Particle Characterization - UvA€¦ · Web viewParticle Characterization Advantages, limitations and applications of modern analytical techniques Joyce Koster 6-7-2015 MSc Chemistry

University of Amsterdam

Particle Characterization

Advantages, limitations and applications of modern analytical techniques

Joyce Koster

6-7-2015

MSc ChemistryAnalytical Sciences

Literature Thesis

Particle Characterization

Advantages, limitations and applications of modern analytical techniques

by

Anna Cornelia Willemina Koster

April 2015

Supervisor:dr. W. Th. Kok

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Preface

This literature study is part of the study program of the MSc Chemistry – Analytical Sciences at the University of Amsterdam. The report contains an informative summary and a critically review of scientific publications on the subject of particle (size) characterization. The study includes the most recent literature and developments, my perception on the subject and suggestions for further research.

Field-flow fractionation will only be briefly discussed, since this is the topic of my final research project. Nonetheless, it is an analytical technique which can be used for particle characterization and, therefore, worth mentioning. Even though microscopy is a commonly used technique for particle size measurement, it will not be discussed in this report. The focus will be on more fundamental analytical techniques.

The techniques discussed in this report are categorized based on their basic operation principles. Each chapter treats a category in which different techniques are addressed. The principles, advantages, limitation and applications of each technique are summarized.

Finally, I would like to thank Remy, who gave me the reason to finish this report.

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Table of Contents

Preface ii

Summary v

1 Introduction 1

2 Basic principles of particle characterization methods 2

2.1 Equivalent sphere theory 2

2.2 Acquired data: Number, length and moment means 2

3 Separation techniques 4

3.1 Sedimentation 4

3.2 Sieve analysis 5

3.3 Size Exclusion Chromatography 5

3.3.1 Principles 5

3.3.2 Advantages and limitations 7

3.3.3 Applications 8

3.4 Hydrodynamic chromatography 9

3.4.1 Principles 9



3.5 Field-flow fractionation 11

3.5.1 Principle 11



4 Mobility techniques 14

4.1 Electrozone sensing 14

4.1.1 Principle 14



4.2 Aerodynamic time-of-flight analysis 16

4.2.1 Principle 16



5 Light scattering techniques 18

5.1 Static light scattering 18

5.1.1 Principle 18

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5.1.2 Right-angle light scattering 19

5.1.3 Low-angle light scattering 19

5.1.4 Multi-angle light scattering 20

5.2 Dynamic light scattering 21

5.2.1 Principle 21



6 Acoustic and Electroacoustic Spectroscopy 24

6.1 Principles 24

6.2 Advantages and limitations 25

6.3 Applications 25

7 Comparison of characterization techniques 27

8 Conclusion and discussion 29

References 31

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Summary

Particle (size) characterization is an important issue in different fields. Particle characteristics influence product properties and, therefore, these characteristics need to be controlled. There is a large variety of different analytical techniques, all based on different principles. Due to this variety it is not always clear which technique is most suitable for a specific application and what the limitations are. This report presents the advantages, limitations and applications of modern and most commonly used analytical techniques used for particle characterization with respect to size and shape. The information in this report is based on scientific publications and includes the most recent literature and developments in the field.

Different techniques measure different characteristics of a particle (e.g. volume, maximum length, weight). The equivalent sphere theory is used in order to unify and simplify these different measuring principles. Furthermore, different means can be used in order to describe a sample.Separation techniques can be used for particle characterization, either for characterization itself, or as ‘sample pretreatment’ for other techniques, such as light scattering. Sedimentation and sieving are conventional methods, whereas size exclusion chromatography, hydrodynamic chromatography and field-flow fractionation are more advanced methods. Then there are mobility techniques which can be used for characterization. The most important ones are electrozone sensing (also called coulter counting) and aerodynamic time-of-flight measurements. Electrozone sensing is performed in a liquid and the sample needs to be dispersed. Aerodynamic time-of-flight can be performed in both gas and liquid samples, but is much applied in aerosol characterization in pharmaceutics.Light scattering techniques, either static or dynamic, are probably the most well-known method for particle characterization. They can be used stand-alone, but also coupled to chromatographic systems. The main problem is the need for dilution and angular dependence of the scattered light. Acoustic- and electroacoustic spectroscopy is less well-known, but also a suitable technique. More concentrated sample can be analyzed than with the light scattering techniques, giving an extended applicability of the technique

Only a fraction of all analytical technique for particle characterization is discussed in this report, but it is clear that there is not one superior technique. Depending on the type of sample and information required, the most suitable analytical technique should be selected. Naturally, every technique has its advantages and limitations and they should be considered during both the selection process and data analysis. It is recommended to select one single techniques of sample comparison, since this is the most accurate.

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1 Introduction

Particle characterization is an important issue in different fields, such as in pharmaceutics, environmental science and the chemical industry. Particle characteristics influence product properties and consequently, these characteristics need to be controlled. In product development it is important to know and understand the effect of particle properties on the behavior of the product. Furthermore, the influence of the production parameters on the particle properties should be known in order to optimize the production process and accurately control the production [1].In order to control particle characteristics and understand their effect and the parameters which will influence them, particle characterization is an essential tool. However, there is a large variety of different analytical techniques, all based on different principles. Since there are different principles on which the analysis can be based, not all techniques will be suitable for each application. Due to the variety of techniques, it is not always clear which technique is most suitable for a specific application and what the limitations are. An unsuitable analytical technique will provide incorrect results and wrong data interpretation.

This report presents the advantages, limitations and applications of modern and most commonly used analytical techniques for particle characterization. It can be used by scientists as background information, method selection and for correct data interpretation.The information in this report is based on scientific publications and includes the most recent literature and developments in the field.Due to the extent of the topic, the scope of this literature study is restricted to the characterization of particles size and shape of particles in the nanometer (nm) to micrometer (μm) range. Additionally, only the most commonly used and modern analytical techniques are discussed. Microscopy is out of scope for this study, since it is a broad subject on its own. The main focus of this report is on the advantages, limitations and applications of the analytical techniques involved. In order to understand these aspects the report only discusses the basic principles of the techniques, no in-depth theory is provided. Consequently, this report provides a more practical, rather than a theoretical insight in particle characterization.

Chapter two will focus on some basic principles and concepts used in particle characterization. Chapter three will first address the separation techniques which can be used for particle characterization. Some conventional methods, such as sedimentation and sieving, will be discussed. Next, chromatographic techniques will be reviewed, such as size exclusion chromatography (SEC), hydrodynamic chromatography (HDC) and field-flow fractionation (FFF). In chapter four mobility techniques will be discussed. Mobility techniques include electrozone sensing and aerodynamic time-of-flight. Light scattering techniques are discussed in chapter 5. This includes static light scattering, dynamic light scattering. Finally, acoustic and electroacoustic spectroscopy will be discussed in chapter 6. The report will be completed with a comparison of all techniques discussed and a conclusion.

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2 Basic principles of particle characterization methods

In order to evaluate the different particle characterization techniques, the basic principles behind the concept of particle characterization need to be known. This chapter contains the basic assumptions made in all particle characterization methods, the different results which can be obtained with it and the conversion of these results. Together, these are the basic principles behind the concept of particle characterization.

2.1 Equivalent sphere theory

Normally, the concept of particles induces a visual image of spherical objects. However, in practice only a minority of the particles are shaped as perfect spheres. This complicates the description of particles, especially concerning the size. A sphere can be described by a single number, but imagine an irregular shaped particle, how can the size of such particle be described? To be absolutely precise, multiple dimensions are needed to describe the entire particle (the length could be different from the height etc.). This would make matters unnecessarily complicated.In order to simplify the description of a particle, the equivalent sphere theory is often used. Using this theory, a specific measured property of a particle (e.g. maximum length, volume, weight, etc.) is converted to the diameter of an equivalent sphere having the same property as the measured particle. By doing this the particle is described as if it was a sphere without actually being a sphere. This forms the foundation for particle size analysis [1] [2]. Using this theory the concept of particle description is simplified, because only a single number is needed to describe the particle. However, the resulting equivalent sphere diameter is dependent of the property which is measured. Since different analytical techniques measure different properties of a particle, they also use different equivalent sphere model and, therefore, provide different results.

Figure 2.1 – The concept of equivalent spheres. Reproduced for ref. [1]

As can be seen in figure 2.1, different properties result in different equivalent sphere models. Consequently, analytical techniques which are measuring different properties cannot be compared directly.

