Project II

Synthesis of a Carbon dots

By
Mahima Baid (12616006029)
Sourav Dutta (12616006053)

Under the supervision of

<Sulagna Chatterjee>
Department of Chemical Engineering
Heritage Institute of Technology
Kolkata

1
Acknowledgement
We express our sincere thanks to our guide Prof. (Dr.) Sulagna
Chatterjee for her inspiring guidance and sharing her experience and for
her exemplary perseverance and hospitality. We would like to admit our
deep sense of gratitude for her constant guidance and cooperation
throughout the work. We sincerely acknowledge her for extending her
valuable guidance, support for literature, critical reviews of reports and
above all the moral support she had provided to us during this project.

2
 Certificate from Supervisor
This is to certify that the Project - I titled “Development of a C-dot
based food sensor” was allotted to Sourav Dutta (12616006053) &
Mahima Baid (12616006029) 4th year students of B. Tech. in Chemical
Engineering of Heritage Institute of Technology. Following the
guidelines as prescribed in the B. Tech Curricula and Syllabi, the
candidates have carried out the project work by occasionally consulting
the undersigned and have prepared the report. In my opinion, this is a fit
piece of project work required to be submitted at the end of B. Tech 7 th
semester.

_______________________________________
Sulagna Chatterjee
Head of Department
Department of Chemical Engineering
Heritage Institute of Technology, Kolkat

1
Table of Contents

Chapter Topic Page No

Aims and Objectives........................................................................................................................3

1 Introduction...........................................................................................................4

2 Literature Review................................................................................................5

3 Material And Methods........................................................................................12

3.1 Materials....................................................................................................12
3.1.1 Reagents used............................................................................12
3.1.2 Equipment used........................................................................12

3.2 Methods....................................................................................................12
3.2.1 Synthesis of carbon-dots............................................................12
3.2.2 Analytical Separation Techniques.............................................13
Electrophoretic Techniques.....................................13
Chromatographic Techniques..................................14
Density Gradient Centrifugation..............................14
Differential Centrifugation......................................14
Solvent Extraction Process......................................15
Dialysis....................................................................15
3.2.3 Separation of carbon dots .........................................................16

4 Results And Discussion..................................................................................19

5 Conclusion........................................................................................................20

Future Scope of The Work...............................................................................21

References.........................................................................................................22

2
Aims and Objectives
An easy bottom–up method for the preparation of carbon dots has been developed by tuning the
carbonization degree of citric acid and dispersing the carbonized products into alkaline solutions
In the last decade, carbon dots (C-dots, CDs) or carbon quantum dots (CQDs) have attracted a
considerable amount of attention from the scientific community as a low cost and biocompatible.
In particular, doped C-dots have excellent fluorescent properties that have been successfully
utilized for numerous applications. We overview the recent advances on the synthesis of doped
C-dots derived from carbon-rich sources. In addition, we a discuss some challenges and outline
some future perspectives of this exciting material. The dispersal of C-dots in solid-state thin
films is important to further expand and reinforce their practical application.

3
Chapter 1

Introduction

Carbon dots were first discovered in 2004 during single-walled carbon nanotubes purification.
These C-dots can be described as quasi-spherical particles with sizes below 10 nm shows that the
number of scientific publications related to C-dots rapidly raised starting from 2005.The
increased interest in these materials is related to several advantages of C-dots, such as carbon
source abundance, simple and low-cost synthesis process, biocompatibility, and excellent
fluorescent properties. In addition, C-dots are chemically stable, inert, possess the attractive
properties of good conductivity, low toxicity, environmental friendliness, simple synthetic
routes, form a stable colloidal solution, and are highly resistant to photobleaching compared to
traditional fluorescent organic dyes and semiconductor quantum dots. The fundamental
mechanisms responsible of the fluorescence capability of carbon-dots are very debated. Some
authors have provided evidence of size-dependent fluorescence properties, suggesting that the
emission arises from electronic transitions with the core of the dots, influenced by quantum
confinement effects, whereas other works have rather attributed the fluorescence to
recombination of surface-trapped charges, or proposed a form of coupling between core and
surface electronic states. The excitation-dependent fluorescence of C-dots, leading to their
characteristic emission tunability, has been mostly linked to the inhomogeneous distribution of
their emission characteristics, due to polydispersity. Therefore, the fluorescent properties of C-
dots are widely utilized for sensing biomedical imaging, catalysis, sensing, optronic, energy
research etc. In recent years, the advances in the development and improvement in C-dots
synthesis, characterization, separation and applications are burgeoning.

