A GUIDEBOOK TO

PARTICLE SIZE
ANALYSIS

Explore the future Automotive | Process & Environmental | Medical | Semiconductor | Scientific
 TABLE OF CONTENTS

1 Why is particle size important?

Which size to measure

3 Understanding and interpreting particle size distribution calculations

Central values: mean, median, mode
Distribution widths
Technique dependence
Laser diffraction
Dynamic light scattering
Image analysis

8 Particle size result interpretation: number vs. volume distributions

Transforming results

10 Setting particle size specifications

Distribution basis
Distribution points
Including a mean value
X vs.Y axis
Testing reproducibility
Including the error
Setting specifications for various analysis techniques

Particle Size Analysis Techniques

15 LA-960V2 laser diffraction technique

The importance of optical model
Building a state of the art laser diffraction analyzer

18 LA-350 laser diffraction technique

Compact optical bench and circulation pump in one system

19 ViewSizer 3000 nanotracking analysis

A Breakthrough in nanoparticle tracking analysis

20 SZ-100V2 dynamic light scattering technique

Calculating particle size
Zeta Potential
Molecular weight

25 PSA300 image analysis technique

Static image analysis

Eyecon2™ in-line image analysis technique

27
In-line and At-line/benchtop

28 CENTRIFUGE centrifugal sedimentation technique

Particle size distribution

29 SA-9600 surface area analysis technique

BET flowing gas surface area

30 Selecting a particle size analyzer

Dynamic range of the HORIBA particle characterization systems
When to choose laser diffraction
When to choose dynamic light scattering
When to choose image analysis

33 References
Why is
particle size important?
Particle size influences many properties of particulate materials and is
a valuable indicator of quality and performance. This is true for powders,
Particle size is critical within
suspensions, emulsions, and aerosols. The size and shape of powders influences
a vast number of industries.
flow and compaction properties. Larger, more spherical particles will typically flow
For example, it determines:
more easily than smaller or high aspect ratio particles. Smaller particles dissolve appearance and gloss of paint
more quickly and lead to higher suspension viscosities than larger ones. Smaller
flavor of cocoa powder
droplet sizes and higher surface charge (zeta potential) will typically improve
suspension and emulsion stability. Powder or droplets in the range of 2-5µm reflectivity of highway paint

aerosolize better and will penetrate into lungs deeper than larger sizes. For these hydration rate & strength of cement
and many other reasons it is important to measure and control the particle size properties of die filling powder
distribution of many products.
absorption rates of pharmaceuticals

appearances of cosmetics
Measurements in the laboratory are often made to support unit operations taking
place in a process environment. The most obvious example is milling (or size
reduction by another technology) where the goal of the operation is to reduce
particle size to a desired specification. Many other size reduction operations and
technologies also require lab measurements to track changes in particle size
including crushing, homogenization, emulsification and microfluidization. Separation
steps such as screening, filtering, cyclones, etc. may be monitored by measuring
particle size before and after the process. Particle size growth may be monitored
during operations such as granulation or crystallization. Determining the particle size
of powders requiring mixing is common since materials with similar and narrower
distributions are less prone to segregation.

There are also industry/application specific reasons why controlling and

measuring particle size is important. In the paint and pigment industries particle
size influences appearance properties including gloss and tinctorial strength.
Particle size of the cocoa powder used in chocolate affects color and flavor. The size
and shape of the glass beads used in highway paint impacts reflectivity. Cement
particle size influences hydration rate & strength. The size and shape distribution
of the metal particles impacts powder behavior during die filling, compaction, and
sintering, and therefore influences the physical properties of the parts created.
In the pharmaceutical industry the size of active ingredients influences critical
characteristics including content uniformity, dissolution and
absorption rates. Other industries where particle size plays an important role include
nanotechnology, proteins, cosmetics, polymers, soils, abrasives, fertilizers, and
many more.

1
 WHICH SIZE TO MEASURE?

A spherical particle can be described using a single number—the diameter—

because every dimension is identical. As seen in Figure 1, non-spherical particles
DIAMETER can be described using multiple length and width measures (horizontal and vertical
projections are shown here). These descriptions provide greater accuracy, but
also greater complexity. Thus, many techniques make the useful and convenient
assumption that every particle is a sphere. The reported value is typically an
equivalent spherical diameter. This is essentially taking the physical measured value

VERTICAL
(i.e. scattered light, settling rate) and determining the size of the sphere that could
PROJECTION produce the data. Although this approach is simplistic and not perfectly accurate,
the shapes of particles generated by most industrial processes are such that the
spherical assumption does not cause serious problems. Problems can arise, however,
if the individual particles have a very large aspect ratio, such as fibers or needles.

Shape factor causes disagreements when particles are measured with different
particle size analyzers. Each measurement technique detects size through the
use of its own physical principle. For example, a sieve will tend to emphasize the
second smallest dimension because of the way particles must orient themselves to
pass through the mesh opening. A sedimentometer measures the rate of fall of the
particle through a viscous medium, with the other particles and/or the container
HORIZONTAL
PROJECTION walls tending to slow their movement. Flaky or plate-like particles will orient to
maximize drag while sedimenting, shifting the reported particle size in the smaller
figure 1
| SHAPE FACTOR direction. A light scattering device will average the various dimensions as the
Many techniques make the particles flow randomly through the light beam, producing a distribution of sizes
general assumption that every from the smallest to the largest dimensions.
particle is a sphere and report the
value of some equivalent
The only techniques that can describe particle size using multiple values are
diameter. Microscopy or
automated image analysis are the microscopy or automated image analysis. An image analysis system could
only techniques that can describe describe the non-spherical particle seen in Figure 1 using the longest and shortest
particle size using multiple values diameters, perimeter, projected area, or again by equivalent spherical diameter.
for particles with larger aspect When reporting a particle size distribution the most common format used even for
ratios.
image analysis systems is equivalent spherical diameter on the x axis and percent
on the y axis. It is only for elongated or fibrous particles that the x axis is typically
displayed as length rather than equivalent spherical diameter.

2
Understanding and interpreting
particle size distribution calculations
Performing a particle size analysis is the best way to answer the question:
What size are those particles? Once the analysis is complete the user has
a variety of approaches for reporting the result. Some people prefer a single
number answer—what is the average size? More experienced particle scientists
cringe when they hear this question, knowing that a single number cannot describe
the distribution of the sample. A better approach is to report both a central point of
the distribution along with one or more values to describe the width of distribution.
Other approaches are also described in this document.

CENTRAL VALUES: MEAN, MEDIAN, MODE

For symmetric distributions such as the one shown in Figure 2 all central values are
equivalent: mean = median = mode. But what do these values represent?

MEAN

Mean is a calculated value similar to the concept of average. The various mean
calculations are defined in several standard documents (ref.1,2). There are multiple
definitions for mean because the mean value is associated with the basis of the
figure 2
| SYMMETRIC DISTRIBUTION
WHERE MEAN=MEDIAN=MODE

distribution calculation (number, surface, volume). See (ref. 3) for an explanation of

number, surface, and volume distributions. Laser diffraction results are reported on a
volume basis, so the volume mean can be used to define the central point although
the median is more frequently used than the mean when using this technique.
The equation for defining the volume mean is shown below. Start by thinking of a
histogram table showing the upper and lower limits of n size channels along with the
percent in each channel. Let these size channels have logarithmic spacing; the ratio
of the limits is constant (e.g., channel 1 is 1, channel 2 is 2, channel 3 is 4, channel
5 is 8…). The Di value for each channel i is the geometric mean, or the square root
of the product of the upper and lower diameter values. For the numerator take
the geometric Di to the fourth power x the percent in that channel, summed over
all channels. For the denominator take the geometric Di to the third power x the
percent in that channel, summed over all channels.

3
 The volume mean diameter has several names including D4,3. In all HORIBA
diffraction software this is simply called the “mean” whenever the result is displayed
as a volume distribution. Conversely, when the result in HORIBA software is
converted to a surface area distribution the mean value displayed is the surface
mean, or D 3,2. The equation for the surface mean is shown below.

The description for this calculation is the same as the D4,3 calculation, except
that Di values are raised to the exponent values of 3 and 2 instead of 4 and 3.

The generalized form of the equations seen above for D4,3 and D3,2 is shown below
(following the conventions from ref. 2, ASTM E 799, ).

Where:
D = the overbar in D designates an averaging process
(p-q)p>q = the algebraic power of Dpq
Di = the diameter of the ith particle
Σ = the summation of Dip or Diq, representing all particles in the sample

Some of the more common representative diameters are:

D10 = arithmetic or number mean
D32 = volume/surface mean (also called the Sauter mean)
D43 = the mean diameter over volume (also called the DeBroukere mean)

The example results shown in ASTM E 799 are based on a distribution of liquid
droplets (particles) ranging from 240 – 6532 µm. For this distribution the following
results were calculated:
D10 = 1460 µm
D32 = 2280 µm
D50 = 2540 µm
D43 = 2670 µm

These results are fairly typical in that the D43 is larger than the D50—
the volume-basis median value.

