Colorization of optically transparent surfactants to track their movement in biphasic systems used for differentiation of nanomaterials

Abstract

Aqueous two-phase extraction (ATPE) is a versatile method for the purification of numerous chemical compounds and materials, ranging from proteins and nucleic acids to cell organelles and various nanostructures. However, despite its widespread use, the underlying extraction mechanism remains unclear, which significantly reduces the utility of ATPE. Many types of surfactants are often added to biphasic systems to enhance the extraction of analytes between phases. Although their role in this process is crucial, it is not entirely understood. In this work, to fill this gap, we adapt and refine a nearly two-hundred-year-old chemical technique for the detection of bile salts in urine, referred to as Pettenkofer's test and monitor the partitioning of single-walled carbon nanotubes (SWCNTs) by ATPE. This approach enabled us to tint the otherwise transparent bile salt surfactants to precisely track their distribution and concentration in the biphasic system, thereby unravelling the modus operandi of this popular purification technique.

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New concepts

Bile salt surfactants are often used to suspend various nanomaterials that are otherwise insoluble in water or organic media. However, these compounds are notoriously challenging to measure because they lack unique functional groups that could enable selective interactions with other substances. As a result, it is uncertain how much the results discussed in the context of nanotechnology are influenced by these solubilizers. This article shows how this ongoing issue can be addressed by making the transparent surfactants colored to enable their straightforward optical tracking. The treatment of bile salt surfactants with 4-hydroxybenzaldehyde under acidic conditions produced a purple product which was noted with the naked eye. To capitalize on this achievement, the newly devised general approach for bile salt quantification was validated by using it to clarify the surfactant-mediated mechanism of the aqueous two-phase extraction of single-walled carbon nanotubes.

1. Introduction

Single-walled carbon nanotubes (SWCNTs) are carbon allotropes that have consistently attracted scientific interest since their discovery. Although all SWCNTs have a cylindrical shape and are composed of a hexagonal carbon sheet, the spatial arrangement of atoms can vary significantly.¹ Consequently, each SWCNT type exhibits different properties, posing a major challenge for their implementation, as there is no commercially viable method for synthesizing monochiral SWCNT products in sufficiently large amounts.² To overcome this problem, various post-synthesis separation techniques have been developed to isolate SWCNTs with specific structure and hence properties. Among these, water-based methods such as density gradient ultracentrifugation,³ electrophoresis,⁴ chromatography,⁵ and aqueous two-phase extraction (ATPE)⁶ are widely employed. The latter, which relies on the partitioning of SWCNTs between two immiscible aqueous solutions, is particularly effective for this purpose as it can deliver fractions enriched with SWCNTs of specific conductivity type,⁷ chirality,⁸ and handedness.⁹

However, due to the highly hydrophobic nature of SWCNTs, their use in a liquid medium requires the application of dispersing agents. Bile salt surfactants, particularly sodium cholate (SC) and sodium deoxycholate (SDC), are often used to achieve individualized, stable SWCNT suspensions.² In ATPE, such suspensions are introduced into a biphasic system, typically composed of dextran (DEX) and poly(ethylene glycol) (PEG).⁶ Depending on the type of surfactant coating, SWCNTs preferentially partition into one of the two phases. For example, with SC or SDC, SWCNTs typically migrate into the DEX-rich bottom phase.¹⁰ To achieve SWCNT separation, surfactants increasing the SWCNT affinity to the top phase (e.g., sodium dodecyl sulfate (SDS) or Triton X-100)^10–12 are gradually introduced. As a result, SWCNTs can be collected stepwise from the top phase. Analogously, SWCNT dispersions encapsulated with surfactants that prefer the top phase can be prepared, and then the SWCNTs are harvested step by step in the bottom phase.

