CHE3243

ADVANCED ANALYTICAL
CHEMISTRY

By

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 & pollution
science, second edition,
Academic Press is an imprint of Elsevier, London
2. 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_
What is chemical analysis?
 General introduction
Is it possible to confirm the purity of water just by looking at it?
I think the answer is No!
Likewise, we cannot confirm the amount of pollutant in our
environment by naked eye.
To make conclusion on the above questions or any other question
that you may have, we use chemical analysis.
Chemical analysis is the process of identifying, separating
and quantifying the components of a sample to understand its
nature and composition.

 General introduction (cont)
Chemical analysis is used in many aspects of daily life, including:
• Food testing: Determines nutritional content, detects
contaminants, and ensures quality.
• Environmental monitoring: Detects pollutants in air, soil, and
water.
• Medicine: Used for blood tests, drug testing, and developing new
medications.
• Forensics: Analyzes evidence
• Consumer products: Tests for safety and effectiveness.
There are different types of chemical analysis, as well as
General introduction (cont)
Steps of chemical
analysis
 General introduction (cont)
Types of chemical
analysis
Chemical analysis is classified into two major types
Qualitative chemical analysis.
Quantitative chemical analysis.
Qualitative chemical analysis is a type of analysis
used to identify the substances within a sample
and determine whether a particular
substance is present or not.
Quantitative chemical analysis is a type of
 General introduction (cont)
Chemical analysis: tests, methods,
Testing forand
pure techniques
substances using
melting and boiling points.
Testing for gases using simple test tube
reactions.
Testing for ions using reactions that you
can carry out in class, such as flame
tests, the sodium hydroxide test, and
tests for anions.
 General introduction (cont)
Instrumental
The tests for gases and the sodium hydroxide test
analysis
are all simple reactions that you can try out in
class.
They are quick and convenient to use, but they
are qualitative, not quantitative.
If we want a quantitative measure of the sample,
we must use certain analytical instruments.
They not only identify
components of a sample but also measure their relative
Sample
preparation
 Sample
preparation
Sample preparation is the most crucial step in any
analytical procedure.
 It is the limiting factor in chemical analysis since it is time
consuming.
 Sample
preparation
Sample preparation can also potentially introduce errors in
the measurement.
 Sample
preparation
No one can doubt that the best approach would be the direct
introduction of the sample to the instrument, however this is
rarely feasible.
Efficient sample pretreatment is inevitably required as the
instrument technology has produced highly sophisticated and
sensitive analytical equipment.
The analytical scientists have developed and apply a suitable
sample preparation protocol that ensures that:
• The composition of the sample remains unchanged.
• No impurities are introduced during handling.
• All interferences have been left back.
• The analytes’ concentration is not only at detectable levels, but it
 Sample
preparation
There are many techniques available for sample preparation, but
extractions and chromatography are exceptionally useful in
analytical methods.
Extraction
 Extraction
 Extraction" refers to a transference of
compound(s) from a solid or liquid into a
different solvent or phase.
 Or simply, the process of moving a species
from one phase to another phase.
The two phases can be liquid-liquid, liquid-
solid, gas-solid, etc…
 For instance, when a tea bag is added to hot
water, the compounds responsible for the
 Extraction
 Extraction technique separate components of a mixture (solutes)
by exploiting differences in their relative solubilities in two
immiscible liquids or between their affinities for a solid sorbent.
 Here are some widely used extraction techniques across various
fields:
 Solvent Extraction (Liquid-Liquid Extraction)
 Solid-Phase Extraction (SPE)
 Soxhlet Extraction
 Supercritical Fluid Extraction (SFE)
 Ultrasound-Assisted Extraction (UAE)
 Microwave-Assisted Extraction (MAE)
 The choice of extraction method depends on the nature of the
Liquid-liquid/
Solvent
extractions
 Liquid-liquid/Solvent extractions
It involves the separation of
impurities/analytes from liquid
solution by contacting it with another
immiscible (solvent) in which the
impurities/analytes have high affinity.
 Liquid-liquid/Solvent extractions
Let's see an example.
 Suppose that you have a mixture of sugar in vegetable oil (it
tastes sweet!) and you want to separate the sugar from the
oil. You observe that the sugar particles are too tiny to filter
and you suspect that the sugar is partially dissolved in the
vegetable oil.
 Liquid-liquid/Solvent
extractions
 How about shaking the mixture with water?
 Will it separate the sugar from the oil? Sugar is much more
soluble in water than in
vegetable oil, and, as you know,
water is immiscible (=not soluble) with oil.
 Did you see the result? The water phase is the
bottom layer and the oil phase is the top layer,
because water is denser than oil.
*You have not
shaken the mixture yet, so sugar is still in the oil phase.
 Liquid-liquid/Solvent
extractions
 Now the water phase tastes sweet, because the
sugar is moved to the water phase upon shaking.
**You extracted sugar from the oil with water !!!!.**
In this example,
 Water was the extraction solvent.
 The original oil-sugar mixture was the solution to be extracted.
 And sugar was the compound extracted from one phase to
another.
 Separating the two layers accomplishesDid theyou get
separation of the
it? .....the concept
sugar from the vegetable oil. of liquid-liquid
extraction?
 Liquid-liquid/Solvent
extractions
Extraction becomes a very useful tool if you choose a suitable
extraction solvent.
You can use extraction to separate a substance selectively
from a mixture, or to remove unwanted impurities from a
solution.
In the practical use, usually one phase is a water or water-
based (aqueous) solution and the other an organic solvent
which is immiscible with water.
The success of this method depends upon the difference in
 Liquid-liquid/Solvent
extractions
How do you select solvents to be used in LLE
A solvent miscibility chart is a
useful aid for determining which
solvent pairs are immiscible and
would therefore be potential
candidates for use in LLE.
More solvent combinations are
miscible than immiscible, and
more solvents are immiscible
with water than with any other
 Liquid-liquid/Solvent
extractions
How do you select solvents to be used in LLE
Data on the
solubility of various
solvents in water
and on the solubility of
water in other solvents
should also be
consulted when
selecting an extraction
solvent pair.
 Liquid-liquid/Solvent
extractions
Partition Coefficient Kp (Distribution Coefficient K
 When a compound is shaken in a separatory funnel with two
immiscible solvents, the compound will distribute itself between
the two solvents.
 Normally one solvent is water and the other solvent is a water-
immiscible organic solvent.