MEDIAN

Median values are defined as the value where half of the population resides above
this point, and half resides below this point. For particle size distributions the
median is called the D50 (or x50 when following certain ISO guidelines). The D50
is the size in microns that splits the distribution with half above and half below this
diameter. The Dv50 (or Dv0.5) is the median for a volume distribution, Dn50 is
used for number distributions, and Ds50 is used for surface distributions. Since the
primary result from laser diffraction is a volume distribution, the default D50 cited
is the volume median and D50 typically refers to the Dv50 without including the
v. This value is one of the easier statistics to understand and also one of the most
meaningful for particle size distributions.

4
MODE

The mode is the peak of the frequency distribution, or it may be easier to visualize MODE

it as the highest peak seen in the distribution. The mode represents the particle
size (or size range) most commonly found in the distribution. Less care is taken to MEDIAN

denote whether the value is based on volume, surface or number, so either run the
risk of assuming volume basis or check to assure the distribution basis. The mode is
MEAN
not as commonly used, but can be descriptive; in particular if there is more than one
peak to the distribution, then the modes are helpful to describe the mid-point of the
different peaks.

For non-symmetric distributions the mean, median and mode will be three different
values shown in Figure 3.

DISTRIBUTION WIDTHS

Most instruments are used to measure the particle size distribution, implying an
figure 3
interest in the width or breadth of the distribution. Experienced scientists typically
shun using a single number answer to the question “What size are those particles?”,
| A NON-SYMMETRIC DISTRIBTION
Mean, median and mode
will be three different values.
and prefer to include a way to define the width. The field of statistics provides
several calculations to describe the width of distributions, and these calculations are
sometimes used in the field of particle characterization. The standard deviation
(St Dev.) is the preferred value in our field of study. As shown in Figure 4, for a
normal (Guassian) distribution, 68.27% of the total population lies within +/- 1 St
Dev, and 95.45% lies within +/- 2 St Dev.

Although occasionally cited, the use of standard deviation declined when hardware
and software advanced beyond assuming normal or Rosin-Rammler distributions. -1 STD 68.27% +1 STD

Once “model independent” algorithms were introduced many particle scientists

began using different calculations to describe distribution width. One of the common
values used for laser diffraction results is the span, with the strict definition shown in
the equation below (2):

95.45%

-2 STD MEAN +2 STD

figure 4
In rare situations the span equation may be defined using other values such as
Dv0.8 and Dv0.2. Laser diffraction instruments should allow users this flexibility. | A NORMAL DISTRIBUTION
The mean value is flanked
by 1 and 2 standard
deviation points.
Be cautious with the term standard deviation. This term is also used to describe
the variation in measurement results as well as the width of a size distribution.
For example, when evaluating repeatability, one commonly uses the Coefficient of
Variation (COV), also referred to as the relative standard deviation. See for example
ISO 13320 (ref. 4). And this is not the width of the distribution.

5
 Another common approach to define the distribution width is to cite three values
on the x-axis, the D10, D50, and D90 as shown in Figure 5. The D50, the median,
Dv0.5 MEDIAN
has been defined as the diameter where half of the population lies below this value.
Similarly, 90 percent of the distribution lies below the D90, and 10 percent of the
population lies below the D10.

TECHNIQUE DEPENDENCE
Dv0.1 Dv0.9
HORIBA Instruments Incorporated offers particle characterization tools based on
10% 50% 90% several principles including laser diffraction, dynamic light scattering, image analysis,
below below below
this size this size this size nanoparticle tracking analysis (NTA), and centrifugal sedimentation. Each of these
techniques generates results in both similar and unique ways. Most techniques
can describe results using standard statistical calculations such as the mean and
standard deviation. But commonly accepted practices for describing results have
figure 5
| THREE X-AXIS VALUES
D10, D50 and D90
evolved for each technique.

LASER DIFFRACTION

All of the calculations described in this document are generated by the HORIBA laser
diffraction software package. Results can be displayed on a volume, surface area,
or number basis. Statistical calculations such as standard deviation and variance
are available in either arithmetic or geometric forms. The most common approach
for expressing laser diffraction results is to report the D10, D50, and D90 values
based on a volume distribution. The span calculation is the most common format to
express distribution width. That said, there is nothing wrong with using any of the
available calculations, and indeed many customers include the D4,3 when reporting
results.

A word of caution is given when considering converting a volume distribution into

either a surface area or number basis. Although the conversion is supplied in the
software, it is only provided for comparison to other techniques, such as microscopy,
which inherently measure particles on different bases. The conversion is only valid
for symmetric distributions and should not be used for any other purpose than
comparison to another technique.

6
DYNAMIC LIGHT SCATTERING

Dynamic Light Scattering (DLS) is unique among the techniques described in

this document. The primary result from DLS is typically the mean value from the
intensity distribution (called the Z average) and the polydispersity index (PDI) to
describe the distribution width. It is possible to convert from an intensity to a volume
or number distribution in order to compare to other techniques.

IMAGE ANALYSIS

The primary results from image analysis are based on number distributions. These
are often converted to a volume basis, and in this case this is an accepted and valid
conversion. Image analysis provides far more data values and options than any of
the other techniques described in this document. Measuring each particle allows the
user unmatched flexibility for calculating and reporting particle size results.

Image analysis instruments may report distributions based on particle length as

opposed to spherical equivalency, and they may build volume distributions based on
shapes other than spheres.

With the ability to measure particles in any number of ways comes the decision
to report those measurements in any number of ways. Users are again cautioned
against reporting a single value—the number mean being the worst choice of the
possible options. Experienced particle scientists often report D10, D50, and D90, or
include standard deviation or span calculations when using image analysis tools.

NANOPARTICLE TRACKING ANALYSIS (NTA)

Like image analysis, the primary results from NTA are based on number
distributions. These are less often converted to a volume basis. In addition, NTA
can be used to determine the particle number concentration or the concentration of
particles in each size class.

CONCLUSIONS

All particle size analysis instruments provide the ability to measure and report the
particle size distribution of the sample. There are very few applications where a
single value is appropriate and representative. The modern particle scientist often
chooses to describe the entire size distribution as opposed to just a single point on it.
(One exception might be extremely narrow distributions such as latex size standards
where the width is negligible.) Almost all real world samples exist as a distribution
of particle sizes and it is recommended to report the width of the distribution for any
sample analyzed. The most appropriate option for expressing width is dependent
on the technique used. When in doubt, it is often wise to refer to industry accepted
standards such as ISO or ASTM in order to conform to common practice.

7
 Particle size result interpretation:
number vs. volume distributions
Interpreting results of a particle size measurement requires an under-
standing of which technique was used and the basis of the calculations.
D = 1µm
Each technique generates a different result since each measures different
VOLUME = 0.52µm
% BY VOLUME = 0.52/18.8 = 2.8% physical properties of the sample. Once the physical property is measured a
calculation of some type generates a representation of a particle size distribution.
Some techniques report only a central point and spread of the distribution, others
provide greater detail across the upper and lower particle size detected. The particle
size distribution can be calculated based on several models: most often as a number
D = 2µm
or volume/mass distribution.
VOLUME = 4.2µm
% BY VOLUME = 4.2/18.8 = 22%

NUMBER VS. VOLUME DISTRIBUTION

The easiest way to understand a number distribution is to consider measuring

particles using a microscope. The observer assigns a size value to each particle
inspected. This approach builds a number distribution—each particle has equal
weighting once the final distribution is calculated. As an example, consider the nine
particles shown in Figure 6. Three particles are 1µm, three are 2µm, and three
D = 3µm
VOLUME = 14.1µm
are 3µm in size (diameter). Building a number distribution for these particles will
% BY VOLUME = 14.1/18.8 = 75% generate the result shown in Figure 7, where each particle size accounts for one
third of the total. If this same result were converted to a volume distribution, the
TOTAL VOLUME
0.52 + 4.2 + 14.1 = 18.8µm result would appear as shown in Figure 8 where 75% of the total volume comes
from the 3µm particles, and less than 3% comes from the 1µm particles.
figure 6
| PARTICLES 1, 2 AND 3µm IN SIZE
Calculations showing percent by
volume for each size range. For
30 70
all three ranges, the percent by
60
number is the same, 3/9 = 33.3%. 25
50
20
40
15
30
10
20
5 10
0 0
1µm 2µm 3µm 1µm 2µm 3µm

figure 7
| NUMBER DISTRIBUTION figure 8
| VOLUME DISTRIBUTION

When presented as a volume distribution it becomes more obvious that the majority
of the total particle mass or volume comes from the 3µm particles. Nothing changes
between the left and right graph except for the basis of the distribution calculation.

