Name: DIONGCO, MARC ANGELO V.

Date Performed: 16 August 2016

MANALO, JEAN LOUISE B. Date Submitted:

Experiment 10
SPECTROPHOTOMETRIC METHODS

I. INTRODUCTION
Colorimetric and spectrophotometric methods are based on the absorption of visible light or
other radiant energy by a solution. The amount of radiant energy absorbed is proportional to the
absorbing material in solution (Skoog et al, 2014). A procedure based on absorption of visible light is
called colorimetry. It is the quantitative determination of a colored substance from its ability to absorb
light. This method relies on the comparison of a colored solution of unknown concentration with one or
more colored solutions of known concentration (Harris, 2007). On the other hand, in photometric
methods the ratio of the radiant energy of incident (P0) to transmitted (P) beam of light (or other radiant
energy) is measured by means of a detector such as photocell. When the ratio is measured at a given
spectral wavelength, the term given is spectrophotometry (Skoog et al, 2014).
The heart of spectrophotometry as applied to analytical chemistry is called the Beer-Lambert law, or
simply Beer’s law. Beer’s Law is the fundamental law on which colorimetric and spectrophotometric
methods are based states that the amount of radiation absorbed or transmitted by a solution is an
experimental function of the concentration of absorbing substances present and the sample path length
(Harris, 2007).

Figure 10.1. Diagram of radiant energy flow

where P0 = incident radiant energy
P = transmitted radiant energy
c = concentration of sample
b = path length
Beer’s Law can be best expressed using two ways: in terms of transmittance, T, or absorbance, A. In
terms of transmittance, the formula is given below:

– log T = abc Equation 10.1

For absorbance,
A = abc Equation 10.2
where a = extinction coefficient or absorptivity (Skoog et al, 2014)
Since absorbance is a unitless quantity, the absorptivity must have units that would cancel the units of b
and c. When c is in moles per liter and b in cm, the proportionality constant is called molar absorptivity
and is given the symbol ε (Skoog et al, 2014).
Absorptivity is a constant and a characteristic of a given absorbing species evaluated at a particular
wavelength. Beer’s law applies strictly for monochromatic radiant energy.

Figure 10.2. Sample Beer’s Law plot (at λmax = 430 nm)
In spectrophotometry, unknown solutions are usually determined by comparison with a set of standards
plotted against absorbance, shown in Figure 10.2. The concentration of the unknown solution is
interpolated from the standard curve.
Aunknown = Astandard
a₁b₁c₁ = a₂b₂c₂ Equation 10.3
Since the path length can be made constant by using the same cuvette or matched cuvettes, and a,
absorptivity is also constant for that particular wavelength then c1 = c2.