2.2 Acquired data: Number, length and moment means

Using the equivalent sphere theory, irregular shaped particles are converted to a simplified model, but there is a second complication which needs to be handled. A sample will contain numerous

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unique particles that can be differently shaped and/or sized in one sample. It is impossible to describe every individual particle; hence the results of particle size analysis are size distributions with the means of these distributions. Different types of means can be calculated based on different properties of the particles. Table 2.1 presents different types of means used in particle sizing.

Table 2.1 – Number, length and moment means [2]Mean Term Formula

Number means

Number length mean D[1,0] ∑ dn

Number surface mean D[2,0] √∑ d2

n

Number volume mean D[3,0] 3√∑ d3

n

Length means

Length surface mean D[2,1]∑ d2

∑ d

Length volume mean D[3,1] √∑ d3

∑ d

Moment means

Surface moment mean D[3,2]∑ d3

∑ d2

Volume moment mean D[4,3]∑ d4

∑ d3

(d) Diameter of equivalent sphere; (n) number of particles

Since the equivalent sphere theory is applied, the calculations involve the diameter of the equivalent sphere (d). Number means are not commonly used in particle sizing since the formula contains the number of particles (n). Normally, particle counting is only applied to samples with a very low concentration of particles, since it would be rather impractical to count millions of particles (especially in suspensions). The type of mean which is obtained depends on the analytical technique which is used to obtain the particle size distribution. Different means and distributions are sensitive to different sized particles (e.g. The surface volume mean is sensitive to small particles while volume moment means are sensitive to large particles) [1]. When means of different techniques need to be compared, they need to be interconverted. This can be done mathematically by using the formulas in table 2.1. However, it should be realized that the error in the measurement is also affected by the conversion. The mean that is used depends strongly on the applications and preferably a suitable technique should be used.

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3 Separation techniques

Separation techniques can be used to determine the particle size distribution of a sample. The most basic techniques are sedimentation and sieving, but also more advanced techniques such as chromatography can be used. Especially Size Exclusion Chromatography (SEC), Hydrodynamic chromatography and Flow-Field Fractionation (FFF) are commonly used for particle analysis. The basic principles, advantages and limitations and applications of these techniques are discussed in this chapter.

3.1 Sedimentation

Sedimentation is a traditional method that is based on measuring the time a particle needs to settle over a known distance in solution. The technique is most commonly used in paint and ceramics industries. The measurement is based on Stokes Law (see equation 3.1).

v s=( ρs−ρf ) d2 g

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Equation 3.1 – Stokes Law. Vs = Settling velocity, ρs = Mass density sphere, ρf = Mass density fluid, d = Sphere diameter, g = Gravitational acceleration, η = viscosity fluid.

Based on the settling velocity of the particles in solution the Stokes diameter can be calculated. For the determination, the viscosity of the solution needs to be known and, consequently, the temperature needs to be controlled during the entire duration of the experiment. Naturally, the method can only be used for suspensions that settle. However, even these suspensions need to be dispersed homogeneously which can be difficult.

There are three common methods to perform a sedimentation experiment. These methods focus on the concentration changes in a specific depth of the sample during the settling process [2]:

Andreasen pipette methodIn this method a suspension is allowed to settle in a temperature controlled environment. At predetermined times a sample is extracted from the suspension at known depths. The sample is dried and weighed to determine the amount of solids in the sample. This way the concentration changes over time are monitored and the size distribution can be calculated [3]. This method can be time-consuming since it takes time for the particles to settle.

X-ray sedimentation methodThis is another gravimetric method which uses an X-ray beam to monitor the concentration changes over time. This decreases the analysis time because the beam can scan the surface. However, the method is limited to materials which can scatter the X-ray beam, thus containing high atomic mass materials.

Centrifugal sedimentation methodBecause gravimetric sedimentation can be time-consuming, the sedimentation can be accelerated using centrifugal forces. As can be seen in equation 3.1, the gravitational acceleration is a factor in the settling velocity. Using centrifugation with a small height of the suspension this constant can be replaced by the centrifugal acceleration [4].

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The concentration changes during the settling time can be monitored using pipetting, photometry or X-ray spectroscopy.

Sedimentation can only be used for particle sizes > 1 μm. Smaller particles also undergo Brownian motion which competes with the gravimetric settling. Moreover, Stokes law is based on spheres and, therefore, irregular shaped particles can results in significant deviations and low reproducibility of the results. The equivalent sphere will be negatively biased, since irregular shaped particle possess more surface area resulting in a lower settling velocity. Furthermore, Stokes law is also limited for polydisperse materials, and results will be biased towards larger particles. The stability of the dispersion should be considered. Aggregate formation should be prevented because the particle size will be change during the measurement resulting in incorrect results with a low reproducibility. Additionally, the concentration influences the viscosity and should not be too high since concentration changes are monitored while the viscosity should be constant.

3.2 Sieve analysis

Sieve analysis is one of the oldest methods used for particle size determination. The material travels through sieves with different pore sizes, by means of shaking the particles travel through the different sieves. When the pores of the sieve become too small for the dimensions of the particles, these particles remain on that particular sieve. Originally, it was done manually but today it can also be automated. The result of the method is a particle size distribution based on the amount of materials going through the individual sieves.

The method is applicable for particles > 10 μm. Both dry and wet samples can be analyzed but the material needs to contain solid particles. As a result emulsions and aerosols cannot be analyzed [1]. It has the advantage of being cheap, however, it can have a low reproducibility especially with wet analysis [2] [5]. Furthermore, during the movement of the sieves, the particles are orientated in the alignment with the sieves. Therefore, only the smallest diameter of the particles is measured [5]. Moreover, the result of the analysis depends on the time and the duration of the movement which is applied to the sieves.

3.3 Size Exclusion Chromatography

3.3.1 PrinciplesUnlike sedimentation and sieving analysis, size exclusion chromatography (SEC) is used for particles in the nm range. SEC is a form of Liquid Chromatography (LC) which is mainly used for the analysis of macromolecules in solution, but it can also be applied to particles <0.5 μm [5]. The molecules or particles are separated based on the difference in hydrodynamic volume. The nanoparticles are passed through a column packed with porous material. Particles with a large hydrodynamic volume cannot enter the pores and do not undergo retention, where particles with a small hydrodynamic volume do enter the pores and as a result, undergo retention. The separation principle will not be discussed in detail but in contrast to LC, the equilibrium in SEC is mainly driven by the entropy term due to the limited mobility of the analytes inside the pores of the stationary phase [6] [7].The hydrodynamic volume can be influenced by the solvent. Consequently, solvent choice is an important parameter for separation. Ideally, retention is only based on the size difference and particles should not have any interaction with the stationary phase (as is the case in other

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chromatographic techniques). Since SEC separation is based on hydrodynamic volume, the molecular weight of the molecules can be determined based on their elution volume. This is done by column calibration with molecular weight standards. Next to molecular weight, the concentration of a component can also be determined. However, SEC is considered to be low resolution chromatography and, therefore, quantification can be less accurate.

SEC can be combined with different detection techniques, depending on the application:- Refractive Index (RI)- Ultra-Violet (UV)- Viscosity (VISC)- Fluorescence- Light Scattering (LS)- Mass Spectrometry (MS)- Inductively Coupled Plasma Mass Spectrometry (ICP-MS)

RI is a universal detection technique which is commonly used in polymer analysis. A majority of the polymers does not contain UV active components and RI is a relative simple technique to use. UV detection is commonly used in protein analysis, since amide bonds have a strong absorbance at low wavelengths around 220 nm [8].Fluorescence detection not as commonly used, since only few components contain fluorescent groups. However, this also means that fluorescence detection is a very sensitive and selective detection technique. It is possible to label analyte with fluorescent probes however, this may influence the separation and is not favorable. If that is the case, post-column derivatization is an alternative. The labeling takes place after the separation, consequently the chromatography is not influenced by the label. However, the reaction time and conditions are limited.Differential viscosity can be used to determine the intrinsic viscosity for each SEC elution increment. In combination with conventional column calibration it provides the absolute molecular weight of a particle. In addition, it provides insight into the conformation of the particle. Meunier et al. used SEC/VISC (among others) in order to characterize cross-linked polymeric nanoparticles [6].

More advanced detection techniques are LS and MS. Online LS detection can be performed in different operation modes (e.g. multi-angle, low-angle, right angle [9]). The principles of this technique will be discussed in chapter 5. LS has the advantages that when coupled to UV, RI or viscosity detection can determine the molecular weight of components independent of the elution volume. MS is another detection technique which determines the molecular weight independently of column calibration. The difficulty with SEC-MS hyphenation is the mobile phase. For organic phases these problems do not occur. However, in protein analysis an aqueous mobile phases is used which contains high concentrations of non-volatile salts. This is not compatible with MS and it should be considered in the method development of SEC-MS. Possible solutions are the use of denaturing mobile phase, desalting or offline MS analysis after fraction collection. Kükrer et al. used the latter approach to characterize antibody aggregates after pH stressing with Electrospray Ionization Time-Of-Flight Mass Spectroscopy (ESI-TOF MS). Even though it was successful for their purpose, it is a time-consuming and labor-intensive method since the LC fractions need to be prepared for MS analysis. ICP-MS is another detection technique. It is a sensitive technique that can measure down to parts-per-trillion (ppt) level. It can be used in combination with SEC to investigate the metal-organic complexes in proteins. Groessl et al. used this technique to study the interaction of metallodrugs

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with serum proteins [10].

