Scanning Ultraviolet/Visible Spectroscopy (Spectronic 20™ and Automated Instruments)

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When measuring the absorbances of several samples that have different concentrations, it is important to identify an appropriate wavelength at which to take measurements. This wavelength is the one at which the samples absorb the most light and is called lambda max, since the Greek letter is used to symbolize wavelength.

The Spectronic 20™ or a scanning spectrophotometer can be used to help generate a plot of absorbance versus wavelength, which can then be used to find lambda max.

To find lambda max, set the wavelength on the Spectronic 20™ to 400 nm and calibrate the maximum and zero absorbance using the reference solution. Insert the sample solution and record its absorbance at this wavelength. These steps are described in the Spectronic 20™ at Fixed Wavelength module.

Increase the wavelength in 20 nm increments and record the absorbance at each wavelength. Remember to recalibrate the maximum and zero absorbance each time you change the wavelength.

When the absorbance begins to rise rapidly, decrease the wavelength increment from 20 nm to 2-5 nm. This smaller increment allows a more accurate determination of the wavelength of maximum absorbance (lambda max). After the absorbance has peaked and again begins to decrease, change the wavelength increment back to 20 nm. Take measurements from 400 nm up to 600 nm.

A plot of absorbance versus wavelength shows lambda max.

Video: Animation of graphing process ( 4.53 M )

Use this wavelength (lambda max) to measure the absorbances of other samples of this solution that have different concentrations.

Below is a graph of absorbance versus wavelength for a solution. What is lambda max? Can you predict the color of this solution? Answer

Below is an absorption spectrum of a solution as measured with a spectroscope. Sketch a graph of absorbance versus wavelength. What is lambda max for this solution? Answer

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A scanning ultraviolet/visible (UV/Vis) spectrophotometer operates on the same principles as a Spectronic 20™. A scanning spectrophotometer's main advantage over the Spectronic 20™ is that the scanning spectrophotometer can rapidly scan a range of wavelengths and record absorbances at each wavelength. In this module, the Perkin Elmer Lambda 11™, a single beam spectrophotometer, is described but similar principles apply to all UV/Vis spectrophotometers. For specific operating instructions, refer to the operator's manual or other instructions.

Double beam and diode array spectrophotometers are also commonly used to obtain spectra.

Use a clean rectangular cuvette that is free of cracks. Plastic cuvettes are acceptable when absorbance measurements will be recorded in the visible range of the spectrum (about 400 to 700 nm wavelengths), but quartz cuvettes are necessary if measurements are made at lower wavelengths or if the sample liquid will destroy a plastic cuvette.

To aid in proper placement of the cuvette within the sample compartment, first cover one face of the cuvette with tape.

Video: Marking with tape ( 2.06 M )

Next, prepare the reference liquid. The reference liquid should be the same as the solution to be analyzed except that it must have zero concentration of the absorbing species. Rinse the cuvette three times with a small volume of the liquid that the sample is dissolved in. Then fill the cuvette about three-fourths full with the liquid. Gently tap the cuvette until any air bubbles are dislodged. Wipe the outside of the cuvette with a laboratory tissue.

Place the cuvette containing the reference liquid into the cell compartment in the spectrophotometer. Make sure that the cuvette's clear sides are perpendicular to the light source when the cuvette is placed into the sample compartment.

Next, set up the wavelength range on the spectrophotometer. Using the computer, set the spectrophotometer to scan the desired wavelengths (usually 400 to 700 nm to scan the visible region of wavelengths). In addition, the step size must also be set. The step size indicates how often the spectrophotometer will record an absorbance measurement. A step size of one nanometer is common.

With the sample compartment lid closed, zero the spectrophotometer.

After the spectrophotometer has been zeroed, remove the reference sample, pour out the reference solution, rinse the cuvette three times with the sample, and place the cuvette into the sample compartment. Why use the same cuvette? Again, be sure that the clear faces of the cuvette are perpendicular to the light source. Close the lid, and use the computer to begin scanning the wavelengths and recording absorbance measurements.

Video: Using the sample ( 5.17 M )

The computer should display a plot of absorbance versus wavelength.

If the spectrum goes off the scale, dilute the sample solution and re-measure the spectrum.

The wavelength at which the absorbance is greatest is lambda max.

Related modules: Spectronic 20™ at Fixed Wavelength, Spectroscope