# Examining the conditions to achieve successful fraction transfer in two-dimensional supercritical fluid chromatography

Laurine Réset, Clément de Saint Jores, Caroline West

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Laurine Réset, Clément de Saint Jores, Caroline West. Examining the conditions to achieve successful fraction transfer in two-dimensional supercritical fluid chromatography. Journal of Chromatography A, 2025, 1762, pp.466355. 10.1016/j.chroma.2025.466355. hal-05421369

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# Examining the conditions to achieve successful fraction transfer in two-dimensional supercritical fluid chromatography

Laurine Réset, Clément De Saint Jores*, Caroline West

Université d'Orléans, CNRS, ICOA, UMR 7311, Orléans, France

**Corresponding author:**

Clément De Saint Jores - University of Orleans, ICOA, CNRS UMR 7311, Pôle de chimie, rue de Chartres - BP 6759 45067, Orléans Cedex 2, France
orcid.org/0000-0002-6763-1216

Phone: +33 2 38 41 99 36

e-mail: clement.de-saint-jores@univ-orleans.fr

## Highlights

* Low impact of valves, loops and tubings on chromatographic performance in 2D-SFC

* Reducing loop dimensions has no significant impact on 2^nd^-dimension performance with pressurized fluids

* Examination of the impact of elution conditions in 1^st^ dimension on 2^nd^-dimension performance

* Large composition differences are deleterious to chromatographic quality
**Abstract**

In this study, the optimization of the conditions necessary to ensure successful fraction transfer in two-dimensional supercritical fluid chromatography (2D-SFC) were examined. Particular attention was placed on the influence of instrumental parameters, including the addition of valves, tubes, and loop volumes, on the chromatographic performance in both dimensions. The impact of these modifications on retention times, gradient delays and peak shape was evaluated. While the first dimension remained unaffected, a slight broadening of the peaks was observed in the second dimension, which did not significantly compromise resolution.

Optimizing the loop volume revealed no benefit in reducing the loop dimensions, as dividing the peaks into several fractions did not improve resolution. Furthermore, combining different proportions of CO2 and methanol between the two dimensions showed that most combinations guaranteed satisfactory performance on polar and non-polar columns. Only the cases where the methanol proportion was greatly different between the first and second dimensions, yielded unsatisfactory peak shapes, but these are quite unlikely in normal SFC operation. Finally, these results provide indications of the operational limits of 2D-SFC systems to guarantee stable and efficient performance.

**Keywords:** gradient delay; injection effect; injection solvent; multidimensional chromatography; supercritical fluid chromatography
# 1. Introduction

Following the current trend on expanding two-dimensional liquid chromatography (2D-LC) applications, two-dimensional supercritical fluid chromatography (2D-SFC) is emerging as an innovative approach to improve the separation of analytes in complex matrices [1,2]. By combining the advantages of supercritical fluid chromatography (SFC) with the increased peak capacity offered by two-dimensional (2D) chromatography, this technique provides better resolution of compounds that would be difficult to separate in one-dimensional (1D) chromatography [3]. One of the main advantages of 2D-SFC lies in the possibility to obtain high orthogonality through the simple use of different stationary phases in both dimensions while maintaining the same mobile phase composition, offering increased selectivity and therefore increased practical peak capacity compared with conventional methods.

However, the development of 2D-SFC presents major challenges, particularly with regard to managing the transfer of analytes between the two dimensions [4–7]. As 2D-LC [3,8,9] and in LC-SFC or LC×SFC coupling [10–13], the transfer conditions directly influence the quality of the separations obtained in the second dimension. Optimization of valves, loop dimensions and storage volumes is essential to minimize dilution and peak distortion [14]. In addition, potential incompatibility between the conditions of the first (dim1) and second (dim2) dimensions, particularly with regard to the percentages of co-solvent [15], may compromise the detection and quantification of analytes.

Another limiting factor is related to the SFC-specific injection effects, which can alter the reproducibility of analyses and quantification of analytes. As observed in 1D-SFC and LC×SFC, significant pressure variations can occur during the injection of polar solvents, particularly those containing water [16–18]. These fluctuations may impact not only the separation performance, notably through alteration of peak shapes, but also the system stability. The volume and composition of the injection solvent must be carefully controlled to ensure optimum resolution [19–21].

The coupling of reversed-phase liquid chromatography (RPLC) and SFC has already demonstrated its potential by improving analytical coverage and exploiting the complementarities between the two techniques [3]. Technical developments have been proposed to improve RPLC and SFC hyphenation, which facilitate the transfer of analytes while taking advantage of the low viscosity of the SFC mobile phase, enabling faster analyses and improved peak capacity [14]. However, unlike traditional liquid-
phase systems, transferring a compressible supercritical fluid from the first to the
second dimension in a 2D-SFC system sets additional challenges compared to
LCxSFC systems, particularly to maintain pressure during the fraction transfer. This
difference requires careful optimization of injection strategies, solvent compatibility and
transfer conditions to ensure robust and reproducible performance in 2D-SFC
instruments.

In this context, our study aimed at optimizing the transfer of analytes in an SFC-SFC
system used in multiple heart-cutting mode, paving the way for future developments of
comprehensive 2D-SFC. First, a comparison was made between a 1D and a 2D
system. Then the impact of elution conditions in the first dimension (dim1) and the
influence of the quantity of solvent stored and transferred to the second dimension
(dim2) were thoroughly examined, using two sets of analytes (non-polar and polar) and
different chromatographic conditions. Parameters were optimized, considering peak
shape and bandwidth. Among the parameters studied were the size of the storage
loops, the system pressure and the composition of the mobile phase in order to
improve compatibility between the two dimensions and ensure the best possible
transfer of analytes in a 2D-SFC system.

**2. Experimental**

**2.1. <u>Chemicals and samples</u>**

HPLC-grade methanol (MeOH) used as the mobile phase and sample diluent, and
ethanol (EtOH) Hipersolv Chromanorm 99.7-100% used for sample preparation were
purchased from VWR (Fontenay-sous-Bois, France). CO2 with a purity of 99.7% was
delivered by Air Liquide (Paris, France). Methanesulfonic acid (MSA) and acetone
were purchased from Sigma-Aldrich (Saint-Quentin-Fallavier, France).

A naringin standard containing identical proportion of the (+) and (-) diastereomers and
standards of pinocembrin, pinocembroside, hesperetin, naringenin, taxifolin,
alphitонin, epigallocatechin and epicatechin gallate were all provided by Extrasynthese
(Genay, France). Standards of caffeine, n-butylbenzene, n-hexylbenzene, n-
octylbenzene, n-decylbenzene and n-dodecylbenzene were obtained from Sigma-
Aldrich. A standard of n-tetradecylbenzene was purchased from Thermo Fisher
(Illkirch, France).

*Citrus aurantium* L. peels were bought from Louis herboristerie (Charleville-Mézières, France).
## 2.2. Standards and sample preparation

Standard solutions of naringin, other flavonoids and alkylbenzenes were prepared respectively at the concentration of 1 mg/mL, 0.5 mg/mL and 0.5 mg/mL in MeOH. Orange peels were finely ground, then 1 g of sample was extracted with 15 mL EtOH for 30 min in an ultrasonic bath. The extract was then centrifuged (20 °C, 8000 rpm) for 5 min and filtered through 0.20 µm, 13-mm diameter PVDF syringe filters from Agilent Technologies (Les Ulis, France) and diluted by 4 in EtOH prior to analysis. Caffeine was directly introduced in the mobile phase co-solvent (MeOH) at different concentrations: 0.025 mg/mL and 0.05 mg/mL.

## 2.3. Chromatography instrument

The SFC system was a Waters Corporation (Millford, MA, USA) ACQUITY Ultra Performance Convergence Chromatography™ (UPC²®). It was equipped with an autosampler (sample manager – SM-FL) with a partial loop volume injection system, a back-pressure regulator, and a 2-position column oven (column manager or CM-A) compatible with 150-mm length columns, which could be accessed via column-switching valves. The system also included a binary solvent delivery pump (identified as ccBSM in Figure 1) compatible with a maximum mobile phase flow rate of 4 mL/min and a maximum pressure of 414 bar. Two detectors were available: a diode-array detector (ACQUITY PDA®) and a single-quadrupole mass spectrometer with an electrospray ionization source (ACQUITY QDa®). An isocratic solvent manager (ISM) was used as a make-up pump and was positioned before the mass spectrometer and with a split, where one portion was sent to the automated back-pressure regulator (BPR, identified as CCM in Figure 1) and the other to the MS. The use of MSA in the mobile phase involved adapting the exhaust valve of the QDa detector as MSA may cause corrosion on certain parts. This unmodified instrument will be designated as sys1D in the following.

In a previous paper, we have proposed a different setup, modifying the 1D instrument for SFC-SFC experiments [5], simply introducing three additional valves. The configuration of this system is shown in Figure 1, and it will be designated as sys2D in the following.

Briefly, the system was equipped with three Rheodyne 6-port, 2-position automatic valves (MXT715-000), supplied by CIL Cluzeau (Sainte-Foy-la-Grande, France). The
fraction storage loops were manufactured in-house from tubes also supplied by CIL
Cluzeau. They were made of stainless steel (OD 1/16") with internal diameters of 0.50
and 0.80 mm. They were cut into 25 cm or 56.5 cm segments using a tube cutter and
then sanded to obtain smooth cuts. Finally, each tube was rinsed with ethanol to
remove particles from sanding and prevent any obstruction of the system.

Between valves 1 and 2, the tubing was 10 cm x 0.18 mm. Between valved 2 and 4
and 2 and 5, the tubings were 20 cm x 0.18 mm.

Empower® 3 was used for chromatographic acquisition and for data treatment.

<figure><img src="image_2.png" /><figcaption>**Figure 1:** Multiple heart-cut SFC-SFC system configuration. The elements in orange rectangles are all contained in the column oven (CM-A). See text for complete description.</figcaption></figure>

## 2.4. Chromatographic conditions

For applications on a real sample (bitter orange peel) and for the analysis of naringin
standard, two different columns were used. The first-dimension (dim1) column was
ACQUITY UPC² Torus DEA (100 × 3.0 mm, 1.7 µm). The second-dimension (dim2)
column was CHIRALPAK IB-3 (150 × 4.6 mm, 3.0 µm) from ChiralTechnologies
(Illkirch-Graffenstaden, France). Additionally, a Torus DEA pre-column (2.1 × 5.0 mm)
from Waters was placed in front of the Torus DEA column. The mobile phase used
was CO₂ with MeOH containing 0.1% MSA. The injection volume was 5 µL. The
gradient used, comprising variation of mobile phase composition, but also back-
pressure and flow-rate (for the first-dimension analysis) is presented in Table 1 (gradient 1). This method was used for the flavonoid samples in: bitter orange peel, naringin and neohesperidin standards.

For the measurement of gradient delay, the tests were carried out on the same columns as those mentioned above, but with MeOH containing 0.1% acetone as mobile phase, and using the gradient 2 presented in Table 1. Back-pressure was set at 150 bar, and flow-rate was respectively 2.0 and 1.5 mL/min in dim1 and dim2 methods.

For the tests with the caffeine standard, the same columns as before were used. The co-solvent for the first dimension was MeOH, containing dissolved caffeine, and for the second dimension MeOH containing 0.1% MSA. The step gradient involving several isocratic steps is shown in Table 1 (gradient 3). Back-pressure was set at 110 bar and 150 bar, and flow-rate was 0.6 and 2.0 mL/min respectively in dim1 and dim2 methods. Tests with alkylbenzene standards were performed with similar stationary phases in both dimensions: XSelect HSS C18 (150 × 4.6 mm, 5.0 µm) and HSS C18 (150 × 4.6 mm, 5.0 µm) from Waters. Isocratic elution with MeOH percentage ranging from 2% to 90% in CO₂ were examined in the first and second dimensions.

Tests with flavonoids standards were performed with the same stationary phases in both dimensions: ACQUITY UPC² Torus DEA (100 × 3.0 mm, 1.7 µm) from Waters. Isocratic elution with MeOH containing 0.1% of MSA, with percentage ranging from 5% to 90% in CO₂ were examined in the first and second dimensions.

For both alkylbenzenes and flavonoids, the back-pressure was set at 110 bar, the flow rate was 1.0 mL/min and the injection volume was 2 µL.

In all cases, the column oven was heated to 30 °C and the sample compartment was kept at 10 °C. All chromatograms were recorded with PDA in the 210-400 nm range, with 1.2 nm resolution, with a scan rate of 20 Hz. Visualization and peak integration were performed at 280 nm for flavonoids standard and peel extract, 272 nm for caffeine and 210 nm for alkylbenzenes.

## 2.5. Mass spectrometry conditions

Mass spectrometry (MS) only served to confirm peak identity, especially for the citrus samples to distinguish target compounds from other peaks. The MS parameters were as follows: make-up fluid MeOH comprising 20 mM NH₄OH and 2% H₂O, pumped at 0.4 mL/min; probe temperature 600 °C; ion source temperature 120 °C; cone voltage
10 V; capillary voltage +/- 0.3 kV; scan rate 3.7 Hz. The mass range for total ion recording was set from m/z 100 to 1000.

# 3. Results and discussion

## 3.1. Comparison of two-dimensional system and one-dimensional system

As appears in Figure 1 describing the modified instrument, the insertion of additional valves and tubing in the 1D instrument shown in Figure S1 (noted as "sys1D" in the following) to obtain a 2D instrument (noted as "sys2D" in the following) results in the addition of dead volumes, which can impact retention time and peak shape in both dimensions. It was therefore essential to assess the impact of these structural modifications on the overall performance of the system.