One of the strengths of SEC is that multi-detection approaches can be applied to obtain more information in one single run. The chromatography applied can be seen as sample preparation for some techniques as ICP-MS, MS or light scattering techniques. Combined these detection techniques can provide information about the composition of the particles and the size and optionally configuration. When RI, UV-Vis or fluorescence are used, SEC can be used for molecular determination. These detectors can be used for concentration determination. Combining both concentrations, composition and size sensitive detectors, provide a more complete inside in the analyzed molecules.

3.3.2 Advantages and limitationsSEC is a chromatographic technique which is relatively easy to use and can be performed on regular HPLC systems. The separation principle relies on size exclusion which can be performed in a short analysis time. Another advantage is that only low sample amounts are required. All these aspects make it an inexpensive analytical technique which makes it very attractive for industrial laboratories [11]. Additionally, SEC analysis is performed in solution, which is an advantage in certain applications.

The advantage of having no interaction with the stationary phase is that the elution times can be related to the molecular weights of the component using column calibration. In practice, this can be challenging since interactions do appear and influence the elution volume hence, incorrect results are obtained. When molecular weight standards of the analyte are available this problem is solved, however, this is often not the case. Another way to solve this problem is to couple SEC to molecular weight sensitive detectors, such as LS detectors. Additionally, SEC has only limited separation power. Small differences in hydrodynamic volume can be hard to separate and broad peaks are often seen, which can result in less precision of quantification. The resolution depends on the analytical column. When solely particle size distribution are of interest, the resolution is often sufficient. SEC is commonly used in polymer and protein analysis. The hydrodynamic size of polymer and the stability of the proteins (aggregation) can be dependent on solvent/buffer composition and the concentration of the solution. Consequently, the choice of eluent, sample preparation and sample dilution in the system should be considered [12] [11].As mentioned in the previous paragraph, SEC can be combined with multiple detectors. This enables scientists to obtain multidimensional information about particles in one single analysis.

One of the characteristics of SEC is that it is only applicable for dissolved molecules and nanoparticles (<0.5 μm). Suspensions contain bigger particles and emulsions cannot be measured and will even plug the chromatographic system. One of the major limitations of SEC analysis of nanoparticles is sorption of the particles to the column packing. This is caused by the high surface area of the stationary phase and the high surface activity of nanoparticle [13]. In order to minimize these interactions, surfactants or capping agents should be added to the eluent. Wei and Liu [14] solved the sorption problem of gold nanoparticles by the addition of sodium dodecyl sulfate (an anionic surfactant) to the eluent. They used a Nucleogel GFC column of 300x7.7 mm with a 100 nm pore size and UV detection for their nanoparticles separation.

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3.3.3 ApplicationsThe most common applications of SEC are synthetic polymer, biopolymer and protein analysis, but it can also be used for nanoparticles separation. For synthetic polymers an organic mobile phase is often used. For biopolymers and proteins, an aqueous eluent is used. Since synthetic- and biopolymer applications are quite common, they will not be discussed here.

In nanoparticles and colloid separation, the analysis of gold nanoparticles is discussed in many applications. Wei and Liu [14] discussed that SEC can also be used to obtain monodisperse fractions from a polydisperse sample. They achieved baseline separation of gold nanoparticles with a 33 nm size difference. But separation can be even more efficient when using a different approach. Colvin et al. baseline separated gold nanocrystals with a 0.6 nm size difference using Recycling Size Exclusion Chromatography (RSEC) [15]. In RSEC the column length of a SEC system is increased by recycling the column output back into the column inlet. In this way the resolution is increased with each cycle, without increasing the backpressure of the system as would be caused by the use of long columns. This can be done on one single column (closed-loop recycling), or on two columns placed in series with a switching valve in between (alternate-recycling). The alternate RSEC is used in Colvin’s research. A porous hydrophobic microgel cross-linked polystyrene column of 300x7.5mm with a 50 or 100 nm pore size is used. UV detection at 520 nm was applied. The size distribution of the nanoparticles in each fraction was analyzed with Transmission Electron Microscopy (TEM). Figure 3.1 illustrates the increased resolution with RSEC of gold nanoparticles at 1 mL/min.

Figure 3.2- Separation of gold nanoparticles with RSEC. Reproduced from Ref. [15].

Furthermore, shape separation of nanoparticles with SEC is reported. Wei et al. [13] were the first to report the SEC separation of different shapes of gold nanoparticles. The separation was obtained on a Nucleogel 300x7.7 mm column with a 100 nm pore size while using a mixed surfactant system as eluent. Rod-shaped particles were separated from spheres, even though, resolution was poor. Using UV detection, the different shapes can be distinguished due to the different surface plasmon resonances (resulting in different absorption maxima). This was confirmed with offline TEM analysis of the fractionated material.

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3.4 Hydrodynamic chromatography

3.4.1 PrinciplesHydrodynamic chromatography (HDC) is another form of chromatography which allows the analysis of particulates and colloids. Unlike LC analyses which are based on interaction with the stationary phase, retention in HDC are based on hydrodynamic processes. HDC uses the velocity gradient created within a capillary channel to separate the dissolved or suspended analytes. Larger particles are excludes from the near-wall regions of the channel due to steric repulsion and, therefore will experience a high average velocity and consequently elute faster than smaller particles. As with SEC, it is important that there is no enthalpy interaction between the analytes and the stationary phase. In aqueous systems salts or surfactants can be added to the eluent to minimize these unwanted interactions [16]. Figure 3.2 illustrates the mechanism of separation of HDC inside the channels.

Figure 3.3 – Mechanism of separation of HDC. (a) Starting mixture. (b) Separation. Reproduced from ref. [16].

The use of channels might suggest that only capillary columns can be used, however, the analysis can also be performed using packed columns, rectangular channels and between plates [17]. The packed columns contain non-porous beads and the velocity gradient is created in between the beads. For the packed columns the applicable size range is 0.03 to 2 μm, while an open-tubular column the can be used for particles between 0.02 to 50 μm [5]. Naturally, this is because the packed column has more restricted channels.

Even though there are similarities in experimental setup, the separation mechanism of HDC is different from the separation mechanism of SEC. However, both mechanisms may occur during SEC analysis [16] [18]. As a consequence, HDC analysis can be performed on a SEC column when the analyte particles are larger are then the largest pores. The maximum size of the particles should be considered in order to avoid blockage within the column. Furthermore, the same detection techniques can be used for HDC. An overview is given in paragraph 3.3.1 and the information will not be repeated here. In addition to these detection techniques, ICP-MS detection can be used for the analysis of metallic nanoparticle with HDC. Hence, it is an interesting technique in environmental studies concerning nanoparticles.

3.4.2 Advantages and limitationsHDC is a flexible technique which is suitable for quite a broad range of applications. The technique has the same advantages as SEC; it is fast, requires only low sample amounts and it can be performed

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on regular LC systems. One of the biggest advantages is that it is not limited to solute species but also suitable for colloids and particulates.

An advantage of HDC over SEC is that, with the use of the most appropriate calculation model [16], the size of the analyte (radius of gyration) can be calculated without the use of column calibration or expensive detectors. Even though this is only a theoretical approach, it has been reported in literature [19]. Another advantage is that it can be performed on regular HPLC system without setup changes. No specialized equipment is needed and this makes it easy to use and cost efficient. As discussed briefly in the previous paragraph, HDC can be performed on a SEC column. The requirement is that the analytes are larger than the largest pores of the stationary phase particles. Due to these instrument and column requirements, HDC is a very accessible technique.The main limitation of HDC is that it has low chromatographic selectivity because of the relative narrow retention window. Moreover, detection can be problematic when using capillary HDC, because of the small detection volumes in the capillary.

Since HDC can be performed with capillaries, the technique can be performed on a chip. Chip technology has the advantages that is reduces solvent consumption, analysis time and improves efficiency. HDC can be used for the separation of large uncharged analytes, which cannot be done by other miniaturized chromatographic techniques [20]. Blom et. al described the first on-chip planar HDC separation combined with UV absorption detection. They used a channel configuration of 1 μm high, 0.5 mm wide and 69 mm long, to separate nanoparticles and biomolecules. The influence of the sidewalls on the dispersion was thoroughly investigated. Using wider channels reduces the contribution of the sidewalls to the dispersion. Furthermore, an optical detection cell is integrated in order to allow UV absorbance detection without extra-column broadening.

