ACTIVITY NO. 1: INTRODUCTION TO UV-VIS ABSORPTION o Familiarize with the spectrophotometer.

SPECTROSCOPY
o Turn on the instrument.
1. INTRODUCTION
o Select wavelength scan.
 UV-Vis spectroscopy measures the absorption or
o Perform blanking with deionized water.
transmission of UV or visible light by a sample.
 The amount of light absorbed or transmitted o Obtain the assigned solution.
depends on the sample's composition. o Transfer the solution to a cuvette.
 Light's energy is inversely proportional to its o Place the cuvette in the sample holder and
wavelength (shorter wavelengths = more energy). start.
 Specific energy is required to excite electrons to o Locate the maximum absorption
higher energy states, which is detected as wavelength.
absorption.
o Record and compare the maximum
 Different substances absorb light at different absorption wavelength with the theoretical
wavelengths. value.
 Visible light ranges from approximately 380 nm o Dispose of the solution and clean the
(violet) to 780 nm (red). cuvette.
 UV light has wavelengths shorter than visible light o Turn off the instrument.
(approximately 100 nm to 400 nm).
6. DATA
 UV-Vis spectroscopy can be used to analyze and
identify substances by finding the wavelengths of  Table with: Color, Theoretical λmax (nm), and Actual
maximum absorbance. λmax (nm) for various solutions.

 UV-visible spectrometers measure absorbance of UV

or visible light.
 The spectrum produced is a plot of absorbance vs.
wavelength.
 Measurements can be taken at a single wavelength
or over a range.
 UV region: 190-400 nm; Visible region: 400-800 nm.
 The technique can be used for both quantitative and
qualitative analysis.
 White light is composed of different wavelengths
that can be dispersed into colors.
 The color of an object depends on the light it
transmits or reflects.
2. OBJECTIVES ACTIVITY NO. 2: SOLVENT EFFECTS ON THE UV-VISIBLE
ABSORPTION SPECTRA
 Familiarize with the parts of a UV-Vis
Spectrophotometer. ACTIVITY NO. 2

 Operate the UV-Vis Spectrophotometer. SOLVENT EFFECTS ON THE UV-VISIBLE ABSORPTION

SPECTRA
 Determine the maximum absorption wavelength of a
given solution. 1. INTRODUCTION

3. REAGENTS Solvent effects on UV-Vis spectra arise due to interactions

between the solvent and the solute (analyte), affecting
 Methyl red solution electronic transitions, absorption maxima (λmax), and
 Dichromate solution spectral bandwidth. These effects are influenced by factors
such as solvent polarity, hydrogen bonding, and solute-
 Methylene blue solution solvent interactions.
 Methyl orange solution 1. Effect of Solvent Polarity
4. MATERIALS NEEDED Solvent polarity can either increase or decrease the λmax of a
 Dropper compound, depending on the type of electronic transition:

 Wash bottle  n → π* transitions (nonbonding to antibonding):

 UV-Vis Spectrophotometer o Observed in compounds with lone pairs

(e.g., carbonyls, amines).
o Polar solvents stabilize the ground state (n
5. PROCEDURE
orbital) more than the excited state (π
 orbital), leading to a hypsochromic (blue)  UV-Vis Spectrophotometer
shift.*
 Quartz/Glass cuvettes
o Example: Acetone in water λmax shifts to a
 Wash Bottle
lower wavelength compared to in hexane.
 Volumetric flasks (10 mL)
 π → π* transitions (bonding to antibonding):
5. PROCEDURE
o Common in conjugated systems (e.g.,
benzene, aromatic compounds).  a. Preparation of Sample Solutions

o Polar solvents stabilize the excited state (π o Prepare diluted solutions: Transfer 1 mL of
orbital) more than the ground state (π the stock solution into three separate 10 mL
orbital), causing a bathochromic (red) shift. volumetric flasks, then dilute each with a
different solvent (hexane, acetone,
o Example: Benzophenone in ethanol λmax ethanol).
shifts to a longer wavelength compared to
in hexane.  b. UV-Vis Spectral Measurement

2. Hydrogen Bonding Effects o i. Turn on the instrument by following

standard protocols in turning on the
 Protic solvents (e.g., water, alcohols) can form solution.
hydrogen bonds with the solute, altering electronic
transitions. o ii. Select wavelength scan.

