Method and device for determining particle characteristics by multiparametric detection of scattered light and extinction signals

Description

[0001] The invention relates to a method and a device for determining parameters of particles of the nano- and microscale size range dispersed in gases or liquids, their number distribution and concentration by means of particle photometry.

[0002] According to the invention, this is achieved by performing scattered light measurements of fractions or individual particles in a photometer for a different number of aperture or reception angles. The particles are counted, and characteristic features (e.g., size, refractive index) are determined for each particle from the determined scattered light intensities using evaluation algorithms. The entirety of the particles contained in the sample is classified accordingly. The determination of particle characteristics over a size range of more than two decades is made possible without the need to replace or adapt the geometry of the sample measurement chamber or optics. State of the art

[0003] There are numerous methods for determining the size of colloidal particles (such as nanoparticles, emulsion droplets) or coarsely dispersed particles.

[0004] Common optical methods include static and dynamic scattered light measurements (ISO 13320, ISO 22412) and gravity- or centrifugation-based (ISO 13317-1, ISO 13318-2) sedimentation methods. The investigations of suspensions and emulsions using volume scattered light or sedimentation methods have in common that the determined particle parameters generally refer to the superimposed scattering behavior of all particles located in the geometric measurement volume (particle ensemble). The resulting scattered light intensity or extinction depends on the particle concentration and the optical particle properties (size, geometry, and refractive index contrast). Using mathematical methods, a particle size distribution is obtained from the superimposed measurement signals. Regardless of the algorithms used, these are always intensity- or extinction-based and can only be calculated by conversion, e.g.Using Mie theory, with knowledge of the optical properties of the particles (assuming identical refractive index and spherical shape), the scattering can be transformed into a volume- or number-weighted distribution, which allows comparison with imaging techniques. A disadvantage is that the physics of these techniques fundamentally does not provide any information about individual particle characteristics. The interaction of acoustic waves and X-ray waves with particles is also used for grain size determination. These techniques also only provide averaged ensemble values, no number-weighted distributions, and no characteristics of individual particles. It should also be noted that the scattering techniques described so far do not provide access to the concentration of particles in individual size classes for polydisperse suspensions and emulsions.

[0005] Various measurement methods aimed at determining number-based particle size distributions are also described in the literature. First and foremost are optical methods that capture the particles statically or dynamically using imaging techniques and determine the size of each imaged particle manually or with the help of computer-assisted image analysis. For submicroscale and nanoscale particles, static transmission or electron microscopy methods, as well as, more recently, force microscopy, are traditionally used.

[0006] In any case, volume-based 3D shape models can only be created based on 2D images and assumptions about the 3D shape.

[0007] Grain size distributions can be calculated. Furthermore, for broad distributions, images at different magnifications are necessary, which significantly complicates calibration and the calculation of cumulative distributions and de facto makes concentration determinations impossible. These methods are also very time-consuming. Furthermore, the methods described can only be used for dry particles (powders). Dynamic imaging techniques (e.g., Flow Cam, Bettersizer, CAMSIZER, QICPIC) are characterized by better statistics. However, dynamic methods are limited to particles larger than 800 nm [ISO 13322].

[0008] The measuring principle of flow cytometers is single-particle scattered light photometry. The state of the art has not disclosed any features that would allow, for example, the experimental simultaneous determination of particle size and refractive index of nano- and microparticles. The analysis of particle size for microscale particles is not always possible due to the ambiguities in the intensity distribution of the scattered light with the particle diameter (see, for example, Fig. 7) and solutions to this problem have not yet been disclosed. Experimental experience also shows that in the described arrangements, particularly for particles with large particle masses, due to geometric size or density, there is a significant under-determination of the particle number, especially for the larger classes (segregation). However, it should also be noted that the superposition of scattered light with very different intensities due to significantly different particle sizes, on the other hand, leads to under-determination of the smaller particles. This can also occur regardless of the size for particles with very different refractive indices or particle fractions whose refractive index contrast is very small.

[0009] A publication EP 2908119 B1 is also known, which deals with a "Method for Detection of Nanoparticles" based on a flow-through principle. The measurement range for small particles (preferably < 100 nm) is to be expanded by reducing the detection zone. The measurements are performed only for one angle in side scattering. The publication describes a method that shifts the typical measurement range of flow cytometry into the nanoscale and is fundamentally unsuitable for microscale particles.

[0010] EP 2388569 A1 describes a method and an apparatus with two sensors, each operating according to physically different measurement principles, for determining the size and number of particles dispersed in liquids. This method is referred to as single particle optical sensing (SPOS). The major difference from the principle of a flow cytometer is that different physical measurement principles are used to analyze widely distributed particle sizes (scattering sensor and extinction sensor). Due to the method, the measurement chamber depth is greatly minimized, and the laser beam has a small focus diameter with a Gaussian intensity distribution across the cross-section.

[0011] A simultaneous measurement of extinction and scattering for the same particle is not disclosed in this document and is not technically possible with the proposed implementation principle.

[0012] A simultaneous determination of several particle characteristics (e.g. size, refractive index, geometry) for all individual particles of the measurement sample is not disclosed.

[0013] Particle tracking analysis (ISO 19430:2016), which has become established on the market in recent years, measures the temporal displacement of nano- and subscale particles using laser-induced scattered light and calculates the particle size from the square of the mean distance traveled per unit time according to Einstein and Smoluchowski. Unresolved deficiencies of this method include the physically based size-dependence of the detection sensitivity, which leads to both losses in the determination of the particle number and a distortion of the particle distribution, particularly in the fine-grain fraction. A further shortcoming lies in the determination of the active measurement volume, which depends on the size and / or refractive index of the dispersed particles. Particle concentration determinations of polydisperse samples are therefore always subject to errors.

[0014] This technique can also only be used for particles dispersed in liquids.

[0015] All of the above-mentioned principles and measuring devices have in common that they cannot simultaneously determine several characteristics (e.g. size, refractive index, geometry, number, concentration) of particles dispersed in air or liquids, i.e. in a suspension, emulsion or an aerosol.

[0016] For the sake of completeness, it should be mentioned that several documents have been published describing the simultaneous determination of size and refractive index of particles in dispersions for special cases. These processes differ from the proposed invention and also have a number of limitations. WO 2017 / 072360 A1 is said to apply primarily to particles smaller than the wavelength of the incident light (preferred wavelength 405 nm or 488 nm). However, this measurement range is said to be extendable to three times the wavelength. The optical scatter ratio (forward scattering / side scattering) is said to be independent of the refractive index in this range. The size can be determined from this, and according to theory, the refractive index can be determined from one of the two scattered light measurements.

[0017] A published table of experimental results documents only results for particles smaller than the incident wavelength (405 nm). Only measurements with PS and SiO2 particles were reported, and the errors of the measurements are all larger than those specified by the manufacturer.

[0018] Our own theoretical calculations confirm the larger error range, although the method fails when the intensity oscillations mentioned above enter the reception angle ranges. Therefore, an extension of this method for particle sizes greater than the wavelength of the incident light will generally not be possible.

[0019] A very complex measurement method according to US Patent 9,068,915 B2 determines the refractive index of a particle type by comparing the forward and side scattering of two samples. The prerequisites for applying this method are, first, that the two samples have different grain sizes, and second, that the particle size and refractive index of a sample (batch) are known. Alternatively, this method can be used to determine the refractive index of particles if their size is known; therefore, it is by no means a simultaneous determination of size and refractive index.

[0020] How to proceed in the case of polydisperse distributions is not disclosed.

[0021] WO 2007 / 100723 A2 and US 2011 / 0058168 A1 describe an optical system for a flow cytometer comprising a flow channel with an interrogation zone and an illumination source illuminating the flow channel in the interrogation zone from a specific direction.

[0022] The optical system preferably comprises a lens system and a detection system. The lens system preferably comprises a plurality of lens surfaces arranged along a plane perpendicular to the flow channel and capable of collecting and collimating light from the interrogation zone. The detection system preferably comprises a plurality of detectors capable of detecting the light from the lens system. Each detector preferably contains a local filter that independently filters for specific wavelengths. This allows the user to exchange the filters in any order to achieve the same detection parameters.

[0023] US Pat. No. 6,067,157 A discloses an optical analyzer with a configuration particularly suitable for use with planar liquid sample flow cells. It includes a polarized light source and at least two wide-angle scattered light photodetectors, each positioned at acute and right or oblique angles to the incident light beams. The differences in light intensities measured at the two photodetectors are used to quantify the sample components.

