Method Development for Laser-Diffraction Particle-Size Analysis

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Method Development for Laser-Diffraction Particle-

Size Analysis
The author examines the process of method development, with reference to ISO
13320:2009 and relevant monographs from the United States and European
pharmacopoeias.

Nov 02, 2010

By Anne Virden [1]
Pharmaceutical Technology

Particle size is a critical quality attribute for a diverse array of pharmaceutical products, from
topical ointments to powders for pulmonary delivery. During recent decades, the unique
attributes of laser-diffraction analysis have positioned it as the particle-sizing technique of
choice for the resulting spectrum of pharmaceutical applications. Fast, nondestructive and
suitable for a broad size range (0.1 to 3000 μm), laser diffraction lends itself to full
automation. As a result, for many routine users, particle-size measurement is now simply a
matter of loading the sample and pressing a button. However, streamlining measurement to
this degree demands the development of a robust secure method that will consistently deliver
reliable and reproducible data. This process is given considerable emphasis in the International
Organization for Standardization's (ISO) 13320:2009 standard for laser diffraction released at
the end of 2009, reflecting that understanding of this technique among industry has grown
significantly during the past decade (1). There is a wealth of information to support method
development, the success of which depends on a rigorous and systematic examination of the
factors known to influence results.

The principles of laser diffraction

Understanding the basic principles of laser diffraction is essential for successful

method development. Laser diffraction is an ensemble particle-sizing technique,
which means it provides a result for the whole sample, rather than building up
distributions from data for individual particles, in the way that, for example, image
analysis or microscopy does. Particles illuminated in a collimated laser-beam scatter
light over a range of angles. Large particles generate a high scattering intensity at
relatively narrow angles to the incident beam, while smaller particles produce a lower
intensity signal but at much wider angles. Using an array of detectors, laser-diffraction
analyzers record the pattern of scattered light produced by the sample.

With the application of an appropriate model of light behavior the particle-size

distribution of the sample can be determined from the scattering data, via a
deconvolution step. The Mie theory and the Fraunhofer approximation (of Mie theory)
are used routinely. ISO 13320:2009 provides a detailed description of both models
but confirms Mie as the method of choice, especially for measurements across a wide
dynamic range. The two models return similar results for large particle sizes, but Mie
offers improved accuracy for finer materials. Furthermore, the inaccuracies that arise
from the use of Fraunhofer are unpredictable. Both models assume that the
measured particles are spherical, so for nonspherical samples the size distribution
returned is one that is based on spherical equivalence.

Method development involves addressing the wider set of practical issues that flow
from this underlying explanation of the basic principles of operation. United States
Pharmacopeia (USP) General Chapter <429> states that laser diffraction involves the
measurement of "a representative sample, dispersed at an adequate concentration in
a suitable liquid or gas" (2). This statement neatly highlights three crucial aspects of
laser-diffraction analysis: sampling, dispersion, and measurement conditions.

Sampling

Obtaining a representative sample from a larger bulk is a major challenge with any
kind of laboratory-based particle characterization technique. Sampling issues
generate the greatest errors in laser-diffraction analysis, especially when measuring
large particles or when the specification is based on size parameters close to the
extremes of the distribution, such as the Dv95 (the particle size below which 95% of
the volume of particles exists). This is because laser diffraction, as a volume-based
technique, is extremely sensitive to small changes in the amount of coarse particles
within the selected sample. ISO 13320:2009 expressly discourages the use of
specifications based on the Dv100 for this reason. The effect of sampling on
reproducibility increases with particle size and the width of the distribution, as the

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Method Development for Laser-Diffraction Particle-Size Analysis

volume of sample required to ensure representative sampling of the coarse particle

fraction increases. For this reason, it may be necessary to measure a large sample
(often greater than 1–2 g) to ensure reproducible results. For wet-dispersion
measurements, this will require the use of a large dispersant volume, to ensure the
concentration of material is within the required range for accurate measurements (i.e.,
to ensure multiple scattering is avoided). A detailed discussion of sampling is beyond
the remit of this paper, but ISO 14488 provides an extremely useful summary of the
requirements for sampling during particle characterization studies (3).

Cohesivity—cohesive and or very fine particles (below 20 μm) can be

Sample difficult to disperse completely using dry techniques
preparation Toxicity—handling samples in the wet state makes control

Laser diffraction is suitable for the analysis of a wide array of sample types, but it is
essential to appropriately tailor the sample-preparation method. For sprays, aerosols,
and gas bubbles in solution, USP <429> strongly discourages sample preparation, or
indeed sampling, because of the difficulty of imposing either step without skewing the
measured particle-size distribution. Laser diffraction instruments designed specifically
for the analysis of sprays have much to offer here, being able to measure relatively
concentrated sprays directly.

