B.

TECH PROJECT REPORT ON

CONTROLLED PRECIPITATION OF DRUG NANOPARTICLES
USING ULTRASONICALLY DRIVEN MIXING DEVICES

Submitted by:

Rakesh Kumar Chaudhary

0800122
Department of Chemical Engineering
Indian Institute of Technology, Gandhinagar

Supervised by:
Dr. Sameer V. Dalvi
Assistant Professor
Department of Chemical Engineering
Indian Institute of Technology, Gandhinagar
1

Acknowledgments
First and foremost, I express my deep sense of gratitude to my project guide Dr. Sameer V.
Dalvi for his valuable guidance and advice. He took plan to go through the gathered information
and presentations on the topic. His guidance fueled my enthusiasm even further and encouraged
me to boldly step into what was a totally dark and unexpected expense before me. I would
especially thank Alpana Thorat for providing innovative ideas to work upon.

I take immense pleasure in thanking everyone working in chemical engineering research

lab for providing me with a good environment and facilities to complete this project. I would like
to thank everyone at IIT Gandhinagar for helping and supporting me during my project. Words
are inadequate in offering my thanks to Dr. Sameer V. Dalvi and IIT Gandhinagar for offering
me the project.

Finally, yet importantly, I would like to express my heartfelt thanks to my beloved

parents for their blessings, my friends for their helps and wishes for the successful completion of
the project.

Rakesh Kumar Chaudhary

ABSTRACT
Process of precipitation of drug nanoparticles through addition of liquid antisolvent can be
controlled either by controlling the mixing of solution and antisolvent or by controlling the
precipitation i.e. by controlling nucleation and growth.
There are two set of experiments covered in this project report. The first part of first set
of experiment done was for the calculation of induction time keeping the mixing rate same
(mixing with ultrasonic energy) and trying to control the rate of precipitation by changing the
degree of supersaturation. The next part of experiment was to vary the rate of mixing by
replacement of ultrasound with stirrer hence controlling mixing while keeping the
supersaturation same.
The first part of second set of experiment consists of calculation of equilibrium solubility
of curcumin in ethanol-water mixture. The second part consists of calculation of metastable zone
width for same mixing condition with different solution antisolvent ratio. From the data of
equilibrium solubility and metastable zone width, graph of variation of solubility and metastable
zone width are drawn and analyzed for different composition of ethanol water solution.

TABLE OF CONTENT
Page No.
1. Introduction ..5
2. Literature Review .....6
2.1. Nucleation...6
2.2. Growth....7
2.3. Induction time.7
2.4. Equilibrium solubility.9
2.5.Metastable zone width9
3. Experiment 1....10
3.1. Materials...10
3.2. Experiments..10
3.3. Apparatus and Experimental procedure...10
3.4. Result....11
3.5. Discussion.14
4. Experiment 2.15
4.1. Materials.15
4.2. Experiment.15
4.3. Apparatus and Experimental procedure.15
4.4. Metastable zone width experiment.17
4.5. Result..18
4.6. Discussion..20
5. References...21

1. Introduction
Curcumin, 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione, is a natural
polyphenolic phytochemical extracted from the powdered rhizomes of the spice turmeric
(Curcuma longa)[1]. It constitutes approximately 3-4% of the composition of turmeric. Along
with the other curcuminoids, curcumin is responsible for the yellow color of turmeric. Turmeric
is prominently used in South and East Asian countries for culinary, medicinal, and cultural uses.
As an additive, turmeric can improve the palatability, aesthetic appeal, and shelf life of
perishable food items. In the Ayurvedic system of medicine, turmeric is used as a tonic, blood
purifier, and topical ointment[2].
Curcumin can exist in at least two tautomeric forms, diketo and keto-enol. The structures are
seen in Fig. 1[3]:

Diketo Form

Keto-Enol Form
Fig.1. Diketo and keto-enol forms of curcumin.

The keto-enol form is strongly favored by intramolecular H-bonding and is more energetically
stable in the solid phase and in solution. The central -diketone moiety is suggested to be likely
responsible for the high beneficial activities of curcumin.
Curcumin is soluble in ethanol while practically insoluble in water.

