Aerosol Science 35 (2004) 509 527

www.elsevier.com/locate/jaerosci

Sizing of powders in inhalers with an Aerosizer J according to

a mixed experimental factorial design
P.J. Mendesa; b , A. Raposoa; b , J.M.M. Sousab , J.F. Pintoa;
a

Dept. de Tecnologia Farmaceutica, Faculdade de Farmacia da Universidade de Lisboa,

Av. Prof. Gama Pinto 1649-003 Lisboa, Portugal
b
Dept. de Engenharia Mecanica, Instituto Superior Tecnico, Universidade Tecnica de Lisboa,
Av. Rovisco Pais 1, 1049-001 Lisboa, Portugal
Received 18 March 2003; received in revised form 18 September 2003; accepted 3 October 2003

Abstract
In the present work, the Aerosizer J with the Aerodisperser J is used to assess the size and size distribution
of two common dry powder inhalers: Ventilan RotacapsTM (salbutamol sulfate) and Bricanyl Turbohaler TM
(terbutaline sulfate), according to a mixed factorial design. The di7erent parameters of the equipment (voltage
applied to the detector, shear force, deagglomeration, feed rate and pin vibration) were the factors considered
in the design, set to di7erent levels. Altogether, a full 41 31 23 factorial was considered and the results
of the experiments analyzed according to the Montgomery technique. Results have shown that samples with
a single population of particles were less a7ected by the changes in the parameters, whereas for mixtures
incorporating di7erent populations results were a7ected upon change of the levels of the factors. The voltage
at the detector was the most important factor, followed by the shear force. By contrast, pin vibration and feed
rate were less signi9cant. The interactions between variables were also responsible for signi9cant changes in
the results and should not be neglected throughout the measurements.
? 2003 Elsevier Ltd. All rights reserved.
Keywords: Aerosizer; Factorial design; Montgomery technique; Dry-powder inhaler; Salbutamol sulfate; Terbutaline sulfate

1. Introduction
In recent years, a great development on aerosols and related technology has been observed and
the dispersion of 9ne powders or liquids found application in the administration of drugs to humans.

Corresponding author. Tel.: +351-217-946-434; fax: +351-217-937-703.

E-mail address: jfpinto@7.ul.pt (J.F. Pinto).

0021-8502/$ - see front matter ? 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jaerosci.2003.10.005

510

P.J. Mendes et al. / Aerosol Science 35 (2004) 509 527

Aerosols have been used in the treatment of diseases, namely those a7ecting the respiratory tract.
The main advantages of the respiratory route, as compared to the oral one, are the lower dose of
drug required to achieve the same therapeutic e7ect, in turn leading to decreased side e7ects, and
an earlier onset of the drugs e7ect (Wierlik, Diepenmaat, & Damhuis, 2002).
Dry powder inhalers (DPIs) play a major role in aerosol therapy because they are chloroFuorocarbon (CFC) free (Wierlik et al., 2002; Dunbar, 2002), less dependent on patients coordination on
inhalation, easy to formulate and less a7ected by environmental changes (e.g. temperature) (Ramon
et al., 2000). Ramon et al., 2000 provided that their components have adequate properties (Hickey,
2002). However, the size of the particles and the way they interact are critical for a proper deposition
in the alveoli (Gonda, 1992), and sizing of the particles is, therefore, of paramount importance for
a good therapeutic e7ect.
There are many in vitro methods for sizing DPI particles. Examples of compendial methods are
the cascade impactor (e.g. Andersen multi-stage impactor), and the multistage liquid impinger (e.g.
twin impinger) (US Pharmacopoeia, 26). Unfortunately, these methods are time consuming (up to
1 h per measurement) by contrast to other analyzers which give measurements in minutes (Peters,
Vanderpool, & Wiener, 2001). Examples of such equipment are the time-of-Fight (TOF) based
aerodynamic particle size analyzers, such as the Aerosizer J , the Aerodynamic Particle Sizer J spectrophotometer or the E-Spart J (Mitchell & Nagel, 1999). These analyzers are often used in the
assessment of medicinal aerosols, either dry powder or pressurized metered dose inhalers (pMDIs).
The particle size distribution spectrum obtained reFects the di7erences in the aerodynamic diameters
of particles present in the aerosol. For instance, a DPI containing lactose (larger particles), as the
drug (salbutamol sulfate, smaller particles) carrier, exhibits a spectrum with two peaks that could
be cautiously related to the individual symmetrical spectra produced for both lactose and salbutamol
sulfate particles (Srichana, Brain, Marriott, & Martin, 2000). However, care must be observed when
measurements are carried out, since when suspensions of pMDIs are considered, the measured values
di7er from those obtained by other techniques (Mitchell, Nagel, & Archer, 1999). This reFects the
vulnerability of the method when disperse systems are considered, with some preferential removal
of larger mass median aerodynamic diameter (MMAD) particles, due to the evaporation of solvent
and/or inertial gravitational deposition (Nagel, Wiersema, Bates, & Mitchell, 2002), thus leading
to di7erent results. Moreover, when DPIs are considered, the ability of the equipment to disperse
particles is highly dependent on the properties of the materials (Laitinen & Juppo, 2003). In a less
conventional application of this technique, the use of a third laser beam has been suggested in order
to avoid the particle density dependency of the method (Oskouie, Noll, & Wang, 2003). The use of
adequate mathematical models, that allow the calculation of the particles lumped density and shape
factors (Oskouie, Noll, & Wang, 2002) have also been suggested.
The technique described above has several advantages as compared to others: (a) the large number
of particles counted per second, (b) the easy operating procedure and maintenance and (c) the small
sample size required. However, some limitations have been pointed out (Mitchell & Nagel, 1996)
such as: (a) the particles are detected on a one at a time basis, therefore some particles may not
be detected particularly at high feed rates; (b) the equipment assumes that all the particles are
spherical and (c) the equipment is unable to distinguish between particles belonging to di7erent
populations (e.g. drug and excipients), so all the particles are detected and treated identically and
(d) the density of the sample must be known. The 9rst limitation a7ects seriously the counting
eKciency and can create false peaks. Since the equipment determines the aerodynamic diameter and