### 3.1.1. Gradient delay

To compare the performance of sys1D and sys2D, the impact on gradient delay was first studied. To measure this delay, a low concentration of acetone (0.1% in methanol) was added to the mobile phase and the UV trace was recorded during gradient operation.

The calculation of the dwell volume (D) is based on several parameters: the flow rate (F), the predefined gradient duration ($t_G$) and the time $t_{0.5}$, corresponding to the moment when the UV absorbance has reached a half of the maximum absorbance that is measured on the final plateau [22]. The formula used is as follows:

$$D = (t_{0.5} - 0.5 \times t_G) \times F \qquad (1)$$

In liquid conditions, the usual practice is to use a wide gradient, covering most of the possible solvent proportions, i.e., from 1 to 99%, to achieve this measurement. The objective is to have a large variation of UV absorbance to offer a good precision in the measurement. However, it is important to note that the choice of conditions to achieve a reliable measurement with supercritical or subcritical fluids is not trivial. Indeed, since the fluid is compressible, the amount of fluid that can be pumped to fill in a defined volume varies with the operating conditions, where the latter include the co-solvent nature and proportion, the temperature, pressure and flow rate [23]. Typically, the apparent void volume was observed to vary depending on operating conditions. Similarly, and relevant to the present case, the measured dwell volume varies
depending on the conditions employed at the beginning of the gradient program. More
precisely, as compressibility is reducing when the proportion of co-solvent increases,
the measured dwell volume will also reduce when the proportion of co-solvent at the
start of the gradient increases. In other words, the amount of fluid that is necessary to
fill in the volume is increasing when the fluid is more compressible, that is to say when
the proportion of co-solvent is less.

In previous works, Sarrut *et al*. had shown that a wide co-solvent gradient (1-99%) in SFC made it impossible to obtain a precise measurement of the dwell volume [22], because the slope of the UV absorbance was changing along the gradient. Instead, they chose to measure the dwell volume based on a gradient program where the proportion of co-solvent was large (69 to 99%), to offer conditions of low compressibility, close to a liquid. In the present study, we chose, on the contrary, to measure the dwell volume with low starting proportion of co-solvent (20%), identical to those chosen for analysis, hoping to provide a value that would be more relevant to our analysis conditions, and to use only a narrow range of co-solvent proportion (20 to 30%) to preserve a constant slope of the UV absorbance.

In sys1D, the normal Acquity UPC² instrument was used, without any modification (Figure S1). In sys2D, the mobile phase would go through different pathways when eluting the samples in dim1 or dim2 (Figure 1).

For dim1 operation, the mobile phase would go through valve 1, the dim1 column, valve 1 again, valve 2 and valve 3. Note that the mobile phase would not go through the loops at this stage since this is the dim1 analysis, that serves to measure collection times. When performing sample collection, a second analysis of the samples is done, going through a different path to store the fractions in the storage loops between valves 4 and 5.

With the initial method we had developed for flavonoids analysis (gradient 1.a in Table 1) [24], accurate determination of the gradient delay in dim1 was not possible due to the complex combination of increasing co-solvent on a large concentration range (20 to 100%), decreasing flow rate and decreasing back-pressure, causing non-linear variation of UV signal. Therefore, a second elution gradient (gradient 2.a in Table 1) was used to facilitate the measurement of gradient delay, where only the proportion of co-solvent was varied: methanol increased from 20 to 30% with the same slope as in gradient 1.a. These conditions were chosen to be similar to the gradient start in method
1, and should therefore adequately represent the case of experiments done with
gradient 1, although the back-pressure and flow-rate remained constant.

Comparing the results obtained with this method for sys1D and sys2D, the measured
gradient delay remained identical for the first dimension (dim1), with a value of around
1.7 mL (see Figure S2.A in supplementary information).

For dim2 operation, as the fluid must go through a storage loop to transfer the fraction
to the second column, the mobile phase would go through valves 1, 2, 4, a 50-µL
storage loop, then valves 5, 2 again and 3, the dim2 column, then again valve 3.

For the second dimension (dim2), gradient 2.b, identical to gradient 1.b (Table 1) was
used. Although a significant amount of tubing and valve volume was added in sys2D,
the measured delays were not significantly different: 3.8 mL for the one-dimensional
system and 3.7 mL for the two-dimensional system. It can be observed in Figure S2.B
that the curves are nearly superimposed.

The reader may however be surprised that a larger extra-column volume would not
yield a larger dwell volume. To understand this point, it is worth reminding that the fluid
is compressible and that the dwell volume was measured as the volume of
compressible fluid required to fill it, not as the true volume that could result from a
measurement in liquid conditions. The longer pathway taken by the fluid in the modified
system may yield a slightly higher internal pressure, resulting in a less compressible
fluid in this system. However, the higher the compressibility of the fluid, the larger the
volume of fluid you would need to pump in the system to fill in the available space.
Hence, even a larger empty volume could yield a lower measured volume of
compressible fluid to fill it in.

Nonetheless, these observations showed that adding the second dimension to the
chromatographic system had no significant impact on the gradient delay, confirming
compatibility between the two configurations under the conditions tested.

3.1.2. Pressure

Because the mobile phase is a compressible fluid, where density may impact
chromatographic retention and band width, internal pressure is an important parameter
that may be impacted by the system modification. The pressure variation induced by
the addition of new components (valves, tubings and second-dimension column) was
measured through the captors of the pumping system during the previous analyses. In
the sys2D, in first dimension (dim1), the addition of components to build up the two-
dimensional system generated no measurable pressure increase (Figure S3.A)
compared with the initial configuration. In the second dimension (dim2), the pressure
variation reached 3 bar (Figure S3.B). Such increase can be considered as negligible
in most usual operating conditions and is therefore unlikely to affect the correct
operation or overall performance of the system. However, it can be related to the
observations in previous section that the fluid may be slightly less compressible in the
2D configuration.

3.1.3. Peak width

Most importantly, it was essential to assess the impact of instrument changes on
chromatographic efficiency, as this would have further consequences on overall
system performance, notably affecting resolution and sensitivity. As all experiments
were done in gradient elution mode, peak width was used as key metrics.

For this study, a solution of naringin standard (containing two diastereomers in identical proportions) was used. In dim1, an achiral stationary phase that is not resolving the diastereomers of naringin (Torus DEA) was used with gradient 1.a (Table 1). In dim2, a chiral stationary phase (CHIRALPAK IB) was used to resolve the diastereoisomers of naringin, with gradient 1.b (Table 1).

The results obtained with sys1D and sys2D are shown in Figure 2.

In the dim1 method, the width at half-height of the naringin peak remained very narrow
in both configurations, close to 0.04 min. In the sys2D configuration, a 12% increase
in peak width was observed.

However, in dim2, a larger increase in peak width was observed in the two-dimensional
system compared with the one-dimensional system. Specifically, the width at half-
height of peak 1 was 0.165 min with sys1D and 0.209 min with sys2D (Table S1), which
represents a 27% increase in peak width. For the second peak, the increase in peak
width was slightly less (18%). However, this reduction did not impair the resolution of
the diastereomers, which was 4.67 in sys1D compared to 4.46 in sys2D.

This small increase of peak width in dim2 was not unexpected and can naturally be
attributed to the addition of dead volumes (tubings and valves) in the two-dimensional
system. Although this difference is significant, it appears that sufficient resolution
between the diastereomers was maintained.
<figure><img src="image_3.png" /><figcaption>**Figure 2:** Chromatograms of dim1 and dim2 showing the analysis of a naringin standard performed on a one-dimensional system (sys1D) and a two-dimensional system (sys2D).</figcaption></figure>

A) Analysis using the dim1 method with sys1D. B) Analysis using the dim1 method with sys2D. C) Analysis using the dim2 method with sys1D. D) Analysis using the dim2 method with sys2D, where the flow is going through a storage loop to mimic the real pathway of a sample when operating the SFC-SFC system.

Analysis conditions for A and B: ACQUITY UPC² Torus DEA (100 × 3.0 mm, 1.7 µm), co-solvent: MeOH + 0.1% MSA, 150-110 bar, 1.7-0.6 mL/min (gradient conditions in Table 1, gradient 1.a)

Analysis conditions for C and D: CHIRALPAK IB-3 (150 × 4.6 mm, 3.0 µm), co-solvent: MeOH + 0.1% MSA 150 bar, 2.0 mL/min (gradient conditions in Table 1, gradient 1.b).

## 3.2. Transfer conditions

### 3.2.1. Storage loop dimensions

In two-dimensional chromatography systems operating in multiple heart-cutting mode, storage loops play an essential role in ensuring accurate transfer of analytes between dim1 and dim2. These loops first serve as storage loops, to collect specific fractions from dim1, then serve as injection loops to re-inject the fractions into dim2 for further analysis. In liquid chromatography, the loop dimensions have a direct impact on band broadening in the second-dimension analysis, since an incompressible fluid is transferred so the loop length has a direct impact on band width, unless some peak compression may be obtained from the eluting phase. However, transferring a
compressible fluid may result in different effects. Thus, in this section, we intended to examine how loop dimensions would influence band dispersion in dim2, and therefore dim2 resolution. Three loop configurations were tested: a 114-µL loop (25 cm × 0.762 mm id), a longer but narrower loop with the same 114 µL volume (56.5 cm × 0.508 mm id), and two identical 50-µL loops (25 cm × 0.508 mm id). The interest in smaller loops was also to examine the possibility of fractionating a large peak into two smaller-volume fractions, as would be done in selective comprehensive 2D methods.

These tests were carried out on a sample of orange peel extract, which we had previously examined [5], to offer a representative example of a complex matrix. The neohesperidin peak (containing two diastereomers and some other compounds) was collected from dim1 and re-analyzed in dim2 to resolve its two diastereomers and the other co-eluting compounds.

The results presented in Figure 3 show that the width of the dim2 peaks remained constant, with an average value of 0.17 min, whatever the loop dimensions. This indicates that, under the operating conditions of this study, neither the volume nor the dimensions of the loops introduced measurable dispersion changes or altered the dim2 resolution. This would probably be a surprising observation for 2D LC chromatographers, but it is important to remember here that the fluid is compressible, so the internal volume of the storage loop does not necessarily translate to the expected band width in the dim2 chromatogram. Indeed, the fluid is compressed again during the transfer, and the analytes get stacked at the column entrance. We had previously demonstrated that transferring a volume of supercritical fluid resulted in much reduced band broadening compared to the same volume of a liquid with comparable polarity [5,26].

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<table><thead><tr><th></th><th>Loading time (min)</th><th>Retention time (min)</th><th>Area (µV*sec)</th><th>Width 50%</th></tr></thead><tbody><tr><td rowspan="2">50 µL – 25 cm × 0.508 mm (1)</td><td rowspan="2">1</td><td>11.766</td><td>8898</td><td>0.136</td></tr><tr><td>12.465</td><td>268 754</td><td>0.171</td></tr><tr><td rowspan="2">50 µL – 25 cm × 0.508 mm (2)</td><td rowspan="2">1</td><td>-</td><td>-</td><td>-</td></tr><tr><td>12.422</td><td>680 439</td><td>0.169</td></tr><tr><td rowspan="2">114 µL – 56.5 cm × 0.508 mm</td><td rowspan="2">1</td><td>11.734</td><td>15792</td><td>0.191</td></tr><tr><td>12.440</td><td>1013 890</td><td>0.179</td></tr><tr><td rowspan="2">114 µL – 25 cm × 0.762 mm</td><td rowspan="2">1</td><td>11.604</td><td>14457</td><td>0.184</td></tr><tr><td>12.242</td><td>997 332</td><td>0.171</td></tr></tbody></table>
**Figure 3:** Second-dimension analysis of dry peel of *Citrus aurantium*, where the peak of neohesperidin was selected from the first dimension and re-analyzed in second dimension. Influence of the dimensions of the storage loop on the peak area and peak width. Green triangles indicate the co-eluting peak in dim1.

Analysis conditions: CHIRALPAK IB-3 (150 × 4.6 mm, 3.0 µm), co-solvent: MeOH + 0.1% MSA (gradient conditions in Table 1, gradient 1.b), 150 bar, 30 °C, 2.0 mL/min.

The impact of loop volume (50 or 114 µL) and added tubings (12.5 µL) was however visible in the values of measured peak areas. First, and as could be expected, the large-volume loops resulted in larger peak areas. In addition, the two loops with 114 µL volume but different dimensions yielded very close peak areas, suggesting that the whole target peak had been correctly collected from dim1, whatever the loop dimensions. Second, and also quite logically, the peak areas from the two 50-µL loops were smaller and different from each other, since they corresponded to two fractions of one large peak from dim1. Furthermore, the peak areas from these two fractions added up to a total amount that was nearly equal to the values measured from the large loops. This again suggests that two small loops recovered the whole target peak from dim1.

In other words, the division of the large peak into two fractions did not improve resolution in the second dimension but only divided peak areas into different fractions. This is an important conclusion from this experiment as it suggests that there would be little interest in further reducing the volume of fractions in a comprehensive 2D-SFC system, as it would mostly impair the method sensitivity. At least, such is the case for a whole peak retrieved from dim1. Naturally, when partial separation occurs in dim1 within the collection time, it could still be a better option to fractionate it in smaller collection loops.