Despite the conceptual simplicity of this approach, the mechanism governing the separation of SWCNTs remains unresolved. One particularly essential but largely overlooked aspect is the distribution of surfactants between the two phases. For an unknown reason, the addition of surfactants to the biphasic system causes the migration of SWCNTs between the phases. As previously stated, bile salt surfactants promote the relocation of SWCNTs to the bottom phase. Hence, is the concentration of these surfactants higher therein, and this increased preference for the bottom phase is the reason why they facilitate the downward extraction of SWCNTs? Considering the dual function of surfactants in this approach (playing the roles of SWCNT solubilizers and partitioning modulators), it is desirable to answer such questions and pinpoint their role in order to come closer to elucidating the workings of ATPE to improve its utility for purification of a broad spectrum of materials, which it can process.^13,14

Unfortunately, this is a challenging endeavor. Although the ATPE samples contain a limited number of components (usually two phase-forming polymers, up to three surfactants, SWCNTs, and water), no straightforward analytical method exists for the quantitative determination of surfactants, as these compounds are typically colorless. Defillet et al. recently noted that it is possible to estimate the concentration of bile salt surfactants by measuring their optical absorption spectra beyond 2000 nm in D₂O.¹⁵ Unfortunately, the method cannot be used to quantify such surfactants at low concentrations since absorption spectra thereof are not sufficiently distinctive. Concomitantly, nuclear magnetic resonance and mass spectroscopy are also not well-suited for quantitative analysis of surfactants present in the matrices of phase-forming components (PEG and Dextran), which are polydisperse and made of the same elements as regular surfactants used in the ATPE framework. One could consider chromatography to isolate surfactant molecules from the phase-forming compounds for analysis, but this tactic would be labor-intensive, hindering the possibility of high-throughput research. Taking into account the widespread use of surfactants, particularly in the context of nanomaterial solubilization and modification,^2,16,17 it would be beneficial to establish a platform for their selective and straightforward determination to improve the comprehension of the phenomena at the nanoscale.

In this work, we present a colorimetric method for the quantitative determination of surfactants in complex chemical environments. By adapting and improving the relatively unpopular Pettenkofer test, first published in 1844,^18,19 we elucidated how to quantify the abundance of bile salt surfactants, such as sodium cholate, directly in the biphasic system composed of poly(ethylene glycol), dextran, SWCNTs, water, etc. The developed method proved to be very robust and compatible with various chemicals present in water. Moreover, it facilitates the detection of bile salt surfactants with the naked eye. Capitalizing on this achievement, we successfully deciphered the mechanism of separating SWCNTs via ATPE with bile salt surfactants. The obtained results reveal meaningful surfactant-SWCNT interactions, paving the way for the development of more effective purification strategies for materials and compounds that can be processed by surfactant-assisted extraction, especially in biphasic systems.

2. Results and discussion

During original Pettenkofer's procedure, sucrose undergoes hydrolysis followed by dehydration to form furfural (FUR), (5-hydroxymethyl)furfural, or other analogous compounds²⁰ which then react with cholic acid to produce a colored compound.²¹ Dextran used in ATPE separation is a glucose-based polymer. Hence, in an acidic environment, it also gradually undergoes hydrolysis and oxidation producing furfural and other aromatic aldehydes,^22,23 which may take part in the Pettenkofer reaction, precluding its use for SC detection. The measurement of SC concentration in situ, either in the top or bottom ATPE phase to unravel its mechanism, using the classical Pettenkofer approach, both of which contain a certain amount of DEX,¹⁴ would not give reliable results. To overcome this problem and liberate the SC detection from the dependence on the complex polysaccharide decomposition pathway, we intentionally added aldehydes into the detection platform instead of sucrose, suspecting that the SC may react directly with them. A selection of derivatives of the simplest aromatic aldehyde, benzaldehyde, were examined, while FUR was evaluated as a reference (Fig. 1a).

Fig. 1 (a) A selection of aromatic aldehydes chosen for conjugation with SC according to the modified Pettenkofer's test procedure, optical absorption spectra of (b) the reaction products between SC and the indicated compounds, (c) stock solutions used for the modified Pettenkofer's test and the products of reaction between SC and (4-OH)BA or sucrose, (d) the products of reaction between (4-OH)BA and SC at various indicated concentrations.

