ISO 13319-1:2021

INTERNATIONAL ISO
STANDARD 13319-1
First edition
2021-03
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
Reference number
©
ISO 2021
© ISO 2021
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ii © ISO 2021 – All rights reserved

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
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
iv © ISO 2021 – All rights reserved

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.
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
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
2 © ISO 2021 – All rights reserved

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)
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 are 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.
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 E.
4 © ISO 2021 – All rights reserved

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
A
p
The size, x, of any particle can be calculated by Formula (2):
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 F). 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
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.
6 © ISO 2021 – All rights reserved

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
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 B.
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 electrical interference and
vibration. If organic solvents are to be used, the area should be well ventilated.
8.3 Linearity of the aperture/amplifier system
The linearity of the aperture/amplifier system can be checked using three materials consisting of near
mono-sized particles with a certified modal diameter. In a suitable electrolyte solution, the instrument
is calibrated with particles at about 0,3 D (see 8.11.2). Two further sizes of particles are then added
to the suspension, one size in the range 0.05 − 0,1 D and one size about 0,5 D. The suspension is re-
analysed and the size corresponding to these extra peaks should correspond to the quoted size of the
particles to within 5 %.
8.4 Linearity of the counting system
The linearity of the counting system can be tested by obtaining three repeat measurements of the total
counts (across all channels) at an arbitrary concentration. The concentration is then reduced and three
further repeat total counts obtained. Coincidence-corrected counts shall be used. The ratio of the mean
of the total counts should be the same as the dilution. If the agreement is not within 5 %, the test should
be repeated comparing the two lowest dilutions. Subsequent analyses should be carried out at the
dilution giving the best results.
8.5 Choice of electrolyte solution
8.5.1 General
An electrolyte solution should be selected in which the sample is stable. The electrolyte solution should
not dissolve, flocculate, react, or once a good dispersion is achieved, not change the state of dispersion
of the sample in the measurement time, typically up to five minutes. Particles insoluble in water can be
analysed in a variety of aqueous electrolyte solutions. Particles soluble in water can often be analysed
in methanol or in isopropanol. See Reference [11] for recommended electrolyte solutions. When using
small apertures (D < 50 μm) or large apertures (D > 400 μm), special care shall be taken due to their
particular characteristics.
8 © ISO 2021 – All rights reserved