2. Literature Review
2.1 Nucleation
Nucleation is the process of formation of initial crystals from a given solution, in which a small
number of ions, atoms or molecules become arranged in a pattern, characteristics of a crystalline
solid. Hence it forms sites on which additional particles can be deposited.
Primary nucleation:
Primary nucleation is the formation of crystals in the initial stage when no other crystals are
present and if present then are in so small amount that they dont influence the formation of new
nuclei.
These are further classified as homogeneous and heterogeneous nucleation, in homogeneous
nucleation; nucleation is not influenced by wall of crystallizer and any foreign substance.
Heterogeneous nucleation includes the enhanced nucleation because of presence of foreign
particles.
For primary nucleation:

Where B = no. of nuclei formed per unit volume per unit time
Kn = rate constant
C= solute concentration
C*= solute equilibrium concentration
N= empirical exponent
Secondary Nucleation:
Secondary nucleation includes nucleation formation in the influence of microscopic crystals. It
occurs because of the fluid shear and other collisions between the already existing nuclei and
newly formed nuclei.
For secondary nucleation:

MTJ =Suspension density

K1 = rate constant
2.2 Growth
The solute molecules present near the nuclei formed get attached to the nuclei and hence
increases the size of the nuclei resulting into its growth. This happens mainly because nuclei
formed are unstable due to super-saturation. The rate of increase of size of the nuclei is known as
growth rate. It is influenced by several factors, such as surface tension of solution, pressure,
temperature, relative crystal velocity in the solution etc.
The Relation between mixing time, induction time and crystal growth time is calculated using
Damkohler number:

for nucleation

for growth

Low Da suggests that mixing will have minimal effect, while increasing Da increases criticality
of mixing. For growth, at low value of Da mixing would have minimal effect on the particle size
distribution. For high values of Da, slow mixing and fast nucleation or crystal growth, mixing
would impact the particle size distribution since localized concentrations would lead to variable
nuclei generation or crystal growth rate throughout the solution [4]
2.3 Induction Time
Induction time is defined as the time difference between reaching super-saturation and formation
of first nuclei in the solution.
But because of measurement difficulties in detecting first few nuclei, another modified definition
of induction time is developed, as the time needed for the number density (Nm/V) of nuclei to
reach a fixed value. This fixed value depends upon the method of detection of nuclei. If the
instrument that is used to detect the nuclei is more sensitive the number density taken will be
small, however for less sensitive device it would be a greater value.

Factors affecting induction time:

Degree of super-saturation: For high super-saturated solution induction time will be less in
comparison to solution with less super-saturation. Since in case of high supersaturation, there
will be more driving force for precipitation because of a bigger change in free energy of solution.
Degree of mixing: For more degree of mixing induction time will be less in comparison to lower
degree of mixing.
Antisolvent Solvent ratio: For more antisolvent degree of precipitation will be higher hence
lower will be the induction time.
Temperature of the solution: For higher temperature the degree of supersaturation will
decrease resulting into higher induction time of precipitation.
Stabilizer: Presence of stabilizer will increase the induction time by reducing the instability of
solution. The change in the value of induction time also depends upon the amount of stabilizer
added to the solution. For higher amount of stabilizer added, the value of induction time will also
increase.
Method of detection used by others to measure induction time
Laser scattering method: This method is based on the principle of scattering of light from the
particles present in the solution. On the formation of nuclei the amount of total light scattered
and degree of scattering will change and hence the formation of particles can be detected.
Visual Appearance: From visual appearance the formation of new particles can be detected by
observing the change in color (turbidity) of the solution. However the sensitivity of this method
is very low, hence it is not a good method of detection of nuclei.
Conductivity measurement: In this method, the conductivity of solution is measured
continuously and the formation of particles is detected by change in the conductivity of solution.
Because of precipitation of nuclei, number of charge carrier present in solution decreases, it
results into significant decrease in conductivity of solution.
Other methods: Other method of detection include Attenuated Total Reflection-Fourier
Transform Infrared Spectroscopy (ATR-FTIR), Focused beam reflectance measurement
(FBRM) and Lasentec Particle Vision and Measurement (PVM).
8

