( ) * ( -) (1)
Where Ce corresponds to the percentage of ACN (for RPLC analysis) and MeOH (for UC analysis) required to elute the peptide; Ci and Cf are the initial and final compositions of the gradient, respectively; tG is the gradient time; tR is the retention time of the peptide and tD is the system dwell time. The UC dwell time was 0.46 min and the RPLC dwell time was 0.51 min.

The plot obtained is presented in Figure 1. Note that less than 43 points are visible on this figure because some analytes eluted with the same elution composition with both techniques (typically, the un-retained analytes in RPLC that were strongly retained in UC).

Firstly, it appears that these small peptides elute very early in the RPLC method, with proportions of organic solvent never exceeding 40%. It must be noted that this method was not specifically purposed for such small peptides but is a generic method used for a variety of peptides with all sizes and polarities. However, small polar molecules are unlikely to be well retained and resolved in RPLC mode, even with an optimized mobile phase. On the contrary, the UC method that was specifically designed for these small peptides shows adequate scattering of the target peaks, with elution compositions varying between 40 and 100% organic solvent. It is also worth noting that the scales of the figure were adjusted to have a better view of the scattering of points. The figure with identical scales is visible in supplementary information Figure S2, where it clearly appears that the points are mostly grouped at the bottom of the figure (low proportion of solvent in RPLC mode). The scattering of linear and cyclic peptides in this figure was also interesting to illustrate that this smaller analyte set (compared to the larger set used in the first part of this study) was however providing sufficient diversity.

Secondly, there appears to be little correlation between the two methods. As could be expected, a negative trend is observed, as those peptides that are less retained in the RPLC method were most retained in UC and vice-versa. The polygon drawn with interrupted lines in Figure 1 is showing the retention space covered by the two techniques, based on 95% confidence limit. Truly orthogonal methods would have a square figure for this polygon, while the present polygon leaves significant areas of white. However, reasonable orthogonality was observed, with a determination coefficient equal to 0.58. This suggests that the two methods will provide adequate complementarity also for the purpose of separating impurities from the main peak.

General chromatographic achievement

The main objective of this study was to compare the performance of the UC and RPLC methods for impurity profiling of short-chain peptide drug candidates. To better understand the purpose of this method, the general context must be explained. In the laboratory where this analysis must be conducted, drug candidates are received from the chemists at an early stage of drug development. At this stage, the possible impurities are unknown and a quick answer is desirable as to the overall purity of the main peak, to decide on the necessity to further purify it. Any information about the number, proportion and identity of impurities is useful and retained for further development. For this purpose, we first considered the success or failure of each analysis method for each of the 43 peptides examined. This achievement was measured based on 3 decision criteria:

(i) First, the peptide needed to be retained in the chromatographic system and eluted within the gradient time frame. In other words, a measurable peak should be observed for the target peptide. In addition, because impurities are expected to be eluted both before and after the main peak, the fact that the main peak elutes with sufficient but not excessive retention should be favorable to the observation of impurities. An elution of the main peptide occurring 1 min after the dead time and initial baseline disturbances was considered as "sufficient" retention. Indeed, the major disturbance caused by DMSO elution occurred at about 2 min in UC, and some baseline disturbances were observed before 2 min in RPLC, thus "sufficient retention" was considered at 2 min in RPLC and 3 min in UC. As no peptide was totally retained on the column, no excessive retention was defined with this set of analytes.

(ii) Secondly, the target peptide should show no co-elution with impurities, based on the examination of UV and MS spectra.

(iii) Finally, the target peak should not be eluted in a chromatographic area where some baseline disturbances occur. Indeed, the presence of additives in the mobile phase, adsorbing on the stationary phase and causing breakthrough waves when the stationary phase is saturated, is responsible for baseline disturbances. In the first part of this study [START_REF] Molineau | Analysis of short-chain bioactive peptides by unified chromatographyelectrospray ionization mass spectrometry. Part I. Method development[END_REF], the UC generic gradient had been particularly optimized to minimize the impact of such baseline disturbances on impurity profiling.

