How can I use a spectrophotometer to determine the concentration of solutes in a solution? Prelab 2.2

BIOL 1406

PreLab 2.2

How can I use a spectrophotometer to determine the concentration of solutes in a solution?

Electromagnetic radiation is a form of energy that travels through space with the characteristics of a wave. Electromagnetic radiation with wavelengths between about 400 nm and 740 nm is detected as visible light. Our eyes detect different wavelengths of visible light as different colors. For example, light with a wavelength of 400 nm appears violet while light with a wavelength of 740 nm appears red. White light is actually a mixture of all the visible wavelengths. When white light passes through a solution, the color of the solution will depend on how much of each wavelength passes through the solution (called optical transmittance) and how much of each wavelength is absorbed (called optical absorbance).

A spectrophotometer is an instrument that can pass light of a single wavelength through a solution and measure the amount that passes through. First, the spectrophotometer must be zeroed by passing light of the chosen wavelength (500 nm for example) through a blank containing only the solvent. Then, the same wavelength of light is passed through the solution. The percentage of light that passed through the solution relative to the amount that passed through the solvent alone is called the percent transmittance at 500 nm. For example, if half as much light passes through a solution as passes through the solvent alone, we would record this as 50% T:

Percent Transmittance (%T) =

(amount of light transmitted through the solution / amount of light transmitted through the solvent) x 100

On the other hand, the amount of light at 500 nm that is absorbed by the solution is called the absorbance at 500 nm and is abbreviated A₅₀₀. Absorbance is the negative logarithm of the percent transmittance divided by 100. Because logarithms have no units, absorbance has no units:

Absorbance (A) = - log (% transmittance / 100)

A spectrophotometer can be set to measure either the percent transmittance or the absorbance of a solution. In lab, you will be using a spectrophotometer called the “Spectronic 20” or “Spec 20” to measure absorbance. Click the play arrow on the right to watch a short video about how to calibrate and measure absorbance with the digital Spec 20.

If you have problems seeing the video, you can view it in an External Viewer

It is extremely important that you know how to calibrate the Spec-20 and use it to measure absorbance.
Use the interactive exercise below to test yourself and see if you are ready to use the Spec-20 in lab.

Examine the graphs of percent transmittance and absorbance shown on the right. Notice that these 2 variables are inversely related to each other; in other words, they move in opposite directions. For example, if solute concentration is increasing, percent transmittance will decrease while absorbance will increase. Also note that the absorbance of a solution is linearly related to its concentration. This means that if the concentration of a solution increases at a steady rate, then absorbance will also increase at a steady rate (within limits.) This is called a linear relationship because if we measure the absorbance of several solutions with different solute concentrations, and then plot a graph with solute concentration on the x-axis and absorbance on the y-axis, our data points should fall on a straight line.

In this lab, you will learn 3 methods for preparing solutions that contain a specific concentration of solutes. You will then use all 3 methods to prepare 14 solutions of KMnO₄, each with a different concentration of solute. Next, you will measure the absorbance of each solution with a spectrophotometer and plot a graph of your data with the "solution concentration" on the x-axis and the "absorbance" on the y-axis. If your solutions were prepared correctly, all of your data points should fall on a straight line. Then you will use a statistical technique called linear regression to determine the equation of the "best-fit" straight line. This equation will tell you the exact mathematical relationship between the 2 variables (solute concentration and absorbance). Finally, you will use this equation to estimate the concentration of a solution of potassium permanganate of unknown concentration.

YOUR TURN
The following scatter diagram was prepared by plotting "Concentration of KMnO₄ (mM)" vs. "A 540 values" for 7 solutions. The data points are shown as dots. A linear regression line was then calculated from the data points and plotted as a black line: Examine the scatter diagram carefully and then answer the questions below:
Different colors of visible light have different .	Hint	Check your answer.
What is a spectrophotometer used for?	Hint	Check your answer.
What is the difference between percent transmittance (%T) and absorbance (A)?	Hint	Check your answer.
If a solution has a percent transmittance of 20% at 595nm, what is its optical absorbance at 595 nm?	Hint	Check your answer.
A solution has a percent transmittance of 30% at 595nm. As you make the solution more and more dilute, what happens to its percent transmittance at 595nm?	Hint	Check your answer.
A solution has an absorbance of 0.62 at 595nm. As you make the solution more and more dilute, what happens to its absorbance at 595nm?	Hint	Check your answer.
According to the linear regression line, what is the predicted A540 value for a 0.6 mM KMnO4 solution?	Hint	Check your answer.
According to the linear regression line, what is the predicted concentration for a KMnO4 solution with an A540 value of 0.4?	Hint	Check your answer.

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