Figure 3.4 – Separation of polystyrene latex standards of (1) 155 nm and (2) 61 nm, (3) 26 nm fluorescent particles and (4) benzoate at a working range of 6.1 bar. Reproduced from Ref. [20].

3.4.3 ApplicationsA recent development in particle characterization with HDC is the multi-detection approach in which triple- and quadruple detector HDC is used. This is attractive because more information of the sample and analyte can be obtained in a single analysis. Brewer and Striegel reported the analysis of polystyrene latexes using HDC coupled to multi-angle light scattering, quasi-elastic light scattering, differential viscometry and differential refractometry. Using this quadruple-detector approach both size and shape of latexes could be characterized in one chromatographic run [21].

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3.5 Field-flow fractionation

3.5.1 PrincipleIn field-flow fractionation (FFF) a sample solution or suspension is introduced into a channel. The channel can either by flat or cylindrical. Perpendicular to the flow through the channel a field is applied. Different sort of field can be applied, based on the analytes which need to be separated:

- Gravitational or centrifugal- Thermal- Cross flow- Electrical - Magnetic- Etc.

In this report, the focus will be on gravitational/centrifugal and cross flow FFF. In figure 3.4 the principle of FFF is illustrated. The application range of FFF depends on the field which is used but can vary between 10 nm to 10 mm particle diameter [22].

Figure 3.5 – Principle of Field-flow fractionation. Reproduced for Ref. [22]

Because of the field analytes are concentrated at the bottom of the channel. The diffusion coefficient of the analyte will determine the layer thickness of the accumulated molecules. As described in paragraph 3.4.1, the average velocity of a component in a laminar flow is determined by its position in the flow channel. Compounds with a low diffusion coefficient will be closer to the wall and have longer elution times, while compounds with high diffusion coefficients will be further away from the wall and have shorter elution times. A additional separation mode which can be important in the analysis of particles is the steric elution mode. When large particles are analyzed with relatively high field strength, the particles reach the accumulation wall and based on steric hindrance the elution order is reversed. For large particles the diffusion coefficient is relatively low and in combination with high field strength, the particles migrate to the accumulation wall. Small particles are closer to accumulation wall since the size of the particle is smaller. They will have low velocity. Large particles have a larger volume and are influenced by higher velocity streams. Dou et al. studied the steric transition point of polystyrene particles in AF4 [23].The effect of the field on different compounds depends on the type of field and the nature of the compounds. The retention parameter is determined by the diffusion coefficient, the field strength and the height of the channel. In order to improve the fractionation of analytes or reduce analysis time gradients can be applied in the field. FFF can be coupled to different types of detectors as described in paragraph 3.3.1.

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3.5.2 Advantages and limitationsThe advantage and limitation of this technique depend on the type field applied, since the field determines the mechanism for fractionation. The field also determines the instrumentation design. Whereas sedimentation FFF requires complicated instrumentation, Flow FFF can be executed with more common equipment [22]. The fact that different types of fields are available is an advantage on its own. Different fields enable the use of different separation principles and therefore, extend the application field of FFF. The main advantage of FFF in general is the absence of stationary phase and/or packing material as is used in chromatography. This enables the analysis of bigger particles and more sticky components which could easily clog a chromatographic column. Furthermore, high ionic strength solutions can be used as elution medium when a non-electrical cross field is used [24]. Especially, for the analysis of biological compounds this could be a requirement. This can be an advantage over capillary electrophoresis, where a high ionic strength environment can cause Joule heating. The carrier fluid in FFF in not bound to many requirements and can be chosen freely for most fields applied. This has the advantage that the optimal environment for the analytes can be used. Another advantage is the extensive dynamic range which can be covered with FFF. This is also depending on the field applied, but even within the use of one field, the dynamic range is significantly bigger than for other separation techniques. Finally, FFF is a non-destructive technique and allows downstream (online or offline) analysis of the fractions.

The limitations of FFF are mainly linked to the design of the instrumentation and the fact that is it is a novel technique, which is still being developed and improved. In FFF modes where membranes are present, the effects of membrane adsorption are not yet fully studied and will be different for each type of material. The instrumentation of some FFF modes, like centrifugal FFF, are quite complex and contain many moving parts. These parts will wear fast, and more maintenance is required for these instruments.Even though the interactions with channel components are less than for example in SEC, and conditions can be optimized for the analytes of interest, it is still challenging to predict the particle size only based on elution time. Based on the principles and formulas the particle size or molecular weight can be estimated, but for accurate determination a suitable detector (such as a light scattering detector) should be used.

3.5.3 ApplicationsFFF knows many applications in environmental sciences. Especially sedimentation FFF is applied in this field. A broad particle size range can be analyzed and for this reason provides a good alternative for membrane filtration which has a cutoff point. Furthermore, it is faster than microscopy which makes it an attractive new technology in the environmental field. Laura et al. used sedimentation FFF to determine the particle size distribution in order to compare centrifugation and membrane filtration techniques used for clay soil suspension [25]. They used a sedimentation FFF instrument from FFFractionation LLC (now PostNova) with a channel of 86.1 cm. Hagendorfer et al. described the analysis of silver nanoparticles with AF4 in combination with multiple detectors [26]. In this study AF4 was compared with transmission electron microscopy (TEM). AF4 is a faster method and has the additional advantage that samples can be analyzed directly in the dispersions. Compared to batch DLS, AF4 is more suitable for the analysis of samples with high polydispersity. Figure 3.5 illustrated the differences found with the different techniques.

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Figure 3.6 – Number size distributions obtained with AF4-DLS-ICPMS, TEM and batch-DLS with corresponding TEM images of silver nanoparticle samples. Reproduced from reference [26].

Dou et al. used their knowledge on the steric transition phenomenon in asymmetrical flow field-flow fractionation (AF4) for the characterization of recrystallized ‘research department explosive’(RDX) particles. They used the steric mode in combination with hydrodynamic lift forces. At the optimal conditions these lift forces are strong enough to counteract the perpendicular field. The advantage of this lift force is the reduction of membrane interactions. Baalousha et al. reviewed the application of flow-FFF in combination with multiple detectors for the characterization of natural colloids and natural and manufactured nanoparticles [27].

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4 Mobility techniques

Mobility techniques are a group of techniques in which the particles travel through an electrolytic solution or gas phase. Either the velocity difference or the displacement of the solution is a measure for the particle size. Although some of the mechanisms will show similarities with separation techniques, mobility techniques are based on single particle counting. Therefore, mobility techniques provide both size and number information. Two important techniques are electrozone sensing and aerodynamic time of flight. The basic principles, advantages and limitations and applications of these techniques are discussed in this chapter.

4.1 Electrozone sensing

4.1.1 PrincipleElectrozone sensing measures the mobility of ions in solution. The apparatus used for the measurement is called a coulter counter and the technique is best known under this name. It is based on a relatively old principle in which the particles are suspended in a conductive fluid.

Figure 4.7 – Schematic overview of the original Coulter Counter design from the original patent. Reproduced from Ref. [28].

The conductive fluid is present in two compartments (21 and 22) which are connected through a small sapphire aperture (23). In each compartment a platinum electrode (24 and 25) is placed and a constant current applied. If a particle passes through the aperture, a voltage pulse is generated due to the change in resistance of the solution in the aperture. This change is proportional to the volume of the particle. Variations in the measurement due to particle orientation are eliminated by using the recorded peak area instead of peak height [2]. The measurement provides both a volume distribution and the number of particles it measures. It can rapidly analyze a large number of particles in one measurement. It is however not an absolute method and it needs to be calibrated with standards. In most applications the aperture ranges from 30 to 200 μm [28].

4.1.2 Advantages and limitationsElectrozone sensing is a rapid and effective method. The sample needs to be suspended in the conductive medium, in order to be measured. Since the medium needs to be conductive, the technique is unsuitable for organic based material. The particle concentration of the solution should not be too high, because multiple particles may pass through aperture. This will induce incorrect results. In the original design of the Coulter counter, there is a trade-off between sensitivity and the chance of clogging. The sensitivity of the measurement is increased by decreasing the size of the

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aperture. This is a result of the amount of conductive medium which is replaced by the particle. The signal is increased when more fluid is replaced. However, when the aperture is very small, big particle could more easily clog it. The operation window of a Coulter counter aperture is around 3-40% of its size [28].Electrozone sensing can measure a wide particle size distribution. However, the aperture needs to be exchanged in between runs when measuring a wide size distribution. This increases the analysis time. Nieuwenhuis et al. solved this by designing Coulter counter chip using a 2-dimensional liquid aperture control [28]. A non-conductive liquid surrounds the conductive sample liquid on three sides to form the aperture. The size of the aperture can be changed by changing the flow rates of both liquids. This creates optimal sensitivity and fast measurement over a wide range of particle sizes.

