## NCL Method PCC-2

Measuring Zeta Potent ial of Nanoparticles

Nanotechnology Characterization Laboratory National Cancer Institute-Frederick SAIC-Frederick Frederick, MD 21702 (301) 846-6939 ncl@mail.nih.gov

April 2008 revised, November 2009

This protocol assumes an intermediate level of sc ientific competency with regard to techniques, instrumentation, and safety procedures. Rudimentary as say details have been omitted for the sake of brevity

.Method w ritten by:

Jeffrey D. Clogston, Ph.D. (NCL)

Reviewed by: Anil K. Patri, Ph.D. (NCL)

NCL Method PCC-2 November 2009 2 Version 1.11. Introduction
This protocol describes the measurement of zeta potential for nanomaterial samples in aqueous solutions. Particle surface characteristic s and charge play an important role in the particle’s physical state, stabil ity in different media, agglomer ation tendencies, and interaction with biological systems. Zeta potential measurement provides an indirect measure of the net charge and as a tool to test batch-to-batch consistency. A particle in solution with a net charge has a layer of ions (of opposite charge) strongly bound to its surface; this is referr ed to as the Stern layer. A second diffuse outer layer is comprised of loosely associated ions. These two layers are collectiv ely called the electrical double layer. As the particle moves (due to gravity, kinetic energy, and/or under an applied electrical field), there exists a boundary between the ions in the di ffuse layer that move with the particle and ions that remain with the bulk dispersant. The electrost atic potential at this “slipping plane” boundary is the zeta potential. In zeta potential measurements, an electrical field is applied acro ss the sample and the movement of the particles (electrophoretic mob ility) is measured by the light scattering of the particles. The Henry equation is then used to calculate the zeta potential, z : 

##  
   3 2 afz U
e 

where U e is the electrophoretic mobility,  is the dielectric constant, η is the absolute zero-shear viscosity of the medium, f( 

a) is the Henry function, and  a is a measure of the ratio of the particle radius to the Debye length. This assay protocol outlines the procedure for sample preparation and determination of zeta potential. Guidelines for making successful zeta potential measurements are provided, as well as
a discussion of relevant standa rds and data analysis. Examples of zeta potential results are illustrated for 30 nm nominal size colloidal gold and a G5 PAMAM amine-terminated
dendrimer.

NCL Method PCC-2 November 2009 3 Version 1.12. Reagents, Materials, and Equipment
Note: The NCL does not endorse any of the su ppliers listed below; their inclusion is for informational purposes only. Equivalent supplies from alternate vendors can be substituted.

2.1 ZetaSizer Nano ZS (Malvern Instruments) 2.2 Folded capillary cell (referred to as zeta cells, polycarbonate cell with gold-plated electrodes; Malvern Instruments, DTS1060C) 2.3 Caps for zeta cells (2 per cell) 2.4 Zeta Potential Transfer Standard (Car boxylated polystyrene la tex dispersed in pH 9.22 buffer; Malvern Instruments, DTS0050)
2.5 10 mM NaCl

3. Methods
3.1 Cleaning and Handling of Zeta Cells 3.1.1 Zeta cells should be rinsed thorough ly before use. This involves rinsing the zeta cells with water, followed by ethanol, and finally water again. As the zeta cells have two ports, it is recommended to flush a minimum of 1.0 mL of each rinsing solvent through each port to thoroughly rinse each electrode. This can be achieved by e ither using a 1 mL disposable syringe
or a wash bottle filled with th e corresponding rinsing solvent.
3.1.2 After the zeta cells are rinsed , visually check the electrodes both inside
and outside of the cell and the m easuring window for any manufacturing
defects such as scratches on or non- transparent measuring windows, dirty or non-homogenous surface coating of electrodes (electrodes are goldplated), or any residual polycarbonate (from the manufacturing process) in
the cell or on the electrodes. New zeta cells should not be used if any of
these defects are found. 3.1.3 The zeta cells, after rinsing, shoul d be dried using a gentle stream of nitrogen attached to a filter to remove any remaining solvent and physically re-examined as above.