[0024] WO 2009 / 151610 A2 discloses a flow cytometer assembly with a fluid controller configured to form a hydrodynamically focused flow stream containing an outer sheath fluid and an inner core fluid. A coherent light source is configured to illuminate a particle in the inner core fluid. A detector is configured to detect a spatially coherent distribution of elastically scattered light from the particle excited by the coherent light source. An analysis module is configured to extract a three-dimensional morphology parameter of the particle from a spatially coherent distribution of elastically scattered light.

[0025] EP 2 634 557 A2 discloses a method and a system for calibrating a flow cytometer using particles with known refractive indices.

[0026] US 2014 / 152986 A1 describes a method and apparatus for improving particle scattered light measurements by controlling multiple scattering and coincidence count levels. The scattering path in the particle dispersion and the particle concentration are adjusted to reduce multiple scattering in total particle scattering measurements. The particle dispersion volume and the particle concentration are adjusted to reduce coincidence counts in single-particle scattering measurements. The alignment of the optical system for measuring the scattered light is maintained by a reflection device.

[0027] The publication "RAPID COMMUNICATION Measurement of droplet velocity, size, and refractive index using the pulse displacement technique" by SM Lin et al., XP020062972, investigated the detection and quantification of gas bubbles during oil exploration. A novel method was developed for the simultaneous measurement of the velocity, size, and refractive index of large, optically transparent bubbles and droplets. The method is based on the temporal displacement of the refracted and reflected rays scattered by the moving particles.

[0028] EP 2 975 378 A1 presents a method for measuring small particles in solution with various structures and sizes down to a few nanometers by light scattering. A technique is described that allows an extension of the traditional Rayleigh-Gans approximation. The described method is applicable for determining the structural features of irregular particles whose scattering depends on their orientation with respect to the direction of the incident light.

[0029] All solutions known from the prior art are based on measurement principles that differ significantly from the subject matter of the disclosed invention and / or have significant limitations and disadvantages, particularly for widely distributed particle samples in the nanoscale or submicroscale and microscale size range, compared to the claimed inventive solution. Task

[0030] Suspensions (e.g., polymer or oxide particles dispersed in aqueous media, as well as biological materials) or emulsions (e.g., nutritional infusions) occur in many areas of nature, medicine, industry, research, and private households, where they play an extremely important role. Unwanted particles in production operations, contaminants in wastewater, or particle loads (e.g., microplastics) in natural waters also require appropriate treatment measures (e.g., drinking water treatment, air purification).

[0031] The quantitative determination of particle characteristics, such as size, number, concentration, or their optical properties, is of great importance from both scientific, product-relevant, and risk-relevant perspectives (e.g., nanomaterial classification). These requirements arise particularly in the field of modern particle technology for nano- and microscale particles dispersed in gases or liquids. In order to better understand and influence dispersion properties or to formulate relevant products, it is essential to know not only the chemical composition of the dispersed phase, but also the size (particle size distribution (PDS), number (concentration), and optical properties, such as shape and refractive index, of the particles.

[0032] Known measurement methods in the claimed TGV range, as exemplified in the "State of the Art" section, have a number of disadvantages. Established reference methods (microscopy or electron microscopy) are very time-consuming, and for dispersed, highly polydisperse particle systems, experimental and metrological difficulties arise that prohibit widespread use, particularly in industrial applications. On the other hand, the widely used static and dynamic light scattering methods (ISO 13320, ISO 22412) are ensemble methods. The physical measurement principles do not provide information about individual particles, and the measured scattering intensity for a particle depends on its size, geometry, and refractive index contrast, as well as the optical characteristics of the measuring instruments. Furthermore, beyond a certain grain size, it is no longer a unique function of the particle size.

[0033] The invention therefore aims to determine several particle parameters, such as particle concentration, size distribution and number of particles per size class, for each individual liquid- or gas-borne particle of a suspension, emulsion or aerosol, using solid angle-dependent scattered light measurements, as well as for particles distributed narrowly as well as over several orders of magnitude, as well as to quantify additional particle characteristics, such as refractive index or non-sphericity, from the measurement signals of the individual particles using evaluation algorithms. The invention further focuses on a high size discrimination of a few nanometers. A further task, in the case of implementation as a single particle scattered light photometer, is to determine a detection rate (events / s, often also referred to as frequency) of the scattered light events, e.g.of at least 10,000 events per second and to control these automatically solely by hydrodynamic or aerodynamic means in order to detect the particle characteristics for each particle of the primary measurement sample over a very large and high concentration range compared to the state of the art (e.g. EP 2388569 A1), e.g. from 10 2< to 10 9< particles / ml, without coincidence falsification and dilution of the primary sample, preferably to analyze them in real-time, to classify them accordingly and to achieve this without exchanging measuring chambers or making geometric changes to the measuring cell.

[0034] The object of the invention is achieved by the features in claims 1 and 15.

[0035] Appropriate embodiments of the invention are contained in the subclaims.

[0036] A particular advantage of the method according to the invention is the effective determination of particle characteristics. For the determination of the properties of microscale and submicroscale particles down to the single-digit nano range in flow-through measuring cells with hydrodynamic focusing by multiparametric detection of extinction and / or scattered light signals, the scattered light and extinction signals of the particles are counted and measured simultaneously in at least two solid angle ranges with a high detection rate (frequency), and compared analogously or digitally with simulation calculations of the scattered light distribution by analytical or numerical methods for the various solid angle ranges in order to determine the individual particle characteristics over a dynamic particle size range of four orders of magnitude without coincidence, even for high particle concentrations (e.g.10 9< particles / ml) of the sample to be measured, whereby different aperture angles for the forward scattering or several detection directions for the scattered light measurement as well as any combination of these are used, whereby the particle size and the refractive index are determined from the respective scattering intensities of the simultaneous measurements; whereby in order to determine the sphericity or the non-sphericity, the entire temporal intensity profile of the scattered light pulse during the passage of the particle through the measuring volume is measured and analysed in real time or stored.

[0037] Detection rates in the sense of this invention mean counting events in the range of less than 10 particles up to one million particles per second, in particular from 100 to 10,000 particles per second with a pulse height above the noise signals. At particle concentrations of preferably 10 10< particles per ml, the particle number is measured largely coincidence-free. For detection rates of, for example, 10 kHz, counting losses of less than 0.0035 (0.35%) occur. In other words, the inventive solution de facto measures all particles of the sample stream and is characterized by an extremely high sensor sensitivity compared to the prior art. For example, in EP 2338569 A1 (e.g. Fig.9 ) Effectiveness factors of only a few percent to a few tenths of a percent are given, which depend on the particle size.

[0038] Further advantages of the invention result from the fact that different aperture angles for forward scattering or several detection directions for scattered light measurement as well as any combination of these are used.

[0039] In the case of particles with size parameters k greater than or approximately equal to 20, for example, different aperture angles are used for forward scattering.

[0040] An additional advantage of the invention results from the fact that in the case of particles with little distinguishable forward scattering light curves in the different reception angles (especially with a size parameter k less than or approximately equal to 20), several detection directions in sideways directions with different sensitivities are used.

[0041] According to the invention, a simulation calculation is carried out using Mie theory or numerical calculations in accordance with the corresponding optical setup, and the comparison between theory and experimental scattering intensity eliminates any ambiguity regarding particle size. This allows even microscale particles, typically down to 100 µm, to be analyzed.

[0042] According to the invention, the particle properties, such as size or optical particle parameters, are determined by checking the consistency of the modeled and experimentally determined intensities of the particle pulses for different aperture angles and / or solid angle ranges.

[0043] If the scattered light intensities measured in the angular ranges do not match the possible theoretical intensities, for example if the size and refractive index of the particles are known, asphericity is inferred and an asphericity index is calculated.

[0044] A further advantage of the invention is that in the case of single-particle scattering, the shape of the particle is classified as spherical or non-spherical by comparing the experimental, digitized pulse shape of a particle with a known refractive index and the corresponding simulation calculations for spherical particles for the same solid angle range.

[0045] Quantitative results are obtained using theories that deal with the relationship between the geometry of a particle (asphericity) and the scattering behavior.

[0046] The invention enables the analysis of all particles present in the sample, excluding particle wall adhesion or separation phenomena, and the classification of cumulative distributions or subfractions of the particles according to, for example, size, shape, and refractive index. The particle counts (concentrations) per characteristic unit are determined, displayed, and output in absolute terms.

