CHE3243 (10CU)

ADVANCED ANALYTICAL CHEMISTRY

By
Mrs Ruth NTIHABOSE
Dr. JMV NGORORABANGA
Dr Emmanuel GAKUBA

University of Rwanda
College of Education
Part1: Chemical analysis
 References
 Core textbooks
1. Skoog Holler, Nieman. 1998., Principles of Instrumental Analysis”, 5th edition,
Harcourt Brace Publishers
2. Jag Mohan, 2014., Organic Analytical Chemistry: theory and practice, 5th
Edition, Narosa Publishing House, India
 Background textbooks
1. Ian L. Pepper, Charles P. Gerba, Mark L. Brusseau., 2006., Environmental &
2. pollution science, second edition, Academic Press is an imprint of Elsevier,
London
3. Kislik, V.S., 2011., Solvent extraction: classical and novel approaches. Elsevier.
 Websites
https://orgchemboulder.com/Technique/Procedures/Extraction/Extraction.shtml
https://www.embibe.com/exams/solvent-extraction/
https://chem.libretexts.org/Bookshelves/Organic_Chemistry/Organic_Chemistry_
Lab_Techniques_(Nichols)/04%3A_Extraction
Activity 1
 In group of 4, discuss about the questions:
 What is a chemical analysis?

 What are the types of chemical analysis?

Differentiate them
 What are analytical methods used in chemical
analysis?
Chemical analysis
 Chemical analysis: determination of the
physical properties or chemical composition of
samples of matter.
 Two categories: . Classical analysis and
instrumental analysis.
Chemical analysis
 Classical analysis or wet chemical analysis, consists of
those analytical techniques that use no mechanical or
electronic instruments other than a balance.
 It usually relies on chemical reactions between the
material being analyzed (the analyte) and a reagent
that is added to the analyte.
 It often depends on the formation of a product of the
chemical reaction that is easily detected and measured.
For example, the product could be coloured or could be
a solid that precipitates from a solution.
Chemical analysis

 Instrumental analysis involves the use of an

instrument, other than a balance, to perform the
analysis.
 spectroscopy, nuclear spectroscopy, mass spectrometry,
crystallography, electrochemical analysis, thermal
analysis, separations, and Microscopy
 A wide assortment of instrumentation is available
to the analyst and is used to
 characterize a chemical reaction between the analyte
and an added reagent;
 measure a property of the analyte.
Contents

Extraction techniques Chromatographic analysis

• Definition • Basics of Chromatography
• Thin layer Chromatography
• Principle of solvent
(TLC)
extraction
• Production of a
• Solvent extraction chromatogram with TLC
methods • Measurement of retention
Liquid/liquid extraction factor
liquid/solid extraction • Visualization of invisible
acid/base extraction substances on TLC plate
• Column Chromatography
• Factors affecting solvent
• Packing the column
extraction
• Running the column
Contents

UV-VIS analysis NMR analysis

• Definition and purpose • NMR spectroscopy
• Uv –vis radiations • Hydrogen atoms as little
• Uv-vis Principle magnets
• Nature of electronic • The hydrogen atom's
excitation environment
• Uv absorption spectrum • Features of an NMR
• Effect of conjugation and spectrum
chromophores • NMR sample analysis
• Absorbance
General introduction
 Every year, millions of chemical analyses of any
kind are performed around the world, and millions
of decisions are taken, based on these analyses
 Examples of analytical questions:
• Have the medicaments, the amount of drugs reported in
their container?
• Can we safely consume this water or these foods?
• Are these alloys suitable for use in the aircraft construction?
• Was the driver drunk, when he crashed?
• Is this sportsman using drugs to enhance his performance?
General introduction (cont)
 All the above-mentioned questions and several
other of this kind are answered with the help of a
chemical analysis and all have consequences in real
life (compensation claims, disease, fines, even
prison)
 Fundamentally every aspect of society is supported
in some way by chemical analytical measurement,
consequently, there is a need these analyses would
be reliable. Instrumental analysis plays a decisive
role in society.
General introduction (cont)
 “Analytical chemistry involves separating,
identifying and determining the relative amounts of
the components in a sample of matter”. This allow to
determine the identity, concentration or properties
of analyte (component of interest in a sample).
 To make this determination, we measure one or
more of the analyte’s chemical or physical
properties.
 Several methods are used in analytical chemistry
and include classical and instrumental methods
 Introduction
 Sample Purity
 Many chemical analysis are not specific for one

compound
- Actually respond to many potential
interferences in the sample

 Often it is necessary to first purify the compound of

interest
- Remove interfering substances before a
selective analysis is possible
- This requires a separation step.
 Introduction
 Techniques available for Chemical Separations:
 Extraction

 Distillation

 Precipitation
 Chromatography

 Many others (centrifugation, filtration, etc)

Extractions and Chromatography are especially useful in

analytical methods
Extraction
Activity 2

1.What is extraction techniques?

2.What are the types of extraction techniques?
3.How each type of extraction is done?
 Introduction
 The extent to which solutes, both inorganic and organic,
distribute themselves between two immiscible liquids differs
enormously.
 These differences have been used for decades to separate
chemical species.
 We call the process of moving a species from one phase to
another phase an extraction.
 The two phases used can be liquid-liquid, liquid-solid,
gas-solid, etc…
 Liquid-liquid is the most common type of extraction
Types of extraction
 Liquid–liquid extraction
 Solid-phase extraction
 Acid-base extraction
 Supercritical fluid extraction
 Ultrasound-assisted extraction
 Heat reflux extraction
 Mechanochemical-assisted extraction
 Maceration
 Microwave-assisted extraction
 Instant controlled pressure drop extraction
 Liquid-liquid extractions

Liquid–liquid extraction (LLE), also known as

solvent extraction and partitioning, is a method to
separate compounds or metal complexes, based on
their relative solubilities in two different immiscible
liquids, usually water (polar) and an organic solvent
(non-polar).
There is a net transfer of one or more species from
one liquid into another liquid phase, generally from
aqueous to organic.
 Liquid-liquid extractions
The transfer is driven by chemical potential, i.e. once the
transfer is complete, the overall system of chemical
components that make up the solutes and the solvents are in
a more stable configuration (lower free energy).
The solvent that is enriched in solute(s) is called extract. The
feed solution that is depleted in solute(s) is called the
raffinate. LLE is a basic technique in chemical laboratories,
where it is performed using a variety of apparatus, from
separatory funnels to countercurrent distribution equipment
called as mixer settlers.
 Liquid-liquid extractions

LLE is a basic technique in chemical laboratories,

where it is performed using a variety of apparatus,
from separatory funnels to countercurrent
distribution equipment called as mixer settlers.
 Liquid-liquid extractions
A liquid–liquid extraction usually is accomplished using a
separating funnel.
 When extracting, solvent is stirred with solution containing solute then
solute from original solvent gets transferred into an extracting solvent.
 When the extraction is complete, we allow the liquids to separate.

 The stopcock at the bottom

of the separating funnel
allows us to remove the two
phases.
 Liquid-liquid extractions
 Compared with other separation methods, it gives a
better separation effect than chemical precipitation, and
a higher degree of selectivity and faster mass transfer
than the ion exchange method.
Compared with distillation, solvent extraction has
advantages such as low energy consumption, large
production capacity, fast action, easy continuous operation
and ease of automation.
 Liquid-liquid extractions
Commonly used solvents
Organic solvents dissolve in water. Here are their solubility
in water at 20oC:
 Ethyl acetate (8.1 %),
 Diethyl ether (6.9 %),
 Dichloromethane (1.3 %) and
 Chloroform (0.8 %) dissolved up to 10 % in water.
Water also dissolves in organic solvents:
 Ethyl acetate (3 %),
 Diethyl ether (1.4 %),
 Dichloromethane (0.25 %)
 Chloroform (0.056 %).
Liquid-liquid extractions
Conditions for Ideal Extraction Solvents
 Immiscible pair of solvents: water and low
polarity organic solvents
 Good solubility of the target compound
 Poor solubility of impurities
 Volatility of the extraction solvent
 Toxicity and safety properties of the extraction
solvent
 Liquid-liquid extractions
 Immiscible pair of solvents: water and low polarity
organic solvents
 The extracting solvent must be immiscible with the solution
to be extracted.
 The more polar the organic solvent, the more it is miscible
(soluble) with water.
 For example, polar solvents such as methanol, ethanol and
acetone are miscible with water, thus not suitable for liquid-
liquid extraction.
 Organic solvents with low polarity such as hexanes, toluene,
dichloromethane and diethyl ether are usually chosen as
the organic extracting solvent.
 Liquid-liquid extractions
 Good solubility of the target compound
 The compound(s) to be extracted, which are
present in a solution, should also be soluble in
the extracting solvent.
 Poor solubility of impurities
 Major impurities (e.g., from a reaction) should
not be soluble in the extracting solvent.
Liquid-liquid extractions
 Volatility of the extraction solvent
 The extracting solvent should be sufficiently
volatile so that it can be removed easily from
the extracted material by distillation.
 Liquid-liquid extractions
 Toxicity and safety properties of the extraction
solvent
 It is usually desirable if the solvent is non-toxic
and not flammable.
 Some solvents are not toxic but flammable (e.g., diethyl
ether, hydrocarbons--petroleum ether, hexanes).
 Some are not flammable but toxic (e.g., dichloromethane,
chloroform).
 Some solvents are both toxic and flammable (e.g., benzene).
 Liquid-liquid extractions
The properties of the solvent used for
liquid-liquid extraction
The solvent should be well miscible with the liquid to be
extracted.
The solvent should not be miscible with the other
components of the mixture or react with the solute.
The boiling point of the solvent should be low enough ( well
below the melting point of the solute) such that it can be
evaporated easily after collection.
It should have a favourable temperature coefficient.
 Liquid-liquid extractions
Distribution coefficient
 When a solution is placed in a separating funnel and shaken with
an immiscible solvent, solutes often dissolve in part into both layers.
 The components are said to "partition" between the two layers, or
"distribute themselves" between the two layers.
 When equilibrium has established, the ratio of concentration of
solute in each layer is constant for each system, and this can be
represented by a value K.
 K is called the partition coefficient or distribution coefficient.