4
Chapter 2

Literature Review
CDs are generally quasi-spherical carbon nanoparticles (<10 nm) comprising amorphous to
nanocrystalline and always consist of sp2/sp3 carbon.19 They were successfully synthesized for
the first time via laser ablation of a carbon target.20 Then a series of Synthetic methods for
CQDs were developed and are roughly divided into two categories, "top-down" and "bottom-up"
routes. In the top-down method, carbon macromolecules are cut into smaller pieces followed by
[1] [2],
various synthetic methods such as chemical etching , electrochemical carbonization laser
ablation [3], microwave irradiation [4], arc discharge [5].
Bottom-up methods synthesize CDs from various small carbon molecules including citrates,
carbohydrates, and other green materials. In this method, it is easy to control the surface state, as
[6-8]
well as the size, of the CDs .The process is initiated from the carbonization of carbon
precursors, which occurs concurrently with dehydration via heat-treatment through
hydrothermal, microwave, or pyrolysis methods in concentrated acids[9-11]and
hydrothermal/solvothermal [12,13].

Top down methods

In the top-down method, carbon macromolecules are cut into smaller pieces using physical
forces. Subsequently, further surface modification is applied to enhance and tune their
fluorescence [14]. Many synthetic procedures have been summarized here.
The electrochemical approach involves a nonselective “top down” chemical cutting process of a
carbon material such as graphite, carbon nanotube, or carbon fibre electrodes. However, C-dots
prepared through the present electrochemical methods have mixed sizes, and require further
separation through filtration or chromatography to obtain monodisperse C-dots. In addition, the
as-prepared C-dots possess low photoluminescence efficiencies (always lower than 10%), which

5
limits further study in this field. Furthermore, the size-controllable preparation of high-quality C-
dots is difficult and complicated with traditional electrochemical methods. From the point of
view of material synthesis, the exploration of new carbon sources for the simple, economical,
and controllable synthesis of such C-dots is highly desirable.

Laser ablation is an efficient physical method for nanomaterial synthesis through laser irradiation
of a solid target in liquid. We utilize a laser with a long-pulse width, that is, a millisecond-pulsed
laser, to prepare C-dots. Such a laser is commonly used for welding but seldom for the synthesis
of nanomaterials. Due to its long-pulse width, the laser induced physical processes are expected
to be easily controlled by the regulation of pulse width. The carbon dots were produced via laser
ablation of a carbon target in the presence of water vapor with argon as carrier gas. The carbon
target was prepared by hot-pressing a mixture of graphite powder and cement, followed by
stepwise baking, curing, and annealing in argon flow. A Q-switched Nd:YAG laser (1064 nm, 10
Hz) was used for the ablation, during which the carbon target was in a flow of argon gas carrying
water vapor (through a water bubbler) at 900 °C and 75 kPa. The as-produced sample, according
to electron microscopy analyses, was dominated by nanoscale carbon particles in aggregates of
various sizes. The sample was treated in an aqueous nitric acid solution (up to 2.6 M) with
refluxing for up to 12 then, the surface passivation by attaching simple organic species to the
acid-treated carbon particles, bright luminescence emissions were observed.

