# Critical parameters for achieving high efficiency and reproducible double-chained cationic surfactant coatings for protein separation by capillary electrophoresis

Chirapha Prakobdi, Laurent Leclercq, Phoonthawee Saetear, Hervé Cottet

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Chirapha Prakobdi, Laurent Leclercq, Phoonthawee Saetear, Hervé Cottet. Critical parameters for achieving high efficiency and reproducible double-chained cationic surfactant coatings for protein separation by capillary electrophoresis. Journal of Chromatography A, 2026, 1768, pp.466705. 10.1016/j.chroma.2026.466705. hal-05478130

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Distributed under a Creative Commons CC BY 4.0 - Attribution - International License
Critical parameters for achieving high efficiency and reproducible double-chained cationic surfactant coatings for protein separation by capillary electrophoresis

Chirapha Prakobdi¹, Laurent Leclercq¹*, Phoonthawee Saetear²,³, Hervé Cottet¹*

¹IBMM, University of Montpellier, CNRS, ENSCM, Montpellier, France

²Flow Innovation-Research for Science and Technology Laboratories (Firstlabs), Ratchathewi District, Bangkok, 10110, Thailand

³Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Mahidol University, Rama 6 Road, Ratchathewi District, Bangkok, 10400, Thailand

* Corresponding authors: laurent.leclercq@umontpellier.fr; herve.cottet@umontpellier.fr

**Keywords:** protein separation; surface-based capillary coatings; double chain cationic surfactant; capillary electrophoresis

**Abstract**

Analyzing intact proteins by capillary electrophoresis (CE) is challenging but highly valuable, as it holds potential for future clinical applications via the separation and quantification of biomarkers. To achieve this, the development of CE methods requires improvements in separation efficiency, reproducibility, and reduction of sample adsorption on the capillary wall. Double-chained surfactant coatings were described as potential candidate leading to high separation efficiency. In this work, a capillary coating based on dioctadecyldimethylammonium bromide (DODAB), a double-chained cationic surfactant, was investigated by assessing the critical parameters affecting DODAB-vesicle preparation and coating performances. These parameters include temperature & sonication of the coating solution, nature of the coating buffer, and capillary inner diameter. The plate height (H) as a function of the linear velocity (u) was systematically plotted to evaluate the separation performances under different kinetic conditions by applying different separation voltages. DODAB coating showed high surface homogeneity leading to low slope of the H vs u curve (2.23 ± 0.99 × 10⁻³ s in average on 3 different capillaries) on 5 model basic proteins (lysozyme, carbonic anhydrase, ribonuclease A, β-lactoglobulin A and myoglobin) with a 40 cm total length and 25 µm inner diameter capillary. Excellent separation performances were achieved reaching up to 606,000 ± 27,000 plates/m at -12 kV for lysozyme, with excellent repeatability (RSD on migration times = 0.21%).
33 Highlights

34 • Separation efficiency of DODAB capillary coatings under various kinetics were assessed

35 • Nature, pH, sonication and temperature of coating solution are critical parameters

36 • DODAB coatings provide highly homogeneous surface with low *H* vs *u* slopes

37 • High separation efficiency with high intra- and inter-capillary precisions were achieved

38 • Lower capillary I.D. provided more homogeneous coating with lower *H* vs *u* slopes

39
## 1. Introduction

Capillary electrophoresis (CE) is a highly efficient separation technique compatible with various detection modes [1], including UV detection [2–6], mass spectrometry (MS) [7–10], and others [11–15]. It is widely employed across multiple separation science fields for analyzing a broad range of samples including small ions & inorganic compounds [16], pharmaceuticals [17,18], peptides & proteins [19,20], monoclonal antibodies (mAbs) [21,22] and biomarkers [23,24]. The application of CE in the clinical field is particularly promising and challenging, offering potential for early disease screening through the analysis of clinically relevant biomarkers [25] or therapeutic proteins such as mAbs. However, variants of intact proteins generally appear under the form of small satellite peaks, close to the main peak (main protein form) and require high separation efficiency.

Achieving high theoretical plate numbers ($N$) requires minimizing peak dispersion, which can result from different sources such as injection, adsorption phenomena, capillary surface inhomogeneity and other parameters [26]. These sources of dispersion are at the origin of the slope ($p$) and the constant value in the expression of the plate height ($H$) as a function of the linear velocity ($u$) [27–29]. The $H$ vs $u$ curve is also used to assess the coating performances under different kinetic conditions. It also allows quantifying the impact of solute adsorption and electroosmotic flow (EOF) inhomogeneity along the capillary on separation efficiency [26,30].

In 2024, Dhellemmes et al. reported the separation performances of cationic successive multiple ionic-polymer layer (SMIL) coatings issued from polydiallyldimethylammonium chloride (PDADMAC) and sodium polystyrene sulfonate (PSS) [26]. Important adsorption on the capillary wall was found in the case of β-lactoglobulin A (β-lac A) ($p = 14.27 \pm 1.98 \times 10^{-3}$ s [30]), while capillary surface inhomogeneity was most likely the main cause for the residual slope ($p \approx 3 \times 10^{-3}$ s for a 60 cm long SMIL-coated capillary [30]) of all the other proteins tested. On a 80 cm long capillary, high plate numbers from 300,000 up to 1 million plates/m were found depending on the protein nature [26]. Although SMIL coatings effectively reduced protein adsorption and provide high repeatability, their intrinsic structural inhomogeneity—including residual roughness of the surface with partially looped polymer chains—was thought to contribute to the remaining slope. It was found that even less than 1% charge inhomogeneity could be significantly impacting the slope [30].

These findings motivated the search for alternative coatings capable of providing a smoother and more homogeneous surface. In the early 2000's, double-chained cationic surfactants such as dimethylditetradecylammonium bromide, dimethyldioctadecylammonium bromide, and didodecyldimethylammonium bromide were investigated as capillary coatings in CE for the separation of basic proteins [31,32]. These surfactants can self-assemble into bilayer structures on the capillary wall, which is believed to result in flat surfaces and improved separation performances [30,31].
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Separation efficiency up to 1 million plates/m were reported proving the high performances that can be achieved with such coatings. The temperature, the composition of coating buffer and the coating time were investigated by Lucy's group [31–37]. Despite this important research work already done, no systematic study on repeatability/reproducibility and on the kinetics effects (influence of applied voltage) on separation efficiency was carried out.

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In this work, we investigate the coating of fused silica capillaries with dimethyldioctadecylammonium bromide (DODAB) to reproducibly achieve very high separation efficiencies. Critical parameters influencing the coating preparation were systematically evaluated, i.e., the temperature of the DODAB coating solution, the number of sonication/stirring cycles, the nature and pH of the coating buffer, and the capillary inner diameter (I.D.). Coating performances were characterized by analyzing five model basic proteins under acidic conditions (2 M acetic acid as background electrolyte, BGE) and by systematically constructing *H* vs *u* curves to determine the slope (*p*) and constant (*cst*) values.

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## 2. Experimental section

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### 2.1. Chemicals

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Acetic acid (AcOH, 99.8% purity), sodium phosphate monobasic dihydrate (≥ 99% purity), and sodium hydroxide (NaOH, ≥ 98% purity) were obtained from Honeywell-Fluka (Seelze, Germany). Orthophosphoric acid (85% purity) was purchased from Prolabo (Paris, France). Dimethyldioctadecylammonium bromide (DODAB, ≥ 99% purity) was purchased from Acros Organics (Geel, Belgium). *N*,*N*-Dimethylformamide (DMF, 99.9% purity) was obtained from Carlo Erba Reagents (Val-de-Reuil, France). Ultrapure water was produced using a MilliQ system from Millipore (Molsheim, France).

