Introduction to Instrumental

Analysis

Fundamentals of Spectrophotometry

Dr. Nermin Salah 1st Lecture

5 Biological PHCM561
 Theoretical Course Calendar of Instrumental Analysis I - WS2019
Dates Lectures Instructor Tutorials Compensations
7-09-2019 to 12-09-2019 1. Lecture 1 (UV-Vis) Dr. Nermin Salah --
14-09-2019 to 19-09-2019 Dr. Nermin Salah
2. Lecture 2 (UV-Vis) Tutorial 1
21-9-2019 to 26-09-2019 Dr. Nermin Salah
3. Lecture 3 (UV-Vis) Tutorial 2
28-09-2019 to 03-10-2019 Dr. Menna
Hammam
4. Lecture 4 (IR) Tutorial 3

05-10-2019 to 10-10-2019
5. Lecture 5 Dr. Menna Sunday 6/10 (Armed Forces
Tutorial 4
(Luminescence) Hammam Day)
12-10-2019 to 17-10-2019 6. Lecture 6 (Introduction Dr. Nermin Salah
Tutorial 5
to Chromatography)
Sat 19-10-2019 to
Midterm exams
Mon 28-10- 2019
Tue 29-10-2019
7. Lecture 7 (HPLC) Dr. Nermin Salah Tutorial 6
Mon 04-11-2019
05-11-2019 to 11-11-2019 8. Lecture 8 (other
chromatographic Dr. Nermin Salah Tutorial 7
methods)
12-11-2019 to 18-11-2019 9. Lecture 9 (Gas
Dr. Nermin Salah Tutorial 8
Chromatography)
19-11-2019 to 25-11-2019 Dr. Nermin Salah
10. Lecture 10 (NMR I) Tutorial 9
26-11-2019 to 02-12-2019 Dr. Nermin Salah
11. Lecture 11 (NMR II) Tutorial 10
03-12-2019 to 09-12-2019 12. Lecture 12
(Analytical method Dr. Menna Hammam Tutorial 11
development and
validation in HPLC and
GC)
10-12-2019 to 16-12-2019 Revision Week
Theoretical Course Calendar of Instrumental Analysis I - WS2019

Dr. Nermin Salah B5-105

(Lectures 1-3)
(Lectures 6-11)
Dr. Nayra Omran B1-203 Assessment
Student assessment Assessment
Dr. Menna Hamam B1-203 methods weighting
(Lectures 4,5,12) Assignments 5%
Miss Triveena Maher B7-206 Oral Presentation 5%
Quizzes (Best 2 out of 3) 20%
Miss Donia Eyad B1-203
NO QUIZ
Miss Heba El Nakib B1-203 COMPENSATION
OFFERED
Miss Monica Akram B7.205
Midterm Exam 30%
Miss Yomna Amr B5.229 Final Exam 40%
Total 100%
Office hrs. are at the office door of the instructors

Dr. Nermin Salah (Practical Course Coordinator)

Dr. Nayra Omran (Theoretical Course Coordinator)

 Course Specs Instrumental Analysis I - WS2018
Instrumental analysis I (PHCM561t) course aims
to provide a thorough overview of the theoretical principles underlying common instrumental
analysis methods of ultraviolet-visible spectroscopy, fluorescence spectroscopy, infrared
spectroscopy, as well as, nuclear magnetic resonance. In addition basics of chromatography
(TLC, GC, HPLC, and SFC) are also introduced.

Learning outcomes

By the end of the theoretical part, the student will be able to:
a. Knowledge & Understanding
a1. Define basic instrumental analysis concepts, and illustrate the concepts of the different
instruments of the course, the properties of the instrumental devices used, and their applications.
a2. Define principles of identification, standardization, qualitative and quantitative analysis,
isolation, purification and separation methods of various pharmaceutical compounds.
a3. State the use of different analytical instruments and the functions of different parts of selected
instruments and their effects on data analysis.
b. Professional & practical skills
b1. Use the proper scientific terms, abbreviations & symbols related to instrumental analysis basics
and devices.
b2. Interpret results obtained from the different instruments, assess theoretically several
experiments and conduct research studies involving some related topics.
b3. Apply different methods of pharmaceutical calculations.
 Course Specs Instrumental Analysis I - WS2019
c. Intellectual skills
c1. Develop an understanding of the analytical capabilities of a number of instrumental
methods.
c2. Suggest suitable instrumental methods for particular analytical problems.
c3. Identify the property or quantity of the chemical system to be measured.
c4. Comprehend the physical and chemical principles upon which the measurement is based.
c5. Analyze the strengths and limitations of each particular instrumental method or approach.
c6. Develop analytical method rationally.
c7. Judge the importance of validation of analytical procedures.
d. General skills
d1. Communicate clearly by verbal & tutorial slides, and to present the project required.

d2. Retrieve & evaluate information from different sources like stationary phase data charts and
eluotropic series sources to improve professional competencies.

d3. Work effectively in team to prepare a power point presentation project.

