Separation by Extraction

What is an extraction?
- Move compounds of interest ‘selectively’ to another media.

-The extent to which solutes, both inorganic and organic, distribute themselves between
two immiscible liquids differs enormously, and these differences have been used for
decades to separate chemical species.

14
 Separation by Extraction

The partition of a solute between two immiscible phases is an equilibrium process that
is governed by the distribution law.
If the solute species A is allowed to distribute itself between water and an organic
phase, the resulting equilibrium may be written as

Aaq Aorg
 Types of extraction

1. Solid-Liquid extractions :
An extraction method that separates analytes using a solid phase and a
liquid phase. (CHROMATOGRAPHY)
 Types of extraction

2. liquid-liquid Extractions (solvent extraction):

Liquid-Liquid extraction is a method by which a compound is pulled from
solvent A to solvent B where both solvents are not miscible.
 liquid-liquid Extractions

Liquid-liquid extraction is based on the transfer of a solute substance from one liquid
phase into another liquid phase according to the solubility.

In the practical use, usually one phase is a water or (aqueous) solution and the other
an organic solvent which is immiscible with water.

The success of this method depends upon the difference in solubility of a compound
in various solvents.

For a given compound, solubility differences between solvents are quantified as the
"distribution coefficient“
 liquid-liquid Extractions

The organic solvent used for extraction must meet a few criteria:

1. Should readily dissolve substance to be extracted.

2. Should not react with the substance to be extracted.

3.Should not react with or be miscible with the second solvent.

4. Should have a low boiling point so it can be easily removed from the

product.

Common extraction solvents are diethyl ether and methylene chloride.

 liquid-liquid Extractions

Factors to be considered:
• Selectivity
• Distribution coefficient
• Insolubility of solvent
• Recoverability of solute from solvent
• Density difference between liquid phases
• Chemical reactivity
• Availability and Cost
• Viscosity, Vapour pressure, Freezing point) must be low),
• Boiling temperature (should be low)
• Safety in use (non-Flammable, non-toxic)
 liquid-liquid Extractions

The partition coefficient law.

K = [S]2 / [S]1 Sorg

where K or KD = partition or distribution coefficient

Saq
S1 = concentration of solute in the phase 1

S2 = concentrations of solute in the phase 2

K = a constant (for ideal solutions) at constant temperature which depends on

the nature of solvents used.

[S] 2
KD =
[S] 1
liquid-liquid Extractions
 Extraction efficiency

Equation for the remaining fraction in phase 1 after specific number of extraction:

n
V1
qn =
V1 + KDV2
Where:

qaq : Fraction of solute remaining in phase 1 after known number of extraction.

Vaq : volume of phase 1 (the solvent that we want to transfer the solute from)

Vorg: volume of phase 2 (the solvent that we want to transfer the solute into)
n : number of extraction
 Extraction efficiency

The fraction present in the organic phase after one extraction, (qorg)1, is

qorg= 1-qaq
 Example : Extraction Efficiency

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?

50
qaq = = 0.40
50 + (5) (15)

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%.
Theory of solvent Extraction

Applications of solvent Extraction

 Successive extractions

Multiple extraction with small volumes of organic solvent is

most efficient than a single extraction with a large volume of
solvent .

Usually don’t do more than three successive extraction

 Successive extractions

Why does many smaller extractions are more

efficient than one large extraction?

Because the percentage of solute moving

from one phase into another is more by using
many smaller extractions.
 Effect of pH

If a solute is an acid or base, its charge changes as the pH is changed. Usually, a

neutral species is more soluble in an organic solvent and a charged species is
more soluble in aqueous solution.

 To extract a basic amine ,B, into water, use a pH low enough to convert B
into BH+ .
 To extract the acid HA into water , use a pH high enough to convert HA
into A- .
 Effect of pH

Acidic compounds
Low pH (acidic) High pH (basic)

nonpolar Polar (charged)

Basic compounds
Low pH (acidic) High pH (basic)

Polar (charged) nonpolar

6
 Distribution ratio

The distribution ratio D is a constant independent of the volume ratio.

However, the fraction of the solute extracted will depend on the volume
ratio of the two solvents.
If a larger volume of organic solvent is used, more solute must dissolve
in this layer to keep the concentration ratio constant and to satisfy the
distribution ratio.
 Distribution ratio

If there are more than one solute (say two solutes A and B), then consideration

should be given to the selectivity of the solvent for solute A as against B.

The selectivity between the 2 solutes A and B is defined as the ratio of the

distribution coefficient of A to the distribution coefficient of B.

DA/DB

For all useful extraction operation, the selectivity (Separation Factor) must exceed

unity. DA/DB >1

If the selectivity is unity, no separation is possible. DA/DB = 1

 Percent Extracted

100 D
%E 
D  (Vaq. /Vorg.. )

If Vaq. = Vorg. then,

%E 100 D
D 1
 Percent Extracted

The distribution ratio between HCl and tri-n- butyl phosphate for
PbCl2 is 2.3, what the percent of PbCl2 will be extracted from
25mL of aqueous solution into 10 mL tri-n- butyl phosphate?