In this review, we focus on the synthesis of multiwalled and single-walled carbon nanotubes
(MWNTs and SWNTs, respectively) by an arc discharge method. MWNTs can be obtained in
the cathode deposit of the dc arc discharge of pure graphite rods. Ambient gas plays an important
role, and pure hydrogen gas is the best gas for obtaining high-crystallinity MWNTs. A thin
innermost tube of less than 0.4 nm diameter and a carbon nanowire including a carbon chain can
be produced by a hydrogen-ambient dc arc discharge method. The Raman spectra of MWNTs
are important because radial breathing modes with frequencies higher than 300 cm –1 and a new
Raman band near 1850 cm –1 appear. In the case of SWNT synthesis by an arc discharge
method, the incorporation of catalytic metal particles in a graphite anode is necessary, and
SWNTs are obtained as soot in an evaporation chamber. By an arc-plasma-jet method wherein
two electrodes are placed at a sharp angle, the yield of soot including SWNTs is increased by

6
decreasing the amount of cathode deposit. In particular, the arc evaporation of a graphite rod
with a pure Fe catalyst in a gas mixture (hydrogen + inert gas) is effective for obtaining
macroscopic SWNT nets. The purification of the SWNTs nets by heat and hydrochloric acid
treatments is easy. Arc-synthesized single-walled carbon nanotubes have been purified through
preparative electrophoresis in agarose gel and glass bead matrixes. Two major impurities were
isolated:  fluorescent carbon and short tubular carbon. Analysis of these two classes of impurities
was done. The methods described may be readily extended to the separation of other water-
soluble nanoparticles. The separated fluorescent carbon and short tubule carbon species promise
to be interesting nanomaterials in their own right.
A one-step microwave-assisted polyol synthesis method was adapted with minor modifications
to prepare Gd-CDs.28 Typically, 0.5 mL of 30% sucrose solution, 100 μL of concentrated
H2SO4, and 0.5 mL of 200 mg/mL GdCl3 (GdCl3 was obtained by dissolving Gd2O3 in
hydrochloric acid at an elevated temperature, followed by evaporating the solvent) were
sequentially added to 3 mL of DEG in a 10 mL glass tube. The mixture was sonicated to form a
homogeneous solution and then heated in a domestic microwave oven (750 W, Galanz, China)
for 50 s. During this process, the mixture changed from colourless to dark brownish, indicating
the formation of CDs. After cooling to room temperature, the resultant suspension was
centrifuged (5000 revolutions/min, for 10 min) to remove large or agglomerated particles. The
CDs in supernatant were retained and purified by the dialysis against double-distilled water using
a Spectra/Por dialysis membrane [molecular weight cut-off (MWCO) = 100−500] for 24 h.
Finally, a clear yellow−brown aqueous dispersion containing CDs was obtained
Bottom-up methods

Bottom-up methods synthesize CDs from various small carbon molecules including citrates,
carbohydrates, and other green materials. In this method, it is easy to control the surface state, as
well as the size, of the CDs [15–17]. The whole synthetic procedure is briefly described. The process
is initiated from the carbonization of carbon precursors, which occurs concurrently with
dehydration via heat-treatment through hydrothermal, microwave, or pyrolysis methods in
[18–20]
concentrated acids (Fig. 3) . The microwave-assisted hydrothermal method is common, and
it is easy to synthesize CDs from various organic sources, including sucrose, glucose,
[21–24]
saccharides, amino acids, and proteins, which can all be used as building blocks for CDs .
Because of the diversity of precursor materials, various functional groups remain after synthesis,

7
and these are beneficial for enhancing the fluorescence of the CDs. It is also possible to create
CDs by refluxing candle soot in a strong acid, where the oxidation by the acid is important for
[25]
the dissolution of the soot . However, the fluorescent products derived from small carbon
molecules limit the mass production of CDs and lack quality control because of the
heterogeneity. Some process are listed below.

Cow milk-derived carbon dots (CMCDs)