NCL Method PCC-2 November 2009 4 Version 1.13.1.4 The zeta cells should be capped until use to prevent any dust

contam ination. 3.1.5 Take care not to touch the measuring window of the zeta cells as well as the electrodes. Use of gloves is highly recommended to avoid any residual oil deposits. 3.1.6 Zeta cells are to be used once per samp le and then disposed of. It is highly
advised to not re-use or re-clean the zeta cells for future measurements. 3.2 Sample Preparation 3.2.1 Samples should be prepared in a biological safety hood to protect the sample from particulate and to mi nimize exposure; appropriate safety precautions and protective gear such as gloves, lab coat and goggles must
be worn. 3.2.2 Samples should be prepared in a lo w ionic strength medium; 10 mM NaCl is highly recommended. Suspendi ng medium should be filtered prior to sample preparation using a 0.1 μ m or smaller pore size membrane. 3.2.3 Sample concentration is particle-dependent. Ideal sample concentration is determined by the nature (light sc attering properties) of the analyte nanoparticle. Metallic nanoparticles s catter light more strongly than hydrocarbon-based ‘soft’ nanoparticles such as dendrimers, proteins and polymers. As a result, the concentration for metallic nanoparticles will be lower. A representative measurem ent on two classes of nanoparticles (colloidal gold and dendrimers) is provided in this protocol. 3.2.4 Zeta potential is dependent on pH and therefore the sample pH should be
measured before and after the ze ta potential read ings. Care and precautions should be taken, such as rinsing the electrode with filtered buffers and working on a clean benc h/biological safety cabinet, to minimize introduction of any new particulate into the sample during the pH measurement as this will effect the zeta potential measurement. 3.3 Loading Sample into Zeta Cells
3.3.1 The zeta cell can accommodate a minimum of 750 μ L of sample. Transfer the sample to a 1 mL disposable syringe, dislodge a ny air bubbles, and

NCL Method PCC-2 November 2009 5 Version 1.1insert the syringe to one of the ports on the ze ta cell. Fill the zeta cell with sample by gently depressing the syringe plunger. Remove the syringe
from the zeta cell after the entire sample has been transferred. 3.3.2 Check for any air bubbles in the zet a cell or on the electrodes; gently tap the zeta cell to dislodge them. Visu ally check that both electrodes are submerged. Add more sample to th e zeta cell if necessary by using a 1 mL disposable syringe; add sample in a drop-wise manner to one port and gently tap the zeta cell until the sample drop clears the port. 3.3.3 Cap both ports simultaneously to en sure even sample levels on both sides
of the zeta cell.
3.4 Measurement Procedure
3.4.1 The zeta cell contains a weld line (a small notch along th e top of the cell) on one side; this is the front of the zeta cell and should be facing the front
of the instrument. Before loadi ng the zeta cell into the instrument, carefully wipe the measuring windows with lens paper. Once the zeta cell is placed in the instrument, check that the electrodes on the zeta cell are in
contact with the leads in the instrument. 3.4.2 The temperature should be contro lled and measured with an accuracy and precision of 0.3°C or better. For a maximum sample volume of 1.0 mL and operation close to room temperature ( Δ T  1oC), an equilibration time of 2 minutes is recommended prior to starting measurements.
3.4.3 Perform a minimum of three runs per sample to establish measurement repeatability. Measurement duration (i .e. # of sub-runs per run) should be set according to the instrument manu facturer's recommendations, and will differ depending on particle size a nd scattering characteristics. 3.4.4 The applied voltage can be set to automatic or manual mode. In automatic mode, the best voltage is determined by the software. However it is
recommended to run in manual mode a nd start at a low voltage, typically 80 V, and increase the voltage gradually for optimal results (maximum voltage is 150 V).