%E  100 D
D

%E  100 x2.3  48%

2.3  (25 / 10)

 Percent Extracted
20 ml of an aqueous solution of 0.1 M butyric acid is shaken with 10mL ether.
After the layers are separated, it is determined by titration that 0.5 mmol
butyric acid remains in the aqueous layer. What is the distribution ratio, and
what is the percent extracted?

The number of mmol butyric acid started= M x V= 0.1 x 20 = 2.0 mmol

We started with 2.0 mmol butyric acid, and so 1.5 mmol was extracted.
The concentration in the ether layer is 1.5 mmol/10 mL=0.15 M.
The concentration in the aqueous layer is 0.5 mmol/20 mL=0.025 M. Therefore,

[S]
D = org

[S]
aq

Since 1.5mmol was extracted, the percent extracted is (1.5/2.0)×100%=75%.

Or
%E  100 x 6.0  75%
6.0  (20 /10)
 Percent Extracted

%E  100 D
D  (Vaq. /Vorg.. )

Above equation shows that the fraction extracted can be increased by decreasing the
ratio of Va/Vo, for example, by increasing the organic phase volume.
However, a more efficient way of increasing the amount extracted using the same
volume of organic solvent is to perform successive extractions with smaller individual
volumes of organic solvent.

For example, with a D of 10 and Va/Vo =1, the percent extracted is about 91%.
Decreasing Va/Vo to 0.5 (doubling Vo) would result in an increase of % E to 95%. But
performing two successive extractions with Va/Vo =1 would give an overall extraction
of 99%.
 Extraction Method

1- Batch extraction
-The simplest and mostly used method, consists of extracting the solute from
one immiscible layer by shaking the two layers until equilibrium is attained,
after which the layers are allowed to settle before sampling.
-This batch extraction process provides rapid, simple, and clean separations,
and is more beneficial when the distribution ratio of the solute of interest is
large.
 Extraction Method

2- Craig Method:

you want to separate two species by solvent extraction but their KDs are not
sufficiently different, So carry out a series of extractions:

17
Applications of
solvent Extraction
 Metal ion Extraction

Most chelating agents are weak acid, ionized in water, the ionizable
proton is displaced by the metal ion when the chelate is formed.

8-hydroxy- quinoline Diphenylthiocarbazone(Dithizone)

Metal ion Extraction
 Metal ion Extraction
The two principle solvent extraction systems for metal ions:
a. Metal chelate

Must create a neutral, hydrophobic complex to extract into an organic

phase.
Complex formation is pH dependent

Al-Chelate
 Metal ion Extraction

b. Ion Association
Some salts form complexes (ion pairs) which can be extracted.
Example:

Au+3 + Cl- = [AuCl4]-

[AuCl4]- + R4N+.Cl- = (R4N+). [AuCl4]-
 Metal ion Extraction

The main organic reagents which use in extraction method.

8-oxyquinoline reacts with more than 50 elements
Acetyl acetonate forms compound with more than 60 elements
Thionyl trifluoride acetone is used for excretion and separation actinoids.
dithizon is used for determination of Pd, Au, Hg, Ag, Cu, Bi, Pt, In, Zn, Cd,
Co, etc.
It is of great importance in the toxicological analysis.
Sodium diethyl dithiocarbamate reacts with several tens of elements
It is of great importance in the toxicological analysis.
 Organic Compound Extraction

Some organic compounds can be made water-soluble.

Organic Acids (e.g. carboxylic acids and phenols) and Organic Bases
(amines) can be converted to their water-soluble salt form.
Usually, a neutral species is more soluble in an organic solvent while a
charge species is more soluble in aqueous solution.
Charged species tend to be more soluble in water than in organic
solvent.
To extract a base into water, use a pH low enough to convert B into BH+.
To extract the acid HA into water, use a pH high enough to convert HA
into A-.
 Organic Compound Extraction

What type of organic compounds can be made water-soluble?

A. Strong organic acids
Carboxylic acids

B. Weak organic acids

phenols
 Organic Compound Extraction

C. Organic bases
amines
How can organic acids or bases
be converted to a water-soluble
form?
 Organic Compound Extraction

A. strong organic acids

Carboxylic Acids (strong organic acids (pKa = 3 to 4)) are converted

to the salt form with:
1. By using 5% NaOH aqueous solution (strong inorganic base).
2. by using ( NaHCO3) aqueous solution )weak inorganic bases(.
 Example

How do you separate a mixture of naphthalene and benzoic acid,

dissolved in dichloromethane.

29
 Example

use an aqueous solution of either 5% NaOH or

NaHCO3, to extract benzoic acid as a salt form

30
 Organic Compound Extraction

B. weak organic acids

Phenols (weak organic acids), the parent compound, is partially
water-soluble whereas substituted phenols are not. They are
converted to the salt form with:
1. NaOH, a strong inorganic base, can change phenol to its ionic
(salt) form.
 Example

How do you separate a mixture of benzoic acid and p-

methoxyphenol, dissolved in dichloromethane.
Introduction to Chromatography
Types of chromatography
 First Chromatography Column

toluene

sample

1903 Tswett - plant pigments separated on chalk columns

 Chromatography

The separation of a mixture by distribution of its components between a

mobile and stationary phase over time. The mobile phase may be either a
liquid or a gas, while the stationary phase is either a solid or a liquid
supported on solid.