Interestingly, among the tested compounds, (4-OH)BA displayed a very promising performance as its reaction with SC in acidic environment produced a distinct optical feature positioned at ca. 570 nm (Fig. 1b). At the same time, other benzaldehyde derivatives containing electron-donating and electron-withdrawing substituents in different positions did not afford colored products. Across the evaluated benzaldehyde analogs, (4-OH)BA was the only one possessing an electron-rich hydroxy group attached in the para position, which is considered as ring-activating.²⁴ Other electron-abundant compounds (3-OMe)BA and (3-OH)BA had the functional groups positioned in the meta position, which cannot increase the electron density in the aromatic system analogously.²⁵ Concomitantly, the (2-NO₂)BA and (2-Br)BA compounds were relatively electron-deficient and functionalized in close proximity to the formyl group, producing steric hindrance. It is important to note that the colored product made from (4-OH)BA and SC had a different wavelength of optical absorption maximum from the products of corresponding reactions of FUR (possible sugar decomposition product, Fig. 1b) or sucrose (Fig. 1c) with SC. Since FUR or sucrose (and, by extension, glucose) can be generated from DEX, the application of (4-OH)BA alleviates the problem of the concurrent side reaction of SC with DEX, making SC quantification robust.

The possibility of precise determination of SC concentration is also supported by the fact that the reagents used for the improved variant of the Pettenkofer reaction do not produce any colored products in the explored spectral range (Fig. 1c). Last but not least, the colored product created in the reaction between (4-OH)BA and SC manifests as a relatively intensive peak in the optical absorption spectra, the intensity of which is linearly dependent on SC concentration (Fig. 1d and Fig. S3), making such quantification straightforward.

Capitalizing on the fact that (4-OH)BA turned out to be very effective for analysis of synthetic solutions containing only SC and water by making a purple-colored solution (Fig. 2a), we extended this concept to investigate much more complex environments containing SC. In the ATPE system, commonly made of immiscible PEG and DEX aqueous solutions, SWCNTs can be extracted stepwise to the bottom phase with increasing SC concentration (Fig. 2b). In such case, SWCNTs with the smallest diameters migrate first, while larger SWCNTs require higher concentrations of SC. In the displayed example, once the volume of SC (10%, aq.) was increased from 0 µL to 450 µL in the system, the smallest available SWCNTs, i.e., (6,5) SWCNTs (d = 0.757 nm) and (8,3) SWCNTs (d = 0.782 nm), were extracted to the bottom phase (Fig. 2c). Further increase in the SC volume to 600 µL caused the emergence of the slightly larger (7,5) SWCNTs (d = 0.829 nm) and (8,4) SWCNTs (d = 0.840 nm) in the bottom phase. Currently, it is unclear how surfactants promote this move in the ATPE framework.

Fig. 2 (a) Generation of a colored product from SC enabling its optical detection, (b) the changed distribution of SWCNTs in the ATPE system as a function of SC surfactant concentration, (c) photoluminescence excitation–emission maps of the top and bottom phases of the corresponding samples shown in panel (b) and (d) binodal curve of the biphasic system made from PEG (6 kDa) and DEX (70 kDa) with the experimental conditions explored indicated by letters (the area above the curve indicates the biphasic region conditions), (e) optical absorption spectra of the products of reactions between the top and bottom phases collected from ATPE experiments performed according to the conditions reported in Table S3. The solid line indicates the measured data, whereas the shaded area corresponds to experimental uncertainty determined in Table S4.

We analyzed three variants of DEX-PEG systems (Fig. 2d), which are the most used in SWCNTs separation with the ATPE. Table S3 lists the volumes of compounds combined to form these systems, which were later examined using the disclosed SC detection approach. The first series (A) corresponds to samples wherein the concentration of both phase-forming components (PEG, DEX) was the same, i.e., 6%. To investigate whether the relative amount of one phase to the other impacts the SC distribution in the biphasic systems, two other series were made: (B) with the concentrations of PEG and DEX equal to 6% and 12%, respectively, while for the (C) series these amounts were inverted giving the concentrations of PEG and DEX equal to 12% and 6%, respectively.