P.J. Mendes et al. / Aerosol Science 35 (2004) 509 527

511

then calculates the geometric diameter using the density of the material, an incorrect value for the
density of the material produces wrong values for the geometric diameters, without a7ecting the
aerodynamic diameter because the Aerosizer J measures the TOF of individual particles which is
dependent on their mass. Moreover, non-spherical particles present di7erent velocities of Fight from
spherical particles due to di7erences on the drag coeKcients. For the above-mentioned limitations,
special care should be taken in the analysis of the results since a DPI can have more than two
di7erent populations of particles with di7erent drag forces, densities and shapes, leading to inaccurate
measurements, a drawback shared with other techniques. It follows that the Aerosizer J provides more
accurate results for single populations of particles rather than for mixtures of di7erent populations of
particles, i.e., populations made of di7erent chemical identities, and therefore showing multimodal
distributions. The equipment may also underestimate the aerodynamic sizes of airborne particles from
both powder and pressurized inhalers, often requiring a comparison with other measuring techniques
(Nagel et al., 2002).
The aim of the present work was the assessment of the performance of the Aerodisperser J
Aerosizer J on the characterization of two DPIs when all the operating parameters were considered
in a factorial experimental design. This design, selected on the ground of its robustness and because
the variables (equipment settings) can be considered simultaneously, allows for the evaluation of the
possible interactions between variables after establishing the relative importance of each parameter,
by the Montgomery method (Montgomery, 1996).
2. Material and methods
In this work, VentilanTM RotacapsTM (GlaxoWellcome) and BricanylJ Turbohaler J (AstraZeneca)
were used as model DPIs, whose particle size a7ect their therapeutic eKcacy. The 9rst is presented
to patients as a hard gelatin capsule containing 200 g of salbutamol sulfate (in 25 mg of powder),
whereas the second contains 500 g of terbutaline sulfate per blister. For calibration purposes, two
sets of spheres were considered: one of nylon (mean diameter standard deviation of 5 1:5 m,
Dantec Measurement Technology GmbH, Germany), and another of glass (mean diameter standard
deviation of 10:0 1:0 m, Duke Corporation, Palo Alto, CA, USA).
In these experiments, the two types of DPIs were analyzed by using an Aerodisperser J Aerosizer J
(TSI, GmbH, Aachen, Germany). BrieFy, this equipment operates by suspending the particles in the
air, using a disperser, a Fuidizer and a deagglomerating device (Aerodisperser J ), before this air
Fow is expanded into a vacuum. This vacuum draws the particles through a Fow nozzle, producing a precisely controlled acceleration to a high-speed (subsonic) aerosol jet (Allen, 1990) in the
Aerosizer J . Fig. 1 presents the assembly considered in the present study. The velocity of the air
remains constant in the measurement region but the particles accelerate at di7erent rates, inversely
related to their aerodynamic size (Barber, 1993). Two laser beams (at a known distance between
them) placed in the nozzles exit allow the measurement of the time required for the particles to
travel between them and, thus, the velocity. The scattering of the beams by the particles induces the
emission of an electrical signal by the detector, made of photomultiplier diodes, starting/stopping a
clock. The particles aerodynamic diameter is calculated from the time elapsed between the two signals (Laitinen & Juppo, 2003). Traveling times are in the order of hundreds of nanoseconds for small
particles and thousands of nanoseconds for larger particles. The measurement of the aerodynamic

512

P.J. Mendes et al. / Aerosol Science 35 (2004) 509 527

Fig. 1. Schematic representation of the Aerodisperser J connected to the Aerosizer J .

diameter of particles is automatically compensated for changes in shape, surface irregularities, mass
and refractive index. Visual inspection of the samples was made by scanning electron microscopy
(Phillips Analytical, UK).
Prior to the analysis of the DPIs, according to a factorial experimental design, the equipment was
calibrated with both types of spheres and validated for reproducibility and e7ectiveness of counting.
These characteristics were assessed by the run time and presentation of the data according to size
distribution by number and by volume, as a function of the aerodynamic diameter. To check the
counting eKciency of the Aerosizer J , lactose monohydrate (Granulac 200, Meggle, Germany) and
microcrystalline cellulose (Avicel PH-101, FMC Corp., USA) were used as received, in the same
quantity (6 mg) as that collected from the DPIs. The distribution of the populations was characterized
by the mode, or, if otherwise referred in the text, by another measure of central tendency.
2.1. Experimental design
Experiments were carried out according to a mixed factorial design and the results were analyzed
by the method described by Montgomery (1996). BrieFy, this statistical technique allows the transformation of an n-level factor (with n 2) into a k 2 factorial design (k factors at two levels each),
and the assessment of the e7ect of each factor and their interactions on the results. In the present
investigation, a 41 31 23 mixed factorial design was considered, where the exponents represent the
equipment variables and the bases stand for the levels of each variable in the experiments (Table 1).
According to this method, the variables with three or four levels can be converted into two variables