NCL Method PCC-2 November 2009 6 Version 1.13.5 Data Analysis 3.5.1 A detailed description of the data an alysis will not be given here. It is left for the reader to check the instrument’s user manual to identify the data analysis algorithm. However, regard less of the instrument used, the Henry equation will be ultimately employed to calculate the zeta potential, z : 

##  
   3 2 afz U
e 

where U e is the electrophoretic mobility,  is the dielectric constant, η is the absolute zero-shear viscosity of the medium, f( 

a) is the Henry function, and  a is a measure of the ratio of the particle radius to the Debye length. The electrophoretic mob ility is determined experimentally and used to calculate the zeta potential. The remaining terms, dielectric constant, viscosity, and Henry function ar e user input values. Table 1 lists
the viscosities at three different te mperatures for 10 mM NaCl and the
dielectric constant at 25°C (1) . The Henry function can be approximated as 1.5 (Smoluchowski model) for measurements made in aqueous medium
with salt concentrations ≥ 10 mM.

Table 1. Viscosities for 10 mM NaCl at three different temperatures. Absolute Viscosityy (mPa · s) Aqueous
Medium 20o C 25o C 37o C Dielectric
Constant

10 mM NaCl 1.003 0.891 0.693 78.6

3.5.2 The zeta potential, at a minimum, is reported with its standard deviation (report number of runs) al ong with the measurement temperature, the pH and concentration of the sample, and the dispersion medium composition.
Additional information should be re ported and include: Henry function approximation employed, viscosity of dispersing medium, dielectric constant, applied voltage, instrument make and model, and type of zeta

NCL Method PCC-2 November 2009 7 Version 1.1cell u sed. Examples of zeta potential results are shown for commercial 30nm nominal size colloidal gold and commercially available G5 amineterminated dendrimer in Figures 1 and 2, respectively. 3.5.3 Zeta potential values in the range between -10 to +10 mV are considered
neutral.

4. Precautions and Guidelines
4.1 All safety precautions per you lab SOPs should be followed for the nanomaterial that is being tested. At a minimum, one should wear a lab coat, gloves and protective goggles and work in a biologica l safety cabinet or a chemical fume hood to minimize exposure to the worker.
4.2 Zeta potential measurements are based on first principles and hence no calibration is required. However, the instrument can be validated by running an appropriate
standard. It is recommended to run su ch a commercial standard along with the samples. One such a standard is av ailable from Malvern Instruments (Zeta Potential Transfer Standard, DTS0050) a nd has a zeta potentia l value of -50 ± 5
mV at 25°C. 4.3 Zeta potential is dependent on sample concentration and pH, and the conductivity of the dispersing medium. Different va lues for zeta potentials are obtained depending on these factors and thus it is important to accurately measure and report them. 4.4 The pH of the sample is reported along with the zeta potential. Sometimes it is useful to adjust the native sample pH or change ionic strength. Care should be taken in this process. Addition of strong acid or base too rapidly may compromise the sample’s integrity and re sult in a polydispersed sample. The zeta potential distribution may contain multiple peaks arising from the presence of differently-charged species. It is reco mmended to titrate with 0.1 M HCl or 0.1 M NaOH in a drop-wise (~1-3 μ L) manner while stirring and monitor the pH throughout. Alternatively, an autotitrator ca n be used to adjust the sample pH. The sample should be physically examined to see if it is unstable under different pH or buffer conditions. Pa rticle instabilities may l ead to phase separation,
NCL Method PCC-2 November 2009 8 Version 1.1precipitation, agglom eration or aggregation. If polydisperse, part of the sample may precipitate under certain pH conditions and the data reported may not be accurate. Precautions should be taken to understand these phenomenon for the
test material. 4.5 The quality of data can be assessed by examining several parameters. Check that the scattering intensity count rate is acceptable based on the instrument manufacturer’s specifications. The phas e plots should have alternating slopes with time followed by either a smooth positive or negative peak (see Figures 1A, 2A). Review the frequency and phase plot s; the baseline in the frequency plots should not be noisy but rather smooth (s ee Figures 1B, 2B). The zeta potential should not change with measurement dur ation (i.e. number of runs) or with different applied voltages. 4.6 Unreliable results as defined by the crit eria above can be due to several reasons. Periodically check that air bubbles do not fo rm on the electrode surface as a result of the applied voltage. Gently tap the zeta cell to dislodge them before proceeding with the measurement. Check th at the zeta cell is pl aced in the correct
orientation and that the m easuring window is clean. Samples should be optically
clear and not turbid. Inconsistent zeta potential values with time can be a result of sample or electrode degradation. Blac kening of the electrode surface or visual evidence of precipitate formation or ch ange of sample color verifies such degradation. Lower applied voltage s are recommended in these cases. Blackening of the electrodes also occurs if the dispersing medium has a high salt concentration (high conductivity). Th e authors highly recommend using 10 mM NaCl as the dispersing me dium. Poor phase plots can be often remedied by increasing the number of sub-runs. Sample concentration is pa rticle-specific and depends on the particle’s scattering propert ies. Low concentrations may seriously degrade the signal-to-noise ratio and will re sult in noisy and inconsistent results. High concentrations can lead to multi ple scattering effects and particle interactions, both of which can produce ch anges in the measured zeta potential. Typically, zeta potential m easurements are made after DLS has been performed.

