# Spherulite formation in green nonaqueous media: The impact of urea on gelation in glycerol

Lauren Matthews

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► To cite this version:

Lauren Matthews. Spherulite formation in green nonaqueous media: The impact of urea on gelation in glycerol. Journal of Colloid and Interface Science, 2024, 676, pp.594-602. 10.1016/j.jcis.2024.07.146. hal-05099489

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Regular Article

# Spherulite formation in green nonaqueous media: The impact of urea on gelation in glycerol

Lauren Matthews¹

ESRF, The European Synchrotron, 71 avenue des Martyrs, Grenoble, 38043, France

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## GRAPHICAL ABSTRACT

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## ARTICLE INFO

**Keywords:**

Spherulites

Nonaqueous polar media

Self-assembly

## ABSTRACT

**Hypothesis:** Large macroscopic assemblies formed by a surfactant, sodium dodecylsulfate (SDS), and glycerol, can be directed to assemble in a hierarchical manner by the addition of a strong hydrogen-bond donor/acceptor, such as urea.

**Context:** Self-assembly in complex media is important to a range of applications, for instance in biological media, which are multi-component, to industrial formulations, where additives are present for flavour, texture, and preservation. Here, the gelation and self-assembly of sodium dodecylsulfate (SDS) in glycerol is explored in the presence of an additive, urea. Urea was chosen due to its importance both fundamentally and industrially, but also because of its ability to form strong H-bonds and interact with both glycerol and SDS.

**Experimental:** To cover the variety of length scales present in the gel-like phase, a combination of optical microscopy and small-angle X-ray scattering techniques were used to probe the micro- to nanoscale.

**Findings:** On the microscale, the formation of a spectacular spherulite phase, even at low urea contents - 0.1 wt%, upon cooling was observed, a stark difference to the microfibrillar phase observed in the absence of urea. Interestingly, the nanostructure of the two crystalline phases were similar and showed negligible differences. This suggests that urea is not involved in the SDS/glycerol microfibril formation but instead directs the assembly of spherulites by bundling the microfibrils. These ternary systems are also probed as a function of urea content. SDS concentration, and temperature. The observations in this work highlight the importance of small molecules in the self-assembly process, which is relevant both fundamentally but also industrially, where small molecules are often added to formulations.
# 1. Introduction

Understanding self-assembly of surface-active molecules in the presence of additives is important for a variety of applications, from biological - where cell culture media are complex [1,2] and can contain a variety of excipients, to industrial - in which formulations often contain salts and other additives [3–5]. It is important to probe such systems, as self-assembly in the presence of salts can lead to micelle transformations, from spherical to rod-like [6,7] or from micellar to vesicle [8,9].

Urea is an interesting organic molecule, with many applications, including: fertilizers [10,11], nonlinear optical crystals [12,13], and protein denaturation [14–17]. Structurally, urea is a diamide and as a result of this has the ability to form complex hydrogen-bonds (H-bonds), such as chelated H-bonds in dimers or also in tetramers [18] in urea alone. This H-bonding ability is important to the crystal structure of urea, which is tetragonal, in which the carbonyl group acts as a H-bond acceptor for four amine H-bond donors [19]. The structure within urea aqueous solutions has also been investigated, where historically there has been some disagreement on the effect of urea on the water structure. The main concepts on the effect of urea on the structure of water are: that urea disrupts the structure, that urea enhances the structure, and that urea has no impact on the structure [20–24].

Urea is also an important molecule in colloid science, where it has found use in being a cross-linker both as molecular urea and as polymerised urea-derivatives [25–28]. Such polymers, which are cross-linked via H-bonds, have found great popularity as candidates for self-healing polymers [29–31], as the H-bonds in urea are able to reform after breaking, repairing any damage to the cross-linked network. The effect of urea on surfactant solutions is also well studied, in particular the effect of urea in aqueous solutions of anionic sodium dodecylsulfate (SDS), where it was found that the critical micelle concentration (CMC) of SDS increased with increasing urea content [32,33]. In the case of cationic surfactants, $C_{12} - C_{16}$TAB, in high urea contents (40 wt%) a unexpected lamellar gel phase was observed [34].