Finally, the reduced internal diameter of some of the loops (0.508 mm vs. 0.762 mm) or their longer length (56.5 cm vs. 25 cm) did not cause any significant changes in system pressure as observed in the pump recordings (Figure S4). This suggests that the loop dimensions cannot cause any significant change in fluid density, thus no significant change in analyte elution either and further confirmed that the loop dimensions could not influence the results obtained in this study. It is however possible that different operating conditions, where the fluid would be more compressible (closer to the critical point or with lower proportions of co-solvent), would have shown more impact of loop dimensions.
In conclusion, loop dimensions had no significant impact on analyte dispersion or dim2 resolution under the specific conditions examined here. Also, loop dimensions showed no impact on system pressure. Furthermore, dividing one peak into two smaller-volume loops did not appear to improve efficiency in dim2 analysis and would therefore bring no benefit in terms of resolution nor sensitivity.

### 3.2.2. Quantity transferred

Transfer issues between the two dimensions can lead to significant biases in quantitative results. In particular, with the present system, it is possible that some portions of a collected peak would remain in the intermediate tubings between valves 2 and 4 and between 5 and 2 thus would not be transferred to the second dimension. To avoid this issue, valve switching must be accurately controlled to ensure that the target fraction from dim1 is entirely stored in the storage loop. In our previous works, we had examined this point and demonstrated that no loss or mixing of successive fractions occurred [5]. However, in our previous works, the transfer conditions were always in the same range of co-solvent proportion. In the present study, we wanted to assess the impact of a wide range of methanol proportion on the amounts collected and transferred, to guarantee the reliability of quantitative analyses.

The compressibility of the CO₂-solvent mobile phase, which is present as a subcritical fluid in the system, is again a crucial element of this investigation. Depending on the proportion of co-solvent, the fluid is more or less compressible and sensitive to operating conditions [23]. While the loops have a fixed volume, different operating conditions resulting in different fluid density could influence the quantity of analyte transferred from one dimension to another.

In this study, caffeine was chosen as the model compound to assess the efficiency and reproducibility of fraction transfer. A step-gradient elution method was used in the first dimension (Gradient 3.a), with increasing proportion of methanol co-solvent containing a fixed concentration of caffeine, resulting in increasing quantity of caffeine being stored in a fixed-volume transfer loop. This method was chosen over gradient elution in order to ensure that the composition of the mobile phase was known precisely throughout collection in the transfer loop. The caffeine collected and transferred to dim2 was then eluted with the same gradient program as previously (gradient 3.b, identical to gradient 1.b and 2.b).
More precisely, caffeine was dissolved in the mobile phase cosolvent of pure methanol (MeOH) and examined at two different concentration levels: 0.025 mg/mL (Figure S5, shown in supplementary information) and 0.05 mg/mL (Figure 4). Co-solvent steps were applied at increasing percentages of methanol in the mobile phase, i.e., 20%, 40%, 60%, 80% and 100%. For each step, a fraction was collected, with a constant loop volume of 114 µL and a fixed loop loading time of 0.19 min. The fraction was then transferred to dim2 to observe the caffeine peak and measure peak area. The chromatograms obtained in the second-dimension analysis, showing the caffeine peaks in all conditions, are shown in Figure 4A and Figure S5A. Moreover, the peak area was plotted against the theoretical amount of caffeine transferred, computed from caffeine concentration in the mobile phase and loop volume (Figure 4B and Figure S5B).

<figure><img src="image_8.png" /><figcaption>**Figure 4:** Observation in dim2 of the transfer of caffeine contained in the mobile phase from dim1. A) Peak of caffeine observed in dim2, with loop loading in isocratic conditions (step gradient) at 20, 40, 60, 80, 100% MeOH containing 0.05 mg/mL caffeine. B) Relation between the area of caffeine peak obtained in dim2 and the actual amount transferred from dim1. C) Superposed peaks of identical quantities of caffeine introduced with different proportions of co-solvent: 40% MeOH containing 0.05 mg/mL caffeine or 80% MeOH containing 0.025 mg/mL caffeine.</figcaption></figure>

Analysis conditions dim1: ACQUITY UPC² Torus DEA (100 × 3.0 mm, 1.7 µm), co-solvent: MeOH containing 0.025 or 0.05 mg/mL caffeine, 110 bar, 30 °C, 0.6 mL/min (gradient conditions in Table 1, gradient 3.a)

Analysis conditions dim2: CHIRALPAK IB-3 (150 × 4.6 mm, 3.0 µm), co-solvent: MeOH + 0.1% MSA (gradient conditions in Table 1, gradient 3.b), 150 bar, 30 °C, 2.0 mL/min.

The results show that, for a constant caffeine concentration, increasing the percentage of co-solvent in the mobile phase, thereby increasing the concentration of caffeine in the collected fraction, has a direct and linear effect on the amount transferred to dim2.
The linear correlation coefficient was excellent for both concentrations of caffeine (Figure 4B and Figure S5B). This confirms the consistency of the quantities transferred between the two dimensions, independently of the composition of the mobile phase in dim1. Thus, under the present experimental conditions, the compressibility of the mobile phase had no measurable impact on the quantity collected and transferred in dim2. This is also confirmed by the comparison of points measured with the two different concentration levels of caffeine. For instance, introducing the 0.025 mg/mL solution at 40% methanol should yield an identical quantity to the 0.05 mg/mL solution at 20% methanol. Similarly, introducing the 0.025 mg/mL solution at 80% methanol should yield an identical quantity to the 0.05 mg/mL solution at 40% methanol. Indeed, the peak areas were very close between the compared experiments. However, the peak shapes were different, as can be observed in Figure 4C and is further discussed below.

This lack of significant effect of fluid compressibility can be explained by the stability of the experimental parameters (pressure, temperature and flow rate) during the collection and transfer stages, as well as by the robust configuration of the transfer loop, designed to minimize volume fluctuations due to the compressibility of the fluid. These observations confirm that, although CO₂ compressibility can be a potential disturbance factor in some systems, it did not interfere with transfer efficiency under the specific conditions of this study.

Another observation from Figure 4A and Figure S5A is that an increase in the percentage of co-solvent appeared to cause a broadening of the caffeine peak in dim2, possibly due to a greater quantity transferred, but also probably due to the composition of the fluid containing an increasing proportion of MeOH. This hypothesis is supported by the shoulder observed on the fronting of the peaks retrieved with the large proportions of MeOH, and by the comparison of peak shapes in Figure 4C. In the latter, identical quantities of caffeine were compared, but the peak shape was much more distorted when the proportion of methanol co-solvent was larger. This highlighted the need to further study the injection effects in the second dimension.

### 3.3. Effect of collection conditions on second-dimension injection

#### 3.3.1. Pressure

As mentioned above, pressure is an essential parameter when operating with supercritical, or in this case, subcritical fluids. Internal pressure should be maintained
above the critical pressure to maintain a high density and thus preserve analyte
solubility. While this is normally the case during the chromatographic separation, where
the fluid state is maintained with the back-pressure regulator, it is also essential to keep
the pressure constant during fraction transfer, to prevent variations that may result in
peak distortion or analyte loss.

In storage loops, the compressibility of the fluid varies depending on the composition of the fluid entering the loop [27]. This composition would differ for different fractions collected from dim1 when a gradient elution is employed. Note that the storage loops were always conditioned before the dim1 transfer with the starting conditions of dim2 gradient. However, different conditioning fluids were explored (dim1 elution composition, corresponding approximately to 75% co-solvent, and initial dim2 conditions, 15% co-solvent)and appeared to have no significant effect on the final result.

A naringin standard, analyzed in dim1 with gradient 1 mobile phase conditions, was used in a series of tests to evaluate the effect of pressure on analytical performance. Initially, the dim1 approach relied on a reversed pressure gradient, where back-pressure varies from 150 down to 110 bar. In this program, as naringin had a retention time of around 6 min (the elution composition was around 79% of MeOH), the back-pressure at the moment of its collection was about 120 bar.

In a first series of experiments, the back-pressure in dim1 was modified to assess its
effect on collection. In the first test, the back-pressure was kept constant at 110 bar
throughout the analysis, meaning that the collection of the naringin peak also took
place at this pressure. The second test maintained the initial back-pressure gradient
(150 to 110 bar), therefore corresponded to collection at 120 bar. For both
configurations, the constant back-pressure applied in dim2 was also varied, and three
values were tested: 110 bar, 150 bar (the standard pressure in this method) and 190
bar, allowing a range of experimental conditions to be examined.

The results, summarized in Table 2, indicated that the area and width at half-height of
the naringin peak in dim1 remained constant, regardless of the pressure conditions
applied (constant back-pressure of 110 bar or gradient from 150 to 110 bar). In dim2,
the widths at half-height of the peaks did not vary significantly either, regardless of the
pressure conditions when the loop was loaded in dim1 or the changes in parameters
in dim2. These widths were measured at around 0.2 min for the first peak and 0.17 min
for the second, confirming the excellent stability of the system under the experimental conditions evaluated. The corresponding chromatograms are shown in Figure S6.

These observations highlight the robustness of the pressure conditions tested but also underline the importance of the composition of the mobile phase in the storage loops, which was the next parameter examined.

### 3.3.2. Mobile phase composition

The influence of elution conditions in the first and second dimensions on analytical performance in the second dimension was evaluated. For this purpose, we examined the effect of the proportion of co-solvent (MeOH) at the moment of peak collection, which is the same as the proportion of co-solvent stored in the loops, on the transfer of analytes to the dim2 column. The aim of this study was to determine the limits of compatibility of co-solvent percentages in order to ensure optimal transfer of analytes between the two dimensions. Since the proportion of methanol co-solvent may impact polar and non-polar compounds differently, two sets of experiments were conducted. The first set of experiments was done with non-polar compounds (alkylbenzenes), and therefore required a non-polar stationary phase (C18-type) to ensure adequate retention. The second set of experiments was done with polar compounds (flavonoids) and therefore required a polar stationary phase (diethylaminopropyl-bonded silica).

#### 3.3.2.1. Non-polar compounds on non-polar column

A mixture of alkylbenzenes (with chain lengths of C4, C6, C8, C10, C12, C14) was injected in dim1 on an XSelect HSS C18 column (150 × 4.6 mm, 5.0 µm) in isocratic mode, with different percentages of methanol in the mobile phase. The conditions tested were 2%, 10%, 30%, 50%, 70% and 90% MeOH.

Analysis of the alkylbenzene mixture under these mobile phase conditions showed little impact of methanol proportion on separation and retention times from 2 to 50% MeOH (Figure 5). Only the largest portions of MeOH (70 and 90%) showed a significant retention increase. The U-shaped retention curves observed are quite typical of non-polar compounds analysis in SFC, where retention first decreases upon addition of a polar co-solvent, then increases again when solubility decreases with higher portions of polar co-solvent. This effect was previously described by Lesellier and co-workers [23,28]. In the present case, the minimal retention variation indicated that all alkylbenzenes had adequate solubility in all mobile phase compositions examined,
which made this set ideal to explore the impact of mobile phase composition on peak
collection and re-injection in isocratic mode.

<figure><img src="image_9.png" /><figcaption>**Figure 5:** Analysis of the alkylbenzene mix on XSelect HSSC18 column (150 × 4.6 mm, 5.0 µm) with varying percentages of MeOH co-solvent in isocratic conditions. 110 bar, 30°C, 1 mL/min.</figcaption></figure>

For each condition employed in dim1 elution, each alkylbenzene peak was collected
in a transfer loop then re-injected and analyzed in dim2 on a very similar stationary
phase, HSS C18 with identical dimensions from the dim1 column. The isocratic
conditions tested in dim2 were 2%, 5%, 10%, 20%, 30%, 40%, 50% and 90% MeOH.
In total, each alkylbenzene molecule was thus analyzed in 48 different conditions (6
conditions of dim1, times 8 conditions of dim2).
First, it is important to note that most of these paired conditions yielded dim2 analyses with adequate chromatographic quality. This is illustrated in Figure 6 as a summary table showing the areas of compatibility between dim1 and dim2. Three cases were distinguished and are illustrated with the chromatograms in Figure 6:

- case A: all peaks were visible and could be integrated;

- case B: all peaks were visible, but not all could be properly integrated;

- case C: some peaks were strongly distorted and could therefore not be properly integrated.

<table><thead><tr><th rowspan="2"></th><th colspan="7">Second dimension (dim2)</th></tr><tr><th>2%</th><th>5%</th><th>10%</th><th>20%</th><th>30%</th><th>40%</th><th>50%</th><th>90%</th></tr></thead><tbody><tr><th rowspan="6">First dimension (dim1)</th><th>2%</th><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><th>10%</th><td></td><td></td><td></td><td></td><td>A</td><td></td><td></td></tr><tr><th>30%</th><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><th>50%</th><td></td><td>B</td><td></td><td></td><td></td><td></td><td></td></tr><tr><th>70%</th><td></td><td></td><td>C</td><td></td><td></td><td></td><td></td></tr><tr><th>90%</th><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

**Figure 6:** Mobile phase composition compatibilities between dim1 and dim2 and typical chromatograms obtained in the different situations. Green = the peak was well resolved and could be integrated (chromatogram A), orange = the peak was visible but could not be integrated (chromatogram B), red = no peak was observed (chromatogram C).

As appears in the table, chromatographic quality was always adequate when the conditions of first and second dimension were close (diagonal area of the table). The quality was also always good when the proportion of methanol in the second dimension was larger than the proportion of methanol in the first dimension. However, issues
occurred in the opposite case, namely when the proportion of co-solvent in the first
dimension was very much larger than the proportion of co-solvent in the second
dimension. It is important to note that the dim2 conditions are actually the ones used
to push to the dim2 column the fraction that was stored in the loop with the dim1
conditions. Therefore, solvent mismatch is particularly important when a large plug of
methanol must be transferred and eluted from the dim2 column with a CO2-rich mobile
phase.