[0047] A laser with variable beam intensity and an aspherical beam cross-section (focus) with constant light intensity at least over the cross-section of the sample stream is used (16, Fig. 1b). Thus, the scattering pulse, in contrast to the one explained in the prior art (SPOS), is not dependent on the trajectory of the measured particle in the measurement volume, and deconvolution is not necessary. In the case of insufficient intensity homogeneity, this is corrected by standardization procedures (e.g., experimental determination of the deviation from a constant intensity or by measuring size-certified monodisperse reference particles). To expand the measurement range, lasers with different wavelengths or multiple lasers including fluorescence can be used.

[0048] The extent of hydrodynamic or aerodynamic measuring current focusing can be adjusted manually or automatically by the ratio of the sheath flow to the sample flow, depending on the initial number concentration of the measuring sample, based on the knowledge or a first measuring cycle.

[0049] A further advantage of the invention is that for the size determination of very broad, polydisperse samples, no exchange of measuring chambers or changes, for example, to the geometry of the flow cell or the optical setup, are necessary. For extremely broad distributions up to the size range of several micrometers, the measurement of different scattering angles or the simultaneous recording of the extinction of individual particles can be used.

[0050] Additional advantages include the possibility of inserting or shifting beam stops one after the other, using ring-shaped, rotatable detectors that each cover a specific angular range, or inserting different apertures into circular sectors. Optical elements with special coatings can also be used, whose transparency can be varied for the respective laser wavelength used, for example, by electric fields without mechanical displacement.

[0051] By using ring-shaped detectors or different apertures in circular sectors, the repeated measurements in other angular ranges described in the examples are eliminated, which advantageously reduces the experimental effort and increases the measurement accuracy by simultaneously measuring different angular ranges for the same particle.

[0052] For extinction applications using hydrodynamic focusing and by masking out the areas illuminated by the primary radiation in the image space next to the hydrodynamically focused particles, the ratio of the light intensity of the light source in the cuvette to an extinction signal is improved in order to detect smaller particles.

[0053] By inserting a pinhole aperture after the lens, which has the diameter of the primary laser beam after the lens, a large portion of the forward scattering that would otherwise reach the receiver is eliminated. This extends the measurement range to include smaller particles and enables more precise particle size calculations. An advantage with reference to document EP 2 388 59 A1 is the fact that, due to the separation of the particles, the transmission in the areas between the particles does not exhibit particle concentration-dependent turbidity.

[0054] The device is used and the method is applied according to the invention for the analysis of several characteristics of individual particles or the classification or identification of particle fractions in the industrial and academic fields, such as W / O or O / W emulsions, aerosols, slurries for waver polishing, ink and pigment suspensions, samples of cellular and subcellular particles (including cells, viruses, bacteria) of biological origin or for applications such as the design of nanoparticles, quantification of the stability of dispersions, investigations into the dissolution, agglomeration and flocculation behavior of disperse phases, quantification of the progress of dispersions.

[0055] The invention will be explained in more detail below with reference to exemplary embodiments shown at least in part in the figures.

[0056] They show: Fig. 1: Extinction of some organic substances (n = 1.43); λ = 532 nm; x = particle diameter [µm]; y = scattering cross section [arbitrary units] Fig. 1a: Basic optical principle of the measurement method; Fig. 1b: Geometry in the sample chamber; Fig. 2a: Hydrodynamic focusing; Fig. 2b: Flow cell; Fig. 3a: Electronic schematic and data flows within the device and the external software SepView; Fig. 3b: Electronic components for operation and data processing; Fig. 4: Analog single pulse of a particle and exemplary characteristic quantitative features for the pulse shape for possible pulse differentiation as a basis for manual or automatic classification of particle features; Fig. 4a: Measurement results for simultaneous scattered light detection in forward (left) and sideways direction (right) of a mixture of differently sized polystyrene particles; Fig.5: Concentration determination for polystyrene particles and count rates for polystyrene microparticles and gold nanoparticles; Fig. 6: Basic sequence of a typical analysis flow for calculating the particle size distribution. Fig. 7: Light scattering curve of polystyrene particles; n = 1.59; reception angle range: 4°–12.33°; λ = 532 nm; x = particle diameter [µm]; y = Mie scattered light intensity [arbitrary units]; Fig. 8: Light scattering curve of polystyrene particles (various reception angle ranges); λ = 532 nm; n = 1.59; a: 4°–12.33°; b: 5°–12.33°; c: 6°–12.33°; d: 8°–12.33°; x = particle diameter [µm]; y = Mie scattered light intensity [arbitrary units] Fig. 9: Scattered light curve of particles with an assumed refractive index n = 1.8; (various reception angle ranges) λ = 532 nm; a: 4°-12.33°; b: 5°-12.33°; c: 6°-12.33°; d: 8°-12.33°; x = particle diameter [µm]; y = Mie scattered light intensity [arbitrary units]; Fig.Fig. 10: Scattered light curve of silicon dioxide with an assumed refractive index n = 1.46 (various reception angle ranges); λ = 532 nm; a: 4°-12.33°; b: 5°-12.33°; c: 6°-12.33°; d: 8°-12.33°; x = particle diameter [µm]; y = Mie scattered light intensity [arbitrary units]; Fig. 11: Scattered light curve of silicon dioxide with an assumed refractive index n = 1.47 (various reception angle ranges); λ = 532 nm; a: 4°-12.33°; b: 5°-12.33°; c: 6°-12.33°; d: 8°-12.33°; x = particle diameter [µm]; y = Mie scattered light intensity [arbitrary units] Fig. 12: Scattered light curve of silicon dioxide with an assumed refractive index n = 1.46; selected particle diameters (various reception angle ranges); λ = 532 nm; a: 4°-12.33°; b: 5°-12.33°; c: 6°-12.33°; d: 8°-12.33°; x = particle diameter [µm]; y = Mie scattered light intensity [arbitrary units] Fig.Fig. 13: Scattered light curve of silicon dioxide with an assumed refractive index n = 1.47; selected particle diameters (various reception angle ranges); λ = 532 nm; a: 4°-12.33°; b: 5°-12.33°; c: 6°-12.33°; d: 8°-12.33°; x = particle diameter [µm]; y = Mie scattered light intensity [arbitrary units]; Fig. 14: Scattered light curve of silicon dioxide (side scattering) with assumed refractive index a: n = 1.47 and b: n = 1.46; selected particle diameter range; λ = 532 nm; half aperture angle: 10.02°; x = particle diameter [µm]; y = Mie scattered light intensity [arbitrary units]; Fig. 15: Scattered light curve of silicon dioxide (side scattering) with assumed refractive index a: n = 1.46 and b: n = 1.47; selected particle diameter range; λ = 532 nm; half aperture angle: 10.02°; x = particle diameter [µm]; y = Mie scattered light intensity [arbitrary units]; Fig. 16: 2D plot: refractive index as a function of particle size; n = refractive index; x = particle size Fig.17: 2D plot: sphericity as a function of the refractive index AI = asphericity index; n = refractive index Fig. 18: Scattered light intensities (side scattering) of silicon dioxide particles dispersed in air a: n = 1.0 and in water b: n = 1.335; aperture angle: 10.02°; x = particle diameter [µm]; y = Mie scattered light intensity [arbitrary units] Fig. 19: Schematic diagram of the measuring zone with particles, seen in the direction of the laser beam; the flow direction of the particles is indicated by the arrow a) volume scattering device; b) single particle scattering photometer with laser focusing and shaping without - and c) with hydrodynamic focusing; Fig. 20: Blocking of scattered light radiation by an aperture (dark gray circle); Laser primary beam with extinction signals (light gray circle) is transmitted through the pinhole; Fig. 21: Extinction measurement of a 552 nm polystyrene latex (with background correction); X = number of channels on the multi-channel analyzer; Y = extinction [arbitrary units]; Fig.22: Absorbance of a mixture of polystyrene (PS) and melamine resin (MF) particles of different sizes a: 0.815 µm PS; b: 1.05 µm PS; c: 1.3 µm MF; X = number of channels on the multichannel analyzer; Y = absorbance [arbitrary units]. Description of the invention 1. Measuring apparatus of the single particle photometer

[0057] The technical realization of the invention will be explained below by way of example. Optical measurement setup:

[0058] Fig. 1a shows a typical setup of the measuring apparatus, focusing on the optics in plan view (z-direction). The device according to the invention comprises at least one laser 1 for generating at least one laser beam 3, at least one optical input module 2, e.g. for shaping the laser beam 3 and designing a focus geometry 16 ( Fig. 1b) or similar), a flow cell 5, advantageously with hydrodynamic focusing, in which forward scattered light radiation 6 and sideward scattered light radiation 7 are generated by the laser beam 3, optical output modules 8 in the forward scattered light beam 6 and optical output modules 9 in the sideward scattered light beam 7, semi-transparent mirrors 10, cameras 12, photomultipliers 11a, 11b, whereby scattered light measurements are carried out for a different number of aperture or reception angles by means of the photomultipliers 11a, 11b and particle features are determined from the determined scattered light intensities of the forward scattered light beam 6 and the sideward scattered light beam 7 using evaluation algorithms.

