1. Introduction

In UV-Vis spectrophotometry, the interaction of light with a sample is probed by measuring the transmittance, or proportion of light passing through a sample, across a range of wavelengths from the ultraviolet to visible regions (e.g., 200 nm to 800 nm). For nanoparticles and colloids, the extinction of light could arise from absorption of light to promote electrons to an excited energy state, scattering of light, or both. While all particles scatter light, some nanoparticle-specific cases of enhanced light absorption and/or scattering include quantum confinement for semiconductor nanoparticles (such as quantum dots) and localized surface plasmon resonance for metal nanoparticles (such as gold and silver nanoparticles).

This laboratory explores the applications of UV-Vis spectroscopy to qualitatively compare two types of nanoparticles (gold and titanium dioxide). Extinction peaks will further be analyzed for their correlation to the average nanoparticle size and the nanoparticle size distribution. Finally, the sensitivity or robustness of the UV-Vis spectral characterization method to surface coatings will be investigated.

The laboratory is supplemented with reading of ISO/TS 17466:2015(E) Use of UV-Vis absorption spectroscopy in the characterization of cadmium chalcogenide colloidal quantum dots. Although the scope of the test standard is limited to the quantum dots indicated in the title of the standard, the concepts will be analogous for evaluating the optical spectra of the gold nanoparticles, whereas titanium dioxide nanoparticles (which do not shown surface plasmon resonance behaviors) are used as a contrasting sample.

2. Relevant Documentary Standards and Reading Exercises

3. Experimental Procedure

3.1 Chemical reagents

• Deionized (DI) water
• 10 nm Au nanoparticle stock suspension (BBI Solutions, EM.GC10)
• 30 nm Au nanoparticle stock suspension (BBI Solutions, EM.GC30)
• 60 nm Au nanoparticle stock suspension (BBI Solutions, EM.GC60)
• 100 nm Au nanoparticle stock suspension (BBI Solutions, EM.GC100)
• Au nanoparticles synthesized in Activity 1 (pink color)
• Au nanoparticles synthesized in Activity 2 (purple color)
• TiO2 nanoparticle stock suspension (Evonik Aeroxide® P25, 2 g/L in DI water)
• Polyvinylpyrrolidone (PVP) stock solution (≈ 40 kDa molar mass, 10 g/L in DI water)

3.2 Equipment and supplies

• Shimadzu UV-2600 double-beam UV-visible spectrophotometer
• Pipettors and pipet tips
• Polystyrene cuvettes (semi-micro volume), 2 cuvettes in total

3.1 Procedures

Sample preparation

Prepare a total volume of 1 mL of each the following samples:

1. 1. 10X dilution of 10 nm Au nanoparticles in DI water (i.e., 0.1 mL of the purchased Au nanoparticles + 0.9 mL of DI water)
   2. 10X dilution of 30 nm Au nanoparticles in DI water
   3. 10X dilution of 60 nm Au nanoparticles in DI water
   4. 10X dilution of 100 nm Au nanoparticles in DI water
   5. Mixture of 10, 30, 60, and 100 nm Au nanoparticles in DI water, with each particle at 40X dilution in the final mixture (i.e., 0.025 mL of each size of purchased Au nanoparticles + 0.9 mL of DI water)
   6. 10X dilution of synthesized Au nanoparticles (pink) in DI water
   7. 10X dilution of synthesized Au nanoparticles (purple) in DI water
   8. 10X dilution of 60 nm Au nanoparticles with 1 g/L of PVP in DI water (i.e., 0.1 mL of purchased Au nanoparticles + 0.1 mL of PVP stock solution + 0.8 mL of DI water)
   9. 50X dilution of the TiO₂ nanoparticle stock suspension in DI water (i.e., 0.02 mL of TiO₂ stock suspension + 0.98 mL of DI water)

Instrument setup

Turn on the lamp on the UV-Vis instrument, and allow a sufficient time for the lamp to warm up and stabilize before collecting measurements. Table L1.1 below specifies the instrument settings to apply for the measurements.

