Ultraviolet-Visible (UV-Vis) Spectroscopy

1. Calibrate the Spectrometer

1. Turn on the UV-Vis spectrometer and allow the lamps to warm up for an appropriate period of time (around 20 min) to stabilize them.
2. Fill a cuvette with the solvent for the sample and make sure the outside is clean. This will serve as a blank and help account for light losses due to scattering or absorption by the solvent.
3. Place the cuvette in the spectrometer. Make sure to align the cuvette properly, as often the cuvette has two sides, which are meant for handling (may be grooved) and are not meant to shine light through.
4. Take a reading for the blank. The absorbance should be minimal, but any absorbance should be subtracted out from future samples. Some instruments might store the blank data and perform the subtraction automatically.

2. Perform an Absorbance Spectrum

1. Fill the cuvette with the sample. To make sure the transfer is quantitative, rinse the cuvette twice with the sample and then fill it about ¾ full. Make sure the outside is clean of any fingerprints, etc.
2. Place the cuvette in the spectrometer in the correct direction.
3. Cover the cuvette to prevent any ambient light.
4. Collect an absorbance spectrum by allowing the instrument to scan through different wavelengths and collect the absorbance. The wavelength range can be set with information about the specific sample, but a range of 200–800 nm is standard. A diode-array instrument is able to collect an entire absorbance spectrum in one run.
5. From the collected absorbance spectrum, determine the absorbance maximum (λ_max). Repeat the collection of spectra to get an estimate of error in λ_max.
6. To make a calibration curve, collect the UV-Vis spectrum of a variety of different concentration samples. Spectrometers are often limited in linear range and will not be able to measure an absorbance value greater than 1.5. If the absorbance values for the sample are outside the instrument's linear range, dilute the sample to get the values within the linear range.

3. Kinetics Experiments with UV-Vis Spectroscopy

1. UV-Vis can be used for kinetics experiments by examining the change in absorbance over time. For a kinetics experiment, take an initial reading of the sample.
2. Quickly add the reagent to start the chemical reaction.
3. Stir it well to mix with the sample. If a small amount is added, this could be done in a cuvette. Alternatively, mix the reagent with sample and quickly pour some in a cuvette for a measurement.
4. Measure the absorbance at the λ_max for the analyte of interest over time. If using up the reagent being measuring (i.e. absorbance is going up because there is less reagent to absorb), then the decay will indicate the order of the reaction.
5. Using a calibration curve, make a plot of analyte concentration vs time, converting the absorbance value into concentration. From there, this graph can be fit with appropriate equations to determine the reaction rate constants.