The samples of the top (PEG) and bottom (DEX) phases subjected to the modified Pettenkofer's test (Fig. 2d – A1) showed only a slight increase in absorbance in case of the latter corresponding to hydrolysis and oxidation of DEX. However, this peak with minor intensity was positioned at ca. 440 nm, so it did not coincide with the spectral feature resulting from the combination of (4-OH)BA and SC. Once SC was added into the PEG–DEX system, the expected absorption band emerged at ca. 570 nm in the case of both phases (Fig. 2d – A2) after the reaction and its intensity increased upon adding a higher amount of SC (Fig. 2d – A3). The situation was analogous when 225 µL of SWCNT dispersion in SC aqueous solution was added (Sample A4) instead of pure 225 µL of SC aqueous solution (Sample A2). Two important conclusions can be drawn based on these outcomes. Firstly, the devised approach can be successfully used to track the distribution of SC between the phases, regardless of the complexity of the system and even in the presence of SWCNTs. The high optical density of the product formed by SC and (4-OH)BA reaction covers all signals coming from SWCNTs, the absorption of which is much smaller, taking into account their concentration. Secondly, it was somewhat surprising that the SC promoting the migration of SWCNTs to the bottom phases^10,11,26 turned out to have the same concentration in both phases. Intuitively, since SWCNTs coated with SC prefer the bottom phase, one could expect that the SC molecules themselves would have a higher affinity to the bottom phase and that is where they should be more abundant.

Considering that the distribution of SWCNTs in biphasic systems is strongly dependent on the type of phase-forming polymers, their molecular weights, and concentrations,^14,27 we performed additional measurements in PEG–DEX systems of different proportions of DEX and PEG. The goal was to gain further insight how the surfactant distribution changes upon modifying the extraction conditions. Regardless whether DEX (Fig. 2e – B1) or PEG (Fig. 2e – C1) was twice as concentrated as the other phase-forming component, the amount of SC in the top and bottom phases was analogous. The small discrepancy (experimental uncertainty, presented as shaded area in all the optical absorption spectra enclosed in this article) was not statistically significant (Table S4). Hence, in a PEG–DEX system containing a single type of surfactant, the obtained results strongly suggest that it is homogeneously partitioned between the two phases.

However, the ATPE systems achieve their best resolution for SWCNT separation when multiple surfactants are used to enable their competitive adsorption on SWCNT surface.⁶ Since, as previously discussed, bile salt surfactants such as SC direct SWCNTs to the bottom, Triton X-100 (TX-100, a non-ionic surfactant)¹⁰ and sodium dodecyl sulfate (SDS, an anionic surfactant)^28–30 were examined due to their capacity to act as counter-surfactants preferring SWCNT migration to the top. In both cases, despite the presence of these compounds in the extraction system, the concentration of SC was again not statistically different between the top and bottom phases (Fig. S5 and S6).

Furthermore, we also evaluated other partitioning systems to prove the utility of the newly developed method for in situ determination of SC concentration. For this purpose, we replaced either DEX or PEG in the DEX/PEG system with Ficoll or two types of Pluronic and quantified the amount of SC therein, respectively (Fig. S7). In the case of the Ficoll/PEG system, the concentration of SC was analogous in both phases once more proving that under such conditions SC does not tend to favor any of the phases. However, the application of DEX/Pluronic systems (based on Pluronic P68 and Pluronic L35) led for the first time to the dissimilar concentration of SC between the top and bottom phases. The reduced amount of SC in the bottom phase finally explains why in DEX/Pluronic systems, the migration of SWCNTs to the bottom phase is more challenging than when classical PEG/DEX systems are employed.³⁰ This effect, probably originating from the possibility of the formation of mixed micelle systems between Pluronic and SC molecules in the top phase³¹ is explained in detail in SI (Fig. S8–S11).

To interpret the reaction between the bile salt surfactant SC and (4-OH)BA, which is at the heart of the reported method, we systematically examined a broad spectrum of bile salt derivatives (Fig. 3a). While the core of these compounds is similar, they differ in the position and the number of hydroxyl groups attached as well as on the chemical identity of the joined chain constituting their hydrophilic head.