The overall achievement considering these three criteria was controlled a first time based on the generic gradient analyses. In cases of failure, a "focus gradient", individually tailored to each peptide, was applied and the achievement was assessed a second time based on this second, supposedly more resolutive analysis.

The results are presented in Figure 2.

In UC with the generic gradient, all target peptides were eluted and were sufficiently retained to ensure adequate measurement of peak purity. A "successful analysis" was obtained for 81% of the peptides (35 out of 43). The 8 failed cases all resulted from co-elution of impurities. Consequently, they were re-analyzed with tailored focus gradients. After this second analysis, only 3 peptides (7% of the cases) remained as "unsuccessful", as co-eluted impurities could still be detected. In real-life operation, the gradient program would be further optimized to achieve complete resolution of impurities [START_REF] Fekete | Negative gradient slope methods to improve the separation of closely eluting proteins[END_REF], but naturally this would require time and efforts, while the objective here was to compare the methods based on minimal effort.

In RPLC with generic gradient, only 42% of the analyses were successful (18 out of 43). Two types of failure were observed. The first case of failure was due to the absence of retention for 8 target peptides, which eluted in the dead volume, or too early to ensure accurate purity measurement (19% of cases). Considering the large proportion of water already present at the beginning of the generic gradient, changes in the mobile phase gradient are unlikely to solve this issue and a change of stationary phase would be required to improve their retention. The second case of failure was due to co-elution of impurities with the target peptide (39% cases). Again, in this case tailored focus gradients were carried out for each peptide with observed co-elution.

After this second set of experiments, the overall success rate increased to 67% (29 peptides), as 14% of cases (6 peptides) still showed co-elution of impurities.

Clearly, the overall success rate of the UC method on this set of peptides was superior to the success rate of RPLC method. However, an interesting point is that the two methods appeared to be complementary. Indeed, after the whole process of generic and focus gradients, only 1 peptide remained as a "failed case", as it was unretained in RPLC but showed co-elution of impurities in UC. However, this peptide was strongly retained in UC method, thus optimization would still be possible in changing the mobile phase composition or other operating parameters. In conclusion, all peptides in this set except one could be successfully analyzed with either one or the other method. This process is summarized in Figure 2.

A final comment is that the UC method also seems more advantageous as it is less labor-intensive to achieve satisfactory results. Indeed, the total number of experiments carried out in UC was equal to 51 (43 generic gradients plus 8 focus gradients), while the total number of experiments carried out in RPLC was 61 (43 generic gradients plus 18 focus gradients). An overall performance criterium, defined as the successful cases divided by the total number of experiments: would yield the following numbers: 40/51 = 0.78 in UC, and 29/61 = 0.48 in RPLC, which is largely in favor of the UC method.

Performance for impurity profiling

After this first general view on the data, it was necessary to go into more details and assess the methods specifically for the purpose of impurity profiling. For this purpose, 12 peptides (3 linear and 9 cyclic ones) were retained, which were all eluted in a reasonable time (not too early nor too late) with the generic gradients in both methods.

More precisely, the peptides retained for this detailed examination were eluted between 5.7 and 12.4 min in UC, and between 2.1 and 8.1 min in RPLC. Thus the performance of the two methods would be evaluated solely on successful cases.

Purity assessment

A first information that is expected from the method is the overall purity measured for the target peptide, expressed as a percentage of all peak areas integrated, measured on the UV chromatograms. Each chromatogram was compared to blank injection acquired in identical conditions (UC or RPLC, generic or focus gradient) to assign the minor peaks as impurities. All peaks with peak area superior to 0.1% of the major peptide were considered. Supposing that all impurities elute within the chromatographic time frame but are not in dead volume nor are totally retained in the column, the lowest value obtained for the overall purity should indicate the best method, because a lower value means that more impurities were resolved from the main peak. In Figure 3, the values of peak purity are compared for the 12 selected peptides. In most cases (with the one exception being far from the first bisector), they appear to be very similar. As they are distributed both sides of the first bisector, none of the method is consistently better than the other to provide the lowest purity value.

