ISO 21501 is the recognised standard for optical particle counter calibration introduced by the International Organization for Standardisation (ISO) in 2007 with the purpose of providing ‘…a calibration procedure and verification method for particle counters, so as to minimise the inaccuracy in the measurement result by a counter, as well as the difference in the results measured by different instruments.’
The publication includes four sections, each dedicated to a specific particle counting technology/application:
- Light scattering aerosol spectrometer
- Light scattering liquid-borne particle counter
- Light extinction liquid-borne particle counter
- Light scattering airborne particle counter for clean spaces.
Part 4, which has the title: ‘Determination of particle size distribution – Single particle light interaction methods’, specifically refers to the Light scattering aerosol particle counter (LSAPC).
Prior to the introduction of ISO 21501, there were two standards:
- JIS B 9921:1997 – Light scattering automatic particle counter. A Japanese standard that defines optical particle counters’ performance and counting efficiency.
- ASTM F 328-98 – Standard Practice for Calibration of an Airborne Particle Counter Using Monodisperse Spherical Particles. This was withdrawn in 2007.
The introduction of ISO 21501–4, in 2007, required the adoption of Pulse Height Analysis (PHA) particle sizing technology and particle standard spheres with international traceability and uncertainty equal to or less than ± 2.5%. The standard defines two main goals: to improve the instrument-to-instrument data correlation and improve the particle count accuracy.
The documentation and approval of ISO 21501 replaces ISO 13323-1:2000 and widens the scope of analysis to include methodology for both airborne and liquid particle counting (light scattering and extinction methods).
The recent release of ISO 14644–1:2015 establishes a stronger relationship between the two standards and proposes the need for ISO 21501–4 compliant particle counters in cleanroom certification and monitoring applications.
Section B.2.2 of the previous standard, ISO 14644–1:1999, required particle counting to be performed using ‘calibrated’ instruments and did not require a specific calibration technique.
B.2.2. Instrument Calibration
The Instrument shall have a valid calibration certificate: the frequency and method of calibration should be based on current accepted practice
ISO 14644–1:2015 subsection A.2.2 requires instrument calibrations specified in ISO 21501–4.
A.2.2 Instrument Calibration
The particle counter shall have a valid calibration certificate: the frequency and method of calibration should be based upon current accepted practice as specified in ISO 21501-4. Note: some particle counters cannot be calibrated to all of the required tests in ISO 21501–4. If this is the case, record the decision to use the counter in the test report.
As the note above states, not all instruments will meet the requirements. Non-compliant instruments will require an additional explanation and instrument approval for the cleanroom’s certification audits.
ISO 21501-4 calibration requirements
The following list provides all the ISO 21501–4 calibration requirements, with a brief description of the test method adopted:
Sampling flow rate: The standard uncertainty of volumetric flow rate shall be equal to or less than 5%. Note: If the LSAPC does not have a flow rate control system this subclause does not apply, however the manufacturer shall specify the allowable limit of its flow rate of the LSAPC.
The flow rate calibration is performed to ensure a known volume is presented for counting particles. Specifically, the standard provides two different requirements based upon the type of optical particle counter under test:
Instrument with a flow control system (i.e. pump or blower)
- Compare the unit under test (UUT) flow to certified reference flow meter
- Passing range is ±5% of nominal
Instrument without a flow control system
- UUT samples from a flow set by certified flow system
- Manufacturer specifies the allowable limits of its flow rate.
Controlling the flow rate: PMS particle counters measure the mass flow using a differential pressure sensor that regulates the pressure drop in the sampling region (see Figure 1).
- Pressure drop across the inlet and outlet jets is measured and provides the most precise measurement of flow
- Mass flow is corrected to volumetric flow by using data from an additional atmospheric sensor, then calculating the volumetric flow using the mass flow measured value and the atmospheric pressure.
Why volumetric flow?: Particle concentrations are characterised as particles per-unit-volume, so controlling volumetric flow is essential. Furthermore, changes in barometric pressure can introduce errors as large as 3% when using a mass flow meter. PMS’s flow control adapts to changes in elevation and atmospheric pressure.
Counting efficiency: The counting efficiency shall be 50% ±20% for calibration particles with a size close to the minimum.
The counting efficiency shall be 100% ±10% for calibration particles with a size of 1.5–2 times larger than the minimum detectable particle size.
The counting efficiency is a ratio of the measured particle data between a UUT and a reference instrument. The test is performed using calibration particle standards, known as polystyrene latex spheres (PSLs), with two sizes: one that is close to the minimum detectable reported size range and another that is 1.5–2 times larger than the minimum detectable size. PMS employs Universal Reference Instrument (URI) particle counters that are calibrated against a Scanning Mobility Particle Sizer and Condensation Particle Counter.
Resolution: The size resolution shall be equal to or less than 15% for calibration particles of a size specified by the manufacturer.
The resolution test verifies the instrument’s ability to resolve small differences in particle size.
Figure 2: Curves showing different levels of resolution
How the resolution is calculated: Simply, the resolution is the standard deviation divided by the mean size of the particle, where σ is the standard deviation reported by the instrument while sampling a particle standard (e.g. PSL); σp is the standard deviation reported by the PSL’s published value; and Xp is the mean particle size for the particle standard being sampled.
The calibrated PHA device analyses the calibration curves and automatically determines optimal resolution by calculating the absolute value of the differences between the PSL particle size standard and the particle size measured by the instrument. Further details can be found in the ISO 21501–4 document.
