An in-process instrument for the measurement of nano- particle size distribution and electrophoretic mobility

Jason CW Corbett, Fraser McNeil-Watson & David McKnight

1 Malvern Instruments Ltd, Grovewood Road, Enigma Business Park, Malvern, Worcestershire, UK, WR14 1XZ , [email protected]

ABSTRACT:

Real-time monitoring of physical parameters is a key requirement of process systems in particle manufacturing for either industrial or academic applications. We present an in- process, high pressure (10 bar) flow cell instrument for the combined measurement of particle-size distribution, electrophoretic mobility & conductivity at nano-scales. The measurement performance is characterised in flow and at high concentration in both forward and backscatter.

Keywords Electrophoretic mobility, high concentration.

1. INTRODUCTION

Real-time and on-line monitoring of physical parameters, such as particle size and electrophoretic mobility, is of primary interest in automated and semi-automated processes such as wet milling, chemical reactors and colloidal and protein manufacturing systems in general. These systems often combine high flow throughput and high concentrations, especially in colloidal applications, making measurements based on light scattering challenging. However, with the ability of dynamic light scattering (DLS) to measure particle size distributions (PSD) repeatably and reproducibly at the physical scales associated with nano-particle manufacture, there is renewed interest in applying it to flowing and/or concentrated systems. This includes interest in both calibrated measurements based, perhaps, on the real-time monitoring of some aspect of a highly concentrated manufacturing process and then associating this measured value with an off-line diluted measurement of the primary PSD or electrophoretic mobility and on partially diluted, direct, measurements. We present a dynamic light scattering instrument fitted with a flow cell, capable of operating at up to 10 bar, for applications requiring live monitoring of particle size, electrophoretic mobility and conductivity in the size range 0.6nm to 6 µm.

The instrument itself is described in detail in section 2. Characterisation of the size performance in flow over various size bands is investigated in section 3 and electrophoretic mobility in section 4.

2. THE INSTRUMENT

2.1 OPTICS AND MEASUREMENT PRINCIPLES

The instrument is based on the Malvern Zetasizer Nano ZS platform. Utilising an avalanche photodiode detector, the default optics are configured to provide a back-scattered size measurement using the non-invasive backscatter (NIBS)[1] technology with a forward scattered measurement of the electrophoretic mobility using the phase analysis light scattering technique (PALS) [2] .

With reference to figure 1(a), the size is measured using photon correlation spectroscopy (PCS) with an onboard correlator. The size is measured in the self beating, homodyne configuration. The time correlated signal is then reduced using a regularised non-negative least squares (NNLS) fitting of multiple exponentials yielding the particle size distribution. PCS is capable of detecting the difference between a fully monomeric and monomeric + higher order oligomer sample at the sub 10nm scale. Further, DLS is capable of detecting each individual oligomer if connected to a size exclusion chromatography system.

The NIBS system uses a translation stage to place the back-scattered, detected volume inside the flow cell over a continuous range. Highly dilute samples are measured in the cell centre so that heterodyning from light scattered from the cell wall is not detected. Concentrated samples are measured nearer to the cell wall where any component of the total detected light scattered from stationary surfaces, such as the cell wall, is a tiny proportion of the light scattered from the concentrated sample. However the much reduced path length of both the incident laser into the cell and the detected path back out of the cell are shortened. This significantly reduces the susceptibility of the measurement to multiple scattering and allows the precise and accurate measurement of far higher concentrations.

(a) (b)

Figure 1 – (a) The size optical geometry (electrodes not shown), (b) the electrophoretic mobility geometry (reference path + modulator not shown)

The electrophoretic mobility is measured in a heterodyne configuration using a Mach-Zender interferometer and the phase analysis light scattering PALS [] technique – In this work we collect and compare data in both forward (17 degrees in air) (figure 1(b)) or in back scatter (173 degrees in air) – using the size detection direction in panel (a). Note that the reference path with optical light modulator are not shown for brevity – See reference [2] for more details. The electrodes form part of the side walls of the flow cell as shown. A fibre interferometer was used in the back-scattered mobility measurement, which proved to be as stable as its fixed optical counterpart with the added convenience of tight packing of the fibre within the instrument.

