Supercritical Micronization of β‐Carotene · The spectrum was traced and Lambert‐Beer law at...

Sofia Antunes Master Dissertation in Biological Engineering September 2007

Supercritical Micronization of β‐Carotene 1

Supercritical Micronization of β‐Carotene

Sofia Inês de Matos Antunes

Instituto Superior Técnico, Universidade Técnica de Lisboa

[email protected]

The aim of the present work was the micronization of β‐carotene, an important carotenoid, due to its

properties as colorant, antioxidant and as a precursor in the synthesis of vitamin A. The micronization of

β‐carotene has as main goal the increase of its dissolution rate in water, allowing, simultaneously, the

preparation of aqueous suspensions containing this carotenoid.

The process was developed in a supercritical micronization apparatus type SAS, using carbon dioxide as

antisolvent and tetrahydrofuran as solvent. The effect of operating pressure in the particle size and

morphology of the final product has been studied. The runs of micronization have also permitted to

study the solubility of β‐carotene in the supercritical mixture under several conditions of pressure,

temperature and flow rate. The micronization of an extract of natural β‐carotene has also been made, in

order to evaluate the purification ability of the process.

Some relationships between experimental variables studied and solubility of β‐carotene in the

supercritical mixture were recognized. It has been concluded that solubility strongly depends on

temperature and on THF fraction and does not depend much on pressure.

It has been shown that particle morphology is changed when the operating pressure changes and it also

depends on the position of the operating point relatively to the mixture’s critical point. Additionally, it’s

been shown that particle size increases with the operating pressure, when one operates over the critical

point, and a relationship between the particle size and the density of the binary mixture has been

established.

The supercritical antisolvent micronization was well done in the processing of β‐carotene and the

control of particle size and morphology through the manipulation of the operating conditions is

possible. Moreover, this process allows the micronization of the natural extract of β‐carotene, although

purification is not achievable.

Keywords: β‐carotene; supercritical fluids; micronization; carbon dioxide; solubility; tetrahydrofuran

Sofia Antunes September 2007


1. INTRODUCTION Particle size plays an important role in several applications among pharmaceutical, chemical and food

industries. Combustion processes of solid explosives, chromatography and drug administration may be

strongly influenced by particle size and size distribution. Coloring efficiency, catalyst activity and

superconducting properties are also enhanced by reducing particle size [1].

Micronization refers to all the processes that aim to reduce the particle size of solid matter. High

pressure homogenization and jet and ball milling are examples of traditional processes of micronization.

However, some disadvantages have been pointed to these techniques, such as product heterogeneity,

instability and decomposition, due to high pressures and friction practiced. More recently, some particle

size engineering techniques have been developed, such as spray drying and precipitation with

supercritical fluids. This kind of processes allow the incorporation of desirable attributes in the product,

for instance, control of particle size and size distribution, stability and bioavailability. Otherwise, these

processes require less handling, that leads to higher yields and simplifies cleaning and sterilization [2].

Most of the biological products are insoluble in water and, consequently, the use of organic solvents is

essential, giving way to product contamination with undesirable substances. Supercritical fluids are an

important solution to this problem, as the micronization processes that employ this technology allow to

obtain a product that is solvent free. Supercritical antisolvent (SAS) micronization stands for the

precipitation process that makes use of an antisolvent – supercritical carbon dioxide – that mixes with

the solvent that contains the substance to micronize. Thanks to ability of the supercritical fluid to diffuse

rapidly into the organic solvent, supersaturation emerges within a thin gap of time and precipitation

takes place. For the micronization to succeed, two main conditions must be satisfied: first, the solute

must be soluble in the organic solvent and insoluble in the supercritical fluid at the operating conditions

of pressure and temperature; second, the solvent and the antisolvent must be miscible [3]. Many

authors have already studied the SAS micronization of several substances [4‐29]. However, modeling

this process is still to be done, despite the effort of several authors in the study of thermodynamics,

hydrodynamics and kinetics of such systems [12, 25, 20, 21, 29].

β‐carotene is a compound of high molecular weight that belongs to the family of carotenoids and is

highly insoluble in water. It is composed of a hydrocarbon chain (C40H56) containing eleven conjugated

double bounds, which form the chromophore. β‐carotene is easily degradable by light, heat and oxygen,

and its color ranges from yellow to dark red, according to its purity, source and location. The main

natural sources of this carotenoid are several plants, algae and fungi, but the bulk of its production is by

synthetic means.

β‐carotene is one of the most important carotenoids, thanks to its properties as an antioxidant, colorant

and precursor of vitamin A. Its efficiency has already been proved in the prevention of some heart and

eye diseases [30]. Some studies also report its importance against several types of cancer [30, 31], but

there is still some controversy around this matter [31].