Figure 4.8 – Coulter Counter chip design with aperture control. The red dye is used as sample liquid and used to illustrate aperture control. Reproduced from Ref. [28].

The difficulty of this design is that the measurement frequency and the electrical resistance of the channel needs to be matched , due to the large capacity of the substrate.Porous particles can pose a problem in electrozone sensing. The measured particle size is less than the actual particle size, since the pores contain the conductive medium, inducing a higher conductivity. This might be problematic in some applications. Yuan et al. described the use of a theoretical model to describe the relationship between the particle size determined with microscopy and electrozone sensing [29]. The model based on experimental data is linear with a slope of approximately two (microscopy size vs. electrozone size). However, the model needs to be based on experimental data rather than theoretical approximation for each type of particles, since this can result in significant deviations.

4.1.3 ApplicationsThe coulter counter principle can be miniaturized and as a result, it is suitable for lab-on-a-chip applications. Jagtiani and coworkers reported the implementation of a multichannel micro-fabricated coulter counter [30]. The problem with multichannel coulter counter devices is the detection, since individual detection for each channel proofs to be problematic. Jagtiani et al. solved this problem by the implementation of multiplexed detection of multiple channels with a single detector. The design consist of 4 parallel micro channels. Multiplexing is achieved by applying a different amplitude of the voltage on each channel. The alternating current sine wave has a known and unique frequency for each channel. A multiplexed signal is obtained which represents a combination response from all channels. By demodulation of this signal, the individual channel signals are recovered. The modulation signal (AC) is applied by an electrode which divides each microchannel into half. The microchannel is only exposed the modulation signal in the center of the channel. By working with multiple channels high throughput analysis can be realized, since the number of channels can be

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increased. However, the design has only been tested on a polystyrene particle standard using only two channel simultaneously. It will need to be further investigated whether it works for more than two channels and real sample applications, such as biological samples.

4.2 Aerodynamic time-of-flight analysis

4.2.1 PrincipleAerodynamic time-of-flight analysis (ATOF) uses the mobility of particles in an expanding stream of air. It relies on the principle that particles with a low mass undergo more acceleration than particles with a high mass. Particles are accelerated over a certain distance and are detected by a dual laser system. The light scattering which is causes by the particles is not used for the determination of the particle size; the laser system is only used for time measurement. From the time of flight of the particles and the particle density, the particle mass can be calculated. This type of analysis falls into the single particle analysis group and generates a number mean.

4.2.2 Advantages and limitationsAerodynamic time of flight analysis is a high resolution technique with a wide dynamic range (0.2 to 700 μm) [31]. The relation between the particle size and time-of-flight is established from a combination of theoretical models and experimental data. The relation depends on the particle density, since the particle mass has the direct relation to the time-of-flight and not the particle size. As shown by calibration curves published by Thomas Fields, care should be taken for the analysis of particles < 1 μm with a low density. The relation between size and time-of-flight is not linear in this region. Sample can be introduced as dry powder or aerosol, depending on the accessories. Only small sample amounts are required for analysis. Actually, the particle concentration is a limitation for accurate measurement. The particles are detected and counted by laser light scattering, when the particle concentration is too high, multiple particles will pass through the measurement zone (between the first and the second laser) simultaneously, which will produce errors. The particle concentration must be controlled. Mitchell and Nagel describe the use of a diluting accessory which uses an additional sheath flow during aerosol introduction as one of the solutions to this problem [32].

4.2.3 ApplicationsAerodynamic time-of-flight analysis can be coupled to fluorescence spectroscopy or mass spectrometry (MS) in order to provide information on the chemical composition of the particles. For the coupling to MS different ionization methods can be applied. Murphy reviews the design of aerodynamic time-of-flight MS (ATOFMS) instruments with laser ablation ionization [33]. The main disadvantage of laser ablation ionization techniques is the systematic error in the composition measurement caused by the particle shape. Non-spherical particles will be biased to spherical particles during transmission to the ionization laser. Murphy also discussed the practical issue of inlet clogging, especially when measuring in humid air. Since this technique has a lot of applications in environmental analysis; this is an important problem. Also the equipment is rather complex, which limits the application. A few years later, Liu et al. performed research towards urban particulate matter in Atlanta using ATOFMS [34]. Even though the measurements were performed during the summer and Atlanta has a hot and humid climate, no technical problems (such as clogging) were reported. The only effect of the high humidity was the detection of numerous water

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clusters found in particular particles. This indicates the technical developments and improvements in ATOFMS equipment over the last years.

Figure 4.9 – Schematic representation of a single particle laser ionization mass spectrometer. Reproduced from Ref. [33].

Another ionization technique used in ATOFMS is flash thermal vaporization followed by electron ionization (EI). The evaporation of particles is done by impact on a heated surface of approximately 873 K. Often this type of ionization method is coupled to the quadrupole MS, but also TOF MS can be used. DeCarlo et al. described the use of high resolution time-of-flight MS (HR-TOFMS) to obtain even better mass resolution in order to separate ions with different elemental composition at a nominal m/z value [35]. The instrument is equipped with two ion optical modes, namely single-reflection mode and double reflection mode. This provides the possibility of measuring with either increased sensitivity or increased resolving power. Besides the use of MS, fluorescence can also be coupled to aerodynamic time-of-flight analysis in order to provide compositional information. Fluorescence can be used for the characterization of biological aerosols. The fluorescence is induced by laser excitation, using a UV laser for bio-molecular applications, e.g. biohazard and toxicology studies, air monitoring and drug studies [36].

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5 Light scattering techniques

Light scattering techniques can be used to determine the particle size distribution of a sample. In this chapter, static light scattering and dynamic light scattering techniques will be discussed as well as their basic principles, advantages, limitations and applications.

5.1 Static light scattering

5.1.1 Principle

In static light scattering (SLS), a beam of polarized light, usually a laser, is focused onto the sample molecule. The incident photon is scattered by the molecule (Rayleigh scattering), this scattered light is detected with a photo detector. Figure 5.1 gives a presentation.

Figure 5.1 – The incident photon induces energy scattering of a molecule. Reproduced from ref. [37]

As the molecular size increases the photons will no longer scatter independently (isotropic scattering) but will start interfering with each other (anisotropic scattering). When the diameter of a molecule becomes bigger than 1/20th of the wavelength of the incident light, the scatter becomes anisotropic and, therefore, angular dependent [9]. Because of the angular dependency of the scattering, the intensity at one certain angle cannot be related to the molecular weight of the scattering of macromolecule. Only at the zero angle, this effect is not present and it is possible to obtain the molecular weight (Mw) of macromolecules. However, measurement at the zero angle is impractical as the light source would outshine the light scattering intensity at zero angle. As an alternative the scatter is measured at multiple angles and extrapolated to the zero angle using a model (Rayleigh, Rayleigh-Gans-Debye, Berry, Mie, etc.) [9]. Equation 5.1 is the most common equation to calculate the molecular weight using light scattering.

Equation 5.2 - Where R(θ) = Rayleigh ratio, C = solute concentration (g/ml), A2 = second viral coefficient, a measure of solute-solvent interaction, P(θ) = form factor or “scattering function”, K is a constant.

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From equation 5.1 it can be seen that the concentration of the sample is required in order to calculate the molecular weight form the light scattering. Consequently, LS detectors should always be accompanied by a concentration detector, such as, UV or RI detectors. For the interpretation of the light scatter which is measured by any light scattering detector, the single scattering regime is used. This means that photons only can be scattered between the light source and the detector. This requires very clean, transparent and highly diluted sample solutions. Care should be taken with this approach, since dilution can change the original particle interaction and shapes present in the concentrated sample [38]. There are two different operation modes in which SLS can operate namely batch mode and continuous flow (chromatographic) mode. Batch mode is used when it is necessary or desirable to make light scattering measurements of an unfractionated sample. Such measurements are collectively termed batch measurements. Batch mode can be performed either by injecting a sample into a flow cell or by the use of cuvettes. The measured quantities are averaged over all masses and sizes present in the sample. Continuous flow mode, also known as chromatographic mode, is used to study the elution of molecules from virtually any chromatography separation system, such as SEC, HDC or FFF which are described in chapter 3. The eluent from a separation system passes through a flow cell in a continuous matter. In that case equation 5.1 can be greatly simplified [9]. After LS analysis, the sample can go to a second detector or it can be collected and stored, since light scattering is a non-destructive analysis.There are three different types of SLS instruments, which are Right-Angle Light Scattering (RALS), Low-Angle Light Scattering (LALS) and Multi-Angle Light Scattering (MALS). The main difference between these instruments is the angle at which the scattered light is detected. Because each collection angle results in its own advantages and disadvantages, the techniques will be discussed separately.