2.4 Equilibrium Solubility

The maximum amount of solute that can be dissolved in a solution of given composition at a
given temperature and pressure is defined as the equilibrium solubility of that solute in given
solution at specified conditions of temperature and pressure.
Factors affecting equilibrium solubility
Temperature: Depending upon the heat involved in dissolution process, solubility of solute
varies with change in temperature. If the dissolution of solute is endothermic, on increasing
temperature solubility increases, while for exothermic dissolution solubility decreases with
increase in temperature.
Particle size: Solubility of solute increases with decrease in the size of solute particles. This
effect remains negligible unless the size of the particle becomes significantly small (smaller than
1 m).
Method of detection used
HPLC analysis: The composition of component present is analyzed using HPLC instrument and
hence solubility of a saturated solution can be measured.
Density difference: Based upon the difference in density of solvent and solution with known
concentrations, a calibration curve can be plotted and based on that solubility of a given solute
can be determined. However this method demands very high sensitivity of measurement as well
as it is feasible only in case where high amount of solute can be dissolved in the solution giving
significant change in density.

Filtration and drying of extra solute: In this method, a supersaturated solution of known
concentration is prepared and then after proper mixing, the precipitated solute is filtered out,
dried and then weighted. From the difference in weight of solute dissolved and precipitated, the
equilibrium solubility is determined.
2.5 Metastable zone width
The solute remains in the solution until a sufficiently high level of super-saturation is developed
to induce the nucleation. The extent of this super-saturation is referred to as metastable zone
width [1].
9

Factors affecting MSZW

Metastable zone width is dependent on saturation temperature, rate of generation of supersaturation, impurity level present in the solution and the history of the solution. Hence it is
important to characterize metastable zone width under a specific set of operating conditions.[5]
Measurement technique used
The most widely used technique to measure the metastable zone width is polythermal technique.
It involves cooling of a saturated solution at fixed rate until nucleation occurs.
The other method used in case of anti-solvent precipitation is amount of anti-solvent added to the
solution (with a fixed rate of addition of antisolvent) until nucleation occurs.

3. Experiment 1
3.1. Materials
Curcumin, Hydroxypropyl methyl cellulose (HPMC) (4000 cPs, F.C.C.), Ethanol (99.8 %
pure) were purchased from Sigma-Aldrich Inc. India. All these chemicals were used without
further purification. Deionized Millipore water was used as an antisolvent.
3.2 Experiment
The first experimental set up was for the measurement of induction time of a solution containing
ethanol and water (solvent and anti-solvent respectively) in the ratio of 1:10 for different
concentration of curcumin in presence of HPMC stabilizer.
3.3 Apparatus and Experimental Procedure
This diagram represents jacketed glass reactor having jacket around its boundary, through
which a cooling or heating liquid flows in order to maintain the desired temperature inside the
reactor according to the condition of the reactions. We have added two transparent tubes one
having LED (light emitting device) and other having light receptor in it as shown in figure.
These two tubes were placed in the reactor in such a fashion that light coming from LED was fell
on the receptor. The terminal of receptor was interfaced with computer terminal such that the
intensity observed by the receptor was stored in the software installed for receptor and the data
were then stored in form of spreadsheet.
Initially in the above explained reactor, 600 ml water with 120 mg HPMC was added and
then it was cooled to 1C temperature, after this 60 ml of ethanol- curcumin solution was added.
10

Since we had already inserted the test tubes having LED and receptor, the receptor measured the
change in intensity due to addition of solution and then it kept on measuring further change in
intensity. When nanoparticles precipitated, since new particles came into the solution it became
more turbid resulting into the decrease in intensity of light received by receptor. All these data
were stored by the software and plotted intensity as a function of time.
All these experiments were done for concentration of curcumin ethanol solution of 5, 7.5, 10,
12.5, 15, 17.5 and 20 mg/ml.
For observing the effect of mixing these experiments were done with mixing devices inserted in
the reactor. Mixing devices includes stirrer with high rpm (2600-2800 rpm), stirrer with low rpm
(600-700) and ultra-sonication with power of 100W. All these experiments were done with
mixing devices inserted and active for 1 hour after the addition of solvent-solute mixture in the
antisolvent.