Fig. 3 (a) Bile salt derivatives examined, (b) optical absorption spectra of the products of reactions between the indicated compounds and (4-OH)BA (dilution factors in brackets).

Unfortunately, the elucidation of the reaction product is also highly problematic. We tried to isolate the reaction product, however, it is only stable in acidic conditions established by the reaction mixture. Attempts to dilute the mixture or transfer it to another solvent failed and resulted in product degradation, judging by discoloration of the solution and formation of a precipitate. Still, there are multiple chemical tests for the analysis of saccharides, which can reveal some insight into the underlying chemistry. It is known that aromatic aldehydes such as FUR formed during dehydration of sugars react easily with various hydroxyl-containing compounds in acidic environment to give characteristic colored products. An example is the Seliwanoff's test, wherein the aromatic aldehyde reacts with two resorcinol molecules, forming a coloured three-membered ring product.³² Similarly, Bial's test based on orcinol³³ and Molisch's test based on napthol³⁴ also transform aromatic aldehydes into particular colored products. The best performing (4-OH)BA aromatic aldehyde is therefore capable of reacting with sodium cholate and its derivatives, both of which contain the necessary hydroxyl groups for the chemical transformation to proceed. In parallel, it is also highly probable that in an acidic environment the 3,7,12-trihydroxycholic framework can be dehydrated and a cation can be formed, similarly to the Liebermann–Burchard reaction, used to detect cholesterol.³⁵ This would explain why the product is stable only under acidic conditions and decomposes when diluted with water or when the solvent is changed.

Finally, capitalizing on the fresh insight about the behavior of surfactants in biphasic systems, it is worthwhile to improve the understanding of the SWCNT partitioning with this method. However, the extraction system also contains SDS, which initially positions the SWCNTs in the top phase. Hence, it is essential to quantify its concentration. To achieve this goal, we exploited the phenomenon of a gradual migration of methylene blue (MB) from aqueous to chloroform (CHCl₃) phase with increasing SDS concentration^36,37 (Fig. 4a). We operated at slightly acidic pH to eliminate the potential interference from SC, whose pK_a is considerably higher than that of SDS,^38,39 by favoring its protonation. The combination of MB and SDS leads to the creation of an MB:DS complex (with simultaneous generation of sodium chloride), preferring the organic phase. The optical absorption spectra of these complexes in chloroform are quite distinct (Fig. 4c), which unlocks the possibility of colorimetric assessment of SDS concentration (Fig. 4d). Blank experiments confirmed that no other components of the biphasic system (PEG, DEX, bile salt surfactants, or combination thereof) produce optical features that could affect the analysis (Fig. S12a–c). Importantly, the addition of SC to SDS does not seem to change its optical absorption intensity (Fig. S12d). In light of the foregoing, the simultaneous application of both analytical techniques enabled precise tracking of surfactant distribution during SWCNT partitioning to better understand the migration of SWCNTs between the phases in the ATPE technique.

Fig. 4 (a) Photographs of gradual migration of MB:DS complexes to the CHCl₃-based phase with increasing SDS concentration, (b) molecular structures of MB and DS (Na⁺ and Cl⁻ counterions not shown), (c) optical absorption spectra of the MB:DS complexes in CHCl₃ as a function of SDS concentration, and (d) relationship between the maximum of absorption centered at 653 nm and SDS concentration.

