# Carbon Dot Synthesis and Purification: Trends, Challenges and Recommendations

Yalei Hu, Océane Seivert, Yue Tang, Hüseyin Enis Karahan, Alberto Bianco

► To cite this version:

Yalei Hu, Océane Seivert, Yue Tang, Hüseyin Enis Karahan, Alberto Bianco. Carbon Dot Synthesis and Purification: Trends, Challenges and Recommendations. Angewandte Chemie International Edition, 2024, 63 (48), 10.1002/anie.202412341. [http://dx.doi.org/10.1002/anie.202412341](http://dx.doi.org/10.1002/anie.202412341)

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# Carbon Dot Synthesis and Purification: Trends, Challenges and Recommendations

Yalei Hu,* Océane Seivert, Yue Tang, H. Enis Karahan, and Alberto Bianco*

Dr. Y. Hu, O. Seivert, Y. Tang, Dr. H. E. Karahan, Dr. A. Bianco

CNRS, Immunology, Immunopathology and Therapeutic Chemistry, UPR 3572, University
of Strasbourg, ISIS, 67000 Strasbourg, France

E-mail: yalei.hu@ibmc-cnrs.unistra.fr; a.bianco@ibmc-cnrs.unistra.fr

**Keywords:** Carbon materials, graphene, photoluminescence, separation, biocompatibility

## Abstract

Carbon dots (CDs) have rapidly emerged as a new family of carbon-based nanomaterials since their initial discovery two decades ago. Numerous appealing properties, such as precursor and synthesis process flexibility, tunable photoluminescence, and good biocompatibility, have enabled their widespread applications in sensing, catalysis, energy, and biomedical fields. As the field expands, notable efforts have recently focused on mechanistically elucidating the structural formation and optical behavior of CDs. However, the absence of “clean” CDs presents a major obstacle to achieving a solid understanding of these aspects. Often, the claimed CDs are, in fact, a mixture of small molecules, oligomers, nano-sized aggregates, or even microparticles. Such coexistence of impurities markedly impacts the physicochemical properties of resulting CD-based mixtures, hampering the resolution of key mechanistic questions. Here, we aim to address this fundamental shortcoming of the field, going beyond the customary focus of the existing reviews that predominantly cover synthesis, optical performance, and application prospects. We begin with an overview of CD synthesis and then thoroughly examine the purification methods, including filtration, dialysis, electrophoresis, and chromatography. The insights provided here will guide the researchers towards obtaining high-quality CDs, employing proper combinations of available tools, and ultimately paving the way for more demanding applications.
# 1. Introduction

Carbon dots (CDs) are carbon-based (quasi)-spherical nanomaterials typically ranging from 1 to 10 nm in diameter.[1] The first member of this emerging class of nanomaterials was serendipitously observed in 2004 during single-walled carbon nanotube purification, thanks to their fluorescent properties.[2] Two years later, using graphite powder and clay as raw materials, similar luminescent nanoparticles were successfully synthesized by laser ablation, which were named “carbon dots”.[3] After an initial rather latent period, the number of CD-related publications has grown drastically in various fields over the last decade.[4] If one can say that the material is the king, the nanomaterial is its queen, carbon-based nanomaterial is the queen's crown, and CDs are the bright jewels on the crown. Nevertheless, the field suffer from several fundamental issues, ranging from the very definition of the materials and perceivable lack of consistency in literature in several fronts, notably including synthesis and purification aspects. We particularly argue that the high variability of the CD literature brings a certain level of vagueness, which is hard to overcome without applying stringent purification conditions.

The complexities arising from the small size and chemical and structural richness of CDs make their characterization rather difficult. Accordingly, the definition of CDs is neither purely chemical, structural, nor functional. The current consensus is that CDs can be classified into four main categories: carbon quantum dots (CQDs), graphene quantum dots (GQDs), carbon nanodots (CNDs), and carbonized polymer dots (CPDs).[5] Among them, CQDs possess a rather defined lattice structure, are almost spherical, and exhibit quantum confinement phenomena. GQDs also exhibit defined lattice structures built upon few-layered very small graphene structures. CNDs, on the other hand, possess amorphous carbon cores and typically lack quantum confinement effects, while CPDs exhibit polymer-aggregate-like characteristics, with weakly crystalline carbon cores and surface-tethered chains decorating around. However, it is worth noting, typical synthesis and purification protocols might yield a mixture of some of those sub-classes at the same time.

Various widely accessible techniques, such hydrothermal/solvothermal treatment, pyrolysis, microwave irradiation, and ultrasonication, are in wide use for CD synthesis. Practically irrespective of the method selection, under the right conditions, virtually all carbon-containing substances can be converted into CDs, including small molecules, polymers, and even biomass.[6] Besides, it is not challenging to incorporate different chemical functionalities to CDs by using simple dopants. As a result, while generally being composed of carbon, hydrogen, and oxygen, with a carbon content of typically over 60%, CDs may include various other elements
(heteroatoms) such as nitrogen, sulfur, phosphorus, fluorine, and boron, further complicating
the picture. Such a variability offers notable opportunities for engineering materials properties.
However, in certain applications, where purity might be of particular importance, such as
biomedical applications, it might be a challenge, as well. While several studies reported CDs
with excellent biocompatibility.[7] Therefore, when exacerbated with sub-optimal purification
practices, the CD literature becomes hard to navigate and reach concrete structure-property-
activity relationships.

CDs possess remarkably tunable luminescence (ranging from ultraviolet to second near-
infrared window) and show resistance to photo-bleaching, which are among the top reasons for
wide attention towards them. However, notably, the combination of materials properties
variability and sub-optimal purification, seemingly also complicates reaching a fundamentally
strong mechanistic understanding of CDs' luminescence behavior. Because of their
considerable affordability and ease of optical characterization at an elementary level, the CD
literature is overpopulated with optical performance measurements, but our mechanistic
understanding is still fairly limited. Besides, being used in various other applications, including
sensing, catalysis, and energy storage, there is no clear-cut structure-property-activity
relationships for different applications to the best of our knowledge.[8] To our understanding,
inadequate purification practices play a major role in this picture.

Here, to address the challenges sketched above, we approach the current CD literature through
the lenses of purification methods. To create a basis for further discussions, we first provide an
overview of CD synthesis. Later, we examine the purification methods ranging from filtration
and dialysis to electrophoresis and chromatography. Overall, we expect to guide researchers in
developing and practicing adequate CD purification protocols to move the field forward.

**1.1. Synthesis of CDs**

Since their unexpected discovery, a series of methods have been used to prepare CDs.[9]
According to the material size changes during the preparation process, traditional synthesis
methods for CDs can be divided into two categories: top-down and bottom-up approaches.[10]
Top-down approaches involve decomposing bulk carbon materials (e.g., graphite, charcoal) or
relatively large carbon nanostructures (e.g., graphene oxide, carbon nanotubes) into CDs.[11]
Arc discharge, laser ablation, chemical oxidation, and electrochemical oxidation are some
representative top-down approaches.[12] Bottom-up approaches involve generating CDs from
small molecular precursors through polymerization and carbonization methods.[13]
Hydrothermal and solvothermal methods, microwave, and pyrolysis are the commonly used
bottom-up methods.[14] To facilitate and improve purification and help determine the synthesis pathway, structural features, and luminescence mechanism of CDs, we recommend conducting deeper studies on the use of small molecules as precursors.

Lately, some nonconventional methods for CD synthesis have been explored, such as electromagnetic induction heating methods,[15] flow synthesis,[16] and machine-learning-enhanced approaches.[17] Electromagnetic induction heating is a non-contact heating alternative to conventional heating that focuses the heat where it is needed. The technology is significantly more efficient than conventional heating, and it is often used for preparing CDs at larger quantities. Flow synthesis of CDs is accomplished using microfluidic reactors.[18] The use of microfluidic reactors can reduce reaction times, allow for rapid and extensive screening of many different conditions involving a variety of precursors and solvents, and have a great potential to optimize the reaction conditions for CD synthesis. With the development of artificial intelligence, machine learning has been used to explore the synthesis of CDs.[19] With data input from the available studies of CDs with different parameter combinations and optical properties, machine learning was used to optimize and predict the optical properties of these materials, demonstrating its potential in guiding the synthesis of desired CDs.[20] In addition, green synthesis of CDs using natural precursors like biomass wastes are considered contributing to sustainability efforts.[9c] Nevertheless, despite these advancements virtually all CD synthesis procedures produce suffer from impurities, which are mainly unconverted precursors and unwanted by-products.