The nature of the peptide (linear or cyclic) seems to have no impact on this measurement.

Detection of impurities

The second information expected is the number and concentration of impurities present in the synthesized peptide. First, we focused on the number of impurities detected with each method, again considering only those peaks with UV peak area superior to 0.1% of the main peptide. The best method should be the one showing the largest number of impurities. In Figure 4a, it appears that, when the number of impurities was low, the two methods were comparable, with a slight advantage to UC.

On the contrary, when the number of impurities was large, the best method was always RPLC. No relation to the nature of peptide (linear or cyclic) was observed in this small set. In the larger set of 43 peptides, more impurities were generally observed in linear peptides than in cyclic ones, possibly because the cyclic peptides are more stable. The largest difference observed was of 10 impurities detected in RPLC versus 1 impurity detected with UC. This most significant difference was observed for the peptide that was the most retained in UC (12.4 min), so it can be supposed that the missing impurities were too strongly retained to be eluted within the gradient time. Perhaps, this is an indication that above 12 min retention in UC, the peptide could be considered as being too much retained to provide meaningful information on purity assessment, but a single case is not enough to be conclusive.

Moreover, to better assess the differences between the two methods, all impurities observed were further categorized depending on their relative peak area with UV detection: abundant impurities (area < 1%) and minor impurities (0.1% < area ≤ 1%).

Impurities below 0.1% were not considered. The results are shown in Figure 4b. It appears that the advantage of the RPLC method seems mostly related to minor impurities. This is consistent with the fact that overall purity measurements did not show much difference between the two methods (Figure 3).

There may be two reasons for the better performance of RPLC method. A first reason is that the resolving power in RPLC would be superior, thus showing more peaks resolved. In UC, these impurities would be either co-eluted with the major peak of the target peptide, or co-eluted with other impurities (thereby reducing the overall number of peaks observed). The impurities might also be eluted in areas where they cannot be measured, very early or very late in the chromatograms, or where a baseline disturbance is complicating accurate measurement. Such cases would be related to the cases in Figure 3 where the measured purity of the target peak was larger in UC than in RPLC (points below the first bisector).

To examine the hypothesis of a higher resolving power in RPLC, peak capacity was measured. The evaluation of peak capacity was carried out on the 12 peptides.

For each of them, the peak width at 50% height was measured. It is interesting to note that peak width in UC showed no relation to retention time and was rather constant (similar to LC), confirming that the strategy of progressively reducing the mobile phase flow rate to better approach the optimum flow rate [START_REF] Molineau | Analysis of flavonoids with unified chromatography-electrospray ionization mass spectrometry-method development and application to compounds of pharmaceutical and cosmetic interest[END_REF][START_REF] Molineau | Analysis of short-chain bioactive peptides by unified chromatographyelectrospray ionization mass spectrometry. Part I. Method development[END_REF] that is decreasing along the gradient [START_REF] Desfontaine | Applicability of supercritical fluid chromatography -mass spectrometry to metabolomics. I -Optimization of separation conditions for the simultaneous analysis of hydrophilic and lipophilic substances[END_REF][START_REF] Losacco | Investigating the use of unconventional temperatures in supercritical fluid chromatography[END_REF] was not detrimental to peak width. In Table 2, the average maximal peak capacity was calculated for both generic gradient methods. A large difference appears, as UC peak capacity (Pc = 189) is significantly lower than RPLC (Pc = 314).

Gradient times were similar (10 vs. 10.9 min) so this significant difference principally issues from the larger peaks observed in UC (about 60% larger, on average). To a certain extent, this may be related to the different column and particle formats employed in this study as the RPLC method was done with sub-2 µm fully porous particles, while the UC method was done with sub-3 µm superficially porous particles.