For other sample types, there is a choice to be made between wet and dry dispersion.
Influencing factors include the natural state of the sample, the ease with which it can
be dispersed and the volume of sample to be measured. Where the sample is dry
powder, dry dispersion may present the simplest option, but there are several reasons
why wet measurement may be preferred. These include:

easier, reducing the risk of inadvertent inhalation

Friability—wet dispersion can provide more gentle dispersion for fragile
particles.

Because wet dispersion is appropriate for so many samples, it is the method most
widely used for laser diffraction measurement.

A further issue to consider alongside the choice of dispersion method is: What should
be the goal of the dispersion procedure? While it is the primary particle size of an
active ingredient for a tablet blend that will influence its in vivo dissolution and
consequent bioavailability, for instance, in other applications, the particle size of
agglomerates may be more relevant. A good example of this is the study of
suspension stability where, if particles are prone to agglomeration, it is agglomerate
size that will control sedimentation rates.

Dispersant choice. The dispersants used in laser-diffraction

Wet measurement range from highly polar water to very nonpolar long-
dispersion chain alkanes and alkenes (see Table I). A candidate dispersant

Factors to address in the development of a robust wet dispersion include dispersant

choice, conditions for stable dispersion, and sample concentration. The aim is to
reliably produce a stable, representative dispersion of suitable concentration for
measurement.

Table I: Dispersants in order of polarity.

should:

Be transparent to the wavelength of light used in the measurement

Be chemically compatible with the materials used in the instrument
Not dissolve the particles
Be free from bubbles and other particles
Favor easy and stable particle dispersion
Have a refractive index different to that of the particles
Have suitable viscosity.

The dispersant must also be able to wet the sample. Wetting can be assessed by

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Method Development for Laser-Diffraction Particle-Size Analysis

mixing sample and dispersant in a beaker and observing the resulting suspension. A
uniform suspension is indicative of good wetting; sedimentation of the sample on the
base of the beaker is undesirable. Wetting depends on the surface tension between
the particles and dispersant and can, therefore, be modified through the use of
surfactants. Other admixtures may also be beneficial, with the pH of the dispersant an
important variable for systems having an iso-electric point (4). ISO 14887 is a useful
source of further guidance for the dispersion of powders in liquids (5).

When characterizing emulsions, choosing a dispersant as close as possible to the

Figure 1: Contrasting emulsion stability in deionized and tap water. (ALL FIGURES ARE
COURTESY OF THE AUTHORS)
continuous phase minimizes the risk of dissolution shock, which is the modification of
droplet size by the dissolution process. Figure 1 contrasts the suitability of tap and de-
ionized water for dispersing an example emulsion; 10 measurements were made with
each system to assess dispersion stability in each case. Figure 2 illustrates the

With tap water, droplet size increases over time, a response attributed to flocculation.
Deionized water prevents this and is therefore more suitable. These data highlight the
importance of making repeat measurements to assess dispersion stability because
initial measurements of particle size are almost identical, with differences becoming
increasingly pronounced over time.

Optimizing conditions for stable dispersion. Energy input maintains a stable
dispersion with the chosen dispersant. With wet dispersion, the sample is typically
made up in a dispersion cell, which is essentially a feed vessel for the analyzer.
Energy for dispersion comes from the agitator in this cell, from the pump that transfers
the sample to the measurement zone, and from sonication if applied. Effective tuning
of these three inputs ensures repeatable dispersion to a size appropriate to the
application, avoiding primary particle breakup.

Both USP <429> and ISO 13320:2009 highlight microscopy as a useful tool for
determining whether or not suitable conditions have been established. Particle
imaging allows for the identification of agglomerates, and can be used to cross-
validate the particle size range of the sample.

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Method Development for Laser-Diffraction Particle-Size Analysis

Figure 2: Optimizing conditions for stable dispersion.

progression of an agglomerated sample toward stable dispersion. Images show
agglomeration when only the dispersion cell stirrer and pump are active; however,
when ultrasound is applied particle size reduces to a stable level. The leveling of
particle size suggests that primary particles are undamaged, and the results provide
an indication of required sonication time. When the ultrasound is switched off, the stable
dispersion persists, and image analysis confirms the presence of discrete primary particles.