The aim of this work is to produce microparticles of β‐carotene, through supercritical antisolvent

micronization. The use of microparticles should enhance β‐carotene dissolution rate in water and the

formulation of aqueous suspensions of this compound might be possible too. Solubility data of β‐

carotene in the supercritical system will also be collected, due to its importance in so many

technological fields of supercritical fluids, such as chromatography, extraction and precipitation.

2. MATERIALS AND METHODS

2.1. Materials

Synthetic β‐carotene was obtained from Sigma (≈95% UV purity) and natural β‐carotene was produced

and provided by BioTrend. Tetrahydrofuran (THF) was the solvent used and was obtained from Merck

(≥99,8% purity stabilized with BHT). Carbon dioxide was supplied by Air Liquide.

2.2. Apparatus

Supercritical antisolvent micronization was performed using an apparatus already described [24]. This is

made of a high pressure vessel made of stainless steel, which is immerged in a thermostatic water bath.

Two streams feed the vessel at its top, the gas stream and the solution stream containing the compound

to micronize. The bottom of the vessel contains a frit that allows the recovery of the dry powder, the

supercritical solution passing through it and being conducted to a microvalve that regulates the flow

rate and, consequently, the system’s pressure. The expansion takes place in an expansion vessel, where

the solvent that still contains a small amount of solute is recovered. The gas phase leaves this vessel and

passes through a flow meter and a gas counter.

All the measures have been made according to a similar procedure. The first step is to pump CO2 until

the desired pressure is reached. Simultaneously, pressure in the expansion vessel is also established. A

previously calculated amount of solvent is then injected into the system, in order to reach its steady

state, and the flow rate at the exit is established. After that, the solution containing the solute is added

and micronization takes place. Finally, CO2 passes through the vessel for about 75 minutes, what should

reduce the solvent concentration in the vessel to 1% of its initial value [10]. Dry powder and liquid

solvent are recovered at the end and put to analyze.

2.3. Analytical methods

The solution of β‐carotene in THF collected in the expansion vessel was analyzed by spectrophotometry

(Shimadzu UV PharmaSpec – 1700). The spectrum was traced and Lambert‐Beer law at 458 nm for this

system was determined before the measures. The absorbance measured allowed to calculate solubilities

of β‐carotene in the supercritical phase and spectra traced permitted to evaluate to some extent the

powder’s and solution’s β‐carotene purity. Degradation of β‐carotene was analyzed by high



performance liquid chromatography (Shimadzu LC.2010CHT with UV/visible detector type and a reverse

phase column Chromolith Performance RP‐18).

The samples of powder were observed by a scanning electron microscope (Phillips XL 30 FEG). Samples

were covered with 250 Å of gold, using a sputter coater (Jeol, model JFC‐1100).

3. RESULTS AND DISCUSSION

3.1. Solubility results

Solubilities of β‐carotene in the supercritical mixture were measured at several conditions of

temperature (35, 40 and 50°C), pressure (75, 100 and 130 bar) and liquid flow rate (0,3, 1, 2 and 3

mL/min). Table 1 presents these results.

Table 1 – Experimental conditions and solubilities measured.

Trial 0 P1 P2 T1 T2 Q1 Q2 Q3

Pressure (bar) 130 100 75 130 130 130 130 130

Temperature (°C) 40 40 40 35 50 40 40 40

xCO2 (mole fraction) 0,956 0,956 0,956 0,956 0,956 0,986 0,916 0,879

xTHF (mole fraction) 0,044 0,044 0,044 0,044 0,044 0,014 0,084 0,121

xβ‐caroteno (mole fraction) ×106 2,29 3,35 1,95 3,25 1,76 0,40 7,34 16,57

Figure 1 shows the mole fraction of β‐carotene as a function of temperature, at constant pressure and

liquid flow rate. It can be observed that solubility decreases as temperature increases and an

exponential relationship has been established, due to the density increase as temperature rises at

constant pressure. Similar conclusions have been reached by other authors [32].

Figure 1 – Mole fraction of β‐carotene in supercritical mixture of CO2/THF as a function of temperature.



Figure 2 shows the solubility of β‐carotene as a function of pressure. No relationship has been

established for two reasons: firstly, at 75 bar system is below its critical point and above this pressure it

is above critical point, so it is not correct to compare, for system behavior is not the same; secondly, it

cannot be assumed that mixture composition is the same in the three trials, for the amount of THF

added to reach steady state was a calculated average, but still there is a big gap between pressures (and

densities), which makes the composition slightly different from trial to trial.

Figure 2 – Mole fraction of β‐carotene in supercritical mixture of CO2/THF as a function of pressure.