This observation is also in line with previous studies comparing RPLC to SFC analysis of small chemical drugs on sub-2 µm fully porous particles [START_REF] Lemasson | Comparison of ultra-high performance methods in liquid and supercritical fluid chromatography coupled to electrospray ionization -mass spectrometry for impurity profiling of drug candidates[END_REF], where the measured efficiency was higher in RPLC than SFC. It is also possible that the slow interaction kinetics due to polar interactions in UC would cause larger band broadening than the hydrophobic interactions in RPLC. It is also worth mentioning that peak asymmetry was not responsible for the larger peaks observed in UC, as the average peak symmetry in this set was AsEP = 1.10, slightly better than the average 1.23 measured in LC. In general, it was observed that the peak symmetry on the main peak was better in UC chromatograms than in LC chromatograms, where peak asymmetry frequently exceeded 1.2. In UC chromatograms, asymmetry was slightly worse for late-eluting peaks than for early-eluting peaks.

A second reason that could explain the superior performance of the RPLC method is that the sensitivity would be higher, which should principally show in the number of minor impurities detected. Indeed, as appears in Figure 4b, the increased number of impurities observed in RPLC was essentially due to minor impurities (0.1% < area ≤ 1%). For the set of 12 well-eluted peptides, the UV signal-to-noise ratio was higher in RPLC than in UC. Independently of the UV detector performance, this is consistent with larger peaks observed in UC. This sensitivity issue is also well known by SFC-UV chromatographers. Indeed, although modern instruments have largely improved sensitivity compared to older instruments, it is still not as good as comparable instruments in liquid conditions. This is partly inherent to the way the system is working: pressure regulation causes a constant variation of fluid density, which is causing a constant variation of refractive index, thus causing baseline noise in UV [START_REF] Berger | Minimizing ultraviolet noise due to mis-matches between detector flow cell and post column mobile phase temperatures in supercritical fluid chromatography: Effect of flow cell design[END_REF].

Baseline compensation is somewhat improving this problem but not enough to reach the sensitivity of LC systems.

Impurity profiling

The 12 peptides were investigated in great details on their impurity profiles, trying to match the information from both methods based on peak area and MS spectra. Two representative examples are shown in Figure 5 with a linear peptide (Figure 5a) and a cyclic peptide (Figure 5b). In these figures, the main peptide is identified with a red square, and was of course detected with both methods. The impurities that were observed with both methods and could be matched are identified with yellow circles.

The impurities that were observed with only one method are identified with green triangles. On this figure, they are placed on the right and top of the figure to indicate that they were seen with UC (abscissa) or with RPLC (ordinates) only.

For the linear peptide, 4 impurities were observed with both methods (yellow circles). In addition, 1 impurity was observed with UC only (green triangle at the bottom), while 4 impurities were observed with RPLC only (green triangles on the left).

It also appears that the impurities were eluted over a wider time frame in UC than in RPLC. This is consistent with the above observation that retention times of the target peptides in UC were scattered in a wider time frame than in RPLC (Figure 1).

For the cyclic peptide, 6 impurities were observed with both methods (yellow circles). In addition, 3 impurities were observed with UC only (green triangles at the bottom), while 5 impurities were observed with RPLC only (green triangles on the left).

For this sample, some impurities appeared at the end of the UC gradient that were difficult to identify. As the proportion of co-solvent is high at this point, MS sensitivity is somewhat less than at the beginning of the gradient. Further improvement of MS parameters might improve identification of late-eluting impurities.

Among the 12 cases examined in detail, the complementarity of the two techniques was evident. While the RPLC method often showed more minor impurities than the UC method, the two methods mostly provided different information.

