UV-Vis spectrophotometry is a well-established, easy to use, and inexpensive technique for measuring the size, concentration, aggregation state, and refractive index of nanomaterials. UV-Vis provides noninvasive and fast, realtime screening and evaluation of nanomaterial characteristics using simple absorption measurements. UV-Vis is exceptionally straightforward.
The samples require little preparation, and the software has few variables and is easy to use. Turn on the UV-Vis spectrometer for at least 20 minutes to allow the lamp to heat up. Load the software and select Connect the instrument Select the option Spectrum Scan from the mode window which displays the operating modes.
In the software, click on Instrument and go to Settings in the command bar, select Parameters for Spectrum Scan, then adjust the settings for measurement. Go to the Instrument tab and select ABS for Data Mode, and Slit width of 1.5. Go to the Wavelength Scan tab and set a Start wavelength of 680 nanometers, End wavelength of 380 nanometers, and scan speed of 400 nanometers per minute.
After setting the parameters, fill two cuvettes with one milliliter of ultra pure water, and place them in the reference and sample cell holder to cover the light path. Close the instrument cover and continue with blank calibration by selecting blank from the command bar, then select OK.Take a 500-microliter subsample of each gold nanoparticle sample, and prepare a dilution with 500 microliters of ultrapure water. Place the dilutions in one-milliliter cuvettes with a final concentration of 25 micrograms per milliliter.
Run a blank calibration. After the blank calibration, replace one of the blank cuvettes in the sample cell holder with a gold nanoparticle sample, keeping the reference cuvette untouched. Select measure Start in the command bar to run the scan.
Run three spectrum scans for each diluted gold nanoparticle sample and unknown sample. Extract the raw experimental data for each measurement in a spreadsheet-compatible file. Select Property on the scan, on the command bar, go to the Print/Export tab, click Data, Spectrum, and parameters, and press OK.Click Save on the command bar and save the data as a xls file.
Note the maximum absorption wavelength and lambda for each of the readings and record them in the provided template. Plot a calibration curve with the average of maximum lambda against nanoparticle size by selecting data, insert graph, scatter plot, add trendline, and polynomial curve. To include the polynomial equation for the calibration curve, select Trendline options and Display equation on chart from the command bar.
Finally, calculate the size of the unknown gold nanoparticle sample by isolating the polynomial equation from the calibration curve to fit the mean value for the unknown maximum lambda using a derivation of the quadratic formula. Among the six laboratories, The maximum wavelength showed close repeatability, whereas the maximum absorbance results exhibited a more scattered range of data values for different gold nanoparticle sizes. The ranges and overall means for each gold nanoparticle size are shown here.
The maximum Z-score value for lambda max was reported by laboratory three for the 5-nanometer-sized nanoparticles, and the highest z-score for maximum absorbance was reported by laboratory one for 40-nanometer-sized nanoparticles. Most of the partners calculated the size of the unknown nanoparticle to be 76 to 80 nanometers, with laboratory five reporting an outlier larger size of 109 nanometers. The Z-scores for unknown sizes were calculated to be between minus 0.25 to minus 0.56 for all the laboratories, with the only exception reported by laboratory five, which displayed the highest positive Z-score of 2.03.
Make sure the parameters and settings have been correctly adjusted. The diluted samples must be prepared fresh before measurement with the UV-Vis. UV-Vis can be easily combined with other tools, such as TEM, DLS, and AFM to measure a large variety of attributes if users want to characterize their sample more comprehensively.