were prepared by hydrothermal treatment of
cow milk, and the as-prepared CMCDs were
further extracted by ethyl acetate to obtain
amphiphilic CMCDs (ACMCDs). Using the
ACMCDs both as a reducing agent and a
template, the ACMCDs-supported silver
nanoparticles (ACMCD-Ag nanocomposites)
were prepared, which showed good biocidal
effect on both Gram-positive (Staphylococcus
aureus) and Gram-negative (Escherichia coli) bacteria. After that, a novel ACMCD-
Ag/polymethylmethacrylate nanocomposite antibacterial film was fabricated by solvent casting
method. Due to the excellent antibacterial, light admitting, and flexible properties, the
nanocomposite antibacterial film is considered to be of great potential in applications
Herein, CQDs were synthesized via a green and economic hydrothermal route utilizing lemon
peel as carbon source, generally discarded as waste. Synthesized CQDs possessed high
photostability with ~ 9 % of quantum yield even after one year of storage as compared to ~ 14 %
of quantum yield for fresh sample. CQDs has been successfully applied for the simple, sensitive
and selective detection of Cr6+ based upon fluorescent “turn off” technique. Further, composites
of CQDs and TiO2 nanofibers were developed at room temperature using 6- amino hexanoic
acid as linker molecule. Photocatalytic activity of TiO2-wsCQDs composites was explored for
the degradation of model pollutant methylene blue (MB) dye and found ~2.5 times higher than
that of TiO2 nanofibers.

8
A simple and facile one-pot synthesis of fluorescent carbon dots from orange waste peels was
performed using the hydrothermal carbonization method at a mild temperature (180 °C). The
chemical composition and morphological feature of the obtained carbon dots (C-dots) were
characterized using various spectroscopies and a transmission electron microscopy. The prepared
hydrothermal carbons were amorphous in nature, and clusters of polyaromatic hydrocarbons
included a large quantity of oxygen functional groups. A composite of C-dot with ZnO was used
as a photocatalyst for degradation of naphthol blue-black azo dye under UV irradiation, and the
superior photocatalytic activity was demonstrated. Overall,the present preparation method of C-
dots takes on meaning in the area of green synthesis in aqueous solutions, and the product has
great potential as a component material in the development of a remarkably efficient catalytic
system

Hair, a kind of natural and nontoxic raw material, was chosen as the precursor to prepare
CDs via a one-step pyrolysis process. The structure and fluorescence properties of the CDs were
thoroughly investigated. The obtained CDs can emit bright blue light under UV light with the
quantum yield of ca. 17%, and exhibit excitation-, pH- and solvent-dependent fluorescence. The
functional groups on the surface of CDs confer these nanomaterials with excellent dispersibility
in water and most polar organic solvents, as well as good compatibility with polymer matrices
such as poly (methyl methacrylate) and polyvinylpyrrolidone. Then multidimensional CD–
polymer hybrid materials, including one-dimensional (1D) microfibers, 2D patterned films and
3D microbeads were constructed with excellent overall performance, which are useful in
fluorescent patterns, flat panel displays and anti-counterfeiting labelling fields.

We report on a simple and eco-friendly approach that employs a domestic pressure cooker as an
inexpensive hydrothermal reactor for the batch synthesis of water-soluble, photoluminescent

9
nanoscale carbon dots derived from benign and cheap commercial starting materials. The
resulting carbon nanodots, which consist primarily of hydrophile-decorated amorphous carbon
and boast bright, stable, excitation wavelength-dependent fluorescence, were shown to be viable
cellular imaging agents for mice embryonic fibroblast cells, displaying little or no cytotoxicity
for carbon dot concentrations up to 0.667 mg/mL. In addition, the carbon dots proved useful as
nanoprobes for the fluorescence-based detection of environmentally-relevant heavy metal ions
such as Cu2+, displaying detection limits below 6 µM, sufficient to determine potable water
safety (20 µM is the limit for safe drinking water set by the U.S. Environmental Protection
Agency). More generally, these results highlight the utility of a household pressure cooker as a
cost-effective hydrothermal vessel relevant to nanocarbon synthesis, opening up other
possibilities for nano synthesis, particularly in resource-limited settings, educational venues, and
the classroom itself