Immiscible
[ S ]2
liquids K
[ S ]1
 The partitioning of solute s between two chemical phases (1 and 2) is described
by the equilibrium constant K (partition coefficient or distribution coefficient)
Liquid-liquid extractions
Extraction Efficiency
 The fraction of moles of S remaining in phase 1 after one extraction
can be determined
- The value of K and the volumes of phases 1 and 2 need to be
known V1
q
V1  KV2 
where: q = fraction of moles of S remaining in phase 1
V1 = volume of phase 1
V2 = volume of phase 2
K = partition coefficient

 The fraction of S remaining in phase 1 after n extractions is

n
 V1 
qn    Assumes V2 is constant

 V1  KV2  
 Liquid-liquid extractions
Extraction Efficiency
 Illustration

Ether layer
Water layer

1M
UO2(NO3)2
(yellow)

After mixing, UO2(NO3)2 After 8 extractions,

Is distributed in both UO2(NO3)2 has been
layers removed from water
 Liquid-liquid extractions
Example #1:
 Solute A has a K = 3 for an extraction between water (phase 1) and
benzene (phase 2).
If 100 mL of a 0.01M solution of A in water is extracted one time
with 500 mL benzene, what fraction will be extracted?

Solution:
First determine fraction not extracted (fraction still in phase 1, q):
n 1
 V1   100 mL 
qn       0.062  6.2%

 1V  KV 
2   100 mL  ( 3 )  ( 500 mL ) 

The fraction of S extracted (p) is simply:

p  1  q  1  0.062  0.938  93.8%
 Liquid-liquid extractions
Example #2:
 For the same example, what fraction will be extracted if 5 extractions
with 100 mL benzene each are used (instead of one 500 mL
extraction)?
Solution:
Determine fraction not extracted (fraction still in phase 1, q):
n 5
 V1   100 mL 
qn        0.00098  0.98%
 V1  KV2    100 mL  ( 3 )  ( 100 mL ) 

The fraction of S extracted (p) is:

p  1  q  1  0.00098  0.99902  99.902 %

Note: For the same total volume of benzene (500 mL), more A is extracted
if several small portions of benzene are used rather than one large portion
 Liquid-liquid extractions
Distribution coefficient
K is called the partition coefficient or
distribution coefficient.
A large value for KD indicates that extraction of
solute into the organic phase is favorable.
To evaluate an extraction’s efficiency we must
consider the solute’s total concentration in each
phase, which we define as a distribution ratio, D.
D = [Sorg]total / [Saq]total
 Liquid-liquid extractions
Distribution coefficient
The partition coefficient KD and the distribution ratio D
are identical if the solute has only one chemical form
in each phase;

If the solute exists in more than one chemical form in

either phase, then KD and D usually have different
values. For example, if the solute exists in two forms in
the aqueous phase, A and B, only one of which, A,
partitions between the two phases, then
 Liquid-liquid extractions
Distribution coefficient
This distinction between KD and D is important. The
partition coefficient is a thermodynamic equilibrium
constant and has a fixed value for the solute’s
partitioning between the two phases. The distribution
ratio’s value, however, changes with solution
conditions if the relative amounts of A and B change.
If we know the solute’s equilibrium reactions within
each phase and between the two phases, we can
derive an algebraic relationship between KD and D.
 Liquid-liquid extractions
Distribution coefficient
Example #3:
A solute has a KD between water and chloroform of
5.00. Suppose we extract a 50.00-mL sample of a
0.050 M aqueous solution of the solute using 15.00
mL of chloroform. (a) What is the separation’s
extraction efficiency? (b) What volume of chloroform
do we need if we wish to extract 99.9% of the
solute?
 Liquid-liquid extractions
Distribution coefficient
Example #3: Solution
 For a simple liquid–liquid extraction the distribution ratio, D,
and the partition coefficient, KD, are identical.
 (a) The fraction of solute that remains in the aqueous phase
after the extraction is

 The fraction of solute in the organic phase is 1–0.400, or

0.600. Extraction efficiency is the percentage of solute that
moves into the extracting phase; thus, the extraction
efficiency is 60.0%.
 Liquid-liquid extractions
Distribution coefficient
Example #3: Solution
 (b) To extract 99.9% of the solute (qaq)1 must be
0.001. Finding Vorg, and making appropriate
substitutions for (qaq)1 and Vaq gives

 This is large volume of chloroform. Clearly, a single

extraction is not reasonable under these conditions.
 Liquid-liquid extractions
Distribution coefficient
Example #4:
 To plan a liquid–liquid extraction we need to know the solute’s
distribution ratio between the two phases. One approach is to carry
out the extraction on a solution that contains a known amount of solute.
After the extraction, we isolate the organic phase and allow it to
evaporate, leaving behind the solute. In one such experiment, 1.235 g
of a solute with a molar mass of 117.3 g/mol is dissolved in 10.00 mL
of water. After extracting with 5.00 mL of toluene, 0.889 g of the
solute is recovered in the organic phase. (a) What is the solute’s
distribution ratio between water and toluene? (b) If we extract 20.00
mL of an aqueous solution that contains the solute using 10.00 mL of
toluene, what is the extraction efficiency? (c) How many extractions will
 Liquid-liquid extractions
Distribution coefficient
Example #4: Solution
 (a) The solute’s distribution ratio between water and
toluene is

 (b) The fraction of solute remaining in the aqueous

phase after one extraction is

The extraction efficiency, therefore, is 72.0%.

 Liquid-liquid extractions
Distribution coefficient
Example #4: Solution
 (c) To extract 99.9% of the solute requires

 a minimum of six extractions.

 Liquid-liquid extractions
 If the solution to be extracted consists of two solutes A and B,
and we have to separate A from B, then we will use
extracting solvent which will dissolve more quantity of A and
very less quantity of B.
 Under this condition, effectiveness of separation is expressed
in terms of separation coefficient or separation factor.
 It is the ratio of distribution coefficients of two solutes A and
B and denoted by -b
KDA
b=
KDB

b should be very high to separate two solutes by solvent

extraction, otherwise clear separation will be very difficult.
 Liquid-liquid extractions
pH Effects in Extractions
 For weak acids (HA) and Bases (B)
- Protonated and non-protonated forms usually have
different partition coefficients (K)
- Charged form (A- or BH+) will not be extracted
- Neutral form (HA or B) will be extracted
 Partitioning is Described in Terms of the Total Amount of a
Substance
- Individual concentrations of B & BH+ or HA & A- are
more difficult to determine
- Partitioning is regardless of the form in both phases
- Described by the distribution coefficient (D)
Total Concentration of A in Phase 2
D
Total Concentration of A in Phase 1
 Liquid-liquid extractions
pH Effects in Extractions
 The distribution of a weak base or weak acid is pH
dependent
For a weak acid (HA) where A- only exists in phase 1:
equilibrium reactions that affect the extraction of the weak
acid, HA, by an organic phase in which ionic species are not
soluble.
 Liquid-liquid extractions
pH Effects in Extractions
To derive an equation for D that shows this dependence, we
begin with the acid dissociation constant for HA.