Another use of urea, becoming commonplace in self-assembly reasonably recently, is in deep eutectic solvents (DES) [35], where in combination with choline chloride, urea forms the DES: reline [36]. The structure of reline is quite complex but based on strong H-bond interactions [37], where there is both a strong H-bond between the choline ion and the chloride ion, and also between urea and the chloride ion. Self-assembly of surfactants is also well studied in reline, where anionic SDS forms cylindrical micelles [38,39] as opposed to the spherical/ellipsoidal micelles formed in water.

In this work, the formation of spherulites is reported in the gel-like ternary system consisting of urea, glycerol, and SDS formed upon cooling from micellar phase to 278 K. The microscopic structure upon cooling was imaged *in-situ* using darkfield and polarising optical microscopy, and the nanostructure was investigated *in-situ* using small-angle X-ray scattering. The nanostructure of the microscopic spherulite assemblies was shown to be lamellar and similar to that of the gel-like system formed in the absence of urea [40]. The results presented here reinforce that urea is an interesting molecule capable of directing self-assembly processes, a result of its ability to form or participate in complex H-bonding networks in polar solvents. It is also demonstrated the importance in choosing an additive or solvent, for obtaining a particular assembled structure, which can affect a variety of industrial applications.

# 2. Materials and method

## 2.1. Materials

Urea (Honeywell; 99.5%) and Glycerol (Sigma-Aldrich; >99.5%) were used as received. SDS (Sigma-Aldrich, 98.5%) was used after recrystallisation from ethanol.

## 2.2. Preparation of ternary mixtures

The preparation of the samples involved combining the SDS and urea powders, before adding glycerol. The ternary mixture was then heated to 80 °C to dissolve the two powders in glycerol. Lastly, the samples were removed from heat and mixed using a vortex mixer, before being allowed to naturally cool down to room temperature.

## 2.3. Methods

### 2.3.1. Optical microscopy

Brightfield and darkfield optical microscopy was carried out in ambient temperature using an Olympus SZX16 microscope. Images were taken using the uEyeCockpit software. Polarising optical microscopy (POM) was performed using an Olympus BX50 microscope with crossed polarisers. Imaging was performed at various temperatures, using a Linkam temperature controller, T96; with liquid nitrogen pump, LNP96; the stage, LTS420; and images were captured using the ToupView software. Both the heating and cooling rates for the imaging were 1 °C/min.

### 2.3.2. Small-angle X-ray scattering (SAXS)

SAXS measurements were performed on the TRUSAXS ID02 beamline at the European Synchrotron Radiation Facility (ESRF, Grenoble, France) [41]. A single sample-to-detector distance of 0.8 m was used, with an X-ray energy of 18 keV ($\lambda$ = 0.069 nm). This yielded a q-range of 0.14 - 10 $nm^{-1}$, where $q = (4\pi/\lambda)\sin(\theta/2)$, and $\theta$ is the scattering angle. SAXS measurements were performed in 1 mm diameter quartz capillaries from WJM-Glas Müller GmbH, with quartz walls of 0.01 mm thickness. The capillaries were placed into a temperature controlled Peltier stage equipped with a PID controller (LFI-3751, Wavelength Electronics), the temperature was varied from ~ 6 - 75 °C (~ 279 - 348 K). The 2D SAXS patterns were collected by an Eiger2 4M (Dectris) pixel array detector. The scattering intensity from the samples was strong, and thus, an attenuator was used (Molybdenum; 50 µm), and the sample transmission was measured simultaneously. After a radiation damage test ensured the exposure time ($t_{exp}$ = 0.1 s here) caused the sample no damage, 20 x 0.1 s measurements were taken, whilst scanning down the capillary, and averaged to improve data quality. The 2D images were then normalised to an absolute intensity scale before azimuthal integration to achieve the 1D SAXS profiles. The corresponding background was subtracted from each of the 1D SAXS profiles, to give the resultant intensity (expressed as I(q)).

### 2.3.3. SAXS data analysis

The SAXS data analyses performed in this work can be summarised by peak fitting of the Bragg peaks seen in SAXS profiles for the gel-like phases. This involved fitting with a Gaussian lineshape to obtain peak parameters such as peak position $q_n$, d-spacing $d$, peak height $I(q)_{peak}$, and full-width at half maximum $\Delta q$, then used in Scherrer analysis [42]. For the liquid-like or micellar phases, a more detailed analysis was performed using a core-shell sphere form factor combined with a Hayter-Penfold rescaled mean sphere approximation structure factor [43–46]. Further details of the data analysis performed here are shown in the supporting information Equations 1 – 7.

