1. The development and validation of a method to characterize nanoparticle
hydrophobicity
Zia Klocke1, Lauren Crandon1, Bryan Harper2, Stacey L. Harper1,2,3
1School of Chemical, Biological and Environmental Engineering, 2Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis OR, 3Oregon Nano science and Microtechnologies Institute,
Eugene OR
Results
Acknowledgements
We would like to thank the Johnson Undergraduate Internship Program, the Harper
Nanotoxicology Lab and URSIC and URSA-Engage from the School of
Undergraduate Research or the support of ZK.
Discussion
Conclusions
Materials
Introduction
• Hydrophobicity determines how a molecule will interact with water and other
liquids or surfaces.
• Hydrophobicity or hydrophilicity of a NP can predict its interaction with the
environment which is important to understanding the fate and transport of NPs.
• Nanoparticles (NPs) are defined as particles with the size 1–100 nm.
• NPs are widely used in commercial and industrial applications, such as water
treatment, food preservatives, antimicrobial purposes, and to provide color and
texture to consumer products.
• NPs with a metal core often have attached functional groups (i.e.. polyethylene
glycol) that can change the surface chemistry of the NP in order to assist in targeted
behavior and stability.
• Current methods are inaccurate for testing hydrophobicity of NPs. The only
standard method is the Octanol/Water Partitioning Coefficient method in which
nanoparticles, dynamic particles do not reach equilibrium.
• The partitioning coefficient (Kow) is used to determine how hydrophobic a
material is and is defined by
• Hydrophobic Interaction Chromatography (HIC) is a current method for
determining the hydrophobicity of biomolecules, it overcomes the equilibrium
barrier at phase interface.
• The objective of this research was to develop and validate a more efficient and
accurate method to determine the relative hydrophobicity of nanoparticles.
• We predict that surface functional groups will influence the relative hydrophobicity
of the NP.
HIC Setup
Five HiTrap Octyl Columns was selected due to its similarity to the Octanol phase in the
Octanol/Water Partitioning assay. HiTrap Octyl Columns are 1 mL, composed of sepharose
beads with octyl ligands attached to the beads.
MATLAB
MATLAB program was used for raw data analysis and graphing purposes. Area Under the
Curve (AUC) was calculated using the trapezoidal approximation and KOW,HIC calculations.
Procedure
• 5mL of 10 ppm NP solution was prepared and loaded into the syringe pump and into
the HIC column at a rate of 1 mL/min.
• .5X PBS Solution (.22 μm filtered) was loaded into the syringe pump and fed to the
HIC column at a rate of 1 mL/min. Each min (or mL) of elucidate was collected. PBS
represents the water phase of an Octanol/Water Partitioning assay.
• 5mL of .1% Triton X-100 (. 22 μm filtered) was loaded into the syringe pump and fed
to the column at a rate of 1 mL/min. Each min (or mL) of elucidate from this phase
was collected. Triton X-100 represents the Octanol phase of an Octanol/Water
Partitioning assay and scrubs out any remaining NPs in column.
• Analyzed with UV-visible spectroscopy to determine optical density of NP in each
fraction.
• Raw data was then input into MATLAB Code which performed graphing and analysis
of the UV-visible spectroscopy Optical Density data.
• Regeneration was achieved by a 20 mL of 20% (.22 filtered) ethanol rinse through
the column.
HIC Column Interaction Site HIC Matrix Material
HIC Column Procedure
Volume (mL)
0 5 10 15 20 25 30 35 40 45
Concentration(mg/L)
0
0.5
1
1.5
2
2.5
3
3.5
4
Bare Au NP through HIC
= PBS Phase
= Triton Phase
Figure 6. Bare Au NP AUC
KOW,HIC (Au NP) = .3688 ± .010
• Gold NP is hydrophilic which is consistent with the literature.
• PEG-Au NP results show that it is relatively very hydrophobic which is contrary
to reported values of larger chains of PEG NP.
• Overall, the HIC column assay is faster and is consistent with other methods.
• HIC produces a replicable results in a timely manner
• Regeneration and storage achieved by Ethanol rinse
Limitations:
• NP concentration needs to be directly measured on UV-visible
spectrophotometer
• This limits the spectrum of materials that can be read in the UV-visible
spectrophotometer’
• Agglomeration could cause physical blockage in column
• This limits the size and aggregation of nanomaterial put through the HIC
• HIC produces a hydrophobicity measurement of bare and functionalized NPs which
compares with reported values of Au NPs
Next Steps:
• Test a hydrophobic control NP through HIC
• Determine the transition phase
• More accurate and precise calculation of AUC
• The relative hydrophobicity
that is measured by the HIC
Column
Figure 5. Raw data from bare Au NP experiment.
The area under the curve (AUC) from each phase is
calculated and used in the KOW,HIC equation. One
point not included is the transition phase between
PBS and Triton, notated by *. The green is the PBS
Phase, and the grey is the Triton Phase.
*
KOW, HIC (PEG Au NP)= 82.09 ± .019
Figure 7. PEG Au NP AUC
Figure 6. AUC data from each HIC phase
represented by concentration. KOW,HIC
reported for the experiment below Figure 6
using Equation 2. KOW, HIC(Au NP) suggests a
relative hydrophilic nanoparticle.
Figure 7. AUC data from each HIC phase
represented by concentration. KOW,HIC reported
for the experiment below Figure 7 using
Equation 2. KOW, HIC(PEG Au NP) suggests a relative
hydrophobic nanoparticle.
[Equation 1]
[Equation 2]
Bare Au NP Raw Data
Concentration(mg/L)
Volume (mL)
Figure 3. Schematic of HIC Column Matrix.
Figure 1. Example of Octanol/Water Partitioning, a molecule in equilibrium between
the octanol and water phases.
Nanomaterials
Gold (Au) NPs (~20nm) suspended in filtered water was selected due to its ease in
detection through the UV-visible spectroscopy and its small size. PEG-Au NP (20kDa, 13nm)
suspended in filtered water was selected due to its similarity to Au NP, short PEG chain length
and common use.
Figure 2. Diagram of PEG NP.
NP
Figure 4. Diagram of procedure through HIC column.

2. 1. Au NP Solution 2. Phosphate buffer Solution (PBS)3. Triton X-100 Solution