PHA 052: Pharmaceutical Analysis 2

Module # 5 Teacher’s Guide

Lesson title: UV-VIS Spectroscopy Materials: Book, pen and notebook,

Learning Targets: index card/class list
At the end of the module, students will be able to:
1. Define the importance of UV-Vis Spectroscopy as a References:
tool in measuring electromagnetic radiation; Watson, David G. (2017).
2. Identify the parts of UV-Vis spectrometry, and Pharmaceuticals analysis: a textbook for
3. Identify the type of wavelength according to its length. pharmacy students and pharmaceutical
chemists, 4th ed. Singapore: Elsevier

A. LESSON PREVIEW/REVIEW

Activity 1
Write T if true, F if false.
_T_1. UV-visible spectroscopy measures the response of a sample to ultraviolet and visible range of
electromagnetic radiation.
_T_2. Beer-Lambert’s Law is a combination of the above laws and relates the power of the incident and
transmitted radiant beams to the thickness and concentration of the solution containing the absorbing chemical
species
_F_3. Wavelength is the number of complete cycles which pass a given point per second (cps). [Frequency]
_F_4. Light source may be designed to split the light beam so that the beam passes through two sample
compartments [Optics]
_T_5. Beer’s Law – states that the power of transmitted radiant beam decreases exponentially as the
concentration of the solution containing the absorbing chemical species increases

B. MAIN LESSON
I. INTRODUCTION

Definition of terms:

● Electromagnetic spectrum – the complete system of energy propagated in wave form

● Radiant Energy – refers to the energy propagated in the UV, Visible, and IR regions of the
electromagnetic spectrum
● Frequency - the number of complete cycles which pass a given point per second (cps). It may also be
expressed in Hertz, where 1 Hertz= 1cps
● Wavelength – the length of a complete wave or cycle, from the peak of one wave to the peak of the
next. The units used in spectrometry are micrometer (10-4 cm), nanometer (10-7 cm), and less
frequently is angstrom (10-8 cm).
➢ UV – 200 to 380 nm
➢ Vis – 380 to 780 nm
➢ Near IR – 780 to 3000 nm

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 PHA 052: Pharmaceutical Analysis 2
Module # 5 Teacher’s Guide

➢ Medium IR – 3.0 to 15 μm

3-8 (group frequency)

8-15 (fingerprint)

➢ Far IR – 15 to 300 μm

Table 1. Summary of wavelengths of different colors in the spectrum

Wavelength of color Color Absorbed Complementary Colors

absorbed (nm) (colors transmitted and seen
by an observer)
380 - 450 Violet Yellow-green
450 - 480 Blue Yellow
480 - 490 Green - blue Orange
490 - 500 Blue - green Red
500 - 570 Green Purple to Red-violet
570 - 590 Yellow Blue
590 - 620 Orange Green-blue
620 - 780 Red Blue-green

IA. UV-Vis Spectroscopy

● Electron Energy Transition

● Used to obtain the absorbance spectra of a compound in solution or as a solid. What is actually being
observed spectroscopically is the absorbance of light energy or electromagnetic radiation, which
excites electrons from the ground state to the first singlet excited state of the compound or material.
● Absorption of photon results in electronic transition of a molecule and electrons are promoted from
ground state to higher electronic states

➢ Principle

UV-visible spectroscopy measures the response of a sample to ultraviolet and visible range of
electromagnetic radiation. Molecules have either π,n,σ electrons. These electrons absorb UV radiation &
undergo transitions from ground state to excited state.
➢ Instrumentation
Parts of UV-Vis Spectrometer
a. Light Source – a deuterium lamp for the UV region from 190 to 350 nm and a quartz halogen or
tungsten lamp for the visible region from 350 to 900 nm.
b. Monochromator – used to disperse the light into its constituent wavelengths, which are further selected
by the slit. The monochromator is rotated so that a range of wavelengths is passed through the sample
as the instrument scans across the spectrum.

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 PHA 052: Pharmaceutical Analysis 2
Module # 5 Teacher’s Guide

c. Optics – may be designed to split the light beam so that the beam passes through two sample
compartments, and in such a double beam instrument, a blank solution can then be used commonly the
solvent in which the sample is dissolved.
Figure 1. Parts of UV-Vis Spectrometer

➢ Application in Pharmaceutical Analysis:

● A method for the quantification of drugs in formulations where there is no interference from excipients
● Determination of the pKa values of some drugs
● Detection of impurities
● Structure elucidation of organic compounds
● Determination of partition coefficients and solubilities of drugs
● Can be used to monitor the reaction kinetics of drug degradation
● The UV spectrum of a drug is often used as one of a number of pharmacopeial identity checks

IB. LAWS ON SPECTROPHOTOMETRY

❖ Notable Persons

● Pierre Bouguer (1698-1758) – astronomer, Light is diminished as it passes through the atmosphere.
● Johan Lambert (1728-1777) – mathematician, first to prove that π is irrational. No absorption coefficient
● August Beer (1825-1863) – added absorption coefficient and related to concentration in solution

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 PHA 052: Pharmaceutical Analysis 2
Module # 5 Teacher’s Guide

❖ Relevant Laws
1. Beer’s Law – states that the power of transmitted radiant beam decreases exponentially as the
concentration of the solution containing the absorbing chemical species increases
2. Lambert’s or Bouguer’s Law – states that the power of a transmitted radiant beam decreases
exponentially as the thickness of the solution containing the absorbing chemical species increases
3. Beer-Lambert’s Law – is a combination of the above laws and relates the power of the incident and
transmitted radiant beams to the thickness and concentration of the solution containing the absorbing chemical
species
This law is expressed through this equation:

A = log10 (I0/I) = εCL

A stands for the absorbance, I0 refers to the intensity of light upon a sample cell, l refers to the intensity of light
departing the sample cell, C stands for the concentration of the solute, L stands for the length of the sample
cell and ε refers to the molar absorptivity.