For certain types of samples, there are additional factors influencing dispersion
conditions. With emulsions for example, excessively high agitation may shear
emulsion droplets, in which case droplet size will decrease with increasing agitator
speed. On the other hand, for samples containing coarser particles, higher agitator
speeds may increase particle size by reducing the tendency to sediment. Finally,
where particles have a high aspect ratio, certain agitator or pump speeds may cause
flow alignment. Nonrandom alignment can affect the measured particle-size
distribution, so this phenomenon should be carefully considered if samples fall into
this classification.

Setting sample concentration. The ideal sample for laser-diffraction analysis is
sufficiently concentrated to give a stable scattering signal but dilute enough to avoid
the issue of multiple scattering. Multiple scattering occurs where the light interacts
with more than one particle before being detected, and leads to an underestimation of
particle size.

Figure 3: Obscuration titrations for submicron and larger particle samples.

Obscuration is a measure of the percentage of emitted laser light that is lost by scattering or
absorption. It is therefore indicative of sample concentration. Carrying out an obscuration
titration and plotting particle size as a function of obscuration is an efficient way of identifying
a concentration range for reproducible measurement, as Figure 3 illustrates. Data are shown
for two samples: one with particles in the submicron region, the other with a Dv50 of > 30 μm.
Figure 4 (a) shows a pressure/particle-size titration for a relatively fragile material. Particle

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Method Development for Laser-Diffraction Particle-Size Analysis

With the submicron-sized sample, particle size begins to decrease at obscurations

above 5%, as multiple scattering begins to have an appreciable effect. For the sample
containing larger particles, particle size is stable over a much wider obscuration
range. Larger particles scatter light at relatively high intensity and narrow angles so
measurement is less influenced by multiple scattering, and the signal-to-noise ratio is
less of a challenge. Where larger particles are present, or the particle size distribution
is particularly wide, concentration may be set on the basis of sampling requirements
(as previously discussed).

size decreases quite steeply as pressure is increased from 0 to 1 bar, but there

Dry is no way of telling simply by looking at this plot if this size reduction is the
dispersion result of agglomerate breakup or the milling of primary particles. Figure 4

Energy to disperse a dry sample is applied by entraining the powder in a compressed

air stream. Similar to wet dispersion, the goal is to disperse to an application-relevant
degree but no further; air pressure is the lever used to control energy input. ISO
13320:2009 notes that a "pressure/particle-size" titration should in the ideal case
identify a region where particle size is nearly constant over a range of pressures,
indicating that agglomerate dispersion has occurred without particle breakup.
However, it makes clear that this is seldom observed, in which case it becomes
important to reference dry results against wet measurements, to avoid breakup and/or
milling of the primary particles.

Figure 4: Method-development data for a fragile powder sample, (a) a pressure/particle-size titration, (b) a comparison of wet-dispersion data with dry
results measured at a dispersion pressure of 0.2 bar.
(b) shows that close agreement between wet results and dry data is achieved at a dispersion
pressure of 0.2 bar, suggesting that pressures above this result in particle milling. As with wet
dispersion, images of the particles can be useful in elucidating the effect of entrainment at
different air pressures. Figures 5 (a) and (b) show strictly analogous data for a different

Figure 5: Method-development data for a pharmaceutical powder, (left) a pressure/particle-size titration, (right) a comparison of wet-dispersion data with dry
results measured at a dispersion pressure of 3 bar.
material—a pharmaceutical powder. Here too, the pressure/particle size titration fails to
plateau, giving an unclear indication of optimal air pressure. A comparison of data measured at
3 bar with those from a wet dispersion suggests that dispersion is inadequate—there are larger
quantities of material toward the coarse end of the particle-size distribution. The dry results
also show, however, a larger proportion of fines. Here then, dispersion and breakup occur
simultaneously, rather than sequentially, making dry dispersion problematic. Further
increasing the dispersion pressure will reduce the amount of agglomerated material present but

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Method Development for Laser-Diffraction Particle-Size Analysis

will also increase particle damage. For this system, wet dispersion is a better option. For a wet
dispersion, measurement duration can be specified to analyze just a small
Measurementfraction of the sample, or, at the other extreme, to repeatedly measure the

The actual process of sample measurement involves recording the scattering pattern
produced as the sample passes through the path of the laser. An initial background
measurement captures scattering from the cell windows, any contaminant present
and, in the case of wet measurement, the pure dispersant. This process assesses
cleanliness and allows precise capture of the scattering pattern relating solely to the
sample.