The concentration of A– in the aqueous phase

The distribution ratio, D, is

The relationship between the distribution ratio D and the pH

of the aqueous solution is
 Liquid-liquid extractions
pH Effects in Extractions
 The distribution of a weak base or weak acid is pH
dependent
For a weak base (B) where BH+ only exists in phase 1:
Total Concentration of Base in Phase 2
D
Total Concentration of Base in Phase 1

0
K BH   0
[ BH  ]1

[ B ]2
D
[ B ]1  [ BH  ]1
 Liquid-liquid extractions
pH Effects in Extractions
 The distribution of a weak base or weak acid is pH dependent

Substitute definition of KB and Ka into D:

[ B ]2 [ B ]2 [ H  ][ B ]
D KB  Ka  Kw Kb

[ B ]1  [ BH ]1 [ B ]1 [ BH  ]
(partition coefficient) (equilibrium constant)

K B Ka
D D is directly related to [H+]

Ka  [ H ]
 Liquid-liquid extractions
pH Effects in Extractions
 A similar expression can be written for a weak acid (HA)

K HA [ H  ] K HA 
[ HA ]2
D where:
 [ HA ]1
Ka  [ H ]

 The ability to change the distribution ratio of a weak acid

or weak base with pH is useful in selecting conditions that
will extract some compounds but not others.
- Use low pH to extract HA but not BH+ (weak acid
extractions)
- Use high pH to extract B but not A- (weak base
extractions)
 Liquid-liquid extractions
Examples
1. Butanoic acid has a partition coefficient of 3.0 (favoring benzene)
when distributed between water and benzene. Find the formal
concentration of butanoic acid in each phase when 100 mL of 0.10
M aqueous butanoic acid is extracted with 25 mL of benzene at pH
4.00 and pH 10.00
2. An acidic solute, HA, has a Ka of 1.00×10−5 and a KD between
water and hexane of 3.00. Calculate the extraction efficiency if we
extract a 50.00 mL sample of a 0.025 M aqueous solution of HA,
buffered to a pH of 3.00, with 50.00 mL of hexane. Repeat for pH
levels of 5.00 and 7.00.
 Liquid-liquid extractions
Examples
1.
As such at pH concentration of butanoic acid as unionized molecules will
be very less because being acid in basic pH it gets ionized to a great
extent. This depends upon the ionization constant of butanoic acid.
Let the butanoic acid be represented by HA for simplicity.
Butanoic acid pKa = 4.82. there4; pKb = 14 - pKa = 9.18
HA⇌H+ + A− at pH10, A−gets hydrolyzed as per its dissociation constant
 Liquid-liquid extractions
Examples
1.
 Liquid-liquid extractions
Examples
1.
Let x M be the amount of butanoic acid in benzene, then in the
water phase amount of butanoic acid present is 6.6069 ⋅10-7 -
xM

25 ml benzene contains 4.9552 ⋅10-7 M and 100 ml water contains

1.6517⋅10-7 M of butanoic acid.
 Liquid-liquid extractions
Examples: Solutions
2. When the pH is 3.00, [H3O+aq] is 1.0×10−3 and the distribution
ratio is

The fraction of solute that remains in the aqueous phase is

The extraction efficiency, therefore, is almost 75%. The same

calculation at a pH of 5.00 gives the extraction efficiency as
60%. At a pH of 7.00 the extraction efficiency is just 3% .
 Liquid-liquid extractions
Factors affecting solvent extraction
 Masking agent -These are chemical species which do not allow
to extract unwanted metal ion with metal ion of interest.
 Modifiers - these are the substances when added into
aqueous phase, they increase the solubility of solute to be
extracted into organic solvent. Usually high molecular weight
alcohols are used as modifiers in solvent extraction.
 Oxidation state - by carrying out redox reaction with suitable
reagent, oxidation state of metal ion can be changed. The
latter may affect the partition of metal ion.
 pH - pH affects stability and charge on the metal complex.
The pH at which metal ion complex is most stable and neutral
is the best pH for extraction of metal ions.
 Liquid-liquid extractions
Factors affecting solvent extraction
 Salting effect - The high concentration of salt sometimes help
to extract metal ions from aqueous phase to organic phase.
Salt increases ionic strength of aqueous phase and thereby
increases the solubility of metal complex into organic phase.

Synergic agents - These are reagents which when added to

organic phase increase the efficiency of extraction. They get
associated with metal complex, make it more soluble into
organic phase.
 Liquid-liquid extractions
Solvent Extraction of Metals
 Metal ions do not tend to dissolve appreciably in the organic
layer. For them to become soluble, their charge must be
neutralized and something must be added to make them
neutral or hydrophobic complex .
 This can be accomplished using one of the following:
 Chelate formation
 Ion- Association method
 Solvation
 Synergic extraction
 Liquid-liquid extractions
Chelate formation
 We use chelating agents
 Metal forms stable and neutral
complexes with chelating agents.
 Such chelates are usually water soluble
 The chelating agents are usually
bidentate or multidentate organic
ligands which provide hydrophobic
pocket to the metal ion.
 The organic part of such ligands strongly interact with
organic solvent and thereby meta-chelate become soluble
into organic phase.
 Liquid-liquid extractions
Chelate formation
Most chelating agents are weak acids that ionize in water;
the ionizable proton is displaced by the metal ion when the
chelate is formed, and the charge on the organic compound
neutralizes the charge on the metal ion.
An example is diphenylthiocarbazone (dithizone), which
forms a chelate with lead ion:
 Liquid-liquid extractions
Ion association method
 The metal ion is incorporated into a bulky molecule and then
associates with another ion of the opposite charge to form
an ion pair, or the metal ion associates with another ion of
great size (organic-like).
 For example, it is well known that iron(III) can be quantitatively
extracted from hydrochloric acid medium into diethyl ether.
 The mechanism is not completely understood but the following
ion-association complex is believed to be involved.

{(C2H5)2O: H+, FeCl4[(C2H5)2O]2ˉ}

 Liquid-liquid extractions
Solvation
 It is the process in which metal ion gets solvated by solvent
molecule and trapped inside the solvent cage.
 The solvent used for solvation is soluble in organic phase,
hence metal ion gets extracted from aqueous phase into
organic phase.
 Carboxylic acids, ternary amines, alkyl substituted
phosphoric acids. etc. can be used as extractants
Liquid-liquid extractions
Synergic extraction
 These are the reagents which when added to
organic phase increase the efficiency of
extraction
 They get associated with metal complex, make it
more soluble into organic phase.
Liquid-liquid extractions
Applications of solvent extraction
 Solvent extraction is used in the processing of
perfumes, vegetable oil, or biodiesel.
 It is also used to recover plutonium from irradiated
nuclear fuel, a process which is usually called
nuclear reprocessing.
 The recovered plutonium can then be re-used as
nuclear fuel.
 Liquid-liquid extractions
 Liquid-liquid extractions have several limitations such
as:
 The solvents that can be used must be immisicible
with water and must not form emulsions.
 Use of relatively large volumes of solvent, which
can cause a problem with waste disposal.
 Most extractions are performed manually, which
makes them somewhat slow and tedious.
 Solid-phase extraction, or liquid-solid extraction, can
overcome several of these problems.
Activity

1.What is solid phase extraction techniques?

2.How is solid phase extraction done?
3.What difference is between solid phase and
liquid-liquid extraction?
 Solid phase extractions
 Solid-phase extraction (SPE) , or liquid-solid extraction is
an extractive technique by which compounds that are
dissolved or suspended in a liquid mixture are separated
from other compounds in the mixture according to their
physical and chemical properties.
 It is used to concentrate and purify samples for analysis;
to isolate analytes of interest from a wide variety of
matrices, including urine, blood, water, beverages, soil, and
animal tissue.
 It is a technique designed for rapid, selective sample
preparation and purification prior to the chromatographic
analysis (e.g. HPLC, GC, TLC).
 Solid phase extractions

 SPE uses the affinity of solutes dissolved or suspended in a

liquid (known as the mobile phase) for a solid through
which the sample is passed (known as the stationary
phase) to separate a mixture into desired and undesired
components.
 The result is that either the desired analytes of interest or
undesired impurities in the sample are retained on the
stationary phase.
 Solid phase extractions
 The portion that passes through the stationary phase is
collected or discarded, depending on whether it contains
the desired analytes or undesired impurities.
 If the portion retained on the stationary phase includes the
desired analytes, they can then be removed from the
stationary phase for collection in an additional step, in
which the stationary phase is rinsed with an appropriate
eluent (a solvent used in order to effect separation by
elution).
 Solid phase extractions
 In a solid phase
extraction of a liquid
sample, we pass the
sample through
a cartridge that
contains a solid
adsorbent.
 The choice of adsorbent
is determined by the Selection of solid phase extraction cartridges for
liquid samples. From left-to-right, the absorbent
species we wish to materials are octadecylsilane, carbon,
separate. octadecylsilane, polyamide resin, and diol
 Solid phase extractions
Two strategies fro elution
 Bind and elute strategy: The analyte(s) are retained and
matrix components do not retain. The analyte subsequently
is washed and eluted with a strong solvent.
 Removal/trapping strategy: The analyte(s) are not
retained and elutes, while the matrix components are
retained.
 Solid phase extractions
 Bind and elute
strategy:
 Solid phase extractions