To solve the problem of high temperature or long reaction time in hydrothermal synthesis of
carbon dots (CDs), a novel method based on the promoting carbonization by hydrochloric acid as
catalysis was developed. The acid catalysed carbon dots (ACDs) were prepared facilely from
tryptophan and phenylalanine at 200 ° C for 2 h. In our findings, the acids could promote
significantly the formation of the ACDs’ carbon core, as a result of the accelerating of the
carbonization due to the easy deoxidation. The ACDs showed an average size of 4.8 nm, and
consisted of high carbon crystalline core and various surface groups. The ACDs exhibited good
optical properties and pH-dependent photoluminescence (PL) intensities. Furthermore, the ACDs
were safe and biocompatible. The experimental results demonstrated that such new ACDs were
connected with DNA-aptamer by EDC/NHS reaction maintaining both the bright fluorescence
and recognizing ability on the cancer cells, which so could be served as an effective PL sensing
platform. The resultant DNA-aptamer with ACDs (DNA-ACDs) could stick to human breast
cancer cells (MCF-7) specifically, and exhibited high sensitivity and selectivity, indicating the
potential applications in the cancer cells targeted imaging fields.

As the fluorescent properties of CDs are closely linked to their particle sizes, surface states and

10
internal structures, the top-down method usually suffers from a lot of disadvantages, such as
requirement of special equipment, low fluorescence quantum yield, poor control of particle size,
and difficult regulation of surface state and inner structure. While the bottom-up method offers
us exciting opportunities to tune CDs with well-defined sizes, various surface states and inner
structures by elaborately designing the molecular structures of precursors and
preparation processes. The precursors have been generally divided into two types of natural
biomasses and organic molecules. Considering the components of biomasses are too complicated
to clarify, the CDs synthesized by organic molecules especially organic molecules containing
hetero-atoms (B, N, P, S, etc.) and specific groups (such as hydroxyl, carboxyl and amine
groups) are expected to find more application potential in fluorescence sensing field, because
the heteroatom doping and specific groups can not only improve the fluorescence properties
of CDs, but also endow CDs-based probes with high selectivity towards specific ions.
Moreover, it is worth noting that most of CDs used for constructing fluorescent probes are
obtained by the hydrothermal method which can provide the milder formation conditions for
CDs than other methods. Therefore, the researches on syntheses of CDs by hydrothermal
treatments of novel organic molecules with heteroatoms and specific groups are extremely
meaningful for widening the application of CDs in fluorescence sensing field.

Concerning the applications of C-dots, significant achievements have come up in a variety of

[26]
scientific fields. Followed by the first report by Cao et al. who demonstrated that C-dots can
penetrate easily through cell membrane and are used for conventional bioimaging, a further
[27]
development occurred at the hands of Yang et al. . Yang et al. succeeded in using C-dots for
the in vivo studies of optical imaging with mice for animal test. Followed by these, as a result of
extensive and multidisciplinary efforts, C-dots have found their place in a wide range of
applications in nanoprobes [28–38], drug delivery [39–43], therapy [44–48], and optoelectronic devices [49–
53]
.

Considering the enormous potential of C-dots for numerous applications, it is extremely

necessary to develop their large-scale synthesis. From this point of view, the hydrothermal

11
synthesis has become the most popular method due to its simplicity, low-cost, and high
efficiency. In this method, one simply heats carbon-rich compounds (sugars, organic molecules)
in a tightly closed vessel to initiate the carbonization process. For example, the hydrothermal
[54] [55]
method was used to prepare highly fluorescent C-dots from orange juice , glucose , banana
juice [56], citric acid [57].

One of the most common synthesis methods for fluorescent carbon dots is the thermal
decomposition; the reaction pathway is, however, quite time consuming. For instance, several
intermediates form during the process and they also give rise to fluorescent species. In the
present work, the formation of fluorescent C‐dots from citric acid has been studied. The
understanding of the citric acid thermal decomposition reaction is expected to improve the
control and reproducibility of C‐dots synthesis.

Chapter 3

Material and methods

3.1. Materials

3.1.1 Reagents used

Citric acid was first isolated in 1784 by the chemist Carl Wilhelm Scheele, who crystallized it
from lemon juice. It can exist either in an anhydrous (water-free) form or as a monohydrate. The
anhydrous form crystallizes from hot water, while the monohydrate forms when citric acid is
crystallized from cold water.

NaOH pellets Sodium hydroxide, also known as lye, glass beads, acetonitrile, deionised water.

