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KlockeZia_USRSPresentation_2016
- 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, 3Oregon Nanoscience and Microtechnologies Institute
- 2. • Hydrophobic: “water fearing” • Hydrophilic: “water loving” • Hydrophobicity or hydrophilicity can predict a compound’s interaction with the environment… Hydrophobicity
- 3. • Typically defined as particles between 1-100nm • Nanoparticles (NPs) can… – Clean water – Preserve food – Provide texture and color in consumer products What are nanoparticles? .1 nm 1 nm 10 nm 100 nm 1 um 10 um 100 um 1 mm Dust miteHuman hairPollenRed Blood CellAspirin Molecule Nanoparticle
- 4. • Current standard method is inaccurate for NPs and time consuming – Octanol Water Partitioning Coefficient (KOW) Current Method
- 5. • Quantify the relative hydrophobicity of metal NPs with outer charge or attached functional groups • NPs are functionalized to prevent aggregation or for targeted drug delivery Objective NP
- 6. • Hydrophobic Interaction Chromatography can be used to determine the relative hydrophobicity of bare and functionalized NPs. • Functional groups attached to NPs will dictate the hydrophobicity. • Gold (Au) NPs were selected for this study – Easily detected – Widely applied – Previously reported as hydrophilic Hypothesis
- 7. • Hydrophobic Interaction Chromatography (HIC) is a current method for determining the hydrophobicity of biomolecules Hydrophobic Interaction Chromatography
- 8. Procedure 1) 10 ppm Au NP Solution 2) 0.5X Phosphate Buffer Solution (PBS) 3) 0.1% Triton X-100 Solution (surfactant) Flow rate: 1 mL/min Au NP Concentration
- 9. Results 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
- 10. Results • KOW, PEG Au NP= 82.09 Bare Au NP Area Under the Curve • KOW,Au NP= .3688 PEG Au NP Area Under the Curve
- 11. Discussion • Gold NP is hydrophilic as is reported in previous literature • 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 • Agglomeration could cause physical blockage in column
- 12. Conclusion • 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 • Better calculation of AUC
- 13. Acknowledgements • We would like to thank the Johnson Undergraduate Internship Program, the Harper Nanotoxicology Lab and URISC and URSA-Engage from the School of Undergraduate Research or the support of ZK.
Editor's Notes
- Hi my name is Zia Klocke, this upcoming school year I will be a Junior in Chemical Engineering and this summer I worked in the Harper Nanotoxicology lab on developing and validating a method to characterize NP hydrophobicity.
- First I’m going to define Hydrophobicity, which is the tendency of a compound to repel water. If a compound is hydrophobic it repels water and is “Water Fearing” and if a compound is hydrophilic it is “Water Loving” and bonds with water. A well-known example of a hydrophobic solution and a hydrophilic solution interacting is olive oil and balsamic vinegar on a salad. The oil is hydrophobic and the balsamic vinegar is hydrophilic and no matter now much you shake them together they always repel each other. Hydrophobicity is a key impact factor when predicting a compound’s interaction with the environment for example whether the compound will settle out of a stream or stay in water and in a biological system whether the compound will stay in the blood or absorb into the fatty tissue.
- Nanoparticles are typically defined as particles between 1 and 100 nanometers. Nanoparticles are both industrially manufactured and naturally occurring and are used in large and small amounts in today’s consumer products. Nanoparticles are currently being used for cleaning water, Preserving food by acting as an antimicrobial, protecting against UV light, and providing texture and color in consumer products.
- The current standard method for measuring nanoparticle hydrophobicity is Octanol Water Partitioning however this is originally used to measure the hydrophobicity of a chemical and doesn’t work on Nanoparticles. Unlike chemicals, nanoparticles are dynamic particles. A limitation of Octanol Water Partitioning is that nanoparticles are usually too heavy and large and settle out of solution or accumulate at the phase interface. Nanoparticles don’t reach an equilibrium between the two phases. Back to the salad dressing analogy, the octanol would be like the olive oil, the balsamic vinegar is like the water and Nanoparticles would be like feta cheese never able to stay in the dressing.