 Hydrogen bonding typically lowers n → π* o iii. Perform blanking on the instrument

transitions (blue shift) due to stabilization of the using the corresponding solvent as the
ground state. blank.

 Example: The UV-Vis spectrum of acetone shows a o iv. Transfer the sample solution to cuvette
significant shift in aqueous solutions compared to using a dropper to avoid spillage.
nonpolar solvents.
o v. Measure the absorbance spectrum of the
3. Dielectric Constant and Refractive Index Effects sample in each solvent.
 Solvents with high dielectric constants (polar o vi. Locate and record the maximum
solvents) enhance solute-solvent interactions, absorption wavelength (λmax) of the
modifying absorption intensity and peak positions. sample solution.
 Solvent refractive index affects peak broadening or o vii. Dispose the solution on the designated
sharpness; polar solvents often cause peak waste bottle and clean the cuvette before
broadening due to increased molecular interactions. keeping it.
4. Specific Solvent-Solute Interactions o viii. Turn off the instrument.
 Charge-transfer complexes, dipole-dipole
interactions, and solvatochromism (solvent-
ACTIVITY NO. 3: CONCENTRATION EFFECTS ON THE
dependent color changes) also contribute to spectral
ABSORBANCE READING OF A SAMPLE
shifts.
1. INTRODUCTION
2. OBJECTIVES
 UV-Vis spectroscopy is used to determine the
 Observe and analyze the effect of solvent polarity on
concentration of colored solutions.
the UV-Vis absorption spectrum of a compound.
 According to Beer-Lambert Law, absorbance is
 Compare hypsochromic (blue) and bathochromic
directly proportional to concentration (when path
(red) shifts in different solvents.
length and molar absorptivity are constant).
 Understand the interaction between solutes and
 Beer-Lambert Law: A = εlC
solvents affecting electronic transitions.
(A = Absorbance, ε = molar absorptivity, l = path length, C
= concentration).
3. REAGENTS
 This experiment investigates the relationship
 Methyl red solution between concentration and absorbance.
 Methylene blue solution 2. OBJECTIVES
 Methyl orange solution  To understand the relationship between
 Salicylic acid solution concentration and absorbance using UV-Vis
spectroscopy based on Beer-Lambert Law.
 Acetone
3. REAGENTS
 Hexane
 Standard solution of a colored compound (e.g.,
 Ethanol potassium permanganate, KMnO4).
4. MATERIALS NEEDED  Distilled water.
4. MATERIALS NEEDED  Common analysis methods include calibration
curves, standard addition, and internal standard
 UV-Vis Spectrophotometer.
method.
 Quartz/Glass cuvettes.
 This experiment uses spectrophotometry to prepare
 Wash Bottle. a calibration curve for KMnO4 quantitative analysis.
 Pipette. 2. OBJECTIVES
 Volumetric flasks (10 mL).  Prepare standard solutions of potassium
 Beakers (20 or 50 mL). permanganate.