Most organic compounds are more soluble in organic solvents,

 Liquid-liquid/Solvent
extractions
Partition Coefficient Kp (Distribution Coefficient K
 Here is the universal rule:
‘‘At a certain temperature, the ratio of concentrations of a
solute in each solvent is always constant’’.
This ratio is called the distribution coefficient, K.
 Liquid-liquid/Solvent
extractions
Partition Coefficient Kp (Distribution Coefficient K
 For example, suppose the compound has a distribution
coefficient K = 2 between solvent1 and solvent2.
 Liquid-liquid/Solvent
extractions
Partition Coefficient Kp (Distribution Coefficient K
 What happens if we extract twice with 100 mL of solvent 2 instead of
200 ml?
 As seen previously, one extraction with 200 mL
gave a TOTAL of 240 particles.
 Once 200 ml is divided in two portions, the first
addition of 100 ml of fresh solvent 2 will
extract 200 particles leaving
behind 100 ml of solvent1 with
100 particles.
 Liquid-liquid/Solvent
extractions
Partition Coefficient Kp (Distribution Coefficient K
 Now lets compare two extraction pathways.
 It evident that is more efficient to
carry out two extractions with 1/2
volume of extraction solvent than
one large volume!
 Thus the greater the number of
small extractions, the greater the
quantity of solute removed.
 However for maximum efficiency
 Liquid-liquid/Solvent
extractions
Partition Coefficient Kp (Distribution Coefficient K
 Illustration

Ether
layer
Water
layer
1M
UO2(NO3)
2
(yellow) After mixing, After 8 extractions,
UO2(NO3)2 UO2(NO3)2 has been
Is distributed in
 Liquid-liquid/Solvent
extractions
Extraction
 In liquid-liquid extraction,
Efficiency
the efficiency of an extraction depends on:
 The value of the distribution ratio, D.
 Relative volumes of the two liquid phases.
 The percentage of a solute extracted, E, is given by the expression:

(1) Where Vaq and Vorg are the

volumes of the
aqueous and
 For solutes with small organic
values of D,phases, respectively.
multiple extractions will improve
the overall efficiency, and an alternative expression enables this to
be calculated. (2) Where Caq is the amount of
analyte in aqueous
 Liquid-liquid/Solvent
extractions
Example
A water sample contains 10 mg each of a halogenated pesticide and an
ionic herbicide which are to be separated by extraction of the pesticide
into methylbenzene. Given that the pesticide distribution ratio, D, for
methylbenzene/water is 50, calculate the extraction efficiency for:
(i) One extraction of 20 cm3 of water with 10 cm3 of
methylbenzene
(ii) One extraction of 20 cm3 of water with 30 cm3 of
methylbenzene
(iii) Three extractions of the same 20 cm 3 of water with 10 cm3
 Liquid-liquid/Solvent
extractions
Solution

It is clear that increasing the volume of organic solvent or extracting

with the same volume divided into several smaller portions, increases
 Liquid-liquid/Solvent
extractions
Chemically active (acid-base)
extraction

Can you change the solubility property of a

compound?
If yes. How?
 Liquid-liquid/Solvent
extractions
Chemically active (acid-base)
extraction
 Most organic compounds are
more soluble in organic
solvents than in water, usually
by the distribution coefficient K
>4
 However, specific classes of
organic compounds can be
This
reversibly alteredtechnique
is a powerful chemicallyand
to allows you to separate
becomecompounds
organic more water-soluble
from a mixture -- if they belong to
 Liquid-liquid/Solvent
extractions
Chemically active (acid-base)
extraction

What type of organic compounds can be made

water-soluble?
 Liquid-liquid/Solvent
extractions
Chemically active (acid-base)
 Compoundsextraction
belonging to the following solubility classes can be
converted to their water-soluble salt form.
1. Organic acids include carboxylic acids (strong organic
acids) and phenols (weak organic acids).
 Liquid-liquid/Solvent
extractions
Chemically active (acid-base)
 Compoundsextraction
belonging to the following solubility classes can be
converted to their water-soluble salt form.
2. Organic bases includes amines.
 Liquid-liquid/Solvent
extractions
Chemically active (acid-base)
extraction

How can organic acids or bases be converted to a

water-soluble form?
 Liquid-liquid/Solvent
extractions
Chemically active (acid-base)
extraction
 Organic Acids can be converted to their salt form when
treated with an aqueous solution of inorganic base
(e.g., NaOH and NaHCO3).
 Salts are ionic, and in general, ions are soluble in
water but not soluble in water-immiscible organic
solvents.
 Remember: water is a very polar solvent thus salts
(i.e., ionic species) are well dissolved in it.
 Liquid-liquid/Solvent
extractions
Chemically active (acid-base)
extraction
 Carboxylic Acids are converted
to the salt form with 5% NaOH
aqueous solution. NaOH is a
strong inorganic base.
 Carboxylic acids are strong
organic acids (pKa = 3 to 4),
so they can also be ionized
with weak inorganic bases
e.g., NaHCO (sodium
 Liquid-liquid/Solvent
extractions
Chemically active (acid-base)
extraction
Let's try a sample problem.
 Here is a mixture of naphthalene and benzoic acid, dissolved in
dichloromethane.

 You want to separate these two compounds. What will

you do?
 Liquid-liquid/Solvent
extractions
Chemically active (acid-base)
 You may useextraction
an aqueous
solution of either 5% NaOH
Add 5%
or sat. NaHCO3, to extract NaOH/
sat.
NaHCO3
benzoic acid as a salt form solution
 NaOH/sat. NaHCO3 will only

react with benzoic acid,

then benzoic acid will get
 Liquid-liquid/Solvent
extractions
Chemically active (acid-base)
extraction
 Phenols are considered to be weak organic acids. Phenol, the
parent compound, is partially water-soluble whereas substituted
phenols are not.
 NaHCO3 aqueous solution, a weak inorganic base, will not
deprotonate phenols to make it ionic, because it is not
strong enough.
 However, treatment with NaOH, a strong inorganic base, can
change phenol to its ionic (salt) form.
 Liquid-liquid/Solvent
extractions
Chemically active (acid-base)
extraction
Let's try another sample problem.
 Here is a mixture of benzoic acid and p-methoxyphenol, dissolved in
dichloromethane.

 You want to separate these two compounds. What will you

do?
 Liquid-liquid/Solvent
extractions
Chemically active (acid-base)
extraction
 You cannot use 5% NaOH
to separate these two
Add 5%
compounds. NaOH
solutio
 NaOH will react with both n

benzoic acid and p-

methoxyphenol, thus both
compounds will be
 Liquid-liquid/Solvent
extractions
Chemically active (acid-base)
Let's try this extraction
problem again.