3.1.2 Equipment used

In this work we have used Weighing machine, Furnace (temperature setpoint 200-300 in ˚C), Ph
meter, reflux boiling apparatus and Magnetic stirrer (temperature range 0-100 in ˚C)

12
3.2. Methods

3.2.1 Synthesis of carbon-dots

The carbon dots were prepared by directly pyrolyzing citric acid (CA). In a typical procedure of,
15g CA was put into a 25ml crucible and heated to 200˚C in a furnace. About 5 min later, the CA
was liquated. Subsequently, the colour of the liquid was changed from colourless to pale yellow,
and then orange in 30 min, implying the formation of carbon dots (If the heating was kept on, the
orange liquid would finally turn to black solid in about 2h, suggesting the formation of Graphene
oxides).The obtained orange liquid was transferred from crucible to a 50ml beaker and kept on a
magnetic stirrer at a temperature 60°C and 900 r.p.m.).Initial pH of the obtained orange solution
was recorded (3.50).NaOH solution was prepared by adding 5g of NaOH pellets in 10ml of
H2O.This above prepared NaOH was added drop by drop into the obtained orange liquid which
is under continuous and vigorous stirring. After adding each drop pH is recorded in the interval
of 5 seconds. It took almost 30 min to neutralize the pH.

3.2.2. Analytical Separation Techniques

Building upon the great interest in developing novel synthetic methods and searching for new
applications for this newly found nanomaterial, one has to fully understand the fundamental
properties of C-dots products. Due to the intensive research efforts carried out in the past
decades, the scientific community nowadays understands much better the structural,
photophysical, and chemical properties of C-dots; however, the complete characterization of C-
dots mixture still remains unachieved and faces noteworthy challenge. This is due to the fact that
a C-dots product exists as a complex mixture comprising components with various sizes and
different surface functionalities, and a polydisperse C-dots sample only represents the summation
or average properties of all individual C-dots species. In this sense, there is an urgent need for
the development of efficient analytical separation methods to isolate high-purity C-dots fractions
for more precious investigation of their unique chemical and photophysical properties. Since the
first isolation of C-dots from SWCNT by polyacrylamide gel electrophoresis (PAGE) in 2004,
the reports associated with analytical separation techniques of C-dots are emerging. Impressive
successes in this area include not only electrophoresis, but also, chromatography, density
gradient centrifugation, differential centrifugation, solvent extraction techniques and dialysis. A
summary of the separation techniques described in this section.

13
Electrophoretic Techniques. Electrophoretic methods are valuable tools for the separation of
water-soluble NP (nano particle) including iron oxide particle, silver, and gold as well as silicon
NP based on particle size, shape, and ionization of the surface functionality of NP. For the
electrophoretic separations of water-soluble C-dots, gel electrophoresis (GE) and capillary zone
electrophoresis (CZE) have been reported.

Chromatographic Techniques. The chromatographic techniques, previously proven to be highly

efficient in the separation of metal-based quantum dot based on their sizes and chemical
properties, have recently been applied to separate and analyse C-dots. Currently,
chromatographic methods available for the separation of C-dots are anion-exchange high
performance liquid chromatography, reversed-phase- (RP-) HPLC, and size exclusion
chromatography.

Density Gradient Centrifugation. The density gradient centrifugation method which is a routine
technique used to separate biomacromolecules also exhibits great potential in isolating NP
Briefly, samples are added on the top of a density gradient formed by sequentially layering
solutions of different densities. Upon centrifugation, the particles deposit in the density gradient
at different speeds on the basis of their sizes, shapes, or densities and eventually form different
hands in the gradient. Usually, the density gradient is prepared with sucrose, glycerol, cesium
chloride, and other aqueous solutions. The advantage of this technique is its nontoxicity.
However, for NP prepared in organic medium, transferring from organic solvent to aqueous
solution is challenging shows that fine separation can be achieved based on the size of C-dots
species. The obtained fractions exhibited green and blue fluorescence under UV light.