SWCNTs injected into a PEG–DEX system containing SDS are initially positioned in the top phase (Fig. 5a). The addition of SC, as discovered in this work, leads to equal concentration of SC in the top and bottom phases, which we determined using the modified Pettenkonfer's test (Fig. 5b and Fig. S13). With the increase in SC concentration, it remains equally distributed between the phases. To deepen the analysis, we complemented the characterization by quantifying the amount of SDS present in the system. Interestingly, at the minimum SC amount introduced by the addition of only the SC@SWCNT dispersion, SDS prefers the top phase (Fig. 5c and Fig. S13). This result stays in line with findings reported by Defillet and colleagues.¹⁵ As the concentration of SC is increased, SDS molecules migrate into the bottom phase up to the point at which the concentration therein is slightly higher than in the top phase. We postulate that the initial positioning of the SC@SWCNT complexes in the top phase in the presence of SDS relates to the fact that SDS interacts very well with PEG since it can form mixed micelles with it.⁴⁰ Besides, SDS creates loose coatings around SWCNTs,^30,41 enabling their better contact with the environment. The top phase is more hydrophobic, and so are SWCNTs, which explains why SDS-rich SWCNT dispersions prefer the more hydrophobic top phase (Fig. 5d). On the other hand, bile salt surfactants such as SC create relatively tight coatings around SWCNTs⁴² with hydroxyl groups extended outward,⁴³ facilitating their suspension in the more hydrophilic dextran-rich bottom phase. Moreover, since the bottom phase has a higher density, the increased density of the closely wrapped SWCNTs with SC further improves the odds of their migration to the bottom phase purely due to density.⁴⁴ Simultaneously, these results once again strongly suggest that the dynamic exchange of surfactant molecules occurs on the SWCNT surface, and this phenomenon can be amplified by increasing the concentration of the desired surfactant type. It should also be noted that, contrary to the sudden transition of SWCNTs between the phases,^15,45 the changes of surfactant concentrations in the phases leading to these migrations seem to be gradual.

Fig. 5 (a) Photoluminescence excitation–emission maps of the top and bottom phases of SWCNTs partitioned in the presence of SDS and SC, (b) quantification of the SC concentration in both phases at various SC addition volumes, (c) SDS concentration in both phases at various amounts of SC introduced, and (d) the hypothesized phenomena occurring during the SWCNT separation process.

Last but not least, one also needs to consider that SDS and SC can form mixed micelle systems, and their degree of hydrophilicity is dependent on the SDS/SC ratio.⁴⁶ We have indicated the possibility of this phenomenon by tracking the amount of SDS in both phases as a function of SC concentration (Fig. 5c). The results obtained suggest that decreasing the SDS/SC ratio favors the migration of these mixed micelle systems into the more hydrophilic bottom phase. While identifying the origin of this case is outside of the scope of this publication, we highlight that the simultaneous application of both SC and SDS quantification methods reported herein can be applied to study such phenomena in greater detail.

3. Conclusions

Despite considerable progress, the mechanism of both the ATPE method discovered in 1896 and the Pettenkofer's test devised in 1844 remain elusive. However, the implementation and improvement of the latter approach carried out in this work enabled us to gain a substantial insight into the principles governing the biphasic extraction. Concomitantly, a straightforward test for the optical detection of bile salt surfactants, commonly used for the dispersion of otherwise insoluble nanomaterials, can be very useful to better understand their behavior in liquid media. The technique proved to work successfully in complex chemical media, enabling precise quantification of such compounds due to the linear dependence of optical absorption of the generated, highly-colorful products on surfactant concentration. In the context of SWCNTs, the obtained knowledge can facilitate the development of more effective methods of their purification, which is necessary to unleash their unique properties beyond the laboratory environment.

4. Experimental

All the information about the materials and methods is given in the SI. Quantification of SDS and SC was carried out on top and bottom phase aliquots collected from respective ATPE separations. This was necessary to ensure that the SWCNT partitioning, or individualization, is not disturbed by introducing the abovementioned compounds. High optical density of the colored surfactants necessitates considerable dilution of the solutions for characterization, which makes the optical transition related to SWCNTs not visible.

Conflicts of interest

There are no conflicts to declare.

Data availability

All data supporting this article have been included in the main text and supplementary information (SI). Supplementary information: experimental details and additional results related to the determination of surfactant concentration. See DOI: https://doi.org/10.1039/d5nh00574d.

Acknowledgements

The authors would like to thank the Polish National Science Centre for the financial support of the research (under the OPUS program, grant agreement 2019/33/B/ST5/00631).

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