 Removal/trapping
strategy:
 Solid phase extractions
 Solid-phase extraction techniques use membranes or small
disposable syringe-barrel columns or cartridges.
 A hydrophobic organic compound is coated or chemically bonded to
powdered silica to form the solid extracting phase or adsorbent.
 Solid phase extractions
Procedure for cartridge system for solid-phase extractions
 The sample is placed in the cartridge and pressure is applied by
the syringe or from an air or nitrogen line.
 Alternatively, a vacuum can be used to pull the sample through
the extractant.
 Organic molecules are then extracted from the sample and
concentrated in the solid phase.
 They can later be displaced from the solid phase by a solvent
such as methanol.
 By extracting the desired components from a large volume of
water and then flushing them out with a small volume of solvent,
the components can be concentrated.
 In some solid-phase extraction procedures, impurities are
extracted into the solid phase while compounds of interest pass
through unretained.
 Solid phase extractions
 In addition to packed cartridges, solid-phase extraction can
be accomplished by using small membranes or extraction
disks.
 These have the advantages of reducing extraction time and
lowering solvent use.
 Solid-phase extraction can also be done in continuous
flow systems, which can automate the preconcentration
process.
 In comparison to a liquid–liquid extraction, a solid phase
extraction has the advantage of being easier, faster, and
requires less solvent.
 Solid phase extractions
Continuous extractions
 Often applied when analyte has an unfavourable partition
coefficient.
 Once analyte’s partition coefficient is unfavourable, a single
extraction will not recover all the analyte.
 A quantitative extraction is achieved when one continuously
pass the extracting phase through the sample.
 A continuous extraction of a solid sample is carried out using
a Soxhlet extractor.
 Solid phase extractions
Soxhlet extraction
 Soxhlet extraction is an exhaustive extraction
technique widely applied to analytes that are
sufficiently thermally stable.
 The extraction solvent is continuously cycled
though the matrix, by boiling and
condensation, with the sample being collected
in the hot solvent.
 It is used for extracting nonvolatile and semi-
volatile organic compounds from solids such as
soils, sludges, and wastes.
 Solid phase extractions
Soxhlet extraction
 It ensures intimate contact of the sample matrix with the
extraction solvent.
 It is applicable to the isolation and concentration of
water-insoluble and slightly water soluble organics in
preparation for a variety of chromatographic
procedures.
 It is restricted to use by or under the supervision of
trained analysts. Each analyst must demonstrate the
ability to generate acceptable results with this method.
 Solid phase extractions
Soxhlet extraction
Procedure
 The extracting solvent is placed in the lower reservoir and heated to
its boiling point.
 Solvent in the vapour phase moves upward through the tube on the
far right side of the apparatus, reaching the condenser where it
condenses back to the liquid state.
 Passes through the sample, which is held in a porous cellulose filter
thimble, collecting in the upper reservoir.
 When the solvent in the upper reservoir reaches the return tube’s
upper bend, the solvent and extracted analyte are siphoned back to
the lower reservoir.
 Over time the analyte’s concentration in the lower reservoir increases.
 Solid phase extractions
Soxhlet extraction
 In some applications, microwave-assisted extractions have
replaced Soxhlet extractions.
 A microwave oven is used to heat the mixture.
 Use of sealed digestion vessel allows the extraction to take
place at a higher temperature and pressure, reducing the
amount of time needed for a quantitative extraction.
 In a Soxhlet extraction the temperature is limited by the
solvent’s boiling point at atmospheric pressure.
e.g.: When acetone is the solvent, a Soxhlet extraction is
limited to 56 oC, but a microwave extraction can reach 150
oC.
 Solid phase extractions
Applications
 SPE is frequently used in the pharmaceutical, clinical and
high-throughput diagnostic testing, forensic, environmental
and food/agrochemical industries for analyses related to:
 Pharmaceutical compounds and metabolites in biological
fluids
 Drug of abuse in biological fluids
 Environmental pollutants in drinking and wastewater.
 Pesticides, antibiotics or mycotoxins in food/ agricultural
matrices.
 Desalting proteins and peptides.
 Fractionation of the lipids
Chromatographic analysis
Basics of Chromatography
It was discovered by the Russian botanist Mikhail
Tswett, 1872-1919
In 1906 Tswett used chromatography to separate
plant pigments. He called the new technique
chromatography because the result of the analysis
was 'written in ‘colour' along the length of the
adsorbent column. The greek words Chroma means
“colour” and graphein means to “write”
Basics of Chromatography(cont)
 In modern laboratories, the colour aspect is no longer
relevant, but the same principles apply. By dissolving
a mixture of interest in a mobile phase and
transporting it through a stationary phase, the
components of the mixture can be separated from
one another based on their different speeds of
travel.
 By varying the mobile phase, the stationary phase,
and/or the factor determining speed of travel, a
wide variety of chromatographic methods have been
created, each serving a different purpose.
Basics of Chromatography(cont)
 Chromatography’ is an analytical technique
commonly used for separating a mixture of chemical
substances into its individual components, so that the
individual components can be thoroughly analyzed.
 Chromatography is a separation technique that
every organic chemist and biochemist is familiar with.
 There are many types of chromatography e.g., liquid
chromatography, gas chromatography, ion-exchange
chromatography, affinity chromatography, but all of
these employ the same basic principles.
Basics of Chromatography (cont)
 In this module, we will focus on types of
chromatography according to the stationary
phase.
 Considering stationary phase (SP), there are
3types of chromatography namely
 thin layer chromatography
 paper chromatography

 column chromatography.
Thin layer Chromatography (TLC)
 Thin layer chromatography is done exactly as its
name says by using a thin, uniform layer of silica
gel or alumina coated onto a piece of glass, metal
or rigid plastic.
 The silica gel (or the alumina) is the stationary
phase. The stationary phase for thin layer
chromatography also often contains a substance
which fluoresces in UV light.
 
.
Fig.TLC Plate
TLC (cont)
Production of a chromatogram with TLC

 Let’s start with a simple case: just trying to show that

a particular dye is in fact a mixture of simpler dyes.
 How this is practically done?

A pencil line is drawn near the bottom of the plate

and a small drop of a solution of the dye mixture is
placed on it. Any labelling on the plate to show the
original position of the drop must also be in pencil. If
any of this was done in ink, dyes from the ink would
also move as the chromatogram developed.
Production of a chromatogram with
TLC (cont)
Production of a chromatogram with
TLC (cont)
 The spot of mixture is air-dried and the plate is
stood in a shallow layer of solvent in a covered
beaker in a TLC tank. It is important that the solvent
level is below the line with the spot on it.
 Production of a chromatogram with
TLC (cont)
 As the solvent slowly travels up the plate, the
different components of the dye mixture travel at
different rates and the mixture is separated into
different coloured spots.
 . click to open the video
Production of a chromatogram with
TLC (cont)
 The solvent is allowed to rise until it almost reaches
the top of the plate. That will give the maximum
separation of the dye components for this particular
combination of solvent and stationary phase.
 Measurement of retention factor
(Rf) values (cont)
 Measurements are often taken from the plate in
order to help identify the compounds present. These
measurements are the distance travelled by the
solvent, and the distance travelled by individual
spots.
 When the solvent front gets close to the top of the
plate, the plate is removed from the beaker and the
position of the solvent is marked with another line
before it has a chance to evaporate.
 Measurement of Rf values (cont)
 . These measurements are then
taken:

The Rf value for each dye is then

worked out using the formula:
 Measurement of Rf values (cont)
 Example:
If the red component travelled 1.7 cm from the base
line while the solvent had travelled 5.0 cm, then the Rf
value for the red dye is:

If you could repeat this experiment under exactly the

same conditions, then the Rf values for each dye would
always be the same.
Visualization of invisible substances
on TLC plate
 Using fluorescence
TLC plate often has a substance added to it which will
fluoresce when exposed to UV light. Therefore if you
shine UV light on it, it will glow (shine). That glow is
masked at the position where the spots are on the final
chromatogram, even if they are invisible with naked
eyes, they will show up as darker patches.
Visualization of invisible substances
on TLC plate
 Showing the spots up chemically
Some times you can make the spots visible by reacting
the analyte with a chemical which produce coloured
product. A good example of this is in chromatograms
produced from amino acid mixtures.
The chromatogram is allowed to dry and is then
sprayed with a solution of ninhydrin. Ninhydrin reacts
with amino acids to give coloured compounds, mainly
brown or purple.
 Visualization of invisible substances
on TLC plate

NB: The paper chromatography is similar to

TLC, but it uses paper instead of TLC plates.
Column Chromatography: intro.
 Activity 4
In groups of 4, watch the video and answer the
following questions:
a. What material is stationary phase made of?
b. What material is mobile phase made of?
c. Describe the procedure of column
chromatography.
Column Chromatography: intro.
 Activity 4
In groups of 4, watch the video and answer the
following questions:
a. What material is stationary phase made of?
b. What material is mobile phase made of?
c. Describe the procedure of column
chromatography.
Column Chromatography:intro. (cont)

Fig. Various columns

Fig. Packing the column with silica gel
Packing the column
Running the column
 Let’s assume that you wanted to separate a mixture
of two coloured compounds - one yellow, one blue.
The mixture looks green.
 Start by making a concentrated solution of the
mixture preferably in the solvent (or solvent system)
to be used in your column.
●First you open the tap to allow the solvent
already in the column to drain so that it levels
with the top of the packing material, and then
add the solution carefully to the top of the column.
Running the column (cont)
●Second, you open the tap again so that the coloured
mixture is all absorbed into the top of the packing
material, so that it might look like this:
Running the column (cont)
 Third, you add fresh solvent to the top of the
column, trying to disturb the packing material as
little as possible. Then you open the tap so that the
solvent can flow down through the column, collecting
it in a beaker or flask at the bottom. As the solvent
runs through, you keep adding fresh solvent to the
top so that the column never dries out.
Running the column (cont)
 As time passes, the separation of the two
compounds of the mixture increases and it will look
like this:
What is happening in the column?
 The blue compound is obviously more polar than the
yellow one - it perhaps even has the ability to hydrogen
bond. You can tell this because the blue compound
doesn't travel through the column very quickly. That
means that it must adsorb more strongly to the silica gel
or alumina than the yellow one. The less polar yellow
one spends more of its time in the solvent and therefore
washes through the column much faster.
 The process of washing a compounds through a column is
known as elution and the solvent used is called eluent.
What if you want to collect the blue
compound as well?
 It is going to take eternities to wash the blue
compound through at the rate it is travelling at the
moment!
 What do you do?