Differential Centrifugation. Differential centrifugation is used to separate the multiple fractions

within a NP sample by size and density; the larger and denser NP precipitate at lower centrifugal
forces while the smaller and lighter NP remain in the supernatant and require more force and
greater times to precipitate. In a typical differential centrifugation process, a NP mixture is
centrifuged multiple times, and after each run the precipitate is removed while the supernatant is

14
centrifuged at a higher centrifugal force. Sahuet al. for the first time reported the use of
differential centrifugation method to obtain different size-range C-dots species synthesized by
hydrothermal treatment of orange juice (Figure 18). In the centrifugation procedure, the aqueous
brown solution containing C-dots was firstly centrifuged at 3000 rpm for 15 min to get the less-
fluorescent deposit coarse nanoparticles (CP). Then, an excess amount of acetone was added to
the upper brown solution and centrifuged at a high speed of 10,000 rpm for 15min. The
supernatant containing the highly fluorescent nanoparticles (CD) has an average sizeof 1.5–4.5
nm. The deposit CP exhibiting weak fluorescence have larger size in the range of 30–50 nm.

Solvent Extraction Process. Solvent extraction, also known as liquid–liquid extraction or

partitioning, is a method used to separate a compound based on its solubility in two liquids that
do not mix (e.g., water and an organic solvent). This method has been widely used in the
isolation of C-dots from the reaction mixtures. Typically, organic solvents (e.g., ethyl acetate,
acetone, chloroform, toluene, and n-hexane) are used to extract the amphiphilic C-dots and
eliminate unreacted reagents in aqueous solution. Recently, Han et al. proposed a novel “gradient
extraction” method to separate C-dots synthesized from hydrothermal treatment of cow milk
based on their surface polarity of the C-dots species. Four organic solvents with different
polarities: hexane (polarity: 0.06), carbon tetrachloride (polarity: 1.6), mixture of carbon
tetrachloride and dichloromethane (v/v = 3: 2), and dichloromethane (polarity: 3.4), were used to
extract C-dots fractions. The Journal of Nanomaterials 17 four fractions obtained were
determined to have different surface polarities and show surface polarity-dependent
photoluminescence. The solvent extraction process is certainly good in roughly isolating C-dots
from the reaction mixtures; however, it does not possess the ability to completely separate and
exactly examine the hidden properties of each individual C-dots species.

Dialysis. Dialysis is a widely used purification method to separate the C-dots from the potential
small fluorescent molecules that can be by-products of the synthetic route. The dialysis process
works on the principles of the diffusion of small fluorescent molecules across a semipermeable
membrane based on the principle that the substances in water tend to move from an area of high
concentration to an area of low concentration. Usually, the as-synthesized fluorescent product

15
was dissolved in distilled water and dialyzed against a semipermeable membrane in purified
water with stirring and recharging with fresh distilled water at different time intervals over a
certain time period. The purification by dialysis is certainly very useful to remove the potential
small fluorescent species from C-dots sample. However, similar to the solvent extraction
process, it is not capable of efficiently separating each individual C-dots species.

3.2.3 Separation of carbon dots by column chromatography and reflux boiling

In present work CDs are synthesized from citric acid via a bottom-up technique such as
hydrothermal or microwave synthesis and reaction products will purify by preparative
column chromatography. During hydrothermal or microwave synthesis, various reaction
pathways are possible. Thus, a distribution of CD structures is expected. In consequence,
exact analysis of the CD structure requires careful separation of the different CD fractions
from the mixture. Chromatography and reflux boiling is a powerful tool for the separation
and purification of organic compounds.

16
17
Small volume of the sample can be separated by column chromatography using a column
with fused-in frit with a length of 200 mm. The silica gel stationary phase material of mean
pore size of 15 nm and a particle size of 37-74 μm. The eluent for the fractionation can be
acetonitrile: millipore-water with a vol. ratio of 1:1.
Here, our sample will be boiled and the resulting vapours travel up a glass tube called a
"fractionating column" and separate. The fractionating column is placed between the flask
containing the mixture and the "Y" adaptor, and is usually filled with glass or plastic beads,
which improves the separation between the liquids being distilled, leads to a better separation
than simple distillation because the glass beads in the fractionating column provide
'theoretical plates' on which the vapours can condense, reevaporate, and then recondense,
essentially distilling the mixture over and over. One theoretical plate is equivalent to one
vaporization-condensation cycle, which is equivalent to one simple distillation. The more
volatile liquids will gradually move towards the top of the fractionating column, while lower
boiling liquids will stay towards the bottom, giving a better separation between the liquids.
The vapor eventually reaches the condenser, where it is cooled and then drips into the
collection vessel.