 Here is another mixture of benzoic acid and p-methoxyphenol,

dissolved in dichloromethane.

 You want to separate these two compounds. What will

you do?
 Liquid-liquid/Solvent
extractions
Chemically active (acid-base)
extraction
 NaOH was too strong a base,
thus it does not differentiate Add
NaHCO
the strong and weak organic 3
solutio
acids. n

 Use of weak inorganic base

such as NaHCO3 will

Strong organic acids such as benzoic acid would be
differentiate deprotonated
between the and ionized, while weak organic acids such
as phenols would NOT be deprotonated.
 Liquid-liquid/Solvent
extractions
Chemically active (acid-base)
extraction
 Organic Bases (amines) can be converted to their salt form
when treated with an aqueous solution of an inorganic acid such
as HCl.
 Recall that salts are ionic and generally soluble in water but not
soluble in water-immiscible organic solvents.
 Liquid-liquid/Solvent
extractions
Chemically active (acid-base)
extraction
Let's try a third sample problem.
 Here is a mixture of benzoic acid and p-chloroaniline, dissolved in
dichloromethane.

 You want to separate these two compounds. What will

you do?
 Liquid-liquid/Solvent
extractions
Chemically active (acid-base)
extraction
 You may use an aqueous
Add
solution of either 5% 5%
HCl
HCl, to extract the solutio
n
amine as a salt form and
benzoic acid has to
remain in the organic
 Liquid-liquid/Solvent
extractions
Chemically active (acid-base)
 Now, You can
extraction
separate four different classes of compounds
from a mixture based on differing solubility properties.
 The four classes are:
• Amines (organic base)
• Carboxylic acids (strong acid)
• Phenols (weak acid)
• Neutral compounds.
Liquid-liquid/Solvent
extractions
Chemically active (acid-base)
extraction
 Liquid-liquid/Solvent
extractions
Chemically active (acid-base)
 After the separation
extraction
of the mixture of four components, we will
have four solutions: each solution contains one component.

 How can we recover each compound obtained from the

separation process?
 Liquid-liquid/Solvent
extractions
Chemically active (acid-base)
1. Isolation extraction
(Recovery) of
 amines
An amine is soluble in acidic aqueous solution because it forms a
salt, an ionic form.
 However, if you change the pH of the solution to basic the
Basification must be
amine can no longer stay dissolved because it is no longer ionic! This
done
carefully, portion by
process is
portion, called basification.
with
swirling each time
because the
acid-base neutralization
reaction
is exothermic.
 Liquid-liquid/Solvent
extractions
Chemically active (acid-base)
2. Isolation extraction
(Recovery) of
 acids
Phenol and carboxylic acid can be returned to the
original form in the same
manner!
 Organic acids are currently dissolved in a basic
aqueous solution, because the acid forms a
salt, an ionic form.
 When you make the aqueous solution acidic (~pH3),
acids no longer remain dissolved because they
are no longer ionic and usually precipitate out of solution.
Liquid-liquid/Solvent
extractions
Separatory Funnel Extraction
Procedure
Liquid-liquid/Solvent
extractions
Separatory Funnel Extraction
Procedure
Liquid-liquid/Solvent
extractions
Separatory Funnel Extraction
Procedure
Liquid-liquid/Solvent
extractions
Separatory Funnel Extraction
Procedure
 Liquid-liquid/Solvent
extractions
Applicati
 Solvent extraction is usedon
to remove phenol from wastewater
 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.
 Activity
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?
2. Using data in the first question, what fraction will be extracted
if 5 extractions with 100 mL benzene each are used (instead of
one 500 mL extraction)?
3. A solute has a K between water and toluene 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 toluene do we need if we wish to extract
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
 Solid-phase are performed
extraction, or liquid-solid
manually,
extraction, which makes
can overcome themofsomewhat
several these
slow and tedious.
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 Selection of solid phase extraction
cartridges for liquid samples. From left-
determined by the to-right, the absorbent materials are
species we wish to octadecylsilane, carbon,
octadecylsilane, polyamide resin, and
separate.
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,
 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
 Often applied when analyte has an
extractions
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
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.
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.
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
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
 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
BrightEmbellishedHerring-mobile.mp4
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
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

ig. 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
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
 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
 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
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?