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The model basic proteins used in this study, i.e., Carbonic Anhydrase from bovine erythrocytes (CA, purity not indicated by the supplier), Myoglobin from equine skeletal muscle (Myo, 95-100% purity), Ribonuclease A from bovine pancreas (RNAse A, purity not indicated by the supplier), β-lactoglobulin A from bovine milk (β-lac A, ≥ 90% purity) and Lysozyme from chicken egg white (Lyz, ≥ 90% purity), were obtained from Sigma-Aldrich (Saint-Quentin Fallavier, France).

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### 2.2. Preparation of the solutions

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1 M NaOH solution was prepared by dissolving 1.0 g of NaOH in 25 mL of ultrapure water and used for capillary pretreatment. 2 M AcOH solution was used as the background electrolyte (BGE), by adding 30 g of AcOH into 250 mL ultrapure water. The measured pH was 2.2. The coating buffer was 50 mM phosphate at pH 3.0 and was prepared by dissolving 1.70973 g of sodium phosphate monobasic dihydrate and 177.66 mg of orthophosphoric acid into 250 mL ultrapure water. The BGE
107 solution and the buffer solution were stored in the fridge.

108 The DODAB coating solution was prepared either using GT SONIC-D6 bath sonicator (Shenzhen, 109 China) at 150 W and 40 kHz, or using a Hielscher UP200HT tip sonicator (Teltow, Germany) at 200 W, 110 and 26 kHz.

111 In the case of using the sonication bath, the coating solution consisted of a 0.1 mM DODAB vesicle 112 solution, prepared by dissolving 2.52 mg of DODAB in 40 mL of preheated coating buffer at 60 °C. The 113 resulting solution was then processed using sonication/stirring cycles (see **Figure 1**). Specifically, it 114 was first sonicated in a bath sonicator at 60 °C for 30 min, followed by stirring at 250 rpm at RT for 15 115 min. This cycle was repeated four times. After the final cycle, the solution was cooled by stirring in a 116 cool water bath (ca. 4 °C) for 6 min. An aliquot of 230 µL of the vesicle solution was then transferred to 117 a 1 mL polypropylene vial, ensuring the liquid level remained below the electrode (as required for 118 Agilent CE instruments equipped with standard electrodes). In the case of the study of the impact of 119 sonication, the DODAB vesicle solution was only heated to 60 °C without ultrasonic treatment before 120 proceeding with the stirring steps, see **Section 3.2**.

121 Alternatively, the coating solution was prepared by tip sonication. Contrary to bath sonicator that 122 provides gentle, uniform sonication through a water bath, a tip sonicator delivers intense, localized 123 energy directly into the sample via a probe, resulting in more efficient disruption. In that case, the 124 preparation procedure included the following steps: (i) preheating the coating buffer, (ii) sonicating with 125 the ultrasonic probe, and (iii) cooling under continuous stirring (see **Figure SI1**). Specifically, the 126 coating buffer was first heated up to ca. 72 °C and then transferred to a plastic container containing 127 2.52 mg of DODAB. At this stage, DODAB was expected to dissolve and begin forming lamellar 128 phases. The DODAB-containing solution was then sonicated using a probe-type ultrasonic processor 129 at 20% of amplitude and 10% of duty cycle for 5 min. Following sonication, the DODAB solution was 130 cooled down in a water bath (at RT) under stirring for 15 min. See more details in **Section SI1**.

131 DMF mother solution was prepared by diluting 10 µL of DMF in 4.99 µL of ultrapure water (in a 5 mL 132 Eppendorf® tube) to obtain 5 mL of 0.2% DMF solution. A diluted solution was prepared by diluting 50 133 µL of the mother solution into 2.55 µL of a twice more concentrated BGE (4 M AcOH) and 2.45 µL of 134 ultrapure water (in a 5 mL Eppendorf® tube) to obtain a 0.002% DMF solution in BGE that was used as 135 the EOF marker.

136 Protein mixtures were obtained from individual stock solutions, each of them being stored in water. To 137 obtain 2 g/L solutions, the proteins were accurately weighed into 5 mL Eppendorf® tubes, followed by 138 the addition of the appropriate volume of ultrapure water. The stock solutions were kept in the freezer 139 before use. Prior to analysis, the protein stocks were thawed, and 10 µL of each protein was 140 transferred into a 1 mL Eppendorf® tube. Then, 50 µL of twice more concentrated BGE (4 M AcOH)
141 was added, and the volume was adjusted to 100 µL with ultrapure water, resulting in a final protein
142 concentration of 0.2 g/L in BGE. The mixed solution was then incubated at 37 °C for 30 min before
143 analysis. The protein mixtures can be stored in the freezer for short-term use; no degradation was
144 observed within one week.

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146 2.3. Coating procedure

147 The DODAB vesicle coatings were prepared based on a simple protocol: (i) preconditioning the bare
148 fused silica capillary with 1 M NaOH, followed by ultrapure water, (ii) coating the capillary with the 0.1
149 mM DODAB vesicle solution, and (iii) rinsing the capillary with BGE (2 M AcOH) to remove the excess
150 of DODAB. Each capillary required adjustments in pressure and flushing time depending on its I.D.
151 and total length (L) for better comparison.

152 Case of DODAB coating solution prepared using the bath sonicator: For a capillary with total length L
153 of 40 cm and I.D. of 10 µm, all flushes were conducted at an external pressure of 6 bar. The capillary
154 was first flushed with NaOH for 30 min, followed by ultrapure water for 6 min, and then with the
155 DODAB vesicle coating solution for 84 min. Finally, the capillary was rinsed with BGE for 6 min.

156 For 40 cm capillary of 25 µm or 50 µm I.D., all flushes were carried out at 1 bar. Preconditioning was
157 done with NaOH for 30 min and ultrapure water for 6 min. The capillary was then flushed with the
158 DODAB vesicle coating solution for 30 min and with BGE for 6 min.

159 Case of DODAB coating solution prepared using the tip sonicator: For 40 cm capillary of 25 µm I.D.,
160 coating procedure was performed at 1 bar for all flushing steps. Preconditioning was performed with
161 NaOH for 30 min, followed by ultrapure water for 6 min. The capillary was then flushed with the
162 DODAB vesicle coating solution for 50 min, and finally with BGE for 6 min (see **Section SI2**).

163 For 80 cm capillary of 25 µm I.D., all flushes were carried out at 2 bar, with NaOH for 60 min, ultrapure
164 water for 12 min, DODAB solution for 100 min, and BGE for 12 min. (See **Section SI2**).

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166 2.4. Capillary electrophoresis

167 Analyses were performed on an Agilent 7100 CE system (Waldbronn, Germany) and 2 M AcOH BGE
168 at pH 2.2. The separation conditions were optimized based on the capillary I.D. and total capillary L.
169 For 40 cm capillaries (31.5 cm to the detector), voltages ranging from -10 kV to -2 kV were applied.
170 For 80 cm capillaries (71.5 cm to the detector) of 25 µm I.D., voltages from -18 kV to -8 kV were
171 applied.

172 The capillaries were flushed between each run with BGE under the following conditions: (i) 10 µm I.D.,
173 40 cm: 6 min at 6 bar; (ii) 25 µm and 50 µm I.D., 40 cm: 6 min at 1 bar; (iii) 25 µm I.D., 80 cm: 12 min
174 at 2 bar.