5.1.2 Right-angle light scatteringRight-angle light scattering (RALS) measures the scattered light at a 90° angle to the incident laser beam. It is the simplest SLS detector and does not use complicated optics. Moreover, RALS is a sensitive technique, because the light passes though the cuvette at 90°. As a result, the noise created by the change in refractive index is minimized. Finally, the sample consumption is low, because only small flow cells are required. The molecular weight of the sample is calculated from the scatter intensity at the 90° angle and the concentration. Because only one angle is used, isotropic scattering is assumed. This is only true for smaller molecules and RALS can therefore not be used for large particles. Another consequence of measurement at only one angle is that the radius of gyration cannot be calculated. Matsuzaki et al. investigated the use of RALS in liposome characterization [39], however, RALS does not seem to be the most suitable technique for this purpose. The results of RALS did not have a good comparison with dynamic light scattering (DLS), which is commonly used for the characterization of liposomes. Additionally, the liposomes do not seem to behave as solid particles and deviate from theoretical behavior.

5.1.3 Low-angle light scatteringLow-angle light scattering (LALS) also measures the light scatter at a single angle, like RALS. Now the light is measured close to the zero degree angle from the incident beam. The measurement is performed at one angle. For this reason only the molecular weight of the sample can be determined, not the radius of gyration. Since the measurement is performed close to the zero angle, there is no

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limit to the molecular weight or size which can be determined with LALS. Furthermore, the measurement is very accurate, because only a small extrapolation is needed. The flow cell can be small too, since only one angle needs to be measured. Due to measurement close to the incident laser light beam, LALS has much more noise compared to RALS and is therefore less sensitive. For this reason LALS is more suitable for large molecules and particles. It is also more sensitive to contamination, since this normally consists of large particles. Combinations of both RALS and LALS can also be used. There are systems available which measure the light scattering in both angles. Because two angles are used, not only the molecular weight but also the radius of gyration can be determined. The hybrid system has also the advantages that it can analyze both isotropic- and anisotropic scatters. These two angle systems can also be applied to microfluidic devices, since small flow cell can be used. Pamme et al. described the use of a two angle light scattering technique in lab-on-a-chip format [40]. They did not use a RALS/LALS hybrid, but measured the scatter intensity at the 45° and 15° angle. The angles were chosen smaller than 90° since only large particles were analyzed. The relatively high scatter angle of 15° was chosen to avoid the high background noise of the system at lower angles. The system was newly developed and needs more optimization. The authors gave a number of improvements to be made, but the principle of lab-on-a-chip light scattering works.

5.1.4 Multi-angle light scatteringA MALS detector consists of a sample cell or flow cell, a laser beam and multiple detectors to collect the scattered light. The detectors are set at specific angles to the incident beam depending on the design of the detector. Since MALS uses multiple detectors, both molecular weight and the radius of gyration of particles can be determined. The use of multiple detectors also requires a slightly different calibration procedure. The determination of the constant K* as described in equation 5.1, is performed on the 90° angle. The response of the other detectors is normalized to the calibrated 90° detector. The advantage of MALS lies in the accuracy of the measurement. Because the scatter is measured in multiple angles, the extrapolation to the 0° angle is more accurate. Still, for large polymers (root-mean-square radius > 100 nm) the relation between molecular weight and light scattering is complex. The extrapolation method should be chosen with care in order to obtain accurate results. [41]. The use of the low scattering angles produces the most accurate and robust results. Consequently, it is important to use a clean system.Furthermore, the performance of each detector can be checked by comparison to the neighboring detectors. Samples of all molecular weight can be analyzed by MALS, since angular dependence is accounted for. By studying the angular dependence, the shape of the particles can be predicted (e.g. sphere, hollow sphere, random coil, etc.). This also induces a disadvantage; since the exact shape is not known it is difficult to predict which extrapolation model should be used. Moreover, the cell is quite complex, because multiple angles need to be analyzed. As a result, the lower angles are often less accurate than the low angle detector in RALS.Normally, MALS is associated with macromolecules and nanoparticles. However, MALS can also be used for the detection of smaller polymers, such as oligomers, due to improvements in the technology. According to Wyatt Technology, 1000 Da can still be measured using an 18 angle detector. However, it should be noted that at lower molecular weight the accuracy could be less than 90% [42] [43]. In most applications MALS is used in combination with chromatography. Picton et al. described the analysis of Arabic gum using both SEC and symmetrical flow field-flow fractionation

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(F4) coupled to online MALS [44]. A MALS with 15 angles and a 633 nm He-Ne laser was used for the analysis of these complex polysaccharides. Characterization was successful for both techniques, but for F4 the separation was better and, therefore, characterization was easier for F4-MALS. It should be noted that the peaks found with F4 are very broad in this particular research. This is probably due to insufficient focusing. Nowadays, focusing can be performed more effective, resulting in better peak width and shape. The coupling of flow-field fractionation (FFF) to MALS is becoming more common in the last years. In 2009 Zattoni et al. described the analysis of nanoparticles using asymmetrical flow field-flow fractionation (AF4) and MALS [45]. They investigated different types of nanoparticles (polymer-coated gold nano-particles, oligothiophene-doped silica nano-particles and quantum dots) and all could be characterized. Even small amount of aggregates could be detected with MALS, as can be seen in figure 5.2.

Figure 5.2 – AF4-MALS fractogram of PAH/PSS/PAH-coated gold NPs. Dashed line = UV/Vis at 230 nm, thin solid line = light scattering at 90°, thick solid line = radius of gyration with MALS. Reproduced from Ref. [45]

5.2 Dynamic light scattering

5.2.1 PrincipleDynamic light scattering (DLS) also relies on the Rayleigh scattering of the incident light by particles and large molecules. The scatter intensity will fluctuate over time because of constructive and destructive interference of light scattered by different particles. These interferences are caused by the Brownian motion of the particles. Brownian motion is the movement of particles suspended in a gas or liquid. This random motion is caused by collisions between the particle and surrounding molecules in the matrix. Light scattered from two particles can enhance each other or cancel each other out. When this happens in a stationary environment (no Brownian motion), this is a constant signal. However, when the particles are moving because of the Brownian motion the distance between the particles changes. This results in the interaction of (both constructively and destructively) of different photons. This can be measured by the monitoring of the scattering intensity over a period of time. The particle size can be calculated via the Stokes-Einstein equation.

Equation 5.3 – Where D = Diffusion coefficient, K = Boltzmann constant, T = temperature, η = dynamic viscosity and R = hydrodynamic radius

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From equation 5.2 it should be noted that the temperature is an important parameter. It is both present directly in the equation and it influences the viscosity of the solution. For this reason, temperature needs to be carefully controlled during the experiment. Additionally, it should be noted that the particle size is measured as the hydrodynamic radius, which will be converted to the particle size via the equivalent sphere theory.The technique is also known as photon correlation spectroscopy (PCS) or quasi-elastic light scattering (QELS). Using dynamic light scattering, particles in the submicron region down to 1 nm can be characterized for monodisperse samples. The sample should be present in a solution, suspension or emulsion, because Brownian motion is needed and the medium needs to be transparent to light. Moreover, laser excitation induced heat, which dissipated heat more effectively than solids. This is a requirement for all light scattering techniques. The scattered light is measured at known angles; this could either be one fixed angle or multiple angles. In order to determine the particle size distribution in a polydisperse sample, multi-angle instruments are required. As explained in paragraph 5.1.1. different particle sizes have different optimal scattering angles. In order to accurately determine particle size distribution, the scatter intensity should be monitored at multiple angles [46].As also described in paragraph 5.1.1, the single scattering regime is also used for DLS data interpretation. This means that also here sample solutions need to be clean, transparent and highly diluted.