8
Another way to visualize the difference between number and volume distributions
is supplied courtesy of the City of San Diego Environmental Laboratory. In this case
beans are used as the particle system. Figure 9 shows a population where there are
13 beans in each of three size classes, equal on a number basis. Figure 10 shows
these beans placed in volumetric cylinders where it becomes apparent that the larger
beans represent a much larger total volume than the smaller ones.

Figure 11 shows a population of beans where it may not be intuitively obvious, but
there is an equal volume of each size, despite the wide range of numbers present.
It becomes apparent in Figure 12 when the beans are placed in volumetric cylinders
that each volumes are equal.
figure 9
| 13 BEANS OF EACH SIZE

TRANSFORMING RESULTS

Results from number based systems, such as microscopes or image analyzers

construct their beginning result as a number distribution. Results from laser
diffraction construct their beginning result as a volume distribution. The software for
many of these systems includes the ability to transform the results from number to
volume or vice versa. It is perfectly acceptable to transform image analysis results
from a number to volume basis. In fact the pharmaceutical industry has concluded
that it prefers results be reported on a volume basis for most applications (ref. 6).

figure 10
|
On the other hand, converting a volume result from laser diffraction to a number THE SAME 39 BEANS PLACED
basis can lead to undefined errors and is only suggested when comparing to IN VOLUMETRIC CYLINDERS

results generated by microscopy. Figure 13 below shows an example where a laser

diffraction result is transformed from volume to both a number and a surface area
based distribution. Notice the large change in median from 11.58µm to 0.30µm
when converted from volume to number.

12
NUMBER

10

8
figure 11
|EQUAL VOLUME OF EACH OF
THE THREE TYPES OF BEANS

AREA VOLUME

0
0.34 0.58 1.15 2.27 4.47 8.82 17.38 34.25
PARTICLE SIZE
NUMBER DISTRIBUTION VOLUME DISTRIBUTION
MEAN = 0.38µm MEAN = 12.65µm

figure 12
|
MEDIAN = 0.30µm MEDIAN = 11.58µm
SA = 13467 cm²/cm³ SA = 13467 cm²/cm³ EQUAL VOLUMES IN
STANDARD DEV = 0.40 STANDARD DEV = 8.29 VOLUMETRIC CYLINDERS

figure 13
| VOLUME DISTRIBUTION CONVERTED
TO AREA AND NUMBER
Conversion errors can result when
deriving number or area values from
a laser diffraction volume result.

9
 Setting particle size specifications
The creation of a meaningful and product-appropriate particle size
specification requires knowledge of its effect on product performance
in addition to an understanding of how results should be interpreted
for a given technique. This section provides guidelines for setting particle size
specifications on particulate materials—primarily when using the laser diffraction
technique, but also with information about dynamic light scattering (DLS) and image
analysis.

DISTRIBUTION BASIS

Different particle sizing techniques report primary results based on number, volume,
weight, surface area, or intensity. As a general rule specifications should be based
in the format of the primary result for a given technique. Laser diffraction generates
results based on volume distributions and any specification should be volume based.
Likewise, a number basis should be used for image analysis and NTA while dynamic
light scattering specifications should be in terms of Z-average and polydispersity
index. Conversion to another basis such as number—although possible in the
software—is inadvisable because significant error is introduced. The exception to
this guideline is converting a number based result from a technique such as image
analysis into a volume basis (ref. 7). The error involved is generally very low in this
scenario.

DISTRIBUTION POINTS

While it is tempting to use a single number to represent a particle size distribution

(PSD), and thus the product specification, this is typically not a good idea. In nearly
every case, a single data point cannot adequately describe a distribution of data
points. This can easily lead to misunderstandings and provides no information about
the width of the distribution. Less experienced users may believe that the “average
particle size” can adequately describe a size distribution, but this implies expecting
a response based on a calculated average (or mean). If forced to use a single
calculated number to represent the mid-point of a particle size distribution, then the
common practice is to report the median and not the mean. The median is the most
stable calculation generated by laser diffraction and should be the value used for a
single point specification in most cases.

10
Rather than use a single point in the distribution as a specification, it is suggested
to include other size parameters in order to describe the width of the distribution.
The span is a common calculation to quantify distribution width: (D90 – D10) /
D50. However, it is rare to see span as part of a particle size specification. The
more common practice is to include two points which describe the coarsest and
finest parts of the distribution. These are typically the D90 and D10. Using the
same convention as the D50, the D90 describes the diameter where ninety percent
of the distribution has a smaller particle size and ten percent has a larger particle
size. The D10 diameter has ten percent smaller and ninety percent larger. A three
point specification featuring the D10, D50, and D90 will be considered complete and
appropriate for most particulate materials.

How these points are expressed may vary. Some specifications use a format where
the D10, D50, and D90 must not be more than (NMT) a stated size.

Example: D10 NMT 20µm

D50 NMT 80µm
D90 NMT 200µm

Although only one size is stated for each point there is an implied range of
acceptable sizes (i.e. the D50 passes if between 20 and 80µm).

Alternatively, a range of values can be explicitly stated.

Example: D10 10 – 20µm

D50 70 – 80µm
D90 180 – 200µm

This approach better defines the acceptable size distribution, but may be
perceived as overly complicated for many materials.

It may also be tempting to include a requirement that 100% of the distribution

is smaller than a given size. This implies calculating the D100 which is not
recommended. The D100 result (and to a lesser degree the D0) is the least robust
calculation from any experiment. Any slight disturbance during the measurement
such as an air bubble or thermal fluctuation can significantly influence the D100
value. Additionally, the statistics involved with calculating this value (and other
“extreme” values such as the D99, D1, etc.) aren’t as robust because there may
not be very many of the “largest” and “smallest” particles. Given the possible broad
spread of D100 results it is not recommended for use in creating specifications
involving a statement that 100% of the particles are below a stated size.

INCLUDING A MEAN VALUE

Ultimately, the sophistication of the specification should be driven by how particle

size influences product performance. Given that some people ask about the “average
size”, it is not surprising that some specifications are based on a mean diameter.
This approach is complicated by the fact that there are several mean values that
can be calculated and reported in the result (ref. 8). The most common mean value
noted when using laser diffraction is the volume mean, or D4,3. The D4,3 is very
sensitive to the presence of large particles in the distribution. It is a good idea to
use or include the D4,3 in the specification if product performance is sensitive to
the presence of large particles. The other mean value occasionally used is the D3,2,
or surface mean. This value is only typically used when the product is an aerosol or
spray.

11
 1.0 X VS. Y AXIS
0.9 Other published specifications are based on the percent below a given particle
0.8 size such as: 50% below 20µm and 90% below 100µm. This type of specification
0.7 is based on points along the y axis (which reports frequency percent) as opposed
undersize error of +/- 20%
to the x axis (which reports diameter) as in the previous examples. Although
0.6
% UNDER

this approach has been used in many specifications, it is important to realize the
0.5
difference between using the x (size) and y (percent) axes. All measurements
0.4
include an error which should always be considered when setting a specification.
0.3

0.2 For the example shown in Figure 14, the D50 is 100µm with an error of +/- 5%
size error
0.1 of +/- 5% on the x (size) axis. This error includes all sources such as sampling and sample
preparation. The same error becomes +/- 20% when translated to the y (percent)
20 40 60 80 100 120 140 axis. Stating an error of +/- 5% is more attractive than +/- 20%, even when
SIZE IN µm
expressing the same actual error range. The degree to which the y axis error is
figure 14
| MEASUREMENT ERROR
Error appears exaggerated on
the Y axis because of the
exaggerated vs. the x axis depends upon the steepness of the distribution curve.

There are applications where the percent below a given particle size is an important
narrowness of the PSD
result. Recently there has been interest in the presence of “nanoparticles” (at least
one dimension smaller than 100nm) in products such as cosmetics. The software
which calculates the PSD should be capable of easily reporting the percent under
any chosen size—in this case the percent below 100nm (Figure 15). In the LA-960V2
software this is displayed as “Diameter on Cumulative %”. In the example below the
value for percent less than 100nm is reported as 9.155%.

Several points are worth mentioning in regards to setting a specification on the

percent below 100nm as in this example specifically and for sub-micron materials
generally. The particle size distribution is dependent upon many factors including
the sample preparation method. The laser diffraction technique works best within
a certain particulate concentration range. This sometimes requires that samples
undergo dilution. In some cases this dilution may change the state of the particles
and affect the apparent size distribution. Additionally, ultrasonic energy can be
applied to improve the dispersion of agglomerates which can significantly change
the result.