Distribution Coefficient (Equilibrium Distribution ) =

Concentration of component A in stationary phase

Concentration of component A in mobile phase
 Important terms

Mobile phase: the solvent moving through the column (liquid or gas)
Stationary phase: a viscous liquid chemically bonded to the inside of a
capillary tube or onto the surface of solid particles packed in the column.
Supporting medium: a solid surface on which the stationary phase is bound
or coated
Elution: The process of passing liquid or gas through a chromatography
column .
Eluent : Fluid entering the column.
Eluate : Fluid emerging from the end of the column .
 Types of Chromatography according to geometry

planner column

P.C T.L.C L.C G.C

 Types of Chromatography according to geometry

1- Column chromatography
The stationary phase is contained in a tube called th e column.

Mobile Phase Stationary Phase

Liquid Solid
GAS
Gas-liquid Gas-Solid
Chromatography
Chromatography Chromatography
GC
GLC GSC
LIQUID Liquid-Liquid Liquid-Solid
Chromatography Chromatography Chromatography
LC LLC LSC
Column
 Types of Chromatography according to geometry

2- Planar chromatography

A. paper chromatography:
a sheet or a narrow strip of paper serves as the stationary phase.

Paper chromatography
 Types of Chromatography according to geometry

2- Planar chromatography
B. Thin-layer chromatography (TLC):
Separations in TLC involve distributing a mixture of two or more substances
between a stationary phase and a mobile phase.
The stationary phase: is a thin layer of adsorbent (usually silica gel or
alumina) coated on a plate. The mobile phase is a liquid
Types of chromatography according to the mechanism of separation

1- Adsorption chromatography
2- Partition chromatography
3- Ion-exchange chromatography
4- Size-exclusion chromatography
5- Affinity chromatography
 Adsorption Chromatography

A solid stationary phase and a liquid or gaseous mobile phase are used.
Solute is adsorbed on the surface of the solid particles.
The more strongly a solute is adsorbed, the slower it travels through the
column.
The most common of adsorbent are Alumina & Silica gel in which the
interactions with solute molecules is due to OH groups present on their surface.
More polar molecules are adsorbed more strongly & thus, will elute more
slowly.
Strength of adsorption of polar groups (solutes) on polar support
is in the following order:
-C=C- < O-CH3 < -COOR < >C = O < -CHO < -NH2 < -OH < -COOH
Adsorption Chromatography
 Partition chromatography

In partition chromatography a solid

support with a high surface area is coated with a high boiling liquid which acts
as the stationary phase.
Separation occurs because of the differences in solubility for the analytes in
the stationary and mobile phases.
 Types of Partition chromatography

There are two types of partition chromatography normal phase and reversed
phase, they are defined by the relative polarities of the mobile and stationary
phase.

Normal phase Reversed phase

The stationary The stationary phase is non polar

phase is polar and and the mobile phase is polar
the mobile phase is phase.(is the most frequently
non-polar. used hplc method. )

For this reason, the use of silica (a polar molecule)

as the stationary phase (as in adsorption
chromatography) is also considered to be a normal
phase separation method.
 Ion-exchange chromatography.

Anionic (-SO3-) or cationic (-N(CH3)3+)

molecules are covalently attached to the
stationary solid phase, usually a resin, in
this type of chromatography. Solute ions
of the opposite charge are attracted to
the stationary phase by electrostatic
force. The mobile phase is a liquid.
 Molecular exclusion chromatography

Called gel filtration or gel permeation chromatography, this technique

separates molecules by size, with the larger solutes passing through most
quickly. In the ideal case of molecular exclusion. The liquid or gaseous
mobile phase passes through a porous gel. The pores are small enough to
exclude large solute molecules but not small ones .Small molecules take
longer to pass through the column because they enter the gel.
 Affinity chromatography.

This most selective kind of chromatography employs specific interactions

between one kind of solute molecule and a second molecule that is covalently
attached (immobilized) to the stationary phase.
1.) Typical response obtained by chromatography (i.e., a chromatogram):

chromatogram :- concentration versus elution time

Wb

Where:
= retention time
= elution time
Wb = baseline width of the peak in time units
Wh = half-height width of the peak in time units
The retention time (tR) is the time between injection of the mixture onto
the column and arrival of that component at the detector.
Elution time (tm): is the time it takes for an unretained species to pass
through achromatographic column.
The (VR) is the volume of the mobile phase required to
elute a particular solute from the column.
 The separation of solutes in chromatography depends on two factors:

a difference in the retention of solutes (i.e., a difference in their time or

volume
of elution).
a sufficiently narrow width of the solute peaks (i.e, good efficiency for the
separation system)
Peak width & peak position
determine separation of peaks
The distribution of analytes between phases can often be described quite simply An
analyte is in equilibrium between the two phases

A mobile phase A stationary phase

The equilibrium constant, , is termed the

Cs
(1)
k =
Cm

Where
Cs is the concentration of solute in stationary phase.
Cm is the concentration of solute in mobile phase.
Paper Chromatography
PC
Thin layer chromatography
TLC
 Column Chromatography
Although there are other types of chromatography (e.g. paper and thin

layer), most modern applications of chromatography employ a column.