Once the yellow has all been collected, replace the

solvent you have been using by a more polar one.
This will have two effects:
What if you want to collect the blue
compound as well? (cont)
√The polar solvent will compete for space on the silica
gel or alumina with the blue compound. Any space
temporarily occupied by solvent molecules on the
surface of the stationary phase isn't available for blue
molecules to stick to and this will tend to keep them
moving along in the solvent.
√There will be a greater attraction between the polar
solvent molecules and the polar blue molecules. This
will tend to attract any blue molecules sticking to the
stationary phase back into solution.
What if you want to collect the blue
compound as well? (cont)
 The net effect is that with a more polar solvent, the
blue compound spends more time in solution, and so
moves faster
 So why not use this alternative solvent in the first

place?
Your reflection is welcome!
 What if everything in your mixture
is colourless?
 Let's assume that everything is colourless. That is all the
compounds to be separated are colourless!
How will you know when the substance you want has
reached the bottom of the column?
There is no quick and easy way of doing this!
What do you do?
• Collect what comes out of the bottom of the column in a

whole series of labelled tubes. How big each sample is will

obviously depend on how big the column is - you might
collect 1 cm3 samples or 5 cm3 samples or whatever is
appropriate
 What if everything in your mixture
is colourless?(cont)
• Then take a drop from each solution (fraction) and
make a thin layer chromatogram from it.
• By doing this for all fractions, you can identify which
of your samples collected contain the desired product,
and only the desired product.
• Once you know this, you can combine all of the
samples which contain your pure product, and then
remove the solvent.
How do you remove the solvent?
Again your reflections are welcome!
Column Chromatography
 Watch the following video to deepen your
understanding on column chromatography
UV-VIS ANALYSIS
UV-VIS analysis : definition
Activity 5
In groups of 4, use different research engines to
answer the following questions:
 What is the UV-VIS analysis?

 Which instrument is used to carry out UV-VIS

analysis?
 How is UV-VIS done? (think about the procedure)
 Analysis by UV-VIS: definition

UV-VIS (Ultraviolet and visible) Spectroscopy (or

Spectrophotometry) is:
 a quantitative technique used to measure how much

a chemical substance absorbs light.

 a quantitative measurement of the absorption/

transmission or reflection of a material as a function

of wavelength.
 the measurement of the attenuation of the beam of light
after it passes through a sample or after reflection from
a sample surface.
 Analysis by UV-VIS: definition

 This is done by measuring the intensity of light

that passes through a sample with respect to the
intensity of light through a reference sample or
blank.
 Absorption measurement can be at a single

wavelength or over an extended spectral range.

 The wavelength range that is typically used ranges

from 190 nm up to 1,100 nm in the near-infrared.

 Analysis by UV-VIS: definition
 Consider electromagnetic spectrum,
 The region beyond red is called infrared while
that beyond violet is ultraviolet.
 The wavelength range of UV radiation starts at
blue end of visible light (400nm) and end at
200nm)
 UV-VIS analysis : device
 Instrument used is Spectrophotometer

 The following video shows

UV-VIS spectrophotometer
parts and functioning.
UV-VIS analysis: purpose

 Detection of functional groups

 Detection of impurities
 Qualitative analysis
 Quantitative analysis
 Single compound without chromophore
 Drugs with chromophoric reagent
 Relationship between groups and detection of
conjugation of compounds.
 UV-VIS analysis: principle
 Ultraviolet absorptions spectraverse from transition
of electron within a molecule from a lower level to a
high level.
 A molecule absorb ultraviolet radiation of frequency
n and the electrons in that molecule undergo
transition from lower to higher level
 The energy can be calculated by the equation:

n=c/l
The nature of electronic excitation

 Ultraviolet light and visible light have just the

right energy to cause an electronic transition:
the promotion of an electron from one orbital
of lower energy to another of higher energy.
 Depending on the energy needed for the
electronic transition, a molecule will absorb
either ultraviolet or visible light.
The nature of electronic excitation(cont)

 If it absorbs ultraviolet light, a UV spectrum is

obtained;
 if it absorbs visible light, a visible spectrum is

obtained.
 Ultraviolet light is electromagnetic radiation
with wavelengths ranging from 180 to 400 nm
(nanometres);
 visible light has wavelengths ranging from 400

to 800 nm. (One nanometre is 10 Å.)

The nature of electronic excitation(cont)
 Wavelength is inversely related to the energy: The
shorter the wavelength, the greater is the energy.
Ultraviolet light, therefore, has greater energy than
visible light.
The nature of electronic excitation(cont)
 Every time a molecule has a bond, the atoms in a
bond have their atomic orbitals merged to form
molecular orbitals.
 The normal electronic configuration of a molecule is
known as its ground state: all the electrons are in
the lowest-energy molecular orbitals.
 When a molecule absorbs light of an appropriate
wavelength and an electron is promoted to a higher
energy molecular orbital , the molecule is then in an
excited state.
The nature of electronic excitation(cont)
 In other words these electrons when impacted energy
in the form of light radiation get excited from the
highest occupied molecular orbital (HOMO) to the
lowest unoccupied molecular orbital (LUMO) and the
resulting species is known as excited state or anti-
bonding state.
 Thus, an electronic transition is the promotion of an
electron to a higher energy molecular orbital.
 The relative energies of the bonding, nonbonding,
and antibonding molecular orbitals are shown in the
following figure:
The nature of electronic excitation(cont)
The nature of electronic excitation(cont)
 Ultraviolet and visible light have sufficient energy to
cause only the two electronic transitions known as
“n to π star "and “π to π star”
 The electronic transition with the lowest energy is the
promotion of a nonbonding (lone-pair) electron (n) into a
antibonding molecular orbital (stated as “n to π star”)
transition.
 The higher energy electronic transition is the promotion of
an electron from a π bonding molecular orbital into a π*
antibonding molecular orbital, known as a π→π* (stated
as “π to π star”) transition (see figure below).
The nature of electronic excitation(cont)

 This means that only organic compounds with π electrons

can produce UV- VIS spectra.
The UV absorption spectrum
 When a sample is exposed to a light energy that
matches the energy difference between a possible
electronic transition within the molecule, a fraction of
the energy would be absorbed by the molecule and
the electrons would be promoted to the higher
energy state orbital.
 A spectrometer records the degree of absorption by
a sample at different wavelengths and the resulting
plot of absorbance (A) versus wavelength λmax
(stated as “lambda max”) is known as spectrum
The uv-vis absorption spectrum(cont)
Significant features:
λmax: wavelength at which there is maximum
absorption
= molar absorptivity (litre mol-1 cm-1)
max: the intensity of maximum absorption
The uv-vis absorption spectrum(cont)
The uv-vis absorption spectrum(cont)

 UV spectrum of acetone is shown in the following

figure. Acetone has both π electrons and lone-pair
electrons.
 Thus, there are two absorption bands: one for the
π→π* transition and one for the n→π* transition.
The uv-vis absorption spectrum(cont)
The uv-vis absorption spectrum(cont)
 The λmax (stated as “lambda max”) is the wavelength
corresponding to the highest point (maximum
absorbance) of the absorption band.
 For the π→π* transition λmax = 195 nm,

for the n→π* transition, λmax =274nmλmax=274

 We know that the π→π* transition in the above figure
corresponds to the λmax at the shorter wavelength
because that transition requires more energy than the
n→π* transition.
Effect of Conjugation on λmax