TESTING REPRODUCIBILITY

figure 15
| REPORTING PSD PERCENTAGE There are currently two internationally accepted standards written on the use of
SMALLER THAN THE GIVEN SIZE laser diffraction: ISO 13320 (ref. 9) and USP<429> (ref. 10). Both standards state
In this example, percentage of
that samples should be measured at least three times and reproducibility must
the PSD is reported at 100nm.
meet specified guidelines. Note that this means three independent measurements
(i.e. prepare the sample, measure the sample, empty the instrument, and repeat).
The coefficient of variation (COV, or (std dev/mean)*100) for the measurement set
must be less than 2.5% for D50, less than 3% for D10, and less than 4% for D90
to pass the ISO 13320 requirements. These guidelines change to less than 10%
at the D50 and less than 15% at the D10 and D90 when following the USP<429>
requirements. Finally, the guidelines all double when the D50 of the material is less
than 10µm.

While following the ISO or USP guidelines to test reproducibility is suggested, it is

typically part of an internal specification or procedure. The specifications shown to
potential customers typically don’t include the reproducibility values.

12
INCLUDING THE ERROR

The reproducibility errors discussed previously should be investigated and minimized

because they play an important role in the final setting of a specification. Once
the specification based on product performance has been determined, then the
final specification must be narrowed by the error range (ref. 11). In the example
shown in Figure 16 the specification for the D50 is 100 +/- 20% (or 80–120µm)
based on product performance. If the total measurement error is +/- 10% (using
USP<429> guidelines for the D50 value), the specification must be tightened to
~90–110µm (rounded for simplicity) in order to assure the product is never out of
the performance specification. For example, if the D50 is measured to be 110µm, we
are certain the D50 is actually less than 120µm even with a maximum 10% error.

This is why it is important to create robust standard operating procedures for

any material we wish to set a published specification for. Any combination of high
measurement error (usually stemming from non-optimized method development)
and tight specifications will make meeting that specification more difficult.
Why make life harder than it need be?

figure 16
| BUILDING SIZE SPECIFICATION
TO INCLUDE ERROR SOURCES
If the total measurement error is
+/- 10%, then the specification
PRODUCT PERFORMANCE SPECIFICATION must be tightened in order to
assure the product stays within
performance specification.
80 85 90 95 100 105 110 115 120

SIZE IN µm

SPECIFICATION INCLUDING ERROR

DYNAMIC LIGHT SCATTERING

The primary results from dynamic light scattering (DLS) systems are typically
reported as an intensity distribution. Key values included in DLS-based specifications
are the intensity-weighted average (often called the “z average”) and the
polydispersity index (PI), which quantifies distribution width. Mean values for one
or more peaks can be calculated and included in the results. The results can be
transformed into a volume-based or number-based distribution when comparing to
other techniques such as laser diffraction or microscopy.

13
 IMAGE ANALYSIS

The primary result reported by image analysis is a number distribution since the
particles are inspected one at a time. Setting specifications based on the number
distribution is acceptable, but this is the one example where conversion to another
basis (i.e. volume) is both acceptable and often preferred. As long as a sufficient
number of particles are inspected to fully define the distribution, then the conversion
from number to volume does not introduce unknown errors into the result. The
pharmaceutical industry discussed the subject at a meeting organized by the AAPS
(ref. 6) and concluded that results are preferably reported as volume distributions.

Particle size distribution specifications based on the image analysis technique often
include the mean, D10, D50, and D90 values. Care should be taken to avoid basing
specifications on the number-based mean since this value may not track process
changes such as milling or agglomeration (ref. 12). Conversion from number to
volume distribution can be performed with high accuracy by specifying the typical
particle shape (spherical, cylindrical, ellipsoidal, tetragonal, etc.).

Particle shape parameters such as roundness, aspect ratio, and compactness are
used to describe particle morphology. Specifications for shape parameters are
typically reported using just the number-based mean value, so this is recommended
for setting specifications.

CONCLUSIONS

The task of setting a particle size specification for a material requires knowledge
of which technique will be used for the analysis and how size affects product
performance. Sources of error must be investigated and incorporated into the final
specification. Be aware that, in general, different particle sizing techniques will
produce different results for a variety of reasons including: the physical property
being measured, the algorithm used, the basis of the distribution (number, volume,
etc.) and the dynamic range of the instrument. Therefore, a specification based on
using laser diffraction is not easily compared to expectations from other techniques
such as particle counting or sieving. One exception to this rule is the ability of
dymanic image analysis to match sieve results.

Attempting to reproduce PSD results to investigate whether a material is indeed

within a stated specification requires detailed knowledge of how the measurement
was acquired including variables such as the refractive index, sampling procedure,
sample preparation, amount and power of ultrasound, etc. This detailed information
is almost never part of a published specification and would require additional
communications between the multiple parties involved.

14
 LA-960V2
LASER
DIFFRACTION
TECHNIQUE
The LA-960V2 combines the most popular modern sizing technique with state of the
art refinements to measure wet and dry samples measuring 10 nanometers to 5
millimeters. The central idea in laser diffraction is that a particle will scatter light at an
angle determined by that particle’s size. Larger particles will scatter at small angles and
smaller particles scatter at wide angles. A collection of particles will produce a pattern
of scattered light defined by intensity and angle that can be transformed into a particle
size distribution result.
RANGE IN MICRONS
10 nm - 5,000 µm
INTRODUCTION
OPTIMAL APPLICATIONS
The knowledge that particles scatter light is not new. Rayleigh scattering of light from POWDERS, SUSPENSIONS,

particles in the atmosphere is what gives the sky a blue color and makes sunsets AND EMULSIONS

yellow, orange, and red. Light interacts with particles in any of four ways: diffraction, WEIGHT 56kG (123 lbs)

reflection, absorption, and refraction. Figure 17 shows the idealized edge diffraction FOOTPRINT

of an incident plane wave on a spherical particle. Scientists discovered more than a WIDTH 705mm (28”)
DEPTH 565mm (22”)
century ago that light scattered differently off of differently sized objects. Only the
HEIGHT 500mm (20”)
relatively recent past, however, has seen the science of particle size analysis embrace
light scattering as not only a viable technique, but the backbone of modern sizing.

figure 17
| DIFFRACTION PATTERN
OF A PLANE WAVE
SCATTERING FROM
A SPHEROID

Bench-top laser diffraction instruments

became practical with the advent of high
intensity, reasonably priced lasers and
sufficient computing power to process
the scattered light data. Once these
barriers to market entry were eliminated
the advantages of laser diffraction over
other techniques were apparent: speed
of analysis, application flexibility, small
particle accuracy, and ease of use. The
ability to measure nano, micro and
macro-sized powders, suspensions,
and emulsions, and to do it within one
minute, explains how laser diffraction
displaced popular techniques such as
sieving, sedimentation, and manual
microscopy.
 Such an instrument consists of at least one source of high intensity, monochromatic
light, a sample handling system to control the interaction of particles and incident
light, and an array of high quality photodiodes to detect the scattered light over a
wide range of angles. This last piece is the primary function of a laser diffraction
instrument: to record angle and intensity of scattered light. This information is then
input into an algorithm which, while complex, reduces to the following basic truth:

LARGE PARTICLES SCATTER INTENSELY AT NARROW ANGLES

SMALL PARTICLES SCATTER WEAKLY AT WIDE ANGLES

The algorithm, at its core, consists of an optical model with the mathematical
transformations necessary to get particle size data from scattered light. However,
not all optical models were created equally.

THE IMPORTANCE OF OPTICAL MODEL

In the beginning there was the Fraunhofer Approximation and it was good. This
model, which was popular in older laser diffraction instruments, makes certain
assumptions (hence the approximation) to simplify the calculation. Particles are
assumed:
• to be spherical
• to be opaque
Scattering Intensity

• to scatter equivalently at wide angles as narrow angles

• to interact with light in a different manner than the medium

Practically, these restrictions render the Fraunhofer Approximation a very poor

choice for particle size analysis as measurement accuracy below roughly 20 microns
is compromised.
scattering angle, θ

The Mie scattering theory overcomes these limitations. Gustav Mie developed a
figure 18
| REPRESENTATIONS OF
FRAUNHOFER AND MIE
SCATTERING
closed form solution (not approximation) to Maxwell’s electromagnetic equations for
scattering from spheres; this solution exceeds Fraunhofer to include sensitivity to
Angle, energy and size are used smaller sizes (wide angle scatter), a wide range of opacity (i.e. light absorption), and
as parameters in these examples.
the user need only provide the refractive index of particle and dispersing medium.
Accounting for light that refracts through the particle (a.k.a. secondary scatter)
allows for accurate measurement even in cases of significant transparency. The Mie
theory likewise makes certain assumptions that the particle:
• is spherical
• ensemble is homogeneous
• refractive index of particle and surrounding medium is known

Figure 18 shows a graphical representation of Fraunhofer and Mie models using

scattering intensity, scattering angle, and particle size (ref. 13). The two models
begin to diverge around 20 microns and these differences become pronounced
below 10 microns. Put simply, the Fraunhofer Approximation contributes a
magnitude of error for micronized particles that is typically unacceptable to the
figure 19
| MIE (RED) AND FRANHOFER
BLUE) RESULTS FOR
SPHERICAL GLASS BEADS
user. A measurement of spherical glass beads is shown in Figure 19 and calculated
using the Mie (red) and Fraunhofer (blue) models. The Mie result meets the material
specification while the Fraunhofer result fails the specification and splits the peak.
The over-reporting of small particles (where Fraunhofer error is significant) is a
typical comparison result.

