UV-Vis Spectroscopy Tutorial Part 1: Measuring Nanoparticle Absorbance

This video demonstrates how to monitor the reaction kinetics between blue food dye and bleach using absorption spectroscopy. The experiment involves preparing 1.5 mL each of blue food dye solution and bleach, combining them in a cuvette, and measuring absorbance at 630 nm every 3 seconds using a spectrometer. By applying Beer's Law (A = εlc), the absorbance data is converted to concentration values, and the reaction order is determined by plotting concentration, ln(concentration), and 1/concentration versus time to identify whether the reaction follows zero-order, first-order, or second-order kinetics.

Nanoparticles tend to aggregate due to different surface energy compared to bulk materials. Aggregation changes the absorption spectrum - when two nanoparticles join (dimerization), the spectrum shifts (redshift). This occurs because the transition probability increases and the absorbing surface area increases. The absorption spectrum provides immediate observation of what happens in the solution, including aggregation states.

Aggregation of nanoparticles can be detected by analyzing changes in UV-Vis spectra. Early-stage aggregation shows a slight elevation in baseline absorbance between 450-550 nm, which indicates formation of larger aggregates that absorb more strongly in the red region of the spectrum. As aggregation progresses, the peak absorbance decreases because some nanoparticles that previously absorbed at the peak wavelength are now aggregated. Simultaneously, absorbance increases in the longer wavelength regions where aggregates absorb. A 20% drop in peak absorbance with baseline elevation indicates mild aggregation, while a 40% drop indicates severe aggregation.

UV-Vis spectroscopy is a fundamental technique for monitoring nanoparticle synthesis in real-time. During synthesis, periodic UV-Vis measurements track changes in the absorption spectrum, allowing researchers to observe formation kinetics and identify optimal stopping points. The technique detects the emergence and evolution of characteristic SPR peaks, providing immediate feedback on particle growth and quality. This enables precise control over the synthesis process and helps in optimizing reaction conditions for desired nanoparticle properties.

Nanoparticle aggregation occurs when particles attach to each other through various interactions. The nanoparticles are not actually fusing with each other but are attaching through hydrogen bonding or electrostatic interactions with the amine groups of polymers. The more polymer present, the more particles will aggregate, and the more pronounced the color change will be. When gold nanoparticles aggregate, they undergo a color change from red to purple and then to blue. This color change indicates that the nanoparticles are aggregating. The aggregation can be observed visually or through UV-Vis spectroscopy. RGB (Red, Green, Blue) colorimetric sensors are used to measure nanoparticle aggregation. Gold nanoparticles are red in color, and when they aggregate, they shift toward blue. The RGB colorimetric app measures the intensity of the red value. As nanoparticles aggregate with increasing polymer concentration, the red intensity decreases. A nanodrop spectrophotometer is used to measure the spectral properties of nanoparticle samples. It requires only small amounts of sample (1-2 microliters). For gold nanoparticles, the maximum peak is initially at 530 nanometers. When nanoparticles are incubated with polymers, the peak shifts toward higher wavelengths (e.g., to 560 nanometers), indicating aggregation. At low polymer concentrations, the absorbance is lower because the SPR peak is at 530 nanometers. As polymer concentration increases, the SPR peak shifts from 530 to 560 nanometers, and the absorbance at 560 nanometers increases. This relationship between polymer concentration and absorbance is used for quantitative sensing applications.

To calculate the concentration of a substance like quinine using extinction coefficient, perform serial dilutions of the sample (e.g., 5, 10, 15, 20, and 25 microliters), measure absorbance at specific wavelengths (such as 257 nm with an extinction coefficient of 19.98 and 376 nm with 21.8), plot absorbance versus dilution volume, apply linear regression to obtain the equation y = mx + c, and use the intercept and slope values along with the extinction coefficient to determine the original concentration through the Beer-Lambert Law (A = εlc), finally averaging results from multiple wavelengths for accuracy.

