Experiment 11: Spectrophotometric Analysis

3rd Shifting | MT635 Laboratory | Alifa Camille N. Santos, M.Sc.

OBJECTIVES

• Learn the basic principles and operation of

UV-VIS spectrophotometer

• Apply the calibration curve method for the

analysis of copper sulfate solutions

• Determine the molar absorptivity using

Beer’s Law
PROPERTIES OF LIGHT

• Light is a form of electromagnetic radiation.

• Electromagnetic radiation is a type of energy that is

transmitted through space as a transverse wave at
enormous velocity.

• Wave Property:

1. Wavelength
C= n l
- the distance between successive maxima or minima of Where,
a wave (nm)
C= velocity of light
2. Frequency V= frequency
l = wavelength
- the number of oscillation of the field per second.
PROPERTIES OF LIGHT

• Light is a form of electromagnetic radiation.

• Electromagnetic radiation is a type of energy that is

transmitted through space as a transverse wave at
enormous velocity.

• Particle Property:

- Electromagnetic radiation of light can be viewed

E = hv
as a stream of discrete wave packets of distinct
particles called photons. Where,

- Energy is inversely proportional to wavelength E= energy of a photon

h = Planck’s constant (6.626 x 10-34 J●s)
E = hc / λ v = frequency
 ELECTROMAGNETIC SPECTRUM

Frequency increases

Energy increases
ABSORPTION OF VISIBLE RADIATION

• Visible light consists of wavelengths ranging from 380 nm

to 720 nm.

• The observed color is the result of chemical species

(atom, ion or molecule) selectively absorbing a certain
color of light that leads to the perception of the
complimentary color.

Example:

If a white light passes through a test tube containing a

solution of CuSO4, the solution will be blue because the
Cu2+ ions strongly absorb “orange” photons of light (λ=
600 nm)
ABSORPTION OF VISIBLE RADIATION

• When a photon of colored light is absorbed by a compound, an electron

transitions from lower energy (ground state) orbital to higher energy orbital
(excited state)

• The wavelength of light required to

promote an electron from the
ground to excited state is specific to ∆Elight = EUO - EOO
each chemical, just as the ∆E is
dependent on chemical identity.
SPECTROPHOTOMETRY

• Light can either be transmitted or absorbed by dissolved substances.

In terms of intensity,

I3 < I2 < I1

Io I1 In terms of absorbance,
Io I2 Io I3
Sol’n 1 < Sol’n 2 < Sol’n 3
Solution 1 (low conc.) Solution 2 (high conc.) Solution 3 (same conc. w/ S2 but
inc. pathlength)

𝐼𝑥 𝐼𝑥
Transmittance (T) = Absorbance (A) = -log = -logT
𝐼0 𝐼0

where, Io= intensity of incident light and Ix= intensity of transmitted light
BEER-LAMBERT’S LAW

A = εcl
Where,

A = absorbance l = pathlength (cm)

c= concentration of the analyte ε = molar absorptivity or molar extinction Exponential curve relationship
coefficient (L.mol-1.cm-1)

• Molar absorptivity is the measure of how strongly the solution

absorbs light in a wavelength. It is unique for a given compound

Linear curve relationship

BEER-LAMBERT’S LAW

• For a standard cuvette, pathlength is usually at 1cm whereas, the pathlength of multi-
well plates will differ depending on the brand used and the total volume in each well.
UV-VIS SPECTROPHOTOMETER

I. Spectrometer - produces a desired range of wavelength of light.

1. collimator (lens): transmits a straight beam of light (photons)

2. monochromator (prism): splits the beam into several component wavelengths (spectrum).

3. wavelength selector (slit): transmits only the desired wavelengths

UV-VIS SPECTROPHOTOMETER

Note: For the light source, a tungsten lamp is used for the visible regions and a
deuterium lamp for the ultraviolet region

II. Photometer - detects the amount of photons that is absorbed and then sends a
signal to a galvanometer or a digital display
UV-VIS SPECTROPHOTOMETER

• a spectrometer capable of producing a variety

of wavelengths is recommended because
different compounds absorb best (λmax) at
different wavelengths. For example, p-
nitrophenol (acid form) has the maximum
absorbance at approximately 320 nm and p-
nitrophenolate (basic form) absorb best at
400nm.

