ISO 13319-1:2021

INTERNATIONAL ISO
STANDARD 13319-1
First edition
Determination of particle size
distribution — Electrical sensing zone
method —
Part 1:
Aperture/orifice tube method
Détermination de la distribution granulométrique — Méthode de
détection de zones électrosensibles —
Partie 1: Méthode d'ouverture/d'orifice du tube
PROOF/ÉPREUVE
Reference number
ISO 13319-1:2021(E)
©
ISO 2021

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ISO 13319-1:2021(E)

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Published in Switzerland
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ISO 13319-1:2021(E)

Contents Page
Foreword .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Symbols . 2
5 Principle . 3
6 General operation . 4
6.1 Response . 4
6.2 Size limits . 5
6.3 Effect of coincident particle passage . 5
6.4 Dead time . 6
6.5 Analysis volume . 7
7 Repeatability and reproducibility of counts . 7
7.1 Instrument repeatability . 7
7.2 Method reproducibility/intermediate precision . 8
8 Operational procedures . 8
8.1 General . 8
8.2 Instrument location . 8
8.3 Linearity of the aperture/amplifier system . 8
8.4 Linearity of the counting system . 8
8.5 Choice of electrolyte solution . 8
8.5.1 General. 8
8.5.2 Special considerations for small apertures (D < 50 µm) . 9
8.5.3 Special considerations for large apertures (D > 400 µm) . 9
8.6 Preparation of electrolyte solution . 9
8.7 Recommended sampling, sample splitting, sample preparation and dispersion . 9
8.7.1 General. 9
8.7.2 Method 1: Using a paste .10
8.7.3 Method 2: Alternative method applicable to low-density particles of less
than 50 µm .10
8.7.4 Suspensions and emulsions .11
8.7.5 Verification of the dispersion .11
8.8 Choice of aperture(s) and analysis volume(s) .11
8.9 Clearing an aperture blockage .11
8.10 Stability of dispersion .12
8.11 Calibration .12
8.11.1 General.12
8.11.2 Calibration procedure — microsphere calibration .13
9 Analysis .13
10 Calculation of results .13
11 Instrument qualification .14
11.1 General .14
11.2 Report .14
Annex A (informative) Derivation of maximum count number to limit coincidence .15
Annex B (informative) Fishbone diagram for method development .17
Annex C (informative) Calibration and control of frequently used apertures .19
Annex D (informative) Mass integration method for calibration and mass balance.20
Annex E (informative) Calibration for the measurement of porous and conductive particles .26
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Annex F (informative) Technique using two (or more) apertures .29
Annex G (informative) Chi-squared test of the correctness of instrument operation or
sample preparation .31
Bibliography .33
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ISO 13319-1:2021(E)

Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www .iso .org/ patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO's adherence to the
World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www .iso .org/
iso/ foreword .html.
This document was prepared by Technical Committee ISO/TC 24 Particle characterization including
sieving, Subcommittee SC 4, Particle characterization.
This first edition of ISO 13319-1 cancels and replaces ISO 13319:2007, which has been technically
revised. The main changes compared to the previous edition are as follows:
— a general update to reflect the needs of modern quality assurance;
— the section on repeatability and inter system variation has been expanded;
— many instruments of this type are under strict controls within the pharmaceutical and related
industries, therefore a new annex has been prepared with details of the factors which should be
considered when developing a validated method in this arena;
— Clause 10 now gives details of the exact parameters which should be reported, in order to present
the method and the key parameters of the result.
A list of all parts in the ISO 13319 series can be found on the ISO website.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www .iso .org/ members .html.
