Prelab Report

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Absorption Spectrum of a Conjugated Dye

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Chem 375-003

Group #5


Introduction & Theory

The electrons of a molecule are typically in their most stable state, known as the ground state. When energy in the form of photons strikes a molecule, that energy may be absorbed by raising the electrons of the molecule to a higher energy state. As energy is quantized however, only photons of certain energies are capable of being absorbed. As a result, the color of a substance is determined by the wavelength of light that corresponds to the energy required to excite its electrons. It should be noted that this excited state is very unstable and is rapidly followed by the emission of a photon (often of a different wavelength) and a return of the electron to the ground state.

Since the energy required to excite an electron depends on the components and configuration of the molecule it is a part of, the amount of light absorbed by a substance at different wavelengths serves as a fingerprint of that substance. As a result, absorption spectrums serve as a tool for the identification of unknown compounds as well as providing qualitative information about the makeup of a molecule. As a simple example of this, an unknown compound with an absorption spectrum similar to NaCl and known to contain sodium is likely to be NaBr due to the geometric and electronic similarities between the two molecules.

In the case of polymethine dyes such as used in this experiment, the absorption in the visible region is a result of mobile electrons along the polymethine chain in the center of the molecule. By treating these electrons as electrons that are free to move along the distance between the two ethyl groups, the quantum mechanical energy levels are modeled by

Where m is an electron's mass, L is the distance between the ethyl groups, and h is Plank's constant. The Pauli exclusion principle states that a molecule with N electrons will have the N/2 lowest energy levels filled if N is an even number. Hence the following equations for the energy change for the transition from ground state to one state higher:

With the speed of light being c and the wavelength being , =hc/. Substituting into eq. (2) and solving for yields

If the number of carbon atoms in the polymethine chain is symbolized by p, then N=p+3 and L=(p+3)l where l is the bond length between atoms in the chain. By using the value of l from benzene, a similarly bound molecule, expressing in nm, filling in the other constants, and simplifying it can be found that

For non-ideal cases where L is not defined sharply by the end of the polymethine chain, an empirical parameter may be added to the p+3 term to correct for this. This parameter would then be constant for dyes in a series and would be between 0 and 1 as the distance that the free electrons would have to move would increase by at most 1 bond and never decrease.


The goal of this experiment is to record and observe the absorption spectrum of a solution of several dyes, as well as determining the extinction coefficient of 3,3 diethylthiatri-carbocyanine iodide dye. This will be accomplished through the use of a single beam spectrophotometer interfaced with a computer.


A computer attached to a Hewlett Packard diode array spectrophotometer will be used to obtain and record the measurements. Sample cells; lens tissue; 10-mL volumetric flasks; a wash bottle; reagent-grade methyl alcohol (150 mL); several milligrams of 3,3 diethylthiatri-carbocyanine iodide dye (C25H25IN2S2); several milligrams of polymethine dyes in a series.

Experimental Observables

In this experiment, the apparatus will obtain the absorption of a solution over the visible spectrum and correct it with regard to the absorption of a standard solution. This corrected absorption will then be used as raw data.

Experimental Method

Spectroscopy is the measure of the amount of light (including IR and UV) that passes through a given sample. The amount of light to pass through a sample is called the transmittance T, and is defined by the following formula:

Where I is the intensity of the light transmitted by the sample and I0 is the intensity of the light before passing through the sample. As a result, a solution that is transparent to the given wavelength would have a transmittance of 1, and an opaque solution would have a transmittance of 0.

Another common measure of the amount of light allowed to pass through a sample is call the absorbance A. It is simply a variation of transmittance that varies from 0 for a transparent solution and for an opaque solution. Absorbance is defined as follows:

Finally, the molar absorption coefficient (also called the extinction coefficient) can be determined from the c, the molar concentration, and d, the path length in cm.

Experimental Procedure

As soon as other lab equipment reaches a state where a stable power supply can be assured, the computer station should be powered up. After the boot process is complete, the spectrometer lamp should be turned on by pressing the appropriate function key. It is important that this be done at least 15 minutes before data is collected to assure consistent results.

Per the preliminary calculations, 0.00545 grams 3,3 diethylthiatri-carbocyanine iodide are to be added to precisely 100 mL of methyl alcohol. If the dye does not dissolve within 10 minutes, sonicate the solution for approximately 1 minute. This will create a 0.1 mM solution of the dye. The exact concentration should be recorded, in order to facilitate the calculation of the extinction coefficient.

After the lamp has been on for at least 15 minutes, a sample blank must be scanned for calibration. This blank should be the done with the cell that is to be used throughout the experiment, and filled only with methyl alcohol.

After the blank has been scanned, prepare 5 mL of ~10-3 M solution of one of the available dyes, using methyl alcohol as a solvent. Only the Mettler or A&N balances should be used for massing the dyes, and not the Fischer balances. Scan across broad bands until the absorption peak is located, then narrow in around the peak until it broadens so that the most precise readings may be obtained. If necessary, dilute the solution until the absorption maximum is between 0.5 and 1.0. Note that any dilution of the 3,3 diethylthiatri-carbocyanine iodide dye must be carefully performed and noted to allow calculation of its extinction coefficient, for the other dyes only the relative shape of the spectrum is of importance. Be sure to save the data to disk after every run that results in useful data. It is also important to note d, the path length in cm for use in later calculations.

Experimental Precautions

Accurate dilution, especially with small samples, can be difficult. Ensure that precision is of high priority in every step when diluting the 3,3 diethylthiatri-carbocyanine iodide dye.

Ensure that all materials are thoroughly rinsed between dyes. Contamination can cause unexpected peaks in the observed absorption.

Preliminary Calculations

The handout gives instructions to make 100 mL of 0.1 mM 3,3diethylthiatri-carbocyanine. This equates to 0.01 millimoles of dye. Given the dyes chemical composition of C25H25IN2S2, it has a molecular weight of 544.51 grams mole-1. Consequently, 0.00545 grams dye to 100 mL water will give the desired solution.


The MSDS for 3,3 diethylthiatri-carbocyanine iodide do not indicate any health risks or special precautions for this material. The identities of the other dyes to be used are not given specifically, only various examples, so the appropriate safety data cannot be located. Shoemaker, Garland, and Nibler(2) list no safety concerns associated with this lab.

Methyl alcohol is highly flammable and may cause asphyxiation if exposed in large amounts in an unventilated area.


1. Safety data taken from MSDS database on the Internet at ""

2. D. P. Shoemaker, C. W. Garland, J. W. Nibler, Experiments in Physical Chemistry, 6th ed., chap. 6, experiment 6, The McGraw-Hill Companies (1996).