8.5.2 Special considerations for small apertures (D < 50 µm)
Where possible, the electrolyte solution should consist of an aqueous 4 % sodium chloride solution or
one of equivalent conductivity. It should be membrane filtered twice at 0,2 µm or 0,05 µm for 10 µm
apertures.
8.5.3 Special considerations for large apertures (D > 400 µm)
To prevent turbulence that can cause noise signals due to fast flow through the aperture, the viscosity
of the electrolyte solution may be increased by the addition of glucose or glycerol; 10 % glycerol is
recommended for 560 µm and 400 µm apertures, and 30 % glycerol for the 2 000 µm and 1 000 µm
apertures.
8.6 Preparation of electrolyte solution
An electrolyte solution should be well filtered with a membrane filter for which the pore size is less
than the diameter of the smallest particle measured, as it is essential that its background count should
be as low as practicable. It should be noted that quoted values for filters are not absolute. Usually a
mean pore size is given. The width of the distribution of pores around this mean varies depending on
filter type and manufacturer. This will affect the choice of filter size used. All glassware and apparatus
used should be pre-rinsed with filtered electrolyte solution or other suitable liquids. Background
counts should not exceed the values given in Table 2 or yield a total equivalent volume in excess of 0,1 %
of the total volume of particles subsequently measured in the analysis volume.
Table 2 — Counts for background for typical aperture diameters
Aperture Analysis Background
a b
diameter volume counts
D V
m
μm ml
1 000 2 2
560 2 10
400 2 25
280 2 75
200 2 200
140 2 600
100 0,5 400
70 0,5 1 200
50 0,05 300
30 0,05 1 500
20 0,05 5 000
a
For other analysis volumes, use pro rata values.
b
Suggested maximum counts.
8.7 Recommended sampling, sample splitting, sample preparation and dispersion
8.7.1 General
[18]
See ISO 14488 for guidance on the sampling and sample-splitting procedure. Select an appropriate
[19]
dispersant and a dispersion method (ISO 14487 and Reference [11] provide guidance in this area).
The expertise of the laboratory performing the analysis with respect to the sample under test may also
be utilized.
Ultrasonic baths and probes are recommended for dispersion of materials. The time taken to disperse
will be sample dependent. It is suggested that to optimize this that the sample be placed in the bath
or probe and regular samples be taken over a time period of several minutes, every 30 s would be a
suitable default. The size distribution over time can be monitored and a time point can be selected at
which full dispersion has been achieved.
NOTE The use of high energy ultrasonic baths and probes, blenders and mixers can cause both agglomeration
and fracture of particles.
8.7.2 Method 1: Using a paste
The sample should be subdivided to about 0,2 cm . If the sample is in the form of a powder, it should
be worked and kneaded gently with a spatula and a few drops of suitable dispersant to break down
agglomerates. Transfer a mass of about 20 mg to 50 mg of the paste to a round-bottomed beaker and add
a few drops of electrolyte solution and thin it with dispersant. Almost fill the beaker with electrolyte
solution and place it in an ultrasonic bath with suitable power and frequency for the time determined
as per 8.7.1, stirring occasionally. A stopwatch is recommended for a reproducible dispersion technique.
One suitable design of beaker of 400 ml capacity with a baffle is shown in Figure 3.
8.7.3 Method 2: Alternative method applicable to low-density particles of less than 50 µm
In this case, the density of the material will be close to the density of the electrolyte. Subdivide the
sample into portions of about 1 g. Mix a portion with the dispersant and add it to the electrolyte
solution. Then place the beaker (see Figure 3) containing the suspension in an ultrasonic bath for the
time determined in 8.7.1. After stirring this stock suspension well, withdraw 5 ml using a pipette and
add to approximately 400 ml of electrolyte solution. Place in the ultrasonic bath for a further 15 s. When
using this method, it is important that at least two samples are withdrawn from the stock suspension
and analysed to ensure repeatability of the aliquot sampling and the analysis.
Key
1 aperture tube
2 stirrer
3 baffle
Figure 3 — Example of a beaker with baffle and stirrer
10 © ISO 2021 – All rights reserved