175 Sample injections were performed hydrodynamically with 0.002% DMF in BGE as EOF marker
176 followed by the protein mixture at 0.2 g/L each in BGE as follows: (i) 10 µm I.D., 40 cm, 100 mbar for
177 11 s (DMF), 1 bar for 3 s (proteins, injected volume $V_{inj}$= 0.34 nL, 1.38% of the volume to detector) ; (ii)
178 25 µm I.D., 40 cm, 60 mbar for 3 s (DMF), 60 mbar for 8 s (proteins, injected volume $V_{inj}$= 1.28 nL,
179 0.8% of the volume to detector); (iii) 50 µm I.D., 40 cm, 30 mbar for 2 s (DMF), 30 mbar for 4 s
180 (proteins, injected volume $V_{inj}$= 5.11 nL, 0.8% of the volume to detector); (iv) 25 µm I.D., 80 cm: 90
181 mbar for 4 s (DMF), 96 mbar for 10 s (proteins, injected volume $V_{inj}$= 1.28 nL, 0.36% of the volume to
detector). Injected volumes in terms of % to the capillary volume to the detector: 0.7% for all
182 capillaries, except for 25 µm I.D. and L = 80 cm capillary (0.3%).

183 All experiments were performed at 25 °C with UV detection at 200 nm. Separation efficiencies were
184 calculated using CEval software [27,38]. The capillary total (L) and effective (l) lengths must be
185 entered, as well as the voltage half ramp time ($t_{1/2}$ = 0.6 s). Peak width at half height (δ) and migration
186 time at the peak apex ($t_{apex}$) were determined by the software and used to calculate N and H values for
187 the different voltages (or u) values using equation 1 to 3 [39]:

$$189 \qquad H = \frac{l}{N} \qquad (\text{eq. 1})$$

$$190 \qquad N = 5.54 \left( \frac{t_{apex}}{\delta} \right)^2 \qquad (\text{eq. 2})$$

$$191 \qquad u = \frac{l}{t_{apex} - t_{1/2}} \qquad (\text{eq. 3})$$

## 3. Results and discussion

194 Small Unilamellar Vesicles (SUV) solution of DODAB, a double-chained cationic surfactant, was
195 prepared via n cycle(s) of sonication/stir method as depicted in **Figure 1**. 2.52 mg of DODAB
196 surfactant (0.1 mM) was dissolved in 40 mL of 50 mM sodium phosphate buffer (pH 3.0) preheated at
197 60 °C in a plastic container. DODAB molecules form lamellar phase at a temperature above the
198 transition temperature (or melting temperature, $T_m$) about 45 °C [39–44]. Then, the resulting solution
199 was continuously sonicated using a bath sonicator in normal mode for 30 min at 60 °C. Sonication
200 helps to break down the lamellar structure and form vesicles of a size that depends on the power of
201 the sonicator [45–47]. Finally, the vesicle solution was cooled down to room temperature for 15 min
202 under stirring. Further sonication/stir cycles were performed until the DODAB solution gets clear.
203 Indeed, the vesicle solution might be cloudy after the first cycle, but become clear after another or a
few more cycles. The clearness of the final solution was checked by measuring the turbidity at 520 nm. The DODAB vesicles size was obtained by Dynamic Light Scattering (DLS) at about 300 ± 100 nm [36]. Four cycles are generally required to get clear DODAB solutions. After the last cycle, the solution was cooled down to room temperature by placing the plastic container in a cold water bath (4°C) for 6 min.

<figure><img src="image_2.png" /></figure>

### Coating solution

- Temperature

- Number of cycle(s)

- Sonication

- Nature & pH

### Capillary inner diameter (I.D.)

- 10 µm

- 50 µm

- 25 µm

**Figure 1.** Procedure of DODAB coating. Preparation of DODAB vesicle coating solution using a certain number *n* of sonication/stir cycles (**A**). Details are: (i) 2.52 mg of DODAB dissolved in 40 mL of preheated coating buffer at 60 °C, (ii) sonication at ca. 60 °C for 30 min, (iii) stirring at room temperature (from ca. 60 to 40 °C) for 15 min, (iv) cooling down (from 40 °C) to room temperature for 6 min in 4 °C water bath. Capillary coating and protein separation by CE (**B**). Data processing of the electropherograms obtained at different voltages and plotting *H* vs *u* curve (**C**).

DODAB coated capillaries were prepared according to the protocol described in **Section 2.3**. The DODAB solution was flushed into the capillary, leading to the formation of a bilayer at the capillary surface via three steps: (i) adsorption of small vesicles on surface; (ii) fusion of small vesicles to become larger vesicles, and (iii) rupture of large vesicles to form bilayer [48–51].

Electrophoretic separation of five basic model proteins (i.e., CA, Myo, RNAse A, β-lac A, and Lyz), covering broad range of molar masses between 10,000 and 30,000 g/mol, isoelectric points between 5
and 11 and hydrodynamic radii between 1.5 and 4.0 nm, was then performed on DODAB coated capillaries (see **Table SI1** for the main characteristics of the proteins). These proteins are generally classified as "hard" proteins, which are able to maintain their conformation upon adsorption and bind primarily through hydrophobic interactions, or through electrostatic interactions [52,53]. These proteins were chosen to allow comparison with results previously obtained on other capillary coatings [27]. The protein solutions were prepared according to a protocol previously described by Dhellemmes et al. showing that protein preheating is important to get high separation efficiency [27]. Proteins were individually prepared at high concentration (2 g/L) in ultrapure water and kept in the freezer. They were then mixed together in concentrated AcOH to get a solution of protein mixture at a final concentration of 0.2 g/L each in 2 M AcOH (pH 2.2). At this pH, the proteins are positively charged and are not supposed to interact electrostatically with the DODAB coating.

Experimental parameters influencing the coating preparation and performances were evaluated, i.e., the temperature of the DODAB coating solution (RT vs 60 °C), the number of sonication/stirring cycles (1 to 4), the sonication (with or without), the nature and pH of the coating buffer (MilliQ water vs acetic acid at pH 2.2 vs sodium phosphate buffer at pH 3.0), and the capillary I.D. (10 vs 25 vs 50 µm). Coating performances were characterized by systematically plotting H vs u curves to determine the slope (p) and constant (cst) values.

Typical electropherograms are given in **Figure 2A** using 5 different negative voltages (from -10 to -2 kV) with n = 5 repetitions for each separation voltage of a 40 cm × 31.5 cm × 25 µm (I.D.) capillary. Proteins are separated in counter electroosmotic mode and were detected according to the following order: (1) CA; (2) Myo; (3) RNAse A; (4) β-lac A; and (5) Lyz. Very good repeatability was obtained as shown by the overlay of the five repetitions in **Figure 2A** and by the RSD (< 0.15%) on the migration times (t<sub>m</sub>) at -2 kV given in **Table 1**. H vs u curves were then plotted (**Figure 2B**) for each model protein based on the experimental separation efficiencies, see eqs (1) to (3), and by curve fitting using eq. 4:

$$H = \frac{2D}{u} + pu + cst \quad (\text{eq. 4})$$

where D is the molecular diffusion coefficient of the corresponding protein (see **Table SI1**). The p and cst values were calculated by the Jackknife method [27].