5. PROCEDURE  Construct a calibration curve based on Beer's Law.

 Prepare Standard Solutions:  Use Beer's Law to determine molar absorptivity.

o Prepare standard solutions of the  Calculate the concentration of the unknown sample
using the calibration curve.
compound with different concentrations
using serial dilution. 3. REAGENTS
o Use distilled water as the solvent.  Standard 0.008 M Potassium permanganate,
KMnO4.
 Blank Calibration:
 Unknown Sample (1 per group).
o Fill a cuvette with distilled water and place
it in the spectrophotometer. 4. MATERIALS NEEDED
o Calibrate the instrument by setting the  UV-Vis Spectrophotometer
absorbance of the blank to zero.
 Quartz/Glass cuvettes
 Measure Absorbance:
 Wash Bottle
o Pour each standard solution into a clean
 Pipette
cuvette and record its absorbance at the
compound's maximum absorption  Volumetric flasks (10 mL)
wavelength (λmax).  Beakers (20 or 50 mL)
o Rinse the cuvette with distilled water 5. PROCEDURE
between measurements.
 Prepare Standard Solutions:
 Data Analysis:
o Prepare standard solutions of the
o Record all absorbance readings in a table. compound with different concentrations
o Plot a graph of absorbance (y-axis) vs. using serial dilution (0.008 M, 0.004 M,
concentration (x-axis). 0.002 M, 0.001 M, 0.0005 M, 0.0001 M).

o Analyze the linearity of the graph and o Use distilled water as the solvent.
determine the equation of the line.  Blank Calibration:
o Fill a cuvette with distilled water and place
ACTIVITY NO. 4: DETERMINATION OF UNKNOWN it in the spectrophotometer.
CONCENTRATION OF AN ANALYTE BY USING THE BEER- o Calibrate the instrument by setting the
LAMBERT LAW absorbance of the blank to zero.
1. INTRODUCTION  Measure Absorbance:
 Spectrophotometry uses the absorbance of light by o Pour each standard solution into a clean
an analyte to determine its concentration. cuvette and record its absorbance at the
 UV/VIS spectrophotometry uses UV and visible light. compound's maximum absorption
wavelength (λmax).
 UV-Vis spectroscopy is used to determine the
concentration of colored solutions. o Rinse the cuvette with distilled water
between measurements.
 Beer-Lambert Law states that absorbance is directly
proportional to concentration (when path length and  Data Analysis:
molar absorptivity are constant). o Record all absorbance readings in a table.
 Beer-Lambert Law: A = εlC (A = Absorbance, ε = o Plot a graph of absorbance (y-axis) vs.
molar absorptivity, l = path length, C =
concentration (x-axis).
concentration).
o Analyze the linearity of the graph and
 Calibration relates the measured analytical signal to
determine the equation of the line.
the analyte's concentration.
o Read the absorbance of the unknown
sample using the generated equation of the
line.
 o Calculate the molar absorptivity using the 11. The shorter the wavelength, the lower the energy of
absorbance reading of the 0.0001 M light.
KMnO4 standard solution.
12. Absorbance and transmittance are inversely related.
13. The Beer-Lambert Law can be used to determine the
UV-Vis Spectroscopy Quiz unknown concentration of a sample.
Section 1: Multiple Choice Questions (MCQs) (10 points) 14. All solvents have the same effect on UV-Vis
absorption spectra.
1. What does UV-Vis spectroscopy measure? a)
Emission of light 15. The absorbance of a solution is always greater than
b) Absorption of UV or visible light 1.
c) Reflection of infrared light
16. A spectrophotometer can measure both absorbance
d) Scattering of X-rays
and transmittance.
2. Which wavelength range does visible light cover? a)
17. Protic solvents cause hypsochromic shifts in n → π*
100-400 nm
transitions.
b) 200-300 nm
c) 400-800 nm 18. UV-Vis spectroscopy can only be used for qualitative
d) 800-1000 nm analysis.