8.7.4 Suspensions and emulsions
Suspensions and emulsions should be diluted by addition of smaller volumes of diluent to the emulsion,
not by addition of the emulsion to a larger volume of diluent. Dilution should be performed stepwise
with mixing performed at each step. To avoid “dilution shock”, oil-in-water emulsions may be initially
diluted with distilled or de-ionized water. If the suspension or emulsion is electrostatically stabilized
the amount of electrolyte used should be minimised. The choice of diluent is important. If it needs
to be diluted it should be diluted with an appropriate buffer which is inert to the particles and the
act of dilution should not change the conductivity of the sample (if it does the system will require
recalibration). Any diluent should also ideally be particle free and certainly at the very least should
be prefiltered to remove any particles that could be measured at the given aperture size, i.e if a 20 μm
aperture is being used, then the diluent should first be filtered at approximately 3 μm prior (0,15 D)
prior to use.
8.7.5 Verification of the dispersion
A small sample of the dispersion may be placed on a microscope slide and used to verify the degree of
dispersion and to estimate the size range of the particles using an optical microscope. If the observed
particles are particularly acicular, there is potential for aperture blockage and incomplete measurement.
For non-spheroidal material the largest diameter should be less than the aperture diameter (so it will
not block the aperture if it passes end on). If an acicular particle with an equivalent spherical diameter
of 0,02 D is considered as the smallest measurable particle, this gives a maximum length to breadth
ratio of 400.
8.8 Choice of aperture(s) and analysis volume(s)
From the microscope examination (see 8.7.5), estimate the diameter of the largest particles present.
Choose an aperture for the size analysis such that the diameter of the largest particles to be analysed
is less than approximately 60 % of the diameter of the aperture, selected to reduce the possibility of
blocking the aperture. For particles that are spherical or nearly spherical, an aperture such that the
diameter of the largest particles is less than 80 % of the diameter of the aperture may be chosen. If
there is a considerable proportion of sample below the lower size limit of that aperture (1,5 % of its
diameter), a second and possibly a third smaller aperture will be needed (see Annex F).
Select a suitable analysis volume with reference to Table 1 or select a suitable time of accumulation. It
may be necessary to analyse a number of analysis volumes or to accumulate for a long time to obtain
an acceptable precision, e.g. 50 000 particles will yield a precision on the median of the distribution
(coefficient of variation) of 0,4 %. Counting fewer particles will reduce the precision, but this may be
necessary when using the larger apertures (see Clause 7 and Annex F).
8.9 Clearing an aperture blockage
Apertures below 100 µm in diameter may become blocked with extraneous particles, particularly
if care is not exercised in the clean handling, careful filtration and thorough rinsing of beakers and
associated equipment. A blockage or a partial blockage may be seen by means of the viewing optics, if
provided with the analyser. A blockage may also be indicated by measuring the flow time through the
aperture or by measuring the electrical resistance of the aperture. A blockage will cause a longer flow
time and a higher resistance. A blockage can also be revealed by an examination of the particle pulse
train, which is recorded with some instruments. A blockage will cause a clearly visible shift in the pulse
train. In some instruments, there are means to automatically detect and remove blockages. Blockages
can also be removed by one of the following techniques.
a) Back flushing: Reversing the flow through the aperture may be sufficient to clear a blockage.
b) Boiling: It is possible to use the heating effect of the current to boil the blockage out. This is done by
using a high aperture current.
c) Brushing: It is often possible to brush the particles off the aperture by using a small high-quality
soft-hair brush with the hair cut short. Care should be taken not to damage the aperture.
d) Air pressure.
e) Ultrasonic cleaning: With the aperture tube filled with electrolyte solution, the end is dipped
into a low-power ultrasonic bath for about 1 s. Repeat this operation as necessary. This method
is very effective but extreme care should be taken as it is possible to damage the aperture.
CAUTION — This method should not be used for apertures of 50 µm or less as can cause
severe damage to the aperture.
8.10 Stability of dispersion
The stability of the dispersion of particles can vary during the analysis time. Make a full-size
distribution analysis as soon as possible after dispersion; then stir the dispersion for 5 min to 10 min
and then reanalyse. When using a stirrer, adjust for maximum effect without creating a vortex that
entrains bubbles. Cumulative counts are recorded at size levels close to 30 % and 5 % of the aperture
diameter (denoted as x and x respectively). Changes in the counts greater than those expected
max min
from statistics will indicate that the dispersion is not stable (see Clause 7 and Annex F). Additional
verification of stability can also be performed in instruments that record raw pulse data. Inhomogeneity
across the pulse train during the time of analysis may indicate a change in the stability of dispersion.
[20]
Table 3 details some possible causes. ISO/TR 13097 provides practical guidelines for the assessment
of dispersion stability and is recommended reading in this area.
Table 3 — Examples of suspected phenomena in dispersion
Change in count at
Suggests
x x
max min
No change No change Stable dispersion
Increase Increase Crystallization, precipitation
Decrease Decrease Dissolution
Decrease Increase Size reduction, deflocculation
Increase Decrease Flocculation, agglomeration
Decrease No change Settling of large particles
8.11 Calibration
8.11.1 General
Electrical sensing zone instruments are typically calibrated using polymer latex microspheres of
known size and narrow size distribution.
Those instruments which use the “constant current” approach should not require recalibration if the
electrolyte conductivity is changed. Instruments using the “constant voltage” approach will require re-
calibration for each electrolyte system to be used.
Another method, which is an absolute method, is the mass integration method (see Annex D). Here the
[12],[13]
weighed mass is compared to the mass found as determined by the instrument . This calibration
method is directly traceable and there is no assumption made about the shape, porosity or electric
conductivity of the particles.
Special care shall be taken for porous particles. Such particles may have an interconnected pore system
which, at least partly, is being filled with electrolyte solution during the sample preparation procedure.
This electrolyte solution will, to a certain extent, not contribute to the impedance change in the sensing
zone when the particle passes through it. Therefore, a porous particle generates a pulse with lower
amplitude than a solid particle of equivalent envelope volume. The difference is not negligible; for some
porous materials the size can be as little as half that of the envelope size. For the calibration for the
measurement of porous particles, see Annex E.
12 © ISO 2021 – All rights reserved

8.11.2 Calibration procedure — microsphere calibration
Microspheres with narrow size distribution with a single mode, characterized by a variety of other
methods, are available. They should be characterized traceably to the metre, either by direct calibration
or by comparison with a certified reference material (CRM) that itself has certified dimensions traceable
to the metre obtained from a national metrology institute or another competent reference material
producer. The calibration
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