Experiment 2

Introduction

Spectrophotometry is a technique that uses the absorbance of light by an analyte (the substance to be analyzed) at a certain wavelength to determine the analyte concentration. UV/VIS (ultra violet/visible) spectrophotometry uses light in UV and visible part of the electromagnetic spectrum. Light of this wavelength is able to effect the excitation of electrons in the atomic or molecular ground state to higher energy levels, giving rise to an absorbance at wavelengths specific to each molecule. 

Spectrophotometer

“The instrument used to measure the amount of electromagnetic radiations absorbed by a compound is called spectrophotometer." For visible spectrophotometer, the light source is a common tungsten light bulb emitting “white” light. The light is collimated and focused on an entrance slit, and then falls on a monochromator, that separates the white light in its constituent wavelengths. The monochromator can be a glass prism, but in modern instruments it will be a grating. From the monochromator the light is sent through the sample, and finally reaches a photocell that measures the intensity of the light at each specific wavelength and recorded (Fig.1).                              

                     Fig. 1: Working of Spectrophotometer                               

Principle

Spectrophotometers have many different designs. In the simplest instrument, for each wavelength λ set by the monochromator, the user first inserts a blank solution in place of the sample. The photocell then records the intensity at that wavelength. This intensity is called I0(λ). Next the blank solution is replaced by the sample solution, the photometer measures the new intensity called I(λ). The Transmittance, T is then displayed on the screen or spectrophotometer output:                                   

T(λ) =   I(λ) / I0(λ)    

Often the Transmittance is expressed as a percentage: 

%T(λ) =   I(λ) / I0(λ) x 100%

This procedure is then repeated for a number of wavelengths.  

More sophisticated instruments “scan” the spectrum over the required wavelength range automatically, and record the transmittance as a function of wavelength. In a “double beam” scanning spectrophotometer, part of the light is reflected to a separate blank cell, and the intensities I and I0 are measured simultaneously at each wavelength, and automatically compared to yield a direct output of T vs λ. Another modern form of the UV/VIS spectrophotometer is the “Diode Array” spectrophotometer. In this instrument the photocell, which measures light intensity at one wavelength at a time, is replaced by a CCD detector similar to the detector in your digital camera, and the instrument can record the spectrum over the full wavelength range (typically 200−700 nm) within one second.

Transmittance T decreases exponentially with increasing pathlength l. This is expressed in the Beer Lambert law, which states:                

  -log (I/I0) = εcl  

where, ε is called the “Molar Absorptivity” of the compound. It is a function of wavelength specific for each molecule. With the pathlength l normally given in cm, and c in Molarity units, mol/L,  ε has the units L.mol−1cm−1 

The Absorbance, A, is defined as 

    A =  -log (I/I0)

Finally, the relation between A and T is given by:

                                                                                                A = -logT

Thus, the Beer-Lambert law, more commonly called Beer’s law, can be written as: 

                                                A = εcl                                              

When c and l are held constant, then ε is constant and hence A varies with λ. The plot of A versus λ at constant c and l gives an absorption spectrum, from which λmax  can be determined. According to the Beer–Lambert Law, absorbance is proportional to concentration, so that at dilute solutions a plot of concentration vs. absorbance would be straight line, but the Law breaks down for solutions of higher concentration, and so you might get a curve under those circumstances. 

Note that A is a dimensionless quantity. Because A is directly proportional to the analyte concentration, it is more often used than T or %T. Most spectrometers can record either A, T, or %T for a given wavelength. 

Procedure

1. Prepare 100 mL of 0.02M KMnO4 solution using distilled water. 

2. Pipette out 10 mL of 0.002M KMnO4 solution and dilute it up to 100mL. 

3. Take 2, 4, 6, 8 and 10 mL of 0.002 M KMnO4 solution and dilute it up to 100 mL in volumetric flask. 

4. Prepare an unknown solution of KMnO4 lying between 1 – 10 mL of 0.002 M KMnO4 solution and dilute it up to 100mL in volumetric flask. 

5. Use 1.2 × 10-4 M solution (6 mL diluted solution) for determination of λmax. 

6. Record the absorbance of solution at 450 nm. Change the wavelength by 10 nm and record the absorbance again; continue the process up to 550 nm wavelength. 

7. Plot a graph between absorbance versus wavelength (Table-1). A wavelength corresponding to highest absorbance represents the λmax of KMnO4 solution. 

1. For second part of the experiment, set the wavelength of instrument on λmax value of wavelength (determined in the first part). Set 100 % transmittance with water at λmax wavelength. 

2. Now, fill one of the cuvette with different diluted solutions (0.4 × 10-4, 0.8 ×10-4, 1.2 × 10-4, 1.6 × 10-4, and 2.0 × 10-4) one by one and measure the absorbance. 

3. Plot a curve between absorbance versus concentration of KMnO4 solution. Find the concentration of unknown KMnO4 solution from the straight line obtained.    

Observation:

Result:

The λmaxof KMnO4 solution is ___________________ nm and concentration of unknown KMnO4 solution is ___________________ M

Reference Materials::

• The Spectrophotometer: A demo and practice experiment

• UV-Visible Spectroscopy

Questions

1. How are concentration and absorbance related?

2. Why are some solutions coloured while others are colourless?

3. How can you determine the concentration of an unknown sample?