# 3. Results and discussion

## 3.1. Surprising formation of spherulite assemblies

The SDS/urea/glycerol ternary systems were prepared by heating up and mixing with a vortex mixer to form a homogeneous mixture, and then, they were cooled down to form gel-like phases depending on surfactant concentration (Figures S1 – S5 in the supporting information). The microstructure of the phases without urea added is well-studied [40,47], where it was determined that on the microscale, microfribrils exist with a lamellar nanostructure. Here, the effect of adding
<figure><img src="image_6.png" /><figcaption>**Fig. 1.** Darkfield microscopy images of the SDS in glycerol microfibrillar phase at an SDS concentration, $c_{SDS} = 4.4$ wt%, for increasing urea content, $c_{urea} = 0.0$ wt% (a), 0.1 wt% (b), 0.2 wt% (c), 0.5 wt% (d), 0.8 wt% (e), 1.0 wt% (f), 2.0 wt% (g), 5.0 wt% (h), 7.0 wt% (i), 10.0 wt% (j), 20.0 wt% (k), 30.0 wt% (l), 40.0 wt% (m), 50.0 wt% (n).</figcaption></figure>

urea to the system was initially investigated with optical microscopy (Fig. 1), which shows the noticeable differences in the microscopic structure with increasing urea content.

At reasonably low urea contents, < 2.0 wt% urea, it was observed that the microfibrillar phase persists with increasing urea content. However, starting at a urea content of 2.0 wt%, the microfibrillar phase is disturbed with few remaining microscopic features until higher urea contents, such as $c_{urea} = 40.0$ wt%, where crystallites of urea are observed. This was confirmed by the microscopy of the control samples imaged in the absence of SDS, which also showed the presence of urea crystallites (Figure S6 in the supporting information). For the systems in the range, $c_{urea} = 2.0 - 30.0$ wt%, the bubble morphology is interesting, as the bubbles appear deformed and less isotropic until $c_{urea} = 10.0$ wt%, where a mixture of anisotropic and isotropic bubbles are observed. The deformed bubbles could be a result of the presence of undissolved SDS crystallites at the lower urea contents, but these are then solubilised as the urea content is increased, leaving behind air bubbles with spherical geometry in the liquid.

The solubilisation of the surfactant with increasing urea content is an interesting phenomenon and can be discussed relating to the Krafft point. The Krafft point is the temperature above which micelle formation is observed, and below which, there are solubility issues [48], resulting in hydrated crystals. Specifically, for SDS, the Krafft points have been determined in both water and glycerol, $T_k = \sim 289$ K and 304 K respectively, and the addition of salts, like sodium chloride, have been shown to shift this temperature [49–51]. Here, it was observed that the Krafft point is reduced with increasing urea content, which could arise from the formation of a strong hydrogen-bonding network formed by glycerol and urea molecules. Thus, the solvophobic, or hydrophobic in water, effect is strengthened and micelle formation is favoured [52–54].

Given the importance of temperature in regards to the Krafft point, and its importance demonstrated by previous works [40,55], polarising optical microscopy (POM) was performed with in-situ heating and cooling to observe any microscopic structural changes. Fig. 2 shows example images from POM for three compositions above and below the physical melting point of the gel. The behaviour during heating aligns with previous work on the system in the absence of urea, and the addition of urea did not seem to force the persistence of any assemblies to higher temperatures. However, the behaviour upon cooling is immediately

<figure><img src="image_7.png" /><figcaption>**Fig. 2.** Polarising optical microscopy images of the SDS in glycerol microfibrillar phase at an SDS concentration, $c_{SDS} = 4.4$ wt%, for increasing urea contents, $c_{urea} = 0.0$ wt% (a,b,c), 0.1 wt% (d,e,f), and 1.0 wt% (g,h,i). Imaging was performed at different temperatures: T = 278 K before heating (a,d,g), 348 K during heating (b,e,h), and 278 K after the cooling process (c,f,i). The scale bars represent 100 µm.</figcaption></figure>

interesting, where a clear difference in the structure when in the presence or absence of urea is seen.

