1. Introduction

Multimodal or highly polydisperse samples of nanoparticles often present challenges for a comprehensive and accurate determination of the particle size distribution. Although “batch” measurements on mixtures of particles can provide an averaged size and polydispersity measure, multimodal size distributions are generally difficult to resolve. Furthermore, the size determination may be biased if the measurement is more sensitive to a subset of the population, e.g., more strongly scattering particles, that obscures other particles in the population. Separation of the nanoparticles would enable different sub-populations to be characterized individually, thus reducing errors or interferences in the measurements. However, sample losses or transformations such as aggregation could occur during the separation process.

Single particle inductively coupled plasma mass spectrometry (spICPMS) is an advanced nanoparticle characterization method that takes advantage of the extremely high sensitivity of ICPMS analysis, along with the ability to selectively monitor specific elements, to evaluate the mass of individual nanoparticles in a population. The method can be applied for metal or inorganic nanoparticles, such as gold, silver, titanium dioxide, etc. The single-particle mode of ICPMS differs from standard ICPMS measurements, where samples are typically digested into dissolved ions for total concentration determination by evaluating the average signal intensity at the MS detector. Rather, the only sample processing for spICPMS is dilution, and individual particles are distinguished from dissolved ions as spikes in intensity, the height of which correlates to the mass of the element detected in the nanoparticle. An equivalent spherical diameter can then be computed if the composition of the nanoparticle (i.e. the molar mass ratio of the element detected versus the overall elemental composition) and the nanoparticle density are known.

Two types of calibration samples must be run to complete the analysis: (1) dissolved ionic standards at known concentrations, to correlate the signal intensity to the mass of the element detected, and (2) a nanoparticle standard of known concentration, to determine the transport efficiency, i.e. the fraction of nanoparticles that reach the detector relative to the known quantity of nanoparticles in the sample. The data processing also requires a routine to separate particle spikes from the background (ionic) signal. Given all the requisite data, the analysis can yield the following results: (1) ionic concentration; (2) particle number concentration; and (3) individual particle masses or sizes, which can be used to generate a histogram of the particle size distribution and determine the number-average size or other statistical features.

This laboratory will apply spICPMS for the analysis of a multimodal sample containing a mixture of commercial Au nanoparticles (nominally 10 nm, 30 nm, 60 nm, and 100 nm) and two Au nanoparticle samples synthesized in class with different colors (pink and purple). The learning objectives of the laboratory include: (1) evaluating advantages or disadvantages of spICPMS to characterize nanoparticle size distributions; (2) evaluating the data quality compared to performance standards in the ISO documentary standard for spICPMS; (3) comparing the observed results to expected results; and (4) critically evaluating the results.

The laboratory is paired with ISO/TS 19590:2017 — Size distribution and concentration of inorganic nanoparticles in aqueous media via single particle inductively coupled plasma mass spectrometry. (It is noted that an updated standard has been published in 2024.)

2. Relevant Documentary Standards and Reading Exercises

3. Experimental Procedure

3.1 Chemical reagents

• Deionized (DI) water
• Concentrated nitric acid (67%-70%), trace metal grade (FisherScientific)
• Dissolved gold standard solution, 1000 mg/mL (Agilent ICP-079)
• 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)
• Compressed argon or liquid argon tank for ICPMS operation

3.2 Equipment and supplies

• ThermoScientific iCAP RQ ICPMS system with high sensitivity skimmer insert
• Pipettors and pipet tips
• Polypropylene centrifuge tubes (15 mL) for samples

3.3 Procedures

Sample preparation

1. Dissolved gold standards: Prepare serial dilutions of the gold stock solution to 2 mL of 10 mg/L Au and 2 mL of 100 mg/L Au, both acidified to ≈ 3 % HNO3 (trace metal grade). Then prepare 10 mL of the calibration standards at (0.2, 0.5, 1.0, 2.0, and 5.0) mg/L Au, also acidified to ≈ 3 % HNO3 (trace metal grade).
2. Gold nanoparticles, 60 nm for transport efficiency determination: Prepare serial dilutions as necessary to dilute the 60 nm gold nanoparticles to a target concentration of 2 × 10⁷ particles/L.
3. Gold nanoparticle mixture (10, 30, 60, and 100 nm): Prepare serial dilutions as necessary to prepare a nanoparticle mixture with a target concentration of 5 × 10⁶ particles/L of each size of particle (i.e. total particle concentration of 2 × 10⁷ particles/L for all four particle sizes).
4. Synthesized gold nanoparticles, Pink: Dilute as necessary (optimized during the data collection to attain particle detections within the range recommended in ISO/TS 19590:2017).
5. Synthesized gold nanoparticles, Purple:
6. Dilute as necessary (optimized during the data collection to attain particle detections within the range recommended in ISO/TS 19590:2017).
7. Prepare 2% to 3% HNO3 (trace metal grade) to use as the rinse solution between samples.

Instrument setup

The instrument settings to apply for the measurements are specified in Table L5.1 below.

