Lambert-Beer's Law UV-Visible Spectroscopy

7/22/2019 Lambert-Beer's Law UV-Visible Spectroscopy

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Determining the Concentration of an Unknown Colored Solution

Experimental Problem

Determine concentration of an unknown colored solution.

Educational Purpose

To familiarize yourself with visible spectroscopy.

The Beer-Lambert Law.

Construct a calibration curve

What to Turn In1. The prelab and the postlab

2. The absorption spectrum and calibration curve

Background

Transmittance and Absorbance

When a beam of radiation (light) encounters a light absorbing substance or a

solution containing a light absorbing substance, some or all of the light may be

absorbed. The rest of the light is transmitted through the sample.

Io is defined as the light entering the sample and It as the light leaving the

sample. The amount of light passing through the sample is usually expressed as the

percent transmittance (%T) where

%T = It x 100

I0


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In the pic I = It

If all of the light is absorbed by the sample I t = 0 and %T = 0. If all of the light istransmitted through the sample It = I0 and %T = 100. Normally, we will work with

values of %T that are between these two extremes and with the most accurate

measurements being made between 10 and 80%T (between 0.1 and 1 absorbance units).

The absorbance (A) of a sample is more difficult to measure directly than

%T, but is a much more useful (see the Beers Law section) parameter.

Absorbance values can be calculated from the %T values using

A = - log %T = 2 log (%T)100

Absorbance spectrum

The wavelengths of light absorbed by a molecule are determined by the

electronic energy levels present. If the incident light has an energy corresponding to the

difference between two of these energy levels, then the light may be absorbed by

the molecule. If it does not correspond to the difference between two energy levels, then

the light cannot be absorbed. We should therefore expect that a molecule would absorb

some wavelengths of light, but not absorb others.

An absorption spectrum, (a plot of A versus is used to show what wavelengths of light

are absorbed by a sample. The peaks in the spectrum correspond to the wavelengthsthat are strongly absorbed. These peaks are often referred to as

absorption maxima, and the wavelengths at which these peaks occur are called

maxs.


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We can use max values to predict what color the sample may look like. When white

light (containing all the visible wavelengths) is shone on a sample, those colors

corresponding to maxswill be absorbed while others pass

through. The complementary color to the max will usually be the colorthat dominates and will therefore determine what color the sample appears.

Looking at the absorption spectrum of the dye, we would expect that the wavelength

of 600nm and higher would be transmitted. With a max corresponding to green light

(525nm) we should expect the dye to appear reddish.

Wavelength (nm) Color of light Complementary color400-435 violet green-yellow

435-480 blue orange

480-500 green-blue red

500-560 green red-violet

560-580 yellow-green violet

580-595 yellow blue-violet

595-650 orange blue

650-750 red blue-green

The absorption spectrum and max values can be used to help identify a substance, but are


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usually used to determine the amount of known substance in a sample. This is done

using Beers Law.

Beer-Lambert Law

Under many circumstances the absorbance of a solution at a particular wavelength of

light is directly proportional to the concentration of the substance in solution. This is the

Beer-Lambert Law (often just called Beers law) and is written as

A = x l x cwhere A = Absorbance

= absorptivity (also represented as

l = path length (distance the light travels through the sample)

c = concentration

Typically, molarity is used for concentration, cm for path length, and liter mole-1

cm-1

for absorptivity. Absorbance has no units.

Calibration Curves

Beers law shows us that, at a given wavelength, the absorbance is proportional to the

concentration of the absorbing compound in solution. This fact can be used to create a

calibration curve. A calibration curve is created by plotting the absorbance of a series of

standards (solutions of known concentrations) at a particular wavelength (usually at max)

as a function of their concentration (use any units you choose as long as they are

consistent). The result will be a straight line. If the unknown samples absorbance is alsomeasured, its concentration can be determined from the calibration curve.

Beers Law/Calibration Curve Example

An analysis was to be performed on a red dye. As 525nm is the max for the dye,

this wavelength was chosen for the analysis.

A series of standard solutions was made by diluting a stock solution and then

measuring the absorbance of each solution. An absorbance of 0.39 was also

determined for a solution with an unknown concentration of dye.

solution Concentration (M) Absorbance

blank 0.00 0.00

Standard#1 0.15 0.24



Standard#4 0.60. 0.99

Unknown ? 0.39


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If the absorbance of the blank and standard solutions are plotted as a function of their

concentration, the equation of the best fit line through the points is y= 1.64x 0.0002.