- The objective of this research is to quantify the relative hydrophobicity of metal Nanoparticles with an outer charge or attached functional group. Nanoparticles are functionalized to help prevent aggregation or to assist targeted drug delivery. Most commonly Nanomaterials are functionalized with Polyethylene glycol, a carbon chain varying in lengths for different purposes.
- We hypothesized that Hydrophobic Interaction Chromatography can be used to determine the relative hydrophobicity of bare and functionalized nanoparticles and that functional groups attached to Nanoparticles will dictate the hydrophobicity. To test this, we selected bare Gold Nanoparticles (14 nm primary particle size) and PEG Gold Nanoparticles (20nm primary particle size) to test on the Hydrophobic Interaction Chromatorgraphy. Gold was the best option to validate this hypothesis because it is easily detected, widely applied in consumer products, specifically electronics, and previously reported as hydrophilic.
- Hydrophobic Interaction Chromatography (HIC) is a current method for determining the relative hydrophobicity of biomolecules. These HIC columns are packed with different materials, we selected the GE HiTrap Octyl Fast Flow Columns, we chose the octyl to have a similar reference as the Octanol Water Partitioning assay. To the left is what the column looks like, it is a 1mL coumn, *CLICK* if we look closer at the materials you can see that the Octyl is attached to a sepharose (sugar) bead and interacts with the biomolecules. *CLICK* And if we look closer at those interaction sites, the octyl looks like this.
- Most of my work this summer was working to optimize this procedure, and recently I have been starting to test nanomaterials such. What we do is LOAD 10 ppm of the gold Nanoparticle solution into the column at 1 mL/min through the syringe pump. All solution is pumped through the column at 1 mL/min in order to ensure constant velocity of solution and interaction with Octyl matrix. Next .5X Phosphate Buffer Solution was rinsed through the column at the same speed and each mililiter of elucidate is collected. PBS was selected because it stabilizes the pH of solution before and after the column. Finally, .1% of Triton X-100 is rinsed through the column and each mililiter of elucidate is collected. Triton X-100 is a surfactant and acts as a heavy duty soap, scrubbing the column of nanomaterials. Each mililiter of elucidate is plated on a clear plastic 96-well plate and read for concentration of Nanoparticle in a UV-visible specrophotometer. During this process, essentially what is happening the nanoparticles are loaded in the column and either interact or flow through the column. The PBS acts like water, rinsing the column and the Triton scrubs the nanoparticles that interacted with the Ocyl matrix out of the column. Nanoparticles that elucidate with the PBS phase would be hydrophilic and the nanoparticles that elucidate with the Triton phase would be hydrophobic.
- This is what the raw data looks like for bare gold NP. For our calculations we take the area under each data set by trapezoid integration and calculate the Kow of the HIC, very similar to the calculation from the Octanol Water Partitioning assay. There is one point we do not include in this calculation which is the transition phase from PBS to Triton where we don’t know the amount of each solution in the column.
- From that raw data, we get these bar graphs that show the Concentration in ppm of gold nanoparticles from both particles in the PBS and the Triton phases. The Kow, HIC is then calculated from each of these data sets. You can very clearly see that the bare gold nanoparticle concentration is higher in PBS than in Triton but for the PEG gold nanoparticles, the concentration is much higher in the Triton than in the PBS.
- We found that bares gold NP is hydrophilic as is reported in previous literature and that PEG gold nanoparticles are hydrophobic. The HIC column produces replicable results in a timely manner and regeneration and storage was achieve by an ethanol rinse. However for this assay to work, the nanoparticle needs to be directly measured on the UV-visible spectrophotometer. An additional limitation is that agglomeration could cause physical blockage in the column hindering interaction with the beads.
- In conclusion, the HIC produces a hydrophobicity measurement of bare and functionalized NPs which compares with reported values of gold nanoparticles. This research is just going through the validation process now so the next steps are testing a hydrophobic control nanoparticle, determining the transition phase composition and getting a more accurate calculation of the Area under the Curve.
- I would like to thank the Johnson Undergraduate Internship Program, the Harper Nanotoxicology Lab, the URISC and URSA Engage for supporting this project. Additionally I would like to thank Mark Surette from the Nason lab for providing some nanomaterials.