5.2.2 Advantages and limitationsThe advantages and limitation of DLS are very similar to the other light scattering techniques. The measurements are fast and require little sample amount. However, the measurements are sensitive to pollution (e.g. dust) and sample solutions need to be transparent. Furthermore, no information of the shape can be obtained, and the equivalent sphere theory is used which automatically introduces inaccuracies.However, DLS has some advantages over static light scattering. DLS measurement is truly absolute, no calibration factors or sample concentration are needed since the diffusion is directly measured from the fluctuation in time. Only the viscosity of the medium is important, no further information of the sample is required. The main disadvantage of DLS is that the resolution is poor. The resolution is dependent on the wavelength of the laser which is used but can range up to 60 nm for polydisperse samples. The poor size resolution is measured at a single angle and is causes by insufficient scattering intensity of the different sized particles. Consequently, it might be assumed that the technique is very suitable for measurement of a wide dynamic range within one sample. However, this is also not the case; if the difference between the particle sizes in a mixture is bigger than a factor of 30, the accuracy decreases significantly when measuring at one fixed angle [46]. This induces problems for polydisperse samples. In order to measure this dynamic range in one sample, a multi angle setup should be used. This effect is caused by the angular dependence of scattered light. The intensity optimum for each particle size is different. When measuring a polydisperse sample at a fixed angle, specific particle sizes scatter the light more intensely than others. The most intense scatter will have the highest contribution to the overall signal. In order to accurately determine other particle sizes, and the particle distribution, the intensity fluctuations at multiple angles need to be determined.

5.2.3 ApplicationsDLS requires high diluted samples. Consequently, it is almost impossible to study dynamic processes (e.g. aggregation, gelation and phase separation). In order to study these processes, an alternative to

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DLS is used, namely diffusing wave spectrometry (DWS). DWS is much like DLS however, it assumes a random walk model of photons through the sample as data interpretation method. For this reason DWS is used for the analysis of turbid or concentrated samples [38]. The principle of DWS will not be described here, but Alexander et al. provide a clear review of the technique and its application in food research. A research area in which DLS is more suitable is nanoparticle characterization. Often real life samples contain a wide size range of nanoparticles; here DLS had the advantage that it can measure a wide size range without performing multiple measurements or sample pretreatments. Currently, toxicology studies on nanoparticles are an important research field. Particle size and size distribution are two of the parameters which can be related to the toxicity of the nanoparticles. Murdock et al. described the characterization of nanoparticles with multiple techniques, among others DLS [47]. They coupled these characteristics to in vitro toxicity studies. Furthermore, dispersion stabilities were investigated. DLS provided information on particle sizes and agglomeration effects. For some nanomaterials significant agglomeration effects were observed. The scattering properties of nanoparticles can also be used in immunoassays. Lui et al. demonstrated that the use of nanoparticle probes and DLS can simplify the traditional immunoassay for detection of cancer biomarkers. The traditional immunoassay is time consuming and labor intensive. Using spherical and rod gold nanoparticles, the antibodies can be detected via light scattering in a single step homogeneous assay. The antibodies bind to both the spherical and the rod nanoparticles, combining both in one complex. This complex has a bigger average diameter, which can be detected by DLS [48]. Transmission Electron Microscopy (TEM) data shows that this process works, however, it is quite remarkable that the DLS measurement provides accurate results. The difference between the free nanoparticles and the complex is only 20 nm, which is not a significant difference when taking into account the low resolution obtained with DLS. Fortunately, bigger complexes are formed, since particle sizes up to 500 nm are found. These can be distinguished from the free nanoparticles with a particle size smaller than 60 nm. Figure 5.3 represents the results.

Figure 5.3 – Hydrodynamic diameter distribution plots determined by DLS. (a) nanoparticle-detector antibody conjugates; (b) nanorod-capture antibody conjugates; (c) mixture with antibodies; (d) the calculated numerical ratio of

nanoparticle oligomers versus individual particles at different antibody levels. Reproduced from Ref. [48]

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6 Acoustic and Electroacoustic Spectroscopy

Acoustic spectroscopy is a technique which relies on the interaction of ultrasonic sound with dispersed material. This technique is relatively unknown in the field of particle characterization. Nevertheless, it is worth mentioning.

6.1 Principles

Acoustic spectroscopy is based on the four most important mechanisms by which the ultrasonic energy is lost after interaction with particles [49]:

- Viscous losses due to shear waves generated by particle oscillation- Thermal losses due to the temperature gradient near the particle surface- Scattering losses similar to light scattering by a particle- Intrinsic losses due to the interaction of the ultrasound wave with the material of the

particle.This is called the ECAH theory. There are more mechanism which play a part in the attenuation of ultrasound, however, only the most significant ones are used in this theory. Different types of particles have different dominant mechanism. For small particles (< 3 μm) the viscous losses are most significant. For soft particles and emulsions the thermal losses of ultrasound are dominant. Scattering losses are similar to light scattering, and, therefore are more important for higher frequencies and particles > 3 μm.The overall attenuation spectra that are obtained are the sum of these four contributions. From the signal detected by the detector, the extinction is calculated. The frequency of the ultrasonic wave is varied during the measurement (1 – 160 MHz [50]), so a spectrum is obtained. The extent of attenuation at different wavelengths is a measure for the particle size. Data interpretation can be rather complex because of these multiple mechanisms which apply.The size range of this technique is 0.01 – 1000 μm depending on the application. Since ultrasound is used, concentrated and opaque sample can be analyzed without any problems. This is an advantage over other particle characterization techniques. Nevertheless, low concentrated samples can also be analyzed with acoustic spectroscopy. Sample concentration can be as low as 0.1% vol. [49]. In the concentration range which overlaps with other analytical techniques, acoustics provide comparable results.Next to particle size acoustic spectroscopy can also provide information about the microstructure of the dispersion itself [49]. This is because in acoustics, the same stresses are applied on the sample as in rheometers in the micro scale.

Another form of acoustic spectroscopy is electroacoustic spectroscopy. Electroacoustic spectroscopy focuses only on one less significant mechanism of ultrasonic energy loss by particle interactions, namely electrokinetic losses. This only works with charged particle and colloids (via the electric double layer). This can be measured in two ways:

- Apply an ultrasonic field to the sample and measure the electric field which is generated- Apply an electric field to the sample and measure the resultant acoustic field

The background and developments of electroacoustic spectroscopy are thoroughly reviewed by Hunter [51].

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6.2 Advantages and limitations

The advantage of acoustic spectroscopy is that it can measure particle sizes of samples which are turbid or non-transparent. This means that concentrated samples can be measured without dilution, which enables the analysis of particles in e.g. aggregation conditions or their original matrix. An extra advantage related to this, is that there is almost no sample preparation. This makes the method time and cost efficient. Another advantage is the wide size range in which particles can be determined. This is not only favorable for a wide application range, but most real-life samples are polydisperse. This requires analytical techniques which can analyze a broad size range. Finally, the technique can be applied in online analysis, due to its non-destructive property. Alba et al. describe that the results obtained with acoustic spectroscopy are in good agreement with results obtained with static light scattering [50]. Unfortunately, they do not specify for what kind of samples this has been investigated. The example which is published is only a reference sample containing of glass beads. This might not be representable for real samples. Acoustic measurements are sensitive to all kind of particles, including air bubbles. This has both advantages and disadvantages. The advantage is that bubbles can be detected in dispersed systems, and, their influence on the stability of the dispersion can be studied. So, when the presents of air bubbles are interesting for the application, acoustic spectroscopy is a suitable technique. However, when air bubbles are not of interest they can interfere with the measurement. In the period 1940 to 1950, it has been experimentally proven that air bubbles affect the acoustic spectrum [49]. Fortunately, the effect of the air bubbles depends on their size and the frequency of the ultrasonic sound. The bubbles have to be < 10 μm in order to affect the complete spectrum. Since, bubbles below 10 μm are very unstable, this is normally not a problem. Under normal circumstances only the low frequencies of the spectra will be affected by the bubbles [49]. As a result, particle size can still be determined in samples containing bubbles. Electroacoustic spectroscopy depends on the generation of an electric field and for this reason is not sensitive to air bubbles. Another limitation of this technique is the use of the ECAH theory. The theory is based on a diluted monodisperse system consisting of only spherical particles. With diluted samples, it is meant that there is no particle-particle interaction. This means that, the measurement of concentrated sample is limited. However, Dukhin et al. describes that for systems in which thermal losses (e.g. emulsions) are dominant, the ECAH theory still applies even in concentrated samples [49].

6.3 Applications

Acoustic spectroscopy has many applications in ceramics. Dohnalova et al. reported the characterization of kaolin, also known as China clay, with acoustic and electroacoustic spectroscopy [52]. They used sample concentration of maximal 10 wt. %, in which the ECAH theory still applies. The stability of the dispersion is investigated with tanning agents and overtime. After four days the dispersion becomes unstable and the mean particle size increases. Another application field of this technique is nanoparticles. Biodegradable nanoparticles, such as nanocrystalline celluloses, are currently under investigation because of growing interest in eco-friendly materials. Colloid stability is a critical parameter in the application of these nanocrystals and Safari et al. investigated this with electroacoustic spectroscopy [53]. They improved the colloid stability of cellulose nanocrystals by using electrostatically stabilized nanocrystalline cellulose.