[0059] The light source 1 is ideally a stable monochromatic, intensity-controllable, and short-wave laser with a power of, for example, 100 mW. Other light sources and configurations are also feasible. According to the invention, all wavelengths of the visible and near UV and IR ranges can be used. The shorter the wavelengths, the smaller the particles that can be measured. The coupling of different or multiple lasers is also possible via the optical module 2 or by means of corresponding optical components via the light beam 3. The optical module 2 is used to design, for example, an elliptical focus geometry (16 in Fig. 1b), which allows for both uniform light intensity in the measurement volume and low particle coincidence. Both means can also be integrated into a single module. The incident or scattered beams can also be "guided" by optical fibers, which is useful, for example, for miniaturizing the setup. The use of micro-optical components is particularly advantageous. The incident light beam 3 is focused on the single-particle stream 4 in the flow cell 5.

[0060] It should be noted that the edges of the measuring cell are not hit by relevant portions of the primary radiation in order to minimize background radiation.

[0061] Fig. 1bshows the conditions as a section seen from the sideways scattering 7 (y-direction). The focus geometry is typically selected such that its width (x) is larger than the diameter of the particle stream. This can be achieved, for example, by a combination of appropriate lenses in module 2 and / or the use of apertures. The focus must also be designed using the optical module 2 so that the laser intensity is as homogeneous (constant) as possible across the cross-section of the particle stream (y-direction) in the optical measuring volume (optical sensing zone). If this is not the case, the deviation can be measured by measuring the location-dependent laser intensity or by a standardization measurement with very monodisperse particles. The particle size distribution can be determined using the determined local intensity dependence by means of correction factors and the assumption of statistically homogeneously distributed particles in the flow path 21. Fig. 2b be corrected.

[0062] When a particle passes through the laser focus, the light is scattered into space. As an example, the optical modules 8 and 9 for forward 6 and sideward scattering 7 are shown for a particle located in the laser focus. They collect the light, stop out the direct beam (e.g., by beam stop) (only 6), contain the receiving optics, and focus the light scattered in a specific area onto, for example, photomultipliers 11a, 11b. Depending on the measurement requirements, the latter can be identical or functionally adapted for the different radiation angles. The use of, for example, photodiodes and avalanche diodes (avalanche photodiodes) is also possible. The semi-transparent mirrors 10 facilitate the adjustment of the beam paths using cameras 12. The camera is used to assess the image of the scattering particle stream in the measuring zone for sharpness and its position relative to the pinhole.Only when the edges of the pinhole and the scattered light signals appear sharp can the image of the measurement volume be narrowed by using the smallest possible aperture. This will mask out some of the background radiation. Furthermore, it must be ensured that the aperture does not obscure any part of the image from particle scattering, to avoid the resulting broadening of the TDE and errors in the particle concentration.

[0063] According to the invention, 6 identical or differently shaped "beam stops" (e.g., with different diameters) can be inserted one after the other in the forward scattered beam—before or after the receiving optics (objective)—or a constant-sized aperture can be moved within the diverging scattered light cone (or focusing cone) to achieve different reception angle ranges. Ring-shaped detectors, each covering a differential angular range, can also be combined. Ring detectors with different radii and beam stops can also be arranged, for example, in one component in four quadrants with associated radiation receivers. This eliminates the need for mechanical placement, e.g., of the "beam stops," for achieving different reception angles.

[0064] Essential for high counting accuracy is the passage of only one particle at a time through the measurement volume covered by the focused laser beam. This can be conveniently achieved by hydrodynamic focusing of the sample for liquid-borne particles and aerodynamic focusing for airborne particles. The maximum sample flow rate as a function of the particle concentration can be estimated using an approach from the literature (Analytical Chemistry 1987 59 (6), 846-850, DOI: 10.1021 / ac00133a013).

[0065] Fig. 2ashows a typical flow arrangement (fluidics) with a vertically aligned flow cell 5 with hydrodynamic focusing. The flow cell 5 is typically cuboid-shaped (external dimensions e.g. 10 mm x 10 mm, height e.g. 30 mm, other dimensions are also possible) and is made of highly transparent material (e.g. quartz glass). The internal cross-section is e.g. 1500 µm x 1500 µm or 200 µm x 200 µm. Other geometries and cross-sections are also usable. The flow cell 5 also has an inlet for the sheath flow 13 and the sample inlet 14 as well as the outlet 15. The sheath flow 13 is pumped from a reservoir 17 via a pressure generation device 18 in a controlled and pulsation-free manner. This is achieved, for example, by an adjustable gravimetrically generated pressure difference. To stabilize a laminar flow, special geometries can be used in the inflow area 13, 5.The sample stream 19 is introduced via a volume-controlled, calibratable syringe pump 20, e.g., with a nominal volume of 0.5 ml - 2 ml. Larger and smaller delivery volumes are also possible. It should be emphasized that this allows all dispersed particles flowing through the measuring cell to be analyzed, and not just a small percentage, as stated in EP2 388 569 A1. In order to minimize the sample volume, e.g., to 80 µL or 400 µL, preferably 250 µL, an additional port, e.g., for Hamilton syringes of different volumes, and a correspondingly dimensioned sample loop can be integrated into the feed line 14. The cross-section of the flow path 21 in the flow cell 5 (. Fig.2b ) can be adjusted manually or automatically over wide limits via the ratio of sheath flow to sample flow on the basis of known sample counts or those collected at the beginning of the experiment (e.g. the diameter of the sample stream (4, Fig. 1b) from 5 µm up to 30 µm diameter can be variably adjusted.

[0066] According to an approach from the literature (Analytical Chemistry 1987 59 (6), 846-850 ,DOI: 10.1021 / ac00133a013), the sample stream diameter can be calculated as a function of the two flow rates for sheath and sample flow. The geometric optical measurement volume is thus, for example, 10 pL and 295 pL for a laser beam height of 15 µm (small radius of the exemplary elliptical beam cross-section (16)) and a sample flow in the measuring cell of, for example, 0.3 µL or 1.2 µL, respectively. These values ​​can be adapted according to the invention using variable fluidics both to smaller volumes, e.g., to shift the measurement sensitivity for smaller nanoparticles, and to larger volumes, e.g., to reduce the measurement time. This means that initial sample concentrations in a concentration range of e.g. 10,000 times higher can be achieved simply by using suitable flow rates of the sample and / or sheath flow and without dilution or changing the mechanical arrangement orthe flow cell geometry can be measured as required. For example, with sample stream flow rates of (300 - 1200) nL / min and concentrations of 10 9< particles per milliliter, it is possible to measure with virtually no coincidence and to determine the particle number very precisely with a relative error of less than 1%. This typically means that samples with a particle concentration of at least 10 9< particles per mL can be analyzed by single particle detection. In order to achieve the necessary wide size measurement range over 3-4 decades, the flow cell 5 and the fluidics are designed and realized in such a way that the sheath flow 13 and the sample feed 14 can be fed in from above or below. This prevents counting losses due to sedimentation of larger particles or creaming of larger droplets.In addition or instead, it is also possible to use devices such as one or more mixers in the sample supply system to feed the sample to the Flowcell 5 without particle losses.

[0067] Instead of hydrodynamic focusing, acoustic centralization in the measuring volume 4 of the particles in the middle of the flow cell 5 is also possible.

[0068] Special devices for sequential sampling from reactors or pipelines and feeding via 14 also enable quasi-continuous measurement of, for example, production processes (online). It is advantageous to monitor the primary initial particle concentration using optical sensors or other suitable measuring sensors. If the initial concentration of the primary sample taken from the process is too high, one or more dilution steps can be implemented using calibratable mixers. These dilution steps can be integrated into the overall measuring system, both technically and in the standard operational procedures (SOPs), e.g., with the aid of appropriate software (e.g., SEPView), and included in the concentration calculations.

[0069] The incident laser beam 3 transmits the flow cell 5 and interacts with the hydrodynamically or aerodynamically separated particles in the sample stream 21. The scattered radiation (e.g. forward 6 and sideward 7) exits the flow cell 5. In principle, the use of, for example, two different lasers with parallel or, for example, 90° offset incident beams 3 is also possible in order to improve the measurement resolution for individual applications. In practice, with parallel incident beams 3, the height difference with respect to the flow cell 5 is very small, but without interactions between the two beams occurring. The intensity-time curves of the scattering events recorded with the two lasers must be synchronized accordingly.