For instance, using 50% MeOH in dim1, the compounds eluted in dim2 with MeOH
percentage of 2 to 10% yielded visible peaks, but that could not be integrated due to
some baseline disturbance at the peak front (case B). In such a case, the analyte could
be detected but could not be quantified. However, all dim2 compositions from 30 to
90% yielded good chromatographic quality (case A). Similarly, for fractions collected
with a high percentage of co-solvent (70% or 90%), it was necessary to use a
sufficiently high percentage of co-solvent in dim2 (≥ 40% when dim1 is eluted at 70%,
≥ 50% when dim1 is eluted at 90%) to guarantee the elution of the analyte with a good
peak shape, thus making it detectable and quantifiable.

Further illustration of these effects is provided in Figure 7, with several chromatograms
of butylbenzene analyzed in dim2 after being collected in a storage loop under isocratic
conditions with 30%, 70% or 90% MeOH. When large proportions of MeOH were used
in the first dimension, i.e., when large quantities of MeOH were transferred to the dim2
column, the baseline was strongly distorted at the beginning of the chromatogram.
Viscous fingering and methanol adsorption on the stationary phase must be largely
responsible for this [29–33]. In the worst cases, when the difference in mobile phase
composition was the largest, the analyte could not be seen as it was included in the
distorted band. On the other hand, when the composition of mobile phase between the
two dimensions got closer, visible and measurable peaks were observed.
<figure><img src="image_13.png" /></figure>

**Figure 7:** Analysis of butylbenzene, loaded from dim1 with A) 30% MeOH, B) 70% MeOH, C) 90% MeOH, and eluted in dim 2 with different co-solvent percentages in isocratic conditions (2, 5, 10, 20, 30, 40, 50, 90%).
Other analysis conditions in dim2: HSSC18 column (150 × 4.6 mm, 5.0 µm), 110 bar, 30 °C, 1.0 mL/min

<figure><img src="image_14.png" /></figure>

**Figure 8:** Analysis of individual alkylbenzenes (from C4 to C14), loaded from dim1 with 90% MeOH in the storage loop then eluted in dim2 with different co-solvent percentages (2, 5, 10, 20, 30, 40, 50, 90%).
Analysis conditions in dim2: HSSC18 column (150 × 4.6 mm, 5.0 µm) with 2% MeOH co-solvent, 110 bar, 30 °C, 1.0 mL/min
As is usually the case in LC and SFC, these effects of injection solvents were of primary importance for the low-retention analytes, but became insignificant when analyte retention increased. This is illustrated in Figure 8, which represents chromatograms of the 6 alkylbenzenes that were eluted from dim1 and collected in the loops with 90% MeOH, but transferred and eluted from the dim2 column with 2% MeOH. It appears from these chromatograms that the short-chain alkylbenzenes yielded strongly distorted baselines where no peak could be discerned, while the long-chain alkylbenzenes yielded perfectly acceptable peaks. With these loop dimensions and this set of operating conditions, for a peak to be visible, the retention factor (k) in dim2 had to be superior to 1, and for integration to be dependable, it had to be more than 1.33. Therefore, it is preferable for the analyte to be properly held on the column to reduce the solvent effect in order to provide accurate analysis with large percentages of methanol in dim1. Finally, and opposite to previous observations [33], possible breakthrough elution of the analytes, where the analyte elutes near dead volume with the solvent front, was not obvious in these experiments.

A peculiar behavior was observed for butylbenzene (C4), and for this compound only. In Figure 7A, when the analyte was loaded in the storage loop with 30% MeOH, the peak was detectable and the peak area was measurable whatever the composition of the mobile phase in dim2. However, when it was eluted in dim2 with 50% MeOH, an unexpected effect was observed: butylbenzene was much more retained than under any other condition. In Figures 7B and 7C, the same retention shift was observed. This strange phenomenon was confirmed by several repetitions of the analyses. No plausible explanation could be found for this peculiar behavior. Note that all other alkylbenzenes followed the trend observed in Figure 5, with no anomalous behavior.

To conclude on this set of experiments, a large part of the mobile phase combinations yielded perfectly acceptable chromatographic quality. Only the largest discrepancies between the mobile phase compositions in dim1 and dim2, where the proportion of co-solvent in dim1 is much larger than the proportion of co-solvent in dim2, caused severe baseline distortion and affected the peaks of the least retained analytes. It is important to note that such large differences in mobile phase composition are unlikely to be useful in normal SFC practice. Large portions of co-solvent (above 50%) are only employed to achieve the elution of most polar compounds, when solubility in CO₂-rich mobile phases is not sufficient. In such cases, it would make no sense to elute them with a low proportion of co-solvent in the second dimension. Another important conclusion to
draw from these experiments is that problems should be easily avoided when sufficient
retention is obtained in dim2, which can usually be solved by an adequate choice of
stationary phase.

3.3.2.2. Polar compounds on polar column

While the above results were highly informative, they may be limited in their applicability since the chosen test set was only composed of non-polar analytes, and the non-polar column employed may not be representative of the majority of current SFC applications [34]. Therefore, we considered repeating these experiments with polar compounds on a polar stationary phase.

Definition of the test set

The ideal behavior observed above, with low retention variation over a wide range of mobile phase compositions (Figure 5) was unlikely to be observed again with polar compounds. Indeed, polar compounds are much more sensitive to mobile phase composition because the methanol co-solvent is interacting with them through hydrogen bonding. This is usually increasing analyte solubility all along, thereby decreasing its retention continuously when the proportion of methanol increases. As a result, it would be impossible to cover the entire range of methanol proportions tested (5 to 90%) with a single series of compounds: the low-polarity compounds would soon be eluted in the dead volume when the proportion of methanol increases, while the high-polarity compounds would never elute with low proportions of methanol. To overcome this difficulty, three sets of test compounds were defined: low, average and high polarity, yielding high, average and low solubility in CO2, therefore resulting in low, average and high retention on a polar stationary phase. The sets were divided according to the co-solvent combinations between dim1 and dim2, as shown in Figure 9. Because flavonoids comprise a large variety of molecules with a range of polarities and solubilities in CO2-methanol mixtures, they were selected for this second set of experiments. The diethylaminopropyl-bonded silica stationary phase employed in our previous 2D analysis of flavonoids in the first dimension (Acquity Torus DEA) was used here in the two chromatographic dimensions (dim1 and dim2).
<figure><img src="image_15.png" /><figcaption>**Figure 9:** Distribution of the different flavonoid sets analyzed in different conditions between dim1 and dim2.</figcaption></figure>

Set no. 1 = pinocembrin, hesperitin, pinocembroside

Set no. 2 = naringenin-7-O-glucoside, taxifolin, naringenin, alphitonin

Set no. 3 = alphitonin, epigallocatechin, epicatechin gallate

### Set no. 1: low retention

The first set consists of pinocembrin, hesperetin and pinocembroside (Figure S7). These compounds have low polarity, and are therefore easily eluted in CO₂-rich mobile phase compositions. For example, pinocembrin (Figure 9) is a simple flavanone with only 2 hydroxyl groups where one is involved in intra-molecular hydrogen bonding, and therefore has good solubility in CO₂ and rather low affinity with the polar stationary phase. At high co-solvent percentages, these three compounds are therefore eluted in the dead time, without significant interaction with the stationary phase. These compounds need to be analyzed at low co-solvent percentages (5-30%) to allow for sufficient retention in a reasonable analysis time (no more than 30 min).

### Set no. 2: moderate retention

The second set consists of naringenin-7-O-glucoside, taxifolin, naringenin and alphitonin. Some of these compounds contain several hydroxyl groups, like taxifolin, or sugar moieties, like naringenin-7-O-glucoside, which increase their polarity. This would both reduce their solubility in CO₂ mobile phases and favor retention on the polar stationary phase. These compounds require a minimum proportion of 30% co-solvent
to elute with a reasonable analysis time. In dim1, where they are injected as a mixture, a large proportion of co-solvent (above 50%) was inadequate as they all eluted in the same retention window and could therefore not be transferred individually to the second dimension. However, in the second injected one by one, it was possible to use high percentages of co-solvent (up to 90%).

### Set no. 3: high retention

This third set comprises alphitonin, epigallocatechin and epicatechin gallate. These compounds are highly hydroxylated, notably due to the trihydroxylated gallic ester function present as on epicatechin gallate. These characteristics greatly decrease their CO₂ solubility and increase their affinity for the polar stationary phase, resulting in very high retention. If they were analyzed at low percentages of co-solvent, elution may require several hours, or would never happen if they would not be properly dissolved. They were therefore analyzed only at high co-solvent percentages (70 to 90%) to limit analysis times.

### Inaccessible combinations

Two combination zones (in gray, Figure 9) were not accessible as we could not identify compounds that would be both sufficiently soluble in low methanol proportion and sufficiently retained at high methanol proportion. At low dim1 and high dim2 percentages, set n°1 compounds would elute in dead time in dim2, and thus yield no measurable peak. Conversely, at high dim1 and low dim2 percentages, the compounds in set n°3 would have excessive retention in dim2 (or even remain on the column), making analysis unfeasible within a reasonable time. To see the impact of different combinations of dim1 and dim2, it is essential that retention remains in a balanced zone, neither too low (co-elution) nor too high (too long analysis time and detection issues). Perhaps these missing combinations are not an issue from a practical point of view, since it is unlikely that any polar compound would require such different mobile phase compositions in a 2D-SFC system.

### Definition of desirability functions and application to different conditions

With the non-polar compounds examined previously, the behaviors were rather consistent between different analytes, and it was thus easy to draw a general conclusion as was presented in Figure 6. With the polar compounds however, different
behaviors could be observed between the different analytes eluted in the same mobile phase conditions. Therefore, a new strategy needed to be developed to draw generally applicable conclusions. Derringer's desirability functions seem relevant in this case [35]. These functions make it possible to compare different co-solvent conditions on the basis of selected criteria. The first step was to choose different criteria that would be relevant to our study. Then the 3 selected criteria had to be translated into desirability functions (d1, d2, d3). These functions are described in Figure 10.

<figure><img src="image_16.png" /><figcaption>**Figure 10:** Derringer functions based on: visibility of a peak shape (d1), ratio of peak width at half height and area in dim2 relative to dim1 (d2) and ratio of asymmetry at 10% peak height in dim2 relative to dim1 (d3).</figcaption></figure>

## Visibility of peaks (d1)

The first criterion is the visibility of the peak represented by the d1 function (Figure 10). This criterion evaluates the presence of an identifiable peak in the chromatogram, regardless of its width or symmetry. For example, some compounds that were highly impacted by the solvent effect were hardly discernible from the baseline, making identification impossible. This criterion is the most important because it determines the ability to detect and possibly quantify the compound of interest. Thus, the presence of a peak was assigned a value of 1 and the absence of a peak a value of 0. All the values for each analyte in each pair or elution conditions are presented in Table S2.

## Ratio of peak width at half height and area in dim2 to dim1 (d2)

The evaluation of the quality of the peaks obtained in dim2 cannot be based exclusively on the width at half height of the peak. Indeed, this measurement loses its significance in the case of partial transfer between dim1 and dim2. In the case of large peaks, at the time of transfer, only a fraction of the peak is collected, and therefore comparing peak width between the two dimensions makes no sense.
This can be explained by the way the instrument operates: the compounds were stored between the two dimensions in 114 µL loops. For example, when hesperetin was analyzed at 5% co-solvent and a flow rate of 1.5 mL/min (Figure 11), the peak width at baseline was about 2 min, whereas the volume sampled corresponded to only 0.08 min, i.e., a very small portion of the initial chromatographic signal. Thus, the widths measured in dim2 represented only a small part of the collected peak, making any direct comparison of peak width irrelevant.

<figure><img src="image_17.png" /><figcaption>**Figure 11:** Chromatogram of dim1 analysis (orange) of the hesperitin peak eluted with 5% co-solvent (isocratic conditions). The superposed chromatogram (blue) shows the same dim1 analysis recorded when the peak is cut to be loaded in a storage loop.</figcaption></figure>

Analysis conditions: ACQUITY UPC² Torus DEA (100 × 3.0 mm, 1.7 µm), co-solvent: 5% MeOH comprising 0.1% MSA, 150 bar, 30 °C, 1 mL/min.

To overcome this bias, the width at half-height was normalized by the area of the peak, in order to calculate a width-to-area ratio. This approach makes it possible to compare peaks corresponding to similar relative quantities, while incorporating a correction linked to the signal intensity.

The ratios obtained for one analyte were then compared between dim2 and dim1. This ratio was integrated into the Derringer desirability function according to the following principles: a low ratio between dim2 and dim1 indicated good focusing of the compound in dim2, and a desirability value close to 1 was then assigned. Conversely, a high ratio indicated broadening or poor focusing, leading to a desirability value close to 0. All measured values are presented in Table S2.
## Ratio of asymmetry at 10% peak height in dim2 to dim1 (d3)

Chromatographic peak asymmetry is another important indicator to consider, in order to evaluate the quality of the peak shape. To assess the impact of the mobile phase compositions on dim2 peak shapes, a ratio of asymmetry values was calculated by comparing the asymmetry at 10% peak height in dim2 to that measured in dim1.

However, in some cases, the peaks were too distorted (highly flattened or irregular) for the asymmetry to be calculated reliably. In such cases, a zero value was assigned to the d3 criterion.

The desirability value d3 was defined according to the ratio of asymmetry values (dim2/dim1). When asymmetry was unchanged or slightly improved, the ratio was less than or equal to 1 and a desirability value of 1 was assigned. When the ratio increased, indicating a deterioration in symmetry in dim2, the desirability value decreased, tending towards 0 and being less restrictive between 1 and 2 (Figure 10).