P.J. Mendes et al. / Aerosol Science 35 (2004) 509 527

513

Table 1
Equipment variables and levels considered in the experiments
Variables

Levels

Photomultiplier voltage (PMV) (V)

Shear force (SF) (Psi)
Deagglomeration (D)
Feed rate 103 (FR) (counts/s)
Pin vibration (PV)

850 1100
0.5 1.5 3.0 4.0
0 1
15 10
0 1

with two levels each (22 ) (Myers & Montgomery, 1995). Therefore, for a variable with two levels,
this technique indicates the e7ect of that variable on the results when it changes from the lowest to
the highest level. For a variable with three levels, it indicates the average e7ect of that variable for
each level (levels 1 to 2 or levels 2 to 3). Thus, when the variable changes from the lowest to the
highest level, the magnitude of the e7ect doubles the value found for the intermediate variations.
With factors at four levels, Montgomerys technique produces three results: the 9rst corresponds to
the average e7ect when the factor changes from levels 1 to 2 or 3 to 4, the second corresponds to
the average e7ect of the change from levels 1 to 3 or 2 to 4 and the third reFects the tendency of
the change. Consequently, the total e7ect is the sum of the 9rst and second results.
The di7erent magnitudes of the e7ects observed were compared by an analysis of variance
(ANOVA) of the factorial design with more than two levels (Armstrong & James, 1996), and
the levels of signi9cance assessed by an F-test. The smallest values of the mean squares were averaged and considered as the experimental error in experiments. Appendix A presents a more detailed
explanation of the method.
3. Results and discussion
3.1. Preliminary experiments
At 9rst, the good performance of the measuring equipment was ascertained with the calibration
spheres. The mean geometric diameters by number measured with the Aerodisperser J Aerosizer J
con9guration were 4.97 and 10:45 m for the spheres of nylon and glass, respectively. The di7erences
to the nominal values of 5.00 and 10:00 m were, therefore, 0:03 m (i.e., 0.6%) and 0:45 m (i.e.,
4.5%), respectively. These di7erences were deemed acceptable and indicative of the ability of the
Aerosizer J to measure the diameter of the particles accurately. Moreover, as shown in Fig. 2,
the narrow distribution of the geometric diameters by number is identical to that reported by the
manufacturers for the calibration spheres.
In a separate set of experiments, it was observed that the run time of the equipment was proportional to the number of particles counted and dependent on the feed rate: the higher the feed
rate, the shorter the run time (Fig. 3). A change from the largest to the lower feed rate decreased
the number of counts per second from 1950 (higher) to 1460 (medium) and 590 (lower). The
e7ect of the number of particles counted on the diameter measured has been studied by using the

514

P.J. Mendes et al. / Aerosol Science 35 (2004) 509 527

Fig. 2. Distribution by number of counts for the calibration spheres: () spheres of nylon, (- - - - - -) spheres of glass.

Fig. 3. Relationship between the run time and the number of particles counted as a function of feed rate: () low, ( )
med, () high.

Fig. 4. Relationship between the aerodynamic diameters (mode) of particles and the number of particles counted: ()
distribution by number, ( ) distribution by volume.

pharmaceutical VentilanTM RotacapsTM (salbutamol sulfate). Once the parameters of the equipment
have been set, it was realized that for a number of counted particles larger than 120,000, the geometric aerodynamic diameter (modes shown) was more uniformly distributed (Fig. 4). This 9gure
takes 54 runs into consideration and shows the results for the aerodynamic diameters obtained in

P.J. Mendes et al. / Aerosol Science 35 (2004) 509 527

515

Fig. 5. Size distributions for microcrystalline cellulose samples detected at 850 V (a) and 1100 V (b): () 1 mg,
( ) 4 mg, () 8 mg, () 16 mg, () 24 mg.

distributions by number and by volume, and the number of counted particles in each run. As anticipated, as the sample size increases (reFected by the number of counts), the precision of the
measurement also increases (reFected by a decrease of the standard deviation). Above 120,000 particles, the dispersion of the mode values was small enough for the measurement to be considered
acceptable. For samples of other materials, a di7erent number of counts was required (results not
shown) for the readings to become reproducible. For instance, when microcrystalline cellulose was
used, a minimum sample quantity of 8 and 4 mg were required when results were detected at 850
and 1100 V, respectively (Fig. 5).
As previously discussed, when DPIs are considered, one should be aware that more than one
population of particles may be present in the sample. There is, usually, one population of drug
particles (small in size) and another of the carrier (larger particles). The proportion between the two
populations has an impact on the therapeutic e7ect of the drug and, consequently, it is important
to characterize them adequately. In this part of the study, carried out using VentilanTM RotacapsTM
(containing particles of both salbutamol sulfate and lactose), the evaluation of both populations was
possible by considering the particle size distribution both by number and by volume. It was possible
to estimate the approximate geometric diameters (modes) of both components in the formulation,
due to the large di7erence in size between the two populations of particles. In short, the averages of
the modes found in 54 runs were calculated and the values of 1:48 m (by number) and 68:9 m
(by volume) were estimated for the drug and carrier particles, respectively. The validity of the