Based on the Beer-Lambert law, it has been established that the greater the number of the molecules that are
capable of absorbing light at a certain wavelength, the greater the extent of the absorption of light.

C. CHECK FOR UNDERSTANDING

The instructor will prepare 10-15 questions that can enhance critical thinking skills. Students will work by
themselves to answer these questions and write the rationale for each question.

Multiple Choice

(For 1-10 items, please refer to the questions in the Rationalization Activity)

RATIONALIZATION ACTIVITY (DURING THE FACE TO FACE INTERACTION WITH THE STUDENTS)
The instructor will now rationalize the answers to the students and will encourage them to ask questions and to
discuss among their classmates for 20 minutes.

1. Radiant energy is the energy propagated from which regions of the electromagnetic spectrum?

I. UV II. IR III. Radiation IV. Optics

a. I only
b. I and II
c. I and III
d. I and IV

Answer: B
Rationale: Regions of the electromagnetic spectrum that propagate Radiant energy are the UV, Visible, and IR .

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 PHA 052: Pharmaceutical Analysis 2
Module # 5 Teacher’s Guide

2. Which part of the UV-Vis Spectrometer is rotated so that a range of wavelengths is passed through the
sample as the instrument scans across the spectrum?
a. Monochromator
b. Optics
c. Light Source
d. Dispersion Device

Answer: A
Rationale: Rotating the Monochromator disperses the light into its constituent wavelengths, which are further
selected by the slit.

3. This law states that as the concentration of the solution containing the absorbing chemical species
increases, the power of the transmitted radiant beam decreases exponentially.
a. Lambert’s Law
b. Beer-Lambert’s Law
c. Bouguer’s Law
d. Beer’s Law

Answer: D
Rationale: Beer’s Law by German Physicist August Beer.

4. The unit that is less frequently used in Spectrometry.

a. Micrometer
b. 10-8 cm
c. Nanometer
d. 10-4 cm

Answer: B
Rationale: Angstrom (10-8 cm) is used less than Micrometer (10-4 cm) and Nanometer (10-7 cm).

5. Frequency is the number of complete cycles which pass a given point per second (cps). How many
Hertz is there in 1 cps?
a. 1
b. 10
c. 1/2
d. 2

Answer: A
Rationale: Frequency can also be expressed in Hertz, where 1 Hertz is 1 cps.

6. If the Wavelength is 495 nm, what is the color absorbed?

a. Green-Blue
b. Blue

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 PHA 052: Pharmaceutical Analysis 2
Module # 5 Teacher’s Guide

c. Green
d. Blue-Green

Answer: D
Rationale: Blue-Green is the color absorbed if the Wavelength is 490-500 nm.

7. The length of a complete wave or cycle, from the peak of one wave to the peak of the next.
a. Frequency
b. Electromagnetic Spectrum
c. Wavelength
d. NOTA

Answer: C.
Rationale: Wavelength is the distance between identical points (adjacent crests) in the adjacent cycles of a
waveform signal propagated in space.

8. The following are applications of UV-Vis Spectrometry in Pharmaceutical Analysis, except:

a. Structure elucidation of organic compounds
b. Detection of purity
c. Determination of pKa values of some drugs
d. Determination of partition coefficients and solubilities of drugs

Answer: B.
Rationale: Detection of Impurities

9. Which of the following is paired correctly?

a. Fingerprint – 8-15 μm
b. Vis – 200 to 380 nm
c. UV – 380 to 780 nm
d. AOTA

Answer: A.
Rationale: Fingerprint – 8-15 μm, Vis – 380 to 780 nm, UV – 200 to 380 nm

10. Which of these electrons absorb UV radiation and undergoes transitions from ground state to an
excited state?
a. Π
b. N
c. Σ
d. AOTA

Answer: D
Rationale: Molecules have either π,n,σ electrons.

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 PHA 052: Pharmaceutical Analysis 2
Module # 5 Teacher’s Guide

D. LESSON WRAP-UP

Teacher directs the student to mark (encircle) their place in the work tracker which is simply a visual to help
students track how much work they have accomplished and how much work there is left to do. This tracker will
be part of the student activity sheet.

PERIOD 1 PERIOD 2 PERIOD 3

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

AL Strategy: MINUTE PAPER

After the instructor collects all papers, he/she will now summarize the topic. Towards the end of the class, ask
the students to bring out and write on a half sheet of paper written feedback to the following questions: Firstly,
what was the most meaningful or important thing they learned during the class. Secondly, the important
question remains unanswered. Make sure to position yourself at the door. Conversely, instruct the students to
file out towards the exit door and collect the “minute papers” as students depart from the room. Respond to
students’ feedback during the next class meeting or as soon as possible

Most meaningful or important thing they learned from this session: (Why did they find it meaningful or
important?)

________________________________________________________________________________________
________________________________________________________________________________________
________________________________________________________________________________________

Question which remains unanswered: (What will they do to find the answer?)
________________________________________________________________________________________
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