This criterion thus makes it possible to integrate the quality of the peak profile into the overall assessment of the system's performance. All values are presented in Table S2.

## Total desirability

In the end, a total desirability value (D) was calculated as the geometric mean of the three functions:

$$D = \sqrt[3]{d_1 \times d_2 \times d_3} \qquad (1)$$

It is possible to add weighting coefficients to add importance to certain criteria. In this case, the choice was made not to add any and instead to assign the same contribution to each criterion. It would also have been possible to calculate an arithmetic mean, but this was considered not restrictive enough. In fact, with the geometric mean, the absence of a peak (d1) is prohibitive, so when d1 is 0, D is also 0.

The desirability value (D) was calculated for each analyte eluted in each dim1/dim2 combination (Table 3). Areas of high desirability (in green) correspond to optimal combinations between the two dimensions. Similar to the observations on non-polar compounds, the diagonal line where the compositions of dim1 and dim2 eluents are close, yields the best results. However, the best results are mostly located around intermediate percentages of co-solvent in the first dimension (10 to 50%). Conversely,
some areas show low or zero desirability (orange or red) and are mostly encountered in cases of strong solvent mismatch, e.g., 50% in dim1 with 5 or 10% in dim2, or 90% in dim1 with 50% in dim2. These conditions should be avoided as they yielded invisible, poorly defined or highly asymmetric peaks.

In addition, again solvent effects were more critical to the least retained compounds. For instance, the combination of 5% in dim1 and 5% in dim2 showed a lower desirability for the first eluted analyte than for the second, due to the high asymmetry of the first. Similarly, the configuration of 70% in dim1 and 50% in dim2 showed a zero-value for total desirability for the first two peaks, but high desirability for the last. So again, and consistent with the literature, most retained compounds are less affected by the injection solvent composition.

Empty cells (light grey) correspond to non-existing cases. For instance, in the 70/50 area, the set no. 3 had only 3 standards, so the 4th column is empty. The analysis of pinocembroside with 5% co-solvent (peak 3 in set no. 1) was not evaluated because its retention was too high (over 35 min) to be relevant in this study.

Overall, it appears that the three series of flavonoids allowed a wide range of dim1/dim2 combinations to be explored. To summarize the observations from Table 3, Table 4 was obtained by averaging the total peak desirabilities for each condition. It shows a clear trend: when dim1 used a higher percentage of methanol than dim2, peak quality was significantly degraded. This was the case, for example, for the combination of 50% in dim1 and 5% in dim2. On the contrary, a reverse transfer, with a lower percentage of methanol in dim1 and a higher percentage in dim2 (e.g., 30% in dim1 and 70% in dim2), gave good results. This is again similar to the observations from the non-polar test compounds.

Finally, although some red zones remain, indicating incompatible conditions for transfer between dimensions, it is important to remember that these configurations are rarely used in SFC. In addition, the incompatibility mostly applies to the least retained compounds.

## 4. Conclusion

This study enabled a detailed assessment of the impact of different parameters of the two-dimensional system on its analytical performance. The addition of extra valves and tubing was examined, to ensure that the additional dead volumes did not significantly increase retention times or alter chromatographic quality in both dimensions. The
results show that the gradient delay varies somewhat between the one- and two-
dimensional systems, possibly due to changes in the internal pressure, but that the
chromatographic quality was well preserved. Actually, the evaluation of peak widths
revealed that, although dim1 was not affected by the switch to a two-dimensional
system, dim2 showed a slight increase in widths at half-height. This phenomenon,
although measurable, remains within acceptable limits and does not alter the overall
resolution of the system.

Regarding the size of transfer loops, it was observed that there would be little interest
in reducing the loop volume, as no resolution gain was observed when dividing a peak
in two sections.

A thorough examination of all possible pairs of dim1/dim2 elution conditions on the
performance of dim2 showed that most combinations of CO2-MeOH compositions
offered satisfying chromatographic quality, but that strong solvent mismatch could
disrupt the detection and integration of the analytes that were least retained on the
column. This effect was particularly marked when peaks were collected and stored in
the loop with a high percentage of MeOH, followed by analysis in dim2 at a much lower
percentage. However, this strong solvent mismatch should have little interest in
practical operation of a 2D-SFC system where (i) the proportion of co-solvent is rarely
raised above 50% in usual practice and (ii) solubility issues would make it difficult to
elute an analyte in both dimensions with strongly different mobile phase compositions.
The case of alkylbenzenes being nicely eluted with a wide range of methanol (5 to
90%) is quite exceptional. Furthermore, adjusting operating conditions in the second
dimension to obtain sufficient retention should largely mitigate this effect in displacing
the analyte elution band out of the injection solvent disturbances.

In conclusion, the two-dimensional system set up is reliable and optimized for a wide
range of analytical conditions. A good understanding of the interactions between the
two dimensions means that undesirable effects can be anticipated and corrected,
ensuring reliable separation of the analytes studied.

Although not explored in this paper, combining different polarities of stationary phases
in the first and second dimensions should yield comparable conclusions, since the
results obtained with non-polar and polar compounds basically yielded the same
observations. Preliminary assumptions suggest that similar performance could be
achieved on different stationary phases, provided that the retention behavior is well
understood and the solvent effects are properly managed.
Finally, these results pave the way for the optimization of two-dimensional chromatographic systems in SFC, guaranteeing stable and efficient performance.

## Acknowledgments

Extrasynthese is acknowledged for the kind gift of flavonoid standards. Isabelle François is acknowledged for fruitful discussions. Région Centre Val-de-Loire is acknowledged for a PhD grant allocated to Laurine Réset. ICOA is supported by the University of Orleans, the National Centre for Scientific Research (CNRS), the projects CHemBio (FEDER-FSE 2014-2020-EX003677), Valbiocosm (FEDER-FSE 2014-2020-EX003202), Techsab (FEDER-FSE 2014-2020-EX011313), QUALICHIM (APR-IA-PF 2021-00149467), Project ESTIM-ICOA (CPER / FEDER-FSE+ 2021-2027-00022860), the RTR Motivhealth (2019-00131403) and the Labex programs SynOrg (ANR-11-LABX-0029) and IRON (ANR-11-LABX-0018-01).

## CRediT author contributions

**Laurine Réset:** Conceptualization, Methodology, Investigation, Writing – Original Draft, Visualization

**Clément De Saint Jores:** Conceptualization, Methodology, Validation, Writing – Review & Editing, Supervision

**Caroline West:** Conceptualization, Resources, Writing – Review & Editing, Supervision, Funding acquisition
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Supporting Information to:

Examining the conditions to achieve successful fraction transfer in two-dimensional supercritical fluid chromatography

Laurine Réset, Clément De Saint Jores*, Caroline West

Université d'Orléans, CNRS, ICOA, UMR 7311, Orléans, France

<table><tr><td>Table S1</td><td>38</td></tr><tr><td>Table S2</td><td>38</td></tr><tr><td>Table S3</td><td>39</td></tr><tr><td>Table S4</td><td>40</td></tr><tr><td>Table S5</td><td>41</td></tr><tr><td>Figure S1</td><td>42</td></tr><tr><td>Figure S2</td><td>42</td></tr><tr><td>Figure S3</td><td>43</td></tr><tr><td>Figure S4</td><td>43</td></tr><tr><td>Figure S5</td><td>44</td></tr><tr><td>Figure S6</td><td>45</td></tr><tr><td>Figure S7</td><td>46</td></tr><tr><td>Figure S8</td><td>47</td></tr><tr><td>Figure S9</td><td>48</td></tr><tr><td>Figure S10</td><td>49</td></tr><tr><td>Figure S11</td><td>50</td></tr><tr><td>Figure S12</td><td>51</td></tr><tr><td>Figure S13</td><td>52</td></tr><tr><td>Figure S14</td><td>53</td></tr><tr><td>Figure S15</td><td>54</td></tr></table>
<table><thead><tr><th>Method</th><th>system</th><th>Peak</th><th>Retention time (min)</th><th>Area (µV*sec)</th><th>Width @50%</th><th>Resolution (min)</th></tr></thead><tbody><tr><td rowspan="2">dim1</td><td>sys1D</td><td>1</td><td>6.032</td><td>4491427</td><td>0.0410</td><td>-</td></tr><tr><td>sys2D</td><td>1</td><td>6.017</td><td>4772346</td><td>0.0461</td><td>-</td></tr><tr><td rowspan="4">dim2</td><td rowspan="2">sys1D</td><td>1</td><td>11.188</td><td>1080833</td><td>0.165</td><td rowspan="2">4.67</td></tr><tr><td>2</td><td>12.418</td><td>967313</td><td>0.146</td></tr><tr><td rowspan="2">sys2D</td><td>1</td><td>11.484</td><td>1178090</td><td>0.209</td><td rowspan="2">4.46</td></tr><tr><td>2</td><td>12.928</td><td>1054079</td><td>0.173</td></tr></tbody></table>

**Table S1:** data measured in dim1 and dim2 from the analysis of a naringin standard performed on a one-dimensional system (sys1D) and a two-dimensional system (sys2D).

Analysis conditions for dim1: ACQUITY UPC² Torus DEA (100 × 3.0 mm, 1.7 µm), co-solvent: MeOH + 0.1% MSA, 150-110 bar, 1.7-0.6 mL/min (gradient conditions in Table 1, gradient 1.a)

Analysis conditions for dim2: CHIRALPAK IB-3 (150 × 4.6 mm, 3.0 µm), co-solvent: MeOH + 0.1% MSA (gradient conditions in Table 1, gradient 1.b), 150 bar, 2.0 mL/min.

<table><thead><tr><th rowspan="3"></th><th rowspan="3"></th><th colspan="24">Second dimension (dim2)</th></tr><tr><th colspan="3">5%</th><th colspan="3">10%</th><th colspan="3">30%</th><th colspan="3">50%</th><th colspan="3">70%</th><th colspan="3">90%</th></tr><tr><th>1</th><th>2</th><th>3</th><th>1</th><th>2</th><th>3</th><th>1</th><th>2</th><th>3</th><th>1</th><th>2</th><th>3</th><th>4</th><th>1</th><th>2</th><th>3</th><th>4</th><th>1</th><th>2</th><th>3</th><th>4</th></tr></thead><tbody><tr><td rowspan="4">d1</td><td>5%</td><td>1.00</td><td>1.00</td><td></td><td>1.00</td><td>1.00</td><td></td><td>1.00</td><td>1.00</td><td></td><td>1.00</td><td>1.00</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>10%</td><td>1.00</td><td>1.00</td><td></td><td>1.00</td><td>1.00</td><td>1.00</td><td>1.00</td><td>1.00</td><td>1.00</td><td>1.00</td><td>1.00</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>30%</td><td>1.00</td><td>1.00</td><td></td><td>1.00</td><td>1.00</td><td>1.00</td><td>1.00</td><td>1.00</td><td>1.00</td><td>1.00</td><td>1.00</td><td>1.00</td><td>1.00</td><td>1.00</td><td>1.00</td><td>1.00</td><td>1.00</td><td>1.00</td><td>1.00</td><td>1.00</td><td>1.00</td></tr><tr><td>50%</td><td>0.00</td><td>0.00</td><td></td><td>0.00</td><td>0.00</td><td>0.00</td><td>1.00</td><td>1.00</td><td>1.00</td><td>1.00</td><td>1.00</td><td>1.00</td><td>1.00</td><td>1.00</td><td>1.00</td><td>1.00</td><td>1.00</td><td>1.00</td><td>1.00</td><td>1.00</td><td>1.00</td></tr><tr><td rowspan="4">d2</td><td>70%</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>1.00</td><td>1.00</td><td>1.00</td><td></td><td>1.00</td><td>1.00</td><td>1.00</td><td></td><td>1.00</td><td>1.00</td><td>1.00</td><td></td></tr><tr><td>90%</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>0.00</td><td>0.00</td><td>0.00</td><td></td><td>1.00</td><td>1.00</td><td>0.00</td><td></td><td>1.00</td><td>1.00</td><td>1.00</td><td></td></tr><tr><td>5%</td><td>0.84</td><td>0.31</td><td></td><td>0.87</td><td>0.08</td><td></td><td>0.91</td><td>0.17</td><td></td><td>0.58</td><td>0.00</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>10%</td><td>0.87</td><td>0.86</td><td></td><td>0.80</td><td>0.56</td><td>0.00</td><td>0.87</td><td>0.63</td><td>0.31</td><td>0.92</td><td>0.91</td><td>0.72</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="4">d3</td><td>30%</td><td>0.05</td><td>0.71</td><td></td><td>0.35</td><td>0.34</td><td>0.00</td><td>0.75</td><td>0.75</td><td>0.72</td><td>0.88</td><td>0.88</td><td>0.82</td><td>0.56</td><td>0.82</td><td>0.85</td><td>0.77</td><td>0.45</td><td>0.91</td><td>0.99</td><td>0.92</td><td>0.61</td></tr><tr><td>50%</td><td>0.00</td><td>0.00</td><td></td><td></td><td></td><td></td><td>1.00</td><td>1.00</td><td>1.00</td><td>0.73</td><td>0.76</td><td>0.73</td><td>0.73</td><td>0.67</td><td>0.79</td><td>0.73</td><td>0.72</td><td>0.88</td><td>0.99</td><td>0.93</td><td>0.80</td></tr><tr><td>70%</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>0.00</td><td>0.00</td><td>0.38</td><td></td><td>0.68</td><td>0.79</td><td>0.74</td><td></td><td>0.87</td><td>0.51</td><td>0.96</td><td></td></tr><tr><td>90%</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>0.00</td><td>0.00</td><td>0.00</td><td></td><td>0.42</td><td>0.00</td><td>0.00</td><td></td><td>0.63</td><td>0.99</td><td>0.91</td><td></td></tr><tr><td rowspan="4"></td><td>5%</td><td>0.08</td><td>1.00</td><td></td><td>1.00</td><td>0.76</td><td></td><td>1.00</td><td>1.00</td><td></td><td>0.25</td><td>1.00</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>10%</td><td>1.00</td><td>1.00</td><td></td><td>1.00</td><td>0.96</td><td>1.00</td><td>0.84</td><td>0.96</td><td>1.00</td><td>1.00</td><td>1.00</td><td>1.00</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>30%</td><td>0.40</td><td>1.00</td><td></td><td>1.00</td><td>1.00</td><td>1.00</td><td>0.44</td><td>0.51</td><td>0.14</td><td>1.00</td><td>1.00</td><td>1.00</td><td>1.00</td><td>1.00</td><td>1.00</td><td>1.00</td><td>1.00</td><td>1.00</td><td>1.00</td><td>1.00</td></tr><tr><td>50%</td><td>0.00</td><td>0.00</td><td></td><td>0.00</td><td>0.00</td><td>0.00</td><td>0.75</td><td>0.62</td><td>0.45</td><td>1.00</td><td>1.00</td><td>1.00</td><td>1.00</td><td>0.92</td><td>1.00</td><td>1.00</td><td>1.00</td><td>1.00</td><td>1.00</td><td>1.00</td></tr><tr><td rowspan="2"></td><td>70%</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>0.17</td><td>0.55</td><td>1.00</td><td></td><td>0.11</td><td>0.00</td><td>0.08</td><td></td><td>1.00</td><td>0.72</td><td>1.00</td><td></td></tr><tr><td>90%</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>0.00</td><td>0.00</td><td>0.00</td><td></td><td>0.60</td><td>0.22</td><td>0.00</td><td></td><td>0.10</td><td>0.17</td><td>0.15</td><td></td></tr></tbody></table>