The column is where the actual separation takes place. It is usually a

glass or metal tube of sufficient strength to withstand the pressures that

may be applied across it

Basic concepts

1- The separation occur by placing the stationary phase, a solid

adsorbent in a vertical glass column

2- The mixture was introduced into the top of the column

3- Mobile phase was eluted to develop the column (by either gravity or
external pressure)

4- The highest effective distribution coefficient substance which is less

polar will move quickly down

5- The separation of these compound will achieved and separated from

that have lower effective distribution coefficient which has high polarity

Different shapes and sizes of column

Columns for chromatography can be small or big, according to the

amount of material which needs to be loaded onto the column
The "column" on the far left in the photo is actually a Pasteur pipet. This
size of column is suitable for 10-125 mg of material
The middle column is a 10 mL disposable glass pipet
The column on the right would be used for many grams of material

Column chromatography is classified into two categories, depending

onhow the solvent flows down the column
1- If the solvent is allowed to flow down the column by gravity, or
percolation, it is called gravity column chromatography

2- If the solvent is forced down the column by positive air pressure, it is

called flash chromatography, a "state of the art" method currently
used in organic chemistry research laboratories

Principle of column chromatography

The principle of separation depicted by considering a column packed

with stationary phase to a height of 5 cm surrounded by the mobile
phase of which there is 1 cm3 per cm of column.
If 32 g of a compound is added in 1 cm3 of solvent then as this 1 cm3
moves onto the column to occupy position A, 1 cm3 of solvent will leave
the base of the column.
If the compound has an effective distribution coefficient of 1, it will
distribute itself equally between the solid and liquid phases.
If a further 1 cm3 of solvent is introduced onto the column, the solvent in
section A will move down to B taking 16 g of the compound with it,
leaving 16 g at A.
The addition of further 1 cm3 of solvent to the column displaces the
solvent in A to B and that in B to C giving the distribution of the
compound; that there is 8 g in the solvent and 8 g in the solid phase
stage 3.
Addition of a further 1 cm3 of solvent leads to the distribution of stage 4,
and a further 1 cm3 aliquot to the situation at stage 5.

The principle of separation

1 2 3 4 5
STAGE

........
A 32 16 8 4 2

B 16 16 12 8

C 8 12 12

D 4 8

E 2
 Fig Principle of column chromatographic separation

It is apparent that after five equilibrations the compound is distributed

through out the whole column, but is maximally concentrated at the
centre of the column.
If a compound had an effective distribution coefficient of less than 1,
more than 50% of the compound would be left on the solid phase after
each equilibration. The concentration peak would be above the centre of
the column.
Alternatively, for a compound with an effective distribution of greater
than 1, the concentration peak after five equilibrations would be below
the centre of the column.
The greater the number of equilibrations that occur on a column, the
greater becomes the concentration of the compound on a certain part of
the column.
There are, therefore, two important factors which influence the pattern
of separation (resolution) of a mixture of compounds.
1- Effective distribution coefficient which effect on the rate of migration
of compounds
2- The number of equilibrations that have taken place which effect on
the sharpness of the spearated bands.
In a real situation, equilibration occurs continuously on a column since
the solvent is being continuously added and thousands of equilibrations
take place.
Columns chromatography are considered to consist of a number of
adjacent zones in each of which there is sufficient space for the solute to
achieve complete equilibrium between the mobile and stationary phases.
The more efficient the column, the greater the number of theoretical
plates that are involved.
The Adsorbent

1- Silica gel SiO2

i- The term silica, silica gel and silicat salt all reffere to the same types
of adsorbent
ii- The adsorptive properties of these various solids depends exclosivly
on the OH groups attached to the silicone atoms and these interacts with
polar or unsaturated compounds by (H) bonding
iii- Silica gel is considered to be the most popular and commonly used
chromatographic adsorbent
2- Alumina (Aluminum Oxide)

i- Alumina or aluminum oxide is the most chromatographic adsorbent

after silica
ii- The most commonly used alumina is the neutral type, in special
groups we recommended to use the acidic or basic alumina
 Al Al Cl Al ONa

O O O O O O O

Al Al Cl Al ONa
Neutral Acidic Basic

iii- Alumina is used more frequently in column chromatography than it is

in TLC
iv- Alumina existed in different grades of activity ranging from grade I
to grade V according to its water content where the grade I contain the
least amount of water (II = 3%, III= 6%, IV= 10%, V= 15%) i.e. Grade I
is the most active one
Vi- Alumina is quite sensitive to the amount of water which is bound to
it: the higher its water content, the less polar sites it has to bind organic