d4. Use numeracy, calculation and statistical methods as well as information technology tools
like the suitable internet databases.

d5. Practice independent learning needed for continuous professional development via exploring
the websites indicated in the lecture slides.

d6. Adopt ethical and legal guidelines; students can use the lecture slides as a role model for
preparing notes with proper referencing.

d7. Implement writing skills via presenting the requested project as a PowerPoint print.

d8. Practice presentation skills via presenting the final project in front of the whole tutorial class,
as well as, the instructor.

d9. Demonstrate critical thinking, and problem-solving regarding different instrumental

applications, and solving extra exercises in tutorial
Learning outcomes
By the end of this lecture, the student should be able to:
1. To determine basic properties of light.
2. To identify the regions of the electromagnetic spectrum.
3. To describe what happens when a molecule absorbs light.
4. To discriminate between different effects of energy absorption on a molecule.
5. To calculate absorbance and transmittance of a sample.
6. To label the main components of optical instruments.
7. To calculate concentrations using Beer’s law.
8. To identify limitations to Beer’s law and reasons for certain deviations from the
expected results.
9. To use the proper scientific terms, abbreviations & symbols in
spectrophotometry.
Spectroscopy is any procedure that uses the interaction of
Electromagnetic Radiation (EMR) with matter to identify and/or
to estimate an analyte.
molecules solid
Qualitative
Quantitative ions liquid Analysis
Analysis atoms gas
Mixtures solutions

Electromagnetic radiation (light)

 EMR can be described in

terms of both particles and
waves (Dual nature of light)

 Light waves consist of

perpendicular and oscillating
electric and magnetic fields
 Light waves can be characterized By:

Wavelength (, Greek lambda):

Distance from one wave peak
to the next.
Units: m, cm, m, nm or A0

Frequency (, Greek nu):

Number of peaks that pass a
given point per second.
Units: Cycles/second or s-1 or
Hertz (Hz)

Wavenumber
Number of waves per cm.
1
υ= cm-1
λ
Wave nature of light can explain phenomena such as reflection, refraction
interference and diffraction.
 Light diffraction
Water diffraction

Water wave interference Light wave interference

 Electromagnetic radiation consists of discrete packets of
energy, which we call photons.

 Photons are the particles of light or the quanta of light.

 Each photon carries the energy, E (Joule).

where h is the Planck’s constant (=6.626x10-34 J.s)
E = hυ
 The all characteristics of light can be related as follows:
c
E = h υ = h = hc υ
λ
The higher the frequency, the --------------------------------- energy
The higher wavenumber, the --------------------------------- energy
The shorter the wavelength, the --------------------------------- energy

The particle nature can explain phenomena like absorption and

emission of light.
 Regions of electromagnetic radiation
Ejection of inner
shell electrons
Molecular
processes that

change in the spin of

occur when
light is absorbed
in each region Change in nuclear
configuration

protons
Near
Ultraviolet
Memorize
200 nm 800 nm
Absorption of light
A molecule that absorbs light photons will end up with increased
energy. The molecule will be promoted to an excited state.
UV/Vis energy promotes electrons into higher orbitals.

ΔE(molecule) =Δ(photon)
absorbed light is quantized

Most excited molecules relax again to the ground state

emitting the excess energy in the form of heat.
 Ultraviolet-Visible Spectrophotometry
What happens when a molecule absorbs UV-Visible radiations?
 When a molecule absorbs light having sufficient energy (e.g. UV-Vis
radiation) to cause an electronic transitions, additional vibration
and rotation transitions also occur

 Molecule can absorb one photon of just the right energy to cause
the following simultaneous changes:

E2
vibrational levels
v2
v1
r2 rotational levels Band
E r1 v0 Spectrum
E1 A
N
E Pure electronic
transition
R v2 DE = hn
G
v1 max 
Y r2
r1 v0 spectrum
E0 electronic levels
 1. A transition from the ground electronic state E0 to the E1 excited
electronic state
2. A change in the vibrational energy from the ground vibrational
state of E0 to an excited vibrational state of E1
3. A transition from one rotational state of E0 to a different
rotational state of E1
4. All the above transitions are quantized which means that they
required certain exact amount of energy
5. Thus, total energy absorbed = Eelec + Evib + Erot
DEelec >> DEvib >> DErot

absorbance
As a result, a large numbers of photons
of certain wavelengths are absorbed by
a molecule. These individual
wavelengths are too numerous and too
close to each other and a spectrum of
broad bands of absorbed wavelengths
Spectrum (a graph that shows
are obtained how absorbance varies with
wavelength)
For purpose of chemical analysis

Beer’s law
A = bc

Absorbance is directly proportional to:

1. Concentration, c, of absorbing species in the sample (A c)

2. Path length of light, b, through the sample (A b)

The previous equation is the heart of spectrophotometry as

applied to analytical chemistry, it is called Beer-Lambert law or
simply Beer’s law
 A=bc
 Concentration of the analyte is given in unit mol/L (M)
 The path length, b, in cm
 , is called the molar absorptivity or molar absorption
coefficient
“Absorbance of 1 M solution measured in a cell of 1 cm
pathlength”
A 1
   L mol  1 cm  1  M  1 cm  1
bc mol
cm
L
 , is characteristic for each substance at a particular
wavelength, .
 Transmittance and Absorbance T = 0.7, %T = 70%, A = 0.155

106 photons 0.7x106 photons


500 nm

There are two quantities that relate the change in the intensity or
radiant power of EMR before, P0 , and after, P, interaction with matter.
1. Transmittance, T, is simply defined as “the fraction of light
that reaches a detector after passing through a sample”
P
T
P 0<T<1
The percent transmittance, %T, is simply 100 T

P
%T  x 100 0 < %T < 100
P log rule
2. Absorbance, defined as:
P P
A=  log T A =  log P A =log ( )
 P
 1
A=bc
Anti


Transmittance, T
certain Log T =- A log
Absorbance, A

constant b
One analyte 0.5 T=10-A

Slope = b T =10- bc

Concentration Concentration

Transmittance decreases
Beer’s law is a relation between
exponentially as concentration
absorbance and concentration
increases
which is a straight line passes by
origin at constant pathlength, b,
and at certain wavelength, .

Beer’s law is obeyed for

monochromatic light
The absorbance
increases linearly with
pathlength.

The transmittance
decreases
exponentially with
pathlength.
Beer’s law proves a direct correlation between the absorbance (A)

of a molecule to the concentration (c) and the path length (b).

Deviation of Beer’s Law.

This relationship is a linear for the most part. However, under certain
circumstances the Beer relationship gives a non-linear relationship.

These deviations from the Beer Lambert law can be classified into three
categories:

Real Deviations - These are fundamental deviations due to the limitations

of the law itself.

Chemical Deviations- These are deviations observed due to specific

chemical species of the sample which is being analyzed.

Instrument Deviations - These are deviations which occur due to how the
absorbance measurements are made.
1- Real Deviation

Beers law is capable of describing absorption behavior of solutions

containing relatively low amounts of solutes dissolved in it (<10-3M).

When the concentration of the analyte in the solution is high (>10-3M),

the analyte begins to behave differently due to interactions with the
solvent and other solute molecules and at times even due to hydrogen
bonding interactions.

It is also possible that the concentration is so high, that the molecules

create a screen for other molecules thereby shadowing them from the
incident light.
2- Chemical Deviations

Chemical deviations occur due to chemical

phenomenon involving the analyte molecules
due to association, dissociation and
interaction with the solvent to produce a
product with different absorption
characteristics.

For example, phenol red undergoes a

resonance transformation when moving from
the acidic form (yellow) to the basic form
(red). Due to this resonance, the electron
distribution of the bonds of molecule changes
with the pH of the solvent in which it is
dissolved.
3- Instrumental Deviations
A] Due to Polychromatic Radiation

Beer’s law is strictly followed when a monochromatic source of radiation

exists. In practice, however, it is common to use a polychromatic source of
radiation with continuous distribution of wavelengths along with a
monochromators to create a monochromatic beam from this source.

B] Due to Presence of Stray Radiation

Stray radiation or scattered radiation is defined as radiation from the

instrument that is outside the selected wavelength band selected, or
any light reaching the detector without passing through the sample.
Usually, this radiation is due to reflection and scattering by the
surfaces of lenses, mirrors, gratings, filters and windows. If the
analyte absorbs at the wavelength of the stray radiation, a deviation
from Beer-Lambert law is observed similar to the deviation due to
polychromatic radiation.
C] Due to Mismatched Cells or Cuvettes [BLANK]

If the cells holding the analyte and the blank solutions are having
different path-lengths, or unequal optical characteristics, it is
obvious that there would be a deviation observed in Beer-
Lambert law.

Deviations from Beer’s Law

 Innovation in Spectroscopy

Reverse vending
machine
for PET bottles

To ensure an economically reasonable recycling process, reverse vending machines

have to be equipped with a cost-efficient material detector which detects PET
bottles and rejects other objects. The project‘s target aimed at the development of a
cost-efficient detector system on spectroscopy basis, including corresponding
automation components, in order to facilitate the extensive set-up of cost-favorable
reverse vending machines.
27
Teaching UV−Vis Spectroscopy with a 3D-
Printable Smartphone Spectrophotometer
 TERMS USED IN UV-VIS
SPECTROSCOPY

Auxochrome

Hypochromic shift

Chromophore
Atomic vs molecular spectroscopy

Hyperchromic shift Bathochromic shift

Hypsochromic shift
 Resources and references
Textbook: Principles of instrumental analysis, Skoog et
al., 5th edition, chapter 13.
Quantitative chemical analysis, Daniel C. Harris, 6th
edition , chapter 18.
 Useful links
http://www.chemguide.co.uk/analysis/uvvisiblemenu.html
#top
http://teaching.shu.ac.uk/hwb/chemistry/tutorials/molsp
ec/beers1.htm