Due to high surface charge density, EOF was found very high (-79.5 ± 0.5 TU, where TU stands for Tiselius Unit; 1 TU = 10<sup>-9</sup> m<sup>2</sup>V<sup>-1</sup>s<sup>-1</sup>). Low values were obtained for p = 1.61 ± 1.01 × 10<sup>-3</sup> s averaged on the five proteins. **Table 1** provides the p and cst values for each individual protein. These results show that DODAB coating could provide lower p value than SMIL coating (av. 3.33 ± 0.93 × 10<sup>-3</sup> s on 40 cm × 31.5 cm × 50 µm capillaries [27]). To assess the inter-capillary intermediate precision, these results
were repeated on 3 different capillaries as presented in **Figure SI2** and in **Tables SI2-4**. In addition, the average RSD on $t_m$ at -10 kV was found similar for 3 capillaries (0.19%).

In this work, our objective was to optimize the coating procedure to maximize *N* and to easily reproduce the results by identifying the operating critical parameters. The impact of the temperature, the number of sonication/stir cycles, the sonication, the nature and pH of the coating buffer, and the capillary inner diameter were investigated in the following sections.

<figure><img src="image_3.png" /><figcaption>**Figure 2.** Repeated electropherograms (n = 5) of a mixture of 5 model proteins (CA, Myo, RNAse A, β-LacA, and Lyz) (A) and the corresponding plate height (H) vs linear velocity (u) plots (B) on a DODAB coated capillary prepared with 4 sonication/stir cycles. Experimental conditions: Capillary: 40 cm (31.5 cm to the detector) x 25 µm I.D. Coating solution: 0.1 mM DODAB in 50 mM phosphate buffer (pH 3.0) with 4 cycles of sonication/stir method. BGE: 2 M AcOH, pH 2.2. Flush before each run: BGE 1 bar, 6 min. Applied separation voltages: -10 kV, -8 kV, -6 kV, -4 kV, -2 kV. Sample mixture: 0.2 g/L of each protein in BGE. Hydrodynamic injection: 60 mbar, 8 s. Hydrodynamic co-injection of 0.02% DMF in BGE: 60 mbar, 3 s. Temperature: 25 °C. Peak identification on electropherograms: (1) CA, (2) Myo, (3) RNAse A, (4) β-lac A, (5) Lyz. Peak identification on *H* vs *u* curves: ● (CA), ■ (Myo), ◆ (RNAse A), ▲ (β-lac A) and ★ (Lyz). For the coating procedure, see **Section 2.3**. EOF: -79.5 ± 0.5 TU, *p* and *cst* values are provided in **Table 1**. Average RSD of $t_m$ of the five proteins tested at -10 kV: 0.19%.</figcaption></figure>

<table><caption>**Table 1.** Typical values obtained from the *H* = *f*(*u*) curves: slope (*p*), constant (*cst*), plate number (*N*) and %RSD on $t_m$ determined at -2 kV (n = 5 repetitions). Experimental conditions as in **Figure 2**. For more details, see the values for each capillary in **Table SI2-4**.</caption><thead><tr><th>Proteins</th><th><i>p</i> (10<sup>-3</sup> s)</th><th><i>cst</i> (µm)</th><th><i>N</i> (10<sup>3</sup>)<br>at -2 kV</th><th>%RSD (<i>t</i><sub>m</sub>)<br>at -2 kV</th></tr></thead><tbody><tr><td>CA</td><td>2.51 ± 0.11</td><td>3.37 ± 0.10</td><td>70.1 ± 1.3</td><td>0.13</td></tr><tr><td>Myo</td><td>1.21 ± 0.33</td><td>3.37 ± 0.23</td><td>77.6 ± 0.2</td><td>0.14</td></tr><tr><td>RNAse A</td><td>2.19 ± 0.20</td><td>2.84 ± 0.16</td><td>70.4 ± 1.1</td><td>0.15</td></tr></tbody></table>
<table><tr><td>β-lac A</td><td>0.02 ± 0.04</td><td>5.36 ± 0.04</td><td>51.3 ± 5.4</td><td>0.14</td></tr><tr><td>Lyz</td><td>2.12 ± 0.36</td><td>2.76 ± 0.27</td><td>77.2 ± 12.3</td><td>0.15</td></tr><tr><td>Average</td><td>1.61 ± 1.01</td><td>3.54 ± 1.06</td><td>69.3 ± 10.7</td><td>0.14</td></tr></table>

The effect of temperature on the preparation of the DODAB coating solution was first investigated, revealing that DODAB fails to dissolve in 50 mM sodium phosphate buffer (pH 3.0) when the temperature is below its melting temperature ($T_m$), i.e., approximately 45 °C. Consequently, DODAB must be dissolved in a coating solution preheated at 60 °C. From a theoretical standpoint, temperature plays a crucial role in the preparation of DODAB by facilitating the phase transition of the surfactant from the solid state to the lamellar phase.

### 3.1. Influence of the number of sonication/stir cycles

The preparation of the DODAB coating solution was conducted using the sonication/stir method. This procedure involved sonication in a bath sonicator maintained at 60 °C in normal mode. Initially, the surfactant was added to the preheated coating solution (60 °C) to induce the formation of the lamellar phase. Subsequently, sonication was applied within a temperature-controlled water bath to disrupt the lamellar structures into vesicles. After sonication, the solution was allowed to cool down to room temperature under stirring. Based on this procedure, it was hypothesized that increasing the number of sonication/stir cycles would yield a more uniform vesicle structure and reduce vesicle size. Consequently, the influence of the number of sonication/stir cycles on coating performance was investigated. Specifically, the coating solutions were compared using one or four sonication/stir cycles in terms of $p$ and $cst$ values derived from the corresponding $H$ vs $u$ curves (see **Figure 3** for 1 cycle and **Figure 2** for 4 cycles).

The results showed slightly better performances in the case of 4 cycles compared to 1 cycle only, with average $p$ values of $2.28 \pm 0.79 \times 10^{-3}$ s and $1.61 \pm 1.01 \times 10^{-3}$ s for 1 and 4 cycles, respectively. This low impact indicates that the number of sonication/stir cycles cannot be considered as a critical parameter affecting DODAB coating performance. Nevertheless, due to slightly lower $p$ value, the sonication/stir method with four cycles was adopted for subsequent preparations of DODAB coating solution. To assess the inter-capillary intermediate precision, these results were repeated on 3 different capillaries, as presented in **Figure SI3** and **Table SI5** (1 cycle) and in **Figure SI2** and **Table SI2-4** (4 cycles).
<figure><img src="image_4.png" /><figcaption>**Figure 3.** Repeated electropherograms (n = 5) of a mixture of 5 model proteins (CA, Myo, RNAse A, β-LacA, and Lyz) (A) and the corresponding plate height (H) vs linear velocity (u) plots (B) on a DODAB coated capillary prepared with one sonication/stir cycle. Experimental conditions: Capillary: 40 cm (31.5 cm to the detector) × 25 µm I.D. Coating solution: 0.1 mM DODAB in 50 mM phosphate buffer (pH 3.0). BGE: 2 M AcOH, pH 2.2. Flush between runs: BGE 1 bar, 6 min. Applied separation voltages: -10 kV, -8 kV, -6 kV, -4 kV, -2 kV. Sample mixture: 0.2 g/L of each protein in BGE. Hydrodynamic injection: 60 mbar, 8 s. Hydrodynamic co-injection of 0.02% DMF in BGE: 60 mbar, 3 s. Temperature: 25 °C. Peak identification on electropherograms: (1) CA, (2) Myo, (3) RNAse A, (4) β-lac A, (5) Lyz. Peak identification on H vs u curves: • (CA), ■ (Myo), ◆ (RNAse A), ▲ (β-lac A) and ★ (Lyz). For the coating procedure, see **Section 2.3**. EOF: -79.9 ± 0.3 TU. *p*: 3.07 ± 0.06 × 10⁻³ s for CA; 2.60 ± 0.19 × 10⁻³ s for Myo; 2.81 ± 0.19 × 10⁻³ s for RNAse A; 1.20 ± 0.31 × 10⁻³ s for β-lac A; 1.72 ± 0.13 × 10⁻³ s for Lyz; *cst*: 3.61 ± 0.05 µm for CA; 2.30 ± 0.11 µm for Myo; 2.72 ± 0.10 µm for RNAse A; 4.81 ± 0.29 µm for β-lac A; 2.92 ± 0.06 µm for Lyz; *N* at -2 kV: 69.6 ± 12.8 × 10³. RSD of *t*<sub>m</sub> of the five proteins tested at -10 kV: 0.28%.</figcaption></figure>