16
BUILDING A STATE OF THE ART
LASER DIFFRACTION ANALYZER

The basics of what needs to be measured and how it’s transformed into particle size
data are understood (ref. 14). What constitutes a basic particle size analyzer has
also been discussed, but there’s a wide gulf between bare minimum and state of
the art. The latter is always the industry leader in accuracy, repeatability, usability,
flexibility, and reliability. The current state of the art in laser diffraction is the Partica
LA-960V2 featuring two high intensity light sources, a single, continuous cast alumi-
num optical bench (Figure 20), a wide array of sample handling systems, and expert
refinements expected from the fifth revision in the 900 series.

3
2
figure 21
| LIGHT SCATTERING PATTERNS
FOR 50nm AND 70nm PARTICLES
USING 650nm LASER

4 4

figure 20
| SIMPLIFIED LAYOUT OF THE LA-960V2 OPTICAL BENCH
1. Red wavelength laser diode for particles > 500nm
2. Blue LED for particles < 500nm
3. Low angle detectors for large particles
4. Side and back angle

Using two light sources of different wavelengths is of critical importance because the
figure 22
| LIGHT SCATTERING PATTERNS
FOR THE SAME SAMPLES
USING 405nm LED
measurement accuracy of small particles is wavelength dependent. Figure 21 shows
the 360° light scattering patterns from 50nm and 70nm particles as generated from
a 650 nm red laser. The patterns are practically identical across all angles and the 40
60
algorithm will not be able to accurately calculate the different particle sizes. Figure 50 LATEX
22 shows the same experiment using a 405nm blue LED. Distinct differences are STANDARDS
(µm)
50
now seen on wide angle detectors which allows for accurate calculation of these 30 70
materials. Integrating a second, shorter wavelength light source is the primary
40
means of improving nano-scale performance beyond that of a bare minimum laser
diffraction analyzer.
30

CONCLUSIONS
20
The HORIBA LA-960V2 particle size analyzer uses the laser diffraction method to
measure size distributions. This technique uses first principles to calculate size using
10
light scattered off the particle (edge diffraction) and through the particle (secondary
scattering refraction). The LA-960V2 incorporates the full Mie scattering theory to q (%)
cover the widest size range currently available. Wide measurement ranges, fast 0.010 0.100 1.000
analyses, exceptional precision, and reliability have made laser diffraction the most DIAMETER (µm)

|
popular modern sizing technique in both industry and academia. figure 23 30, 40, 50 AND 70 NANOMETER
MATERIALS MEASURED
INDEPENDENTLY ON THE LA-960V2
USING THE BLUE LED

17
 LA-350
LASER
DIFFRACTION
TECHNIQUE
The LA-350 Laser Diffraction Particle Size Distribution Analyzer excels in applications as
diverse as slurries, minerals, and paper chemistry. Based on the advanced optical design
of previous LA-series analyzers, the LA-350 strikes a harmonious balance between high-
functionality, easy operation, and low maintenance. The optimized design allows for a
compact optical bench, resulting in an efficient use of bench space, while preserving the
accuracy, precision and resolution that HORIBA’s analyzers are famous for.

RANGE IN MICRONS
0.1 - 1,000 µm SMALL AND POWERFUL

OPTIMAL APPLICATIONS The combination optical bench and circulation pump in one system is one of HORIBA’s
POWDERS, SLURRIES, most popular designs. Now this design has a much smaller footprint which allows the
AND EMULSIONS
analyzer to be moved where it is needed. This is especially valuable for quality control
FOOTPRINT situations when the locations of sampling and analysis need to be separate to avoid
WIDTH 297 mm (12”)
contamination. The optical
DEPTH 420 mm (17”)
bench and circulation pump
HEIGHT 376 mm (15”)
are combined into a single
compact system. The compact
size and low weight make this a
convenient analyzer for today’s
crowded laboratories.

Instant automatic alignment function with blanking

and sample measurement ensures reproducible
measurements and reliable performances. The laser
diode light source provides stable performance
throughout the long lifetime of the analyzer. The
detectors, lens, and mirrors are protected within
the interior of the instrument. The design has been
rigorously tested for durability and robustness.

SIMPLE OPERATION. EXQUISITE PERFORMANCE.

The Partica mini covers a wide range of sizes from

100nm to 1000 microns. The analysis guarantees
that production quality and development process will
be accurate. Measurement accuracy support:±1.4%
guaranteed data accuracy with specified NIST
traceable standard materials.

The Fraction cell optional accessory enables

measurement of very limited sample amounts and
collecting them after the measurement. The stir
bar inside of the cell of the cell prevents the particle
segregation and sedimentation.
 ViewSizer 3000
NANOTRACKING
ANALYSIS
TECHNIQUE
A BREAKTHROUGH IN NANOPARTICLE TRACKING ANALYSIS
As with conventional NTA, the instrument visualises scattering from individual
nanoparticles in suspension. This data is then used to determine particle movement
and infer particle size using the Stokes Einstein relationship. Then, by using the known
illuminated and imaged sample volume, particle number concentration is readily
determined. Thus, from a single measurement, two critical pieces of information are
determined: particle size distribution and particle concentration.
RANGE IN MICRONS
10 nm - 15 µm
By incorporating three variable light sources (blue, green and red) the instrument
OPTIMAL APPLICATIONS
is able to select the optimum conditions for any sample analysis, overcoming the POWDERS
limitations of NTA when analysing polydisperse sample and enabling a much larger
WEIGHT 27kG (59.5 lbs)
range of particle sizes to be visualised.
FOOTPRINT
WIDTH 550mm (22”)
Furthermore, by introducing the sample in easy-to-use (and clean) cuvette, DEPTH 660mm (26”)
the ViewSizer 3000 is able to repeatedly ‘analyse and stir’, giving a much more HEIGHT 350mm (14”)

reproducible result. And because the sample is viewed in a vertical orientation, it is

perfect for visualising processes such as protein aggregation or crystal dissolution.

HOW IT WORKS
The instrument characterizes nanoparticles by analyzing their thermal-induced motion
(Brownian motion) and larger, micron-sized particles by analyzing gravitational
settling. The optical system includes innovative multispectral illumination and
detection techniques that enable video recording of scattered light from wide-ranging
sizes of individual particles simultaneously.

The obtained video shows each individual nanoparticle. By taking

advantage of modern high resolution video cameras and computer
graphics processing speed, the motion of each particle is tracked
to determine the diffusion coefficient, and, from that, the size
of each particle.

A key advancement of this system is its ability to work

with the very large dynamic range of scattered light
intensity produced by differently-sized nanoparticles
coexisting in a polydisperse sample. This technical
feat is accomplished by combining clever software
with advanced optics and multiple light sources. The
ViewSizer 3000 technology is an elegant and absolute
method that does not require knowledge of particle
material properties such as refractive index.
 SZ-100V2
DYNAMIC LIGHT
SCATTERING
TECHNIQUE
The SZ-100V2 nanoPartica Dynamic Light Scattering (DLS) system measures particle

PARTICLE SIZE size, zeta potential, and molecular weight from 0.3 nm to 8 µm at concentrations ranging
from 0.1 mg/mL of lysozyme to 40% w/v. This section explains the underlying principles
ZETA POTENTIAL used by the SZ-100V2 DLS system.

MOLECULAR PARTICLE SIZE

WEIGHT Particle size can be determined by measuring the random changes in the intensity of
light scattered from a suspension or solution. Small particles in suspension undergo
random thermal motion known as Brownian motion. This random motion is measured to
RANGE IN MICRONS
calculate particle size using the process described below. A top view of the optical setup
0.3 nm - 10 µm
for particle size measurements in the SZ-100V2 is shown in Figure 24.
OPTIMAL APPLICATIONS
NANOSUSPENSIONS
AND EMULSIONS UNDER 10 µm,
ZETA POTENTIAL AND
MOLECULAR WEIGHT

WEIGHT 25kG (55 lbs)

Backangle Right angle
FOOTPRINT detector detector
WIDTH 528 mm (21”)
DEPTH 385 mm (18”)
HEIGHT 273 mm (11”)
laser
Sample

|
figure 24 DYNAMIC LIGHT
SCATTERING LAYOUT
FOR THE SZ-100V2

Light from the laser light source

illuminates the sample in the cell. The
scattered light signal is collected with one
of two detectors, either at a 90 degree
(right angle) or 173 degree (back angle)
scattering angle. The obtained optical
signal shows random changes due to the
randomly changing relative position of the
particles. This is shown schematically in
Figure 25.
 figure 25 | LIGHT SCATTERING
FLUCTUATIONS DUE TO
INTENSITY (arb. units)

BROWNIAN MOTION VS. TIME

1.0 The optical signal shows

random changes due to the
randomly changing relative
position of the particles.