**Table S2:** synthesis of the different criteria (d1, d2, d3) of the Derringer functions for each flavonoid in dim1 and dim2 condition. Data are calculated per peak in each condition. d1 = peak visibility, d2 = ratio of peak width at half height and area in dim2 relative to dim1, d3 = ratio of asymmetry at 10% peak height in dim2 relative to dim1.
<table><caption>**Table S3:** Complete data from the analyses of set n°1 under dim1 and dim2 conditions: retention time, peak area, peak width at half-height (w50%), peak width at half-height/area ratio (w50%/area), and number of theoretical plates (N). peak 1 = pinocembrin, peak 2 = hesperitin, peak 3 = pinocembroside.</caption><tbody><tr><td rowspan="20">Set n°1</td><td rowspan="5">5%</td><td>tR</td><td>4.00</td><td>28.58</td><td rowspan="5">5%</td><td>tR</td><td>3.80</td><td>23.81</td></tr><tr><td>area</td><td>2760728.00</td><td>2505869.00</td><td>area</td><td>640972.00</td><td>110453.00</td></tr><tr><td>w50%</td><td>0.25</td><td>1.20</td><td>w50%</td><td>0.09</td><td>0.44</td></tr><tr><td>asym 10%</td><td>0.85</td><td>1.22</td><td>asym 10%</td><td>3.07</td><td>1.77</td></tr><tr><td>w50%/area</td><td>0.00</td><td>0.00</td><td>w50%/area</td><td>0.00</td><td>0.00</td></tr><tr><td>N</td><td>298.04</td><td>670.88</td><td>N</td><td>1980.98</td><td>3436.10</td></tr><tr><td rowspan="5">10%</td><td>tR</td><td>4.15</td><td>29.66</td><td rowspan="5">10%</td><td>tR</td><td>1.67</td><td>6.24</td></tr><tr><td>area</td><td>1117047.00</td><td>997584.00</td><td>area</td><td>645243.00</td><td>99437.00</td></tr><tr><td>w50%</td><td>0.11</td><td>0.66</td><td>w50%</td><td>0.03</td><td>0.10</td></tr><tr><td>asym 10%</td><td>1.20</td><td>1.49</td><td>asym 10%</td><td>1.52</td><td>1.36</td></tr><tr><td>w50%/area</td><td>0.00</td><td>0.00</td><td>w50%/area</td><td>0.00</td><td>0.00</td></tr><tr><td>N</td><td>1780.59</td><td>2359.59</td><td>N</td><td>3464.73</td><td>4933.13</td></tr><tr><td rowspan="5">50%</td><td>tR</td><td>4.07</td><td>29.15</td><td rowspan="5">30%</td><td>tR</td><td>1.13</td><td>1.65</td></tr><tr><td>area</td><td>1123859.00</td><td>1004745.00</td><td>area</td><td>1017293.00</td><td>102663.00</td></tr><tr><td>w50%</td><td>0.11</td><td>0.67</td><td>w50%</td><td>0.02</td><td>0.02</td></tr><tr><td>asym 10%</td><td>1.16</td><td>1.49</td><td>asym 10%</td><td>1.52</td><td>1.46</td></tr><tr><td>w50%/area</td><td>0.00</td><td>0.00</td><td>w50%/area</td><td>0.00</td><td>0.00</td></tr><tr><td>N</td><td>1732.89</td><td>2260.26</td><td>N</td><td>4406.47</td><td>5487.00</td></tr><tr><td rowspan="5">10%</td><td>tR</td><td>4.07</td><td>29.15</td><td rowspan="5">50%</td><td>tR</td><td>0.97</td><td>0.92</td></tr><tr><td>area</td><td>1123859.00</td><td>1004745.00</td><td>area</td><td>181766.00</td><td>24314.00</td></tr><tr><td>w50%</td><td>0.11</td><td>0.67</td><td>w50%</td><td>0.02</td><td>0.02</td></tr><tr><td>asym 10%</td><td>1.16</td><td>1.49</td><td>asym 10%</td><td>1.36</td><td>1.01</td></tr><tr><td>w50%/area</td><td>0.00</td><td>0.00</td><td>w50%/area</td><td>0.00</td><td>0.00</td></tr><tr><td>N</td><td>1732.89</td><td>2260.26</td><td>N</td><td>3666.87</td><td>3721.77</td></tr><tr><td rowspan="20">30%</td><td rowspan="5">5%</td><td>tR</td><td>1.53</td><td>6.91</td><td rowspan="5">5%</td><td>tR</td><td>3.77</td><td>23.99</td></tr><tr><td>area</td><td>2913601.00</td><td>2635776.00</td><td>area</td><td>1749131.00</td><td>668936.00</td></tr><tr><td>w50%</td><td>0.10</td><td>0.26</td><td>w50%</td><td>0.21</td><td>0.55</td></tr><tr><td>asym 10%</td><td>2.14</td><td>0.71</td><td>asym 10%</td><td>1.08</td><td>1.38</td></tr><tr><td>w50%/area</td><td>0.00</td><td>0.00</td><td>w50%/area</td><td>0.00</td><td>0.00</td></tr><tr><td>N</td><td>269.40</td><td>851.92</td><td>N</td><td>366.00</td><td>2271.75</td></tr><tr><td rowspan="5">10%</td><td>tR</td><td>1.53</td><td>6.91</td><td rowspan="5">10%</td><td>tR</td><td>1.63</td><td>6.09</td></tr><tr><td>area</td><td>2913601.00</td><td>2635776.00</td><td>area</td><td>774721.00</td><td>233040.00</td></tr><tr><td>w50%</td><td>0.10</td><td>0.26</td><td>w50%</td><td>0.06</td><td>0.11</td></tr><tr><td>asym 10%</td><td>2.14</td><td>0.71</td><td>asym 10%</td><td>1.42</td><td>1.17</td></tr><tr><td>w50%/area</td><td>0.00</td><td>0.00</td><td>w50%/area</td><td>0.00</td><td>0.00</td></tr><tr><td>N</td><td>269.40</td><td>851.92</td><td>N</td><td>1009.43</td><td>3652.97</td></tr><tr><td rowspan="5">50%</td><td>tR</td><td>1.60</td><td>7.01</td><td rowspan="5">30%</td><td>tR</td><td>1.12</td><td>1.65</td></tr><tr><td>area</td><td>1081386.00</td><td>965078.00</td><td>area</td><td>851464.00</td><td>257777.00</td></tr><tr><td>w50%</td><td>0.05</td><td>0.12</td><td>w50%</td><td>0.02</td><td>0.03</td></tr><tr><td>asym 10%</td><td>1.22</td><td>1.17</td><td>asym 10%</td><td>1.94</td><td>1.67</td></tr><tr><td>w50%/area</td><td>0.00</td><td>0.00</td><td>w50%/area</td><td>0.00</td><td>0.00</td></tr><tr><td>N</td><td>1473.62</td><td>3777.38</td><td>N</td><td>2819.82</td><td>4437.93</td></tr><tr><td rowspan="5">50%</td><td>tR</td><td>1.60</td><td>7.01</td><td rowspan="5">50%</td><td>tR</td><td>0.96</td><td>1.08</td></tr><tr><td>area</td><td>1081386.00</td><td>965078.00</td><td>area</td><td>1686765.00</td><td>632807.00</td></tr><tr><td>w50%</td><td>0.05</td><td>0.12</td><td>w50%</td><td>0.02</td><td>0.02</td></tr><tr><td>asym 10%</td><td>1.22</td><td>1.17</td><td>asym 10%</td><td>1.58</td><td>1.56</td></tr><tr><td>w50%/area</td><td>0.00</td><td>0.00</td><td>w50%/area</td><td>0.00</td><td>0.00</td></tr><tr><td>N</td><td>1473.62</td><td>3777.38</td><td>N</td><td>2868.52</td><td>3621.48</td></tr><tr><td rowspan="20">50%</td><td rowspan="5">5%</td><td>tR</td><td>0.89</td><td>1.53</td><td rowspan="5">5%</td><td>tR</td><td>3.17</td><td>24.32</td></tr><tr><td>area</td><td>1618543.00</td><td>1513334.00</td><td>area</td><td>959051.00</td><td>902875.00</td></tr><tr><td>w50%</td><td>0.03</td><td>0.04</td><td>w50%</td><td>0.85</td><td>1.54</td></tr><tr><td>asym 10%</td><td>1.42</td><td>1.34</td><td>asym 10%</td><td>2.11</td><td>0.31</td></tr><tr><td>w50%/area</td><td>0.00</td><td>0.00</td><td>w50%/area</td><td>0.00</td><td>0.00</td></tr><tr><td>N</td><td>836.47</td><td>1531.70</td><td>N</td><td>16.52</td><td>294.69</td></tr><tr><td rowspan="5">10%</td><td>tR</td><td>0.89</td><td>1.53</td><td rowspan="5">10%</td><td>tR</td><td>1.51</td><td>5.96</td></tr><tr><td>area</td><td>1618543.00</td><td>1513334.00</td><td>area</td><td>915552.00</td><td>445454.00</td></tr><tr><td>w50%</td><td>0.03</td><td>0.04</td><td>w50%</td><td>0.22</td><td>0.31</td></tr><tr><td>asym 10%</td><td>1.42</td><td>1.34</td><td>asym 10%</td><td>1.18</td><td>0.52</td></tr><tr><td>w50%/area</td><td>0.00</td><td>0.00</td><td>w50%/area</td><td>0.00</td><td>0.00</td></tr><tr><td>N</td><td>836.47</td><td>1531.70</td><td>N</td><td>56.54</td><td>430.83</td></tr><tr><td rowspan="5">10%</td><td>tR</td><td>0.88</td><td>1.51</td><td rowspan="5">30%</td><td>tR</td><td>1.07</td><td>1.58</td></tr><tr><td>area</td><td>1570452.00</td><td>1459786.00</td><td>area</td><td>1473408.00</td><td>1327649.00</td></tr><tr><td>w50%</td><td>0.03</td><td>0.04</td><td>w50%</td><td>0.08</td><td>0.09</td></tr><tr><td>asym 10%</td><td>1.42</td><td>1.35</td><td>asym 10%</td><td>2.51</td><td>2.28</td></tr><tr><td>w50%/area</td><td>0.00</td><td>0.00</td><td>w50%/area</td><td>0.00</td><td>0.00</td></tr><tr><td>N</td><td>816.68</td><td>1518.95</td><td>N</td><td>222.40</td><td>336.72</td></tr><tr><td rowspan="20">50%</td><td rowspan="5">5%</td><td>tR</td><td>0.68</td><td>0.85</td><td rowspan="5">5%</td><td>tR</td><td></td><td></td></tr><tr><td>area</td><td>1650917.00</td><td>1302196.00</td><td>area</td><td></td><td></td></tr><tr><td>w50%</td><td>0.04</td><td>0.03</td><td>w50%</td><td></td><td></td></tr><tr><td>asym 10%</td><td>0.62</td><td>1.49</td><td>w50%/area</td><td></td><td></td></tr><tr><td>w50%/area</td><td>0.00</td><td>0.00</td><td>w50%/area</td><td></td><td></td></tr><tr><td>N</td><td>356.25</td><td>788.82</td><td>N</td><td></td><td></td></tr><tr><td rowspan="5">10%</td><td>tR</td><td>0.68</td><td>0.85</td><td rowspan="5">10%</td><td>tR</td><td>1.49</td><td>5.95</td></tr><tr><td>area</td><td>1650917.00</td><td>1302196.00</td><td>area</td><td>810872.00</td><td>536986.00</td></tr><tr><td>w50%</td><td>0.04</td><td>0.03</td><td>w50%</td><td></td><td></td></tr><tr><td>asym 10%</td><td>0.62</td><td>1.49</td><td>asym 10%</td><td></td><td></td></tr><tr><td>w50%/area</td><td>0.00</td><td>0.00</td><td>w50%/area</td><td></td><td></td></tr><tr><td>N</td><td>356.25</td><td>788.82</td><td>N</td><td></td><td></td></tr><tr><td rowspan="5">10%</td><td>tR</td><td>0.69</td><td>0.86</td><td rowspan="5">30%</td><td>tR</td><td>1.01</td><td>1.45</td></tr><tr><td>area</td><td>1825579.00</td><td>1536434.00</td><td>area</td><td>3228821.00</td><td>3765773.00</td></tr><tr><td>w50%</td><td>0.04</td><td>0.03</td><td>w50%</td><td>0.18</td><td>0.30</td></tr><tr><td>asym 10%</td><td>0.86</td><td>1.52</td><td>asym 10%</td><td>2.06</td><td>2.13</td></tr><tr><td>w50%/area</td><td>0.00</td><td>0.00</td><td>w50%/area</td><td>0.00</td><td>0.00</td></tr><tr><td>N</td><td>382.19</td><td>762.25</td><td>N</td><td>37.26</td><td>27.36</td></tr></tbody></table>