In the absence of urea, microfibrillar, feather-like assemblies are observed, however, in the presence of urea, spherulite formation is seen. The lower urea content system shows better defined spherulites, and the higher urea content system shows numerous less well-defined smaller spherulite assemblies. The differences in microstructure between the
<figure><img src="image_8.png" /><figcaption>**Fig. 3.** Polarising optical microscopy images of the cooling process in a system with the composition: $c_{SDS} = 4.4$ wt% and $c_{urea} = 0.1$ wt%, images were taken at the following times: 0 (a), 100 (b), 200 (c), and 300 s (d). The scale bars represent 100 µm.</figcaption></figure>

two urea-containing systems are explained, if urea is considered the director of spherulite formation. As the system with a higher urea content has a larger number of smaller spherulites, the number of spherulite “seeds” must increase with the number of urea molecules. The importance of in-situ heating and cooling for this system is demonstrated, where none of the microstructure observed before heating is seen once the sample is cooled back to 278 K. The spherulites are not initially seen as a result of the sample preparation itself for the microscopy techniques. Placing the gelled sample onto a slide and covering with a coverslip generates a high shear, which could disturb the spherulites. This was confirmed with darkfield microscopy, performed by cooling a hot sample, and observing the evolution of the microstructure with time. Then, the coverslip was placed whilst the sample was still hot and the formation of spherulites was seen, before gently moving the coverslip to apply a shear (Figures S7-S9 in the supporting information).

The formation of spherulites is indicative of a striking self-assembly behaviour directed by urea molecules, similar to their cross-linking ability in polymer networks [25] or as a H-bond network disruptor [56,57]. This ability to create or disrupt H-bonds makes urea interesting in a polar solvent like glycerol, not only as glycerol has a large H-bond density ($\rho_{HB} = 76$ nm⁻³, vs 24 nm⁻³ and 134 nm⁻³ for ethanol and water respectively) [40], but also due to the polymeric-like self-assembly of glycerol and SDS resulting in the formation of microfibrils. The effect of urea in this case would be as a cross-linker, where urea is able to form bridges between fibrils via H-bond interactions. These bridges result in the formation of initial axialites, transitioning to sheaves, and finally to the spherulites. These assemblies then grow in diameter as a function of time (Fig. 3), where the whole process follows a nucleation and growth mechanism, like any other crystallisation. During the growth, the spherulites initially grow as large seemingly spherical assemblies, but due to confined space, this isotropy is lost as the growth of the spherulite is blocked.

The effect of heating and cooling in-situ was shown to be important, but a second cycle was performed to ensure there was no hysteresis and that spherulite formation was still seen (Figures S10 and S11 in the supporting information). The microstructural changes during the heating process (Figure S13 in the supporting information) show an initial dis-ordering of the spherulites into randomly oriented crystalline assemblies before disappearing, either becoming fully solvated or into an assembly smaller than the resolution of the microscope. Upon cooling a second time, the formation of spherulite assemblies was seen, confirming the microstructure.

<figure><img src="image_9.png" /><figcaption>**Fig. 4.** Flattened 1D SAXS profiles demonstrating the effect of urea on the SDS and glycerol system measured at 278 K.</figcaption></figure>

## 3.2. Nanostructural investigation of SDS/urea/glycerol spherulites

The addition of urea showed the formation of some interesting structures on the microscale, but to confirm the effect of urea as a director and not participating in the nanostructure of the microfibrils, small-angle X-ray scattering (SAXS) was performed. Fig. 4 shows the flattened intensity 1D SAXS profiles as a function of urea content, and overlaid 1D SAXS profiles are shown in Figure S14 in the supporting information. In the SAXS profiles, comparing both in the absence of urea and low urea contents, the Bragg peak positions do not vary much, which suggests the same nanostructure. This confirms the indirect participation of urea in the self-assembly through bridging rather than direct participation in the assembly of the lamellar nanostructure forming the fibrils. Fig. 4 also shows the effect of urea content, where there is an initial shift of the Bragg peak position, from $q_0 \sim 1.15$ nm⁻¹ to $q_0 \sim 1.98$ nm⁻¹, indication of a transition from the microfibrillar phase to solvated crystals, the calculated *d*-spacing as a function of urea concentration is shown in Figure S15 of the supporting information. There was an additional small initial swelling $\sim 1$ Å in total - determined from a small shift in the peak position, as the urea content increases from 0 - 1 wt%, suggesting that some solvent is taken in the microfibrillar/spherulite structure before the transition to solvated crystals.

