4. Materials and Methods
4.1. Powder Characterization
4.1.1. Scanning Electron Microscopy (SEM)
SEM images of the powder as well as of the granules were taken using an ESEM Philips XL 30 (Philips, Eindhoven, Netherlands) at a voltage of 10 kV after sputtering with gold.
4.1.2. Density
True density of the powders and granules, in triplicate, was measured by AccuPyc 1330 V2.02 (Micromeritics Instrument Corporation, Norcross, USA). A known weight of the samples was placed into the sample cell. Helium was used as a measuring gas and values were expressed as the mean of five parallel measurements.
Bulk and tap density of powder mixtures and granules were determined using an apparatus Type STAV 2003, (Engelsmann AG, Ludwigshafen, Germany). Measurements were done according to the following method: 50 g of powders and granules gradually were filled in a 250 ml glass cylinder. A volume (V0) at the beginning was noted and bulk density ρbulk (g/cm3) was calculated. After that, the cylinder was tapped for 1250 times, and using this volume (V1250) tap density ρtapped
Materials and Methods
(g/cm3) was calculated. Bulk and tap density were used to calculate Carr and
Hausner index, see equation 13 and equation 14, respectively.
bulk bulk tapp
CI
ρ
ρ
ρ
−
=
(13) bulk tappHI
ρ
ρ
=
(14) Where: CI – Carr index [%]ρbulk – bulk density [g/cm3] ρtapp – tapped density [g/cm3] HI – Hausner index
4.1.3. Moisture content
Moisture content of the materials was measured by Karl fisher titration (Apparatus Karl fisher Titrando, 836 Methrohm, UK). The measurements were carried out with 0.2 g substance according Ph. Eur.
4.1.4. Particle Size Distribution
Particle size and its distribution in volume for all samples were measured by laser diffraction (Malvern Mastersizer 2000, Scirocco 2000). For all samples dry measurement method was done. An adequate amount of each powder was introduced as dispersion produced by air pressure. According to the material properties different pressures were used: for Theophylline anhydrate powder pressure of 0.5 bars, for Theophylline anhydrate powder pressure of 2.0 bars, for Theophylline monohydrate pressure of 2.0 bars and for Cellulose microcrystalline pressure of 2.0 bars. Each sample was measured in triplicate.
4.1.5. Specific Surface Area
Specific surface area was determined by the multipoint (5 points) BET method using Surface area and pore size analyzer (Quantachrome NOVA 2000 E, Florida, USA). Accurately weighed samples were degassed under vacuum at room temperature for 24 h, and measurements were made using nitrogen as the adsorbate and helium as the carrier gas. The amount of gas was measured by volumetric flow procedure. The data are treated according to the Brunauer, Emmet and Teller (BET) adsorption isotherm equation 15 47: mC
V
P
P
VmC
C
P
P
V
1
1
1
1
0 0+
−
=
⎥
⎦
⎤
⎢
⎣
⎡
⎟
⎠
⎞
⎜
⎝
⎛
−
(15) Where:P – partial pressure of adsorbate [Pa]
P0 – saturated of adsorbate at experimental temperature [Pa] V – volume of gas adsorbated at pressure [cm3]
Vm – volume of gas adsorbed in monolayer [cm3]
C – dimensionless constant that is related to the enthalpy of adsorption of the adsorbate gas on the powder sample
The volume of gas absorbed at monolayer Vm was obtained from the slop and
intercept of BET plot according toequation 16:
Intercept Slope
Vm
+
= 1 (16)
The total surface area of the sample is calculated using equation 17:
M
A
VmN
St
=
a cs (17) Where:St – total surface area Na – Avogadro’s number
Materials and Methods Acs – cross-sectional area of the adsorbate
The specific surface area S is finally obtained by dividing total surface area by the sample mass equation 18:
W
S
S
=
t (18)4.1.6. Solubility
Solubility of THAP, THAFP and THMO was determined using the shake flask method at speed of shaking 35 rpm. To assure work under sink conditions, saturated solutions of the model drugs were prepared at a temperature at 25˚C. The kinetic of the solubility was monitored by sampling at certain time interval to check transformation of anhydrate to monohydrate in order to monitor differences in solubility of anhydrate and monohydrate form of Theophylline. Aliquots of the solutions were withdrawn and after filtration and appropriate dilution drug content was monitored by UV at 272 nm. The measurement had 72 h equilibration time.
4.1.7. Contact Angle
For the measurement of contact angle the sorption method was used. Measurement was done by Tensiometer K10 (Krüss GmbH, Hamburg, Germany) in combination
with Krüss LabDesktTM software (Version 3.0.1.2509, Krüss GmbH, Hamburg,
Germany). The constant weight and volume of the powder were placed in a glass cell with a porous glass base. The measurement of every sample was done in triplicate. The glass cell was fixed to electronic balance integrated in the tensiometer, and brought in contact with vessel containing the test-liquid. Measuring the increase in weight as a function of time and applying the modified Washburn equation (19) allows calculation of the contact angle of the material 48.
η
θ
γ
cos
c
t
h =
(19) Where:t – time [s]
c – constant
γ – surface tension of the liquid [mN/m]
θ – contact angle
η – viscosity of the liquid [mPa s]
Due to the fact that measurement is based on the increasing in mass of sample as function of time, equation (19) can be modified to equation (20):
η
θ
γ
ρ
2cos
2c
t
m =
(20) Where:m – mass of adsorbed liquid [g] ρ – density of the liquid [g/cm3]
To determine constant c, measurement with a liquid (n-hexane) that completely wets the sample was carried out, and this constant was entered in to the Washburn equation. For all samples distilled water was used as test liquid.
4.1.7. X – Ray Diffractometry
This method is widely used for the identification of solids phases. The X - ray powder pattern of every crystalline form of compound is unique making this technique particularly suited for the identification of different polymorphic forms of the material. The samples of powder and granules were analyzed by X-Ray diffractometer (Model D 5005 Siemens) with Cu–Kα radiation (45 kV x 40 mA). The instrument was operated in a step scan mode and in increment of 0.01°2θ. The angular range was 5 to 40° 2θ and counts were accumulated for 10 s at each step.
4.1.8. Differential Scanning Calorimetry (DSC)
DSC is a thermal analysis in which the properties of the material can be defined in function of external applied temperature. This method can be used to determine
Materials and Methods some very important characteristics of the material: melting and boiling point, glass transition, vaporization, solid–solid phase transformation, crystallization decomposition, etc 49.
DSC measurements of THAP, THAFP and THMO powder were performed (Pyris Diamond 1, Perkin Elmer, Switzerland) in order to characterize and examine polymorphic form of the materials.
Approximately 4 mg of the sample were weighed into 30µl aluminum pan with hole and heated in the DSC from 30˚C to 300˚C. Heating rate was set to 10˚C/min under nitrogen purge.