Resolution of isomers

The separation of isomeric species is of interest as isomeric impurities are often present in synthesized peptides. In the set of 43 peptides that could be analyzed with both UC and RPLC methods, no isomeric pairs were present for the linear peptides, but 10 isomeric pairs or trios were present for cyclic peptides. When enough retention could be obtained in RPLC, the resolution of isomeric pairs was generally good with RPLC method. In UC, where all peptides were eluted in an adequate retention time, some isomeric pairs were well resolved while others remained co-eluted. Because the set of compounds was very small for this comparison, it is impossible to draw any conclusion as to the reason for success or failure of isomers separation in one or the other method. Sample chromatograms are presented in Figure 6, to illustrate the different capabilities for isomer separation. in Figure 6a, a case where UC offered superior resolution, is observed with positional isomers: CP 18791 was -R1-R2-R3-R4-R5-while CP 18787 was -R1-R4-R3-R2-R5-, i.e. the positions of R2 and R4 were exchanged. In Figure 6b a case where RPLC offered superior resolution is observed with a diastereomeric pair, where CP 19846 was -R1-(D-R2)-R3-R4-R5-and CP 19850 was -R1-(L-R2)-R3-R4-R5-, i.e. one amino acid had opposite absolute configuration in the two peptides (D/L). Figure 6 also shows that the two techniques provided comparable chromatographic quality, as previously mentioned when comparing significant features. Finally, only one isomeric pair could not be resolved with any of the two methods, again supporting the high complementarity of the UC and RPLC methods.

Robustness

The robustness of the well-established LC method was not assessed. The UC method, however, required robustness assessment as it was little explored so far. For this purpose, a Doehlert design of experiment [START_REF] Ferreira | Doehlert matrix: a chemometric tool for analytical chemistry-review[END_REF] was achieved with three variable parameters: back-pressure, column temperature and final gradient composition. The limit values of the three parameters were voluntarily chosen to be large (20 bars, 20°C and 20 % of co-solvent). Indeed, they are much larger than what would occur in normal operation of the instrument, where the accepted variations of instrument control are about 2 bar, 0.5°C and 2% respectively for back-pressure, temperature and co-solvent proportion. The DoE matrix is detailed in in Figure S3 (supplementary information). Retention factor for one peptide eluting about mid-gradient (with retention time of about 7 min) was examined to assess its variability with these three parameters. The results are presented in Figure 7 and Table S2. The model was wellfitted, as appears in Figure S3 with R 2 = 0.982 and a confidence interval of 0.2.

Unsurprisingly, the most influent parameter was the solvent proportion, while backpressure and temperature were found to be insignificant. Indeed, the large proportion of co-solvent along the gradient and particularly at the end of the gradient make it a low-compressibility fluid, on which pressure and temperature can have only little impact. Whatever the fluid density and compressibility, the effect of changing mobile phase composition is still significant as it will impact the elution strength through changes in polarity, similar to what would be observed in liquid chromatography.

However, this impact is largely acceptable as it appears that the normal range of variation that is considered acceptable in instrument control (typically less than 2% variation in the proportion of co-solvent) would yield a variation of retention factor of about 0.1. This corresponds to a variation in retention time of about 0.06 min. As a result and taking account of the instrument reliability, the method looks sufficiently robust to be applied as a generic method in the research and development context.

Operational cost comparison

Finally, comparing the operational cost of the two techniques may be of use. For this purpose, the solvent consumption per analysis was computed for the generic gradient methods. The volume of solvent used (and thus requiring waste treatment after analysis) with the RPLC method was about 10 mL acetonitrile-water, while the volume of solvent used (and requiring waste treatment) with the UC method was 11.9 mL methanol-water. Because acetonitrile is much more expensive than methanol and because carbon dioxide is relatively cheap, the final cost per sample was nearly the same: about 2.3 € per sample. Of course, the UC equipment is somewhat more expensive than the RPLC equipment. However, we must point out that the high success rate of the UC generic method is also providing operational cost savings as it does not require additional expenses to re-analyse the sample with focus gradient or another analytical method.

Conclusions and perspectives

Orthogonality between the UC and RPLC methods on this set of peptides is somewhat less significant than was previously observed when small chemical drugs were compared between SFC and RPLC. However, the UC method is particularly interesting for the cases when RPLC retention is too low, which occurred frequently for these small, rather polar molecules. In the present test set, 19% of the samples could not be analyzed with the RPLC method while all of them had adequate retention in UC. When adequate retention could be obtained, less effort was required with UC than with RPLC to obtain satisfying results, as the generic gradient was most successful while the RPLC method often required a second experiment with focus gradient. In terms of capability for impurity profiling, the RPLC method seems superior as it was capable to detect more minor impurities, either due to higher peak capacity or possibly

Figure captions Figure 1 .

 captions1 Figure captions

Figure 2 .Figure 3 .