3- Other adsorbents

There are many other types of adsorbents which are used for special
group of compounds such as:

3.1- Magnesium silicate (Florisil)

The adsorption properties of water deactivated florisil are intermediate

between those of silica and alumina.
Florisil is an acidic adsorbent and has been used instead of alumina for
base sensitive samples

3.2 Charcoal

It was extensively used, prior to 1955 in the chromatograph separation

for a wide variety of sample types. In recent years its application was
limited and used only in clean up natural product mixtures, separation of
sugars according to M. Wt.
3.3- Fuller's earth (hydrous aluminum magnesium silicate)

Suitable grades of chrom. fuller's earth are available commercially under

the trade names (Florex xxs) moderately course powder and (Florex
xxx) a very fine powder

3.4- Diatomaceous earth (celite, Kieselguhr)

It is used occasionally as an adsorbent, it is extremely week and

therefore employed only for very polar samples.
Relative adsorbent on it resembles that on alumina and silica. It is most
frequently used in admixture with active adsorbent to promote faster
column flow rates so it act as inert diluents

3.5- Polyamides (Nylon):

It is synthetic adsorbent which particularly suitable for the separation of

phenolic compounds
Packing of Column

Column Chromatography Procedures

Procedure for Gravity Column Chromatography

There are (at least) two ways to pack a gravity column: the slurry (wet)
method and the dry pack method

Liquid Filled or Slurry Method for Packing

Mix the adsorbent with the solvent and then pour this slurry into the
prepared column
The advantage of slurry methods is that they eliminate air bubbles from
forming in the column as it packs

Dry-pack Method for Packing

The stationary phase was dry added directly inside the column which
contain suitable amount of mobile phase
This method is easier, but can lead to bubbles in the column

Application of Sample

1- A Simple Way

This was achived by removing most of the solvent from above the
column by suction or via open the column tap and just to drain the
remainder into the column bed
The sample solution is then carefully applied by pipette and it allowed
just to run into the column
A small volume of solvent is then applied in a similar manner to wash
final traces of the sample into the bed
More solvent is then carefully added to the column to a height of 5 to 10
cm
The column is then connected to a suitable reservoir which contains
more solvent, so that the height of the solvent in the column can be
maintained at 5 to 10 cm

2- An alternative procedure

By sulrring the sample with an inert support (celite or sucrose) or with a

small amount of the adsorbent before addion to the column which avoids
the necessity to drain the column to the surface of the bed.
 Gas-Liquid Chromatography (GLC(

Introduction

This technique based upon the partitioning of compounds between a

liquid and a gas phase, is a widely used method for the qualitative and
quantitative analysis of a large number of compounds since it has high
sensitivity, reproducibility and speed of resolution.
A stationary phase of liquid material such as a silicone grease is
supported on an inert granular solid.
This material is packed into a narrow coiled glass or steel column 1 to
10 m long and 2 to 4 mm internal diameter through which an inert
carrier gas (the mobile phase) such as nitrogen, helium or argon is
passed.
GLC may also be performed using capillary columns which are made of
glass or metal with diameters of between 0.03 to 1.0 mm and which may
be up to 100 metres in length.
The basis for the separation is the difference in the partition coefficients
of the volatilised compounds between the liquid and gas phases as the
compounds are carried through the column by the carrier gas.

Gas chromatography

Specifically gas-liquid chromatography - involves a sample being

vaporized and injected into the head of the chromatographic column.
The sample is transported through the column by the flow of inert,
gaseous mobile phase. The column itself contains a liquid stationary
phase which is adsorbed onto the surface of an inert solid.
Instrumental Components

Carrier gas

1- The carrier gas must be chemically inert .

2- Commonly used gases include nitrogen, helium, argon, and carbon
dioxide.
3- The choice of carrier gas is often dependant upon the type of detector
which is used.
4- The carrier gas system also contains a molecular sieve to remove
water and other impurities .

Sample injection port

For optimum column efficiency, the sample should

a- Not be too large amount

b- Should be introduced onto the column as a "plug" of vapour - slow
injection of large samples causes band broadening and loss of resolution.
The most common injection method is where a microsyringe is used to
inject sample through a rubber septum into a flash vapouriser port at the
head of the column.
The temperature of the sample port is usually about 50° C higher than
the boiling point of the least volatile component of the sample.
For packed columns, sample size ranges from 0.1 - 20 micro liters.
Capillary columns, on the other hand, need much less sample, typically
around 0.1-3 micro liters.

Columns

There are two general types of columns

1- Packed 2- Capillary
Packed columns contain a finely divided, inert, solid support material
(commonly based on diatomaceous earth) coated with liquid stationary
phase. Most packed columns are 1.5 – 10 m in length and have an
internal diameter of 2 – 4 mm.
Capillary columns have an internal diameter of 0.03 to 1.0 mm and 100
m length.

