# AF4-UHPLC: Two-dimensional separation of macromolecules in four white wines from South-Western France

Auriane Figué, Marianne Gosset, Frédéric Violleau

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Auriane Figué, Marianne Gosset, Frédéric Violleau. AF4-UHPLC: Two-dimensional separation of macromolecules in four white wines from South-Western France. Journal of Chromatography A, 2024, 1738, pp.465456. 10.1016/j.chroma.2024.465456. hal-04952197

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# AF4-UHPLC: Two-dimensional separation of macromolecules in four white wines from South-Western France

Auriane Figué<sup>a,b,c</sup>, Marianne Gosset<sup>c,*</sup>, Frédéric Violleau<sup>a,b</sup>

## ARTICLE INFO

### Keywords:

- Proteins
- White wines
- AF4-UHPLC

## ABSTRACT

Proteins in wines contribute to "protein haze," which, while not affecting health or taste, can detract from the visual appeal of wines to consumers. To mitigate this issue, winemakers commonly use fining agents such as bentonite, despite the high costs involved. To overcome these challenges, numerous studies employ various analytical methods to better understand the behaviour of proteins. A novel technique which separates compounds by size, Asymmetrical Flow-Field Flow Fractionation (AF4), allows for studying proteins without denaturing them. Given that most proteins share similar sizes, identification remains challenging. The aim of this work was initially to develop a new system enabling simultaneous analysis of the macromolecular profile of wines (proteins, mannoproteins) using AF4 and real-time protein nature analysis (hydrophobicity properties) using Ultra-High Performance Liquid Chromatography (UHPLC). By injecting two standards of different sizes and chemical nature, thaumatin protein and mannoproteins, the system was validated. The subsequent application of this system to Southwestern wines revealed distinct profiles across monovarietal white wines from four grape varieties. While the Colombard (COL) and Gros manseng (GM) varieties showed similar protein compositions with varying concentrations, the Len de l'el (LL) variety had only two types of protein and no protein was detected in the Mauzac (MZ) variety. Despite these variations, all varieties contained mannoproteins. This system shows promise for studying wine protein composition and could potentially find applications in other matrices.

## 1. Introduction

White wine is a complex matrix primarily composed of water, alcohol, and organic acids, along with various other compounds, including macromolecules such as proteins, phenolic compounds, and polysaccharides. Although present in small quantities, these compounds have been identified as having an influence on taste perception [1]. The phenolic compounds in wine primarily consist of non-flavonoid compounds such as hydroxycinnamates, and flavonoid compounds such as flavanols and anthocyanins [2–3]. The polysaccharides in wine are predominantly arabinogalactans, arabinogalactan proteins (AGP), and ramnogalacturonan II (RGII) [4]. Furthermore, another category of polysaccharides known as mannoproteins, as they are characterized by mannose polymers in their chemical structure, are present in wines [5].

Proteins are present in low concentrations in white wines, typically not exceeding 300 mg/L in wines that are not fined [6]. The protein content of wines depends on several factors, including grape variety,

grape ripeness, and the winemaking process [7]. Their impact on taste perception has been understudied to date [8]. Wine proteins are primarily known to play a significant role in wine instability, notably in a phenomenon known as "protein haze" [8–9–10]. This phenomenon causes haze formation in bottles exposed to heat [3]. Phenolic compounds interact with wine proteins, leading to haze formation that may aggregate over time during storage or transportation [11]. Proteins implicated in this role include thaumatin-like proteins (TLPs) and chitinases [8]. Importantly, this phenomenon poses no health risk [12] and does not affect the olfactory and gustatory characteristics of the wine [13]. Nevertheless, the presence of turbidity or deposits in bottled wines can diminish their commercial appeal, making them visually unappealing to consumers [14]. To address this issue, winemakers primarily use bentonite as a fining agent [3]. This negatively charged clay interacts non-specifically with proteins, which exhibit a positive charge at the acidic pH of wine, to induce their sedimentation [15]. However, this process is costly and has several quality drawbacks, so alternatives are

<footnote><sup>a</sup> Plateforme TFFFC, Université de Toulouse, INP-PURPAN, 75 Voie du Toec, Toulouse 31076, France</footnote>

<footnote><sup>b</sup> Laboratoire de Chimie Agro-industrielle, LCA, Université de Toulouse, INRA, 4 Allée Emile Monso, Toulouse 31000, France</footnote>

<footnote><sup>c</sup> Département sciences de l'agroalimentaire et de la nutrition, Université de Toulouse, INP-PURPAN, 75 Voie du Toec, Toulouse 31076, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: marianne.gosset@purpan.fr (M. Gosset).</footnote>
sought [16].