[0070] The design of aerodynamic focusing is similar to hydrodynamic focusing. The aerosol jet is surrounded by a clean air jacket and constricted by the ratio of sample flow rate to the sheath flow volume of the air jacket, as well as the shape of the suction nozzle in Flowcell 5.

[0071] The invention is characterized by a non-spherically shaped laser beam (16, Fig. 1b ) is characterized by a very low intensity dependence across the cross-section of the beam compared to the typically occurring Gaussian distribution of a spherically focused laser beam (see EP 2388569 A1). In the exemplary Fig. 16For the elliptical beam cross-section shown, a sufficiently homogeneous laser intensity can be achieved across the optical measuring range by using a large axial ratio. On the other hand, by simulating the optical conditions with a known intensity distribution of the primary beam of laser 1 or based on an experimental determination of the intensity distribution in the recorded measuring current, the technically realized intensity curve can be corrected, e.g., by integrating over the measurable range and determining the deviation factors from a constant intensity distribution, and thus obtaining precise grain size distributions.

[0072] To extend the sensitivity of the measuring system into the lower nanorange, the optical measuring volume must be reduced, the scattered radiation from component surfaces in the optical range must be minimized, and a low-noise laser 1 must be used. An encapsulated laser module (1, 2) with integrated micro-optics and a minimized exit window enables a significant reduction of unwanted scattered radiation.

[0073] Inventive solutions for improving the signal-to-noise ratio are also used in the area of ​​flow cell 5 and scattered beam detection optics. In the flow cell 5, for example, only the necessary entrance and exit windows for the radiation need to be made transparent. This can be achieved, for example, by using glasses of varying transparency or partially coated interior walls. Additional optical components, such as multilevel diffractive optical elements or absorbing coatings, such as Vantablack®, can also be used in the detection optics (e.g., 8, 9, 10, 11a, and 11b). Electronic structure:

[0074] Fig. 3a provides an overview of the main functional elements of the electronic structure developed according to the invention based on an embedded board with operating interface BO, boot media, at least one microcontroller supported by one or more FPGA(s) and fast mass storage devices (e.g. SSD). Fig. 3a also reflects the basic principles of networking and the flow of information between the PC or server-based SEPView software and the electronic structure, which enables real-time, high-speed communication in both directions.

[0075] The most important electronic information processing components are in Fig. 3b The diagram shows, for example, two scattered light sensors, which may differ in scattering angle or aperture angle. The use of additional sensors, e.g., for temperature, flow rates, fill level of storage vessels, laser intensity, optical adjustment, cameras, etc., is also appropriate.

[0076] The analog signals of the intensity pulses emanating from the individual particles, delivered by the sensors, are first amplified by a low-noise, preferably linear, two-stage amplifier with automatic switching and then digitized in real-time by an analog-to-digital converter. Due to the fact that the scattering intensity decreases significantly with increasing particle size, very broadband amplifiers of, for example, 120 dB must be used. These must be designed in such a way that registered scattering events from the sensors can be processed with very variable frequencies (clock frequencies), for example from 20 Hz to 10 kHz, advantageously up to very high frequencies in the range of 50 - 100 kHz, in order to be able to record statistically reliable counts (number of particles of the respective size classes, for example from 80 nm to 100 nm) in a short measuring time and, on the other hand, to also record changes in the sample during the measuring time, for exampleby sedimentation or particle-wall contact, particularly for concentration determinations. On the other hand, rapid changes in the dispersed phase, e.g., dissolution behavior or agglomeration rates, can also be measured. The amplifiers developed for the invention can be operated in both linear mode (for high-resolution pulses or monomodal particles) and logarithmic mode (broad distributions over several orders of magnitude). To achieve the high resolution required for pulse evaluation, AD converters with, for example, 20 to 24 bits and a sampling rate range of 0.1 to 25 MS / s, preferably 1-5 MS / s, must be used.

[0077] During the preprocessing of the digital values, very short interference pulses and invalid converter results are filtered out and discarded using special algorithms. Offset processing can also be implemented here.

[0078] Special pulse detection electronics / firmware evaluates the digitized data stream in real time and identifies evaluable pulses. Invalid pulses are discarded. Invalid data includes, among others, pulses that are too long, too short, or pulses below the trigger threshold. After digitizing valid pulses, characteristic values ​​of the pulse of the respective individual particle, such as the maximum of the pulse, intensity ( Fig. 4a), duration of the pulse and area under the pulse signal for each particle in the FPGA are determined. The digitized pulse with, for example, 100 sampling points including an adjustable time lead, as well as the determined parameters are saved or transferred to the embedded board in real time and classified highly sensitively in up to 10 6< bins. The BO can be used to advantageously select the number of bins between, for example, 10 and 10 6<, allowing particle classes to be grouped together as required. The pulse classification is displayed on the BO in real time. Additional supporting electronics provides the parameters for pulse detection and sensor settings. These SOP parameters are requested from the SEPView server or created via the BO and transferred from the embedded board to the electronics. All data streams from the various sensors carry time stamps and allow synchronization for visualization and analysis. Software:

[0079] SEPView ®< consists of a total of three functional software components. The first integral component of SEPView ®< is a platform-independent application server that communicates with the measuring device described above. Communication takes place via a specially developed communication protocol, which, according to the invention, enables parallel data entry and SOP programming / modification both via the server and directly via the BO by the measuring device's embedded computer. The central component of the SEPView ®< server is a document-based database in which the complete master and transaction data from SEPView ®< is persistently stored.

[0080] The second integral component of SEPView ®< is the SEPView ®< Explorer, which represents the user interface and is advantageously implemented as a platform-independent web interface. Using the web browser, comprehensive data visualizations, such as individual scattered light pulses and the classification of the various measured particle parameters, are realized using the graphics processor. These are analyzed in real time during the measurement and displayed on the operating system. Access to the SEPView ®< Explorer is only authorized.

[0081] The third component is the SEPView ®< recorder. It is launched from the SEPView ®< explorer, controls the entire measurement according to the SOP adopted by the user, e.g., from the server, or specifically programmed via the BO, visualizes the synchronized scattered light pulses, e.g., for different aperture angles or scattered light directions, as well as specified measurement parameters in real time, and records the processed SOP, all functional states, and the measurement data. The client-server architecture decouples the administrative, procedural, and analytical levels from one another, allowing them to be adapted to the respective customer processes and enabling distributed collaboration.

[0082] Fig.4shows typical results of the simultaneous registration of single-particle scattering in the forward (left) and sideward directions (right) for a mix of monomodal particle types (material: polystyrene) with a nominal diameter range of 143 nm to 3000 nm (logarithmic representation). According to the invention, the differently sized particles of the mixed sample with identical SOP were measured simultaneously and without any changes / adjustments to the measuring chamber geometry. For improved analysis, the forward and sideward scattering, for example, are also displayed as a 2-dimensional plot (axes: forward (Y-axis) versus sideward (X-axis) or vice versa). Each particle then corresponds to a point in the graph, which quantifies the measured intensities in the selected scattering or extinction channels. The point density corresponds to the respective number of particles with these characteristics.This also makes it possible to advantageously detect particles of the same size made of different materials as well as coated or uncoated particles.

[0083] Fig.5 (left) shows the results for determining the concentration of polystyrene particles with a size of 726 nm. The mean values ​​with standard deviation for five replicates (measurement time 1 minute each) are shown. The mean concentration is 108,960 + / - 640 particles per microliter. The standard deviation is only 0.6%, demonstrating the high counting stability of the developed method. Fig.5(right) shows the pure count rate of polystyrene (80 nm) and nanogold particles (50 nm). As can be seen, the inventive solution enables the recording of a constant, drift-free count value over the measurement time (one minute in the example image), even at a very high count rate of approximately 9000 Hz (events / s). Thus, after calibrating the syringe pump transporting the sample, the proposed method enables a very effective concentration determination (particles per mL) of microscale and, in particular, nanoscale dispersed particles.