18
Chapter 4

Results and Discussion

C-dots can be a versatile material for future biomedical and sensing applications thanks to the
unique fluorescent properties, excellent biocompatibility, and high aqueous stability. The
development of carbon dots was satisfactory. But neutralizing the pH of the orange solution was
a tedious process and needs a standardisation which can be achieved only by repeating the
experiment and setting a NaOH concentration instead of trial and error. In present work, the
obtained orange solution emitted green fluorescence when excited by UV light. the general
approaches for analytical separation of C-dots is outlined. Given the complexity of C-dots
components and the inherent drawbacks of each technique, only one technique is not enough to
capture the complete picture of the C-dots properties. For most of time, multiple characterization
and analytical separation techniques described here are used in complementary ways. Though
significant progress has been made in recent years, we are still far from achieving full
understanding of C-dots properties. Bearing this in mind, much effort is required. On one hand,
despite the size and chemical structure of C-dots having been estimated by a number of
researchers, the exact molecular weight of C-dots still remains debated, and, therefore,
developing new methods with improved resolution and sensitivity based on new or original
concepts is essential to progress the characterization of C-dots. On the other hand, carefully
checking and refining the existing methods to overcome the limitations and to further improve
their working efficiency is of equal importance. While any tiny improvement may not
significantly matter, the cumulative effects of a series of such advances will make a substantial
change. Furthermore, among the numerous techniques applied to separate or to characterize C-
dots, efforts have been seldom used in coupled ways. It is anticipated that the coupled techniques
could be further explored in the near future to achieve integrative separation-characterization of
C-dots in an efficient way. Also, some issues such as the exact fluorescence, the location of ions
in C-dots were not resolved yet. Thus, these issues should be addressed for fundamental
understanding and control of the fluorescence in doped C-dots.

19
Chapter 5

Conclusion
In this report, we outline the general approaches for the synthesis, characterization and analytical
separation of C-dots. Much research into carbon-dots has been reported in the last few decades,
and a wide range of synthetic methods and characterization techniques have been used. In most
cases, studies of these fluorescent materials have focused on their bioimaging applications. The
recent progress in fluorescent CDs has opened the possibility of developing portable detectors.
Moreover, the progress in biocompatible fluorescent CDs enables harmless onsite detection as
well as the colour-mediated analysis provides easy interpretable read-out even for non-
professional persons. However, relatively low solubility of CDs in water remains challenges and
low cost for fabricating devices is another requirement for the use of CDs in various fields, even
though many synthetic methods have been developed. In addition, the exact mechanism for
different photoluminescent which depends on the synthetic method and raw carbon sources
should be more cleared. We hope that this review will inform researchers about the recent
progress in carbon-dots, leading to develop new eco-friend and cost-effective synthetic methods
and practical use.

20
Future Scope of the work

Though significant progress has been made in recent years, we are still far from achieving full
understanding of C-dots properties. Bearing this in mind, much effort is required. On one hand,
despite the size and chemical structure of C-dots having been estimated by a number of
researchers, the exact molecular weight of C-dots still remains debated, and, therefore,
developing new methods with improved resolution and sensitivity based on new or original
concepts is essential to progress the characterization of C-dots. On the other hand, carefully
checking and refining the existing methods to overcome the limitations and to further improve
their working efficiency is of equal importance. While any tiny improvement may not
significantly matter, the cumulative effects of a series of such advances will make a substantial
change. Furthermore, among the numerous techniques applied to separate or to characterize C-
dots, efforts have been seldom used in coupled ways. It is anticipated that the coupled techniques
could be further explored in the near future to achieve integrative separation-characterization of
C-dots in an efficient way.

21
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