Figure 10.3. Schematic diagram of a double-beam scanning spectrophotometer

Light can be considered as a stream of particles called photons. The amount of energy associated with
each photon is called a quantum, given by the following equation:
E = ћν Equation 10.4
where E = energy
ћ = Planck’s constant (6.626 x 10⁻³´ J•s)
ν = frequency of radiation
Since the velocity of light, c (3 x 1010 cm/sec), is given by the equation:
c = λν Equation 10.5
where λ = wavelength
Then,
E = ћc/λ Equation 10.5
Thus, the shorter the wavelength of the light, the greater the energy associated with each photon
(Skoog et al, 2014).
The ultraviolet (180–380 nm) and visible regions (380–780 nm) of the electromagnetic spectrum can
give rise to electronic transitions in ions or molecules (Skoog et al, 2014). Any molecule is capable of
absorbing only certain discrete amounts of energy, which varies with frequency. Thus, a solution will
only absorb light energy at wavelengths corresponding to those energy levels that cause electronic
transitions within the molecules or ions contained in the solution (Harris, 2007). Therefore, the
absorption of light by ions and molecules serve as the basis for numerous analytical methods.
Qualitative analysis identifies the components of a sample while quantitative analysis determines the
concentration of a specific component (Skoog et al, 2014).
Qualitative spectrophotometric analysis involves the determination of the wavelength where there is
maximum absorption of light. The wavelength is specific for a particular analyte (Harris, 2007).
Coordination complex ions involve a transition metal surrounded by anions and/or neutral molecules
called ligands (Housecroft and Sharpe, 2005). In this exercise, the coordination compound
tetraamminecopper (II) sulfate monohydrate *Cu(NH₃)₄+SO₄•H₂O will be synthesized by adding
concentrated ammonia, NH₃, to copper (II) sulfate pentahydrate solution, which will displace the water
molecules bound in the original copper complex, and a dramatic color change occurs. The color of a
complex is due to an electron in the metal ion absorbs the energy of visible light causing it to "jump"
from one energy level to a higher one. This subtracts that color from the visible spectrum as the light is
absorbed (Miessler and Tarr, 2004).
The reaction of ammonia with the copper(II) sulfate pentahydrate is a two-step process. In the first step,
solid copper (II) hydroxide is formed which is light blue in color. The Cu(OH)₂(s) dissolves as additional
quantities of ammonia are added in the second step to form the dark blue *Cu(NH₃)₄+²⁺ complex
(Miessler and Tarr, 2004). The equations for the process are below:
*Cu(NH₃)₄+SO₄•H₂O (aq) + 2 NH₃ → *Cu (H₂O)₂(OH)₂+SO₂ •H₂O(s) + 2 NH₄⁺
light blue
*Cu (H₂O)₂(OH)₂+SO₂ •H₂O(s) + 2 NH₄⁺(aq) + 2 NH₃(aq) → *Cu(NH₃)₄+ SO₄•H₂O (aq) + 4 H₂O(l)
light blue royal blue
In this experiment, the wavelength of maximum absorption will be determine by plotting the absorption
curves of some substances. Beer’s Law plot for a given analyte will be prepared and from the plot, the
concentration of analyte in an unknown sample will be determined.

Prelab question

Using 1.00 mL of 0.0400 M CuSO4 stock solution:
(0.0400 M)(0.0010 L) = x (0.050 L)
x = 0.0008 M
Using 7.50 mL of 0.0400 M CuSO4 stock solution:
(0.0400 M)(0.0050 L) = x (0.050 L)
x = 0.0040 M
Using 2.50 mL of 0.0400 M CuSO4 stock solution:
(0.0400 M)(0.0025 L) = x (0.050 L)
x = 0.0020 M
Using 10.00 mL of 0.0400 M CuSO4 stock solution:
(0.0400 M)(0.075 L) = x (0.050 L)
x = 0.0060 M
 Using 7.50 mL of 0.0400 M CuSO4 stock solution:
(0.0400 M)(0.0100 L) = x (0.050 L)
x = 0.0080 M

II. SCHEMATIC DIAGRAM OF PROCEDURE

A. PREPARATION OF CuSO4 SOLUTION

FOR BLANK SAMPLE:

B. PLOTTING THE ABSORPTION CURVE

 C. PLOTTING BEER’S LAW PLOT

III. DATA AND RESULTS

Unknown No.: __________________

Table 10.1. Preparation of CuSO4 solutions

For CuSO4 solutions
Volume of original stock, mL 1.00 2.50 5.00 7.50 10.00
Volume of solution prepared, mL 50.00 50.00 50.00 50.00 50.00
[CuSO4], M 0.0008 0.0020 0.0040 0.0060 0.0080

Table 10.2. Determination of λmax

For 0.0080 M CuSO4 solution
λ, nm Abs λ, nm Abs
700 540
680 520
660 500
640 480
620 460
600 440
580 420
 560 400
λmax

Table 10.3. Determination of concentration of unknown at λmax using Beer’s Law Plot
[CuSO4], M Abs
0.0008
0.0020
0.0040
0.0060
0.0080
Using linear interpolation
Abs of unknown
[CuSO4]unknown, M
Using linear regression
slope, m
y-intercept, b
r
Equation of the line
Abs of unknown
[CuSO4]unknown, M

% difference

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