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INTERNATIONAL STANDARD ISO 13319-1:2021(E)
Determination of particle size distribution — Electrical
sensing zone method —
Part 1:
Aperture/orifice tube method
1 Scope
This document specifies the measurement of the size distribution of particles dispersed in an
electrolyte solution using the electrical sensing zone method. This can include biologics such as cells,
but also industrial particles such as carbon, cement, ceramic powders, metal powders, pigments and
polymer powders. The method measures pulse heights and their relationship to particle volumes or
diameters, and is applicable over the range (implementation dependant) from approximately 0,5 μm tο
above 1 mm. This document does not address the specific requirements of the measurement of specific
materials.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
3.1
dead time
time during which the electronics are not able to detect particles due to the signal processing of a
previous pulse
3.2
aperture
small diameter hole through which suspension is drawn
3.3
sensing zone
volume of electrolyte solution within, and around, the aperture in which a particle is detected
3.4
analysis volume
volume of suspension that is analysed
3.5
size bin
size interval to distinguish particle size for size distribution measurement
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3.6
envelope size
external size of a particle as seen in a microscope
3.7
envelope volume
volume of the envelope given by the three-dimensional boundary of the particle to the surrounding
medium
3.8
effective density
density of a porous particle where open pores are filled with liquid and closed pores are not (so included
in the density)
4 Symbols
For the purposes of this document, the following symbols apply.
A amplitude of the most frequent pulse
p
A amplitude of the electrical pulse generated by an arbitrary particle
x
D aperture diameter
d certified mean diameter of the microspheres used for primary calibration
m
d mean diameter of the sieved fraction as determined using microscopy
micr
d mean diameter of the sieved fraction as determined using the ESZ instrument
ESZ
df degrees of freedom
d particle diameter at the lower boundary of a size interval or channel
L
d modal diameter of a certified particle size reference material
p
d reference diameter of the microspheres
ref
d particle diameter at the upper boundary of a size interval or channel
U
f response factor
resp
K calibration constant of diameter
d
K arbitrary calibration constant of diameter of any value to start the mass calibration procedure
da
m mass of sample
M mass balance, percentage of particles accounted for in a measurement in comparison to input
b
particle mass
M mass of particles measured by the instrument
m
n number used to signify the maximum of an integral be it channel number [(in Formula (D.1)] or
number of repeat measurements [in Formulae (G.1) and (G.2)]
n counted particle number
c
N mean of a Poisson distribution, used to describe the temporal spread of counts within a size bin
c
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ISO 13319-1:2021(E)

N count for 5 % coincidence
c5
N
total number of counts across all size intervals
i
ΔN number of counts in a size interval i
i
mean of particles in counts N (i = 1, 2, 3…n)
N
i
p significance level of statistical test
V analysis volume
m
V volume of electrolyte solution in which a mass, m is dispersed
T
arithmetic mean volume for a particular size interval i
V
i
x diameter of a sphere with volume equivalent to that of the particle
x maximum particle size that can be obtained on a specific aperture
max
x minimum particle size that can be obtained on a specific aperture
min
ρ immersed density/effective density (solid density including eventual closed pores, but excluding
open pores within the particles)
2
χ chi-squared statistical distribution
5 Principle
A dilute suspension of particles dispersed in an electrolyte solution is stirred to provide a homogeneous
mixture and is drawn through an aperture in an insulating wall. An electric current applied across
two electrodes, placed on each side of the aperture, enables the particles to be sensed by the electrical
impedance changes as they pass through the aperture. The impedance pulses generated by particle
passage are amplified and digitally captured, and the pulse height and shape is analysed, yielding
particle count data. The pulse height is regarded directly proportional to particle volume. After
employing a calibration factor, a distribution of the number of particles against the volume-equivalent
diameter is obtained. The size range of particles to be measured depends upon the size of the aperture.
Conventionally, particles having a size greater than around 0,5 µm are measured by the technique. A
schematic of the instrumentation is given in Figure 1.
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Key
1 volumetric metering device 7 output
2 valve 8 stirred suspension of particles in electrolyte solution
3 pulse amplifier 9 aperture
4 oscilloscope pulse display 10 counter start/stop triggered by the volumetric device
5 counting circuit 11 electrodes
6 pulse-height analyser
Figure 1 — Diagram illustrating the principle of the electrical sensing zone orifice/tube method
6 General operation
6.1 Response
The response (i.e. the electrical pulse height generated when a particle passes through the aperture)
has been found both experimentally and theoretically to be proportional to the particle volume if
[1]-[3]
the particles are spherical . This has also been shown to be true for particles of other shapes;
[4]
however, the constant of proportionality (i.e. the instrument’s calibration constant) may be different .