516

P.J. Mendes et al. / Aerosol Science 35 (2004) 509 527

Fig. 6. Relationship between the diameter (mode) expressed by volume and by number of counts.

procedure is supported on the ground that di7erent measurements have shown that the diameters by
number and by volume are not related (Fig. 6). In fact, in this 9gure, a scattered distribution of
the readings is observed, with no relationship between the geometric diameters by number and by
volume, as expected.
Thornburg, Cooper, and Leith (1998) observed that the counting eKciency (ratio of the number
of particles detected to the number of particles passing through the detector) of the Aerosizer J is
variable. The same authors (Thornburg et al., 1998) stated that counting eKciencies below 100%
or, variations dependent upon particle size, would give inaccurate size distributions. Furthermore,
factors, such as the concentration of the sample, the model of the equipment, the voltage at the
detector, the measurement of particles with diameters below 7 m or acceptance of counting rates
above 20,000 particles per second, were also described as a7ecting the counting eKciency of the
equipment. These observations were con9rmed in the present work, when particles of lactose were
considered. In fact, for samples with the same mass and equivalent number of counts, the size
distribution of the particles varied at small number of counts.
After performing a few tests with VentilanTM RotacapsTM , it was noticed that the size distribution
of the particles was not constant between runs performed under the same conditions, even for samples taken from the same capsule, although the modes of the distributions (peaks) remained constant.
This fact shows that the counting eKciency changes with the size of the particles in di7erent runs
and samples. Furthermore, we noticed that the values read in the equipment were discontinuous,
i.e., identical values were registered several times, whereas in between values were never found.
This happens because the Aerosizer J collects the TOF of the particles in 2048 linearly spaced time
channels, which are converted by the software into 500 logarithmically spaced sized bins (Amherst
Process Instruments). Consequently, the equipment presents the rounded value in the bin instead
of showing the true measurement. This e7ect of rounding the results is diKcult to assess because the software of the equipment shows the results already in bins and, therefore, that some
lack of consistency in the measurements is expected. Results are presented in a more discrete way
than the measurements, and consequently, some lack of accuracy, reproducibility or sensitivity can
be anticipated.

P.J. Mendes et al. / Aerosol Science 35 (2004) 509 527

517

Fig. 7. E7ect of the voltage in the detector over the size of the particles. Insets show scanning electron photomicrographs
of BricanylJ Turbohaler J and VentilanTM RotocapsTM , () BricanylJ TurbohalerJ (1100 V), (- - -- - -) BricanylJ
TurbohalerJ (850 V), ( ) VentilanTM RotacapsTM (1100 V), (- - - - - -) VentilanTM RotacapsTM (850 V).

Results present in both Figs. 4 and 6 stress these observations since the results are organized
according to horizontal and vertical lines, rather than a scattered distribution. This problem may a7ect
the analysis of the samples, especially of those populations with large particles. When the results
are represented as percentages this di7erence is less signi9cant, but di7erences in the aerodynamic
and geometric diameters can still be observed, even though the samples have been prepared under
the same conditions.
3.2. Factorial design
The performance of the Aerosizer J changed with the voltage applied to the detector, as observed
when the voltage was changed between measurements, both for the calibration spheres and the
aerosols. A narrower distribution curve was obtained when the voltage was increased (Fig. 7), and
in all cases, the number of counted particles was higher for the highest voltage, as observed already
by Thornburg et al. (1998). In close agreement with the work of Mitchell and Nagel (1996), di7erent
sensitivities of the detector did not a7ect the mean geometric diameter for the calibration spheres
(nylon) with a very narrow size distribution as opposed to the results observed for the inhalers (large
sizes distributions).
In the case of BricanylJ Turbohaler J , the distributions had identical shapes for the two recommended values of the photo-multiplier voltage (850 and 1100 V), although di7erent geometric mean
diameters and smaller base lines of the peaks were observed for the highest voltage (Fig. 7, open