With the incident laser and detected beam direction in the scattering plane (horizontal in figure 1), the q vector is given by,

q = 2knsin (θ ) (1) 2

Where, θ is the detection angle, with respect to the incident laser beam, n the refractive index of the medium in which the angle is measured and the wavelength of the incident light λ = 2π /k. At 633 nm , the wavelength used in the system described above, |qback | ≈ 8.817 |qforward | .

* PALS samples the (pseudo-stationary) phase, ϕI, of the light scattered from the fringes by the charged particles under motion due to the electric field E in the detection volume. Each sample of ϕ consists of a small time resolved measurement of the intensity at the detector I(t) th of duration δt. The i sample, Ii(t) obeys,

∝ + ∈[ + δ ] I i (t) sin( φi ωt) t,t t (2)

where ω is the modulator angular frequency. Many such consecutive samples of ϕi yield a phase change per unit time, φ = ϕ( t), to which an electrophoretic mobility, µ (µmcm/Vs) in a known field E ( V/cm), can be assigned via

φ = q µE (3)

By inspection of equation (2), in order for phase demodulation to work efficiently the phase value ϕi must be assumed to be pseudo-stationary on the sample, δt. Since the detected phase change per unit time is dependent on | q| then the sampling must also increase in the back-scattered regime to compensate – i.e. the interval δt must decrease to maintain the pseudo-stationarity of ϕi on the interval. Again for demodulation to work, the modulated phase component, ωt, must also be at least Nyquist sampled and the modulator frequency must then be increased in backscatter to be adequately sampled within δt. The sampling was optimised separately for the forward and back scattered cases. However, the response of the sample to the applied electric field is not dependent on the detection angle so the field reversal settings (required to stop bulk charge migration and subsequent field cancellation between the electrodes) are identical for both cases.

2.2 FLOW CELL

The same cell, shown in figure 2 prior to mounting in the unit, is used for size, mobility and conductivity measurements and is arranged so that the flow is orthogonal to the plane of scattering [3]. The cross section is chosen based on the results shown in the following sections, as 3.5mm (electrode spacing) x 7.8mm (optical depth). This is large enough to tolerate commonly found flow rates but at low enough velocity past the detection volume that the measurement is not effected over the target size range, but small enough that optical depth allows the measurement of electrophoretic mobility of high concentration samples at the forward angle.

The cell is capable of being operated at up to 10 bar and has removable windows for cleaning and an embedded temperature probe capable of monitoring the temperature to within +/- 0.1 Celsius. The plunger on the cell top operates a cleaning wiper inside the cell used to remove contamination from both the windows and electrodes.

* The reference and detected beams are actually interfered at the detector in the instrument. Showing the fringes created within the sample is physically equivalent and more instructive. Cleaning plunger

Measurement Window

Inlet pipe

Figure 2 – The cell – prior to fitting into the unit

The conductivity is measured using a high frequency field technique, used in order to avoid the build up of back EMF effects, and testing has shown the technique accurate to within +/- 10% up to 200mS/cm This is adequate for many applications.

2.3 INTERFACING & CONTROL

The driver software is loaded onto a computer running Windows 2000/XP/Vista and the computer connected to the unit via a USB cable. It is likely that the detector will form part of some larger controlled system, e.g. a reactor & stirrer ,a pump, a dilution loop for very high concentration applications, for instance. With this in mind, the Zetasizer driver software is COM client enabled so that an external control system can maintain complete and live control over the instrument. All relevant functions of the software suite are exposed to the COM interface so that the unit can be enabled, the measurement sequence defined and run and the subsequent data mined for analysis by the server application, such as Labview TM, for instance. The software driver can be operated as a fat or thin client. For instance a single standard operating procedure (SOP) can be invoked or indeed an entire playlist (a pre-defined list of SOPs and macro’s), in order, for example, to measure size and zeta immediately after each other under the control of the instrument rather than the server application.