The concentration of a solution can be calculated from its absorbance using the Beer-Lambert law (A = εcl), where concentration equals absorbance divided by the product of the extinction coefficient and path length; for example, an absorbance of 0.445 measured at 423 nm with a 1 cm path length and extinction coefficient of 3739 M⁻¹cm⁻¹ yields a concentration of 1.19 × 10⁻⁴ M, which can also be expressed as 0.119 mM or 119 μM.

This segment demonstrates how to calculate extinction coefficients (epsilon values) from standard solutions. The instructor explains that extinction coefficient equals absorbance divided by concentration (ε = A/C). For each component (acetone, ethyl acetate, toluene), the instructor shows how to calculate epsilon values at specific wavelengths by dividing the absorbance of the standard solution by its known concentration. The instructor notes that negative values in some regions are non-physical and should be replaced with zero. These extinction coefficients are then used in the matrix method to determine concentrations in the unknown mixture.

This section teaches Excel formulas for concentration calculation using the extinction coefficient. The instructor demonstrates creating formulas for cuvette concentration: = absorbance / (extinction coefficient × path length), and original concentration: = (cuvette concentration / volume of dye in μL) × 1000. Absolute referencing is used for the extinction coefficient cell ($L$4) when dragging formulas. The instructor shows how to copy formulas between different unknown samples and how Excel automatically updates cell references when copying.

When the extinction coefficient is unknown, it can be determined graphically by creating a calibration curve. Prepare solutions with known concentrations of the substance, measure their extinction values, and plot concentration on the x-axis against extinction on the y-axis. The resulting straight line's slope equals the extinction coefficient. This allows determination of unknown concentrations by measuring their extinction and reading off the corresponding concentration from the graph.

This video explains three fundamental techniques for characterizing nanoparticles: Dynamic Light Scattering (DLS) measures particle size and distribution in liquids by analyzing how light scatters from particles undergoing Brownian motion; Scanning Electron Microscopy (SEM) provides high-resolution imaging of nanoparticle shape, morphology, and elemental composition using electron beams; and UV-Vis Spectroscopy detects optical properties and surface plasmon resonance of nanoparticles. Together, these complementary methods allow scientists to fully characterize nanoparticles for applications in medicine, electronics, and materials science.

This tutorial introduces six essential nanoparticle characterization techniques used by nanoComposix: Transmission Electron Microscopy (TEM) for imaging particle size, shape, and crystallinity; UV-Vis spectrophotometry for evaluating optical properties, size, shape, and concentration; Endotoxin Assay for detecting bacterial contamination using horseshoe crab blood reagents; ICP-MS for measuring metal concentration through plasma atomization and mass analysis; Dynamic Light Scattering (DLS) for determining hydrodynamic diameter via Brownian motion analysis; and Zeta Potential measurement for assessing electrostatic repulsion/attraction between particles to evaluate colloidal stability.

Nanoparticle size can be estimated from UV-Vis absorbance data using the Haiss equation: d = ln((λ_SPR - λ₀)/L₁)/L₂, where λ_SPR is the wavelength at maximum absorption, λ₀ is the wavelength at minimum absorption at the start of the SPR peak, and L₁=6.53 and L₂=0.0216 are empirically derived fitting parameters from transmission electron microscopy versus UV-Vis data comparisons; this method is accurate for spherical colloidal metal nanoparticles with diameters ranging from 25 to 110 nm.

Dynamic light scattering (DLS) measures hydrodynamic radius through Brownian motion analysis, but its extreme sensitivity to dust and concentration-dependent behavior requires strict laboratory protocols including filtration with 1.2-2 micron filters, inspection of count rates and correlation functions, accumulation of at least 5 million counts, and use of number-weighted distribution functions rather than Z-average values for accurate comparison with transmission electron microscopy (TEM) results; researchers must also account for radiation damage in TEM samples and perform concentration extrapolation to infinite dilution to obtain true particle sizes.