There are two types of capillary column systems

1- Wall Coated Open Tubular (WCOT) columns
The stationary phase is coated directly onto the walls of the capillary
tubing
2- Support Coated Open Tubular (SCOT( also known as porous layer
open tubular (PLOT) colums.
The inner wall of the capillary is lined with a thin layer of support
material such as diatomaceous earth, onto which the stationary phase has
been adsorbed.

Comparison between the SCOT and WCOT

a-The capacity of SCOT columns is considerably higher than that of

WCOT.
b- SCOT columns are used for quantitative analyses than WCOT
systems.
C- SCOT systems efficiency is less than WCOT systems but
considerably better than conventional GLC columns.
* There is an optimum carrier gas flow rate for maximum column
efficiency (i.e. minimum HETP (
In 1979, a new type of WCOT column was devised - the Fused Silica
Open Tubular (FSOT) column;
These have much thinner walls than the glass capillary columns, and are
given strength by the polyimide coating.
These columns are flexible and wound into coils.
They have the advantages of physical strength, flexibility and low
reactivity.

Column Temperature

Column temperature must be controlled to within 0.1 degree.

The optimum column temperature is dependant upon the boiling point of
the sample.
Minimal temperatures give good resolution, but increase elution times.
If a sample has a wide boiling range, then temperature programming can
be useful.
The column temperature is increased (either continuously or in steps) as
separation proceeds.

Preparation and Application of Sample

The majority of non-and low-polar compounds are directly amenable to

GLC, but other compounds possessing such polar groups as —OH, -NH2
—COOH are generally retained on the column for excessive periods of
time if they are applied directly.
Methylation and silanisation are common derivatisation methods for
fatty acids, carbohydrates and amino acids. This increases the volatility
and effective distribution coefficients of the compounds.
The sample for chromatography is dissolved in a suitable solvent such as
ether, heptane or methanol .
Chlorinated organic solvents are generally to be avoided as they may
contaminate the detector.
The sample is injected onto the column using a micro-syringe through a
septum in the injection port which is attached to the top of the column.
Normally between 0.1 and 10 cm3 of solution is injected. It is common
practice to maintain the injection region of the column at a slightly
higher temperature than the column itself. This helps to ensure rapid and
complete volatilisation of the sample.

Separation Conditions

Nitrogen, helium and argon are the three most commonly used carrier
gases.
They are passed through the column at a flow rate of 40 to 80 cm3 min.
The column temperature must be within the working range of the
particular stationary phase and is chosen to give a balance between peak
retention time and resolution.
In isothermal analysis a constant temperature is employed. In the
separation of compounds of widely differing polarity or molecular
weight it may be advantageous to gradually increase the temperature.
type of gas chromatography detectors
 GC Detectors

Detector:
A device that detect the presence of solutes vapours as they are eluted from the
column and converts the response into a measurable signal.
Therefore, the signal is directly propotional to the solutes ’concentration.

The following devices are common types of GC detectors:

1. Thermal Conductivity Detector (TCD)
2. Flame Ionization Detector (FID)
3. Nitrogen-phosphorus Detector
4. Electron Capture Detector (ECD)
5. Mass Spectrometers
 GC Detectors

properties of an Ideal detector:

1. Sensitive.
2. Good stability and reproducibility
3. A linear response .
4. Fast response.
5. Uniform response to all analytes.
6. Simple (reliable)
7. Nondestructive of sample.
 Thermal Conductivity Detector (TCD)

The device consists of heated source may be a fine platinum , gold or tungsten
wire.
The temperature of this heated source depends on the thermal conductivity of
the surrounding gas.
The thermal conductivities of helium and hydrogen are roughly six to ten times
greater than those of most organic compounds.
Thus, even small amounts of organic species cause relatively large decreases in
the thermal conductivity of the carrier gas, resulting in a marked rise in the
temperature of the wire.
The difference between reference and sample wire temperature is measured,
and a signal is generated.
Temperature : 150 – 250 ℃
 Thermal Conductivity Detector (TCD)

Reference
channel Sample channel

Pure carrier gas carrier gas+

sample

6
 Thermal Conductivity Detector (TCD)

Advantages:
 Simple and inexpensive.
 Posses long life.
 Accurate results.
 Non-selective, hence known as universal detectors.
 The detector is nondestructive.
Disadvantages:
 Low sensitivity.
 Affected by fluctuation in temperature and flow rate.

7
 Flame ionization Detector (FID)

 Organic compounds burning in the flame produce ions and electrons,

which can conduct electricity through the flame .
 These ions are collected towards respective electrode and were
recorded on recorder due to electric current.

8
Flame Ionization Detector (FID)
9
 Flame Ionization Detector (FID)
advantages:
universal detector for organics
mobile phase impurities not detected
carrier gases not detected
limit of detection: FID is 1000x better than TCD
linear and dynamic range better than TCD
disadvantage:
Destructive detector
Doesn’t respond to common inorganic compounds
Dynamic range is limited.