<table><caption>**Table S4:** Complete data from analyses of set n°2 under dim1 and dim2 conditions: retention time, peak area, peak width at half-height (w50%), peak width at half-height/area ratio (w50%/area), and number of theoretical plates (N). peak 1 = naringenin-7-O-glucoside, peak 2 = taxifolin, peak 3 = naringenin, peak 4 = alphitomin.</caption><thead><tr><th rowspan="2"></th><th colspan="4">dim1</th><th colspan="4">dim2</th></tr><tr><th>pic1</th><th>pic2</th><th>pic3</th><th>pic4</th><th>pic1</th><th>pic2</th><th>pic3</th><th>pic4</th></tr></thead><tbody><tr><td rowspan="12">Set n°2</td><td rowspan="6">30%</td><td>tR</td><td>1.67</td><td>5.27</td><td>6.91</td><td>16.35</td><td>tR</td><td>1.09</td><td>1.45</td><td>1.85</td><td>2.89</td></tr><tr><td>area</td><td>1962938.00</td><td>1096118.00</td><td>1264545.00</td><td>1600717.00</td><td>area</td><td>1705395.00</td><td>770202.00</td><td>664185.00</td><td>346852.00</td></tr><tr><td>w50%</td><td>0.05</td><td>0.10</td><td>0.12</td><td>0.29</td><td>w50%</td><td>0.03</td><td>0.03</td><td>0.04</td><td>0.05</td></tr><tr><td>asym 10%</td><td>1.38</td><td>0.86</td><td>1.02</td><td>1.03</td><td>asym 10%</td><td>1.61</td><td>1.41</td><td>1.39</td><td>1.34</td></tr><tr><td>w50%/area</td><td>0.00</td><td>0.00</td><td>0.00</td><td>0.00</td><td>w50%/area</td><td>0.00</td><td>0.00</td><td>0.00</td><td>0.00</td></tr><tr><td>N</td><td>1554.93</td><td>3072.89</td><td>3990.17</td><td>3674.26</td><td>N</td><td>1487.87</td><td>2713.59</td><td>3191.35</td><td>4048.07</td></tr><tr><td rowspan="6">50%</td><td>tR</td><td>1.67</td><td>5.27</td><td>6.91</td><td>16.34</td><td>tR</td><td>1.51</td><td>1.64</td><td>1.87</td><td>2.21</td></tr><tr><td>area</td><td>1961726.00</td><td>1086993.00</td><td>1264096.00</td><td>1636887.00</td><td>area</td><td>2821080.00</td><td>1334018.00</td><td>1286208.00</td><td>678036.00</td></tr><tr><td>w50%</td><td>0.04</td><td>0.10</td><td>0.12</td><td>0.29</td><td>w50%</td><td>0.04</td><td>0.03</td><td>0.04</td><td>0.04</td></tr><tr><td>asym 10%</td><td>1.31</td><td>0.85</td><td>1.04</td><td>1.02</td><td>asym 10%</td><td>1.30</td><td>1.39</td><td>1.34</td><td>1.26</td></tr><tr><td>w50%/area</td><td>0.00</td><td>0.00</td><td>0.00</td><td>0.00</td><td>w50%/area</td><td>0.00</td><td>0.00</td><td>0.00</td><td>0.00</td></tr><tr><td>N</td><td>1709.08</td><td>3241.74</td><td>4100.83</td><td>3678.68</td><td>N</td><td>2078.91</td><td>2769.53</td><td>3251.56</td><td>4032.82</td></tr><tr><td rowspan="6">50%</td><td>tR</td><td>1.67</td><td>5.27</td><td>6.90</td><td>16.33</td><td>tR</td><td>1.42</td><td>1.42</td><td>1.52</td><td>1.61</td></tr><tr><td>area</td><td>1948432.00</td><td>1032679.00</td><td>1234330.00</td><td>1518817.00</td><td>area</td><td>2813697.00</td><td>1234412.00</td><td>1177637.00</td><td>639109.00</td></tr><tr><td>w50%</td><td>0.05</td><td>0.10</td><td>0.12</td><td>0.34</td><td>w50%</td><td>0.03</td><td>0.03</td><td>0.03</td><td>0.03</td></tr><tr><td>asym 10%</td><td>1.41</td><td>0.91</td><td>1.03</td><td>0.82</td><td>asym 10%</td><td>1.41</td><td>1.56</td><td>1.45</td><td>1.40</td></tr><tr><td>w50%/area</td><td>0.00</td><td>0.00</td><td>0.00</td><td>0.00</td><td>w50%/area</td><td>0.00</td><td>0.00</td><td>0.00</td><td>0.00</td></tr><tr><td>N</td><td>1560.35</td><td>2974.13</td><td>3866.40</td><td>2664.20</td><td>N</td><td>2059.59</td><td>2150.16</td><td>2397.20</td><td>2573.61</td></tr><tr><td rowspan="12">Set n°2</td><td rowspan="6">30%</td><td>tR</td><td>0.86</td><td>1.32</td><td>1.76</td><td>2.93</td><td>tR</td><td>1.03</td><td>1.41</td><td>1.78</td><td>2.82</td></tr><tr><td>area</td><td>2004977.00</td><td>1168716.00</td><td>1385011.00</td><td>1888954.00</td><td>area</td><td>1727685.00</td><td>1042625.00</td><td>1073405.00</td><td>1118849.00</td></tr><tr><td>w50%</td><td>0.03</td><td>0.04</td><td>0.04</td><td>0.06</td><td>w50%</td><td>0.07</td><td>0.08</td><td>0.09</td><td>0.10</td></tr><tr><td>asym 10%</td><td>1.47</td><td>1.34</td><td>1.26</td><td>1.16</td><td>asym 10%</td><td>3.27</td><td>2.90</td><td>2.75</td><td>2.30</td></tr><tr><td>w50%/area</td><td>0.00</td><td>0.00</td><td>0.00</td><td>0.00</td><td>w50%/area</td><td>0.00</td><td>0.00</td><td>0.00</td><td>0.00</td></tr><tr><td>N</td><td>977.51</td><td>1465.02</td><td>2272.61</td><td>2792.02</td><td>N</td><td>251.29</td><td>359.87</td><td>512.24</td><td>978.21</td></tr><tr><td rowspan="6">50%</td><td>tR</td><td>0.86</td><td>1.33</td><td>1.77</td><td>2.95</td><td>tR</td><td>1.47</td><td>1.60</td><td>1.84</td><td>2.20</td></tr><tr><td>area</td><td>2029178.00</td><td>1173391.00</td><td>1397001.00</td><td>1897748.00</td><td>area</td><td>2981837.00</td><td>1594578.00</td><td>1669474.00</td><td>1821120.00</td></tr><tr><td>w50%</td><td>0.03</td><td>0.04</td><td>0.04</td><td>0.06</td><td>w50%</td><td>0.07</td><td>0.05</td><td>0.05</td><td>0.05</td></tr><tr><td>asym 10%</td><td>1.51</td><td>1.35</td><td>1.28</td><td>1.19</td><td>asym 10%</td><td>1.67</td><td>1.41</td><td>1.46</td><td>1.37</td></tr><tr><td>w50%/area</td><td>0.00</td><td>0.00</td><td>0.00</td><td>0.00</td><td>w50%/area</td><td>0.00</td><td>0.00</td><td>0.00</td><td>0.00</td></tr><tr><td>N</td><td>933.75</td><td>1390.69</td><td>2177.35</td><td>2741.52</td><td>N</td><td>556.80</td><td>1038.28</td><td>1330.35</td><td>2096.32</td></tr><tr><td rowspan="6">50%</td><td>tR</td><td>0.87</td><td>1.35</td><td>1.80</td><td>2.99</td><td>tR</td><td>1.41</td><td>1.41</td><td>1.52</td><td>1.62</td></tr><tr><td>area</td><td>2035816.00</td><td>1184709.00</td><td>1401511.00</td><td>1922355.00</td><td>area</td><td>2859533.00</td><td>1574145.00</td><td>1483391.00</td><td>1347361.00</td></tr><tr><td>w50%</td><td>0.03</td><td>0.04</td><td>0.04</td><td>0.06</td><td>w50%</td><td>0.05</td><td>0.04</td><td>0.04</td><td>0.04</td></tr><tr><td>asym 10%</td><td>1.50</td><td>1.36</td><td>1.29</td><td>1.18</td><td>asym 10%</td><td>1.43</td><td>1.49</td><td>1.45</td><td>1.46</td></tr><tr><td>w50%/area</td><td>0.00</td><td>0.00</td><td>0.00</td><td>0.00</td><td>w50%/area</td><td>0.00</td><td>0.00</td><td>0.00</td><td>0.00</td></tr><tr><td>N</td><td>945.75</td><td>1401.34</td><td>2226.74</td><td>2787.38</td><td>N</td><td>1058.72</td><td>1584.21</td><td>1780.83</td><td>2159.40</td></tr></tbody></table>
<table><caption>**Table S5:** Complete data from analyses of set n°3 under dim1 and dim2 conditions: retention time, peak area, peak width at half-height (w50%), peak width at half-height/area ratio (w50%/area), and number of theoretical plates (N). peak 1 = alphitomin, peak 2 = epigallocatechin, peak 3 = epicatechin gallate.</caption><thead><tr><th rowspan="2"></th><th colspan="3">dim1</th><th colspan="3">dim2</th></tr><tr><th>pic1</th><th>pic2</th><th>pic3</th><th>pic1</th><th>pic2</th><th>pic3</th></tr></thead><tbody><tr><td rowspan="10">Set n°3</td><td rowspan="5">70%</td><td>tR</td><td>1.87</td><td>3.22</td><td>5.61</td><td rowspan="5">50%</td><td>tR</td><td>2.67</td><td>1.39</td><td>2.21</td></tr><tr><td>area</td><td>3332511.00</td><td>101308.00</td><td>1505052.00</td><td>area</td><td>1357998.00</td><td>913106.00</td><td>1300667.00</td></tr><tr><td>w50%</td><td>0.06</td><td>0.08</td><td>0.12</td><td>w50%</td><td>0.28</td><td>0.39</td><td>0.49</td></tr><tr><td>asym 10%</td><td>1.38</td><td>1.29</td><td>1.30</td><td>asym 10%</td><td>2.68</td><td>2.13</td><td>1.49</td></tr><tr><td>w50%/area</td><td>0.00</td><td>0.00</td><td>0.00</td><td>w50%/area</td><td>0.00</td><td>0.00</td><td>0.00</td></tr><tr><td rowspan="5">N</td><td>1083.73</td><td>2132.62</td><td>2500.46</td><td>N</td><td>106.14</td><td>15.45</td><td>23.73</td></tr><tr><td>tR</td><td>1.84</td><td>3.18</td><td>5.52</td><td rowspan="5">70%</td><td>tR</td><td>2.11</td><td>1.52</td><td>1.81</td></tr><tr><td>area</td><td>3380455.00</td><td>100961.00</td><td>1503558.00</td><td>area</td><td>2624408.00</td><td>2388646.00</td><td>1950776.00</td></tr><tr><td>w50%</td><td>0.06</td><td>0.07</td><td>0.11</td><td>w50%</td><td>0.13</td><td>0.19</td><td>0.16</td></tr><tr><td>asym 10%</td><td>1.32</td><td>1.19</td><td>1.29</td><td>asym 10%</td><td>3.29</td><td>nd</td><td>3.36</td></tr><tr><td>w50%/area</td><td>0.00</td><td>0.00</td><td>0.00</td><td>w50%/area</td><td>0.00</td><td>0.00</td><td>0.00</td></tr><tr><td rowspan="5">N</td><td>1216.06</td><td>2253.47</td><td>2759.71</td><td>N</td><td>326.84</td><td>77.54</td><td>150.85</td></tr><tr><td>tR</td><td>1.84</td><td>3.18</td><td>5.52</td><td rowspan="5">90%</td><td>tR</td><td>1.58</td><td>1.89</td><td>2.14</td></tr><tr><td>area</td><td>4991911.00</td><td>5624298.00</td><td>8280789.00</td><td>area</td><td>3729625.00</td><td>23766.00</td><td>3279327.00</td></tr><tr><td>w50%</td><td>0.06</td><td>0.07</td><td>0.12</td><td>w50%</td><td>0.07</td><td>0.05</td><td>0.05</td></tr><tr><td>asym 10%</td><td>1.32</td><td>1.19</td><td>1.29</td><td>asym 10%</td><td>1.79</td><td>2.03</td><td>1.71</td></tr><tr><td>w50%/area</td><td>0.00</td><td>0.00</td><td>0.00</td><td>w50%/area</td><td>0.00</td><td>0.00</td><td>0.00</td></tr><tr><td>N</td><td>1216.10</td><td>2403.59</td><td>2677.45</td><td>N</td><td>690.29</td><td>1604.53</td><td>1913.56</td></tr><tr><td rowspan="10">90%</td><td>tR</td><td>1.22</td><td>1.48</td><td>1.83</td><td rowspan="5">50%</td><td rowspan="5">peak in the baseline</td><td rowspan="5"></td><td rowspan="5"></td></tr><tr><td>area</td><td>3372597.00</td><td>124763.00</td><td>1665280.00</td><td>area</td><td>1665280.00</td><td>1665280.00</td><td>1665280.00</td></tr><tr><td>w50%</td><td>0.05</td><td>0.05</td><td>0.06</td><td>w50%</td><td>0.06</td><td>0.06</td><td>0.06</td></tr><tr><td>asym 10%</td><td>1.62</td><td>1.40</td><td>1.51</td><td>asym 10%</td><td>1.51</td><td>1.47</td><td>1.47</td></tr><tr><td>w50%/area</td><td>0.00</td><td>0.00</td><td>0.00</td><td>w50%/area</td><td>0.00</td><td>0.00</td><td>0.00</td></tr><tr><td rowspan="5">N</td><td>800.79</td><td>951.61</td><td>1093.44</td><td>N</td><td>1130.94</td><td>1130.94</td><td>1130.94</td></tr><tr><td>tR</td><td>1.22</td><td>1.48</td><td>1.83</td><td rowspan="5">70%</td><td>tR</td><td>1.26</td><td>1.51</td><td>not integrable</td></tr><tr><td>area</td><td>3406766.00</td><td>122165.00</td><td>1638520.00</td><td>area</td><td>1139748.00</td><td>23191.00</td><td></td></tr><tr><td>w50%</td><td>0.05</td><td>0.05</td><td>0.06</td><td>w50%</td><td>0.10</td><td>0.05</td><td></td></tr><tr><td>asym 10%</td><td>1.55</td><td>1.35</td><td>1.47</td><td>asym 10%</td><td>2.15</td><td>2.42</td><td></td></tr><tr><td>w50%/area</td><td>0.00</td><td>0.00</td><td>0.00</td><td>w50%/area</td><td>0.00</td><td>0.00</td><td></td></tr><tr><td rowspan="5">N</td><td>785.36</td><td>948.48</td><td>1130.94</td><td>N</td><td>189.69</td><td>1232.03</td><td></td></tr><tr><td>tR</td><td>1.22</td><td>1.48</td><td>1.83</td><td rowspan="5">90%</td><td>tR</td><td>1.46</td><td>1.28</td><td>2.00</td></tr><tr><td>area</td><td>3307975.00</td><td>104252.00</td><td>1609410.00</td><td>area</td><td>2878092.00</td><td>4174333.00</td><td>3359519.00</td></tr><tr><td>w50%</td><td>0.05</td><td>0.05</td><td>0.06</td><td>w50%</td><td>0.15</td><td>0.17</td><td>0.11</td></tr><tr><td>asym 10%</td><td>1.55</td><td>1.35</td><td>1.47</td><td>asym 10%</td><td>3.95</td><td>2.92</td><td>3.35</td></tr><tr><td>w50%/area</td><td>0.00</td><td>0.00</td><td>0.00</td><td>w50%/area</td><td>0.00</td><td>0.00</td><td>0.00</td></tr><tr><td>N</td><td>792.23</td><td>1062.88</td><td>1142.32</td><td>N</td><td>112.70</td><td>67.36</td><td>367.98</td></tr></tbody></table>
<figure><img src="image_18.png" /><figcaption>**Figure S1:** System configuration of the normal Acquity UPC²</figcaption></figure>