3. CHARACTERISATION – SIZE

In this section we discuss the measurements taken of particle size using samples of various sizes and over a range of flow rates.

The pump used was an Äkta chromatography pump capable of flow rates up-to 10.000ml/min to a precision of 0.001ml/min. The reservoir, pump and the instrument were connected in series. Various duke polystyrene latices were prepared and tested in the system with the results shown in figure 3.

(a) (b)

(c)

Figure 3 – Polystyrene latex size measurements in flow (a) 200nm particle diameter at 0.01% solids and 2.1% solids, (b) 325nm at 0.01% solids, (c) 450nm at 0.01% solids.

The 220nm dilute sample shows a flat response out to 10.000ml/min so we would expect particle sizes smaller than this to behave in the same way – a 60nm latex showed the same behaviour – not shown. A 135nm silica sample was also tested at 15ml/min with only a 2 % drop in the mean value. This highlights that high flow rates of possibly many 10’s of ml/min are likely to yield accurate results in the nano-scale regime. The limiting flow rate at the cell centre at 325nm is approximately 1.000ml/min before the mean falls outside the uncertainty of the zero flow value. The 450nm data show a maximum flow rate of 0.500ml/min. In both cases accurate, good quality data were recorded much closer to the cell wall (which is at 0.00mm). This is attributed to the flow profile across the cell, which in an ideal system would be (negative) parabolic and therefore slower nearer to the wall. This appears to significantly increase the performance of the cell in the dilute limit. With this data in hand, we measured a 2.1 % 220nm latex with only half of the detection volume in the sample (the other half residing entirely within the window). The more highly concentrated sample shows a significant increase in the susceptibility to flow with a maximum unaffected flow rate of 4.000ml/min. 2.1% is not considered high enough for particle-particle interactions to play a significant role however, multiple scattering will have an effect. We know this since measurements, at say 0.50mm or further, into the cell, in zero flow, also show reductions in size – a known effect of multiple scattering. It is possible that concentration fluctuations near to the wall increase with increasing flow rate. For now, we note it as an effect to be aware of in concentrated systems and also note that the behaviour needs further investigation and will likely be highly sample dependent.

In summary we have found that flow rates in excess of 10.000ml/min are possible in the nano-scale regime with flow rates an order of magnitude slower than this in the lower micro scale case. Dilute systems can be measured at higher flow rates near to the wall as long as not dominated by scattering from static surface such as the cell wall itself.

4. CHARACTERISATION – ELECTROPHORETIC MOBILITY AND CONDUCTIVITY

We now turn to the characterisation of electrophoretic mobility at high concentration and in flow. The NIBS technique measures the particle size distribution in the back scattered regime in order to reduce the optical depth of the sample allowing the measurement of concentrated samples. Could this approach be used for zeta potential? In order to properly compare the measurement in performance in forward and back scattering angles, the Malvern dip cell, with its equal path lengths in both measurement directions was used as the test cell. As noted in section 2.1, the sampling was altered to match the higher q- vector magnitude and the data in figure 4 recorded using the same sample in both forward and backscatter.

(a) (b)

Figure 4 – (a) Zeta potential as a function of concentration – Coffeemate at pH4.1 – Forward and Backscatter in a Malvern dip cell (b) Phase plot of backscatter 0.075% solids (Red) and 0.4% solids (Green)

The sample used was coffeemate due its ease of preparation at various mobilities and concentrations. The backscattered signal is far more dependent on the concentration than the forward measurement despite the fact that the forward and back-scatter path lengths are matched in the dip cell. The reason for the fall off in performance in backscatter is under investigation and a full analysis of this phenomenon is beyond the scope of this work. The forward scattered regime was thus implemented and the 10 mm path length of the dip cell was reduced to 7.8 mm in the flow cell and all subsequent work performed at the forward scattering angle. Faster flow velocity and wall conduction effects prevented the reduction of the path length further.