The challenge of discovering new, more cost-effective fining methods with fewer adverse effects on wine is of great importance to the local economy of the Southwestern region of France. This region is among the leading producers and exporters of dry white wines in France, utilizing numerous characteristic grape varieties for wine production. There are several distinct wine-growing regions in the South-West. The present study focused on two major viticultural areas: Gers (20,362 hectares in 2022) [17] and Tarn (6274 hectares in 2022) [17]. These areas specifically cultivate four typical grape varieties: COL and GM in Gers, and MZ and LL in Tarn.

Wine proteins have been the subject of extensive research. Numerous studies have employed various chemical methods to investigate them, such as ion exchange [18,19,20], size exclusion chromatography [21], protein blotting [22], two-dimensional electrophoresis [23]. Currently, most studies [24] primarily utilize reversed-phase liquid chromatography. These techniques are not the most suitable for studying proteins in a non-denatured state. Due to their three-dimensional structure, proteins can interact with other biological molecules [25]. Consequently, analytical techniques must preserve supramolecular and non-covalent interactions while being able to characterize proteins in their native, unaltered state. This is why, recently, a novel technique called Asymmetric Flow Field-Flow Fractionation (AF4) has been employed to examine the macromolecular composition of wines [26,27, 28,29], particularly of proteins [30]. For instance, Daniel E. Osorio-Macías et al. [28] utilized AF4 to assess the effectiveness of seven common fining agents. The primary advantage of this method lies in its absence of a stationary phase and low-pressure conditions, enabling separation without degradation of complexes or co-aggregates [31]. The separation channel features an upper wall that is solid and a lower wall that is permeable in design. AF4 enables the separation of analytes based on their size. The analytes are subjected to a laminar elution flow with a parabolic profile within a narrow channel, alongside a transverse flow that is perpendicular to the direction of elution. This transverse flow initially draws the analytes towards a membrane forming the lower wall of the channel, after which its strength is reduced to allow diffusion back towards the centre of the channel, where the solutes are exposed to a stronger elution flow. In this configuration, the rate of solute diffusion is inversely proportional to their hydrodynamic size; consequently, smaller solutes move further from the wall, experience greater elution flows, and thus elute earlier than larger solutes [32]. AF4 can be coupled with a range of detectors including UV/Vis, fluorescence, multi-angle light scattering (MALS), and refractive index detection (dRI), allowing for simultaneous spectroscopic analysis and size characterization. This technique offers invaluable insights into sample composition, stability, and the dynamics of aggregation or disaggregation phenomena [33].

However, the size-based separation of macromolecules poses a challenge for protein study in wines, particularly because the majority of proteins in white wines exhibit similar sizes. Moreover, isolating, separating, and characterizing proteins in musts and wines are challenging tasks due to their low concentration and pronounced interactions with endogenous polyphenols and other non-protein compounds. To date, only one study [34] has explored the integration of AF4 into HPLC analyses. Johannes [34] investigated proteins in egg whites by developing a new system combining AF4 with liquid chromatography. This system has great potential for separating proteins according to their size and hydrophobicity. However, the fractions collected at the output of AF4 are generally diluted by a factor of 10. Additionally, two-dimensional analyses using liquid chromatography are time-consuming. To address these issues, a new two-dimensional system incorporating AF4 and UHPLC has been developed in this study. This system enables the separation of samples based on their size using AF4. The fractions exiting the AF4 are subsequently concentrated using cartridges and then eluted onto a UHPLC column, thereby facilitating rapid analysis. In this study, the system was first validated and then applied to a complex matrix, wine, with the aim of differentiating

various wines from different grape varieties based on their macromolecular profiles.