3. What is the purpose of blanking in UV-Vis 19. The calibration curve in spectrophotometry is always
spectroscopy? a) To clean the cuvette a straight line.
b) To set a baseline for accurate absorbance 20. The molar absorptivity constant (ε) is the same for all
measurement compounds.
c) To increase absorbance values
d) To calibrate the instrument with a sample solution Section 3: Fill in the Blanks (10 points)

4. The Beer-Lambert Law states that absorbance (A) is 21. The UV region ranges from ______ to ______ nm.
directly proportional to: a) Solvent density 22. _______ is used to set the spectrophotometer’s
b) Concentration and path length baseline before taking absorbance measurements.
c) Temperature
d) Wavelength 23. In UV-Vis spectroscopy, the spectrum is a plot of
______ vs. ______.
5. What type of transition is associated with a
bathochromic (red) shift? a) n → π* 24. The absorption maximum (λmax) of a compound
b) π → π* may shift due to changes in ______.
c) σ → σ* 25. ______ shifts occur when the absorption maximum
d) d → d* moves to a longer wavelength.
6. A cuvette used for UV measurements should be 26. The Beer-Lambert Law equation is _______.
made of: a) Plastic
27. The color of a solution is due to the ______
b) Quartz
wavelengths of light it transmits or reflects.
c) Aluminum
d) Copper 28. A spectrophotometer uses a ______ as a light source
in UV-Vis analysis.
7. Which solvent would likely cause the greatest
hypsochromic (blue) shift for n → π* transitions? a) 29. The part of the spectrophotometer that holds the
Water sample is called a ______.
b) Hexane
30. An unknown sample's concentration is determined
c) Chloroform
using a ______ curve.
d) Benzene
Section 4: Short Answer Questions (10 points)
8. What is the unit of absorbance? a) Molarity (M)
b) Wavelength (nm) 31. Explain the importance of blanking in a UV-Vis
c) Absorbance has no unit spectrophotometer.
d) Joules (J)
32. What happens to the absorbance reading if the
9. In UV-Vis spectroscopy, which factor does NOT affect cuvette is not properly cleaned?
absorbance? a) Concentration of the analyte
33. Describe how solvent polarity affects the absorption
b) Path length of the cuvette
maximum of a solute.
c) Wavelength of the light source
d) Temperature of the sample 34. How does increasing concentration affect the
absorbance of a solution?
10. Which of the following reagents would be used in an
experiment involving solvent effects? a) Potassium 35. What is the purpose of using different solvents in
permanganate UV-Vis spectroscopy experiments?
b) Acetone 36. Why is quartz used instead of glass for UV
c) Sodium chloride measurements?
d) Iron sulfate
37. How do hydrogen bonds between a solvent and
Section 2: True or False (10 points) solute affect UV-Vis absorption?
 38. What does it mean if a sample has an absorbance
greater than 1?
39. Describe how a calibration curve is created.
40. How can UV-Vis spectroscopy be applied in real-
world analysis?
Section 5: Calculation-Based Questions (10 points)
41. A solution has an absorbance of 0.45, a molar
absorptivity of 5000 M⁻¹cm⁻¹, and a path length of 1
cm. Calculate the concentration.
42. If a compound has a λmax of 450 nm in ethanol but
480 nm in hexane, explain the solvent effect
observed.
43. A sample has a transmittance of 20%. Calculate its
absorbance.
44. The concentration of an unknown solution is
determined using the equation of a calibration
curve: A = 2.5C. If absorbance is 0.75, what is the
concentration?
45. If a sample is diluted by a factor of 2 and its original
absorbance was 1.0, what would be the new
absorbance?
46. Calculate the absorbance of a solution with a
transmittance of 10%.
47. A solution follows Beer’s Law with ε = 2000 M⁻¹cm⁻¹
and a path length of 2 cm. If the concentration is
0.005 M, find the absorbance.
48. If a UV-Vis spectrophotometer detects a peak at 250
nm, what type of electronic transition is most likely
occurring?
49. A standard KMnO4 solution has an absorbance of
0.60 at 540 nm. If the unknown solution gives an
absorbance of 0.30, what is its relative concentration
compared to the standard?
50. A calibration curve equation is given as A = 0.85C +
0.02. If an unknown sample has an absorbance of
0.50, determine its concentration.