 The n→π* transition for methyl vinyl ketone is at

324 nm, and the π→π* transition is at 219 nm.
 Both λmax values are at longer wavelengths than the
corresponding values of acetone because methyl
vinyl ketone has two conjugated double bonds.
Effect of Conjugation on λmax (cont)
Effect of Conjugation on λmax
Effect of Conjugation on λmax
 The above figure shows that: conjugation raises the
energy of the highest occupied bonding molecular
orbital (HOMO) and lowers the energy of the
lowest unoccupied antibonding molecular orbital
(LUMO) so less energy is required for an electronic
transition in a conjugated system than in a
nonconjugated system.
Effect of Conjugation on λmax(cont)
 The more conjugated double bonds there are in a
compound, the less energy is required for the
electronic transition, and therefore the longer is the
wavelength at which the electronic transition occurs.
 The λmax values of the π→π* transition for several
conjugated dienes are shown in the following table.
Effect of Conjugation on λmax(cont)
Effect of Conjugation on λmax
 Notice that both the λmax and the molar absorptivity
increase as the number of conjugated double bonds
increases. Thus, the λmax of a compound can be used
to predict the number of conjugated double bonds
in the compound.
Effect of Conjugation on λmax
 If a compound has enough conjugated double bonds,
it will absorb visible light (λmax >400) and the
compound will be coloured.
 Carotene, a precursor of vitamin A, is an orange
substance found in carrots, apricots, and sweet
potatoes. Lycopene is red and is found in tomatoes,
watermelon, and pink grapefruit.
Effect of Conjugation on λmax
Effect of conjugation
Chromophores
 Chromophore is that part of a molecule that
absorbs UV or visible light.
 The carbonyl group is the chromophore of
acetone.
 The following four compounds all have the
same chromophore, so they all have
approximately the same λmax.
Chromophores (cont)
Class transition
λmax (nm) log

R2C=CR2 π→π* 175 3.0

R-CC-R π→π* 170 3.0
R-CN n→π* 160 ˂1.0
R-N=N-R n→π 340 ˂1.0

R-NO2 n→π* 271 ˂1.0

R-CHO π→π* 190 2.0

n→π* 290 1.0
R2CO π→π* 180 3.0
n→π* 280 1.5
RCOOH n→π* 205 1.5
RCOOR n→π* 205 1.5
Chromophores (cont)
 The attachment of a substituent to a chromophore in
place of hydrogen can modify the absorption of
the principle chromophore
 The substituents increasing the intensity of
absorption and possibly the wavelength are called
auxochromes, ex: methyl, hydroxyl, alkoxy, halogen,
amino groups
Chromophores(cont)
 Other substituents may have any of four kinds of
effects on the absorption:
►bathochromic shift (red shift): shift to lower frequency
or longer wavelength
►hypsochromic shift (blue shift): shift to higher
frequency or shorter wavelength
►hyperchromic effect: an increase in intensity
►hypochromic effect: a decrease in intensity
Chromophores(cont)
Chromophores(cont)
 EX: Because the anilinium ion does not have an
auxochrome, its λmax is similar to that of benzene.
Absorbance
 The Beer-Lambert law relates the attenuation of
light to the properties of the material through which
the light is traveling.
 Beer’s Law or Beer-Lambert Law states that the
amount of energy absorbed or transmitted by a
solution is proportional to the solution’s molar
absorptivity and the concentration of solute.
Absorbance
 The Beer-Lambert law relates the attenuation of
light to the properties of the material through which
the light is traveling.
 Beer’s Law or Beer-Lambert Law states that the
amount of energy absorbed or transmitted by a
solution is proportional to the solution’s molar
absorptivity and the concentration of solute.
 In the following slides we are going to look at the
Beer-Lambert Law and explains the use of the terms
absorbance and molar absorptivity relating to UV-
VIS absorption spectrometry.
 Absorbance (cont)

 For each wavelength of light passing through the

spectrometer, the intensity of the light passing
through the reference cell is measured. This is
usually referred to as Io (I for Intensity).
I0 I

 The intensity of the light passing through the

sample cell is also measured for that wavelength -
given the symbol, I.
Absorbance (cont)
 If I is less than I0, then the sample has
absorbed some of the light (neglecting
reflection of light off the cuvette surface).
 If I = I0, the sample compound does not absorb
light of a given wavelength and there is no
absorption occurred.
 A simple bit of math is then done in the

computer to convert this into something called

the absorbance (A) or transmittance (T) of the
sample.
Absorbance (cont)
 The absorbance (A) is defined via the incident
intensity Io and transmitted intensity I by

 The transmittance is T = I/I0

 If the sample compound does not absorb light of

a given wavelength, there is no absorption, thus
I=I0, A=0 and T=1.
Absorbance (cont)
The absorbance of a transition depends on two
external assumptions:
 The absorbance is directly proportional to the

concentration (c) of the solution of the sample

used in the experiment and to the light path:

 The absorbance is directly proportional to the

length of the light path (L), which is equal to the
width of the cuvette.
Absorbance (cont)
 Combining the first assumptions, we deduce:

 This proportionality can be converted into an equality

by including a proportionality constant (ϵ).

This formula is the common form of the Beer-Lambert

Law, although it can be also written in terms of
intensities:
Absorbance (cont)
 The constant ϵ is called molar absorptivity or molar
extinction coefficient and is a measure of the
probability of the electronic transition.
 On most of the diagrams you will come across, the
absorbance ranges from 0 to 1, but it can go higher
than that.
 An absorbance of 0 at some wavelength means that
no light of that particular wavelength has been
absorbed. The intensities of the sample and reference
beam are both the same, so the ratio Io/I is 1 and
the log10 of 1 is zero.
Absorbance (cont)
 Note that the larger the molar absorptivity, the
more probable the electronic transition.
 In UV spectroscopy, the concentration of the sample
solution is measured in mol L-1 and the length of the
light path in cm.
 Thus, given that absorbance is unitless, the units of
molar absorptivity are L mol-1 cm-1. However, since
the units of molar absorptivity is always the above,
it is customarily reported without units.
Absorbance (cont)
Example 1:
Guanosine has a maximum absorbance of 275 nm.
ϵ275=8400M−1cm−1 and the path length is 1 cm. Using a
spectrophotometer, you find that A275=0.70 .
What is the concentration of guanosine?
Solution:
To solve this problem, you must use Beer's Law:
0.70 = (8400 M-1 cm-1)(1 cm)( c )
C = 0.70/(8400 M-1 cm-1)(1 cm)
c = 8.33x10-5 mol/L
Finding concentration by standard curve

 Using graphic plotting of concentration vs

absorbance of known solutions. Once you find that,
you can compare the absorbance value of an
unknown sample to figure out its concentration.
Finding concentration by standard curve (cont)

 You should have a data set which was used to create a

standard curve. The graph should plot concentration
(independent variable) on the x-axis and absorption
(dependent variable) on the y axis.
 You'll need to add a line of best fit to the data points and

determine the equation for the line. The equation should be

in y= mx + b form.
Where: y = absorbance (A) (no units for absorbance)
x = concentration (C) (unit is M or mol/L)
 Exercises

1. Which molecule absorbs at the longest wavelength?

a) 1,3-hexadiene or 1,4-hexadiene?

b)

c)
 Exercises

 Answers:
1) 1,3 hexadiene (conjugated).
2) B (the ketone participates in conjugation, while the
carbon with the alcohol does not).
3) B (three pi bonds) should absorb at a higher
wavelength than A (2 pi bonds). Molecule A (ergosterol)
absorbs UV light and undergoes a transformation to
molecule B (ergocalciferol) -> this is the first step in
Vitamin D biosynthesis. Hence, this is why sunlight is
needed to produce vitamin D.
 Exercises

2) The ultraviolet spectrum of benzonitrile shows a

primary absorption band at 224 nm. If a solution
of benzonitrile in water, with a concentration of
1x 10-4 molar, is examined at a wavelength of
224 nm, the absorbance is determined to be
1.30. The cell length is 1 cm. What is the molar
absorptivity of this absorption band?
 Exercises

3) The ultraviolet spectrum of benzonitrile shows a

secondary absorption band at 271 nm. If a
solution of benzonitrile in water, with a
concentration of 1×10-4molar solution is
examined at 271 nm, what will bethe absorbance
reading (ℇ= 1000) and what will be the intensity
ratio, IO/I, respectively?
 Exercises

4) A UV/vis spectrum of a 0.0001 M solution of a

compound in methanol in a 0.5 cm pathlength
quartz cuvette is recorded. The spectrum shows
λmax = 235 nm with an absorbance of 1.05.
a) Calculate εmax at 235 nm. b) What absorbance
would be measured if the solution was contained in a 0.1
cm quartz cuvette?
c) What absorbance would be measured if the solution
was diluted 1:1 with more methanol?
 Exercises

5) A student records a UV/vis spectrum of p-nitroaniline

from a benzene solution. The student is confused by the
spectrum and takes it to a professor for advice. The
professor is at first sad, then sighs, and sends the student
away to record the spectrum from a solution in a
different solvent.
(a) Give two likely reasons why the professor was sad
that the student used benzene as a solvent in this work.
(b) Suggest a solvent that the student could use to
appease the professor's sadness.
NMR ANALYSIS
NMR spectroscopy
Activity 6
In groups of 4, use different research engines to
answer the following questions:
 What is the NMR spectroscopy?

 Which kind of nuclei are involved in the NMR?

 How are molecules recognized using NMR?