## 2. Materials and methods

### 2.1. Samples

In this study, four samples from four indigenous grape varieties from the Southwest of France were analysed. These samples were chosen based on their key physicochemical properties — alcohol by volume (ABV), pH, titratable acidity, and concentrations of malic, lactic, and tartaric acids — to eliminate bias in analytical assessments (Table 1). The properties were measured utilizing Fourier-transform infrared (FTIR) spectroscopy with a WineScan analyzer (Foss, France SAS) in accordance with OIV/OENO Resolution 390/2010. The monovarietal wines from 4 native grape varieties, COL, GM, MZ, and LL, all from the 2021 vintage, were obtained from various wineries. Samples were collected directly from tanks post-alcoholic fermentation (AF). Throughout the study, the wines were maintained at 4 °C, with a minor addition of potassium bisulfite ($SO_2$) to avert oxidation and microbial contamination, and nitrogen was used to inert the samples.

To ensure the repeatability of each analysis, a reference wine was established, comprising of a blend of different grape varieties, primarily from the French Southwest.

### 2.2. Chemicals

Supergradient acetonitrile was procured from Carlo-Erba© (France), and trifluoroacetic acid was sourced from Fisher Chemical © (France). Both chemicals were of industrial-grade purity, rated at 99% [HPLC grade]. Ultrapure water was produced using a Merck® MilliQ Integral 15 system. Ethanol and formic acid, also of 99% industrial-grade purity (HPLC grade), were acquired from Carlo Erba© (France). Potassium hydroxide was obtained from Merck© (USA). Additionally, a 10 kDa pullulan from Merck© (USA) was used.

### 2.3. Two-dimensional separation of macromolecules using AF4 and UHPLC

To ascertain the macromolecular composition of different grape varieties, a two-dimensional AF4-UHPLC system was developed (Fig. 1). The objective was to fractionate the sample using AF4 to collect fractions at one-minute intervals from the fractogram onto cartridges, which facilitates the concentration of the various fractions. These will subsequently be eluted onto a UHPLC column. The AF4-UHPLC system was assembled from an AF4 device, an HPLC system, and one interfacing 10-port, high-pressure, two-position valve (C2-1000EP, VICI Valco).

To validate the system, two standards were injected: Thaumatin protein (Merck©,USA) and mannoprotein (Martin Vialatte©, France).

#### 2.3.1. 1D analysis using AF4: fractionation of the sample according to its size

A Thermo Scientific© Ultimate 3000 HPLC System, featuring a degasser, isocratic pump, autosampler and fraction collector, was used to inject samples and transport the mobile phase through the system. Samples were introduced with the autosampler maintained at 4 °C. The pump was coupled with an Eclipse AF4 (Wyatt technology©, Santa Barbara, USA) which regulated the flow in AF4 separation channel (long channel, Wyatt technology©, Santa Barbara, USA). A 10 kDa regenerated cellulose membrane (Consensus©, Germany) was used in this work. The height of the channel was set at 490 µm and its dimensions L, $b_0$ and $b_L$ were 23.5, 2.1 and 0.6 cm respectively. This setup was connected to a UV-visible Ultimate 3400 RS detector. UV-visible detector was set at $\lambda$ = 220 nm to increase the sensitivity of the signal. Acquisition of UV ($\lambda$ = 220 nm) was performed by the Astra© 8.2.2 software. The AF4 system was calibrated using a 10 kDa pullulan solution (4 mg/mL) prepared in a
<table><caption>**Table 1**
Summary table of the classical oenological parameters of all the samples used in the study.</caption><thead><tr><th>Grape variety</th><th>Wine production area</th><th>Sampling stage</th><th>Vintage</th><th>Alcohol by volume (ABV) %</th><th>Titratable acidity g.L<sup>-1</sup> as Tartaric acid</th><th>pH g.L<sup>-1</sup></th><th>Tartaric acid g.L<sup>-1</sup></th><th>Malic acid g.L<sup>-1</sup></th><th>Lactic acid g.L<sup>-1</sup></th></tr></thead><tbody><tr><td>COL</td><td>GERS</td><td>AF</td><td>2021</td><td>12</td><td>5,44</td><td>3,1</td><td>1,95</td><td>5,27</td><td>0</td></tr><tr><td>GM</td><td>GERS</td><td>AF</td><td>2021</td><td>12</td><td>5,17</td><td>3,2</td><td>1,92</td><td>4,59</td><td>0</td></tr><tr><td>LL</td><td>TARN</td><td>AF</td><td>2021</td><td>13</td><td>3,81</td><td>3,3</td><td>1,64</td><td>2,79</td><td>0,6</td></tr><tr><td>MZ</td><td>TARN</td><td>AF</td><td>2021</td><td>12</td><td>3,10</td><td>3,4</td><td>1,51</td><td>1,56</td><td>0,75</td></tr></tbody></table>