The best relationship established in this study is shown in Figure 3 and relates the solubility of β‐

carotene with the mole fraction of THF. The presence of an increasing amount of a co‐solvent (THF)

enhances solubility of β‐carotene, for the affinity of the solute for supercritical mixture rises

exponentially, as can be seen in Figure 3. Another important conclusion taken from the analysis of this

figure is the independency of pressure at constant temperature. It’s easy to see in Figure 3 that the

solubility point at 100 bar correlates with the solubility data at 130 bar. Thus, the difference between

the solubility at these pressures in Figure 2 results from the difference in the mixture composition,

meaning that THF fraction is much more relevant than pressure, at constant temperature.

The effect of co‐solvent was also observed in other studies [33]. For this system, the presence of 5% of

THF means an increase in β‐carotene solubility of about two orders of magnitude, relatively to pure

supercritical CO2 [32].



Figure 3 – Mole fraction of β‐carotene in supercritical mixture of CO2/THF as a function of molar fraction of THF.

3.2. Micronization results

Micronization of synthetic β‐carotene was conducted, in order to evaluate particle size and morphology

under different operating pressures. Micronization took place all around in the precipitation vessel,

starting from its walls, except when the pressure was 75 bar, below mixture critical point, where crystals

formed and accumulated at the bottom of the vessel. These observations are shown in Figure 4.

Macroscopically, crystals appear to be like plain leaves colored dark red with some silver reflection,

which was not verified in the initial product.

(a) (b)Figure 4 – Precipitation vessel after micronization: above critical point (a); below critical point (b).

3.2.1. Product purity

Product purity has been analyzed by HPLC and spectrophotometry and micronized and soluble product

have been compared with raw β‐carotene. Two different wavelengths have been used to detect β‐

carotene isomers (454 nm) and degradation oxides (260 nm), according to Randolph et al. [13].



Through HPLC, it has been detected no degradation or isomerization, as no peak was observed at 260

nm and one only peak was detected at 454 nm, which corresponds to all‐trans‐β‐carotene, by

comparison with raw β‐carotene. Additionally, no significant difference between micronized, soluble

and raw β‐carotene has been observed in their spectra. These results prove that isomerization and

product oxidation don’t happen in this process, as was also verified by Miguel et al. [16]. This can be

explained by two reasons. First, CO2 is inert. Second, even if there is a small amount of oxygen mixed

with CO2, its contact with β‐carotene only happens in the short period of time before micronization

takes place. After that, oxidation reaction should proceed as an heterogeneous reaction, which is much

slower than the homogeneous reaction.

3.2.2. Effect of operating pressure on particle size and morphology

Micronization of β‐carotene has been successful for pressures above 75 bar, at 40°C. A trial has been

made at 65 bar, but no powder has been obtained and the initial β‐carotene was recovered in the liquid

solution in the expansion vessel. Thus, three different samples were analyzed, which were processed at

75, 100 and 130 bar, the former being formed below critical point.

The initial product and processed powders were analyzed by scanning electron microscopy (SEM) and

their size, size distribution and morphology were compared. Observing Figure 5, one can easily confirm

the tridimensional aspect of raw β‐carotene crystals and their narrow particle size distribution. On the

other hand, processed β‐carotene looks completely different in shape and size uniformity.

(a) (b)Figure 5 – Raw β‐carotene (SEM images, zoom 1000× (a) and 3000× (b)).

Figure 6 shows the aspect of processed β‐carotene when the operating pressure is 75 bar (below critical

point). In this figure, it’s particularly evident the almost bidimensional structure of crystals, as well as its

irregular leaf‐like shape, which can prove the aggregation of small particles in further stages of crystal

growth, as proposed by Bristow et al. [34]. Particles formed below critical pressure aggregate more

easily than those formed at higher pressures, which can be explained by their precipitation that occurs

inside THF‐rich droplets, where submicrometric particles start to nucleate, coalesce and fuse during the

growth stage that proceeds the nucleation step. Additionally to particle shape change relatively to raw

β‐carotene, it has been observed that particles formed at 75 bar show a porous structure, which,



despite the increase in particle size, increases the surface area and can, thus, give way to interesting

applications.

(a) (b)Figure 6 – β‐carotene processed at 75 bar (SEM images, zoom 100× (a) and 500× (b)).

For the two trials above 75 bar, the mixture where precipitation occurred was already in a supercritical

state, completely miscible and there exists only one phase inside the precipitation vessel. The

precipitation mechanism is now gas phase nucleation, but one can easily assume that crystal growth has

also occurred, because of the particle size obtained. At 100 bar, particle size distribution is relatively

uniform, as can be observed in Figure 7, and particle size and morphology are quite different from those

obtained at 75 bar. Particles formed at 100 bar show irregular rectangular or triangular shape, smooth

surfaces and they are thin but still tridimensional. Cocero & Ferrero [14] have obtained similar particles

in their study of β‐carotene precipitation.