Table L5.1. iCAP RQ Measurement Settings for Particle Size Distribution Analysis by spICPMS

Sample analysis

1. Determine the volumetric flowrate provided by the peristaltic pump by measuring the mass of a tube of deionized water before and after 1 minute of pump time.
2. Turn on the instrument, and run the ICPMS instrument tune procedures to optimize signal intensity and verify mass calibration.
3. Following the method in Table L5.1, Analyze the dissolved standards and the four nanoparticle samples, with blanks injected and measured between each sample. Triplicate measurements are collected on each nanoparticle sample.

Shutdown procedure

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

4. Data Analysis

The linked Excel file (UH – Exptl Methods Nano – Laboratory 5 Data Analysis Template) contains a set of example data to process.

1. Open the data spreadsheet template and follow the linked instructions (UH – Experimental Methods Nano – Laboratory 5 Data Processing Instructions) to process the dissolved ion calibration curve, the 60 nm gold nanoparticle sample for the transport efficiency, and the other three gold nanoparticle samples.
2. From the recommended reading of ISO/TS 19590:2017, performance criteria were reported for the following items:
   1. Minimum acceptable correlation coefficient (R²) on the calibration slope
   2. Minimum acceptable transport efficiency
   3. Maximum acceptable number of particle detections per minute

Report the criterion from the ISO standard, the actual results for the three measurands, and your assessment of whether or not the criteria were met.

3. Report the number-averaged particle diameter for each of the four nanoparticle samples (including the 60 nm used for the transport efficiency).
4. For the mixture of four sizes of gold nanoparticles, inspect the histogram to evaluate how many peaks you observe in the particle distribution. Assign the most likely particle size (10 nm, 30 nm, 60 nm, or 100 nm) corresponding to each observed peak. For any peak that is observed, report the size bin corresponding to the mode of the peak (i.e. where the maximum number of particles was observed for that peak).

5. Discussion Questions

1. Theoretical vs. observed nanoparticle sizes
   The manufacturer of the purchased nanoparticles reports the following mean diameters for the batches used in the example data set, as determined by transmission electron microscopy (TEM) (Table L5.2):

Table L5.2. Properties reported by BBI Solutions for purchased Au nanoparticles

(a) Based on your results for the Au nanoparticle mixture: Is there any systematic deviation (i.e., consistently higher or lower values) between the spICPMS modal average size for each nanoparticle observed in the mixture, relative to the TEM size?

(b) Consider all of the input or calculated parameters that were required to compute the particle size by spICPMS – errors in any of these parameters could propagate to errors in the particle size. For each of the following parameters, inspect the spICPMS data equations presented in ISO/TS 19590:2017, and identify any equations in which errors in the parameter values would propagate to error in the computed size. Assuming the TEM size measurements are accurate, indicate whether any observed deviations in part (a) could be indicative of the following input parameter values being too low or too high:

• 1. Dwell time
  2. Sample flowrate
  3. Calibration slope (i.e., response factor)
  4. Transport efficiency

1. Effect of polymer surface coatings
   The PVP-coated 60 nm gold nanoparticles were not evaluated in this lab. However, consider the principle of the spICPMS analysis. If all the input parameters (molar mass ratio, particle density) are kept the same as for the uncoated gold nanoparticles, would you expect the computed spICPMS size to differ significantly for the PVP-coated gold particles versus the uncoated particles? Explain why or why not.
2. Particle size detection limit
   The ISO standard discusses that there is a minimum particle size that can be detected, based on the ion/particle cutoff – any particles whose signal height is below the cutoff value are not included in the particle analysis. Review the minimum particle sizes observed in your analysis of the mixture of the four sizes of gold nanoparticles. Were any of the nominal sizes (10, 30, 60, or 100 nm) below the particle size detection limit?
3. Comparison of spICPMS to other sizing methods
   Recall the batch UV-Vis, DLS, and AF4 analyses on the mixture of the four Au nanoparticle sizes in the prior laboratories. Based on the actual results observed in Laboratories 1, 2, 4, and 5 (this laboratory), describe any major advantages or limitations of spICPMS versus the batch analyses to acquire a complete and accurate size distribution on the mixture of four particle sizes. Note that you should be able to identify both an advantage and a limitation in the spICPMS data.
4. Unknown (pink and purple) samples
   (a) The pink and purple gold nanoparticles were evaluated in Laboratories 1, 2, 4, and 5. Tabulate the estimated mean size for each sample in each lab (for Laboratory 4, you can make a rough estimate from the plots shown for the last discussion question). Compare the results and describe any major discrepancies between the different measurements (e.g. differences in the average sizes reported in your table, differences in the polydispersity or number of peaks in the size distribution, the relative size difference for whether “pink” or “purple” corresponds to a larger or smaller particle size, etc.).
   (b) Rank your level of confidence in each of the four measurements (UV-Vis, DLS, FFF, and spICPMS) for determining the size of these completely unknown particle samples. Explain your reasoning for your confidence or uncertainty in the measurement. You can consider the principle of how the measurement works, quality issues noted in the measurements taken during the lab session, etc.
   (c) You should have noted some discrepancies between the mean sizes acquired using the different instruments. Develop a conceptual description or diagram of possible differences in physical particle properties between the pink and purple samples that could be consistent with the results observed across all four labs.