model wine solution containing 12% ethanol and adjusted to a pH of 3.2. The mobile phase consisted of 88% water and 12% ethanol, with formic acid added to adjust the pH to 3.2. A potassium salt was included at a concentration of 1 g/L (K⁺ equivalent). The eluent was first filtered through a 0.1 µm PTFE Millipore filter (Merk, Darmstadt, Germany). During the focus-inject step, 850 µL of wine sample was injected into a 1 mL sample loop with a focus flow rate of 0.8 mL/min and the focusing/relaxation step was performed for 3 min. During the entire elution period, the channel flow was maintained at 0,1 mL/min whereas the crossflow gradient varied. The first step of the elution was the increase of the crossflow at 1 mL/min then decreased with a linear decay to a constant crossflow of 0.05 mL/min during 56 min. Then, the crossflow at 0,05 mL/min was maintained for 20 min. At the end of the run, the crossflow was cut at 0 mL/min. To integrate the one-dimensional (1D) and two-dimensional (2D) systems, the 1D system was connected at the UV outlet to port number 1 of the interfacing valve (C2–1000EP, VICI Valco). Chromeleon® 7 software was used to control the autosampler, pump and Eclipse flows, and to activate 2D analysis.

### 2.3.2. Two-dimensional analysis using UHPLC

#### 2.3.2.1. Concentration of AF4 fractions on a cartridge.
To concentrate the AF4 fractions, two loops each consisting of a cartridge (C8, 2.1 mm x 4.6 mm ID, Phenomenex®) were placed on the selective valve (Fig. 1). Every minute of the fractogram, a fraction from the AF4 was injected into the inlet port of the selection valve (port 1). Subsequently, the fraction was concentrated in a cartridge. While the first cartridge released the concentrated material, the second cartridge was filled with the new fraction. Then, the concentrated samples were eluted on the UHPLC column.

#### 2.3.2.2. Elution on the UHPLC column.
The macromolecule content was determined by UHPLC using a modified method [24]: conversion from HPLC to UHPLC. The analysis time was reduced to 1.30 s instead of 35 min in the reference method to increase the 2D resolution. Indeed, if the method lasts 35 min, all analytes will be eluted at the same time in a single HPLC run.

UHPLC was done using an Ultimate 3000 Series instrument (Thermofisher®, France). The separation was done by using a Kinetex® (C8), particle size 100 A (50 mm x 2,1 mm x 1,7 µm) thermostated at 35 °C.

Two eluents were used as mobile phases: mobile phase A consisted of 80% acetonitrile, 20% mQ water with 0,1% TFA and mobile phase B consisted of 8% acetonitrile, 92% mQ water and 0,1% TFA. The flow rate was of 0.86 mL/min. Table 2 shows the gradient profile. A DAD detector monitoring at 220 nm was used for detection.

### 2.4. 2D contour plot processing

To process all the data, each of the 2D fractions collected using Chromelon® were exported as a text file and then processed using the LC x LC image software (2024). Once the fractions are imported into the software, the 2D image appears. First, the correction of the baseline with the command Baseline Correction' is necessary. The areas of the various blots were analysed using the 'Blot' function of the software.