## 1.2. Impurities coexisting with CDs

Regardless of the synthetic method used, as-prepared crude CDs inevitably contain a certain amount of small molecular carbonaceous impurities and large-sized carbonaceous nano- or micro-structures.[21] When top-down approaches are utilized, materials such as graphene oxide and carbon nanotubes are cut into CDs during laser ablation or chemical oxidation processes.[22] During these processes, some intact and incompletely decomposed large-sized derivatives often exist as impurities alongside with CDs product. In addition, event fairly large materials, such as layered materials, may also be down-sized into very small fragments, such as aromatic molecules.[23] When bottom-up approaches are employed, small molecules form CDs through various steps, such as dehydration, condensation, and carbonization.[24] In these processes, relatively low temperatures are preferred to pursue high fluorescence quantum yield (QY). However, low reaction temperatures also lead to incomplete conversion rates, resulting in some unreacted small molecules and low-molecular-weight oligomers present in the crude CDs.[25]
Sometimes, relatively high temperatures are used to obtain highly crystalline CDs. In these cases, there are instances that small molecules assemble into micrometer-sized layered structures. Overall, the diversity of the synthesis methods and the abundance of the precursors make the impurities in CDs diverse, as well.

**1.3. Why purity matters?**

As a novel class of nanomaterials, CDs have attracted notable attention, largely due to their superior performance and applications in diverse fields. However, given the potential impurities discussed above, strict attention to their purification is mandatory. On the one hand, the purification ensures that CDs synthesized using the same method maintain consistency in terms of size, surface chemistry, and optical properties across different batches. Consistency is crucial for the reproducibility of results and further potential industrial applications. On the other hand, the impurities in CDs are likely to affect their materials properties. For example, impurities such as strongly luminescent small molecular fluorophores may coordinate, complex, adsorb, or covalently bound to the functional groups on the surface of CDs, leading to electron or energy transfer that influence the luminescent performance of CDs.[26] Larger impurities (e.g., nanoaggregates and microparticles) may instead affect the way CDs interact with cells, lowering their targeting efficiency, for example.

Researchers have recently realized that it is essential to understand why CDs exhibit their unique behaviors.[27] The focus of CD-related research has shifted from the use of various raw materials to prepare CDs to the exploration of their luminescence mechanisms and to the investigation of unique applications. Until now, many types of CD luminescence mechanisms have been reported, including the quantum confinement effect, surface state, edge state, carbon-core state, molecule state, and crosslink enhanced emission effect.[28] There are many reasons for such a multitude of complex luminescence processes, one of which is that CDs have only undergone sub-optimal purification, leading to the use/analysis of CD-based composites rather than pure CDs. From an application perspective, to achieve specific uses, unique CDs must be designed and purified carefully. In addition, the required degree of purity might also be different from one application to another.

**2. Purification methods**

The history of CD purification has evolved along with their development. The purification of CDs refers to the process of removing impurities, such as unreacted precursors, oligomers, and
other aggregates, separating CD species with different properties, thus obtaining highly pure
CDs, which is hard to standardize as different samples contain those at different ratios. In the
early stage of CD development, some of the CDs reported in the literature have not been purified
in any way. Even worse, it has been claimed that in some cases, CDs were the sole products.
Later, researchers started to use simple and rapid techniques like centrifugation and filtration to
purify their CDs.[29] When it marks around ten years of CDs' existence, it has been reported
syntheses of CDs with high mass yields and high fluorescence QY through hydrothermal
treatment of citric acid and ethylenediamine.[30] In this article, dialysis was used for purification.
Since then, the hydrothermal treatment followed by dialysis became popular for CD synthesis
and purification. In 2015, another group reported the synthesis of multi-color emissive CDs,
purified by column chromatography, which opened the way for using chromatography in CD
purification.[31] Later, several other techniques have been used to purify CDs, including the
combinations of centrifugation, filtration, dialysis, chromatography, electrophoresis.[32] Based
on their working principles, these techniques can be categorized into two main sets: one set
comprises the removal techniques, including centrifugation, filtration, and dialysis while the
other set includes separation or isolation techniques, like chromatography and electrophoresis.
In the following sections, we will review in detail the principles, the advantages, and the
disadvantages of these techniques.

## 2.1. Centrifugation

Centrifugation is widely used with CDs due to its timesaving character and ease of operation (Figure 1a).[33] It is a straightforward technique and can be scaled up for industrial applications. In one study, CDs were synthesized by refluxing carbon nanoparticles in thionyl chloride, followed by centrifugation at 25000 g.[34] The resulting CDs were not only brightly fluorescent in solution, but also behave well as contrast agents once administered in mice. In another report, a facile hydrothermal synthesis was developed to produce S and N co-doped CDs.[35] The as-prepared CDs exhibited a mean diameter of 3.10 ± 0.54 nm after centrifugation at 5000 rpm for 5 min. It was then demonstrated that these CDs possessed excellent photocatalytic performance, indicating their great potential for applications in environmental purification and energy conversion. Repeated centrifugation is also commonly used to enhance the purification of CDs. Commercial honey containing organic macromolecular passivating agents was heated in a domestic microwave oven for 30 min (Figure 1b).[36] Subsequently, the as-synthesized nanoparticles were purified by repeated centrifugation from water. The resulting CDs displayed an averaged diameter of 9 ± 3 nm.
The use of a single purification method often yields unsatisfactory outcomes. Recently, combined purification routines have emerged as a promising strategy, involving two or more purification techniques a coordinated manner to improve the purity. Compared to using a single method, combined approaches can greatly improve the degree of purity, thus minimizing unwanted effects. Centrifugation can be effectively combined with dialysis or column chromatography. For example, CD aggregates with enhanced deep red fluorescence and long-wavelength absorption bands were synthesized by heating in a microwave oven (Figure 1c).[37] The resulting solid was then dissolved in water and centrifuged to remove aggregated particles at a speed of 8000 rpm for 20 min, three times. The solutions were further purified by dialysis against deionized water using a membrane with a molecular weight cut-off (MWCO) in the range of 0.1-0.5 kDa. Finally, clear and yellow-brown CD solutions were obtained and further freeze-dried for use. The combination of centrifugation and dialysis led to a significant increase in purity. When centrifugation is combined with column chromatography, each component can be separated to enhance the optical properties of the CDs, thereby increasing their efficacy in multifunctional applications. For instance, a series of solvent mixtures with different polarities were used to separate out each of the CD components from crude hydrothermal product (Figure 1d).[38] The crude products were purified by centrifugation several times followed by chromatography. This indicates that centrifugation can be combined with other purification methods to achieve CDs of better quality.
<figure><img src="image_2.png" /><figcaption>**Figure 1.** a) Schematic representation of centrifugation procedure. b) SEM image of honey derived CDs after repeated centrifugation. Reproduced with permission.[36] Copyright 2013, Tsinghua University Press. c) Schematic illustrating the evolution from green emissive CDs to fused CDs aggregates *via* the solvothermal fusion. Reproduced with permission.[37] Copyright 2021, Wiley-VCH. d) Schematic of the synthesis and purification of CDs from o-phenylenediamine. Reproduced with permission.[38] Copyright 2021, Royal Society of Chemistry.</figcaption></figure>

In summary, although centrifugation is highly useful and effective for the removing impurities effectively, it still comes with several challenges. For instance, several CD impurities are still typically nano-objects, rendering them less prone to complete sedimentation during centrifugation, especially when their density is close to CDs. In the reverse scenario, it is also practically challenging to recover a pure CD phase as a sediment for similar reasons. Additionally, centrifugation processes may impact the structure and optical properties of CDs, necessitating careful control of centrifugation conditions to preserve materials properties and functionality. To address these challenges, we need to explore new purification strategies and conditions for CDs to achieve higher efficiency and improved quality.
## 2.2. Filtration

Filtration is an established method for separating mixtures, whereby a mixture of dispersed particles and molecules are separated based on the selective transport/retention of substances through porous media. Known for its simplicity and ease of operation, filtration is widely used for purifying CDs (Figure 2a). It does not require highly specialized equipment and can be easily implemented in both laboratory and industrial settings. Filtration routines are commonly executed using regular filters/membranes, such as filter papers and non-isoporous membranes, whose pore sizes vary in effective diameter. Yet still, even a regular filter/membrane can often successfully block large solid impurities while allowing CDs to pass through, making filtration a very common initial method in CD purification protocols.