**3.2. Influence of sonication**

Sonication plays a crucial role in disrupting the lamellar structures of surfactants to promote the
formation of SUVs. To evaluate whether SUVs could form in the absence of sonication, DODAB
solution was prepared following the same 4-cycle sonication/stirring protocol described in **Section 2.2**
without the sonication step, i.e., the surfactant solution vial was placed in a 60 °C bath sonicator
without activating the ultrasonic function. This approach ensured that all parameters were kept
constant except for the application of sonication.

The DODAB solution prepared without sonication exhibited poor separation performances, as shown
in the electropherograms (Figure 4A) and H = f(u) curves (Figure 4B), with average p and CST values
much higher compared to those obtained using sonication (1.61 ± 1.01 × 10⁻³ s and 3.54 ± 1.06 µm
with sonication vs 5.69 ± 1.08 × 10⁻³ s and 2.60 ± 0.64 µm without sonication (n = 1 capillary)). Inter-
capillary intermediate precision was also investigated on 3 different capillaries as presented in Figure
SI2 and Tables SI2-4 (with sonication) and Figure SI4 and Table SI6 (without sonication). These
results indicated that sonication is essential for the preparation of SUVs in DODAB vesicle solutions, confirming that sonication is a critical parameter. During the procedure, while simple stirring might induce some vesicle formation, the mechanical energy provided is insufficient to achieve the formation of SUVs [44,47]. Most vesicles formed without sonication are multilamellar vesicles (MLVs) [42,54].

343

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

344

345

**Figure 4.** Repeated electropherograms (n = 5) of a mixture of 5 model proteins (CA, Myo, RNAse A, β-LacA, and Lyz) (A) and the corresponding plate height (H) vs linear velocity (u) plots (B) on a DODAB coated capillary prepared with 4 cycles of sonication/stirring without sonication. Experimental conditions: Capillary: 40 cm (31.5 cm to the detector) × 25 µm I.D. Coating solution: 0.1 mM DODAB in 50 mM phosphate buffer (pH 3.0). BGE: 2 M AcOH, pH 2.2. Flush between runs: BGE 1 bar, 6 min. Applied separation voltages: -10 kV, -8 kV, -6 kV, -4 kV, -2 kV. Sample mixture: 0.2 g/L of each protein in BGE. Hydrodynamic injection: 60 mbar, 8 s (0.8% of the volume to detector). Hydrodynamic co-injection of 0.02% DMF in BGE: 60 mbar, 3 s. Temperature: 25 °C. Peak identification on electropherograms: (1) CA, (2) Myo, (3) RNAse A, (4) β-lac A, (5) Lyz. Peak identification on H vs u curves: • (CA), ■ (Myo), ◆ (RNAse A), ▲ (β-lac A) and ★ (Lyz). Temperature: 25 °C. For the coating procedure, see **Section 2.3**. EOF: -71.1 ± 0.2 TU. p: 7.56 ± 0.07 × 10⁻³ s for CA; 5.33 ± 0.12 × 10⁻³ s for Myo; 5.60 ± 0.31 × 10⁻³ s for RNAse A; 5.00 ± 0.45 × 10⁻³ s for β-lac A; 4.96 ± 0.39 × 10⁻³ s for Lyz; CST: 3.03 ± 0.06 µm for CA; 2.26 ± 0.10 µm for Myo; 2.56 ± 0.17 µm for RNAse A; 3.40 ± 0.15 µm for β-lac A; 1.77 ± 0.22 µm for Lyz; N at -2 kV: 67.1 ± 6.8 × 10³. RSD of tₘ of the five proteins tested at -10 kV: 0.51%.

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Additionally, tip sonication was tested and compared to bath sonication. Tip sonication is known to promote vesicle formation and has the advantage of reduced preparation time (see protocol in **Sections 2.2** and **SI1** for more details). Post-sonication centrifugation, which is commonly applied to remove potential metal residues originating from probe erosion, was intentionally omitted [55–57]. Indeed, under the experimental conditions used, the absence of that step did not negatively affect protein separation performance (see **Figure SI5**), thereby enabling a more time-efficient preparation protocol. Results showed that DODAB SUVs formed exhibited an average size of approximately 300

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367 nm at a 10% of duty cycle (see **Figure SI6**). Moreover, the coating duration was found to impact the 368 capillary surface homogeneity, with longer coating times resulting in improved homogeneity, as 369 reflected by reduced *p* values (see **Figure SI7**). In the optimal conditions, DODAB vesicle solutions 370 prepared by tip sonication led to similar *p* and *cst* values as those using bath sonication. The *p* value 371 for tip sonication was 1.99 ± 1.16 × 10⁻³ s (see **Figures SI6A, B**) compared to 1.61 ± 1.01 × 10⁻³ s for 372 bath sonication (see **Figures 2A, B** and **Table 1**). The *cst* value for tip sonication was 3.51 ± 0.95 µm 373 (see **Figures SI6A, B**) compared to 3.54 ± 1.06 µm for bath sonication (see **Figures 2A, B** and **Table 1**). The reported *p* and *cst* values for tip and bath sonication were averaged over all proteins and 374 based on measurements from a single capillary. 375

376

### 3.3. Influence of nature and pH of the coating solution

378 One of the most critical factors affecting the performance of the DODAB coating is the nature and pH 379 of the coating solution. Indeed, significant differences in separation performances were observed 380 when the DODAB coating solution was prepared using different media: 50 mM phosphate buffer (pH 381 3.0), ultrapure water (pH 7.0), and 2 M acetic acid (AcOH, pH 2.2). Among these, the DODAB coating 382 prepared in phosphate buffer showed the lowest *p* value of 1.61 ± 1.01 × 10⁻³ s (see **Figure 2B** and 383 **Table 1**), compared to coatings prepared in ultrapure water (5.33 ± 1.98 × 10⁻³ s, see **Figure 5B** and 384 **Table SI7**) and AcOH (126±5 × 10⁻³ s, see **Figure 5D** and **Table SI7**).

385 Similarly, electroosmotic flow (EOF) values followed the same trend, with -79.5±0.5 TU, 386 -52.2±0.1 TU, and -45.9±0.3 TU for DODAB coating solutions prepared in phosphate buffer, 387 ultrapure water, and AcOH, respectively (see **Figures 2A, 5A**, and **5C**). The highest EOF that was 388 obtained with phosphate buffer suggests enhanced packing of cationic domains on the capillary 389 surface, leading to stronger EOF and improved surface coverage. Phosphate buffer (pH 3.0) was also 390 reported to enhance the adsorption rate of the surfactant ($k_{ads}$) [36]. In contrast, coatings prepared in 391 water and AcOH exhibited reduced EOF, indicating lower surface charge and less homogeneous 392 surface. It is worth noting that the hydrodynamic diameters of DODAB vesicles were approximately 393 200 nm (phosphate buffer with filtration), 300 nm (phosphate buffer without filtration), 400 nm (AcOH), 394 and 600 nm (ultrapure water), further supporting the importance of solution composition on vesicle 395 size alone (see **Figure SI8**).