## 3. Results and discussion

### 3.1. Validation of the 2D system

As part of the validation of our AF4-UHPLC system, a mixture containing two standards was injected: mannoproteins and thaumatin protein. Mannoproteins, polysaccharides produced by yeasts during alcoholic fermentation, are present in white wines. These polysaccharides primarily consist of mannose groups associated with approximately 10–20% proteins with a molar mass reach up to 450 kDa [35]. Their concentration varies significantly depending on the vinification process [5]. Thaumatin was not described in wine but, there are proteins in wines from the same family called thaumatin-like proteins [8]. These proteins originate from the grape and constitute one of the two principal groups of proteins, alongside chitinases. These proteins are characterized by relatively low molecular masses, typically around 20–30 kDa [36].

Following the injection of these standards into the AF4-UHPLC system, two distinct peaks were observed on the fractogram (Fig. 2) at different retention times according to the first dimension of the axis: tr1D= 20 min and tr2D= 38 min. This first dimension corresponds to the fractionation of macromolecules in an AF4 channel, a technique enabling separation based on macromolecule size [32,25]. This explains why our protein standard, with a lower particle size than mannoproteins, exhibited a shorter retention time. Consequently, our 1D system effectively separated the two standards according to their size. Regarding the second dimension, the thaumatin standard had a retention time of 35 s. This compound appeared to be more retained by the C8-type column, unlike the mannoprotein standard, with a retention time of 10 s. Mannoproteins are hydrophilic mannose polymers covalently linked to the main protein chain [37,38,39]. When the mobile phase was primarily composed of water, the mannoprotein standard was eluted. Indeed, it associated with water by forming hydrogen bonds, which gave it a high affinity for the mobile phase and caused its movement along the stationary phase (C8). However, the compound was still retained by the column due to its protein component, which may contain non-polar amino acids. Thus, a part of the mannoproteins could also be hydrophobic. Thaumatin proteins, due to their complex structure [40], are inherently more hydrophobic compared to mannoproteins, which predominantly consist of a highly polar sugar component. They were eluted after mannoproteins using a mobile phase composed primarily of acetonitrile. Indeed, this solvent exhibits a specific affinity for proteins in chromatographic methods [41], owing to its ability to interact effectively with proteins, solvate molecules efficiently, and control hydrophobic interactions. These properties make it an effective solvent for separating and analysing proteins in chromatographic techniques.

The feasibility of the AF4-UHPLC system was successfully demonstrated. The various standards were effectively separated according to their molecular size through AF4. Furthermore, they were distinctly separated based on their hydrophobicity in the second dimension. This technique proved itself extremely useful for the separation of complex mixtures. Indeed, the separation of compounds by size using AF4 allowed for the distinction of compounds with the same hydrophobicity, thereby creating a unique identity profile for each sample. This is why this method was then applied to white wines to study the
<figure><img src="image_4.png" /><figcaption>Fig. 1. Diagram of the AF4-UHPLC system.</figcaption></figure>

<table><caption>**Table 2**
Eluent gradient for UHPLC determination of macromolecules in white wines.</caption><thead><tr><th>Time (in min)</th><th>0</th><th>0,08</th><th>0,23</th><th>0,29</th><th>0,90</th><th>0,98</th><th>1,00</th><th>1,30</th></tr></thead><tbody><tr><th>%A</th><td>17</td><td>49</td><td>57</td><td>65</td><td>81</td><td>81</td><td>17</td><td>17</td></tr><tr><th>%B</th><td>83</td><td>51</td><td>43</td><td>35</td><td>19</td><td>19</td><td>83</td><td>83</td></tr></tbody></table>

macromolecular composition of various indigenous grape varieties within a single population.