Then, the Bragg peaks begin to lose intensity, starting at a urea content of 5 wt%, eventually leaving solely a single broad peak, $q \sim 1.2$ nm⁻¹, at the highest urea contents, arising from the formation of micellar aggregates. This micellar phase could be modelled as core-shell spheres (depicted by the illustration in Fig. 5), and as SDS is an anionic surfactant, the micelle model accounts for intermicellar interactions. The modelling of the profiles as a function of urea content showed that the micelle size did not change significantly as the urea content is increased: the thickness remained constant and the core radius varied within $\sim 1$ Å. Generally, this behaviour is similar to what was observed microscopically, where at higher urea contents, no microstructure could be seen until urea crystallites were observed at the highest urea contents. This confirms the microscopic observations were a result of urea shifting the Krafft point, favouring micelle formation at higher urea contents.

## 3.3. The effect of SDS concentration

Here, the discussion so far has focused the effect of urea on a reasonably concentrated SDS/glycerol system. However, other concentrations of SDS were also investigated here (Fig. 6), to observe whether lower concentrations were also affected by urea in the same manner. The SDS concentrations were chosen so that some compositions fell above and below the critical gelation concentration [40], to have an understanding of the effect urea has on both the liquid and gel phases. In the case
<figure><img src="image_10.png" /><figcaption>**Fig. 5.** 1D SAXS profiles of compositions that show the presence of a micellar phase, with an illustration of the micellar phase shown in the graphic (top). The solid black lines are representative of a model comprising a core-shell sphere form factor and the Hayter-Penfold rescaled mean spherical approximation structure factor, Equations 6 & 7 in the supporting information. Offsets are shown in the legend.</figcaption></figure>

of $c_{SDS} = 0.5$ wt%, the lack of any structure is apparent, shown by the SAXS profiles of the controls performed in the absence of the surfactant (Figure S16 in the supporting information). This demonstrates that urea does not aid the formation of the nanostructure comprising the microfibrils or other large scale structures in systems where there are no pre-existing large scale structures in the absence of urea.

For the higher SDS containing systems (1.1 and 2.3 wt%), the microfibrillar/spherulite phase can be initially seen and as the urea content is increased, there is a transition to a micellar phase. Microfibrils also exist in these concentrations in the absence of urea, which confirms that urea does not support the microfibril assembly. This is similar to the behaviour observed in the highest SDS containing compositions, where the transition first begins at 5 wt% and then, finishes at 10 wt% urea. There is an interesting deviation in the transition morphologies compared to the higher concentration. For the higher SDS containing systems, a transition first to solvated crystals was observed - however, for 1.1 and 2.3 wt% SDS, this transition did not occur. This unusual behaviour could be a result of the higher number density of fibrils, in the $c_{SDS} = 4.4$ wt% case, allowing for solvated crystals to form, as the microfibrils would be closer together.

<figure><img src="image_11.png" /><figcaption>**Fig. 6.** 1D SAXS profiles of the SDS/glycerol/urea ternary system with varying urea content and SDS concentration, $c_{SDS} = 0.5$ wt% (a), 1.1 wt% (b), and 2.3 wt% (c). All profiles are subtracted with the corresponding background except the profiles where $c_{SDS} = 0.5$ wt%, due to the scattered intensity being very close to the background level. The corresponding 1D SAXS profiles are shown in Figure S15 in the supporting information.</figcaption></figure>

### 3.4. The effect of temperature

The effect of temperature on the SDS in glycerol binary system in the absence of urea has been shown previously [40], where the transition from microfibrillar to micellar was observed with SANS. It has also been shown in this study to be an important parameter, especially when the observed behaviour is considered a phenomenon related to the Krafft point. There are three main phases noted in this measured section of the temperature-concentration phase diagram (Fig. 7), which are the microfibrillar/spherulite phase, solvated crystals, and micellar phase. There also are two “transition” phases, which are a mixture of either solvated crystals or microfibrils/spherulites with micelles. In all cases, no matter the initial phase, at higher temperatures ($T > 324$ K), only the micellar phase exists. This existence of a micelle phase is due to the system temperature surpassing the Krafft point and thus, micelle formation is favoured. As the viscosity of the crystalline phases in glycerol is much higher than in water, it is not surprising that this transition is completed over a temperature range of 5 - 20 K and not completed in
<figure><img src="image_12.png" /><figcaption>**Fig. 7.** The temperature-concentration phase diagram for the system where $c_{SDS}$ = 4.4 wt%, with illustrations depicting the different phases observed from SAXS measurements. Urea compositions over $c_{urea}$ = 10 wt% were not shown as these were composed of the micellar phase alone.</figcaption></figure>

one temperature step. The phase diagrams for other SDS concentrations are shown in the supporting information in Figure S18.