518

P.J. Mendes et al. / Aerosol Science 35 (2004) 509 527

circles). Like the calibration spheres, this DPI presents only one population of particles (Fig. 7 and
micrograph associated with curves), but a wider particle size distribution. In both cases, only one
peak was observed. When the sensitivity of the equipment was increased, smaller particles were
detected more easily, and, thus, a shift of the distribution of the curve to smaller sizes was observed
(mean diameters di7ered by 34%, whereas the mode remained constant). However, when the shear
force was changed from peak to low, the aerodynamic diameters (modes) increased from 0.76
to 0:79 m (0.3% increase). As this product does not contain any excipient, it can be assumed that
the shear force only promotes a minor separation of the particles of terbutaline, the only component
in the formulation (Fig. 7, open circles and micrograph).
When VentilanTM RotacapsTM was considered, the aerodynamic diameters expressed by the number
of counts originated three peaks within the distribution. This type of distribution is due to the presence
of several groups of particles present in the product (Fig. 7, open squares and micrograph). For this
product, a higher voltage led to a shift of the peaks towards smaller sizes (Fig. 7). On the other
hand, when the results (aerodynamic diameter) were expressed by volume, only a slight shift of the
results towards smaller sizes was observed, suggesting a higher sensitivity of the detector to smaller
particles, as mentioned previously. For VentilanTM RotacapsTM , the di7erences between the means
(aerodynamic diameter) of the particles observed for high and low voltages on the detector were
0:52 m (33%) and 23:02 m (33%) for number and volume distributions, respectively (Table 2).
When all the results of the factorial design were considered, it was possible to express them in
terms of modes of the aerodynamic diameters, either by number or by volume. Results of the e7ects
are shown both in Tables 2 and 3. Table 2 presents the variations on the distribution by number and
by volume of the aerodynamic diameters observed for the di7erent samples of the pharmaceuticals
considered. The upper part refers to the main e7ects or individual e7ects when a single variable
was changed at a time. Therefore, it is possible to assess the impact of each operational parameter
(variable) over the size distribution of samples. Table 3 presents the results of the ANOVA performed
on the experimental data emphasizing their degree of signi9cance as reFected by the F-values. An
increase on the voltage of the photomultiplier promoted a decrease on the aerodynamic diameter
(mode) of 0.52 and 23:02 m, respectively by number and by volume (Table 2). This variable is
the most signi9cant one with a value of p 0:001 (Table 3). The sensitivity of the detector a7ects
the number of counts, and, thus, the size of the sample. In order to obtain consistent and reproducible
results, the magnitude of this parameter must be carefully chosen and, thus, special care should be
taken on the selection of the photo-multiplier voltage.
When the shear force applied (Fig. 8a and b) was increased from low to peak, an increase on
the measured diameter (by number) was observed. In fact, when two levels were considered (low
to high and medium to peak), the e7ect was signi9cant (SF2 , Table 2 and p 0:01, Table 3).
This increasing tendency is a7ected only in a few cases by an increase on the shear force from
high (3 Psi) to peak (4 Psi), resulting in measured diameters that were equal or smaller (Fig. 9a
and b). Results in Table 2 do not show this linear tendency, but it should be borne in mind that
the di7erence between levels low and medium and high and peak is only 1 Psi, whereas the
di7erence between medium and high is 1:5 Psi. On the other hand, an increase on the shear force
(Fig. 8a and b) often leads to a decrease on the measured diameter for larger particles (upper parts
of the graphs in Figs. 8 and 9). Although the observation is not signi9cant (Table 3, distribution by
volume) and the results do not show a particular tendency (Fig. 9a and b), it is possible to suggest
that by increasing the shear force, the separation between the particles was promoted, as mentioned

P.J. Mendes et al. / Aerosol Science 35 (2004) 509 527

519

Table 2
Results of the main e7ects and interactions from the calculations, carried out according to the Montgomery technique
Variables and interactions

Distribution by number (m)

Distribution by volume (m)

Main e9ects
Photomultiplier voltage (PMV)

0.52

23.0

Shear force
Total (SFtotal )
1 level (SF1 )
2 levels (SF2 )
Shape of tendency line

0.17
0.05
0.12
0.01

1.15
0.49
0.66
0.52

Deagglomeration (D)

0.00

1.65

Feed rate
Total (FR total )
1 level (FR 1 )
Shape of tendency line

0.02
0.01
0.00

1.92
0.96
3.51

Pin vibration (PV)

0.02

0.23

Binary interactions
PMVSFtotal
PMVD
PMVFR total
PMVPV
SFtotal D
SFtotal FR total
SFtotal PV
DFR total
DPV
FR total PV

0.12
0.03
0.01
0.01
0.03
0.03
0.02
0.02
0.01
0.01

0.26
0.19
0.06
0.05
0.58
0.90
0.20
1.06
0.66
1.06

elsewhere (Houzego, 2002). Because larger particles present higher contact areas, the adhesion and
cohesion between them are larger too. Consequently, the e7ect of the shear force is observed mainly
on the smaller particles of the drug with a shift of the peak observed in the distribution by number to
larger sizes. This variable is particularly important for samples containing more than one population
but, even for a single population, the shear force changes the values measured, as a consequence of
the additional deagglomeration produced.
Regarding the deagglomeration, when this parameter is set to high, the particles are transported
from the sample cup to the dispersion pin at a higher velocity. Hence, a stronger impact on the pin
occurs generating a larger force to separate the particles (Houzego, 2002). It can be assumed that
the same sort of explanation given for the e7ect of the shear force applies to the deagglomeration
variable. For smaller particles, a change from normal to high in deagglomeration failed to produce
a major di7erence between the results (Table 2), whereas for larger particles a reduction on the
measured sizes was observed (Fig. 8a and b), although not statistically signi9cant (Table 3).