396
<figure><img src="image_6.png" /><figcaption>**Figure 5.** Repeated electropherograms (n = 5) and the corresponding plate height (H) vs linear velocity (u) plots of a protein mixture in DODAB capillary coatings prepared in different buffers with 4 cycles of sonication/stirring with sonication. Electropherograms (A) and H = f(u) curves (B) for DODAB coatings prepared in ultrapure water at pH 7.0. Electropherograms (C) and H = f(u) curves (D) for DODAB coatings prepared in 2 M AcOH at pH 2.2. Experimental conditions: Capillary: 40 cm (31.5 cm to the detector) × 25 µm I.D. Coating solution: 0.1 mM DODAB in 50 mM phosphate buffer (pH 3.0) with 4 cycles of sonication/stir method. BGE: 2 M AcOH, pH 2.2. Flush before each run: BGE 1 bar, 6 min. Applied separation voltages: -10 kV, -8 kV, -6 kV, -4 kV, -2 kV. Sample mixture: 0.2 g/L of each protein in BGE. Hydrodynamic injection: 60 mbar, 8 s. Hydrodynamic co-injection of 0.02% DMF in BGE: 60 mbar, 3 s. Temperature: 25 °C. Peak identification on electropherograms: (1) CA, (2) Myo, (3) RNAse A, (4) β-lac A, (5) Lyz. Peak identification on H vs u curves: ● (CA), ■ (Myo), ◆ (RNAse A), ▲ (β-lac A) and ★ (Lyz). For the coating procedure, see **Section 2.3**. For MilliQ water, EOF: -52.2 ± 0.1 TU; *p*: 8.19 ± 0.75 × 10⁻³ s for CA; 4.23 ± 0.40 × 10⁻³ s for Myo; 3.83 ± 0.55 × 10⁻³ s for RNAse A; 6.62 ± 0.59×10⁻³ s for β-lac A; 3.77 ± 0.70 × 10⁻³ s for Lyz; *cst*: 3.99 ± 0.31 µm for CA; 3.32 ± 0.18 µm for Myo; 4.27 ± 0.28 µm for RNAse A; 5.00 ± 0.23 µm for β-lac A; 3.05 ± 0.25 µm for Lyz; *N* at all voltages: 49.5 ± 9.6 × 10³. RSD of *t*<sub>m</sub> of five proteins tested at -10 kV: 0.24%. For AcOH, EOF: -45.9 ± 0.3 TU; *p*: 126.27 ± 9.05 × 10⁻³ s for CA; 120.77 ± 9.08 × 10⁻³ s for Myo; 131.70 ± 9.84 × 10⁻³ s for RNAse A; not detected for β-lac A and Lyz, *cst*: 16.00 ± 3.95 µm for CA; 10.68 ± 3.41 µm for Myo; 0.96 ± 2.12 µm for RNAse A; not detected for β-lac A and Lyz; *N* at -2 kV: 6.0 ± 0.8 × 10³. RSD of *t*<sub>m</sub> of the five proteins tested at -10 kV: 0.44%.</figcaption></figure>

**3.4. Influence of the capillary inner diameter**

Capillary inner diameter (I.D.) was reported to be an important parameter influencing the coating quality and separation performance of DODAB-coated capillaries [58]. However, no quantitative data
420 were reported so far on the impact of capillary I.D. on separation efficiency. In this work, we were able
421 to quantify an increase of the average p values with increasing capillary diameter: p = 1.81 ± 0.57 x
422 10⁻³ s for 10 µm, 2.23 x 0.99 10⁻³ s for 25 µm, and 5.36 ± 9.30 x 10⁻³ s for 50 µm capillaries (average
423 values on all proteins tested, n = 3 capillaries, see Tables SI2 and Figures 6D, 2B, 6B). It is worth
424 noting that not only the slope p but also the cst value increased with increasing capillary diameter:
425 1.40 ± 0.40 µm for 10 µm I.D., 2.78 ± 1.10 µm for 25 µm I.D., and 4.56 ± 1.14 µm for 50 µm I.D.
426 (average values on all proteins tested, n = 3 capillaries, see Tables SI4). This general trend indicates
427 a significant decrease in coating homogeneity on the capillary surface with increasing I.D. that could
428 be explained by a very slow desorption of DODAB surfactant inside the capillary for higher capillary
429 I.D. [59]. Even a slight desorption can lead to strong negative impact on p value due to coating
430 inhomogeneities [30]. In the theoretical model that we proposed [30], the surface charge heterogeneity
431 generates hydrodynamic flow contributions in the capillary that increase the p-term due to Taylor
432 dispersion. Regarding the constant term, these experiments were performed by adjusting the injection
433 parameters so that the injected plug length was kept similar. Therefore, the contribution due to
434 injection cannot be the cause of the increase of the constant term observed for increasing capillary
435 I.D.. It is not clear if the impact of capillary I.D. on the cst term is related to the coating itself, or if it is
436 mainly due to the capillary dimensions on the constant term (e.g. detection, coiling).

437 EOF values remained relatively constant across different I.D.s: -77.7 ± 3.9 TU for 10 µm,
438 -78.3 ± 1.0 TU for 25 µm, and -76.0 ± 0.9 TU for 50 µm capillaries (n = 3 capillaries) (see Figures 6C,
439 2A, 6A). Similarly, the relative standard deviation (RSD) on the protein migration times (t<sub>Δ</sub>) at -10 kV
440 remained very low either on 10 µm capillary (0.62%, n = 3 capillaries), or on the 50 µm capillary
441 (2.14%, n = 3 capillaries), suggesting that the desorption occurring with the wider I.D. remains very
442 low but still impacting the p slope (see Table 2). Moreover, DODAB coating showed higher separation
443 efficiency when I.D. was narrower. This is reflected by the average values of plate numbers (N) for
444 three different I.D. of DODAB coated capillaries: 120,000 ± 18,000 for 10 µm; 77,600 ± 14,400 for 25
445 µm; 52,500 ± 9,300 for 50 µm (n = 3 capillaries) (see Table SI3 and the corresponding
446 electropherograms and H = f(u) curves in Figures SI9, 2, 10, respectively).