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Figure 6.10 – Change in the mean particle size d1 of kaolin slurry in water. Reproduced from Ref. [52]

As mentioned in the previous chapter, food science is of growing interest and it can be quite difficult to characterize these samples. This is mainly because the applications need to be characterized in the undiluted form, in order to maintain sample characteristics. DLS already proved to be insufficient, and DWS needed to be used. Acoustic- and electroacoustic spectroscopy are important techniques in analysis of food emulsions. It has been reported by Dukhin et al. that acoustic spectroscopy provides a reproducible method for the analysis of several dairy products [49]. Figure 6.2 illustrate the results for ranch dressing and light cream.

Figure 6.11 – Attenuation spectra of ranch salad dressing and light cream. Light cream was measured several times for determination of the reproducibility. Reproduced from Ref. [49]

Here it was interesting to see that the attenuation of the ultrasound was increased with increasing fat content. This could be explained by the characteristics of the emulsion. From this data, the particle or droplet size could be calculated. However, is should be noted that certain parameter should be known in order to do make these calculations.Since acoustic spectroscopy is nondestructive, it can be used for in-line applications. The instrumentation can be made into a probe which can be built directly into a production process. This enables real-time monitoring in the production process and can be steered based on the information obtained by this measurement.

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7 Comparison of characterization techniques

As is described in the previous chapters, there is a wide variety of analytical techniques which can be used for particle characterization. Techniques described in this report are only a fraction of all techniques available. It is not difficult to imagine that it is difficult to choose the most suitable technique for a certain application or for a specific laboratory. In order to compare these techniques, the principles, advantages and limitation should be compared. It should be stressed, that these techniques are based on the equivalent sphere theory and that this introduces inaccuracies by definition. Additionally, it should be noted that care should be taken when results of different analytical techniques are compared. This is because techniques might use different statistical means (volume, number, length, etc.).There is not one superior technique to be used for particle characterization. All have their own strengths and weaknesses. Different applications will require different measurements and techniques. In order to provide a comparison of the techniques mentioned in this report, table 7.1 provides an overview of the analytical techniques which are reviewed. Different factors will be addressed for comparison purposes. Specifications of the techniques are checked with instrument manufactures.Table 7.1 only provides a general overview, for more details the corresponding chapters should be consulted. It should be considered that factors are sample and application dependent. The most distinct differences between analytical methods for particle characterization that should be noted:

- Chromatographic techniques (SEC, HDC, FFF), have the longest analysis time- Sieving and (electro) acoustic spectroscopy are the only techniques which can be used for

the analysis of concentrated samples.- Most techniques use ‘wet’ samples. Only few can handle powders, other dry samples or

aerosols.- More than half of the techniques discussed in this report can be applied on-chip. This could

indicate a growing interest in on-chip particle analysis, which can be interesting for in-field analysis.

- Generally, the sample preparation for these techniques is quite simple and fast, making the techniques accessible of all sorts of laboratories.

- Since only small sample amount are required for most techniques, it is important that attention is paid to the sampling procedures. A representative sub-sample is needed in order to assess a total sample. When only small samples are required and used for analysis, this becomes even more important.

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Table 7.2 – Overview of analytical particle characterization techniquesAnalytical Technique Size range Sensitivity Concentration

rangeSample

preparationsSample Sample

sizeChip

possibilitiesAnalysis

timeChapter or paragraph

Sedimentation > 1 μm Dependent on detection technique

Variable NoneDilution

Dispersion ~100 μg No Sample and method

dependent

3.1

Sieving > 10 μm Low Concentrated Variable DryDispersion

Variable No Few minutes 3.2

SEC < 0.5 μm Dependent on detection technique

< 500 μg1 DilutionOther2

Solution < 1 mL No ~ 30 min 3.3

HDC 0.03 – 2 μm3

0.02 – 50 μmDependent on

detection technique

< 500 μg1 DilutionOther2

SolutionDispersion

< 1 mL Yes ~ 30 min 3.4

FFF 10 nm – 10 mm Dependent on detection technique

< 500 μg0.5 – 5 mg4

NoneDilution

SolutionDispersionEmulsion

< 1 mL Yes 10 – 90 min 3.5

Electrozone sensing 10 nm – 100 μm Good5 Diluted Dilution Dispersion (aq.) < 1 mL Yes Few minutes 4.1ATOF 3 nm – 3 μm5 Variable < 1010

(particles/m3)None Aerosols

DryVariable No < 1 min 4.2

RALS Approx. < 30 nm6 Good Diluted Dilution SolutionDispersion

> 12 μL Yes < 1 min 5.1.2

LALS nm - mm Low Diluted Dilution SolutionDispersion

> 12 μL Yes < 1 min 5.1.3

MALS nm - mm Good Diluted Dilution SolutionDispersion

> 12 μL Yes7 < 1 min 5.1.4

DLS 1 nm – μm Good Diluted Dilution Solution DispersionEmulsions

> 12 μL Yes < 1 min 5.2

(Electro-) Acoustic spectroscopy

10 nm - 1 mm Good 0.1 – 50% vol.2 None SolutionDispersionEmulsion

~ 1 mL No < 1 min 6

1 Dependent on column- and pore size2 Dependent on sample matrix3 Dependent on column type4 In semi-preparative mode5 Dependent on instrument design6 Dependent on wavelength incident light7 Number of measuring angles limited

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8 Conclusion and discussion

Even though, only a fraction of all analytical technique for particle characterization is discussed in this report, it is clear that there is not one superior technique. Depending on the type of sample and information required the most suitable analytical technique should be selected. Whichever technique is chosen, the equivalent sphere theory is used and this will induce errors. Moreover, results from different techniques cannot be compared directly due to different statistical means result from different techniques. The results should be interconverted, but this will increase the error of the analytical method itself. Sedimentation and sieving are the most simple and cheap methods for particle size analysis. However, only relatively large particles can be sized using these methods. For particle separation, chromatographic methods such as SEC and HDC are the more advanced methods. They can separate the other side of the particle range (macromolecules and very small particles). If the molecules and particles become too large, they cannot be analyzed and may clog the system. Multiple detectors can be used to characterize the separated materials, which is a big advantage. However, analysis times are relatively long and shear forces and unwanted interactions may appear. FFF provides a more extended range for particle characterization with only limited shear forces and interactions. Furthermore, different field can be applied which extends the applicability. However, the instrumentation can be rather complex and the technique is relatively new and is still in development. In the mobility techniques different advantages and limitations are seen. Electrozone sensing is a fast analytical technique with a wide size range. A conductive medium is required for the analysis, which does not make it suitable for samples which require organic solvents. The limitation of electrozone sensing is the trade-off between sensitivity and the chance of clogging. Aerodynamic time-of-flight analysis is one of the few methods used for aerosols and has a main application field in pharmaceutics. The technique has a high resolution and multiple detectors can be used for characterization of the particles. The limiting factor is that the concentration of the sample can limit the accuracy. High dilutions are required for accurate analysis.For light scattering there are multiple options which can be used, depending on the applications. In static light scattering the most important variants are RALS, LALS and MALS. The sensitivity of these methods depends on the measurement angle of the scattered light, as well as the size range which can be measured. For static light scattering the concentration should be known and the sample should be transparent and diluted. Dilution might change the characteristic of the particles and sample (e.g. aggregation). Dynamic light scattering has the advantage over this, because for this technique the concentration does not need to be known and the measurement is truly absolute. Only the viscosity of the sample solution needs to be known and the temperature should be controlled during the measurement. However, the resolution of the techniques is poor and consequently, less suitable for polydisperse samples.Finally, (electro) acoustic spectroscopy can be used where the light scattering techniques fail. The results of this technique are in agreement with light scattering results. However, acoustic spectroscopic techniques can analyze the particle size in non-transparent and concentrated samples over a wide size range. The technique is sensitive to the presence of bubbles, which can be both an advantage and a disadvantage. The requirement for the analysis of concentrated samples is the absence of particle-particle interactions. As a result, knowledge of the sample characteristics and

29

compounds should be present in order to provide accurate results.

It is recommended to carefully select the most suitable analytical technique for particle characterization. It is better to use one single technique for sample comparison, since this is the most accurate. Naturally, every technique has its advantages and limitations and they should be considered during both the selection process and data analysis. Additionally, the use of the equivalent sphere theory (and possibly other theories depending on the analytical technique) should be considered and the meaning of this should be understood.

30

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