0.0
TIME (microseconds)

The signal can be interpreted using an autocorrelation function. Incoming data is

processed in real time with a digital signal processing device known as a correlator
and the autocorrelation function, shown in Figure 26 as a function of delay time, Τ, is
extracted.

2.0

|
figure 26 AUTOCORRELATION FUNCTION
FROM DYNAMIC LIGHT
SCATTERING
For a sample where all of
ACF

1.5
the particles are the same
size.

1.0
0 100 200 300 400 500

DELAY TIME (µsec)

The autocorrelation function from dynamic light scattering in Figure 26 shows

a sample where all of the particles are the same size, the baseline subtracted
autocorrelation function, C, is simply an exponential decay of the following form:

EQUATION 1 C = exp(-2 Γ)

Γ is readily derived from experimental data by a curve fit. The diffusion coefficient
is obtained from the relation Γ=Dt q 2 where q is the scattering vector, given by
q= (4πn/λ) sin (θ/2). The refractive index of the liquid is n. The wavelength of the
laser light is λ, and scattering angle, θ. Inserting Dt into the Stokes-Einstein equation
then solves for particle size Dh is the final step.

kBT
EQUATION 2 Dh =

3πηDt
Where:
Dh = the hydrodynamic diameter
Dt = the translational diffusion coefficient
kB = Boltzmann’s constant
T = temperature
η = dynamic viscosity

21
 ZETA POTENTIAL

Zeta potential is a measure of the charge on a particle surface in a specific liquid

medium. This value of surface charge is useful for understanding and predicting
interactions between particles in suspension. Manipulating zeta potential is a
method of enhancing suspension stability for formulation work, or speeding particle

slipping flocculation in applications such as water treatment. Zeta potential is measured on

pane the SZ-100V2 using the technique of electrophoretic light scattering where particle
motion is detected in an applied electric field.

The charge on the surface of a particle influences the ionic environment in the
region close to the particle surface. This ionic environment is typically described
using a double layer model – the Stern layer of ions firmly attached adjacent to
the particle surface, and the diffuse layer further away from the particle surface,
but still attracted to the particle such that these ions will move with the particle.
zeta
mV potential The boundary between the electric double layer and the ions in equilibrium in the
negatively charged solution is called the slipping plane, as shown in Figure 27. Zeta potential is defined
particle surface
dispersion as the potential measured in mV at the slipping plane distance from the particle
surface.

To measure zeta potential a small quantity of sample is injected into a cell

containing two electrodes that are used to create an induced electric field. Once

|
figure 27 ZETA POTENTIAL
The zeta potential is the the electric field is applied the particles move toward either the anode or cathode
charge in mV measured at depending on whether the surfaces are positively or negatively charged. The
the slipping plane. direction of the motion indicates positive vs. negative charge. The speed of the
particle motion is used to calculate the magnitude of the charge.

TRANSMITTED LIGHT
ELECTRODE PARTICLE MONITOR (PD)

LASER LIGHT SOURCE

CELL
REFERENCE BEAMS
ZETA POTENTIAL
MEASUREMENT

FORWARD DETECTOR
(PMT)

MODULATOR

|
figure 28 OPTICAL DIAGRAM OF THE SZ-100V2
CONFIGURATION FOR ZETA POTENTIAL

As shown in the top view, above, of the optical setup for zeta potential
measurements in the SZ-100V2, the particles are illuminated with laser light and,
therefore, the particles scatter light. A second beam of light (the reference beam)
is mixed with the scattered beam in order to sensitively extract the frequency shift
in the scattered light. The measured magnitude of the frequency shift is then used
to determine the particle velocity. Equation 1 is used to calculate the electrophoretic
mobility (µ) using the measured frequency shift.

22
 Δωλ0
EQUATION 1 μ =
θ’ )
4πnE sin( —
Where : 2
μ = the electrophoretic mobility
ω = the measured frequency shift
λ = the laser wavelength
n = the refractive index of the medium
θ’ contains the angular light scattering information

After the electrophoretic mobility is determined using equation 1, the zeta

potential (ζ) is calculated using equation 2.

2ζε
EQUATION 2 μ = ƒ(κr)
3ηο
Where:
µ = the electrophoretic mobility
ζ = the zeta potential
ε = the dielectric permittivity of the medium
ηo = the viscosity
f=(κr) = a function describing the ratio of the particle radius to the double layer

Zeta potential is often measured as a function of pH (or other additive property)

in order to determine the conditions at which there is zero zeta potential, also
known as the isoelectric point (IEP).

MOLECULAR WEIGHT
The SZ-100V2 can also be used to measure the molecular weight of proteins,
starches, polymers, dendrimers and other large molecules. The data can be ob-
tained by two different methods: dynamic light scattering and static light
scattering. Both methods are discussed below.

Dynamic Light Scattering

There is a well-known empirical correlation between the diffusion coefficient of a
macromolecule and its molecular weight known as the Mark-Houwink-Sakurada
equation.
a
Dt = kM

Where:
Dt = diffusion coefficient
k = empirical constant
M = molecular weight
α = an empirical constant

The values for k and a are found empirically for polymer/solvent pairs. That is,
they must be specified for the polymer, solvent, and temperature. These values can
be found in the literature.

The advantages of this technique are that polymer concentration need not be
known and that molecular weight can be determined rapidly. It does, however, rely
on empirical constants and the nature of the average molecular weight.

23
 Static Light Scattering
The SZ-100V2 can also be used in a static light scattering mode to measure the
molecular weight of proteins, small particles, and polymers. These results are
generated using a Debye plot (Figure 29) created by measuring the scattered
light at a single angle (90°) at multiple sample concentrations. The intercept of
the Debye plot is used to determine the molecular weight and the slope is used to
calculate the second virial coefficient.

Molecular weight from static light scattering experiments uses the Rayleigh
equation given below:

lim Kc = 1 + 2A 2c
θg0 ΔRθ Mw
Where:
K = the Debye constant
C = the sample concentration
Rθ = the Rayleigh ratio
Mw = the weight average molecular weight
A2 = the second virial coefficient

The Debye constant is given by K= 4π2n2 (dn/dc)2/(λ4NA) where n is the refractive

index of the liquid, (dn/dc) is the refractive index increment, λ is the wavelength of
light in vacuo, and NA is Avogadro’s number. In most cases, all of these values are
independent of molecular weight.

The limit given in the equation above deserves special attention. The equation only
works at the limit of zero angle. One practice required for larger macromolecules is
to use a multi-angle scattering instrument and extrapolate the result to zero angle.
For smaller molecules (Rg<20nm), this is not necessary and data at a single angle
can be used. However, this does introduce a systematic error that increases with
angle used. That is, measurement results using back angle have about twice the
systematic error compared to results obtained using scattering at right angle (90°).
For this reason, the SZ-100V2 collects light scattering data at 90°.

The advantage of this technique is that the results are well-defined and not reliant
on empirical correlations, although it requires careful sample preparation and is a
more time-intensive process.

CONCENTRATION OF POLYSTYRENE 1kDa (mg/mL)

KC/R OF POLYSTYRENE 96kDa (10-5mol/g)

KC/R OF POLYSTYRENE 1kDa (10-3mol/g)

0 5 10 15 20 25 30 35

|
figure 29
3.5 1.4
DEBYE PLOTS TO MEASURE
MOLECULAR WEIGHT OF
SEVERAL SAMPLES
AND CHLOROPHYLL

3.0 1.3

POLYSTYRENE (1kDa)
2.5 1.2
CHLOROPHYLL

POLYSTYRENE (96kDa) 2.0 1.1

1.5 1.0

1.0 0.9
0 1 2 3 4 5

CONCENTRATION OF POLYSTYRENE 96kDa (mg/mL)

AND CHLOROPHYLL

24
 PSA300
IMAGE
ANALYSIS
TECHNIQUE
The microscope has always been the referee technique in particle
characterization since it is accepted as the most direct measurement
of particle size and morphology. Automating manual microscopy has
been driven by the desire to replace a tedious, somewhat subjective
measurement with a sophisticated technique for quantifying size and
shape of a sufficient number of particles to assure statistical confidence
with the end result. Analysts performing manual microscopy tend to describe RANGE IN MICRONS
particle shape using language such as round, blocky, sharp, fibrous, etc. By 0.5 nm - 1,000 µm
assigning quantitative values rather than qualitative to various shape descriptors, OPTIMAL APPLICATIONS
image analysis systems provide numerical distributions of well defined shape POWDERS AND SUSPENSIONS
parameters WEIGHT 34kG (75 lbs) w/o computer

FOOTPRINT
Two distinct development paths have emerged over time differing in how the WIDTH 686mm (27”)
sample is introduced to the measurement zone: dynamic image analysis where DEPTH 483mm (19”)
HEIGHT 446mm (17.5”)
particles flow past one or more cameras and static image analysis where particles
sit on a slide moved by an automated stage for inspection by camera and
microscope.