447
<figure><img src="image_7.png" /><figcaption>**Figure 6.** Repeated electropherograms (n = 5) and the corresponding plate height (H) vs linear velocity (u) plots of a protein mixture in DODAB capillary coatings prepared in 50 mM phosphate buffer at pH 3.0 with 4 cycles of sonication/stirring with sonication and coated in different capillaries inner diameters (I.D.). Electropherograms (A) and H = f(u) curves (B) for DODAB coatings coated in 50 µm. Electropherograms (C) and H = f(u) curves (D) for DODAB coatings coated in 10 µm. Experimental conditions: Capillary: 40 cm (31.5 cm to the detector) × 25 µm I.D. Coating solution: 0.1 mM DODAB in 50 mM phosphate buffer (pH 3.0) with 4 cycles of sonication/stir method. BGE: 2 M AcOH, pH 2.2. Flush before each run: BGE 1 bar, 6 min for 50 µm, and 6 bar, 6 min for 10 µm. Applied separation voltages: -10 kV, -8 kV, -6 kV, -4 kV, -2 kV. Sample mixture: 0.2 g/L of each protein in BGE. Hydrodynamic injection: 30 mbar, 4 s for 50 µm (0.8% of the volume to detector), and 1 bar, 3 s for 10 µm (1.38 % of the volume to detector). Hydrodynamic co-injection of 0.02% DMF in BGE: 30 mbar, 2 for 50 µm, and 100 mbar, 11 min for 10 µm. Temperature: 25 °C. Peak identification on electropherograms: (1) CA, (2) Myo, (3) RNAse A, (4) β-lac A, (5) Lyz. Peak identification on H vs u curves: ● (CA), ■ (Myo), ◆ (RNAse A), ▲ (β-lac A) and ★ (Lyz). For the coating procedure, see **Section 2.3**. For 50 µm, EOF: -77.0 ± 1.3 TU, *p*: 5.27 ± 0.70 × 10⁻³ s for CA; 4.92 ± 0.25 × 10⁻³ s for Myo; 4.27 ± 0.16 × 10⁻³ s for RNAse A; not reported for β-lac A and Lyz due to peak overlapping, *cst*: 5.13 ± 0.56 µm for CA; 3.44 ± 0.25 µm for Myo; 3.44 ± 0.08 µm for RNAse A; not reported for β-lac A and Lyz due to peak overlapping, *N* at -2 kV: 58.7 ± 7.1 × 10³. RSD of *t*<sub>m</sub> of five proteins tested at -10 kV: 0.30%. For 10 µm: -79.8 ± 0.2 TU, *p*: 2.27 ± 0.38 × 10⁻³ s for CA; 1.66 ± 0.13 × 10⁻³ s for Myo; 1.63 ± 0.05 × 10⁻³ s for RNAse A; 1.75 ± 0.21 × 10⁻³ s for β-lac A; 1.40 ± 0.03 × 10⁻³ s for Lyz, *cst*: 1.47 ± 0.22 µm for CA; 1.02 ± 0.13 µm for Myo; 1.51 ± 0.04 µm for RNAse A; 1.71 ± 0.12 µm for β-lac A; 0.96 ± 0.02 µm for Lyz, *N* at -2 kV: 124.0 ± 15.2 × 10³. RSD of *t*<sub>m</sub> of the five proteins tested at -10 kV: 0.07%.</figcaption></figure>
<table><caption>**Table 2.** Average values of slope (*p*), constant (*cst*) and %RSD on *t*<sub>m</sub> for three different DODAB coated capillaries (unless otherwise indicated). The *p* and *cst* values were calculated from average values of *H* = *f*(*u*) curve over 5 runs for each voltage and standard deviation (SD) of values determined with the Jackknife resampling method. Experimental conditions, same as **Figure 6** (for 10 and 50 µm) and **Figure 2** (for 25 µm).</caption><thead><tr><th rowspan="2">Protein</th><th colspan="3">p (10<sup>-3</sup> s)</th><th colspan="3">cst (µm)</th><th colspan="3">RSD on t<sub>m</sub> (%) at -10 kV</th></tr><tr><th>10 µm</th><th>25 µm</th><th>50 µm</th><th>10 µm</th><th>25 µm</th><th>50 µm</th><th>10 µm <sup>a</sup></th><th>25 µm</th><th>50 µm</th></tr></thead><tbody><tr><td>CA</td><td>2.53 ±<br>0.24</td><td>3.00 ±<br>0.44</td><td>6.52 ±<br>1.47</td><td>1.46 ±<br>0.06</td><td>2.87 ±<br>0.43</td><td>5.66 ±<br>1.02</td><td>0.56</td><td>1.67</td><td>2.28</td></tr><tr><td>Myo</td><td>1.61 ±<br>0.30</td><td>1.95 ±<br>0.64</td><td>5.51 ±<br>0.88</td><td>1.05 ±<br>0.04</td><td>2.49 ±<br>0.77</td><td>3.87 ±<br>0.60</td><td>0.60</td><td>1.75</td><td>2.40</td></tr><tr><td>RNAse A</td><td>1.28 ±<br>0.31</td><td>2.49 ±<br>0.27</td><td>4.05 ±<br>0.19</td><td>1.83 ±<br>0.30</td><td>2.26 ±<br>0.51</td><td>4.16 ±<br>1.02</td><td>0.61</td><td>1.76</td><td>2.44</td></tr><tr><td>β-lac A</td><td>1.82 ±<br>0.55</td><td>0.79 ±<br>0.91</td><td>N.D.</td><td>1.69 ±<br>0.35</td><td>4.41 ±<br>0.97</td><td>N.D.</td><td>0.64</td><td>1.85</td><td>N.D.</td></tr><tr><td>Lyz</td><td>1.80 ±<br>0.71</td><td>2.90 ±<br>0.67</td><td>N.D.</td><td>0.95 ±<br>0.11</td><td>1.84 ±<br>0.84</td><td>N.D.</td><td>0.66</td><td>1.87</td><td>N.D.</td></tr><tr><td>Average</td><td>1.81 ±<br>0.57</td><td>2.23 ±<br>0.99</td><td>5.36 ±<br>9.30<sup>b</sup></td><td>1.40 ±<br>0.40</td><td>2.78 ±<br>1.10</td><td>4.56 ±<br>1.14<sup>b</sup></td><td>0.62</td><td>1.78</td><td>2.14</td></tr></tbody></table>

N.D.: peak overlapped, not determined; <sup>a</sup>: *n* = 2 capillaries; <sup>b</sup>: average value calculated only from CA, Myo, and RNAse A.

### 3.5. Comparison with SMIL coating

In this section, a long capillary (80 cm) was used to prepare DODAB coating in order to compare with SMIL coated capillaries of similar length. DODAB coating solution was prepared by tip sonication. The electrophoretic separation of the 5 model proteins was performed at applied separation voltages ranging from -18 kV to -8 kV. **Figure 7** compares the electropherograms obtained for DODAB coating at -8 kV (in blue) and for a 5-layer of polydiallyldimethylammonium chloride (PDADMAC) and sodium polystyrene sulfonate (PSS) SMIL coating at -15 kV (in green, data from [26]) under the same acidic conditions (2 M AcOH) and for a given constant injection plug length (2.6 mm, i.e. 0.36% volume to detector). DODAB coating required the separation of proteins into two sets (i.e., set 1: CA, RNAse A, Lyz; set 2: Myo, β-lac A) to avoid peak overlaps between Myo and RNAse A, and between β-lac A and Lyz. In the case of SMIL coating, five different proteins were separated, i.e., CA, Myo, RNAse A, Lyz, and trypsin inhibitor (TI), and the applied electric fields varied from -25 kV to -10 kV since the EOF was much lower compared to DODAB coating [26].