Early in the development of CDs, filtration was often used alone as a CD purification method. For instance, a green preparative strategy toward water-soluble nitrogen-doped fluorescent CDs by hydrothermal treatment of soy milk was reported (Figure 2b).[³⁹] Carbonization, surface functionalization and doping occurred simultaneously during the hydrothermal treatment, leading to the formation of the doped CDs. After synthesizing these CDs, the mixture was filtered to remove larger particles. CDs were predominantly found within 20 to 30 nm, indicating that the filtration process effectively removes impurities outside this size specification. The acquisition of CDs within this defined size range demonstrates that the filtration is an efficient method in size-selective separation. Similarly, milk underwent hydrothermal treatment at 180 °C in an autoclave reactor, resulting in the obtention of a yellow solution (Figure 2c).[⁴⁰] This solution was then filtered through a 0.22 µm Millipore syringe filter, yielding monodispersed, highly fluorescent CDs with an approximate size of 3 nm. According to these studies, although the widely used filtration process is known for its simplicity and ease of operation, it involves precise control of filtration conditions to ensure that the desired size range of CDs is collected while removing undesirable large particles. However, due to its very nature, small molecular impurities will inevitably remain in the resulting mixture in case no further purification measures are put in place.

As mentioned above, filtration has been widely employed for the purification of CDs, and it is a practice well-acknowledged. However, traditional single filtration methods often lack the necessary precision for effective separation. Recently, a shift towards combining filtration with other purification methodologies to attain higher levels of purity has been witnessed. By integrating diverse purification strategies, the thorough removal of impurities is facilitated, thereby yielding CD samples of higher quality.
The integrative purification approach is increasingly prevalent in CDs research, thereby enhancing the quality and application potential of resulting materials. For instance, a series of multicolor emitting chiral CDs (Ch-CDs) were synthesized using as common precursors L- or D-tryptophan and *o*-phenylenediamine *via* solvothermal method in three solvent systems (HCl/ethanol, H₂O, and H₂SO₄/H₂O), followed by filtration through a 0.22 µm membrane filter, then dialyzed and lyophilized (Figure 2d).[41] TEM images showed homogeneous size distributions of approximately 2.5 nm, 3.0 nm, and 4.6 nm for blue, yellow, and red Ch-CDs, respectively. The combination of filtration and dialysis ensured the effective purification of CDs, efficiently removing large-sized particles (filtration) and small-molecular impurities (dialysis). In addition, it is necessary to dialyze against organic solvents when there are a large number of hydrophobic molecules in the crude products.

To further improve the purity of CDs, studies sometimes integrate three or four purification methods in a synergistic manner to achieve the highest possible level of purity. This strategy was successfully applied to synthesize red-emitting CDs (r-CDs) through the hydrothermal protocol, utilizing three distinct precursors.[42] During the purification process, the post-reaction mixture was initially filtered through a 0.22 µm membrane to effectively remove large particles. This purification step was followed by a silica gel column chromatography treatment, using a dichloromethane/ethanol mixture as eluent, which facilitated the refined separation of the different components. For certain CD samples, a combination of precipitation and centrifugation was also employed. The samples were precipitated from EtOH and water with acetonitrile and ethanol and then separated through centrifugation. This procedure was repeated multiple times to ensure a thorough removal of the precursor residues and the small derivatives, resulting in high-purity CDs. Specifically, for the r-CDs mixtures obtained using formamide as the solvent, ion-exchange column chromatography was applied for further purification. Hydrochloric acid solutions of varying concentrations were also utilized to separate the CDs. However, it was found that under acidic conditions, these r-CDs were damaged, indicating that the ion-exchange column purification process should be avoided with sensitive CDs. These findings highlight the challenges in purifying CDs and underscore the need to adapt the purification techniques to the type of CDs.
<figure><img src="image_3.png" /><figcaption>**Figure 2.** a) Schematic representation of filtration procedure. b) Schematic illustration of the preparation of CDs by hydrothermal treatment of soy milk. Reproduced with permission.[39] Copyright 2012, Royal Society of Chemistry. c) Illustration of the synthesis of CDs from milk by hydrothermal treatment. Adapted with permission.[40] Copyright 2014, American Chemical Society. d) Schematic of the preparation of multicolor-emitting Ch-CDs from L-/D-tryptophan and o-phenylenediamine. Reproduced with permission.[41] Copyright 2021, Wiley-VCH.</figcaption></figure>

Overall, filtration is an essential technique for purifying CDs, as it effectively removes large particulate impurities, thereby enhancing the purity and quality of the CDs. This method is notable for its short operation times, low cost, environmental friendliness, and scalability for large-scale processing, making it highly suitable for envisioned industrial applications. However, filtration has its limitations in effectively separating small impurities or CDs of varying sizes. There are risks like membrane clogging, which can reduce separation efficiency and require frequent replacement of the filters. Additionally, relying solely on filtration will result in insufficient purity, raising concerns about the reliability of data generated, especially
in fluorescence applications. Consequently, filtration is often combined with other purification methods, such as dialysis and column chromatography, to achieve a more effective purification.

## 2.3. Dialysis

Dialysis is a commonly employed post-purification method for CDs, primarily utilized to remove organic by-products generated during the synthesis, unreacted precursors, and other small molecular impurities (Figure 3a).[43] This technique utilizes semipermeable membranes with specific pore dimensions, enabling small molecules to diffuse from the CD solution into the dialysis liquid (i.e., water) while retaining larger CDs within the dialysis bag. Through periodic replacement of the external solution, trans-membrane diffusion of solvents and small molecular solutes is further facilitated, effectively eliminating impurities and residuals from the solution. It is a gentle process that avoids harsh conditions that could damage CDs.

The dialysis purification process of CDs encompasses several essential steps to ensure the efficient removal of the impurities. Initially, a dialysis membrane with the appropriate MWCO is selected based on the expected size of the CDs. This step is crucial to ensure that CDs are retained within the membrane while smaller molecules and impurities are able to pass through. A dialysis bag of appropriate length is then prepared, and the reaction solution containing CDs is carefully introduced into the bag. Both ends of the dialysis bag are meticulously sealed to prevent any leakage. The sealed dialysis bag is subsequently immersed in a container filled with solvent, usually deionized water, with the volume of deionized water substantially exceeding that of the solution within the dialysis bag to promote effective diffusion (usually 50 mL of CD dispersion is immersed in 5 L of water backer). The dialysis process typically spans from 2 to 7 days. During this period, it is imperative to periodically replace the deionized water to sustain a high concentration gradient, thereby enhancing the removal of the impurities. Upon completion of the dialysis process, the purified solution containing the CDs is retrieved from the dialysis bag, concluding the dialysis procedure.

To further elucidate the impact of dialysis on the quality of CDs, a series of syntheses were conducted using hydrothermal and microwave routes. For instance, citric acid was combined with urea or ethylenediamine as a nitrogen source, and dialysis was implemented subsequently to the synthesis process (Figure 3b).[44] After careful comparison and analysis, notable differences were observed in the optical and morphological properties between the dialysate (components transported into water bath) and retentate fractions (solution remaining inside the dialysis membrane) obtained during dialysis. These findings confirmed that dialysis can effectively separate small fluorescent molecules from CDs, demonstrating its efficacy in the
purification process. However, when dialysis membranes with different MWCO (1 kDa versus 50 kDa) were utilized, the retentate fractions from exhibited distinct properties. Notably, although retentate fractions contained CDs as observed by microscopy, CDs in the retentate from 1 kDa MWCO membrane were predominantly surrounded by organic impurities. This indicated that a 1 kDa dialysis membrane cannot completely remove organic residues, and that MWCO of the membrane can significantly impact the purification of CDs. Therefore, selecting an appropriate membrane is of paramount importance. Considering the particle size of CDs, it is not recommended to use a membrane under 20 kDa because there is the potential for retention of molecular byproducts in addition to the CDs fraction.

As mentioned above, it is important to use a dialysis membrane with an optimal MWCO, as this parameter directly affects the purity and properties of CDs. For example, it has been reported that synthesizing fluorine and nitrogen co-doped CDs with an average size of 1.38 nm through microwave-assisted pyrolysis, the purification process involved three-day consecutive dialysis using a dialysis membrane with MWCO of 0.5-1 kDa (Figure 3c).[45] These CDs exhibit photo-activated fluorescence enhancement upon exposure to ultraviolet light irradiation and demonstrate reversible force-induced coloration behavior under varying pressure conditions. In addition, an innovative method was reported for synthesizing CDs by solvent-thermal fusion of highly conjugated dihydroxyindole derivative (PTCDA) with urea.[46] The purification process involved centrifugation at 8000 rpm for 10 min and dialysis for 24 h using dialysis membranes with MWCO of 1 kDa and 20 kDa to remove residual reactants and small molecules. These CDs exhibited up to 18.8 % QY absorbing in the NIR region and demonstrated excellent photostability and low toxicity, making them ideal probes for biological imaging.