Why resolution is not perfect: The laser beam’s intensity is highest in the centre and degrades towards the edges, as shown in Figure 3. As a result, particles size larger when crossing through the beam’s centre than similar particles crossing the beam’s edge. The Gaussian-shaped curve (shown below the beam pattern) illustrates the resultant light scattering peak as a particle passes through the laser beam.
Figure 3: Optimising resolution
Resolution is improved if the particles are illuminated by the most uniform and highest intensity light. During instrument design and development, PMS optimises resolution by integrating beam-shaping lenses and masks that can produce more consistent particle sizing, with accurate and repeatable results.
The Pulse Height Analyser (PHA): Particle counters employ solid-state photodetectors that convert scattered light energy into electrical signals. The signals, known as pulses, are proportional amplitudes of energy that represent the particles or photons witnessed by the detectors. The pulses are sent to the PHA, and the PHA correlates the relative pulse amplitude to the instrument’s particle size thresholds or bins (see Figure 4). Each particle channel is represented by a counting bin and totalled with other pulses that are of the same amplitude.
Figure 4: Pulse amplitude correlation
False count rate: The false count rate is determined by measuring the particle number concentration in the unit of counts/m3 at the minimum reported size range when sampling clean air.
The zero count test is a measurement of ‘electrical or signal noise’ (see Figure 5). ISO requires these specifications to be met when performing the zero count test:
‘...the measured particle number concentration per m3, when the LSAPC is set to the minimum detactable size and particle free air flows to the LSAPC.’
Figure 5: Zero count test
In addition, the ISO 21501–4 standard requires that only the count rate be reported. Some standards, including the Japanese Industrial Standard (JIS), do not have this requirement.
Going beyond this requirement, PMS particle counter calibrations include a final zero count test, which challenges the instrument to achieve restrictive limits on false counts. The use of restrictive limits, even when not required by the standard, guarantee higher accuracy in the instrument performance and avoids false counts that appear as particle counts.
Particle size setting: The error in the particle sizes shall be less or equal than 10%.
The particle sizing error is calculated for the minimum detectable size and additional sizes. The test demonstrates that if an instrument accurately sizes particles, it will also accurately count particles. ISO 21501–4 requires the use of a PHA to determine the particle distribution of three different calibration particles that span the range of particle sizing.
During instrument design, PMS develops a particle sizing curve. The curve defines calibration points and their associated threshold voltages or values. The curve also shows the associated values for other particles, across the entire sampling range of the instrument. Figure 6 shows threshold/voltage values between the ‘as found’ condition and the calibration’s final result. The sizing error is the difference between the instrument’s reported particle size range and the calculated particle size.
Figure 6: Calibration points and associated threshold voltages or values
This graph shows threshold/voltage values between the ‘as found’ condition and the calibration’s final result. The sizing error is the difference between the instrument’s reported particle size range and the calculated particle size
Additional requirements
Further calibration requirements determined during the design, development and proof of concept include:
Coincidence loss at maximum particle concentration: The coincidence loss, at the maximum measurable particle number concentration specified by the manufacturer, shall be ≤10%.
Coincidence loss is the percentage of error when counting large particle concentrations and is typically, accompanied by a perceived shift in size distribution. There are two primary contributors to coincidence loss:
- Optical coincidence – caused by having more than one particle in the laser ‘viewed volume’ at the same time and
- Electronic coincidence – which limits the number of particle events that can be processed within the system electronics per unit of time (in modern particle counter electronic designs this typically exceeds optical limitations by a wide margin).
Except in the most extreme cases, the presence of coincidence loss is visible as a shift in particle distribution.
Figure 7: Optical coincidence is the percentage error when counting large particle concentrations
Maximum particle number concentration: = as specified by the manufacturer.
The maximum particle number concentration occurs at 5–10% coincidence loss. The value is calculated using the particle transit time through the laser beam and the instrument’s flow rate.
Sampling time: The standard uncertainty in the duration of sampling time shall be ≤±1%.
Response rate: The response rate of the LSAPC shall be ≤0.5%.
In summary, procedures to improve yield and increase throughput are adopted in cleanrooms that manufacture products. Consequently, many manufacturers follow ISO 14644–1 for determining particle counts and classifying cleanrooms (based upon airborne particle data) and ISO 14644–2 for instrumentation guidelines that demonstrate continued compliance.
Pharmaceutical manufacturers in the US follow the guidelines set down under current Good Manufacturing Practice (cGMP), while those within the European Union (EU), follow the EU’s Good Manufacturing Practice (EU GMP) guidelines. Both guidelines define limits for airborne particle counts, which are based upon specific cleanroom/clean space classification (e.g. Grade A, Grade B, Grade C, Grade D). However, the guidelines do not provide:
- The methods to determine the particle counts within a clean space
- The instrument to measure particle counts within a clean space
- The calibration methods for particle counters to assure data accuracy
ISO 14644–1:2015 introduces the need for particle counters calibrated to ISO 21501–4. The calibration methods assure data accuracy and repeatability. Cleanroom users therefore should look to ISO 21501 as a method to meet cGMP, EU GMP, ISO 14644–1 and other requirements. The methods outlined in ISO 14644 and ISO 21501–4 represent an important step in more accurate evaluations of cleanroom contamination and improved process control.
The authors
Daniele Pandolfi, Global Product Specialist, Aerosol, Life Science Division, PMS
Steve Kochevar, Senior Global Applications Engineer Electronics Division, PMS