10
 Electron Capture Detector (ECD)

The sample eluate from a column is passed over a radioactive β-emitter,

usually 3H tritium or 63Ni .
An electron from the emitter causes ionization of carrier gas( often
nitrogen gas ) and production of a burst of electrons.
In the absence of organic species, a constant current between a pair of
electodes results from this ionization process.
In presence of organic molecules , the current decreases markedly by the
electronegative functional groups that tend to capture electrons.

11
Electron Capture Detector (ECD)

12
 Electron Capture Detector (ECD)

advantages:
useful for environmental testing‚ detection of chlorinated pesticides or herbicides‚
detection of polynuclear aromatic carcinogens‚ detection of organometallic
compounds
selective for halogen- (I, Br, Cl, F), nitro-, and sulfur-containing compounds
detects polynuclear aromatic compounds, anhydrides and conjugated carbonyl
compounds

disadvantages:
It is insensitive to functional groups such as amines, alcohols, and hydrocarbons.
The linear response is limited

13
 GC- Mass Spectrometers (GC-MS)

A sample with a moderaely high vapour pressure is introduced in an inlet

system, operated under vaccum( 10-4-10-7 torr) and at high temperature (up to
300◦C
It vaporizes and is carried to the ionization source (nonvolatile compounds may
be vaporized by spark).
Analyte molecules are nutral and must be ionized .So they are bombarded with
high energy electrons in an electron- impact source .
Separation actually accomplished based on the mass-to-charge (m/z( ratios of
the ions.

14
 Mass Analyzers
Mass spectrometers based on magentic sectors are widely used by organic chemists to
determine molecular structure.

They deflect ions down a curved tube in a magnetic filed based on their kinetic energy
determined by the mass, charge, and velocity.

The most common analyzers for GC/MS are the quaderupole mass filter and the ion trap.

15
GC- Mass Spectrometers (GC-MS)

16
GC- Mass Spectrometers (GC-MS)

17
 Applications Of GC

1- It is used in…………

 the separation and analysis of multi component mixtures such as essential

oils, hydrocarbons and solvents.

 pollution studies, forensic work and general trace analysis.

 biochemical separation .

2- It can quantitatively determine materials present at very low

concentrations.

18
 Advantages of Gas Chromatography

Requires only very small samples with little

preparation

Good at separating complex mixtures into

components

Results are rapidly obtained (1 to 100 minutes)

Very high precision

High sensitivity to detect volatile organic mixtures of

low concentrations

Equipment is not very complex (sophisticated oven)

 Detectors characteristics (‫)مهم‬
Detector Principle Responds Sensitivity Linearity Carrier °C. Advantages
to gas Limit Positive Negative
-7 4
Thermal Measure Organic & 0.5 X 10 10 He, 450 Easy to Limited
Conductivity thermal inorganic -2
H , Ar use sensitivity and
to 10 g 2
TCD conduct. of compound , linearity
one gas
supply,
gases responds to
all
compound
nondestructiv
e
3
Gas Density Difference All gassesVariable 10 N2, 150 Good for Limited
Balance DBD of Mol. Wt. and vapors Ar, corrosive sensitivity and
of gases CO2 comp. linearity
nondestructiv
e
-13 -2 7
Flame Measure All org. 10 - 10 10 He, N2 400 Very sensitive, Require 3
Ionization ionization comp. wide gases N2, H2,
linea
r
FID of org. except range air destructive
comp. by HCOOH
flame CS2 &
HCHO
-14 -8 4
Electron Measure Halo. 10 - 10 10 N2 225 Very sensitive, Easily
Capture loss of e- Compounds g Ar + 350 nondestructive contaminated
ECD absorbed as 10% ,easily cleaned ,difficult to
by herbicides use
halides & & CH4
other pesticides
electrophilic
comp.
Flame Measure P or S org. P 5 X10 -11 103 N2; Ar 300 Very selective Response
Photometer emission Comp. eg. -6 4 and sensitive Affected
-10 10
FPD ions of light herbicides -10 2 contamination
S 3X10 - conc ,
-6
curing & 10 Destructive
combustion pesticides
of P & S
Liquid chromatography
HPLC
 Liquid Chromatography

Liquid chromatography (LC) is a chromatographic technique in which the

mobile phase is a liquid.
The technique of LC is much older than GC but was overshadowed by the
rapid development GC in the 1950’s and 1960’s.
However, LC is currently the dominate type of chromatography and is
even replacing GC in some of GC’s more traditional applications.
Liquid chromatography is important because most compounds are not
sufficiently volatile for gas chromatography
 Liquid Chromatography

In choosing a mobile phase for LC, several factors need to be considered:

• Type of stationary phase used .

• Solubility of the solutes.
• Viscosity of the mobile phase
• Type of detector used .
• Purity of the solvents.
• Miscibility of the solvents (for gradient elution)
 Liquid Chromatography

Types of LC:
1. Partition chromatography
2. Adsorption, or liquid-solid chromatography
3. Ion exchange chromatography
4. Size exclusion, or gel, chromatography
 partition LC

Types of partition chromatography based on the type of

stationary phase:

1- Normal Phase liquid Chromatography (NPLC)

Polar stationary phase: silica
Nonpolar mobile phase: hexane , ethyl acetate.
The least polar compound comes out first.