Experimental Set-up
3.4 Result
After addition of solvent-solute mixture in the antisolvent, the graph of intensity of light and time
was plotted. In order to see the effect of mixing device and the formation of particle as a function
of time, the product solution was taken after each 15 min after addition of solvent-solute mixture
in the reactor.

11

This is the graph of light intensity detected by the receptor vs time for 5 mg/ml ethanol curcumin
solution added to water (with ethanol : water :: 1:10). Here we can see that the first sharp drop in
the intensity is because of the addition of ethanol curcumin solution. Since the color of ethanol
curcumin solution is yellow, while initially there was only water present in the solution, adding
this yellow solution causes a sharp drop in intensity of light received. The second sharp drop was
observed when the formation of particles occurred. Since more particles were formed and
suspended into the solution, the solution became more turbid adding into another drop in light
intensity received.
Now, from the difference of time between formation of particle and addition of solution (solventsolute mixture) induction time was calculated and the result was as follow:
For mixing device as ultrasonication:
S.No.

Curcumin (soln. in

Induction Time (with US)

ethanol) (in mg/ml)

1

836 s ( 13 min 56 s)

7.5

770 s (12 min 50 s)

10

606 s (10 min 6 s)

12

12.5

498 s (8 min 18 s)

15

357 s (5 min 57 s)

17.5

227 s (3 min 47 s)

20

151 s ( 2 min 31 s)

After observing the effect of concentration change, chnge in type of mixing devices was done.
Here to see the effect of mixing devices, these experiments were performed with stirred with
high rpm, stirrer with low rpm and with US of 100W for the solution (ethanol-curcumin) of 10
mg/ml. The results obtained were as follow:
S.No.

Degree of mixing

Induction time

UV probe sonication (100 W)

606 s (10 min 6 s)

Stirrer with 2600-2800 rpm

769 s (12 min 49 s)

Stirrer with 500 rpm

No particle formation

The sizes of particles formed, were measured for each of the experiments and were found to be
as follow:
Conc./time

15 min

30 min

45 min

60 min

5 g/l

0.731

7.5 g/l

0.601

0.517

0.414

10 g/l

10 g/l (low rpm)

8.521

0.769

0.613

0.721

10 g/l (high rpm)

0.697

10.85

11.31

11.26

12.5 g/l

13

15 g/l

1.450(ZLS)

1.389(ZLS)

3.336

4.129

17.5 g/l

4.372

4.370

1.776

4.129

20 g/l

3.475

6.286

4.051

4.065

3.5 Discussion
From the obtained result, it is clearly visible that on increasing the concentration of curcumin in
the solution, the induction time for the precipitation of particle keeps on decreasing linearly.
Since on increasing the concentration of curcumin, amount of supersaturation increases, which
results in the increase of driving force for precipitation. From this linear decrease it can also be
observed that after a certain value of concentration of curcumin in solution there will be instant
precipitation and induction time will be negligible.
From the result for the mixing devices, it can be observed that induction time for ultrasound
probe sonication is lower in comparison to stirrer with high rpm mixing. In case of low rpm
mixing no induction time was observed, this is because in case of low rpm induction time was
very high and it was not comparable to induction time for high rpm stirrer and US probe
sonicator. The increase in induction time in case of stirrer clearly indicates that mixing in
ultrasound probe sonication is much better than the mixing in case of stirrer. Since in case of
ultrasound probe sonication, mixing is at molecular level and is supported by high ultrasonic
energy mixing is better in comparison to stirrer.

In comparison of stirrer with sonication, it was observed during the experiment that in case of
sonicator, maintaining the temperature of the reactor was difficult as high heat energy was added
through the sonicator and it heated the solution to a higher temperature. While in case of stirrer
maintaining temperature was very easy since stirrer does not increase the temperature of the
solution.

Hence it can be concluded that for higher concentration solution induction time is lesser and US
probe is better mixing device in comparison to stirrer.

