Primary Determination of Particle Number Concentration with Light Obscuration and Dynamic Imaging Particle Counters

Abstract

Accurate number concentrations of particles in liquid media are needed to assess the quality of water, pharmaceuticals, and other liquids, yet there are limited reference materials or calibration services available with clear traceability to the International System of Units. We describe two methods, based on very simple modifications of commercial particle counter instruments, that can provide traceable number concentration measurements. One method used a light obscuration counter. Fitting a model to the data enabled correction for timing and coincidence errors, and gravimetric calibration of the syringe pump gave a traceable determination of measured volume. Other potential biases were diagnosed by analysis of the particle size distribution. The other method used a dynamic imaging particle counter (a flow imaging microscope). The instrument was intentionally configured so that each particle passing through the flow cell was imaged multiple times. Following the particle image acquisition runs, runs with a rinse solution released and counted microspheres adsorbed to tubing or flow-cell walls. Software assembled the redundant particle images into tracks, and the total number of tracks was assigned as the number of particles counted. Both light obscuration and dynamic imaging methods, when applied to polystyrene microspheres of approximately 4 µm diameter, achieved expanded uncertainties (k = 2) of approximately 2 % of number concentration and agreed to within a difference of 1.1 %.

Expanding NIST Calibration of Fluorescent Microspheres for Flow Cytometry to More Fluorescence Channels and Smaller Particles

“…A LO-based liquid particle counter was used to determine the bead number concentration of suspensions for beads with mean diameters greater than or equal to 2 μm [ 15 , 16 ]. The LO counter was a PAMAS model SVSS-C with an HCB-LD-25/25 sensor head, S/N U32757.…”

“…A LO-based liquid particle counter was used to determine the bead number concentration of suspensions for beads with mean diameters greater than or equal to 2 μm [15,16]. The LO counter Fluorescence intensity was measured by integrating a fluorescence spectrum (fluorescence intensity versus emission wavelength) over the fluorescence emission wavelength range determined by the emission filter corresponding to a particular fluorescence channel of a flow cytometer.…”

“…Traceability to the SI was assured by determining the confidence that all particles within the sample volume were counted, and by determining the actual sample volume. Qualification of the particle counter for high accuracy measurements and determination of uncertainties included (1) gravimetric calibration of volume, (2) pump volume dependence of particle counts to determine timing error, and (3) concentration dependence of particle counts to determine the linear range, correct for coincidence and determine sampling error due to bead adsorption to surfaces [15].…”

Evaluating changes to Ralstonia pickettii in high-purity water to guide selection of potential calibration materials for online water bioburden analyzers

“…Fluorescent microspheres were characterized for their count, size, morphology, purity and fluorescence using flow-imaging and stop-flow microscopies. The flow-imaging microscope (Micro-Flow Imaging DPA-4200, ProteinSimple [4]) was calibrated for counts using previously calibrated microsphere solutions [20]. The microspheres were characterized at three target concentrations ranging from (≈ 3 × 10 4 to ≈ 9 × 10 4 ) 1/mL.…”

Detection and Sizing of Submicron Particles in Biologics With Interferometric Scattering Microscopy

“…12-14 Subvisible particles above 1 mm in size are characterized with optical microscopy techniques such as microflow imaging and light obscuration. 15,16 Despite intense research in the area of subvisible particle characterization, there remains a characterization gap for submicron particles in the range of 100 nm-1mm. 6,17 Techniques such as nanoparticle tracking analysis (NTA), 18,19 laser diffraction, 20 and resonant mass measurement 21 are currently under development to fill this gap.…”