In addition to the selection of the dialysis membrane, the duration of dialysis is also a critical factor affecting the efficiency of the purification process. Observations made through the temporal evolution of high-performance liquid chromatography (HPLC) chromatograms indicated that a significant period, approximately 120 h, is necessary to ensure the complete removal of small molecular impurities from the CD dispersions.[47] It has been highlighted that extending the dialysis duration is accompanied by a decrease in the fluorescence QY of the retained solution.[44] This observation indicates that a dialysis of only 6 h is grossly insufficient, regardless of the MWCO of the dialysis membrane used. Typically, a dialysis period exceeding 24 h is necessary, along with frequent refreshing of water bath, to ensure the adequacy and effectiveness of the dialysis process. Occasionally, to achieve a higher degree of purification, we should extend the dialysis duration to a week or even more. For instance, a type of CDs
were synthesized by microwave heating a mixture of ethylenediamine and phosphoric acid in deionized water, followed by a week-long dialysis (using a MWCO of 500 Da) and freeze-dried to obtain pure CD powder.[48] Furthermore, by ensuring a sufficiently prolonged duration of the dialysis and complementing it with other purification techniques, such as filtration, centrifugation, and column chromatography, a significant enhancement in purification efficiency can be achieved. This integrated approach effectively elevates the purity and quality of CDs, making them more suitable for a diverse range of applications. For example, the synthesis of yellow-emitting CDs with high QY was achieved *via* a solvothermal method, employing citric acid and urea as precursors and conducting the reaction in toluene (Figure 3d).[49] The purification process involved the use of silica gel column chromatography combined with dialysis against dichloromethane to obtain high-purity CDs. Prolonged dialysis time led to an increase in the QY of the CDs, indicating that extended dialysis time contributes to enhancing the purity and fluorescence performance of the CDs. The resulting CDs exhibited significant optical characteristics, including a large Stokes shift and excellent photostability. These CDs demonstrate up to 98 % QY in the solid state, showcasing high potential as yellow fluorescent powders for use in luminescent solar concentrators and illumination.

While certain publications omit explicit details regarding the dialysis duration and the specific characteristics of the dialysis membrane employed, it is still advisable to meticulously record these critical parameters in research endeavors. These details are essential for a better understanding and investigation of the luminescent and synthetic mechanisms of CDs. Furthermore, dialysis plays also a crucial role in the separation and purification process of Ch-CDs, facilitating the collection of CDs with desired properties and high purity. A green synthesis has been developed for Ch-CDs (QY ca. 41 %) based on hydrothermal treatment of L- or D-cysteine at temperatures of 60 °C, much lower than commonly reported temperatures, and crude product has been subjected to dialysis.[50] L-CDs showed chirality-dependent enhancement in cellular glycolysis but did not influence the cellular ATP levels of human urinary bladder cancer T24 cells, while D-CDs had no similar effects. Similarly, CDs with tunable oxidation/reduction potentials were prepared by adding commercially available quinones to the reaction mixture, employing a fast microwave-assisted hydrothermal treatment and a facial method with a filter membrane (0.1 µm) followed by dialysis.[51] By carefully adjusting the electrochemical properties of CDs, it is possible to tailor-design CDs for specific energy conversion and catalytic applications. In brief, within the field of CDs, numerous successful studies have substantiated the efficacy of dialysis as a purification technique. While
these cases are commendable, the omission of critical details, such as the duration of dialysis
and the dialysis membrane MWCO, in the literature may impede the replicability of the results
and limit a comprehensive understanding of the impact of the dialysis process on the properties
of CDs. To advance CD research, it is imperative that future studies meticulously document the
dialysis conditions, including the duration of dialysis and the MWCO of the dialysis membrane
used. In addition, dialysis against organic solvents and more organic solvent-resistance
membranes are required for raw products containing hydrophobic molecules. However, the
dialysis also comes with certain disadvantages such as being time-consuming and lacking
control over removal rate.

<figure><img src="image_4.png" /><figcaption>**Figure 3.** a) Schematic representation of dialysis procedure. b) Sequence of images showing the progress of CD dialysis and TEM image of retentate. Reproduced with permission.[44] Copyright 2018, American Chemical Society. c) Schematic of the microwave synthesis and purification of F,N-doped CDs from poly(ethylene imine) and levofloxacin. Reproduced with permission.[45] Copyright 2020, Wiley-VCH. d) Schematic of the solvothermal preparation and</figcaption></figure>
purification of CDs from citric acid and urea. Reproduced with permission.[49] Copyright 2022, Wiley-VCH.

## 2.4. Chromatography

Chromatography is an analytical and purification technique widely used in chemistry for the separation, identification, and quantification of the components of complex mixtures.[52] It relies on the principle of component distribution between a mobile phase and a stationary phase (Figure 4a). When introduced into the mobile phase, the mixture passes through a stationary phase. Each component interacts differently with the stationary phase, resulting in their separation based on their physicochemical properties. It provides thorough separation and can be tailored to specific needs through various stationary and mobile phases.

Silica gel chromatography is one of the most commonly used purification methods for organic compounds. Its application in purifying CDs is helpful to reveal the presence of undesired fluorophore formed alongside CDs during the hydrothermal carbonization process, particularly in bottom-up synthesis using small organic molecules. This has been reported in several studies. For example, a work on “ultrahigh” QY CDs was revisited.[30, 53] Upon further investigation, the authors discovered that the elevated QY was coming from a fluorophore (imidazo[1,2-a]pyridine-7-carboxylic acid, 1,2,3,5-tetrahydro-5-oxo-, IPCA) formed by the condensation of ethylene diamine and citric acid during the hydrothermal process. The identification of IPCA was enabled by purification using silica gel chromatography. Initially, the authors overlooked the presence of this unforeseen molecule. Their initial purification through dialysis (MWCO: 3.5 kDa) removed most of the unreacted product but also most of blue emissive products. The authors noticed that the solutions inside and outside the dialysis bag emitted blue fluorescence. Upon analyzing the isolated CDs with mass spectrometry, they detected a prominent peak at 180 Da. This result encouraged the authors to undertake further purification using chromatography column on silica gel, ultimately revealing the presence of IPCA within the “purified” CDs. This underscores the importance of chromatography in discerning and isolating CDs from the undesired fluorophores.

Similarly, a recent paper reported the formation and purification of multiple different fluorophores during CD synthesis from *p*-/*m*-/*o*-phenylenediamine.[54] A previously published article describing red, green, and blue emissive CDs was revisited.[31] Unlike the first reported purification using silica gel chromatography alone, purification was repeated combining silica gel chromatography with dialysis (MWCO 0.5-1 kDa, 72 h, buffer changed every 24 h). The
analysis of the inner dialysis bag solution and the external buffer revealed the presence of
fluorophores in the dialysis buffer which were responsible for the red, green, and blue emission
previously attributed to CDs. This article highlighted how a single starting compound could
yield a complex mixture with multiple fluorophores that cannot be purified using only a
chromatography column. The purification of CDs using silica gel chromatography is not always
a straightforward and rapid process allowing the attainment of "truly purified" CDs. This raises
important questions about how to accurately determine when CDs are properly cleaned and
whether the observed fluorescence can be attributed to only CDs or to residual fluorophores. It
is important to emphasize that these fluorophores may exist individually or attached to CDs by
covalent bonds, or *via* intermolecular interactions.

In another study, the purification of CDs synthesized *via* hydrothermal carbonization of urea and p-phenylenediamine was reported.[55] The complete fractionation of each population of CDs required over 10 h due to the necessity of maintaining a slow flow rate to avoid the cross-contamination between the different CDs populations. The eluted CDs were surprisingly spanning from blue to red light emission. The authors proved that this elution was not related to the size effect but rather to different oxidation states at the surface of CDs, leading to differences in polarity.