In general, particles should have a low conductivity with respect to the electrolyte solution, but
[5] [6]
particles with high conductivity can be measured e.g. metals , carbon , silicon and many types of
[7],[8]
cells and organisms, such as blood cells . For porous particles, the response may vary with the
[9],[10]
porosity . Recommendations for the measurement of conducting particles and porous particles
are given in Annex D.
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As the response is proportional to the volume of particles, the pulse amplitude provides a relative scale
of particle volumes. By calibration, this scale may be converted to spherical diameter. The calibration
constant based on diameter may be calculated by Formula (1):
d
p
K = (1)
d
3
A
p
The size, x, of any particle can be calculated by Formula (2):
3
xK=⋅ A (2)
d x
Typical apertures have a length to diameter ratio of 0,75. This causes some variation in the electrical
field within the aperture, which leads in turn to some deviations in the particle sizes measured. This
can be countered by increasing the aperture length.
6.2 Size limits
The lower size limit of the electrical sensing zone method is generally considered to be restricted
only by thermal and electronic noise. It is normally stated to be about 0,6 µm but, under favourable
conditions, 0,4 µm is possible. There is no theoretical upper size limit, and for particles having a density
similar to that of the electrolyte solution, the largest aperture available (normally 2 000 µm) may be
used. The practical upper size limit is about 1 200 µm, limited by particle density.
The size range for a single aperture is related to the aperture diameter, D. The response has been
found to depend linearly in volume on D, within about 5 % under optimum conditions, over a range
from 0,015 D to 0,8 D (i.e. 1,5 µm to 80 µm for a 100 µm aperture) although the aperture may become
prone to blockage at particle sizes below the maximum size where the particles are non-spherical. In
practice, the lower limitation is due to thermal and electronic noise and the upper limitation is due to
non-spherical particles passing through the aperture. This restricts the operating range to be within
2 % to 60 % of the aperture size. This size range can be extended by using two or more apertures (see
Annex B). In practice, this procedure can be avoided by the careful selection of the diameter of one
aperture, to achieve an acceptable range.
Sedimentation of particles becomes important when the particles are large and have a high density (for
example, 100 µm quartz particles have a sedimentation rate in water of about 1 cm/s). Large apertures
are available, up to 2 000 µm. In such applications, the viscosity and the density of the electrolyte
solution should be increased, for example, by addition of glycerol or sucrose, in order to prevent particle
sedimentation and to increase the possibility of keeping the particles in homogeneous suspension.
The homogeneity may be checked by repeated analyses at a range of stirrer speeds. The results of this
should be compared to establish the lowest stirrer speed at which recovery of the largest particles is
maintained.
6.3 Effect of coincident particle passage
Ideal data would result if all particles traversed the aperture singly and, thus, would produce single
pulses. However the opportunity exists, especially at increased concentrations, that two or more
particles arrive in the sensing zone more or less together, which would result in a complex pulse.
Several possibilities exist, i.e (a) two particles pass the sensing zone at the same time, leading to a
pulse height equal to the sum of both pulse heights, and to a loss of counts; (b) two particles pass the
sensing zone at slightly different times but within the same measurement period of the larger particle,
leading to the same pulse height for the larger particle but a distorted pulse shape, and to a loss of
counts; (c) two particles, which are individually too small for measurement but have together sufficient
volume, pass the sensing zone at the same time, leading to an extra pulse of measurable height, and to
an increase of counts. This occurrence is named coincidence. Its effects will distort the size distribution
obtained but can be minimized by using low particle concentrations. The probability of coincidence
may be described by a Poisson distribution (see Annex A). Table 1 shows counts per millilitre for the
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coincidence probability to be 5 % as well as the corresponding analysis volumes to count 100 000
particles.