NCL Method PCC-2 November 2009 9 Version 1.1If this is the case, normally the optimal concentration for DLS works well for zeta potential measurements. 4.7 A final note on zeta potential values is worth mentioning. The zeta potential
value obtained from the measurement is an average value. Thus if the sample is monodispersed, this value corresponds to the zeta potential of the single component in the sample. However, if the sample is polydispersed (i.e. contains several different charged species), the zeta potential distribution plot will contain multiple peaks corresponding to the multiple components. The zeta potential in this case can be reported as either an av erage across all charged species or (if the instrument software allows) the zeta potential for each peak. In the former case, it should be noted that the sample is polydispersed.

NCL Method PCC-2 November 2009 10 Version 1.1Figure 1. The averaged (n = 5) phase (A), fre quency (B), and zeta potential distribution (C) for 30 nm colloidal gold. Sample concentration was 1.3 μ g/mL in 10 mM NaCl with a pH value of 6.5. The sample was transferred to a zeta cell (Malvern Instruments, DTS1060C) and measured at 25°C using a Malvern ZetaSize r Nano ZS and an applied voltage of 150 V. The zeta potential was -33.0 ± 0.5 mV (n = 5). A viscosity of 0.891 cP, a dielectric constant of 78.6, and Henry function of 1.5 were used for the calculations.

NCL Method PCC-2 November 2009 11 Version 1.1Figure 2. The averaged (n = 4) phase (A), fre quency (B), and zeta potential distribution (C)
for a G5 PAMAM amine-terminated dendrimer. Sample concentration was 2 mg/mL in 10 mM NaCl with a pH value of 7.4. The sample was transfe rred to a zeta cell (Malvern Instruments, DTS1060C) and measured at 25°C using a Malvern ZetaSizer Nano ZS and an applied voltage of 120 V. The zet a potential was 41.9 ± 5.5 mV (n = 4). A viscosity of 0.891 cP, a dielectric constant of 78.6, and Henry f unction of 1.5 were used for the calculations.

NCL Method PCC-2 November 2009 12 Version 1.15. Waste Disposal Information
Follow your facility's recommended disposal procedure for your specific nanomaterial
and solvents.

6. References

1. Dukhovich, F. S.; Darkhovskii, M. B.; Gorbatova, E. N.; Kurochkin, V. K. Molecular Recognition: Pharmacological Aspects. New York: Nova Science Publishers; 2003, p. 35.

7. Abbreviations

C Celsius
cP centipoise
DLS dynamic light scattering



dielectric constant
f(



a) Henry function
G5 PAMAM fifth-generation polyamidoamine
HCl hydrochloric acid



a ratio of the particle radius to the Debye length

M molar

mL milliliter

mM millimolar
mPa millipascal

mV millivolt

 L microliter

 m micrometer

 M micromolar
NCL Method PCC-2 November 2009 13 Version 1.1NCL Method PCC-2 November 2009 14 Version 1.1 η absolute zero-shear viscosity

NaCl sodium chloride
NaOH sodium hydroxide

nm nanometer

s seconds
T temperature

U

e electrophoretic mobility

V volts
z zeta potential