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

ResultsAcknowledgements

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 SetupFive 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.

MATLABMATLAB 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

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 rinseLimitations:• 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

Con

cent

ratio

n (m

g/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.

NanomaterialsGold (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.

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