Table L1.1. Shimadzu UV-2600 Measurement Settings

Sample analysis

1. Autozero the UV-Vis instrument with the cover closed and no cuvettes present.
2. Rinse two cuvettes, then add 1 mL of DI water to each cuvette. Ensure there are no bubbles in the liquid and that the outside of the cuvette is dry and dust-free.
3. Place one of the cuvettes in the back “reference” compartment and the other in the front “sample” compartment as the solvent blank. Ensure the cuvettes are oriented properly with respect to the light beam and inserted all the way to the bottom of the cell holder. Close the lid of the spectrophotometer.
4. Collect a baseline spectrum on the Blank.
5. Remove the sample cuvette and discard the water. (Do not remove the “reference.”)
6. Add the first sample (sample i above) into the sample holder, and press Start to collect the spectrum. Save the data.
7. Discard the sample, rinse the cuvette with DI water, and load the next sample (ii) to measure. Continue with the remaining samples (iii to ix).

Shutdown procedure

Save and export all of your data. Discard all samples to the waste containers provided.

4. Data Analysis

The attached Excel file (UH – Exptl Methods Nano – Laboratory 1 Data Analysis Template) contains and example data set to process and analyze following the steps listed below.

Gold Nanoparticles

1. Input the UV-Vis data collected in lab into the appropriate columns for “Raw Data” in the spreadsheet template.
2. The extinction peak corresponding to the localized surface plasmon resonance (LSPR) of the Au NPs should appear somewhere between 450 nm and 700 nm. Based on the documentary standard (ISO/TS 17466) for quantum dots, the location of this peak along the x axis (wavelength) is related to the size of the nanoparticles.
   1. Report the observed LSPR peak wavelength (nm) for each of the gold nanoparticle samples.
   2. Compare the spectra of the individual particles of known size (Samples i to iv). Does the peak wavelength increase or decrease with increasing gold nanoparticle size, or show no correspondence?
3. Because the height of the extinction peak differs between samples, it can be difficult to visually compare the peak shape using the raw data.
   1. Prepare a set of “normalized” data in the spreadsheet template by dividing the extinction at each wavelength by the maximum extinction (i.e., the y axis value at the extinction peak). This maximum should be selected separately for each individual particle size. The height of the LSPR peak should be 1 for each sample after normalization. The provided plot should populate with the normalized spectra for all of the gold nanoparticles samples.
   2. Compare the spectra of the individual nanoparticles (Samples i to iv) to the spectrum of the mixture of sizes (Sample iv). Does the peak broadness increase or decrease in the polydisperse mixture?
4. Compare the spectra for the unknown gold nanoparticle samples synthesized in Activity 1 – i.e., the “pink” and “purple” particles – against the known samples. Based on the spectra:
   1. Estimate the average particle size for the two unknown samples. Briefly describe how you estimated the size.
   2. Would you expect the “purple” sample to have a broader or narrower size distribution than the “pink” sample? Briefly explain why.
5. Compare the spectra for the 60 nm gold nanoparticles without vs. with the PVP coating (Sample iii vs. Sample viii). Does the coating cause the peak wavelength to increase or decrease, or is there no change?

TiO2 nanoparticles

1. Input the UV-Vis data collected in lab directly as the raw extinction data (do not normalize). Copy the data into the spreadsheet template. The plot for the TiO2 spectrum should populate. Describe the appearance of the spectrum: Is there any special feature (e.g., a peak) in the spectrum? How does extinction vary with wavelength (e.g. does it follow a linear, exponential, power law function, etc.)?

5. Discussion Questions

1. Multimodal particle size distribution analysis:
   Consider the spectrum of the mixture of 10, 30, 60, and 100 nm Au nanoparticles from (Sample v). Presume you were provided this spectrum with no information about how it was prepared (i.e., that 4 individual monodisperse nanoparticle stocks were mixed together). Would it be possible to resolve (distinguish) that there are four discrete particle sizes in the sample, and what exactly those sizes are, given only the UV-vis spectrum? Explain why or why not, based on your data measured in the laboratory.
   
2. Sensitivity/robustness to surface coatings:
   Presume you are only interested in determining the size of the gold nanoparticle without any interference from the surface coatings on the particles. If there is no available uncoated nanoparticle spectrum and you only received the spectrum of a PVP-coated nanoparticle, would it be possible to use UV-Vis spectroscopy accurately determine the size of the gold nanoparticle core? If not, would you overestimate or underestimate the particle size? Explain why, based on your data measured in the laboratory
3. Qualitative applications of UV-Vis spectrophotometry for sample identification:
   Presume you have a completely unknown sample of particles that could be of any composition (e.g., Au, Ag, TiO2, SiO2, etc.). Considering the experimental results and introductory theory with this laboratory, would you be more confident in identifying Au nanoparticles, or identifying TiO2 nanoparticles, based solely on the shape of the UV-Vis spectrum of the sample (i.e., which spectrum is more unique, and why)?