14

4. Experiment 2
4.1 Materials
Curcumin, Ethanol (99.8 % pure), Deionized Millipore water used as antisolvent.
4.2 Experiment
The second experimental set up was for the measurement of equilibrium solubility of curcumin
in the solution of different composition of ethanol and water. The objective of this experiment
also included the calculation of metastable zone width for different composition of ethanol and
water, and hence to draw the equilibrium solubility and metastable zone width curve of curcumin
for ethanol water system.
4.3 Apparatus and Experimental Procedure
The diagram shows the jacketed glass reactor, the jacketed part is filled with cooling
liquid and the temperature of that cooling liquid can be kept as desired for the reactor. Here it
had been taken as 1C. At the condition of 1C and atmospheric pressure, excess curcumin was
added in the solution of ethanol and water (total of 100 ml). The composition of ethanol water
solution was varied from pure ethanol to pure water with increase of 0.1 volume fraction of
water in each experiment. The first experiment was done with pure ethanol, second experiment
with 0.9 volume fraction ethanol and 0.1 volume fraction water and so on.

Experimental Set-up

15

After adding excess curcumin, the solution was stirred for minimum of 12 hours, hence to
make sure that the solution has reached the equilibrium and maximum possible curcumin is
dissolved in the solution. After that the solution was filtered with the help of filter paper,
filtration equipment and vacuum pump.
After filtration the saturated solution at 1C was brought down to room temperature. At
room temperature it was diluted for 100-200 times. Then its absorbance was calculated by UV
spectrophotometer. The solution was diluted to 100-200 times just to ensure that the absorbance
of the solution comes in the range in which Beer Lamberts law is valid. It was also found that in
case of solution with high volume fraction of water, after diluting for 100 times the absorbance
went in the range which is not in the detectable range for the UV spectrophotometer. Hence in
order to make sure that we had right value of absorbance, the solution was diluted to 10, 100 and
500 times and absorbances of all these solution were measured.

After getting the absorbance of all solutions, in order to interperate it in terms of concentration,
we were in need to make the calibration curve. The calibration curves were made for all 11 types
of solution (having volume fraction of water from 0 to 1). For making calibration curve solution

16

of curcumin in ethanol-water mixture were formed, where concentration of curcumin was in the
range of g/ml.
From Beer Lamberts law, we know

Where A=absorbance, L= width of cuvette (through which light passes)

c = concentration of solution.
Since absorbance is linearly proportional to concentration of solution, our calibration curve must
be straight line and from calibration curve by putting the value of absorbance of saturated
solution, concentration of saturated solution can be obtained.
1
0.9

Absorbance (nm)

0.8
0.7

y = 0.0869x - 0.0019
R = 1

0.6
0.5
0.4
0.3
0.2
0.1
0
0

Conc. of curcumin (g/ml)

10

12

Calibration curve for curcumin in 50% Ethanol, 50% water (vol%) for 400 nm wavelength light
4.4 Metastable zone width Experiment
For metastable zone width determination, the saturated solution was again put back to the reactor
and then in the reactor, two test tubes one having LED and other having light receptor were
added in the same fashion as used in experiment 1. Now here, water was added to this solution
such that the flow rate of water was maintained at 0.1 ml/sec. Water was added till the solution
became turbid due to presence of excess antisolvent. The extra amount of water added till the
solution become turbid is termed as metastable zone width. The turbidity of solution was again
measured by detecting change in intensity of light received by the receptor.
17

4.5 Results
Since the concentrations were measured using UV Spectrophotometer, we need to select the
wavelength of UV light which gives the maximum absorbance. Here in case of curcumin it was
found to be in the range of 390-460nm. Hence concentrations were calculated using wavelengths
390, 400, 410, 420, 430, 440, 450 and 460 nm.
Here is the table containing equilibrium solubility of curcumin (in mg/ml) in solution containing
different vol% of ethanol and water.
(vol %
100%
Ethanol)
(Abs
nm)
390 3.816
400 3.678
410 3.589
420 3.566
430 3.556
3.55
440
450 3.585
460 3.744