[0084] Fig.6 shows a possible sequence of analysis steps of a downstream analysis of a sample measurement with the aim of calculating particle parameters (here e.g. grain size) using the measuring method according to the invention. 2. Examples of implementation

[0085] In the following, individual embodiments of the inventive solution for determining particle parameters are presented and explained in more detail. All of the following calculations refer to a laser wavelength of 532 nm. Other wavelengths, e.g. in the NIR, SL or UV, can also be used and enable the resolution / sensitivity or the dynamic measurement range to be improved for selected applications. Reducing the wavelength enables smaller particles and increasing the wavelength enables larger particles to be detected without entering the ambiguous areas of Mie theory. The particles are typically dispersed in water. However, depending on the materials used for the fluidics and the optical requirements, any liquids, solutions, solvents, etc. or gases can also be used as the carrier medium.For certain applications, the refractive index contrast can be increased by the choice of liquids, for example, and thus the detection of nanoparticles can be shifted into the single-digit nanometer range or the resolution for particles with very small size differences (e.g. < 5 nm) can be enabled.

[0086] The scattered light curves necessary for the description of the invention were calculated for the example particles according to Mie theory. The calculations are terminated at a particle diameter of 10 µm, as this size range is sufficient to explain the inventive concepts. The reception angles for the scattered light measurements, selected in terms of number and angular range, are arbitrary and can be adjusted as desired. 10.33° is the upper fixed limit angle, for example, of a non-commercial photometer, which is used for simplicity.

[0087] For the example calculations, four angles were selected for the lower variable reception angle limits: φ = 4°, 5°, 6°, and 8°, so that the angular ranges between φ and 10.33° can be used for the evaluation as needed. It should be emphasized that these angles can be set very variably. With appropriate optical arrangement, the lower limit angle can also be below 1°. a) Expanding the scope of application of

[0088] Scattered light measurements for the size determination of microscale particles with known refractive index.

[0089] "Normally," so-called forward scattering is used to determine the size of particles that fall outside the Rayleigh scattering range, and the intensity is measured in a specific angular range. Since the scattered light intensity initially increases monotonically as a function of particle size, but exhibits maxima and minima for larger particles ( Fig. 7 - 13), and thus does not allow a clear assignment to the particle size, a clear determination of the size depending on their optical properties is not possible for dispersions for size classes from a few micrometers.

[0090] How Fig.7 As shown, the particle size of frequently used reference particles (polystyrene) can only be clearly determined up to a diameter of 1.17 µm at an aperture angle of 4°-10.33°. The scattered light intensity in Fig.7 is then at I = 1.2. With a slightly higher scattered light intensity, a particle diameter of 2.36 µm according to Mie is also possible. According to the invention, this ambiguity is eliminated by measurements in several angular ranges ( Fig.8 ).

[0091] For a scattered light intensity of I = 2, for example, in the angle range 4°-12.33°, Fig.8The particle sizes 1.20 µm, 2.05 µm and 2.63 µm can be read. To resolve the ambiguity, the angle range 6°-12.33° can be used for an additional measurement ( Fig.8) to determine the actual size of the particle. In this angular range, the scattering intensities based on Mie calculations for the possible particle sizes determined in the angular range 4°-12.33° are 1.59 (1.20 µm), 1.04 (2.05 µm) and 1.34 (2.63 µm). The intensity differences are sufficiently large; accordingly, a clear (correct) diameter can be assigned to the particle. If an intensity of, for example, 1.04 is measured in the experiment for the second angular range, the particle size is 2.05 µm. If the intensities determined for the second angular range do not agree with the calculations, it is reasonable to assume that the observed particle is aspherical. Precise measurement and evaluation of the pulse shape can quantify this optical particle characteristic. In addition to the Rayleigh-Debye-Ganz theory (only valid for a limited application), other theories that deal with asphericity (e.g.discrete dipole approximation) can be used to obtain concrete quantitative parameters.

[0092] A similar procedure can be followed after a measurement in the angular range of 4°-12.33° in the particle size range of 4.0 µm - 5.0 µm. A combination of the angular ranges of 6°-12.33° and 8°-12.33° is recommended here. The equally possible particle size of 5.9 µm is determined by the latter curve. Here, the measured intensity should be 3 if the particle has this diameter.

[0093] As a further example, a scattered light intensity of I = 14 for a dispersion with particles with a refractive index of 1.8 is to be investigated ( Fig.9 ).

[0094] In the angular range of 4°–12.33°, the possible particle diameters are 8.1 µm, 8.5 µm, and 9.0 µm. To eliminate ambiguity, the invention requires measurements in a second angular range. In the angular range of 6°–12.33°, the corresponding intensities are 6.6, 9.5, and 10.1. If the difference of 9.5 and 10.1 does not appear sufficient for determination, the angular range of 8°–12.33° can be used. Here, the intensities are 4.8 and 7.1.

[0095] The examples also show that the theoretical Mie calculations can be used to determine which angle ranges can still be used for experimental decision after an initial measurement.

[0096] It is also clearly visible that for particles with a diameter greater than 10 µm, the scattered light curves in the four angular ranges considered here differ more from each other, making classification even easier. b. Simultaneous determination of particle size and refractive index

[0097] This part of the invention description extends the application a) in the case of an unknown refractive index of the respective particle for the simultaneous experimental determination of refractive index and particle size.

[0098] The procedure described under a) remains fundamentally the same. Even in the four arbitrarily selected reception angle ranges of the exemplary embodiment a), there is no four-matrix of the scattered light intensities in the angular ranges with the same values ​​for different refractive indices and particle sizes. If the experimentally existing measurement error of the scattered intensity is larger than the required accuracy, it is also possible to use a wider angular range (e.g., less than 4°) or, as Fig. 1ashown, to additionally use the side scattering, e.g. at an angle of 90°, to determine the particle parameters.

[0099] Here too, as in the examples already explained, inconsistent intensity values ​​must be used to conclude that the particles are not spherical, quantifiable, for example, by pulse shape analysis.

[0100] The refractive index of many particles of a material varies due to manufacturing processes, such as SiO 2 (porosity). The proposed invention makes it possible to determine even small refractive index differences between analyzed particles (here, silicon dioxide). This will be demonstrated using corresponding scattered light curves for n = 1.46 and n = 1.47.

[0101] For particles larger than 3 µm, the classification is unproblematic ( Fig.10 and Fig.11 ). An overlay, for example, of the Fig. 10 and 11proves that the intensities between the angular ranges are sufficiently different.

[0102] The inventive unambiguous assignment of the refractive index for particles of unknown size for arbitrarily selected diameters, e.g. of 2 µm and 0.5 µm, is now described, whereby for the sake of simplicity SiO 2 was also used as an example ( Fig.12 and Fig.13 or Fig.14 and Fig.15 .

[0103] As in Fig.12 As shown, particles with a diameter of 2.00 µm and a refractive index of n = 1.46, for example, have a Mie scattering intensity of I = 5.60 in the reception angle range of 4°-12.33°. However, there is an ambiguity, since this intensity is also present in particles with a refractive index of n = 1.47 and a particle diameter of 1.95 µm ( Fig. 13). If the reception angle range 8°-12.33° is also used, particles with a diameter of 2 µm and n = 1.46 scatter with an intensity of I = 1.84 ( Fig.12 ). The intensity ratio is 3.044. In the case of an assumed refractive index of n = 1.47, the scattering intensity is I = 1.91 ( Fig.13 ) and a

[0104] Intensity ratio of 2.93. To confirm / confirm the determined refractive index, the side scattering could also be used ( Fig.14 ).

[0105] To calculate the Mie intensity of the side scattering, for example, half the aperture angle (10.02°) of the Fig.1 sketched photometer at λ = 532 nm.

[0106] For a particle diameter of 2.00 μm, the scattered light intensity is calculated as I = 0.00116. The scattered light intensity for a particle with n = 1.47 and a diameter of 1.95 μm is approximately 20% higher (I = 0.00133). Generally speaking, for particles with a size parameter of approximately k less than or equal to 20, side scattering should be used. The size parameter k is calculated as follows: k = π n x / λ x: particle diameter n: refractive index of the continuous phase (dispersion medium) λ: wavelength of the radiation in vacuum

[0107] The determination of both size and refractive index (1.46 or 1.47) will now be demonstrated for particles of the exemplary size of 0.5 µm. In the angular range 8°-12.33°, an intensity of I = 0.00765 is measured. This corresponds to either 0.500 µm (n = 1.46) or 0.487 µm (n = 1.47). In the three other reception angle ranges considered here for forward scattering, the corresponding intensities differ by only about 1%. Using side scattering at an angle of 90°, one obtains Fig.15 : I = 5.68 10 -5< (0.500 µm; n = 1.46) and I = 5.91 10 -5< (0.487 µm; n = 1.47). The intensity differences are large enough to allow a decision to be made.