Finally, purifying CDs with commonly used organic solvent can be challenging, as CDs
typically exhibit better solubility in water. For instance, this issue was addressed by employing
pure water as the eluant for CD purification by chromatography column (Figure 4b).[56] A study
was conducted and proved to be efficient using 19 different starting molecules. Across each
synthesis, varying numbers of distinct CD populations were obtained, each characterized using
diverse techniques, notably TEM. The characterization revealed that small blue-emissive CDs
were eluted first and, while larger red-emissive CDs were eluted last. This observation was
confirmed across all syntheses. The separation mechanism is not explained by traditional
polarity principle but rather by the role of water in facilitating size-based separation.

To summarize, the purification of CDs using silica gel chromatography is not a straightforward
method and requires complex and long preparations, involving careful considerations from
selecting the appropriate eluent to determining the column size and the flow rate. In addition,
silica gel chromatography sometimes reduces the yield.

Size-exclusion chromatography is another type of chromatography. This technique separates
molecules based on their hydrodynamic dimensions, encompassing molecular size and in some
cases, molecular weight. This technique presents many advantages for the purification of CDs as their size varies in a single batch.

The purification of CDs synthesized from dextran sulfate sodium salt was performed using a Sephadex G-25 gel column (desalting column) (Figure 4d).[57] The authors successfully fractionated the CDs into 48 fractions, with smaller size being eluted the last. The particle size was determined by TEM. This study highlights again that the majority of reported studies on CDs may contain multi-component systems, where multiple subspecies coexist, thus, not accurately reflecting the behavior of individual subpopulations of CDs. Moreover, the isolation of 48 fractions of differently sized CDs may raise the question of what degree of purification should be required for specific applications. This above mentioned study shows a high degree of purification that might not be needed for each application of CDs, but it should be kept in mind and taken into consideration when one purifies CDs. By comparison, the purification methods of CDs using gel permeation chromatography, which is a type of size-exclusion chromatography can be considered.[58] A study using this technique showed that only three fractions with different photoluminescent colors were isolated, contrasting with the 48 fractions obtained from the aforementioned article.[57] TEM characterization revealed a broader size distribution within each fraction, once again prompting considerations regarding the necessary degree of purity for specific applications.

Additionally, less commonly employed types of chromatography can be used to purify CDs (Figure 4c).[59] For example, the fractionation of CD mixtures using the combination of filtration, dialysis, and anion-exchange HPLC as purification methods was reported.[60] The authors successfully isolated 12 individual fractions. Interestingly, initial complex mixture exhibited a QY of 1%, whereas certain fractions showed an increase of QY 6 to 7%, suggesting the presence of undesirably absorbing species in the mixture. The analysis of each fraction by TEM and capillary electrophoresis revealed the persistence of multiple size CDs with one predominant species in each fraction. Moreover, the complex mixture exhibited a higher toxicity than the fractionated CDs underscoring the importance for high-resolution separation techniques particularly for further biological or catalysis applications. Furthermore, the necessity of high-resolution separation techniques is paramount for understanding the photoluminescence processes of CDs, as well as for their further regulatory approval in various applications.
<figure><img src="image_5.png" /><figcaption>**Figure 4.** a) Schematic representation of an HPLC setup. b) Representation of a chromatography on silica gel using water as a solvent. Reproduced with permission.[56] Copyright 2020, Elsevier. c) Photoluminescence of the different fractions obtained after purification by ion-exchange HPLC of CDs synthesized from candle soot. Reproduced with permission.[59] Copyright 2012 American Chemical Society. d) Graphical representation of CDs purification using a desalting column. Reproduced with permission.[57] Copyright 2018, Elsevier.</figcaption></figure>

In summary, it is important to highlight that chromatographic methods are usually integrated into a multi-step protocol for reaching molecular-level purity, alongside techniques like filtration, dialysis, or centrifugation. Relying solely on chromatography might be more difficult, more complex and might present limits to prove the quality of CDs. Moreover, chromatography purification requires skilled operator as the techniques can be complex (such as choice of solvents, columns, and gradients). Additionally, chromatography can be expensive and requires large amounts of solvents.

## 2.5. Electrophoresis

Purification by electrophoresis is based on the migration of electrically charged particles under the influence of an electric field (Figure 5a). The separation of the ionic particles is dependent on their mobility, which is determined by several factors, including particle size, shape, and charge. The electrophoretic parameters are defined by various electrical parameters such as current voltage, and power, alongside other factors like ionic strength, pH value, viscosity, and
pore size. In order to retrieve the particles, they must undergo elution from the gel followed by dialysis. Electrophoresis is a powerful and versatile method for the separation and analysis of molecules. Its advantages include high resolution, cost-effectiveness, and rapidity.

As mentioned before, CDs were accidentally discovered during the purification of single-walled carbon nanotubes by electrophoresis (Figure 5b).[2] In a 1% agarose gel, three distinct materials were observed, one of which exhibited remarkable fluorescence. This highly fluorescent material was then fractionated into different components, each one emitting distinct color ranging from green-blue to yellow and orange when exposed to UV light. To isolate each material, the authors had to perform, in addition to the agarose gel electrophoresis, a glass bead electrophoresis column followed by dialysis, yielding highly purified materials.

Electrophoretic separation often reveals the presence of subspecies within a batch of CDs, not always noticeable from an absorbance and an emission spectrum, from a dynamic light scattering measurements or from X-ray photoelectron spectroscopy. For example, it was reported that electrophoretic analysis of uniform size CDs generated from citric acid and ethylenediamine, varying the synthesis time, displayed the presence of three distinct subpopulations with different surface charge (Figure 5c).[61] The individual absorbance and emission spectra of each subpopulation were similar among themselves and when compared to the spectra prior to purification, indicating that the initial spectra contain contributions from all components that were separated in the gel. This conclusion was further corroborated in other studies, as well (Figure 5d).[62]

<figure><img src="image_6.png" /></figure>
**Figure 5.** a) Schematic representation of an electrophoresis setup. b) Purification of single-wall carbon nanotubes allowing the discovery of CDs. Reproduced with permission.[2] Copyright 2004, American Chemical Society. c) Electrophoresis of CDs synthesis from citric acid and ethylenediamine. Reproduced with permission.[61] Copyright 2017, American Chemical Society. d) Electrophoresis picture of CDs synthesized from candle soot showed the multiple species present within one batch. Reproduced with permission.[62b] Copyright 2007, Wiley-VCH.

Purifying CDs through electrophoresis is an unconventional approach, with only a few publications reporting its use. Electrophoretic separation method nevertheless requires meticulous sample preparation and environmental control (e.g., temperature, buffer composition, pH) and possesses a limit on separation capacity. Similarly to chromatography, purifying CDs through electrophoresis revealed the complexity and non-uniformity within a CD batch, although in a rapid and straightforward way. Standard methods of characterizations fail to detect the presence of multiple subpopulations within a single batch and commonly employed purifications techniques (i.e., dialysis, filtration, centrifugation) will not permit the separation of these subpopulations that constitute a complex mixture with properties that might vary comparing to individual subpopulations. In addition, electrophoresis requires specialized equipment, which may limit its practicality for some applications.

## 2.6. Other techniques

While common purification techniques effectively separate CDs from their starting materials, they frequently fail to isolate distinct subpopulations present within a batch. Additional purification methods not mentioned in the previous section will be presented in this section.
As an alternative approach, pH modification of the solution of as-prepared CDs help isolate/extract CDs with different charges within a single batch. For example, an article reported the synthesis of CDs from the pyrolysis of citric acid and their purification by using pH-controlled cloud-point extraction.[63] This method offers several advantages, including its simplicity, its cost-effectiveness, and its rapidity while excluding the need for toxic solvents or reagents. The technique relies on the use of a surfactant that forms globular micelles at high temperatures, inducing a phase separation and the capture of subpopulations of CDs depending on the solution's pH and the CDs' solubility at that pH. From a single batch, 12 different subpopulations of CDs were separated. Comparative analysis of the fluorescence spectra of both the as-prepared and purified CDs revealed similar excitation wavelength-dependence. However, additional characterization via XPS showcased different proportions of functional
groups on their surface explaining the difference in surface charge and so the different solubility behavior. This example highlights the existence of subpopulation with small differences from one synthesis, raising questions about the reproducibility of CD synthesis.

In addition to dialysis, CDs can be directly extracted from their medium. This technique, more commonly used in organic chemistry, is less time and water consuming compared to dialysis.

