ANALYTICAL

CHEMISTRY

By

Dr. Emmanuel GAKUBA and Dr. Pacifique

Felicite MUTUYIMANA
University of Rwanda
College of Education
Part1: Chemical analysis
Chemical separation
 “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
What are the techniques for chemical separation?

❑ Techniques available for Chemical Separations:

➢ Extraction
➢ Distillation

➢ Precipitation
➢ Chromatography

➢ Many others (centrifugation, filtration, etc)

Extractions and Chromatography are especially useful in

analytical methods. These will be detailed in this module
Extraction
 Extraction

❑ 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
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 separatory 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
The properties of the solvent used for
liquid-liquid extraction
❑The solvent should be well immiscible 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 been 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 8 extractions,
After mixing, UO2(NO3)2 UO2(NO3)2 has been
Is distributed in both removed from water
layers
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
❑ 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 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
Example
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
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.
Solid phase 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.
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
❑ 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
❑ 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
➢ Water- and fat-soluble vitamins.
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)
 In this module, we will focus on types of chromatography according to
the stationary phase.
 Considering stationary phase (SP), there are 3 types of
chromatography namely thin layer chromatography, paper
chromatography and 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.
 In TLC, the stationary phase is a thin layer of silica gel or alumina on a
glass, metal or plastic plate. Column chromatography works on a much
larger scale by packing the same materials into a vertical glass
column.
 Various sizes of chromatography columns are used depending on the
amount of material to be loaded in it.
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!