At 130 bar, the mixture inside the precipitation vessel is also in a supercritical state, but its density is

higher than at 100 bar. Particles obtained in this trial show morphology similar to those obtained at 75

bar: they’re thin and leaf‐like, particle size distribution is relatively large, but their surface is smooth like

those obtained at 100 bar. Some particles also reveal possible aggregation during the process, due to

their shape and size.




Particle size distributions obtained in different trials are shown in Figure 9. It’s easy to verify again that

particles obtained at 75 and 130 bar have similar particle size distributions. Smaller particles were

obtained at 100 bar, but particle size distribution was less narrow.

Figure 9 – Particle size distributions obtained at different pressures.

Above the critical point, there is a clear tendency for the increase of particle size with the increase of

pressure (14 to 78 μm when pressure rises from 100 to 130 bar). Similar tendencies have been verified

by other authors [16, 25, 27, 28, 29, 34]. The reason why this happens is the increase in density that

comes from the increase of pressure. At higher densities, solubility of β‐carotene is also higher and

supersaturation is lower. As particle size increases when supersaturation decreases, particle size

increases with pressure. Through Peng‐Robinson equation of state, it is possible to estimate densities of

the mixture where precipitation happens, at different pressures. Representation of average particle size

as a function of density is in Figure 10, which shows a strong relationship between particle size and

density of the mixture. Additionally, this relationship is extended to all the experimental points,

including that one below critical point.



Figure 10 – Average particle size as a function of density of the mixture where precipitation occurs.

Former affirmations about the similarity between particles obtained at 75 and 130 bar can now be

confirmed by the comparable densities of mixtures where micronization takes place. Higher densities

provoke not only lower supersaturation but also more aggregation, leading to bigger particles.

The influence of pressure on particle size below critical point has not been studied. In this case, there

are two unmixable phases and micronization occurs in the liquid THF‐rich phase by a mechanism of

volumetric expansion, when CO2 dissolves into THF. The higher the pressure, the larger the volumetric

expansion of THF‐rich droplets and this fact leads to higher supersaturation and formation of smaller

particles. Thus, if the micronization of β‐carotene at 65 bar had been successful, particles obtained

would be even bigger than those obtained at 75 bar. This thesis is also supported by Figure 10, because,

below critical point, higher pressure means lower density of droplets, and it was confirmed by other

authors [4, 8, 19].

3.2.3. Precipitation of a natural extract of β‐carotene

In order to evaluate micronization as a purification process, a trial has been run using a natural extract

of β‐carotene. The resulting micronized product was analyzed by HPLC and compared to the liquid.

The micronized crystals of the extract look like the micronized synthetic β‐carotene, but their color is

orange, instead of red. Due to the nature of the extract – produced by microorganisms – one can

conclude with some conviction that impurities present in the extract are mostly low molecular weight

compounds, typically products of microbial metabolism, that do not absorb at the same wavelength β‐

carotene does. Otherwise, it was not possible to quantify β‐carotene present in this extract, because its

real composition is unknown and there may also be some carotenoids or other impurities absorbing at

β‐carotene wavelength. Thus, Lambert‐Beer law determined for synthetic β‐carotene is not appropriate.

HPLC analyses (not shown) proved that liquid and micronized products obtained in this trial are quite

similar, meaning that purification didn’t occur. Spectra analyses don’t allow to conclude anything.



4. CONCLUSION Supercritical antisolvent micronization of synthetic and natural β‐carotene has been successful in this

work. It has been shown that it is possible to micronize this carotenoid under experimental conditions

below or above the mixture critical point and the minimal pressure for the precipitation to occur equals

75 bar.

Depending on the pressure, it was possible to obtain microparticles ranging from 14 to 100 μm.

Morphology and crystal aggregation also depends on pressure. It has also been established a linear

relationship between average particle size and the density of the mixture where precipitation occurs,

meaning that it’s possible to control particle size by manipulating experimental conditions.

A solubility study has also given way to some interesting data about this system’s thermodynamics. Two

main results show the exponential decrease of β‐carotene solubility in the supercritical mixture CO2/THF

with the temperature increase, as a consequence of the decrease of density, and the exponential

increase of solubility with the increase of THF molar fraction, independently of pressure, due to β‐

carotene affinity for this organic solvent. Additionally, the presence of a co‐solvent (THF) enhances β‐

carotene solubility by a factor of about two orders of magnitude, relatively to pure CO2.

This micronization process also allowed the precipitation of a biological extract of β‐carotene, but no

purification was detected. This fact shows that this process can be applied in food and drug industries,

for processing biological products, because carbon dioxide is inert, non toxic and low cost and the final

product is free of organic solvents. However, experimental conditions must be optimized, in order to

obtain some purification.

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