It relies on the use of an immiscible solvent in which the impurities are more soluble than in the starting solvent, inducing the separation of CDs in one solvent and the impurities in the other, or vice versa. Indeed, while CDs typically show a high solubility in water, it is also possible to solubilize them into an organic solvent for extraction from the impurities that remain in the aqueous layer. For example, the synthesis of CDs from mulberry leaves and their purification through filtration followed by extraction using a mixture of dichloromethane and water was reported (Figure 6a).[64] The authors successfully isolated red-emissive CDs in the organic layer, while green-emissive CDs were obtained in water. The red-emissive CDs exhibited promising properties and were further used for biological application and bio-imaging.

In a similar approach, CDs were purified by extraction into an organic solvent (Figure 6b).[65]

However, this technique may not always be applicable. Finding an organic solvent with a higher solubility of CDs compared to water remains challenging.

Purification of CDs can also be performed by combining chromatography with extraction methods (Figure 6c).[66] The reported method involved the rapid elution of impurities with an organic solvent through alumina column chromatography while CDs migrate slowly through the column. Subsequently, the purified CDs were extracted from the column by stirring the alumina in water.

We can highlight that the purification of CDs by extraction can be performed in two main ways: utilizing a solvent in which the CDs present a higher solubility than in water or finding a solvent in which the impurities have a higher solubility than in water. The first method may be challenging and may necessitate the use of toxic solvents, while the latter method might allow the obtaining of CDs with higher purity but will require a longer purification time.

Furthermore, precipitation is another technique to purify CDs. This method is based on the addition of a solvent to transit a substance from the dissolved state to the non-dissolved state (Figure 6d). The high-yield synthesis of nitrogen-doped CDs from chitosan with ethanol-precipitation-based purification was reported.[67] The obtained CDs were initially extracted, filtered, and precipitated with ethanol before performing a final centrifugation step. In a similar study, the synthesis of CDs from chiral precursors *via* microwave irradiation and followed by
precipitation with diethyl ether was described.[68] After dissolving the crude product in chloroform, the organic layer was extracted with water, and CDs were finally precipitated from the organic layer with diethyl ether. However, compared to the previously described method, this technique presents a lower resolution separation technique, as the induced precipitation may also capture impurities or side products. As mentioned above, precipitation alone is rarely sufficient and is often coupled with other purification methods.

<figure><img src="image_7.png" /><figcaption>**Figure 6.** a) Schematic representation of the synthesis and purification of CDs from mulberry leaves. Reproduced with permission.[64] Copyright 2022, Wiley-VCH. b) Schematic representation of purification of CDs using solvent extraction method. Reproduced with permission.[65] Copyright 2018, Royal Society of Chemistry. c) TLC representing the fast migration of impurity and the low migration of CDs. Reproduced with permission.[66] Copyright 2018, Royal Society of Chemistry. d) Schematic representation of the precipitation process.</figcaption></figure>

Overall, due to the semi-stochastic nature of CD synthesis, there is a wide variety of by-products and impurities coexisting. High-purity CDs can hardly be obtained by a single technique like dialysis or chromatography, while multi-step or combined techniques demonstrate their superiority. The selection guidance about purification techniques follows: 1) identifying whether the impurities are large particles, small molecules, or specific types of aggregates; 2) considering the properties of impurities and CDs, especially their solubility/dispersity in water and organic solvents; 3) matching the techniques with the needs (defined in steps 1 and 2); 4)
using centrifugation and filtration for preliminary purification to remove large particles; 5) applying dialysis (against water for water-soluble impurities and organic solvents for water-insoluble impurities) for removing small molecular impurities, and 6) employing chromatography or electrophoresis for fine separation. According to the difference between the CDs and the impurities in particle size and molecular weight, we propose a flow chart showing the combined techniques for the purification of CDs (Figure 7). Firstly, we recommend removing the large impurities (e.g., aggregates, bulk precursors) using centrifugation or filtration then proceeding with the removal of the smaller species (e.g., unreacted molecular precursors, oligomers), eventually separating potential different CD species with chromatography and electrophoresis. In addition to commonly used purification methods, there are some alternative nonconventional strategies available. For example, magnetic separation is suitable for CDs with magnetic properties or when magnetic nanoparticles can selectively interact with the impurities. Field-flow fractionation could be also used to separate CDs based on size or other properties. For large-scale purification of CDs, continuous flow solvent extraction or continuous flow electrophoresis could be suitable and effective strategies.

<figure><img src="image_8.png" /><figcaption>**Figure 7.** A flow chart showing the suggested procedures to obtain highly pure CDs.</figcaption></figure>

### 3. CD preparation and purification challenges
In the last decade, there has been a huge development in the CD synthesis and purification. For the synthesis aspect, red and NIR emissive single or two photon CDs with high mass yield have been prepared. New techniques like flow chemistry and machine learning have been employed for developing alternative synthesis routines. For the purification, combined techniques are increasingly preferred for obtaining high-quality CDs. However, due to the chemical and structural complexity of CDs, there are still some challenges lying around synthesis, purification, and purity assessment.

**3.1. Synthetic methods**

As pointed out above, no matter which type of synthetic method is utilized, there will consistently be by-products produced. If CDs could be synthesized directly and easily without any impurities, then purification steps can be ideally omitted, which will greatly facilitate their large-scale industrial applications. Recently, a facile synthesis of CDs by stirring acetaldehyde with sodium hydroxide at room temperature, followed by HCl washing, was reported.[69] Moreover, the approach was claimed to work for kilogram-scale synthesis of CDs. Despite the progress made in the direct synthesis of high-purity CDs, the following challenges remain bound to the preparation process:

(i) Synthesis of CDs with controlled structure and desired properties,

(ii) Exploration of efficient methods for CD synthesis with both high mass yields and conversion yields,

(iii) Understanding CDs synthesis mechanism and unraveling CD synthesis-structure-function relationship.

**3.2. Purification methods**

As discussed above, nearly all crude CD products are obtained with some impurities. To acquire high-quality CDs, a series of methods have been employed for purification (Table S1). However, the above-mentioned methods have their own drawbacks. The following challenges still need to be overcome to obtain high-purity CDs:

(i) Efficient combination of multiple techniques for the purification of CDs prepared by different methods with various precursors,

(ii) Exploring new techniques that enable both purification and separation of CDs species according to different application scenarios.

**3.3. Purity and quality assessment**

Purity evaluation is extremely important for developing high-quality CDs. Though several techniques have been used to prove the successful preparation of CDs, a standard and widely
recognized quality-control evaluation procedure is still lacking. This has led to uncertainties regarding the purity of the resulting products and the efficiency of the purification processes, which also largely limit the application prospects. Recently, Prato and colleagues called for evaluation of the CD purity by NMR due to their polymer-like nature, which is a highly promising proposal.[70] Moreover, diffusion-ordered spectroscopy can assess the size homogeneity and molecular weight of CD samples by revealing their diffusion coefficients, distinguishing between monomers, polymers, and nanoparticles, and can also check for residual molecules and validate post-functionalization. However, the following challenges remain unmet for purity evaluation:

(i) Establishing a standard technique or protocol that adequately characterizes purified CDs, such as CDs content, content of different size or class impurities,

(ii) Finding the combination of appropriate analytical techniques for a fast quality check,

(iii) Building criteria that allow for a comprehensive assessment of purification efficiency, based on yield, cost, time, and environmental sustainability.

## 4. Conclusion

In this review, various purification techniques for CDs are presented, with emphasis on their principles, strengths, and weaknesses. In most raw CD products, two classes of impurities can be found. The first category includes big particles or aggregates larger than CDs. These impurities can be removed by centrifugation and filtration. the other category mainly small molecules and oligomers, which are smaller than CDs in size or molecular weight. These impurities can be removed through dialysis. Further separation of CD species can be achieved by chromatography and electrophoresis. We would also like to emphasize that, due to the coexistence of diverse impurities, the combination of several purification techniques is necessary to obtain high-purity CDs.

Since the quality of the prepared CDs and the accompanying impurities strongly depends on the experimental method and the synthetic conditions, it is difficult to propose a universal purification approach. Indeed, the purification strategies must vary according to the characteristics of the crude CDs desired. Multi-step approaches combining the advantages of different purification methods offer a solution to obtain high-quality CDs. For CD purification, high yield, low cost, and environmental friendliness are essential. Establishing standards for assessing the purity of CDs is necessary to evaluate and improve the effectiveness of purification procedures. Interdisciplinary collaborations are also essential to advance CD
purification aspects. Combining the expertise in chemistry, chemical engineering, and materials science is essential to develop efficient and scalable strategies. In addition, the contribution of computational approaches and industrial partners will expectedly help optimize the processes and overcome existing difficulties to achieve high-quality CDs, eventually facilitating practical applications. Undoubtedly, the development of efficient purification techniques enables the production of superior CDs, which will permit a full understanding of the synthesis-structure-function relationship of CDs, eventually paving a safer way for their practical applications.