• Measurement of concentration based on

absorbance requires a wavelength that is
absorbed well enough (λmax) such that
Absorption Spectra of p-nitrophenol and p- changes in intensity can be easily detected.
nitrophenolate solutions
CALIBRATION CURVE METHOD

• For two solutions of the same compound that are analyzed under identical conditions,
the concentration of the unknown can be determined as:
𝐴𝑢𝐶𝑠
Cu =
𝐴𝑠

Where, Cu= conc. Of unknown, Cs= conc. of Std., Au = Abs. of unknown, and As= Abs. of Std.

• If more than one samples are being run, constructing a standard calibration curve is more
efficient wherein the concentration (x) of the unknown is calculated based on the
produced linear regression equation (y= mx + b) where, y= absorbance of the unknown.
CALIBRATION CURVE METHOD

The absorbance of unknown solution at

470 nm is 0.0798. Concentration (x) is
calculated as:

0.0798 = 27.2 (x) + 0.0052

0.0798 – 0.0052 = 27.2 (x)

x = 0.0746 / 27.2

x = 0.0027 M Rutin
Rutin standard calibration curve
MATERIALS AND REAGENTS

• Copper sulfate pentahydrate

• 96-well microplate

• Test tubes and rack

• Serological pipettes

• Rubber aspirator

• Beaker

• 100 mL Volumetric flask

• 20-200 uL capacity micropipettor and tips

PROCEDURE

I. Preparation of Stock Solution (GROUP 8)

Transfer the sol’n to a Transfer the

Weigh 100mL volumetric flask. prepared sol’n
24.97 in a glass
grams Rinse the beaker thrice reagent bottle
CuSO4● with ~10mL DH2O and label as:
5H2O in a (Transfer rinsing to the
beaker. VF)
Dissolve
in ~20mL Using your previous
DH2O. beaker, fill the VF with
DH2O an inch below
the mark. Use your
wash bottle to carefully
fill up to the mark.

MM of CuSO4 ● 5H2O = 249.685 g/mol

PROCEDURE
* Use C1V1=C2V2 to calculate the molar
II. Preparation of Standard Solutions concentration of each solution

S1 S2 S3 S4 S5

1 mL Stock 1 mL S1 + 1 mL S2 + 1 mL S3 + 1 mL S4 +
+ 1 mL DH2O 1 mL DH2O 1 mL DH2O 1 mL DH2O
1 mL DH2O
PROCEDURE
III. Plate Set-up for Spectral Scan, Curve, and Unknown

Blk Blk Blk Blk Blk Blk Blk Blk

S1 S1 S1 S1 S1 S1 S1 S1
Unk Unk Unk Unk Unk Unk Unk Unk Column numbers
corresponds to the
S1 S1 S1 S1 S1 S1 S1 S1 group number

S2 S2 S2 S2 S2 S2 S2 S2
Blk = 200 uL of distilled
S3 S3 S3 S3 S3 S3 S3 water
S3
S4 S4 S4 S4 S4 S4 S4 S4
S5 S5 S5 S5 S5 S5 S5 S5
PROCEDURE
* Correct the absorbance
IV. Reading of 96-well Microplate readings by subtracting
the absorbance of the Blk
1. Place 200 uL of the solutions in their respective wells well from the analyte well.

2. Conduct spectral scan for Row B (500 to 700 nm at 5nm interval)

3. Read Row A and Row C to H at 635 nm.

4. Determine the λmax based on absorption spectra (step 2)

5. Construct a standard calibration curve for S1 to S5 based on absorbance readings in

step 3.

6. Calculate for the molar concentration of the unknown solution using the produced linear
regression equation from step 5 and the absorbance of the unknown from step 3.
POINT SYSTEM FOR UNKNOWN SOLUTION

± 0.1 from Actual Value = 10 points

± 0.2 from Actual Value = 7 points
more than ± 0.2 from Actual Value = 5 points
Absent = 0 points
SEATWORK:

1. Calculate for the molar absorptivity of a 0.002 M sample with an absorbance of 0.058
at 470 nm (Assume that the pathlength is 1 cm).

2. Light is passed through a solution with a pathlength of 1 cm where the absorbance

was recorded to be 0.15. What would be the absorbance if the light is passed through
the same solution at the same wavelength but the pathlength was increased to 2 cm?

3. A compound has a λmax of 275 nm and ε= 8400 L.mol-1 cm-1. Spectro reading resulted
to an absorbance of 0.70. What is the concentration of the compound if l = 1 cm?

4. The absorption coefficient of a complex is 0.20 at a wavelength of 450 nm. What is the
concentration of the complex when the transmittance is 40% in a cuvette of 2 cm?
END