With a dry measurement, duration is set to ensure analysis of the entire sample to
avoid analyzing an unrepresentative subsample. Obscuration limits can be set to
prevent measurement when the powder density is too low to give a reliable signal-to-
noise ratio, or, conversely so high that multiple scattering is likely.

Figure 6: Results of tracking Dv90 and measurement variability as a function of measurement

duration.
same sample, because material being measured can be recirculated through the measurement
cell many times. Excessively long measurement times are inefficient but an overly short
measurement time may give unrepresentative data, especially if the sample contains coarser
particles and/or the distribution is broad, as illustrated by Figure 6. For this sample, the poor
repeatability and smaller particle size recorded at low measurement times are attributable to
insufficient sampling of the large particles, an issue resolved by extending measurement
duration.

Method
validation +/– 3% for Dv10 (and all other value of cumulative undersize

When assessing the validity of measured results and determining whether a defined
procedure and associated system are fit for purpose, two concepts are central:
repeatability and reproducibility. Assessing repeatability involves duplicate
measurements of the same sample. It therefore tests the precision of the instrument
and the consistency of the sample. Reproducibility is a broader concept that also
encompasses sampling from the bulk.

One aspect of repeatability is the performance of the analyzer. ISO13320 (2009)

provides revised accuracy acceptance criteria for performance verification, which
typically involves measurement of an appropriate standard. Because laser diffraction
is a volume-based measurement technique, sampling errors for large particles will
cause greater uncertainty in the Dv90 than in the Dv10. The revised acceptance
criteria for reference materials reflect this and are:

distribution between the 10th and 30th percentiles)

+/– 2.5% for Dv50 (and all other value of cumulative undersize distribution
between the 30th and 70th percentiles)
+/– 4% for Dv90 (and all other value of cumulative undersize distribution
between the 70th and 90th percentiles).

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Method Development for Laser-Diffraction Particle-Size Analysis

To test repeatability for a given application, duplicate measurements of the same

sample are performed. The precision of laser diffraction measurements is usually
assessed using the term coefficient of variation (%COV) which is defined according to
the following equation (1):

ISO 13320:2009 states that repeatability tests should show a %COV of less than 3% on Dv50
and below 5% for Dv10 and Dv90 but indicates that these values can be doubled for samples
containing particles smaller than 10 μm, because of the difficulties of dispersion. In ideal
conditions, however, much better performance is readily achievable: a %COV of less than
0.5% for samples larger than 1 μm and below 1% for samples finer than this, is realistic.

Reproducibility is assessed by measuring several samples from the same batch of material and
therefore tests how representative the sampling procedure is. Both USP and the European
Pharmacopoeia recommend acceptance criteria for reproducibility testing of a %COV of less
than 10% on Dv50 or any similar central value and less than 15% on values toward the edge of
the distribution such as Dv10 and Dv90 (2, 6). Once again, these limits are doubled for
samples containing particles smaller than 10 μm.

Conclusion References

The highly valued simplicity of routine laser-diffraction measurement belies the relative
complexity of method development. During the last decade, considerable progress
has been made toward securing a comprehensive understanding of how best to
define measurement methods for laser-diffraction analysis and implement them. The
new ISO standard and relevant chapters of the USP and the European
Pharmacopoeia provide useful summaries of the significant guidance now in place.
Instrument manufacturers recognize that helping users to access all available
information—through education, direct support and smarter software—is the way to
maximize the benefits of this vital analytical technique.

Anne Virden is a product technical specialist in diffraction at Malvern Instruments,
Enigma Business Park, Grovewood Road, Malvern, Worcestershire, WR14 1XZ, UK,
tel. +44 (0)1684 892456, fax + 44 (0)1684 892789, [email protected] [2]
.

1. ISO 13320:2009 Particle Size Analysis—Laser Diffraction Methods. Part 1: General

Principles (2009).

2. USP30–NF25 General Chapter <429>, "Light Diffraction Measurement of Particle

Size," pp. 1235–1241.

3. ISO 14488:2007 Particulate materials—Sampling and sample splitting for the

determination of particulate properties.

4. Application note MRK373: The Use of Zeta Potential Measurements for Improving
Dispersion During Particle Size Measurements,
www.malvern.com/appnote_particle_size_determination [3], accessed Sept. 18, 2010.

5. ISO 14887:2000 Sample Preparation—Dispersing procedures for powders in

liquids (2000).

6. Ph.Eur. 6.6 General Chapter 2.9.31, "Laser Diffraction Measurement of Particle

Size," (EDQM, Strasbourg, France) p. 5103.

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