**Supporting Information**

Supporting Information is available from the Wiley Online Library or from the author.

**Acknowledgements**

This work was supported by the Centre National de la Recherche Scientifique (CNRS), by the Interdisciplinary Thematic Institute SysChem, through the IdEx Unistra (ANR-10-IDEX-0002) and by the CSC Graduate School (CSC-IGS ANR-17-EURE-0016) within the French Investments for the Future Program. Y.T. is indebted to the Chinese Scholarship Council for support. H.E.K. and Y.H acknowledge the support of Horizon Europe under Marie Skłodowska-Curie Actions (MSCA) European Fellowship program.

**Conflicts of interest**

The authors declare no conflict of interest.

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<figure><img src="image_9.png" /></figure>

**Yalei Hu** received his doctoral degree in 2022 from Free University of Berlin. He is currently a postdoctoral researcher in the group of Dr. Alberto Bianco at the Institute of Cellular and Molecular Biology, CNRS (Strasbourg). His research interests focus on the preparation of multifunctional carbon dots, unraveling the luminescence mechanism, and exploring their application in the biomedical field.

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

**Océane Seivert** received her bachelor's degree in chemistry and her master's degree in chemistry, biology and medicine from the University of Strasbourg. She is currently pursuing a PhD in therapeutic chemistry under the guidance of Dr. Alberto Bianco. Her research focuses on the development of new antibacterial and antiviral compounds by functionalization of different types carbon materials.

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

**Yue Tang** obtained her master's degree in organic chemistry in 2023 under the supervision of Dr. Zebing Zeng from Hunan University in Changsha. She is currently a PhD student under the supervision of Dr. Alberto Bianco at the Institute of Cellular and Molecular Biology (CNRS, Strasbourg). Her research interests focus mainly on synthesis, characterization and biomedical applications of chiral carbon dots.

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

Nanyang Environment & Water Research Institute, The University of Sydney, Kyoto

**H. Enis Karahan** completed his Ph.D. in Bioengineering at Nanyang Technological University. He holds BSc degrees in Chemical Engineering, and Molecular Biology & Genetics from Istanbul Technical University (ITU), and an MSc degree in Materials Science & Engineering (MSc, Koç University). His research interests include nanocarbons, coatings, self-assembly, cellular toxicity, and membrane technology. He gained postdoctoral experience in
University (JSPS Fellow), ITU (BIDEB-2221 Fellow), and Arizona State University (Fulbright Fellow). He received the Brian Kelly Award and Carbon Journal Prize. Currently, he is an MSCA Fellow at CNRS.

<figure><img src="image_13.png" /></figure>

**Alberto Bianco** received his Ph.D. in 1996 from the University of Padova. As a visiting scientist, he worked at the University of Lausanne, the University of Tubingen (as an Alexander von Humboldt fellow), the University of Padova, and Kyoto University. He is currently research director at the CNRS in Strasbourg. His research interests focus on the design of multifunctional carbon-based and 2D materials for therapy, diagnostics, and imaging. He is Fellow of the European Academy of Science and Academia Europaea, and in 2019 he obtained the CNRS Silver Medal. Since 2011 he is the Editor of the journal Carbon.
Supporting Information

Carbon Dot Synthesis and Purification: Trends, Challenges and Recommendations

Yalei Hu,\* Océane Seivert, Yue Tang, Huseyin Enis Karahan, and Alberto Bianco\*

This file includes:

Supplementary Table S1
<table><caption>Table S1. Overview of purification techniques.</caption><thead><tr><th>Precursor</th><th>Synthetic conditions</th><th>Purification</th><th>Purification time</th><th>Excitation/Emission</th><th>Size</th><th>Ref.</th></tr></thead><tbody><tr><td>Carbon nanoparticles and PEG1500N</td><td>Hydrothermal method (110 °C for 72 h)</td><td>Centrifugation at 25000 g</td><td>Short</td><td>545 nm/620 nm</td><td>-</td><td>[1]</td></tr><tr><td rowspan="2">Citric acid and thiourea; citric acid and urea</td><td>Hydrothermal method (160 °C for 4 h);</td><td rowspan="2">Centrifugation at 5000 rpm for 5 min</td><td rowspan="2">Short</td><td>360 nm/435 nm;</td><td>3.10 ± 0.54 nm;</td><td rowspan="2">[2]</td></tr><tr><td>Hydrothermal method (160 °C for 4 h)</td><td>360 nm/435 nm</td><td>2.69 ± 0.42 nm</td></tr><tr><td>Honey and an macromolecular passivating agent</td><td>Microwave oven (1200 W for 30 min)</td><td>Repeated centrifugation in water</td><td>Short</td><td>-</td><td>9 ± 3 nm</td><td>[3]</td></tr><tr><td>Citric acid and urea</td><td>Heated in a domestic 750 W microwave oven for about 5 min.</td><td>Centrifugation at a speed of 8000 rpm for 20 min ; Dialyzed (MWCO = 0.1-0.5 kD)</td><td>Long</td><td>405 nm/-</td><td>3-6 nm</td><td>[4]</td></tr><tr><td>OPDA</td><td>Hydrothermal method (180 °C for 10 h)</td><td>Centrifugation at 16000 rpm for 20 minutes and column chromatographic purification</td><td>Medium</td><td>579 nm/650 nm</td><td>-</td><td>[5]</td></tr><tr><td>Soy milk</td><td>Heated at 180 °C for 3 h.</td><td>Filtration</td><td>Short</td><td>350 nm/426 nm</td><td>13-40 nm</td><td>[6]</td></tr><tr><td>Milk</td><td>Hydrothermal method (110 °C for 7 h)</td><td>Filtration through 0.22 µm Millipore syringe filters</td><td>Short</td><td>360 nm/454 nm</td><td>2-4 nm</td><td>[7]</td></tr><tr><td rowspan="2">L-/D-tryptophan and o-phenylenediamine</td><td>Hydrothermal method (110 °C for 7 h)</td><td>Filtration with a filter membrane (0.22 µm) and dialysis.</td><td rowspan="2">Long</td><td>365 nm/450 nm;</td><td>2.5 ± 0.5 nm;</td><td rowspan="2">[8]</td></tr><tr><td></td><td>365 nm/550 nm;</td><td>3.0 ± 0.4 nm;</td></tr><tr><td>p-phenylenediamine</td><td>Solvothermal method (110 °C for 24 h)</td><td>Filtration (0.22 µm membrane) and silica gel column chromatography (Dichloromethane/EtOH = 9.5:0.5)</td><td>Medium</td><td>510/-</td><td>-</td><td>[9]</td></tr><tr><td>p-phenylenediamine and citric acid</td><td>Hydrothermal method (210 °C for 10 h)</td><td>Filtration (0.22 µm membrane)</td><td>Short</td><td>510/-</td><td>-</td><td>[9]</td></tr><tr><td>p-phenylenediamine and urea;</td><td>Hydrothermal method (160 °C for 10 h)</td><td>Filtration (0.22 µm membrane)</td><td>Short</td><td>510/-</td><td>-</td><td>[9]</td></tr><tr><td>CA and urea</td><td>Hydrothermal method (180 °C for 12 h)</td><td>Filtration (0.22 µm membrane), centrifugation, and ion-exchange column</td><td>Medium</td><td>560 nm/600 nm</td><td>-</td><td>[9]</td></tr></tbody></table>
<table>
  <tr>
    <td>Levofloxacin and PEI</td>
    <td>Microwave-assisted thermal decomposition method</td>
    <td>Filtration with a 0.22 µm filter membrane and dialysis (MWCO 0.5-1 kDa) for three days</td>
    <td>Long</td>
    <td>298 nm/508 nm</td>
    <td>1.38 nm</td>
    <td>[10]</td>
  </tr>
  <tr>
    <td>PTCDA and urea</td>
    <td>Solvothermal method (160 °C for 5 h)</td>
    <td>Centrifugation at 8000 rpm for 10 min and dialysis (MWCO = 1 kDa for 24 h)</td>
    <td>Long</td>
    <td>725 nm/745 nm</td>
    <td>3.3 ± 0.7 nm</td>
    <td>[11]</td>
  </tr>
  <tr>
    <td>Citric acid</td>
    <td>Microwave heating for 7 minutes with 700 W</td>
    <td>Filtration through filter paper (pore size = 5 µm) and dialysis</td>
    <td>Long</td>
    <td>375 nm/460 nm</td>
    <td>Ca. 1 nm</td>
    <td>[12]</td>
  </tr>
  <tr>
    <td>Ethylenediamine, phosphoric acid, tetraethoxysilane, ammonia</td>
    <td>Heated in a microwave oven (750 W) for 130s.</td>
    <td>Centrifugation and filtration through 0.22 µm membrane, dialysis (MWCO = 0.5 Da)</td>
    <td>Long</td>
    <td>365 nm/410 nm</td>
    <td>4-5 nm</td>
    <td>[13]</td>
  </tr>
  <tr>
    <td>Citric acid and urea</td>
    <td>Solvothermal method (210 °C for 12 h)</td>
    <td>Silica column chromatography (dichloromethane/methanol=50:1), followed by dialysis (MWCO = 3 kDa / 1 kDa / 0.5 kDa)</td>
    <td>Long</td>
    <td>442 nm/553 nm</td>
    <td>1.77 nm</td>
    <td>[14]</td>
  </tr>
  <tr>
    <td>Citric acid and ethylenediamine</td>
    <td>Hydrothermal method (150, 200, 250, and 300 °C for 5 h)</td>
    <td>Dialysis</td>
    <td>Long</td>
    <td>360 nm/443 nm</td>
    <td>2-6 nm</td>
    <td>[15]</td>
  </tr>
  <tr>
    <td>L-/D-cysteine</td>
    <td>Hydrothermal method</td>
    <td>Dialysis</td>
    <td>Long</td>
    <td>405 nm/510 nm</td>
    <td>5-7 nm</td>
    <td>[16]</td>
  </tr>
  <tr>
    <td>Arginine and EDA, quinones</td>
    <td>Microwave heating</td>
    <td>Filtration (0.1 µm microporous membrane) and dialysis for 48 h</td>
    <td>Long</td>
    <td>300 nm/350 nm</td>
    <td>-</td>
    <td>[17]</td>
  </tr>
  <tr>
    <td>Citric acid and ethylenediamine</td>
    <td>Hydrothermal method (140 °C for 10 h)</td>
    <td>Chromatography column (gel silica)</td>
    <td>Medium</td>
    <td>360 nm/443 nm</td>
    <td>2-6 nm</td>
    <td>[18]</td>
  </tr>
  <tr>
    <td>Para-, ortho, meta-benzene diamine</td>
    <td>Hydrothermal method (180 °C for 12 h)</td>
    <td>Dialysis, chromatography column,</td>
    <td>Long</td>
    <td>365 nm/-</td>
    <td>8.1 nm (para), 4.3 nm (ortho), 5.3 nm (meta)</td>
    <td>[19]</td>
  </tr>
  <tr>
    <td>Para-, ortho, meta-benzene diamine</td>
    <td>Hydrothermal method</td>
    <td>Chromatography column (gel silica)</td>
    <td>Medium</td>
    <td>365 nm/435 nm<br>365 nm/535 nm<br>365 nm/604 nm</td>
    <td>10.0 nm (para), 8.2 nm (ortho), 6.0 nm (meta)</td>
    <td>[20]</td>
  </tr>
  <tr>
    <td>Nineteen kinds of molecules</td>
    <td>Hydrothermal method (180 °C for 12 h)</td>
    <td>Filtration, dialysis, drying, chromatography column</td>
    <td>Long</td>
    <td>-</td>
    <td>-</td>
    <td>[21]</td>
  </tr>
</table>
<table>
  <tr>
    <td>Dextran sulfate sodium salt</td>
    <td>Hydrothermal method (220 °C for 3 h)</td>
    <td>Centrifugation, desalting chromatography column</td>
    <td>Long</td>
    <td>-</td>
    <td>-</td>
    <td>[22]</td>
  </tr>
  <tr>
    <td>Citric and 1,2-phenylenediamine</td>
    <td>Microwave method (7 min, 700 Watt)</td>
    <td>Dissolution in water, filtration, size exclusion chromatography column</td>
    <td>Long</td>
    <td>-</td>
    <td>4.48 ± 1.38 nm (fraction 1),<br>3.61 ± 0.92 nm (fraction 2),<br>2.24 ± 0.45 nm (fraction 3)</td>
    <td>[23]</td>
  </tr>
  <tr>
    <td>Soot produced by the flame of a paraffin oil lamp</td>
    <td>Refluxing (oil bath) (130 °C for 24h)</td>
    <td>Dialysis, Filtration, HPLC</td>
    <td>Long</td>
    <td>325 nm/534 nm</td>
    <td>-</td>
    <td>[24]</td>
  </tr>
  <tr>
    <td>Carbon nanofiber, HNO<sub>3</sub>, H<sub>2</sub>SO<sub>4</sub>,</td>
    <td>Oil bath (160 °C for 6 h)</td>
    <td>Centrifugation, filtration, dialysis, HPLC</td>
    <td>Long</td>
    <td></td>
    <td>7.1 ± 0.6 nm (fraction 7),<br>9.4 ± 0.5 nm (fraction 10),<br>10.1 ± 0.8 nm (fraction 5),<br>14 ± 1 nm (fraction 9)</td>
    <td>[25]</td>
  </tr>
  <tr>
    <td>Citric acid and ethylenediamine</td>
    <td>Hydrothermal method (200 °C for 15 min to 10 h)</td>
    <td>Dialysis, gel electrophoresis</td>
    <td>Medium</td>
    <td>350 nm/450 nm</td>
    <td>-</td>
    <td>[26]</td>
  </tr>
  <tr>
    <td>Citric acid and ethylenediamine</td>
    <td>Hydrothermal method (200 °C for 3 h)</td>
    <td>Centrifugation, Electrophoresis</td>
    <td>Medium</td>
    <td>350 nm/450 nm</td>
    <td>-</td>
    <td>[27]</td>
  </tr>
  <tr>
    <td>Candle soot</td>
    <td>Refluxing (12 h)</td>
    <td>Centrifugation, dialysis, denaturing PAGE</td>
    <td>Long</td>
    <td>315 nm/415-615 nm</td>
    <td>1 nm height (AFM)</td>
    <td>[28]</td>
  </tr>
  <tr>
    <td>Citric acid</td>
    <td>Refluxing (200 °C until formation of a juice)</td>
    <td>Cloud point extraction and centrifugation</td>
    <td>Short to medium</td>
    <td>360 nm/440 nm</td>
    <td>Ca. 5 nm</td>
    <td>[29]</td>
  </tr>
  <tr>
    <td>Mulberry leaves</td>
    <td>Hydrothermal method (150 °C for 4 h)</td>
    <td>Filtration, evaporation, extraction</td>
    <td>Short</td>
    <td>350 nm/500 nm;<br>370 nm/676 nm</td>
    <td>Ca. 3 nm</td>
    <td>[30]</td>
  </tr>
  <tr>
    <td>Carbon black (HIBLACK Orion) pigment</td>
    <td>Refluxing (250 °C for 2 h)</td>
    <td>Extraction</td>
    <td>Short</td>
    <td>350 nm/400-450 nm</td>
    <td>2 to 4 nm</td>
    <td>[31]</td>
  </tr>
  <tr>
    <td>Citric acid and diamines</td>
    <td>Hydrothermal method (150 °C)</td>
    <td>Solid phase extraction column, extraction, filtration</td>
    <td>Medium to long</td>
    <td>380 nm/428 nm;<br>370 nm/433 nm</td>
    <td>2 to 3 nm</td>
    <td>[32]</td>
  </tr>
  <tr>
    <td>Citric acid and chitosan</td>
    <td>Hydrothermal method (180 °C for 10 h)</td>
    <td>Extraction, filtration, precipitation, centrifugation</td>
    <td>Short to medium</td>
    <td>310 nm/418 nm</td>
    <td>2 to 10 nm</td>
    <td>[33]</td>
  </tr>
  <tr>
    <td>Citric acid and (S)- or (R)-2-(Aminomethyl)-1-Boc-pyrrolidine</td>
    <td>Microwave (240 °C and 150 W for 180 s)</td>
    <td>Precipitation, centrifugation</td>
    <td>Medium to long</td>
    <td>360 nm/456 nm</td>
    <td>2.7 ± 0.4 nm (AFM)</td>
    <td>[34]</td>
  </tr>
</table>
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