[0108] When initially measuring in the range 4°-12.33°, it should be noted that, as is generally the case with measurements, measurement inaccuracies may occur.

[0109] In general, it is necessary to ensure that the requirements for the measurement accuracy of the intensity of the scattered light curves are met by the dependence of the change in the scattered light intensity on the particle size (e.g. Fig. 14 ) can be determined. For larger dependencies of the scattering intensity on the particle size (slopes, e.g. Fig. 15 In the size range (0.70µm-0.75µm) errors of a few percent can be neglected.

[0110] If the required measurement accuracies are not achieved, additional angular ranges must be considered.

[0111] The smaller the particles become, the smaller the intensity differences become in the reception angle ranges. In the Rayleigh range, they no longer exist.

[0112] For particles with larger refractive indices than SiO 2 (n = 1.46 - 1.47), the determination of both the size and the refractive index tends to be easier for small particles (around 0.5 µm).

[0113] The refractive indices determined for each particle are to be sorted or classified according to size and result in a number-based distribution of the refractive index characteristic for the entire measured population. If the refractive index for each particle is plotted against the respective scattering intensity or the resulting particle size in a 2D plot (see also Fig. 16 ), subpopulations can be identified in the entirety and conclusions can be drawn about the homogeneity of the particle types in the sample.

[0114] The determination of the extinction coefficient (n") is also possible in principle. The presence of an additional variable requires more reception angles to be used for the measurements to resolve the increased ambiguities, as the number of possible combinations is then significantly increased.

[0115] For the invention, it is irrelevant how the different reception angle ranges are realized.

[0116] In a photometer, beam stops of varying diameter can be inserted one after the other—before or after the receiving optics (objective). A constant-size aperture can be moved within the diverging scattered light cone (or focusing cone) to achieve different receiving angle ranges.

[0117] It is also possible to combine ring-shaped detectors, each covering a different angular range. Different apertures in circular sectors can also be introduced. The scattered light intensities for the realized arrangements would then have to be measured separately and experimentally, e.g., using a mirrored prism or a position-sensitive photomultiplier. The invention advantageously eliminates the need for repeated measurements in different angular ranges described in the previous examples, which advantageously reduces the experimental effort and increases measurement accuracy through the simultaneous measurement of different angular ranges for the same particle.

[0118] In principle, multiple devices could be used. However, even with identical technical design, adjustment, and calibration, the selectivity is expected to be lower. c) Determination of sphericity in the case of a single-particle light scattering photometer

[0119] In addition to the refractive index, the optical properties of a particle are also determined by its geometry (e.g., spherical, prismatic, ellipsoidal, cylindrical, or irregular, etc.). Mie theory, most commonly used for particle size determination using light scattering, applies only to spherical particles. It is known from the theory of light scattering that the scattering behavior of spherical and aspherical particles of equal volume differs.

[0120] Aspheric particles cannot be described using the methods disclosed in sections a) and b). If they are applied, the theoretical Mie scattering intensities will not agree at both different scattering angles and aperture angles. This means, on the other hand, that the particle under investigation must be qualitatively classified as aspheric. This has already been pointed out several times in sections a) and b). The quantitative "asphericity index" (AI) can be defined, for example, as the percentage difference between the ratio of the theoretical Mie scattering intensities calculated for two (or more) aperture angles and the ratio of the scattering intensities determined experimentally at the same aperture angles.

[0121] Other defined metrics are also possible. Similarly, the "asphericity index" can be determined from theoretical calculations of the intensity for two scattering directions and the experimentally determined intensity ratio for the corresponding scattering directions.

[0122] The inventive solution also allows a second procedure for determining the "asphericity" of the particles. To determine the sphericity, the entire temporal intensity profile of the scattered light pulse during the passage of the particle through the measurement volume ( Fig. 1b ) is measured by the scattered light apparatus and analyzed or stored in real time. Saving the temporal progression of the scattered intensity for subsequent evaluation is also possible.

[0123] If the intensity distribution in the laser focus is known, the sphericity of the particle can be derived from the determined experimental intensity profile as the particle passes through the laser focus and through unfolding. Furthermore, according to the invention, a theoretical pulse duration for the particle can be calculated from the comparison of the determined size according to Mie (sphere assumption) and taking into account the experimentally calibrated sample volume flow. The comparison of the pulse duration measured experimentally for each particle after high-resolution digitization ( Fig. 4a ) with the theoretically calculated sphere-equivalent time span allows the invention to detect even small deviations and thus to quantify asphericities with high resolution

[0124] The quantitative particle geometry features defined in this way are to be sorted by intensity or size, resulting in a number-based distribution of the feature in the measured total population. By plotting the "asphericity index - AI" for each particle against the scattering intensity or the resulting particle size, the distribution of "AI" in the sample can be quantified or possible subpopulations can be identified within the sample as a whole. In addition to the particle size determined according to Mie, the distribution can also be plotted against the determined refractive index. Fig. 17 suggest that the disperse phase consists of two particle subfractions with different refractive indices (different material or non-uniform core-shell-particle) and that the fraction with the lower refractive index shows a much stronger scattering with respect to the asphericity index.

[0125] Application examples a) to c) were described for hydrodynamic focusing, as the process engineering challenges are considerably greater due to the significantly lower scattered light intensities (lower refractive index contrast for liquid particles compared to air particles). However, applications for aerodynamic focusing essentially follow the same inventive approaches. In particular, the measurement limits will advantageously shift to much smaller particle sizes down to the single-digit nanometer range, as the refractive index contrast increases several times over and the signal noise component is significantly minimized due to the higher scattered light intensities. This is explained in Fig.18 for silicon dioxide particles in air (n = 1,000) and water (n = 1,334). A 20 nm particle has approximately 100 times the scattered light intensity. d) Extinction modules with hydrodynamic focusing

[0126] The invention under a) - c) also has the inventive task of dispensing with the error-prone extinction measurements (Fraunhofer's approximate solution). However, if doubts remain for non-spherical particles using the described methods and calculations, it is advisable to resort to extinction measurements.

[0127] For this purpose, the primary beam can be deflected after flow cell 5, before the beam stop, and evaluated as usual. A second possibility for omitting this additional measuring arm and shifting the measurement limit to smaller particle sizes is shown in the solution described below.

[0128] An extension of the measuring range to smaller particles is opposed by the sensitivity of the known receivers, which are designed to detect small temporal shadows (negative light pulses or "extinction signals") from a large amount of light when a particle passes through the relatively large measuring zone.

[0129] For the measurement of smaller particles, the ratio of constant light intensity of the light source in the cuvette (preferably a laser 1) to an extinction signal is particularly unfavorable.

[0130] Hydrodynamic focusing helps to improve this ratio. In contrast to the state of the art, this technique reduces the original measurement zone for the extinction signal by imaging the entire measurement zone through an optical arrangement onto an aperture (preferably a pinhole or rectangular aperture) in such a way that only the area containing the extinction signals is not blocked by this aperture, which is then directed to a receiver. The light adjacent to the hydrodynamically focused particles is thus largely blocked in the image space. The measurement zone is now determined only by the particle stream diameter and not by the entire illuminated area in the cuvette.

[0131] With this arrangement ( Fig.19 ) it is possible to extend the lower measurable particle size to smaller diameters.

[0132] In a), the entire focus is filled with particles or is perfused. In b), the disadvantage is that a greatly reduced particle concentration must be used to ensure single-particle detection, since coincidences occur proportionally to the measurement volume. Hydrodynamic focusing in c) allows higher concentrations (e.g., up to 10 10< particles per ml) to be measured. The possible particle flow is marked by the dashed lines. The area outside the particle flow is shaded.

[0133] With this setup, the task arises of placing the image of the particle stream (i.e., the extinction pulses) precisely into the pinhole or rectangular aperture so that the pulses can also be registered by the receiver (optically, the brightness fluctuations of smaller particles passing through the laser beam are too weak to be detected by a camera). The target can be determined either by prior adjustment when using a scattered light photometer arrangement (similar Fig.1a) and subsequent removal of the aperture on the lens or displacement of the pinhole by means of x,y adjustment (i.e., perpendicular to the laser beam propagation) to achieve a maximum extinction deflection on a reference latex. In general, an extinction measurement has the disadvantage that a large part of the forward scattering also hits the receiver and attenuates the extinction pulses. Some of these scattered light pulses can be eliminated by inserting a pinhole in the beam path after the lens, following the adjustments described above, which only allows the primary laser beam to pass through ( Fig.20 This arrangement is particularly effective for determining the size of small particles.