NMR spectroscopy
 Nuclear magnetic resonance (NMR) spectroscopy is
the study of molecules by recording the interaction
of radiofrequency (Rf: 60 - 100 MHz)
electromagnetic radiations with the nuclei of
molecules placed in a strong magnetic field.
 Nuclear magnetic resonance (NMR) is concerned
with the magnetic properties of certain nuclei.
 It is used to determine the structure of organic
compounds.
NMR spectroscopy
 NMR uses a large magnet (Magnetic) to probe the
intrinsic spin properties of atomic nuclei.
 Like all spectroscopies, NMR uses a component of
electromagnetic radiation (radio frequency waves)
to promote transitions between nuclear energy
levels (Resonance).
 Most chemists use NMR for structure determination
of small molecules.
NMR spectroscopy
 Some types of atomic nuclei (isotopes) act as they
spin on their axis similar to the earth since they are
positively charged. They generate an
electromagnetic field and act as tiny bar magnetics.
NMR spectroscopy
 In nature these nuclei are oriented in a random
fashion, pointing in all different directions.
NMR spectroscopy
 Once placed in a strong applied magnetic field
(external magnetic field (B0), they align with the
external field and.
NMR spectroscopy
 There are 2 basic orientations, (two spin states exist):
Alpha (+1/2) and Beta (-1/2).
 Alpha: aligns ‘with’ the field (north of the nucleus to
south of the field and vice versa) is the low energy
orientation.
 Beta: opposed to the external field (north poles and
south poles facing) is the high energy orientation.
 NMR spectroscopy
 The difference in energy between the two spin states
is dependent on the external magnetic field strength,
and is always very small.
ΔE : energy difference or radio
frequency applied
µ : magnetic moment of particular nuclei
Bx : external magnetic field
I : spin state (1/2)
The two spin states have the same energy when the
external field is zero, but diverge as the field increases.
The international unit for magnetic flux is the tesla (T)
NMR spectroscopy
 For NMR purposes, this small energy difference (ΔE)
is usually given as a frequency in units of MHz (106
Hz), ranging from 20 to 900 Mz, depending on the
magnetic field strength and the specific nucleus being
studied.
 Irradiation of a sample with radio frequency (Rf)
energy corresponding exactly to the spin state
separation of a specific set of nuclei will cause
excitation of those nuclei in the +1/2 state to the
higher -1/2 spin state.
NMR spectroscopy
 All nuclei with an odd number of protons (e.g.
1H, 2H, 14N, 19F, 31P ...) or nuclei with an odd number

of neutrons (e.g. 13C) show the magnetic properties

required for NMR.
 Only nuclei with even number of both protons and
neutrons (12C and 16O) do not have the required
magnetic properties.
 This module focuses on the magnetic behaviour of
hydrogen nuclei; hence the term proton NMR or 1H-
NMR.
Proton NMR Spectroscopy
 Hydrogen atoms as little magnets
 We all know that compass needle, normally lines up with
the Earth's magnetic field with the north-seeking end
pointing north. You can twist the needle around with your
fingers so that it can point south - lining it up opposed to
the Earth's magnetic field.
 It is very unstable opposed to the Earth's field, and as
soon as you let it go again, it will flip back to its more
stable state.
Hydrogen atoms as little magnets(cont)
 Hydrogen nuclei also behave as little magnets and
a hydrogen nucleus can also be aligned with an
external magnetic field or opposed to it. Again, the
alignment where it is opposed to the field is less
stable (at a higher energy).
 It is possible to make it flip from the more stable
alignment to the less stable one by supplying
exactly the right amount of energy.
Hydrogen atoms as little magnets(cont)
Hydrogen atoms as little magnets(cont)

 The energy needed to make this flip depends on the

strength of the external magnetic field used, but is usually in
the range of energies found in radio waves (see
electromagnetic spectrum on next slide)- at frequencies of
about 60 - 100 MHz.
 It's possible to detect this interaction between the radio
waves of just the right frequency and the proton as it flips
from one orientation to the other as a peak on a graph.
 This flipping of the proton from one magnetic alignment to
the other by the radio waves is known as the resonance
condition.
Hydrogen atoms as little magnets(cont)
The hydrogen atom's environment

 What we've said so far would apply to an isolated

proton, but real protons have other things around
them - especially electrons. The effect of the
electrons is to cut down the size of the external
magnetic field felt by the hydrogen nucleus.
The hydrogen atom's environment
 Since electrons are charged particles, they move in
response to the external magnetic field (Bo) so as to
generate a secondary field that opposes the much
stronger applied field. This secondary field shields
the nucleus from the applied field.

 Bo must be increased in order to compensate for the

induced shielding field and achieve resonance
(absorption of Rf energy).
The hydrogen atom's environment(cont)
 Suppose you were using a radio frequency of 90 MHz,
and you adjusted the size of the magnetic field so that
an isolated proton was in the resonance condition.
 If you replaced the isolated proton with one that was
attached to something, it wouldn't be feeling the full
effect of the external field any more and so would stop
resonating (flipping from one magnetic alignment to the
other).
 The resonance condition depends on having exactly the
right combination of external magnetic field and radio
frequency.
The hydrogen atom's environment(cont)
 How would you bring it back into the resonance
condition again?
You would have to increase the external magnetic
field slightly to compensate for the effect of the
electrons.
• Now suppose that you attached the hydrogen to
something more electronegative. The electrons in the
bond would be further away from the hydrogen
nucleus, and so would have less effect on the
magnetic field around the hydrogen.
The hydrogen atom's environment(cont)
.
The hydrogen atom's environment(cont)

 Hence the external magnetic field needed to bring

the hydrogen into resonance will be smaller if it is
attached to a more electronegative element,
because the hydrogen nucleus feels more of the
field.
 Even small differences in the electronegativities of
the attached atom or groups of atoms will make a
difference to the magnetic field needed to achieve
resonance.
Summary

 For a given radio frequency (say, 90 MHz) each

hydrogen atom will need a slightly different
magnetic field applied to it to bring it into the
resonance condition depending on what exactly it is
attached to - in other words the magnetic field
needed is a useful guide to the hydrogen atom's
environment in the molecule.
Features of an NMR spectrum
 A simple NMR spectrum looks like this:
 Features of an NMR spectrum(cont)
√The peaks
 Referring to the above-mentioned spectrum:

• There are two peaks because there are two different

environments for the hydrogens - in the CH3 group and
attached to the oxygen in the COOH group.
• They are in different places in the spectrum because they
need slightly different external magnetic fields to bring
them in to resonance at a particular radio frequency.
• The sizes of the two peaks gives important information
about the numbers of hydrogen atoms in each environment.
Features of an NMR spectrum(cont)
Cont.
• It isn't the height of the peaks that matters, but the
ratio of the areas under the peaks. If you could
measure the areas under the peaks in the diagram
above, you would find that they were in the ratio of
3 (for the larger peak) to 1 (for the smaller one)
• That shows a ratio of 3:1 in the number of
hydrogen atoms in the two environments - which is
exactly what you would expect for CH3COOH.
Features of an NMR spectrum(cont)
√The need for a standard for comparison - TMS
Again consider NMR spectrum for ethanoic acid above:
• The horizontal scale has a zero point at the right-hand end.

• The zero is where you would find a peak due to the

hydrogen atoms in tetramethylsilane (TMS). Everything else
is compared with this (Usually small amount of an inert reference
standard TMS is added to the sample tube containing the compound
whose NMR spectrum is to be taken).
Features of an NMR spectrum(cont)
 You will find that some NMR spectra show the peak due
to TMS (at zero), and others leave it out.
 If you have to analyse a spectrum which has a peak at
zero, you can ignore it because that's the TMS peak.
 TMS is chosen as the standard for several reasons. The
most important are:
• It has 12 hydrogen atoms all of which are in exactly the same
environment. They are joined to exactly the same things in
exactly the same way. That produces a single peak, but it's
also a strong peak (because there are lots of hydrogen atoms).
Features of an NMR spectrum(cont)
Cont.
 The electrons in the C-H bonds are closer to the

hydrogens in this compound than in almost any other

one. That means that these hydrogen nuclei are the
most shielded from the external magnetic field, and
so you would have to increase the magnetic field by
the greatest amount to bring the hydrogens back
into resonance
→The net effect of this is that TMS produces a peak on the
spectrum at the extreme right-hand side. Almost everything
else produces peaks to the left of it.
Features of an NMR spectrum(cont)
√Solvents for NMR spectroscopy
• NMR spectra are usually measured using solutions

of the substance being investigated (sample being

analysed).
• It is important that the solvent itself doesn't contain

any simple hydrogen atoms, because they would

produce confusing peaks in the spectrum.
• You can also use the solvent in which any ordinary

hydrogen atoms are replaced by its isotope,

deuterium (D).
Features of an NMR spectrum(cont)
√Solvents for NMR spectroscopy
• Unfortunately, CCl4 is a poor solvent for many polar

compounds and is also toxic.

• Deuterium labeled compounds, such as deuterium
oxide (D2O), chloroform-d (DCCl3), benzene-
d6 (C6D6), acetone-d6 (CD3COCD3) and DMSO-
d6 (CD3SOCD3) are now widely used as NMR
solvents. Since the deuterium isotope of hydrogen
has a different magnetic moment and spin, it is
invisible in a spectrometer tuned to protons.
Features of an NMR spectrum(cont)
√The chemical shift
 The chemical shift is the resonant frequency of an

atomic nucleus relative to a standard in a magnetic

field.
 In absolute terms, it is defined by the frequency of

the resonance expressed with reference to a

standard compound which is defined to be at 0
ppm.
 The scale is made more manageable by expressing
it in parts per million (ppm) and is indepedent of
the spectrometer frequency.
Features of an NMR spectrum(cont)
√The chemical shift
 The NMR spectrum is displayed as a plot of the

applied radio frequency versus the absorption.