The Krafft point of the crystalline phase was also affected by both the surfactant concentration and also the urea content. For all SDS concentrations, the Krafft point generally decreases with increasing urea content until only the micellar phase is observed (shown in Figure S19 of the supporting information). The shifting of the Krafft point was also noted in a system comprising a nonionic surfactant in water, where the cloud point was reduced as a function of increasing concentration of small molecules/salt, such as glycerol, sodium chloride, and phenol [58,59]. The observation in this case can be explained through consideration of the intermolecular interactions occurring in the system. Both urea and glycerol are excellent H-bond donors and acceptors, which should make them compatible - confirmed by the high solubility of urea in glycerol, > 540 g L⁻¹. The strength of the solvent network is then increased by interaction of urea and glycerol, favouring micelle formation to reduce unfavourable interaction/disturbance to this complex solvent network.

The phase diagram above only describes the behaviour upon heating, as cooling is more complicated due to the long timescales or cold temperatures necessary to reform the crystalline phase. Whilst heating microfibrils/spherulites persist to temperatures, $T > 300$ K, during cooling microfibrils/spherulites only reform at temperatures, $T < 285$ K, in the time scales measured here. If the cooling was performed whilst equilibrating at each temperature, it should be possible to see the reverse behaviour, this is also the same for the case in the absence of urea. The SAXS profiles of a ternary phase containing a low urea content, $c_{urea}$ = 0.1 wt%, during the cooling process, are shown in Figure S20 in the supporting information. These demonstrate the presence of micelles alone at each temperature, which can be modelled in the same way as shown in Fig. 5. From the analysis, it was demonstrated that the micelle radius increases a little with decreasing temperature, by ~1.2 Å. The micelle charge also increases with decreasing temperature as expected due to the micelle volume, which increases with decreasing temperature, observed also in aqueous SDS solutions [60].

The transition from the micellar to crystalline phase was observed when the system was left to cool for 1 hr at 279 K, Fig. 8a, where SAXS profiles were captured every 20 minutes. After the first 20 minutes had passed, a co-existence of the lamellar phase and the micellar phase could

be seen. The micellar phase contribution to this mixed system could be modelled using Equations 6 and 7 (supporting information), and showed an increase in the volume fraction of micelles, continuing the trend during cooling of the micellar phase from 348 K to 279 K. The crystallisation kinetics are also interesting, the plot in Fig. 8b shows the change in correlation length as a function of time. The dashed line is representative of the Avrami model, a simple sigmoidal relation typically used to describe phase transformations [61]. The few data points taken from experiment seem to follow the model well, but this should be pursued further for confirmation.

## 4. Conclusions

In this work, the unusual hierarchical self-assembly behaviour of a crystalline physical gel in the presence of urea was investigated. Understanding the assembly of surfactants in more complex systems is important both fundamentally, but also industrially - where formulations or more biologically-relevant media often are complex. Also it is particularly interesting in cases where the effect of an additive causes an extreme structural transformation. Urea was selected as it is an industrially relevant molecule, particularly in formulation science, but also for a fundamental purpose, as it is a strong H-bond donor and acceptor.

On the microscale, microfibril growth was seen, noted previously in the absence of urea, however, further assembly directed by urea was also observed, transforming these microfibrils into spherulites. Whilst urea causes a spectacular change on the microscale, on the nanoscale there were negligible differences, suggesting that the spherulites and microfibrils consist of the same SDS/glycerol lamellar structure. The formation of the spherulites is proposed to start with the bundling of the SDS/glycerol microfibrils, directed by urea, to form initial axialites, before further bundling into sheaves and then continuing until spherulite formation occurs. Changes on both the micro- and nanoscale were probed using *in-situ* techniques, as temperature was shown to be an important parameter, and the generated shear during sample preparation causing structural disturbances.