[0134] Using the experimental setup described above, polystyrene particles with a diameter of 0.726 µm were resolved well compared to the noise level. When the noise level was largely suppressed mathematically using suitable smoothing algorithms, PS and SiO2 particles with a diameter of 0.55 and 0.50 µm, respectively, were clearly recorded ( Fig.21 ). With lower-noise electronics, it can be expected that the accessible particle measurement range can be further expanded.

[0135] A mixture of 3 different latexes in the size range from 0.8 µm to 1.3 µm (peaks a, b and c in Fig.22 ) could be separated with good resolution.

[0136] For a more accurate calculation of the particle diameter (not a Fraunhofer approximate solution), a spherical shape and a known refractive index are assumed. With the photometer described above, these calculations can be performed without extensive calibration curves, simply by measuring a size standard, since the angular range in which scattered radiation also reaches the detector is known. By calculating the scattered light incident at the known entrance angle, the particle diameter can be calculated more accurately.

[0137] To simplify particle size determination, or even to make it possible, the particles must be exposed to a nearly constant laser intensity. Therefore, it may be necessary to increase the laser focus by removing the relevant lenses behind the light source.

[0138] An existing single particle scattered light photometer (according to Fig.1a) can be expanded into a combined device using the modifications described above. The existing aperture in front of or behind the objective (or entrance lens) for blocking the primary beam is used for adjustment and then removed from the beam path in a suitable manner (e.g., by folding it up). The pinhole aperture for reducing stray light signals is placed in the beam path (it must be removed for adjustment).

[0139] The primary beam path with the extinction signals is either directed to the photomultiplayer after attenuation, e.g. by an optical neutral filter, or deflected, possibly without a filter, to another receiver.

[0140] The device is used for the analysis of several characteristics of individual particles or the classification or identification of particle fractions in industrial and academic areas, such as W / O or O / W emulsions, aerosols, slurries for waver polishing, ink and pigment suspensions, samples of cellular and subcellular particles (including cells, viruses, bacteria) of biological origin or for applications such as the design of nanoparticles, quantification of the stability of dispersions, investigations into the dissolution, agglomeration and flocculation behavior of dispersed phases, quantification of the progress of dispersions.

Claims

1. A method for determining particle parameters such as particle concentration, particle size, size distribution and particle count per size class, refractive index, and non-sphericity of microscale and submicroscale particles in flow measuring cells with hydrodynamic focusing by multiparametric acquisition of absorbance and / or scattered light signals, wherein the scattered light and absorbance signals of each individual dispersed particle that are generated by means of a laser with variable beam intensity and an aspherical beam cross-section (focus) are counted and simultaneously measured at a high detection rate (frequency) in at least two solid angle ranges, and compared in analog or digital manner using simulation calculations of the scattered light distribution by analytical or numerical methods for the different solid angle ranges in order to determine therefrom the individual particle parameters over a dynamic particle size range of four orders of magnitude without coincidence, even for high particle concentrations (e.g. 109 particles / ml) of the measurement sample, wherein use is made of different aperture angles for forward scattering or a plurality of detection directions for the scattered light measurement and of any desired combinations of these, wherein the particle size and refractive index are determined from the respective scattering intensities of the simultaneous measurements; wherein, in order to determine sphericity or non-sphericity, the entire temporal intensity profile of the scattered light pulse during passage of the particle through the measuring volume is measured and analyzed or stored in real time.

2. The method according to Claim 1, characterized in that, in the case of particles with size parameters k greater than or approximately equal to 20, different aperture angles are used for forward scattering.

3. The method according to Claim 1, characterized in that, in the case of particles with poorly distinguishable forward-scattered light curves at the different receiving angles (in particular in the case of a size parameter k of less than or approximately equal to 20), one or more detection directions in sideways directions with different sensitivities are used.

4. The method according to at least one of Claims 1 to 3, characterized in that the simulation calculation is carried out using Mie theory or numerical simulation calculations in accordance with the corresponding optical setup and the comparison between theory and experimental scattering intensity eliminates ambiguities with regard to particle size and thus microscale particles of up to 100 µm can also be measured.

5. The method according to at least one of Claims 1 to 4, characterized in that optical particle properties, such as size or refractive indices, are determined by checking the consistency of the modeled and experimentally determined intensities of the particle pulses for different aperture angles and / or solid angle ranges.

6. The method according to at least one of Claims 1 to 5, characterized in that, in the event of the scattered light intensities measured in the angular ranges not matching the possible theoretical intensity matrixes of the corresponding particle, asphericity is inferred and quantified by means of an asphericity index.

7. The method according to at least one of Claims 1 to 6, characterized in that, in the case of single particle scattering, the shape of the particle is classified as spherical or non-spherical on the basis of the comparison of the experimental, digitized pulse shape of a particle having a known refractive index with the corresponding simulation calculations for spherical particles for the same solid angle range.

8. The method according to at least one of Claims 1 to 7, characterized in that, in the case of single particle scattering, quantitative results are obtained with the assistance of theories dealing with asphericity and / or in that, in the case of single particle scattering, the particle fractions are classified according to features, e.g., according to size, shape and refractive index, and the particle counts per feature unit are determined, displayed and output and / or in that, in the case of single particle scattering, a laser with variable beam intensity and an aspherical beam cross-section (focus) with constant light intensity is used at least over the cross-section of the sample stream or, in the case of inadequate homogeneity of the intensity, the latter is corrected by mathematical normalization methods.

9. The method according to claim 1, characterized in that the extent of hydrodynamic or aerodynamic measurement current focusing can be adjusted manually or automatically from the ratio of sheath flow to sample flow as a function of the known initial count concentration of the measurement sample or on the basis of an initial measurement cycle.

10. The method according to Claim 1, characterized in that measuring chambers need not be changed nor flow cells modified for the size determination of very wide polydisperse samples.

11. The method according to at least one of Claims 1 to 10, characterized in that, in succession, beam stops are inserted or shifted, or annular detectors, each covering an angular range, are used, or different apertures in circular sectors are introduced, wherein the different scattered light signals are separately measured and evaluated.

12. The method according to at least one of Claims 1 to 11, characterized in that, by using annular detectors or different apertures in circular sectors, the repeat measurements in other angular ranges described in the examples are not necessary according to the invention, so advantageously reducing experimental effort and increasing measurement accuracy through the simultaneous measurement of different angular ranges for the same particle.

13. The method according to at least one of Claims 1 to 12, characterized in that, for absorbance applications, the ratio of the light intensity of the light source in the cuvette to an absorbance signal is improved by hydrodynamic focusing and by masking out the regions illuminated by the primary radiation in the image space adjacent to the hydrodynamically focused particles in order to detect smaller particles.

14. The method according to at least one of Claims 1 to 13, characterized in that, for absorbance applications, a pinhole inserted downstream of the objective lens and having the diameter of the primary laser beam downstream of the objective lens eliminates a major proportion of forward scattering that would also impinge on the receiver, so extending the measurement range to smaller particles and permitting more accurate particle size calculations.

15. An apparatus for determining particle parameters such as particle concentration, particle size, size distribution and particle count per size class, refractive index, and non-sphericity of microscale and submicroscale particles in flow measuring cells with hydrodynamic focusing by multiparametric acquisition of scattered light and absorbance signals, comprising: at least one laser (1) for generating at least one laser beam (3) with variable beam intensity and an aspherical beam cross-section (focus), at least one optical input module (2) for shaping the laser beam (3) and creating the aspherical focus geometry (16), a flow measuring cell (5) with hydrodynamic focusing, in which forward-scattered light radiation (6) and sideward-scattered light radiation (7) is generated by the laser beam (3), and that has an inflow for the sheath flow (13), a sample inflow (14) and an outflow (15), wherein the sheath flow (13) is delivered from a reservoir (17) in controlled and pulse-free manner via a pressure generation apparatus (18), optical output modules (8) in the forward-scattered light beam (6) and optical output modules (9) in the sideward-scattered light beam (7), semitransparent mirrors (10), cameras (12), photomultipliers (11a, 11b), wherein, by way of the photomultipliers (11a, 11b), scattered light measurements can be carried out for a differing number of aperture or receiving angles and, using evaluation algorithms, the particle parameters can be determined from the determined scattered light intensities of the forward-scattered light beam (6) and of the sideward-scattered light beam (7), wherein the particle size and refractive index can be determined from the respective scattering intensities of the simultaneous measurements, and wherein, in order to determine sphericity or non-sphericity, the entire temporal intensity profile of the scattered light pulse during passage of the particle through the measuring volume can be measured and analyzed or stored in real time.