The mobility as a function of concentration (of coffeemate at pH 4.0) was measured in the forward angle, in the flow cell, with the result shown in figure 5. The upper limit on zeta potential concentration at this size (~300nm) is about 0.5% solids. Further work with a 135nm silica showed excellent quality data at 1% and so we might expect far higher concentrations to be measurable in the forward scattered direction at smaller sizes still.

Figure 5 – Zeta potential as a function of concentration – Coffeemate at pH 4.0 – Forward scatter in the flow cell The flow characterisation of the cell was based on the mobility measurement of polystyrene latices prepared to have surface potentials of -50 mV , -10 mV and -5mV . The results are shown in figure 6.

(a) (b)

(c)

Figure 6 – Zeta potential of (a)-50mV latex, (b) -10mV latex and (c) -5mV latex. In each panel – Mean value in bottom curve set, Standard deviation in top curve set.

As the flow rate increases, so the variation in the value of ϕi on the sampling interval δt increases. The power spectrum of all motion (electrophoretic and diffusive) is shown in figure 7, for the -50mV (140s integration time) case, showing that as the flow increases so does the width of the spectrum about the central frequency shift (from the modulator frequency – not shown) of the particles. The uncertainty in the reported value therefore increases at increasing flow rate as shown in figure 6.

Figure 7 – Broadening of the frequency spectrum at increasing flow rate (140s measurement of -50mV sample)

The integration times and mobilities tested and the resulting uncertainties indicate that good quality data can be recorded over a very broad range of mobilities, including very near to the iso-electric point. For instance, even a -5mV mobility flowing at 4.000ml/min requires only a 1 minute integration time for a <10% uncertainty and this is well within the required sampling duration of many manufacturing processes, such as wet-milling, etc…..Further, for applications where faster measurements are required (for the same uncertainty) the field can be increased at lower mobilities. The residence time for particles within the field decreases with increased flow and effects such as Joule heating should be reduced.

6. CONCLUSIONS

We have demonstrated an on-line instrument for the measurement of the particle size and electrophoretic mobility distributions in the nano-scale, using photon correlation spectroscopy and PALS, respectively.

The unit is shown to be tolerant of flow rates of up to 10ml/min at less than 200nm in size (increasing to 325nm at 1.000ml/min) and where the uncertainty in low values of electrophoretic mobility increases. This uncertainty can be reduced back to acceptable values by increasing the integration time.

The increased susceptibility of concentrated size measurements to flow and back-scattered mobility measurements to concentration are work in progress.

Two of these process enabled units are currently undergoing trials on a Silica wet mill at the University of Leeds – The results will be reported at a later date.

7. ACKNOWLEDGEMENTS

We would like to thank Alison Lockton, Mark Shadbolt, Andrew Buckmaster, Karl Baines, Dr Richard Tweedie, Dr Carlos Rega, Dr Robert Jack and Prof Xue Wang and Dr Ruifa Li for their efforts, help, support and good advice during the project. This project was part funded by the UK Dept of Business Enterprise and Regulatory Reform (formerly the Dept of Trade and Industry) whom we gratefully acknowledge.

8. REFERENCES

1) "Accessing Lysozyme Nucleation with a Novel Dynamic Light Scattering Detector", R. Peters, Y. Georgalis, W. Saenger, , Acta Crist. D54, 873-877, (1998) 2) ‘Measurement of small electrophoretic mobilities by light scattering and analysis of the amplitude weighted phase structure function’, K Schatzel & J. Merz, J.Chem Phys 81 (5) (1984). 3) Application of photon correlation spectroscopy to flowing Brownian motion system - Chowdhury, D P; Sorensen, C M; Taylor, T W; Merklin, J F; Lester, T W - Applied Optics, Vol. 23 Issue 22, pp.4149-4154 (1984)