520

P.J. Mendes et al. / Aerosol Science 35 (2004) 509 527

Table 3
Results of the ANOVA for the di7erent experiments, according to the Montgomery technique
Degrees of
freedom

Distribution by number

Distribution by volume

Sum of
squares

Mean
squares

Sum of
squares

Mean
squares

Variables
Photomultiplier voltage (PMV)
Shear force (SF)
Deagglomeration (D)
Feed rate (FR)
Pin vibration (PV)

1
3
1
2
1

6.82
0.53
0.00
0.01
0.01

6.82
0.17
0.00
0.00
0.01

9443.0
245.0
0.00
4.85
11.08

11806.4
41.21
26.15
323.0
24.63

11806.4
13.73
26.14
161.5
24.63

1793.5
2.09
3.97
24.54
3.74

Binary interactions
PMVSF
PMVD
PMVFR
PMVPV
SFD
SFFR
SFPV
DFR
DPV
FRPV

3
1
2
1
3
6
3
2
1
2

0.13
0.03
0.02
0.00
0.07
0.05
0.09
0.01
0.00
0.03

0.04
0.03
0.01
0.00
0.02
0.01
0.03
0.00
0.00
0.01

52.15
13.39
136.1
3.95
119.8
242.8
9.87
88.41
18.62
80.80

17.38
13.39
68.08
3.94
39.92
40.47
3.28
44.20
18.61
40.39

59.08
42.92
15.23
5.54
31.85
12.00
39.23
3.46
20.08

2.64
2.04
10.34
6.07
6.15
6.72
2.83
6.14

Blank spaces represent results associated with the experimental error.

p 0:05,

p 0:01,

p 0:001.

The feed rate directly inFuences the number of particles counted by unit of time. The
Aerodisperser J adjusts automatically the air jet to satisfy the value selected for the aforementioned
parameter. Table 2 and Fig. 8 (a and b) show a general decrease on the size of both small and large
particles when the feed rate was increased. However, the di7erences in the particles characterized
by smaller diameters are not very signi9cant, although this variable promoted a signi9cant decrease
(p 0:01, Table 3) on the aerodynamic diameter by volume, i.e., over particles with larger sizes.
Eventually, a synergistic e7ect occurred at high feed rates due to the presence of more particles,
promoting cluster formation and the detection of apparently larger particles. Fig. 10(ad) shows the
diameter of the particles as a function of feed rate for the di7erent values of the voltage applied to
the detector when the pin vibration was on and o7. Pin vibration a7ects the deposition of particles
on their way to the detector. Data clearly show that a more consistent pattern was observed when a
distribution by number of counts was considered, as compared to the distribution by volume (see,
respectively, lower and upper parts of Fig. 10). Pin vibration did not produce a major e7ect on the
size distribution (Fig. 8a and b, Table 2) and distribution by number was barely a7ected (Table 3)
by a change on the pin vibration status. Even though, if a distribution by volume is considered, two
remarks have to be made: (a) some of the observed di7erences were not due to the Aerosizer J but
to variations in the content of the capsules and (b) the most important component in the capsules

P.J. Mendes et al. / Aerosol Science 35 (2004) 509 527

521

Fig. 8. Aerodynamic diameter of the particles as a function of parameters level: low (a) and high (b) voltage in the
detector. () shear force, (
) deagglomeration, () feed rate, ( ) pin vibration.

(salbutamol sulfate) was assumed to show the smallest particles, while the excipient (lactose) was
associated with the largest particles and less uniform size distribution.
The interactions of the variables at two levels (binary interactions) were more relevant for the
results presented by number of counts (smaller particles) than for the results presented by volume,
suggesting that the distribution by number is more sensitive to changes of these variables than the
distribution by volume. These interactions can occur between 2, 3, 4, or 5 parameters (results not
shown). However, as the number of interactions increases, the interpretation of the e7ect becomes
more diKcult because the variables contribute di7erently to the 9nal result. Results not presented
have shown that as the level of the interaction increases, the module of the result tends to decrease
even if the result is highly signi9cant. Thus, interactions with higher number of levels (more than
3) have proved to be more diKcult to discuss based solely on the data available.
Interesting to point out are the results for the interaction between PMV and SF (p 0:001,
Table 3). A positive value for the distribution by number (Table 2) suggests an interaction between
the two variables, whereby the overall decrease on the value of the mode (balance between the
e7ect of PMV and SF) is about half of the value expected. For the interaction between PMV and
D and PMV and FR although an interaction was expected this time, the value is smaller for the
9rst interaction and larger for the second when the balance between the main e7ects of the variables
are considered. These interactions are signi9cant at p 0:001 and 0:01, respectively (Table 3).
It can be assumed that these variables change the sample in the way it is presented to the detector.
Also signi9cant are the interactions between the shear force and other variables (SF and D, FR and
PV). On the contrary, interactions such as PMVPV, DFR or DPV are not signi9cant. A possible
explanation for these observations is that the shear force transforms agglomerates in individual
particles that are measured by the detector. Since neither the pin vibrator nor the deagglomeration
directly a7ect the process of counting, their interactions are not signi9cant.
Due to the fact that the signi9cant interactions were observed for the measurements by number,
the results presented by volume were not discussed in detail.

522

P.J. Mendes et al. / Aerosol Science 35 (2004) 509 527

Fig. 9. E7ect of the shear force on the measured diameter of the particles with pin vibrationOn (a) and O7 (b).
(- - -- - -) deagglomeration: normal; feed rate: low; () deagglomeration: high; feed rate: low; (- - -
- - -)
deagglomeration: normal; feed rate: med; (
) deagglomeration: high; feed rate: med; (- - - - - -) deagglomeration:
normal; feed rate: high; () deagglomeration: high; feed rate: high.