 23 Figure 2. Cases of failures observed among the 43 peptides analyzed with both gradient methods (generic and focus) in UC and RPLC

Figure 4 .

 4 Figure 4. Comparison of the number of impurities observed with both generic gradient methods, for 12 peptides that were successfully analyzed with both methods. (a) Total number of impurities observed for linear peptides (blue dashes) and cyclic peptides (orange circles). (b) Impurities with large concentration (>1%, black diamonds) and low concentration (<1%, open squares). The interrupted grey line is the first bisector, indicating identical values measured with both methods.

Figure 5 .

 5 Figure 5. Comparison of retention times obtained for the API (red square) and impurities. Yellow circles are impurities observed with both RPLC and UC methods, green triangles are impurities observed with only one method: green triangles at the top of the figure were seen with UC only, green triangles at the right of the figure were seen with RPLC only.

Figure 6 .

 6 Figure 6. Chromatograms for two pairs of isomers in UC and RPLC under generic gradient conditions (a) cyclic pentapeptides better resolved with UC (b) cyclic pentapeptides better resolved with RPLC.

Figure 7 .Figure 1 .

 71 Figure 7. Design of experiment to assess the variation of retention factor in UC conditions. (a) Effect of back-pressure vs. final gradient composition. (b) Effect of oven

Figure 2 .Figure 3 .Figure 4 .

 234 Figure 2. Cases of failures observed among the 43 peptides analysed with both gradient methods in UC and RPLC.

Figure 5 .Figure 6 .Figure 7 .

 567 Figure 5. Comparison of retention times obtained for the API (red square) and impurities. Yellow circles are impurities observed with both RPLC and UC methods, green triangles are impurities observed with only one method: green triangles at the top of the figure were seen with UC only, green triangles at the right of the figure were seen with RPLC only.

Table 1 .

 1 Four gradient methods employed in this study.

	(a) UC Generic gradient
	Gradient time	Cosolvent	Flow rate
	(min)	proportion (%)	(ml/min)
	0	5	3
	0.5	5	3
	10.5	80	1.5
	13.5	80	1.5
	14	5	1.5
	15	5	3
	(b) UC Focus gradient
	Gradient time	Cosolvent	Flow rate
	(min)	proportions (%)	(ml/min)
	0	5	1.5
	0.5	5	1.5
	1.5	C e -5%	1.5
	11.5	C e + 5%	1.5
	11.6	100	1.5
	14.6	100	1.5
	14.7	5	1.5
	15	5	1.5
	(c) RPLC Generic gradient

Gradient time (min) A% (H 2 O/ACN/TFA) B% (ACN/H 2 O/TFA)

	0	100	0
	0.6	100	0
	11.5	40	60
	11.6	100	0
	14.3	100	0
	(d) RPLC Focus gradient
	Gradient time	A%	B%
	(min)	(H 2 O/ACN/TFA)	(ACN/H 2 O/TFA)
	0	100-(C e -3%)	C e -3%
	0.6	100-C e	C e
	11.4	100-(C e +3%)	C e +3%
	11.5	100-(C e -3%)	C e -3%
	14.3	100-(C e -3%)	C e -3%

Table 2 .

 2 Determination of peak capacity with the two generic methods based on 12 peptides well eluted with both methods

	Method	UC	RPLC
	Gradient time (min)	10	10.9
	Average peak width at 50% height	0.032	0.021
	Peak capacity	189	314
	RSD%	30.4%	29.9%

Funding

This work was funded by Servier Research Institute.

due to higher sensitivity with UV detection. However, the UC and RPLC methods are most complementary as different impurities could be observed between the two methods. As a conclusion, the joint use of the two methods is advisable to improve the chances of successful analysis with a minimum effort and to obtain a maximum amount of information on a single sample.

Compliance with ethical standards

The authors declare they have no conflict of interest.

Compliance with ethical standards

The authors declare they have no conflict of interest.

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