As mentioned, temperature was demonstrated to be an important parameter, as seen in previous works [40,55]. But the composition of the SDS/urea/glycerol ternary system was also investigated in detail, as
<figure><img src="image_13.png" /><figcaption>**Fig. 8.** (a) 1D SAXS profiles of a ternary system containing 4.4 wt% SDS and 0.1 wt% urea after being left to cool at 279 K for 60 mins. (b) The effect of cooling on the correlation length in the lamellar phase. The black dashed line represents Avrami growth. (c) Illustrations of the micellar phase and lamellar phase. Offsets are shown in the legend.</figcaption></figure>

composition is a primary consideration in various applications, particu-
larly in industrial use. The temperature studies demonstrated that for all
compositions, in which a crystalline phase was present, increasing tem-
perature causes a transition from a crystalline phase to a micellar phase.
This was considered analogous to the Krafft point, a property common-
place in colloid science, above which micelle formation is favoured.
In SDS/glycerol binary systems, the Krafft point was investigated and
shown to be 304 K [51], compared to 289 K in water. The melting of the
crystalline phase was similar for both microfibrillar and spherulite mor-
phologies, the only difference was the melting point. This phenomenon
was considered an effect of the strong H-bond network formed by glyc-
erol and urea, strengthening the solvophobic effect, which is made pos-
sible by the high compatibility of urea and glycerol. This would favour
micelle formation, by reducing unfavourable contact between the non-
polar surfactant tails and polar urea/glycerol molecules.

With these transformations in the heating process being investigated,
the cooling of these complex systems was also interesting, demonstrat-
ing that the crystallisation was not instantaneous and not in the same
time scales as the melting. This is not surprising, given the change in
viscosities whilst heating and cooling, η<sub>gly</sub> = 12070 cP, 3900 cP and
50.6 cP, at 273 K, 283 K, and 343 K respectively [62]. This can be com-
pared with water, where η<sub>water</sub> = 1.792 cP, 1.308 cP, and 0.4061 cP at
273 K, 283 K, and 343 K respectively [62]. Whilst the sample is heated,
the large reduction in viscosity would allow for processes to be faster,
whilst the reverse, with an increase in viscosity, > 10000 cP, would slow
down such transitions.

However, despite the requirement of a longer time needed to re-
assemble the crystalline phase, the reassembly mechanism could be
investigated by holding the system at ~ 279 K for an hour. When keep-
ing the ternary system at T = ~ 279 K, the lamellar structure starts to
return after 20 minutes, and contribution from the micellar phase dis-
appears between 20 and 40 minutes. The crystallisation process, only
touched upon in this work, appeared to follow the crystallisation kinet-
ics of the Avrami model [61]. This aligns with previous work on the
crystallisation kinetics of SDS in water carried out at 20 wt% SDS [63],
where the authors also used the Avrami model to describe these kinetics
in water. Though as mentioned in the text, this is an initial investigation
and would require more measurements for confirmation.

To conclude, this work shows that the assembly of amphiphiles
and additives in nonaqueous media is complex on both the nanoscale
and more strikingly, on the microscale. A small molecule, like urea,
can cause hierarchical assembly in a crystalline physical gel system,
changing drastically the microstructure. The temperature-dependent be-
haviour on the nanoscale was also interesting, where the transition from
crystalline to micellar is analogous to the Krafft point, which can be
reduced as a function of urea content, due to the strong intermolecu-
lar interactions between urea and glycerol. There is also an emphasis
on the role of intermolecular interactions at play here, and how small
changes in the H-bonding properties, such as H-bond density, causes
such structural differences. Further investigation into the crystallisation
kinetics in these media, combined with studies on the effect of other
small molecules would help further our understanding in this area. Such
self-assembled structures are important for a variety of applications from
biological, where complex media are often present in physiological con-
ditions, to industrial, in which using aqueous solutions is not always
possible.

CRediT authorship contribution statement

**Lauren Matthews:** Writing – review & editing, Writing – original draft, Visualization, Validation, Software, Resources, Project administration, Methodology, Investigation, Formal analysis, Data curation.

Declaration of competing interest

The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.

Data availability

Data will be made available on request.
Acknowledgements

I acknowledge Dr. T. Narayanan and the rest of the ID02 beamline staff for their support and advice. The ESRF is thanked for providing beamtime, financial support and facilities. E.A. Bersenev is thanked for useful discussion. I acknowledge the partnership for soft condensed matter (PSCM) for provision of the polarising optical microscope. Professor W.H. Briscoe is thanked for his advice and support. SasView, originally developed under NSF award DMR-0520547, contains code developed with funding from the European Union's Horizon 2020 research and innovation programme under the SINE2020 project, grant agreement No 654000. The experimental data are available upon request to the author.

Appendix A. Supplementary material

Supplementary material related to this article can be found online at
https://doi.org/10.1016/j.jcis.2024.07.146.

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