Lab 5

Lab 5.
Quantitative analyses
Purpose: To understand (1) the basic spectroscopy; (2) the beer’s law; (3) quantitative analyses
using UV-vis absorbance
Introduction
Electromagnetic radiation is a type of energy that takes numerous forms, the most easily
recognizable being visible light and radiant heat. Electromagnetic radiation is made up of packets
of energy called photons. The energy of a photon depends on the frequency of the radiation and is
given by E = h. Where h is the plank’s constant (6.63  1034J.s) and  is the frequency. The
energy can also be defined as E = hc/. Where  the wavelength and c is the speed of light 3.00 
108 m/s.
Electromagnetic radiation is classified in terms of the frequency of wave. The electromagnetic

spectrum is the range of all possible frequencies of electromagnetic radiation. The visible portion
of the spectrum to which the human eye is sensitive is tiny when compared with other spectral
regions.
Absorption of Radiation
Absorption is a process in which a chemical species in a transparent medium selectively absorbs
certain frequencies of electromagnetic radiation. Every particle (atom, ion or molecule) has a
unique set of energy state, the lowest of which is the ground state. When a photon of radiation
passes near an elementary particle, absorption becomes probable if the energy of the photon
matches exactly the energy difference between the ground state and one of the higher energy states
of the particle. The energy of the photon is transferred to the atom, or ion, or molecule, converting
it to the higher energy level, which is termed as an excited state. Excitation of a species M to its
excited state M* can be depicted by the equation M + h = M*
After a brief period, the excited species relaxes to its original, or ground state, transferring its
excess energy to other atoms or molecules in the medium. This process is called relaxation.
M*  M + heat
Two terms are commonly used as quantitative measures of beam attenuation: transmittance and
absorbance.
When sample molecules are exposed to light having an energy that matches a possible electronic
transition within the molecule, some of the light energy will be absorbed as the electron is
promoted to a higher energy orbital. The schematic above depicts a beam of a parallel radiation
before and after it has passed through a medium of thickness b-cm and a concentration c of an
absorbing species. As a consequence of interactions between the photons and absorbing particles,
the power of the beam is attenuated from P0 to P. The transmittance T of the medium is the fraction
of the incident radiation that is transmitted by a sample, T = P/P0.
Absorption spectra
An optical spectrometer records the wavelengths at which absorption occurs, together with the
degree of absorption at each wavelength. The resulting spectrum is presented as a graph of
absorbance (A) versus wavelength, as in the isoprene spectrum shown below.
For monochromatic radiation, absorbance is directly proportional to the path length l through the
medium and the concentration c of the absorbing species. Therefore, it is necessary to correct the
absorbance value for this and other operational factors if the spectra of different compounds are to
be compared in a meaningful way. This relationship is defined by the
Beer’s law: A = bc. where  is the molar absorptivity and has the units of L.mol-1.cm-1.
b is the path length of the sample - that is, the path length of the cuvette in which the sample is
contained. We will express this measurement in centimetres. c = sample concentration in
moles/liter.
The corrected absorption value is called "molar absorptivity", and is particularly useful when
comparing the spectra of different compounds and determining the relative strength of light
absorbing functions (chromophores). Molar absorptivity () is defined as:
() = A/cb. In some old literatures, it is sometimes called the extinction coefficient.
Instrument components
Most spectroscopic instruments for use in the UV/visible are made up of five components: (1) a
stable source of radiant energy; (2) a wavelength selector that isolates a limited region of the
spectrum for measurement; (3) sample container; (4) a radiation detector, which converts radiant
energy to a measurable electrical signal; and (5) a signal processing and readout unit, usually
consisting of electronic hardware and a computer.
Absorption intensity
• The same substance exhibits different absorption intensities at different wavelengths. The
wavelength corresponding to the highest absorption is called maximum absorption max.
• Changing the concentration of the substance does not affect the shape and max of the
spectra.
• When we change the concentration, the intensity at max change more
Experiment
Objective: Determine the molar absorptivity for the analyte.
Approach:
 Measure the intensity of transmitted light for solutions with various concentrations
of analyte.
 For each solution, calculate the absorbance of the solution.
 Construct a plot of A vs c.
 Plot the line-of-best-fit through the experimental points. The slope of this line is the
molar absorptivity. The line should pass through the origin; that is, the intercept of
this line should be zero.
Procedure
Preparing the stock solution and four standard solutions
The stock solution is prepared by the technician in advance. (100 mL of a stock standard solution
of 0.008M KMnO4: Accurately weigh 126 mg solid KMnO4. Transfer quantitatively to a 100 mL
volumetric flask and fill to the mark with water. This is the stock solution. The technician will
prepare this stock solution for you before experiment. The students can use this stock solution to
prepare five standards:
Prepare five standards in 10.0 mL volumetric flask with concentrations of 0.00032 M (solution
#1), 0.00024 M (solution #2), 0.00016 M (solution #3), 0.00008 M (solution #4), and 0.00004M
(solution #5) by diluting the stock solution using 100μL or 1000μL (1mL) micropipette. You
need to calculate how many μL stock solutions are needed to prepare 10 mL standard solution
#1#5.
Measuring the absorption spectrum and determining max.

Rinse one of the cuvettes with distilled water and fill it with water. Put the cuvette in the sample
compartment. This is the reference solution. Rinse a second cuvette once with distilled water and
once with standard solution #1, then fill it with standard solution #1 (0.00032 M KMnO4). Place
the cell in the sample compartment, measure the Absorbance at different wavelengths such as 460,
480, and 525 nm, etc and record the file name in your notebook (such as Measure 460-1, 460-2,
etc).
Draw a plot having X-axis as concentration (mole/L) and Y-axis as absorbance at 525 nm (the
peak wavelength of KMnO4).
Using beer’s law to calculate  for KMnO4, given the cell width (path length l) to be 1 cm.
CALCULATIONS
1. To prepare 10 mL of the following standard solutions, how much stock solution is needed?
For standard solution 1, ______μL
2. The absorption spectrum of KMnO4: (using solution 1)

Make a graph of A vs  at 20 nm wavelength intervals from 460 nm to 600 nm (as measured whole
laboratory group). Attach the graph with this lab report.
(nm) Absorbance
460
480
500
520
540
560
580
600
The calibration curve at max = 525 nm

Make a graph of A (Y-Axis) vs M (x-axis). Attach the graph to this report.
Standard Concentration Absorbance
Calculation of the molar absorptivity () at 525 nm.

Use the A-value of solution 1:
 = A/(C.l), with l = 1 cm
=
Repeat the calculation for other standards
Average  =

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Lab 5.

Electromagnetic radiation is classified in terms of the frequency of wave. The electromagnetic

• When we change the concentration, the intensity at max change more

Objective: Determine the molar absorptivity for the analyte.

Measuring the absorption spectrum and determining max.

2. The absorption spectrum of KMnO4: (using solution 1)

The calibration curve at max = 525 nm

Standard Concentration Absorbance

Calculation of the molar absorptivity () at 525 nm.

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