## 3.2. Application of the 2D system on four native grape varieties

Each grape variety possesses its own unique identity. COL variety (Fig. 3) was composed of two distinct populations observed in the first
STANDARD OF THAUMATIN PROTEIN AND
MANNOPROTEIN

<figure><img src="image_5.png" /><figcaption>Fig. 2. 1D and 2D signals of two standards samples.</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>Fig. 3. 1D and 2D signals of Colombard grape variety.</figcaption></figure>

dimension: one at a retention time of tr<sub>1D</sub> = 26 min and the other at tr<sub>1D</sub> = 40 min. These two populations showed UV absorption (λ=220 nm), with higher absorption for the first population (Fig. 3). Thanks to the injection of various standards naturally present in wine, a first hypothesis for the identification of these compounds has been suggested: the

first population would be mainly composed of proteins, while the second
would be composed of mannoproteins. The second-dimension analysis
revealed differences in composition within the same population. For the
first population fractionated at tr<sub>1D</sub> = 26 min and hypothetically
composed of proteins, COL variety exhibited three types of macromol-
ecules (proteins). These three protein groups had different affinities with
the stationary and mobile phases, as they were eluted at different
retention times: the first one eluted (a) at tr<sub>2D</sub>=29 s, the second one
eluted (b) at tr<sub>2D</sub>=34 s and the third one (c) at tr<sub>2D</sub>=43s. Moreover, these
components did not absorb with the same intensity (Table 3). Indeed,
the component (a) had a pixel area of 972, component (b) had a pixel
area of 650, and the final component (c) had a pixel area of 291. A first
hypothesis can be proposed: the first (a) and the second (b) might have
been more concentrated in the sample than the third one (c), but these
compounds could also exhibit different absorption coefficients. The
potential identification of these proteins might have been indicative of
thaumatin-like proteins (22 kDa) or chitinase (30 kDa), primarily pre-
sent in white wines. Some studies have examined the impact of these
proteins on wines, revealing a significant diversity of isoforms for both
chitinase and thaumatin-like (TL) proteins [24,42]. The three distinct
macromolecules could potentially be isoforms of thaumatin-like pro-
teins. Regarding the second population at tr 1D= 40 min, COL was

<table><caption>**Table 3**

Pixel area of the different macromolecules in different grape varieties.</caption>
  <thead>
    <tr>
      <th rowspan="2">Area (pixel)</th>
      <th rowspan="2"></th>
      <th colspan="4">Grape varieties</th>
    </tr>
    <tr>
      <th>COL</th>
      <th>GM</th>
      <th>LL</th>
      <th>MZ</th>
    </tr>
  </thead>
  <tbody>
    <tr>
      <td rowspan="3">1st population</td>
      <td>1st protein</td>
      <td>972</td>
      <td>330</td>
      <td>810</td>
      <td>/</td>
    </tr>
    <tr>
      <td>2nd protein</td>
      <td>650</td>
      <td>615</td>
      <td>174</td>
      <td>/</td>
    </tr>
    <tr>
      <td>3rd protein</td>
      <td>291</td>
      <td>marks</td>
      <td>/</td>
      <td>/</td>
    </tr>
    <tr>
      <td rowspan="2">2nd population</td>
      <td>Mannoproteins</td>
      <td>160</td>
      <td>385</td>
      <td>287</td>
      <td>442</td>
    </tr>
    <tr>
      <td>Protein complex</td>
      <td>295</td>
      <td>130</td>
      <td>/</td>
      <td>/</td>
    </tr>
  </tbody>
</table>
composed of two compounds. The first one, with tr<sub>2D</sub> =10 s, certainly corresponding to mannoproteins. Indeed, upon injecting a mannoprotein standard, the macromolecule is eluted at the same retention time in both first and second dimensions as observed in our wines. The second one with tr<sub>2D</sub> =31 s, seems to have the same hydrophobicity properties as the first population hypothetically composed of proteins. However, this macromolecule has a higher molar mass (eluted in AF4 at a longer retention time). This observation may be explained by complexes formation with other protein components or with polyphenols like hydroxycinnamic acids.