90%

80%

70%

60%

50%

40%

30%

20%

10%

0%

2.897
2.805
2.852
2.734
2.801
2.811
2.833
2.911

2.086
2.086
2.124
2.073
2.073
2.083
2.097
2.113

1.44
1.447
1.471
1.45
1.453
1.45
1.448
1.45

0.721
0.718
0.721
0.722
0.726
0.727
0.729
0.735

0.201
0.201
0.2
0.2
0.199
0.199
0.198
0.205

0.0446
0.0442
0.0437
0.0431
0.0424
0.0419
0.0412
0.0401

0.01396
0.0137
0.01333
0.01287
0.01249
0.01222
0.01204
0.01167

0.008774
0.008361
0.007463
0.007
0.006742
0.006732
0.006831
0.007454

0.001025
0.001133
0.001061
0.001029
0.001046
0.00114
0.001228
0.001513

0.00003
0.00002857
0.0000225
0.00002143
0.00002095
0.0000175
0.0000145
0.00001

However among these values of absorbance, the maximum absorbance is for 430 nm, for each
calibration curve ranging from 100% (vol.%) ethanol to 0% ethanol.
1
0.9

Absorbance (nm)

0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
380

390

400

410

420
430
Wavelength (nm)

440

450

460

470

18

This is the graph of absorbance vs wavelength for 50% (vol%) ethanol-water solution.
Hence the final equilibrium solubility of curcumin is:
(vol %
Ethanol)
Solubility
(mg/ml)

100%

90%

3.556 2.801

80%

70%

2.073 1.453

60%

50%

40%

30%

20%

0.726 0.199

0.0424

0.01249

0.006742

10%

0%

0.001046 0.00002095

Metastable zone width

Metastable zone width is the amount of extra water added to the saturated solution till the
precipitation occurs. Here is the result of metastable zone width in ml of water added per ml of
solution.
(vol % Ethanol)
MSZW (ml/ml soln.)

100%

90%

80%

70%

60%

50%

1.446809

1.1

0.888889

0.717647

0.6

0.477778

Metastable zone width was observed only till we had saturated solution of curcumin in 50%
ethanol & 50% water solution. After that there were no change in intensity of light were
observed for addition of any amount of antisolvent.
Equilibrium solubility and MSZW curve
For making equilibrium solubility curve and MSZW curve, all the values from mg/ml were
converted to mole taking the basis of 1 mg for solution.
Assuming 1ml of saturated solution was taken:
Solubility of curcumin in pure ethanol = 3.556 mg/ml = (3.556/368.38)*1000 = 9.653 mol
Moles of water = 0 (since no water is in solution)
Moles of water for solution at MSZ = (1.446809/18.015)*106 = 80311.32
Moles of Ethanol = (vol. of ethanol*density/mol. wt.) = (1*0.789/46.07)*106 =17126.11
According to the convention we put
x1 = moles of antisolvent (water)
x2 = moles of solvent (ethanol)
x3 = moles of solute (curcumin)
Now, we define mole fraction on antisolvent free basis:
X1= x1/ (x2 + x3)
X2= x2/ (x2 + x3)
X3= x3/ (x2 + x3)
19

For equilibrium solubility and metastable zone width, X3 vs X1 was plotted.

0.0006

0.0005
Saturated Soln.
0.0004

X3

MSZW

0.0003

0.0002

0.0001

0
0

X1

10

12

14

4.6 Discussion
From the result obtained, it is clear that on decreasing the amount of ethanol in the solution, the
solubility of curcumin also decreases. Since on decreasing the % of ethanol, the other component
(water) % increases, while curcumin is not soluble in water, hence on increasing the amount of
antisolvent results into precipitation of more particles and dissolve lesser particles, which is
reflected in terms of decrease in solubility.

Metastable zone width decreases with increase in mole-fraction of water. Here as we keep on
increasing the amount of water, amount of ethanol decreases and in the saturated solution it
already have some amount of water as antisolvent, hence it require lesser antisolvent to
precipitate.

20

5. References

[1] N.M. Khanna, Turmeric - Nature's precious gift., Current Science, 76 (1999) 1351-1356
[2] B. Joe, M. Vijaykumar, B.R. Lokesh, Biological Properties of Curcumin-Cellular and Molecular
Mechanisms of Action, Critical Reviews in Food Science and Nutrition, 44 (2004) 97-111.
[3] T.M. Kolev, E.A. Velcheva, B.A. Stamboliyska, M. Spiteller, DFT and experimental studies
[4] David J. am Ende, Chemical engineering in the Pharmaceutical industry, R & D to
manufacturing, Wiley Publication
[5] P. Barret & B. Glennon, Characterizing the metastable zone width and solubility curve using
Lasentec FBRM and PVM, 2002

21