4. Conclusions
The study has proved the ability of the Aerosizer J to determine the aerodynamic and geometric
diameters both by number and by volume of counts. Although the equipment performs in a fairly
constant way, its calibration is convenient prior to measurements. As anticipated, the measurements
made by the equipment are more accurate for powders containing single populations of particles,
rather than with samples made up of two or more populations, which are more sensitive to changes
in the parameterization of the equipment. However, if two populations of particles with distinct size
distribution are present, it is still possible to characterize each population in the mixture, provided
adequate analysis of the results is made.
It was observed that the larger the number of particles counted, or the lower the feed rate, the
larger the required run time of the equipment. The counting eKciency and reproducibility were
a7ected by the number of counts performed. For the two pharmaceuticals considered in the study,
for example, a minimum of 120,000 counts were necessary. Since a di7erent number of counts
is required when other materials are considered, a preliminary evaluation of these parameters is
recommended before starting the analysis.

P.J. Mendes et al. / Aerosol Science 35 (2004) 509 527

523

Fig. 10. Measured diameter of the particles as a function of feed rate at di7erent voltages applied to the detector: (a)
850 V and pin vibrationOn; (b) 850 V and pin vibrationO7; (c) 1100 V and pin vibrationOn; (d) 1100 V and pin
vibrationO7. (- - - - - -) Shear force: low; deagglomeration: normal; (- - -
- - -) shear force: med; deagglomeration:
normal; (- - - - - -) shear force: high; deagglomeration: normal; (- - - - - -) shear force: peak; deagglomeration: normal;
() shear force: low; deagglomeration: high; () shear force: med; deagglomeration: high; ( ) shear force:
high; deagglomeration: high; () shear force: peak; deagglomeration: high.

524

P.J. Mendes et al. / Aerosol Science 35 (2004) 509 527

The equipment used presents several advantages, but care must be taken with its parameterization.
Special attention should also be paid to the counting eKciency and type of distributions of the
particles under analysis.
The experimental design considered was able to ascertain not only the main e7ects of the variables
considered but also their relative importance and interactions at di7erent levels. It was observed that
the voltage at the detector is the most important factor: the higher the voltage, the lower the mode
observed for the distribution of particle size reFecting a higher sensitivity to smaller particles. The
shear force had an impact over larger particles a7ecting their contribution to the overall distribution
and, consequently, distributions by volume have shown a decrease on the mode. Deagglomeration
and the vibration of the pin have not shown signi9cant e7ects. Regarding the interactions at two
levels (higher order interactions produced minor contributions to the understanding of the analysis),
it was found that the voltage on the detector interacted signi9cantly with the feeding rate and
shear force.
In conclusion, the correct parameterization of the equipment and an adequate preparation of a
sample minimize most of the limitations of the equipment, providing reproducible and accurate
results. If results are to be compared between laboratories and researchers, a thorough understanding
of the equipment settings and sample preparation is required.
Acknowledgements
The work was supported by a grant (POCTI/FCB/41628/01) from Fundac<a o para a Ciencia e a
Tecnologia, Portugal.
Appendix A. Brief description of the statistical method considered
A.1. The 2k factorial designed
Very often in research the use of factorial designs is required. The simplest factorial design
considers the study of k factors considered at two levels (2k -factorial design). A higher number
of levels can be considered but the experimental part becomes time consuming. Mixed factorial
designs, whereby di7erent factors are present in di7erent levels can also be considered. However, in
these cases the analysis of the results is not a straightforward process and the following procedure
summarizes a possible technique to use.
One can start to make a table of signs with k columns and 2k rows (Table 4). Each column
corresponds to the main e7ect of a factor and each of the rows to individual experiments. The
geometric notation uses the symbols () and (+) as follows: the symbol () is used when the
factor considered in the run is at the lower level and the symbol (+) when the factor is at its
higher level. The column on the left (Table 4) refers to an increase of the level of the variable
in the experiment (row). Thus, in the 9rst row (individual factors), the factors are at the lower
levels. Then, in order to accommodate the interactions between the factors, this table is expanded to
a table with 2k 1 columns. Interactions are found upon multiplication of the signs for individual

P.J. Mendes et al. / Aerosol Science 35 (2004) 509 527

525

Table 4
Layout of a table for a 23 factorial design

(1)
A
B
C
AB
AC
BC
ABC

AB

AC

BC

ABC

+
+

+
+

+
+
+

+
+

+
+

+
+

+
+
+

Table 5
Conversion of a three level factor into factors A and B
Three level

Two level

x1
x2
x2
x3

+
+

e7ects. E7ects for the factors and interactions are found by application of the signs to the set of
experimental results.
To 9nd the main e7ect or the interaction e7ect, the signs in the corresponding column are
multiplied by the corresponding combination, added and the sum divided by 2k 1 .
For example, the e7ect of A is
1
A = 31 [(a + ab + ac + abc) ((1) + b + c + bc)]
2
and the interaction ABC is
1
ABC = 31 [(abc + a + b + c) ((1) + ab + ac + bc)]:
2
A.2. More than two levels for each factor
The method just described cannot be used with three or four levels for each factor. To overcome
the problem, Montgomery has developed a technique that allows the analysis of these factorials.
Let X be a three level factor (x1 ; x2 ; x3 ), it can be decomposed into two factors with two levels
each, factors A and B (Table 6):
Let Y be a four level factor (y1 ; y2 ; y3 ; y4 ), it can be decomposed into the factors C and D,
(Table 5):