The GM variety exhibited a fractogram profile similar to COL (Fig. 4), with two distinct macromolecular populations eluting at the same retention times. Moreover, the two-dimensional profiles were identical for both populations. They appeared to consist of the same kind of macromolecules. However, the pixel area of the three components were different (Table 3). The volume under the peak was smaller compared to COL, suggesting that the potential protein content in the first population of GM (a) may be less concentrated or have a less absorption coefficient than in COL. Furthermore, the second set of potential proteins in the GM variety (b) was present in higher concentrations or had a greater absorption coefficient than in the first set. The second population, composed of mannoproteins, was identical to that of COL but more concentrated in the GM variety (Table 3). Mannoproteins are synthesized by yeast during the alcoholic fermentation process. These polymers are found in notable quantities in wines, and their levels vary depending on the specific methods used in winemaking [43]. Furthermore, the second macromolecule, which had the same retention time in the first dimension as the mannoproteins (tr<sub>1D</sub> = 40 min), appeared to be identical to that observed in COL. These molecules also shared the same retention time in the second dimension (tr<sub>2D</sub> = 31 s), indicating similar hydrophobic properties. However, the pixel area corresponding to the macromolecules present in the wines was smaller than that observed in COL.

The fractogram profile of LL variety was different to that of COL and GM (Fig. 5); it shows higher UV absorption for the first population compared to the other two. Moreover, the macromolecular composition (hypothetically of proteins) in the first population differed. It included two types of macromolecules (proteins). Specifically, these macromolecules corresponded to those found in the first and second positions in GM and COL. The concentration of the first set of macromolecules

appeared to have a pixel area very close to that of the COL variety (Table 3). However, the second set of macromolecules exhibited much lower intensity compared to the second set in COL and GM. Additionally, the third set of macromolecules was absent in this grape variety. The second macromolecular population, consisting of mannoproteins, was lower in LL than in GM but higher than in COL.

Unlike other grape varieties, MZ stood out for its very weak UV signal between 26 and 38 min in AF4 (Fig. 6), which could correspond to the hypothesis of the presence of proteins at very low concentrations. Indeed, in this grape variety, proteins are scarce, mainly due to the vinification process. Additionally, this slight absorbance may be due to the presence of compounds that absorb slightly in the UV range at a wavelength of 220 nm but have no affinity with the 2D solvent.

<figure><img src="image_7.png" /><figcaption>Fig. 4. 1D and 2D signals of Gros manseng grape variety.</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>Fig. 5. 1D and 2D signals of Len de l'el grape variety.</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>Fig. 6. 1D and 2D signals of Mauzac grape variety.</figcaption></figure>
However, it also contains mannoproteins in higher concentrations than the other grape varieties.

This method enables us to characterize the composition of macro-molecules within a specific population qualitatively and tentatively. However, white wines consist of macromolecules beyond proteins and mannoproteins; they also include polysaccharides. These compounds lack chromophore groups and thus are not detectable under UV light.

## 4. Conclusion

In conclusion, this feasibility of the two-dimensional system incorporating offline AF4-UHPLC analysis has been successfully demonstrated through the injection of two standards. This technique proves to be highly promising for studying complex mixtures such as wines. Indeed, this setup allows for the differentiation of various grape varieties based on their macromolecular composition. Moreover, it could present considerable advantages over classical UHPLC, as the samples are initially concentrated on a cartridge, which seems to prevent material loss and to avoid sample denaturation. The fractionation of samples via AF4 further enables the separation of analytes based on their size, facilitating the identification of compounds with similar hydrophobicity, which would be difficult to observe with a simple UHPLC analysis. Thus, this tool could prove to be a significant asset for the wine industry by providing a better understanding of the proteins responsible for the formation of protein haze and, ideally, by reducing the costs associated with this phenomenon. It could also be applied to other complex matrices such as must, beer or milk.

### CRediT authorship contribution statement

**Auriane Figue:** Writing – original draft, Methodology, Conceptualization. **Marianne Gosset:** Writing – review & editing, Validation, Methodology, Conceptualization. **Frédéric Violleau:** Writing – review & editing, Validation, Supervision, Funding acquisition, Conceptualization.

### Declaration of competing interest

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.

### Acknowledgements

This work was made possible thanks funding allocated by the Occitanie region, France, and Ecole d'ingénieurs de Purpan, Toulouse, France. We thank all the wine estates for providing wine samples. We would also like to thank Mr. Freeman for taking the time to review the English text of our article.

### Data availability

Data will be made available on request.

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