Many basic functions operate the same with either approach (Figure
29): particles are presented to the measurement zone, images are
captured with a digital (CCD) camera, the particles are distinguished
from the background, various size and shape parameters are
measured for each particle, and a result report is generated. Additional
features built into modern image analysis software include the ability
to automatically separate two particles touching each other, filling
holes, smoothing or removing small protuberances,
separating overlapping circular objects, and
keeping track of incomplete objects in a field
in order to recombine them once all fields
are analyzed.

The use of automated image analysis is

increasingly being recognized as an
attractive technique to characterize
particle shape and size distribution.
Traditional methods, such as laser
diffraction, although highly efficient,
give limited information on particle
shape proving image analysis as one
of the best tools for performing
particle analysis.
 STATIC IMAGE ANALYSIS

The samples measured by static image analysis typically rest on a slide that is
moved by an automated stage. With the PSA300 static image analysis system
a microscope and digital camera collect images of the particles as the slide is
scanned. Samples prepared on slides can include powders, suspensions, or creams.
Aerosol delivery forms such as metered dose inhalers or dry powder inhalers can
be inspected using static image analysis by actuating the device onto a slide for
measurement. In addition, particles in suspension (such as parenterals) can be
collected on a filter for characterization.

The majority of static image analysis measurements are made on powders, typically
used for solid oral dosage forms. Most powders require a sample preparation
step prior to analysis. Powder preparation devices—using either positive pressure
to impact on a hard surface or pulling and releasing a vacuum—break apart
agglomerates and create an even dispersion on the slide. After the sample has
been prepared and the automated stage has presented multiple fields to the
optics and camera for capture, a series of image processing steps occur in the
software. The first step is to separate the particles from the background by setting a
parameter with some threshold value. Setting this threshold can be done manually
or automatically based on phases in the grayscale image or through a contrast
threshold function based on the particle/background contrast.

After the threshold operation is completed several functions may be applied to the
image to improve the edge definition. The basic functions of erosion and dilation
improve edge definition by performing opposite tasks of removing or adding dark
pixels at the particle edge. Advanced functions using combinations of erosion and
dilation steps such as delineation and convex hull improve the edge definition of
particles, leading to accurate area and perimeter determinations that are critical for
shape factor calculations. Other software functions perform the task of separating
touching particles including the crossed fibers in order to quantify fiber length
distributions and aspect ratios.

75 100

60 80

45 60

30 40

15 20

0 0
1 2 5 10 30 50 100

IMAGE THRESHOLDING CALCULATIONS

ACQUISITION Separates particles from Generation of results
Captured with a digital the background
(CCD) camera

|
figure 30 BASIC IMAGE ANALYSIS FUNCTIONS
Both static and dynamic image analysis
involve these basic steps.

26
 Eyecon2 ™ IN-LINE
IMAGE
ANALYSIS
TECHNIQUE
In-line image analysis shares many of the features of laboratory analysis.
The major difference is that the measurement takes place directly in the
process and this poses several challenges. First, the process must have an
appropriate window for imaging. Second, the window location must have a
sufficient number of particles visible for good statistics and those particles
must be replaced over time to accurately reflect the current state of the RANGE IN MICRONS
process. Finally, the instrument, both camera and illumination, need to 50 - 5500 μm

be optimized for this application. If these conditions are met, then in-line OPTIMAL APPLICATIONS
analysis gives real-time feedback that enables better process monitoring POWDERS AND BULK SOLIDS

and control for higher yields. WEIGHT 4kG (8.8 lbs)

FOOTPRINT
WIDTH 250mm (9.8”)
DEPTH 128mm (5”)
HEIGHT 132mm (5.2”)

IN-LINE AND AT-LINE/BENCHTOP

The Eyecon2™, developed by Innopharma Technology, is a real-time, direct imaging

particle analyzer built to meet the standards of the pharmaceutical industry but can
be used anywhere powders and bulk solids flow. It enables faster understanding of
particle size, shape and variation with real-time particle size distribution and shape
data updated every ~ 5 sec. With a measurement range from 50 - 5500 μm, it
is well-suited for use in the food, chemical, agricultural, cosmetics and veterinary
industries. Non-product contact ensures proper measurements every time.

Direct imaging with the Eyecon2 enables users to understand particle size and shape
variation and in turn determine why a process is failing, why yield is reduced, what
the source of product variation is, and whether or how a
process can be best scaled up to commercial manufacturing.

The AI upgrade vastly expands the range of material types

that can be measured in real time and drastically improves
particle size and shape detection for materials previously
unviable for image analysis. The EyePASS software performs
particle segmentation i.e., the detection and delineation
of particles from the images sent by the Eyecon2. All
measurements reported by the software are based on the
results of the particle segmentation step.
 CENTRIFUGE
CENTRIFUGAL
SEDIMENTATION
TECHNIQUE
The centrifugal sedimentation method is a method for determining the
particle size from the speed of particles moving by centrifugal force (settling
velocity). When a centrifugal force is applied to particles, larger particles settle
faster and smaller particles settle slower. The settling velocity is calculated
from the distance traveled by the particle (from the initial position to the
detection position as shown in the figure below) and the time required for the

RANGE IN MICRONS
movement. It is possible to measure particles while classifying them.
10 nm - 40 μm

WEIGHT 100kG (220 lbs)

FOOTPRINT
WIDTH 634mm (25”)
DEPTH 550 mm (21.7”)
HEIGHT 341mm (13.4”)

PARTICLE SIZE DISTRIBUTION

With a measurement range of 10 nm - 40 μm, the CN-300 Partica CENTRIFUGE applies
centrifugal force of up to 30,000g and uses temperature control to produce accurate
measurement results for a variety of samples. The key features of centrifugation is that
the particle size is measured following classifying by size. As a result, a wide range of
high-precision results can be obtained in a single analysis.

With its high resolution, small amounts of foreign

particle or agglomeration can be captured. This
instrument obtains reliable measurement results
throughout the range of the particle size distribution,
including small populations, as well as to the low area
of the distribution.

The cooling function of the sample chamber and

the rotor prevents temperature increases of sample
fraction during rotation. This improves the reliability
of the measurement results by keeping the viscosity
constant.

Partica CENTRIFUGE is designed for quieter, easier,

and safer operation. Simply insert a cell filled with
sample into the rotor. The cuvette-type cell is easy to
clean and replace, reducing the risk of contamination.
Additionally, it complies with international safety
standards (IEC6101-1/2-020).
 SA-9600
SURFACE AREA
ANALYSIS
TECHNIQUE
The surface area of a material is, in many cases, as important as the
chemical properties. As particle size decreases, surface area increases.
The interface at the surface is what defines how a solid reacts to other
substances, be they gases, liquids, or solids.

Surface area can impact shelf life, stability, dissolution and efficacy of
pharmaceutical powders and tablets. Likewise, surface area can affect the RANGE
rheological properties and hiding powers of pigments, paints, and coatings. It has Total Surface Area: 0.1 to 50 m2
a significant impact on the ability for materials like catalysts, adsorbents, filtration Specific Surface Area: ~ 0.01 – 2,000 m2/g

materials and air separation products to react in the designed application. OPTIMAL APPLICATIONS
Ceramics used in applications ranging from; dinner plates, to dental implants, to POWDERS

electronics, all are affected by surface area. WEIGHT 14.5 - 18.14kG (34.5 - 42lbs)

FOOTPRINT
While particle size is frequently used to control size reduction and milling WIDTH 356mm (14”)
DEPTH 356mm (14”)
of minerals and other substances, surface area can provide substantial size
HEIGHT 508mm (20”)
reduction feedback. Many times, a material which may have the same particle
size across different batches may reveal completely different surface areas due to
small changes in processing.

BET FLOWING GAS SURFACE AREA

The SA-9600 series of surface area analyzers brings exceptional speed,

convenience, and low cost-per-analysis to surface area measurement on a
wide variety of materials. Now ultra-fast single or multi-point surface area
measurements can be performed with push-button ease.

These tools use the robust and proven flowing gas method to
acquire gas adsorption and desorption data. This information
is then used to calculate total surface area utilizing the well-
known BET method. The advantage of the flowing gas method
is most evident in single-point mode where up to thirty sample
analyses can be performed per hour.