<figure><img src="image_19.png" /><figcaption>**Figure S2:** Measurement of gradient delay. Comparison of a one-dimensional SFC system (sys1D, in blue) and a two-dimensional SFC-SFC system (sys2D, in black). A) Gradient delay in dim1. Analysis conditions: ACQUITY UPC² Torus DEA (100 × 3.0 mm, 1.7 µm), co-solvent: MeOH + 0.1% acetone, 150 bar, 1.5 mL/min (Table 1, gradient 2.a). B) Gradient delay in dim2. Analysis conditions: CHIRALPAK IB-3 (150 × 4.6 mm, 3.0 µm), co-solvent: MeOH + 0.1% acetone, 150 bar, 2.0 mL/min (Table 1, gradient 2.b).</figcaption></figure>
<figure><img src="image_20.png" /><figcaption>**Figure S3:** Pressure monitoring during the analysis of a naringin standard on a dim1 and a dim2 method performed on a one-dimensional system (syst1D) and a two-dimensional system (syst2D).</figcaption></figure>

A) dim1 method. Analysis conditions: ACQUITY UPC² Torus DEA (100 × 3.0 mm, 1.7 µm), co-solvent: MeOH + 0.1% MSA, 150-110 bar, 1.7-0.6 mL/min (gradient conditions in Table 1, gradient 1.a)

B) dim2 method. Analysis conditions: CHIRALPAK IB-3 (150 × 4.6 mm, 3.0 µm), co-solvent: MeOH + 0.1% MSA (gradient conditions in Table 1, gradient 1.b), 150 bar, 2.0 mL/min.

<figure><img src="image_21.png" /><figcaption>**Figure S4:** System pressure monitoring during the analysis with the flow going through different storage loops of 50 µL and 114 µL volume and going through the dim2 column. Analysis conditions: CHIRALPAK IB-3 (150 × 4.6 mm, 3.0 µm), co-solvent: MeOH + 0.1% acetone (gradient conditions in Table 1, gradient 2.b), 150 bar, 2.0 mL/min.</figcaption></figure>
<figure><img src="image_22.png" /><figcaption>**Figure S5:** Observation in dim2 of the transfer of caffeine contained in the mobile phase from dim1. A) Peak of caffeine observed in dim2, with loop loading in isocratic conditions (step gradient) at 20, 40, 60, 80, 100% co-solvent in the mobile phase. B) Relation between the area of caffeine peak measured in dim2 and the actual amount transferred from dim1.</figcaption></figure>

Analysis conditions dim1: ACQUITY UPC² Torus DEA (100 × 3.0 mm, 1.7 µm), co-solvent: MeOH containing 0.025 mg/mL caffeine, 110 bar, 0.6 mL/min (gradient conditions in Table 1, gradient 3.a.)

Analysis conditions dim2: CHIRALPAK IB-3 (150 × 4.6 mm, 3.0 µm), co-solvent: MeOH + 0.1% MSA (gradient conditions in Table 1, gradient 3.b), 150 bar, 2.0 mL/min.
<figure><img src="image_23.png" /><figcaption>**Figure S6:** pressure variation in dim1 and dim2 observed with naringin standard.</figcaption></figure>

A) Analysis of dim1 with a pressure of 110 bar and analysis of dim2 at 150 bar.

B) Analysis of dim1 with a gradient pressure of 150 to 110 bar and analysis of dim2 at 150, 110 and 190 bar.

Analysis conditions dim1: ACQUITY UPC² Torus DEA (100 × 3.0 mm, 1.7 µm), co-solvent: MeOH + 0.1% MSA, 1.7-0.6 mL/min (gradient conditions in Table 1, gradient 1.a)

Analysis conditions for dim2: CHIRALPAK IB-3 (150 × 4.6 mm, 3.0 µm), co-solvent: MeOH + 0.1% MSA (gradient conditions in Table 1, gradient 1.b), 2.0 mL/min.
<figure><img src="image_24.png" /><figcaption>**Figure S7:** Structure of flavonoids used for screening chiral columns, divided into 3 sets. Set no. 1: Pinocembrin, Hesperetin, Pinocembroside. Set no. 2: Naringenin-7-O-glucoside, Taxifolin, Naringenin, Alphitonin. Set no. 3: Alphitonin, Epigallocatechin, Epigallocatechin Gallate. (TPSA values were calculated with MolDesc at [moldesc.icoa.fr](http://moldesc.icoa.fr))</figcaption></figure>
<figure><img src="image_25.png" /></figure>

**Figure S8:** Chromatogram of SFC-SFC analysis of flavonoid set n°1 (pinocembrin, hesperitin, pinocembroside) loaded with 5% co-solvent (CS) in dim1.

A) dim1 analysis at 5% CS (black) or 10% CS (blue).

B) dim2 analysis at 5% CS.

C) dim2 analysis at 10% CS.

D) dim2 analysis at 30% CS.

E) dim2 analysis at 50% CS.

Analysis conditions in both dimensions: ACQUITY UPC² Torus DEA (100 × 3.0 mm, 1.7 µm), co-solvent: MeOH + 0.1% MSA, 1.5 mL/min.
<figure><img src="image_26.png" /></figure>

**Figure S9:** Chromatogram of SFC-SFC analysis of flavonoid set n°1 (pinocembrin, hesperitin, pinocembroside) loaded with 10% co-solvent (CS) in dim1.

A) dim1 analysis at 5% CS (black) or 10% CS (blue).

B) dim2 analysis at 5% CS.

C) dim2 analysis at 10% CS.

D) dim2 analysis at 30% CS.

E) dim2 analysis at 50% CS.

Analysis conditions in both dimensions: ACQUITY UPC² Torus DEA (100 x 3.0 mm, 1.7 µm), co-solvent: MeOH + 0.1% MSA, 1.5 mL/min.
<figure><img src="image_27.png" /></figure>

**Figure S10:** Chromatogram of SFC-SFC analysis of flavonoid set n°1 (pinocembrin, hesperitin, pinocembroside) loaded with 30% co-solvent (CS) in dim1.

A) dim1 analysis at 30% CS (black) or 50% CS (blue).

B) dim2 analysis at 5% CS.

C) dim2 analysis at 10% CS.

D) dim2 analysis at 30% CS.

Analysis conditions in both dimensions: ACQUITY UPC² Torus DEA (100 × 3.0 mm, 1.7 µm), co-solvent: MeOH + 0.1% MSA, 1 mL/min.
<figure><img src="image_28.png" /></figure>

**Figure S11:** Chromatogram of SFC-SFC analysis of flavonoid set n°1 (pinocembrin, hesperitin, pinocembroside) loaded with 50% co-solvent (CS) in dim1.

A) dim1 analysis at 30% CS (black) or 50% CS (blue).

B) dim2 analysis at 5% CS.

C) dim2 analysis at 10% CS.

D) dim2 analysis at 30% CS.

Analysis conditions in both dimensions: ACQUITY UPC² Torus DEA (100 × 3.0 mm, 1.7 µm), co-solvent: MeOH + 0.1% MSA, 1 mL/min.
<figure><img src="image_29.png" /><figcaption>**Figure S12:** Chromatogram of SFC-SFC analysis of flavonoid set n°2 (naringenin, naringenin-7-O-glucoside, taxifolin, alphitinon) loaded with 30% co-solvent (CS) in dim1.</figcaption></figure>

A) dim1 analysis at 30% CS (black) or 50% CS (blue).

B) dim2 analysis at 50% CS.

C) dim2 analysis at 70% CS.

D) dim2 analysis at 90% CS.

Analysis conditions in both dimensions: ACQUITY UPC² Torus DEA (100 × 3.0 mm, 1.7 µm), co-solvent: MeOH + 0.1% MSA, 1 mL/min.
<figure><img src="image_30.png" /><figcaption>**Figure S13:** Chromatogram of SFC-SFC analysis of flavonoid set n°2 (naringenin, naringenin-7-O-glucoside, taxifolin, alphitон) loaded with 50% co-solvent (СS) in dim1.</figcaption></figure>

A) dim1 analysis at 30% CS (black) or 50% CS (blue).

B) dim2 analysis at 50% CS.

C) dim2 analysis at 70% CS.

D) dim2 analysis at 90% CS.

Analysis conditions in both dimensions: ACQUITY UPC² Torus DEA (100 x 3.0 mm, 1.7 µm), co-solvent: MeOH + 0.1% MSA, 1 mL/min.
<figure><img src="image_31.png" /><figcaption>**Figure S14:** Chromatogram of SFC-SFC analysis of flavonoid set n°3 (alphitin, epigallocatechin, epicatechin gallate) loaded with 70% co-solvent (CS) in dim1.</figcaption></figure>

A) dim1 analysis at 70% CS (black) or 90% CS (blue).

B) dim2 analysis at 50% CS.

C) dim2 analysis at 70% CS.

D) dim2 analysis at 90% CS.

Analysis conditions in both dimensions: ACQUITY UPC² Torus DEA (100 × 3.0 mm, 1.7 µm), co-solvent: MeOH + 0.1% MSA, 0.6 mL/min.
<figure><img src="image_32.png" /><figcaption>**Figure S15:** Chromatogram of SFC-SFC analysis of flavonoid set n°3 (alphitонin, epigallocatechin, epicatechin gallate) loaded with 90% co-solvent (CS) in dim1.</figcaption></figure>

A) dim1 analysis at 70% CS (black) or 90% CS (blue).

B) dim2 analysis at 50% CS.

C) dim2 analysis at 70% CS.

D) dim2 analysis at 90% CS.

Analysis conditions in both dimensions: ACQUITY UPC² Torus DEA (100 × 3.0 mm, 1.7 µm), co-solvent: MeOH + 0.1% MSA, 0.6 mL/min.