526

P.J. Mendes et al. / Aerosol Science 35 (2004) 509 527

Table 6
Conversion of a four level factor into factors C and D
Three level

Two level

y1
y2
y3
y4

+
+

In practice, these new factors substitute the three or four levels of individual factors and can be
used as mentioned previously, including the calculation of the e7ects and interactions.
In the analysis of the results the e7ect of A has to be added to the e7ect of B to 9nd the e7ect
of X, and the e7ect of C to the e7ect of D to show the e7ect of Y.
References
Allen, T. (1990). Sampling and sizing from the atmosphere. In: T. Allen (Ed.), Particle size measurement (4th edn)
(pp 112115). New York, USA: Chapman & Hall.
Amherst process instruments, API Aerosizer LD, User Manual (V7.02), Hadley, MA, USA.
Armstrong, N. A., & James, K. C. (1996). Pharmaceutical experimental design and interpretation (pp. 131167). London,
UK: Taylor & Francis.
Barber, T. A. (1993). Particle population analysis. In: T. A. Barber (Ed.), Pharmaceutical particulate matter, analysis
and control (pp. 281298). Bu7alo Grove, IL, USA: Interpharm Press.
Dunbar, C. (2002). Dry powder formulations for inhalation. Drug Delivery Systems & Science, 2, 7880.
Gonda, I. (1992). Targeting by deposition. In A. J. Hickey (Ed.), Pharmaceutical inhalation aerosol technology
(pp. 6182). New York, USA: Marcel Dekker Inc.
Hickey, A. J. (2002). Delivery of drugs by the pulmonary route. In G. S. Banker, & C. T. Rhodes (Eds.), Modern
pharmaceutics (pp 484 490). New York, USA: Marcel Dekker Inc.
Houzego, P. (2002). Deaggregation mechanisms in dry powder inhaler. Drug Delivery Systems & Science, 2, 8185.
Laitinen, N., & Juppo, A. M. (2003). Measurement of pharmaceutical particles using a time-of-Fight particle sizer.
European Journal of Pharmaceutics and Biopharmaceutics, 55, 9398.
Mitchell, J. P., & Nagel, M. W. (1996). An assessment of the API Aerosizer J for the real-time measurement of medical
aerosols from pressurized Metered-Dose Inhaler (pMDI) systems. Aerosol Science and Technology, 25, 411423.
Mitchell, J. P., & Nagel, M. W. (1999). Time of Fight aerodynamic particle size analyzers: Their use and limitations for
the evaluation of medical aerosols. Journal of Aerosol Medicine, 12, 217240.
Mitchell, J. P., Nagel, M. W., & Archer, A. D. (1999). Size analysis of a pressurized metered dose inhaler delivered
suspension formulation by the API Aerosizer time-of-Fight aerodynamic particle size analyzer. Journal of Aerosol
Medicine, 12, 255264.
Montgomery, D. C. (1996). Factorials with mixed levels. In: D. C. Montgomery (Ed.), Design and analysis of experiments
(4th ed.) (pp. 461 465). New York, USA: Wiley.
Myers, R. H., & Montgomery, D. C. (1995). Response surface methodology: Process and product optimization using
designed experiments (pp. 3256). New York, USA: Wiley.
Nagel, M. W., Wiersema, K. J., Bates, S. L., & Mitchell, J. P. (2002). Size analysis of a pressurized metered dose inhaler
delivered solution formulation by an Aerosizer-LD time-of-Fight aerosol particle size spectrometer. Journal of Aerosol
Medicine, 15, 7585.
Oskouie, A. K., Noll, K. E., & Wang, H. C. (2002). Determination of particle diameter and lumped density and shape
factor using a three beam time-of-Fight (TOF) instrument. Journal of Aerosol Science, 33, 11251138.

P.J. Mendes et al. / Aerosol Science 35 (2004) 509 527

527

Oskouie, A. K., Noll, K. E., & Wang, H. C. (2003). Minimizing the e7ect of density in determination of particle
aerodynamic diameter using a time-of-Fight instrument. Journal of Aerosol Science, 34, 501506.
Peters, T. M., Vanderpool, R. W., & Wiener, R. W. (2001). Methodology for measuring PM 2.5 separator characteristics
using an Aerosizer. Aerosol Science & Technology, 34, 398406.
Ramon, M., Juan, G., Ciscar, M. A., Amrti-Bonmati, E., Morcillo, E. J., & Main, J. (2000). InFuence of low temperature
on bronchodilatation induced by terbutaline administered by metered dose or dry powder inhalers in asthmatics.
Fundamentals of Clinical Pharmacology, 14, 133138.
Srichana, T., Brain, A., Marriott, C., & Martin, G. P. (2000). A study of drug-carrier interactions in dry powder inhaler
formulations using the Andersen cascade impactor, X-ray microanalysis and time-of-Fight aerosol beam spectrometry
(TOFABS). Chemical Pharmaceutical Bulletin, 48, 167174.
Thornburg, J., Cooper, S. J., & Leith, D. (1998). Counting eKciency of the API Aerosizer. Journal of Aerosol Science,
30, 479488.
US Pharmacopoea 26The United States Pharmacopoeial Convention (2003). USP Convention, Rockville, MD, USA.
Wierlik, H., Diepenmaat, P., & Damhuis, R. (2002). Formulation of lactose for inhaled delivery systems. Pharmaceutical
Technology Europe, 14, 4752.