Both coatings show very good separation performances with similar plate numbers: DODAB coatings provided average plate numbers (N) of 314,000 ± 60,000 at -8 kV (see **Table SI8**) vs 389,000
± 104,000 at -10 kV for SMIL [26]. However, DODAB coating exhibited an EOF approximately twice (-78.7 ± 0.1 TU) as high as that of SMIL (-45.0 ± 0.61 TU) [26], indicating the formation of stronger positive surface charge on the capillary surface. By comparing *p* values between DODAB and SMIL coatings, DODAB showed average *p* values of 0.85 ± 0.28 × 10⁻³ s (see **Figure SI11**) significantly lower than for SMIL coating (2.15 ± 1.50 × 10⁻³ s) [26]. This suggests that DODAB inherently provides higher surface charge homogeneity than SMIL. The DODAB surface is supposed to be better organized with well-ordered lines of surfactant cationic heads as observed in AFM compared to SMIL surface that inherently present a certain roughness (typically about 0.20 nm) [31,60]. The *p* value of β-lac A obtained with the DODAB coating (**Figure SI11B**) was significantly lower (0.79 ± 0.69 × 10⁻³ s) compared to that of the SMIL coating (1.91 ± 0.31 × 10⁻³ s) [26] due to the absence of interaction of β-lac on the DODAB coating. However, SMIL coating achieved a lower average *cst* value (≈ 0.4 µm) compared to DODAB coating (≈ 1.6 µm) (see **Figure SI11**). In terms of repeatability, although both coatings produced excellent results, DODAB demonstrated better repeatability for protein separation, with an RSD on *t*<sub>m</sub> of 0.03% under -10 kV (see **Figure SI11**), compared to 0.21% for SMIL coating under the same conditions [26]. Additionally, the best separation efficiency obtained with DODAB coating was found 606,000 ± 27,000 plates/m for Lyz at -12 kV (**Table SI9**), while SMIL coating provided better performance for Myo at -12.5 kV (933,000 ± 24,000 plates/m) [26].

510

<figure><img src="image_8.png" /></figure>

511

**Figure 7.** Repeated electropherograms (*n* = 5) of a mixture of 6 model proteins (CA, Myo, RNAse A, β-LacA, Lyz, and trypsin inhibitor, TI) on a DODAB coating prepared under the optimum condition (blue) and SMIL coating (green). Experimental conditions: Capillary: 80 cm (71.5 cm to the detector) × 25 µm I.D for DODAB coating; and 50 µm for SMIL coating. Coating solution: 0.1 mM DODAB in 50 mM phosphate buffer (pH 3.0) for DODAB coating solution; and (PDADMAC/PSS)<sub>2.5</sub> for SMIL coating solution. BGE: 2 M AcOH, pH 2.2. Flush between runs: BGE 2 bar, 12 min for DODAB coating; and 1 bar, 5 min for SMIL coating. Applied separation voltages: -8 kV for DODAB coating; and -15 kV for SMIL coating. Sample mixture: 0.2 g/L of each protein in
BGE. Hydrodynamic injection: 96 mbar, 10 s (0.36% volume to detector) for DODAB coating; and 30 mbar, 8 s (0.36% volume to detector) for SMIL coating. Hydrodynamic co-injection of 0.02% DMF in BGE: 90 mbar, 4 s for DODAB coating; and 30 mbar, 4 s for SMIL coating. Temperature: 25 °C. Peak identification on electropherograms: (1) CA, (2) Myo, (3) RNAse A, (4) β-lac A, (5) Lyz, (6) TI. For the coating procedure, see **Section 2.3** for DODAB coating and for SMIL coating [26]. For DODAB, EOF: -78.7 ± 0.1 TU, *p*: 1.20 ± 0.25 × 10⁻³ s for CA; 0.61 ± 0.26 × 10⁻³ s for Myo; 1.07 ± 0.22 × 10⁻³ s for RNAse A; 0.79 ± 0.69 × 10⁻³ s for β-lac A; 0.56 ± 0.52 × 10⁻³ s for Lyz, *cst*: 1.94 ± 0.17 µm for CA; 1.37 ± 0.24 µm for Myo; 1.17 ± 0.14 µm for RNAse A; 2.28 ± 0.59 µm for β-lac A; 1.03 ± 0.42 µm for Lyz, *N* at -8 kV: 313.9 ± 60.5 × 10³. RSD of *t*<sub>m</sub> of the five proteins tested at -8 kV: 0.04%. For SMIL, EOF: -45.0 ± 0.6 TU, *p*: 0.95 ± 0.39 × 10⁻³ s for CA; 4.76 ± 0.21 × 10⁻³ s for TI; 1.63 ± 0.26 × 10⁻³ s for Myo; 1.91 ± 0.31 × 10⁻³ s for RNAse A; 1.52 ± 0.07 × 10⁻³ s for Lyz, *cst*: 1.07 ± 0.22 µm for CA; 0.26 ± 0.13 µm for TI; 0.15 ± 0.10 µm for Myo; 0.43 ± 0.11 µm for RNAse A; 0.001 ± 0.001 µm for Lyz, *N* at -10 kV: 388.7 ± 104.2 × 10³. RSD of *t*<sub>m</sub> of the five proteins tested at -15 kV: 0.39%.

531

## 4. Conclusion

In this work, various parameters for the preparation of DODAB capillary coatings were investigated and their kinetic properties were systematically assessed in CE. We found that several critical parameters affect the preparation of DODAB vesicle solutions and therefore, the coating properties for the CE separation of intact proteins. Among them, temperature showed the strongest impact on the phase transition of DODAB to form vesicles and must be maintained above the melting temperature (ca. 45 °C). The sonication step was also identified as an essential factor: mechanical energy from sonication helps disrupt the bilayer (lamellar phase) of DODAB and promotes the formation of SUVs which are initiating the bilayer coating onto the capillary surface. Another key parameter was the nature and pH of the coating buffer, with phosphate buffer at pH 3.0 giving the best results. Last but not least, capillary inner diameter (I.D.) was found to influence charge surface homogeneity of the coating. Narrower I.D. provided more effective coatings than wider I.D., likely due to reduced desorption of the DODAB surfactants in thinner capillaries. Therefore, typical 50 µm I.D. capillary is not recommended under these conditions and 10 or 25 µm I.D. capillary should be preferred. In contrast, the number of sonication/stir cycles did not show strong impact, and 4 cycles were used here to ensure that all lamellar phases were disrupted.

Under the optimized conditions, DODAB coating demonstrated satisfactory protein separation performances. DODAB provided higher repeatability and lower *p* values than SMIL, reflecting greater coating homogeneity. It is suggested that DODAB vesicles form a well-organized bilayer on the capillary surface, whereas SMIL presents a certain residual roughness (even at low ionic strength). Moreover, DODAB showed no evidence of protein adsorption for all the investigated proteins. Particularly for β-lac A, DODAB significantly reduced protein adsorption compared to SMIL, as
reflected in the *p* values of *H* vs *u* curves. The *p* value for DODAB was very low (0.79 ± 0.69 × 10⁻³ s) compared to SMIL (15.3 ± 0.8 × 10⁻³ s) [26]. However, DODAB provided higher *cst* values than SMIL, which counter-balanced the lower *p* values.

At low electric fields, DODAB coating (80 cm total length) yielded an average plate number of 314,000 ± 9,000 at -8 kV. This was comparable to SMIL, which provided on average for all the 5 proteins 389,000 ± 104,000 at -10 kV at the same total capillary length. However, when considering the best *N* value, SMIL still outperformed DODAB. For instance, DODAB coating separated best at -12 kV in Lyz (*N* = 606,000 ± 27,000 plates/m), while SMIL achieved better performance for Myo at -12.5 kV (*N* = 933,000 ± 24,000 plates/m). Thus, although DODAB showed better repeatability and lower *p* values than SMIL, peak efficiency was lower due to higher constant terms. To conclude, DODAB coating is promising, but further optimization is required, particularly in modulating EOF. Due to the strong positive charges on the capillary surface, DODAB generates very high EOF, nearly twice as fast as SMIL, which reduces the apparent selectivity.

## Acknowledgements

The authors are grateful for the partial funding of doctoral scholarship to C.P. from the Franco-Thai program (Campus France).

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