 The applied frequency increases from left to right,
thus the left side of the plot is the low field
(downfield or deshielded side) and the right side
of the plot is the high field (upfield or shielded
side).
Features of an NMR spectrum(cont)
√The chemical shift
Features of an NMR spectrum(cont)
√The chemical shift
 The horizontal scale is shown as δ (delta) (in ppm).

 δ is called the chemical shift and is measured in

parts per million - ppm.
 The delta (δ) scale is independent of the

spectrometer frequency .
Features of an NMR spectrum(cont)
√The chemical shift
 For e.g. A peak at a chemical shift of, say, 2.0

means that the hydrogen atoms which caused that

peak need a magnetic field two millionths less than
the field needed by TMS to produce resonance.
 A peak at a chemical shift of 2.0 is said to be
downfield of TMS. The further to the left a peak is,
the more downfield it is.
NMR sample analysis
 To obtain an NMR spectrum, one dissolves a small
amount of a compound in about 0.5 mL of solvent
and puts the solution into a long, thin glass tube,
which is placed within a powerful magnetic field
(figure below).
 Spinning the sample tube about its long axis
averages the position of the molecules in the
magnetic field and thus greatly increases the
resolution of the spectrum.
 NMR sample analysis

An NMR tube filled with a colorless sample, sealed with a green/yellow polyethylene
cap and Parafilm
NMR sample analysis
Experimental analysis: NMR spectrometer

 A sample (in a small glass tube) is placed between

the poles of a strong magnetic.
 A radio frequency generator pulses the sample and
excites the nuclei causing a spin-flip.
 The spin flip is detected by the detector and the
signal sent to a computer where it is processed.
Experimental analysis: NMR spectrometer(cont)

Autosampler
of NMR
spectrometer
loaded with
samples for
analysis.
 NMR spectrum : Interpretation
 Dissolve
 Summary
A quick recap of the NMR process.
1
 Dissolve the sample in solvent without H and add in a small
amount of TMS (a reference molecule).
 Apply radio waves to the solution.

 Some of the hydrogen atoms in the sample absorb the energy

from the radio waves and flip to their antiparallel, spin-

opposed state.
 A spectrum is produced. It shows chemical shift, a property
related to resonance frequency.
 Compare chemical shift values to those in a data table to
work out the environment of the hydrogen atoms present in our
sample.
Problems
 1. Determine the structure of the following
unknown saturated compound based on its
molecular formula (C6H12) and its 1HNMR spectra
Problems
 2. Characterise an unknown alkene with a
molecular formula C6H12 and showing the following
1HNMR spectrum:
Problems
 3. The 1HNMR spectrum of unknown 6C
hydrocarbon compound shows a single signal.
Characterize the compound if its spectrum is the one
given below:
Problems
 4. Characterise the unknown organic compound
whose molecular formula is C2H5Br and 1HNMR is
shown below:
Problems
 5. The molecular formula of an unknown organic
compound is C3H7Br and its 1HNMR is shown below.
Determine its structural formula.
Problems
 6. 2-cholorobutene shows 4 different hydrogen
signals. Explain why this is.
 7. How many non-equivalent hydrogen are in the
following molecules; how many different signals will
you see in a H1 NMR spectrum.
A. CH3CH2CH2Br
B. CH3OCH2C(CH3)3
C. Ethyl Benzene
D. 2-methyl-1-hexene
Problems
 8. Identify the different equivalent protons in the
following molecule and predict their expected
chemical shift.
Problems
 9. Predict how many signals the following molecule
would have? Sketch the spectra and estimate the
integration of the peaks.
Problems
 10. Determine the structure for the following
spectrum corresponding to the molecule C3H8O.
IR ANALYSIS
 Basics of IR spectroscopy
 You probably know that visible light is made up of
a continuous range of different electromagnetic
frequencies and each frequency can be seen as a
different colour. Infra-red radiation also consists of
a continuous range of frequencies. It so happens
that our eyes can't detect them.
 If you shine a range of infra-red frequencies one at
a time through a sample of an organic compound,
you find that some frequencies get absorbed by the
compound.
Basics of IR spectroscopy (cont)
 A detector on the other side of the compound would show
that some frequencies pass through the compound with
almost no loss, but other frequencies are strongly
absorbed.
 How much of a particular frequency gets through the
compound is measured as percentage transmittance.
 A percentage transmittance of 100 would mean that all of
that frequency passed straight through the compound
without any being absorbed. In practice, that never
happens - there is always some small loss, giving a
transmittance of perhaps 95% as the best you can achieve.
Basics of IR spectroscopy (cont)
 A transmittance of only 5% would mean that nearly
all of that particular frequency is absorbed by the
compound. A very high absorption of this sort tells
you important things about the bonds in the
compound.
How does IR spectrum look like?

 A graph is produced showing how the percentage

transmittance varies with the frequency of the infra-
red radiation.
 How does IR spectrum look like?(cont)

 You can see that an unusual measure of frequency is

used on the horizontal axis. Wavenumber is defined
like this:
 Why some frequencies are absorbed
by the sample?
 Each frequency of light (including infra-red) has a
certain energy. If a particular frequency is being
absorbed as it passes through the compound being
investigated (or analysed), it must mean that its
energy is being transferred to the compound(or
sample).
 Energies in infra-red radiation correspond to the
energies involved in bond vibrations
 Why some frequencies are
absorbed by the sample? (cont)
Bond stretching
 In covalent bonds, the two atoms are held together

because both nuclei are attracted to the same pair

of electrons. The two nuclei can vibrate backwards
and forwards or towards and away from each
other, around an average position
 For example, let’s take the carbon-oxygen bond in

methanol, CH3OH.
Why some frequencies are
absorbed by the sample? (cont)
 The energy involved in this vibration depends on things
like the length of the bond and the mass of the atoms at
either end. That means that each different bond will
vibrate in a different way, involving different amounts of
energy.
 Normally bonds vibrate all the times, but if you shine
exactly the right amount of energy on a bond, you can
kick it into a higher state of vibration.
 The amount of energy it needs to do this will vary from
bond to bond, and so each different bond will absorb a
different frequency (and hence energy) of infra-red
radiation.
 Why some frequencies are
absorbed by the sample? (cont)
Bond bending
 Bonds can also bend. The diagram shows the

bending of the bonds in a water molecule.

 The effect of this, of course, is that the bond angle
between the two hydrogen-oxygen bonds fluctuates
slightly around its average value
Why some frequencies are
absorbed by the sample? (cont)
 Again, bonds will be vibrating like this all the time
and, again, if you shine exactly the right amount of
energy on the bond, you can kick it into a higher
state of vibration
 Since the energies involved with the bending will be
different for each kind of bond, each different
bond will absorb a different frequency of infra-red
radiation in order to make this jump from one state
to a higher one.
 Why some frequencies are
absorbed by the sample? (cont)
Tying both stretching and bending together
 Look again at the infra-red spectrum of propan-1-

ol, CH3CH2CH2OH:
 Why some frequencies are
absorbed by the sample? (cont)
 In the diagram, three sample absorptions are
picked out to show you the bond vibrations which
produced them. Notice that bond stretching and
bending produce different troughs in the spectrum.
What are the regions of IR spectrum?
 Two pats:
• The functional group regions
The left-hand two-thirds of an IR spectrum is where
most of the functional groups show absorption bands.
It is called the functional group region.
• The fingerprint regions
 The right-hand third of the IR spectrum is called the
fingerprint region because it is characteristic of the
compound as a whole, just as a fingerprint is characteristic of
an individual.
 What are the regions of IR
spectrum? (cont)
 The IR spectrum is very complex due to the
stretching and bending of each bond giving rise to
different bands.
 Organic chemists generally do not try! to identify
all the absorption bands in an IR spectrum
 Some characteristic bands allows to tell something
about the structure of a compound that gives a
particular IR spectrum
 Some useful absorptions

No Type of bond Wave number (cm-1) region

1 O-H 2500 - 3300 cm-1 Functional group
2 C=O 1680 - 1750 cm-1. Functional group
3 C-H 2853 - 2962 cm-1 Functional group
4 C-O 1000 and 1300 cm-1 Fingerprint
5 C-C Difficult to pick due to Fingerprint
wide range
Interpreting infrared spectrum
 The infra-red spectrum for a simple carboxylic acid
Ethanoic acid has the structure:

You will see that it contains the following bonds:

carbon-oxygen double, C=O
carbon-oxygen single, C-O
oxygen-hydrogen, O-H
carbon-carbon single, C-C
carbon-hydrogen, C-H
Interpreting infrared spectrum (cont)
 The IR for ethanoic acid looks like this:
Interpreting infrared spectrum

 Try to find O-H, C-H and C-O in IR of ethanol

below
Part 2: STATISTICAL ANALYSIS
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End