Spectrophotometry

Absorbance Spectrum

The extent to which a sample absorbs light depends strongly upon the wavelength of light. For this reason, spectrophotometry is performed using monochromatic light. Monochromatic light is light in which all photons have the same wavelength.

In analyzing a new sample, a chemist first determines the sample's absorbance spectrum. The absorbance spectrum shows how the absorbance of light depends upon the wavelength of the light. The spectrum itself is a plot of absorbance vs wavelength and is characterized by the wavelength (l_max) at which the absorbance is the greatest.

The value of l_max is important for several reasons. This wavelength is characteristic of each compound and provides information on the electronic structure of the analyte. In order to obtain the highest sensitivity and to minimize deviations from Beer's Law (see subsequent pages on this topic), analytical measurements are made using light with a wavelength of l_max.

Experiment

Objective:

Determine the absorbance spectrum of a sample solution.
Determine the wavelength of maximum absorbance.

Approach:

Measure the intensity of transmitted light for various wavelengths of light.
For each wavelength, calculate the absorbance of the solution.
Construct the absorbance spectrum by plotting A vs l.
Determine l_max by locating the wavelength at which the absorbance is greatest.

Part 1

The absorbance spectrum for a sample is shown below. For this sample, l_max = 563 nm.

To explore the significance of the absorbance spectrum, perform a series of experiments. For each experiment:

Enter a wavelength between 380 nm and 780 nm (this is the visible range of the spectrum).
Run the simulation long enough to detect at least 1000 photons.
Use the intensity of the transmitted light to calculate the absorbance of the sample at that wavelength. (For a blank, the intensity of the transmitted light is 100.0 photons/sec.)
Plot the l,A point on the graph containing the spectrum.

Notice how the wavelength of the photons dictate the color of the photons.

Not all of the photons are shown in the simulation.

Before beginning a new simulation (for example, using a new wavelength), remember to reset the simulation.


Light Source	Cell	Detector
Wavelength: nm Number of Photons Detected = t = sec I = photons/sec			l = nm A =

Part 2

In this part of the experiment, you must construct the absorbance spectrum for an unknown sample.

Run the simulation for various wavelengths of light. Your goal is to determine l_max to within 5 nm.

IMPORTANT: If you collect 1000 photons, the uncertainty in the final absorbance is around 0.014! When you get near the absorbance maximum, it is desirable to repeat measurements. It is also a good idea to acquire more than 1000 photons. If 4000 photons are detected, for example, the uncertainty in the absorbance is reduced to about 0.007.

For a blank, the intensity of the transmitted light is 100.0 photons/sec.

Not all of the photons are shown in the simulation.

Before beginning a new simulation (for example, using a new wavelength), remember to reset the simulation.


Light Source	Cell	Detector
Wavelength: nm Number of Photons Detected = t = sec I = photons/sec			l = nm A =

Spectrophotometry Effect of Cell Path Length

Spectrophotometry Home Page