Substituting the meaning of x and y in to equation tells us that:

Absorbance = 1.64 x concentration0.0002

1.2

A1

b0.8

s

o0.6

r

b0.4

a

n0.2

c

e0

-0.2

Series1

Linear (Series1)

y = 1.64x - 0.002

R = 0.9993

0 0.2 0.4 0.6 0.8

concentration (M)

To solve for the concentration of dye in the unknown, simply put in the absorbance value

0.39 = 1.64. x concentration -0.002

and solve for the concentration. In this case it is equal to 0.24M.

Prelab

1. Define absorption, %T, absorptivity, calibration curve and Beers law.

2. Determine how to make each of the standard solutions listed in part 2 of the procedure.

3. Predict the max value for a blue dye and that for a yellow dye.4. A series of stock solutions were prepared and the absorbances measured. The best fit

line to the calibration curve (absorbance vs concentration in mg/l) was y= 4.56x +0.0078.

What is the concentration of an unknown solution that has a %T of 45.7?

5. Fill in the blanks to explain the requirements regarding the preparation of solutions for

this experiment.

To make a calibration curve, the concentration of the standards should

concentration anticipated for the unknown.

the


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To make accurate spectroscopic measurements the absorbance of the solution measured

should be between and absorbance units.

Requirements: Standard flask, micropipette, standard stock solution (Potassiumpermanganate),, beaker, Distilled water, Funnel, UV-visible cuvettes, beaker 250 mL (2)

Procedure

Taking Absorbance Measurements

The instrument used to take an absorbance measurement is a scanning UV-Vis

spectrometer. Please read the tutorial on the webpage regarding the use of the instrument;

the use will be explained again in the lab by the faculty taking the lab section.

Procedure Work in groups of 4

Part 1: Obtaining the Absorption Spectrum of KMnO4

1 Take the blank reading (with solvent, which is distilled water in your case).

Use the UV-Vis spectrometer the measure the absorption spectrum of the KMnO4

solution.

2. Print the spectrum and determine the value of max.

3. Measure the absorption spectrum for all subsequent solutions at this max.

http://www.youtube.com/watch?v=rdY41FPI9iE:Learning beers law

http://www.youtube.com/watch?v=WFLcxxSuXiAhow to use UV vis spectrometer
http://www.youtube.com/watch?v=rdY41FPI9iEhttp://www.youtube.com/watch?v=rdY41FPI9iEhttp://www.youtube.com/watch?v=WFLcxxSuXiAhttp://www.youtube.com/watch?v=WFLcxxSuXiAhttp://www.youtube.com/watch?v=WFLcxxSuXiAhttp://www.youtube.com/watch?v=rdY41FPI9iE


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Procedure Work in groups of 4

Part 1: Obtaining the Absorption Spectrum of KMnO4

1. Use the UV-Vis spectrometer the measure the absorption spectrum of the KMnO4

solution.

2. Determine the value of max.

3. Measure the absorption spectrum for all subsequent solutions at this max.

Part 2: Preparing Standard Solutions

1. Obtain approximately 25ml of the stock solution (0.1 M) to be shared by the group.

2. Prepare a series of standard solutions by diluting the stock solution. The standards

should have the following concentrations. Make 25 ml of each standard of the following

concentrations.

Concentration (M)

Stock solution 0.1

Standard solution 1 0.001Standard solution 2 0.00075

Standard solution 3 0.0005

Standard solution 4 0.00025

Part 3: Measuring the Absorbance of the Unknown solution

1. Set up the spectrometer

2. Calibrate it at 0%T transmittance and 100% transmittance3. Measure the absorbance of all the standards and the unknown.


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Part 4: Determining the Concentration of the Unknown solution

1. Plot a calibration curve using the solutions of known concentration. Determine the best

fit straight line using your points. The unknown is not plotted as a point on this graph.

2. Use your calibration curve to calculate the concentration of KMnO4 in the unknown

solution.

Postlab

1. Report the concentration KMnO4 in the solution.2. What would happen to the if the concentration of dye were to increase in the

stock solution?

a) max

b) the intensity of the color in the stock solution

c) the absorbance of the stock solution

d) the molar absorptivity at max

3. Look at your best fit line. Relate the equation of the best fit line to Beers law in 2 or 3sentences.

4. If the cell path length is 1 cm, what is the molar absorptivity for KMnO4 at max?

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Lambert-Beer's Law UV-Visible Spectroscopy