Table 1 — Counts for 5 % coincidence probability and analysis volumes for 100 000 counts
Aperture diameter Maximum counts for 5 % Analysis volume for
a 5 b
coincidence 10 counts
D
N V
5 % a
µm
#/ml ml
1 000 5,0E + 01 2 000
560 2,8E + 02 351
400 7,8E + 02 128
280 2,3E + 03 44
200 6,3E + 03 16
140 1,8E + 04 5,5
100 5,0E + 04 2
70 1,5E + 05 0,69
50 4,0E + 05 0,25
30 1,0E + 06 5,4E - 02
20 6,3E + 06 1,6E - 02
10 5,0E + 07 2,0E - 03
a 10 3
Calculated using formula N = 5·10 /D particles per ml.
5 %
b
Use pro rata values for other analysis volumes and count numbers.
Counts per millilitre should always be less than these quoted values. Since particle size distributions
should not be a function of concentration, the effect of coincidence can be tested by obtaining a
distribution at one concentration and comparing it with that obtained when the concentration is halved.
In such a test, repeat such dilutions until the reduction in count in a channel with the largest number
decreases in proportion to the dilution. This should always be done when analysing very narrow size
distributions, as this is where the effect of coincidence is most noticeable.
6.4 Dead time
In instruments using digital pulse processing routines, the signal is scanned at high frequency.
Information on pulse parameters, such as maximum pulse height, maximum pulse width, mid-pulse
height, mid-pulse width and pulse area is stored for subsequent analysis. In this case, analogue-to-
digital conversion of the pulse with storage of the size value for the pulse is not performed in real time
and dead time losses are avoided.
To minimize the effect of dead time, the analyser should be used with the lower threshold set to exclude
thermal and electronic noise, as indicated at A in Figure 2. Additionally, the concentration of particles
should be maintained below 5 % coincidence levels.
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Key
X channels
Y counts
NOTE Counts at channels below A are noise counts. True particle counts are at the higher channels.
Figure 2 — Typical results
6.5 Analysis volume
The analysis volume should be chosen based on the following requirements:
a) allow a representative sample of the suspension;
b) allow a sufficient number of particles to be counted and measured in relation to the required
quality of the size distribution; and
c) have sufficient precision for the number of particles to be counted if particle concentration is of
interest.
Typical values of the analysis volume are given in Table 1.
Table 1 shows that the analysis volumes become excessive for counting this particle number when
the aperture diameter becomes greater than 140 µm. Then, counting less particles means that less
information on the size distribution will become available, so consideration should be taken into taking
a representative sample.
7 Repeatability and reproducibility of counts
7.1 Instrument repeatability
In a correctly performed analysis, the number of counts in a size interval is a random variable which
follows a Poisson distribution. In this, the variance is equal to the expected (mean) value. This indicates
that the standard deviation of a number of counts, n , with mean, N , approximates to N . Both the
c c c
variance and the standard deviation can be used in statistical tests on the correctness of instrument
operation or sample preparation. The statistical chi-squared test can be used to test whether obtained
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data follows a Poisson distribution or not. In this, the apparent and the theoretical variance for a given
number of measurements and a given probability are related. An example is given in Annex G. This
statistical test can be performed on single size intervals, groups of size intervals, or on the total
particle count.
7.2 Method reproducibility/intermediate precision
The reproducibility and intermediate precision will be influenced by several factors (in addition to
those dealt with in 7.1). They are covered in detail in Clause 8, specifically in 8.3, 8.4 and 8.11.
8 Operational procedures
8.1 General
A summary of all the key factors that can influence the quality of the final result is given in Annex A.
This could be used as the basis for setting a method in accordance with the theory of “quality by design”,
where the variance (or lack of variance) of these factors on the final result is considered as part of
method development and validation and a control structure is put in place for the critical parameters.
8.2 Instrument location
The instrument should be sited in a clean environment that is free from electrica
...