nanoparticles precipitation

8/10/2019 Nanoparticles Precipitation

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B.TECH PROJECT REPORT ON

CONTROLLED PRECIPITATION OF DRUG NANOPARTICLES

USING ULTRASONICALLY DRIVEN MIXING DEVICES

Submitted by:

Rakesh Kumar Chaudhary

0800122

Department of Chemical EngineeringIndian Institute of Technology, Gandhinagar

Supervised by:

Dr. Sameer V. Dalvi

Assistant Professor

Department of Chemical Engineering

Indian Institute of Technology, Gandhinagar


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Acknowledgments

First and foremost, I express my deep sense of gratitude to my project guide Dr. Sameer V.

Dalvifor 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 Thoratfor 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 Gandhinagarfor helping and supporting me during my project. Words

are inadequate in offering my thanks to Dr. Sameer V. Dalvi andIIT Gandhinagarfor 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


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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 theprecipitation 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.


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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


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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.


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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:


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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 theinstrument 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.


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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).


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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 than1 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].


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Factors affecting MSZW

Metastable zone width is dependent on saturation temperature, rate of generation of super-

saturation, 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 differentconcentration 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.


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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 functionof time, the product solution was taken after each 15 min after addition of solvent-solute mixture

in the reactor.


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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 (solvent-

solute mixture) induction time was calculated and the result was as follow:

For mixing device as ultrasonication:

S.No. Curcumin (soln. in

ethanol) (in mg/ml)

Induction Time (with US)

1 5 836 s ( 13 min 56 s)

2 7.5 770 s (12 min 50 s)

3 10 606 s (10 min 6 s)


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4 12.5 498 s (8 min 18 s)

5 15 357 s (5 min 57 s)

6 17.5 227 s (3 min 47 s)

7 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

1 UV probe sonication (100 W) 606 s (10 min 6 s)

2 Stirrer with 2600-2800 rpm 769 s (12 min 49 s)

3 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 - - - -


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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


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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


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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.

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.

y = 0.0869x - 0.0019

R = 1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.80.9

1

0 2 4 6 8 10 12

Absorbance(nm)

Conc. of curcumin (g/ml)


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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 %

Ethanol)

100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0

(Absnm)

390 3.816 2.897 2.086 1.44 0.721 0.201 0.0446 0.01396 0.008774 0.001025 0.000

400 3.678 2.805 2.086 1.447 0.718 0.201 0.0442 0.0137 0.008361 0.001133 0.00002

410 3.589 2.852 2.124 1.471 0.721 0.2 0.0437 0.01333 0.007463 0.001061 0.0000

420 3.566 2.734 2.073 1.45 0.722 0.2 0.0431 0.01287 0.007 0.001029 0.00002

430 3.556 2.801 2.073 1.453 0.726 0.199 0.0424 0.01249 0.006742 0.001046 0.000020

440 3.55 2.811 2.083 1.45 0.727 0.199 0.0419 0.01222 0.006732 0.00114 0.0000

450 3.585 2.833 2.097 1.448 0.729 0.198 0.0412 0.01204 0.006831 0.001228 0.0000

460 3.744 2.911 2.113 1.45 0.735 0.205 0.0401 0.01167 0.007454 0.001513 0.000

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.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

380 390 400 410 420 430 440 450 460 470

Absorbance(nm)

Wavelength (nm)


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This is the graph of absorbance vs wavelength for 50% (vol%) ethanol-water solution.

Hence the final equilibrium solubility of curcumin is:

(vol %Ethanol)

100% 90% 80% 70% 60% 50% 40% 30% 20% 10%

Solubility(mg/ml) 3.556 2.801 2.073 1.453 0.726 0.199 0.0424 0.01249 0.006742 0.001046 0.00002

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) 100% 90% 80% 70% 60% 50%

MSZW (ml/ml soln.) 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 basisof 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)


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For equilibrium solubility and metastable zone width, X3 vs X1was plotted.

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 toprecipitate.

0

0.0001

0.0002

0.0003

0.0004

0.0005

0.0006

0 2 4 6 8 10 12 14

X3

X1

Saturated Soln.

MSZW


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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

nanoparticles precipitation

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