The patented SA-9600 technology provides routine total

surface area determination in as little as two to six minutes
with all analysis and preparation stations in one small, well-
designed cabinet. If expansion is needed, additional stations
are added in the same footprint – not by adding more devices
which consume more space on the lab bench. The SA-9600
may also be controlled from the built-in computer and
keyboard to save additional space.
 Selecting a particle size analyzer
Beginning the selection of a particle size The decision process may be different if the instrument is being purchased
analyzer should start with asking basic for a specific application as opposed to a general analytical technique for
questions including: many possible samples. For specific application it makes sense to search the
industry literature to determine if a particular technique is favored over others. If
Why am I making the measurement?
for example the application is liposomes and 90% of all literature found in this field

Must the new instrument match is DLS, then the decision is simple. On the other hand, if this is the first particle size
historic data? analyzer bought by a company for general purpose use, then flexibility and a wide
dynamic range should be important factors.
Do I need only particle size
distribution, or do I need additional
Sometimes the goal to buy a new instrument includes being able to correlate
information such as shape or surface
to existing data. Accomplishing this goal can range from easy to difficult. Just
charge?
upgrading from an older to newer model diffraction analyzer could cause a change in
results. The changes originate from many sources including differences in dynamic
range, advances in algorithms, and mechanic improvements to samplers. Switching
from an existing technique such as sieving to newer techniques like laser diffraction
or dynamic image analysis could also lead to changes in results. Data from sieves
are typically smaller than data from laser diffraction depending on the shape of the
particles. The less spherical the particle, the greater the difference will likely be.

RANGE OF THE HORIBA

PARTICLE CHARACTERIZATION SYSTEMS

1nm 10nm 100nm 1µm 10µm 100µm 1mm

LA-960V2 10nm 5mm

Laser Diffraction

LA-350 0.1µm 1mm

Laser Diffraction

ViewSizer 3000 10nm 15µm

Nanotracking Analysis

SZ-100V2 0.3nm 10µm

Dynamic Light Scattering

PSA300 0.5µm 1000µm

Image Analysis

Eyecon2 50µm 5.5mm

In-Line Image Analysis

CENTRIFUGE 10nm 40µm

Centrifugal Sedimentation

30
Particle size distribution is sufficient information for the majority of particle char-
acterization applications. But some techniques are higher resolution than others.
Ensemble technologies such as laser diffraction and dynamic light scattering are
powerful techniques than are “resolution limited” compared to high resolution
techniques which are based on particle counting (such as electro zone counting
or image analysis). If the goal of the measurement is finding small populations of
particles larger or smaller than the main distribution, then an investigation of the
sensitivity to second distributions should be part of the selection process.

Particle shape information may be either desirable or critical depending on the de-
gree to which shape affects product performance. Particle shape influences bulk
properties of powders including flow and compaction behavior and the viscosity
of suspensions. For specific application such as glass beads used in highway
paint, shape is a critical factor for reflectivity. When particle shape information is
required, microscopy and image analysis are the only techniques that delivery
the desired data. Manual microscopy provides basic qualitative size and shape
information, but automated image analysis generates quantitative data that is
statistically significant. For this reason, both dynamic and static image analysis
are growing techniques replacing manual microscopy.

Surface charge or zeta potential of suspensions is important information for

formulators or chemists working on dispersion stability. For these applications a
DLS system providing both particle size and zeta potential (along with other such
as pH or conductivity) may be the best option.

Consider the application of wanting to measure the particle size distribution of

50nm colloidal silica. Just considering the size range of the sample indicates that
possible techniques include laser diffraction or DLS. One question worth asking
would be will I need other capabilities in the future? If I might need zeta potential
in the future, this removes laser diffraction from the list of possible techniques.
If I might have particles > 1µm in the future, this would eliminate DLS. Be
forewarned that future requirements can be difficult to ascertain and additional
capabilities always carry incremental cost.

WHEN TO CHOOSE LASER DIFFRACTION

Laser diffraction is the most popular particle size technique for reasons including
speed, ease of use, and flexibility. The most basic laser diffraction system can
measure solid particles in suspensions and emulsions. With the addition of a dry
powder feeder the instrument can then also measure dry powders in air. This is a
low concentration technique, so dilution is often required. The complex refractive
index of the sample and diluent must be known for optimum accuracy, but this
information is easier to obtain than is often indicated (more often by competi-
tors than informed scientists). The HORIBA LA-960V2 has a wide dynamic range
capable of measuring down to 30nm and up to 5000µm. This unique ability to
measure particles < 100nm as well as agglomerates as large as hundreds of
microns makes this a credible choice even for nanotechnology applications. Since
this is such a powerful, flexible technique laser diffraction is often the best option
for companies buying their first analyzer, or hoping to satisfy multiple needs and
applications.

31
 WHEN TO CHOOSE DYNAMIC LIGHT SCATTERING

Dynamic Light Scattering (DLS) can measure suspensions and emulsions from 1nm
to 1µm. Both the lower and upper limits are sample dependent. The lower limit
is influenced by concentration and how strongly the particles scatter light. A low
concentration sample of weakly scattering particles near 1nm can be extremely
difficult or at least difficult to reproduce. The upper size limit is determined mainly
by the density of the particles. DLS algorithms are based on all particle movement
coming from Brownian motion. Motion due to settling is not interpreted correctly
by DLS systems. In addition, particles settled on the bottom of the sample cuvette
can not be inspected by the laser light source. Particles with a high density will
settle more quickly than low density particles. The upper limit of DLS may be 8µm
for emulsion samples where the two phases have similar density. The upper limit
of uranium particles may be as small as 300nm. The upper limit of particles with a
density of 1.7 may be around 1µm.

Using DLS does not require any knowledge of the sample RI (it would be required
to convert from intensity to volume distribution), or concentration. What is required
is viscosity, especially for higher concentration samples. Although most modern
DLS systems claim the ability to work at higher concentrations, this is again sample
dependent. Serious DLS work could involve a dilution study to determine the nature
of the particle-particle interactions and presence of multiple scattering. Easy samples
are simply a matter of pipetting the sample into a cuvette and clicking one button.
More sophisticated DLS systems can also measure other sample characteristics
including zeta potential, molecular weight, and second virial coefficient. Generating
this additional information may require a greater skill set of the operator.

WHEN TO CHOOSE IMAGE ANALYSIS

Many laboratories are now replacing manual microscopy with automated image
analysis. While microscopy provides qualitative accuracy and shape information, it
requires automated image analysis to inspect the number of particles requited to
obtain statistically valid quantitative results. Choosing image analysis is often driven
by the desire to generate results that are accurate, sensitive to second populations,
contains shape information, and includes images of the particles. Dynamic image
analysis is used in both research and QC laboratories for particles ranging from
30µm to 30mm. Static image analysis is typically a research tool for measuring
particles in the 0.5 to 1000µm range. Deciding between dynamic or static image
analysis is seldom difficult, as the applications are typically better served by one
technique or the other, as proven through application development studies.

32
REFERENCES

1 (PAGE 3) 11 (PAGE 13)

ISO 9276-2:2001 : Representation of results Wheeler, D., How to Establish Manufacturing
of particle size analysis – Part 2: Calculation of Specifications, posted on spcspress.com at
average particle sizes/diameters and moments ht__tp://www.spcpress.com/pdf/Manufactur-
from particle size distributions ing_Specification.pdf

2 (PAGE 3, 4) 12 (PAGE 14)

ASTM E 799-03 Standard Practice for Determin- Neumann et. al. “What does a mean size
ing Data Criteria and Processing for Liquid Drop mean?” 2003 AIChE presentation at Session 39
Size Analysis Characterization of Engineered particles Novem-
ber 16–21 San Francisco, CA
3 (PAGE 3)
TN154, Particle Size Result Interpretation: 13 (PAGE 16)
Number vs. Volume Distributions, available at ISO 13320, Particle size analysis – Laser diffrac-
www.horiba.com/us/particle tion methods – Part 1: General principles

4 (PAGE 5) 14 (PAGE 17)

ISO 13320-1 Particle size analysis – Laser dif- “Understanding Calculation Level and Iterative
fraction methods Deconvolution.” www.horiba.com/us/particle

5 (PAGE 7)
ISO 13322-2 Particle size analysis – Image
analysis methods – Part 2: Dynamic image
analysis methods

6 (PAGES 8-9, 14)

Burgess, J., Duffy, E., Etzler, F., Hickey, A.,
Particle Size Analysis: AAPS Workshop Report,
Cosponsored by the Food and Drug Administra-
tion and the United States Pharmacopeia, AAPS
Journal 2004; 6 (3) Article 20 (http://www.
aapsi.org)

7 (PAGE 10)
TN154, Particle Size Result Interpretation:
Number vs. Volume Distributions, available at
www.horiba.com/us/particle

8 (PAGE 11)
TN156, Particle Size Result Interpretation:
Understanding Particle Size Distribution Calcula-
tions, available at www.horiba.com/us/particle

9 (PAGE 12)
ISO 13320-1 Particle size analysis – Laser dif-
fraction methods

10 (PAGE 12)
USP<429> Light Diffraction Measurement of
Particle Size

33
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