2- Reversed Phase liquid Chromatography (RPLC)

Nonpolar stationary phase : C8 , C18
Polar mobile phase : water , methanol
The most polar compound comes out first.
 High Performance Liquid Chromatography (HPLC)

 High-performance liquid chromatography (HPLC) uses high pressure to force

solvent through closed columns containing fine particles that give high-
resolution separations

 Increasing the rate at which solute equilibrates between stationary and mobile
phases increases the efficiency of chromatography.

 The efficiency of a packed column increases as the size of the stationary phase
particles decreases.
 High Performance Liquid Chromatography
(HPLC)

Columns for HPLC

1. stainless steel tubing, 25 cm long, i.d. = 3.9-4.6 mm. A well-packed 4.6-

mm column with 5-µm-diameter particles should give 60,000 - 90,000
plates/meter .
2. Atypical 15-cm-long with 4.6 mm i.d. will give 15,000 plates with 3-µm
particles, 9000 plates with 5-µm particles, and 5000 plates with 10µm
particles.
 High Performance Liquid Chromatography
(HPLC)

Advantages:
fast analysis time
ease of automation
good limits of detection

Disadvantages:
greater expense
lower sample capacities
Solvents consuming
 High Performance Liquid Chromatography
(HPLC)

Why small particles give better resolution ?

1- they provide more uniform flow through the column, thereby reducing the

multiple path term.

2- The smaller the particles, the less distance solute must diffuse.
 High Performance Liquid Chromatography (HPLC)

Effect of temperature:

Heating a chromatography column usually decreases the viscosity of the

solvent, thereby reducing the required pressure or permitting faster flow.
Increased temperature decreases retention times and improves resolution by
hastening diffusion of solutes.
However, increased temperature can degrade the stationary phase and
decrease column lifetime. When column temperature is not controlled, it
fluctuates with the ambient temperature.
Using a column heater set 10℃ above room temperature improves the
reproducibility of retention times and the precision of quantitative analysis
 Composition of Liquid Chromatography
System

Solvent
Solvent Delivery System (Pump)
Injector
Sample
Column
Detectors (Diode Array)
Waste Collector
Recorder (Data Collection)
 Elution Types

1. Isocratic elution:
Use of a constant mobile phase composition to elute solutes.

2. Gradient elution:
Changing composition of mobile phase with time like pH , ionic
concentration or polarity  solvent programming ( automatically)
 Liquid Chromatography Detectors

Common types of LC Detectors

1- Refractive Index Detector
2- Conductivity Detector
3- UV/Vis Absorbance Detector
4- Electrochemical Detector
5- Fluorescence Detector

As in GC, the choice of detector will depend on the analyte and how the LC
method is being used (i.e., analytical or preparative scale)
 Liquid Chromatography Detectors

1) Refractive Index Detector (RI)

Measures the overall ability of the mobile phase and its solutes to refract or
bend light.

Aadvantages:
non-destructive and universal detector
applicable to the detection of any solute in LC

Ddisadvantages:
High limits of detection (10-6 to 10-5 M)) low sensitivity(
Difficult to use with gradient elution
 Liquid Chromatography Detectors

2) UV/Vis Absorbance Detector

Measures the ability of solutes to absorb light at a particular
wavelength(s) in the ultraviolet (UV) or visible (Vis)
wavelength range.

Advantages:
most common type of LC detector
Relative inexpensive
It can be use with gradient elution
Higher sensitivity than R (Ilimits of are ~ 10-8 M)
 Liquid Chromatography Detectors

3) Fluorescence Detector
A selective LC detector that measures the ability of eluting solutes to
fluoresce at a given set of excitation and emission wavelengths

Advantages:
limits of detection for a fluorescence detector are ~ 10-10 M
Can be used with gradient elution
 Liquid Chromatography Detectors

4) Conductivity Detector
Current conducted within the cell will depend on the number and types of
ions present in the mobile phase .
Used in analytical applications of ion-exchange chromatography for the
detection of ionic compounds
Advantages:
high selectivity, low background signal
Limits of detection for a conductivity detector are ~ 10-6 M
It can be used with gradient elution
 Liquid Chromatography Detectors

5) Electrochemical Detector
Generally, it includes two or more electrodes which monitor the current that is
produced by the oxidation or reduction of eluting compounds at a fixed potential.

Advantage:
Limits of detection for a conductivity detector are ~ 10-11 M
 Applications of Liquid Chromatography

Purification of biological and organic compounds.

Pharmaceutical analysis.
Protein & peptide mapping.
Analysis of soil and water samples.
Clinical analysis of blood and urine samples.
Purification of synthetic organic and inorganic compounds.
Separation of polar/non-polar compounds.
Separation of geometrical isomers.
Comparison between LC and GC
 Lecture 5 Contents:

Ion-exchange chromatography
 Principle of Ion-exchange chromatography (IEC)

It is a process that allows the separation of ions and polar molecules

based on the charge properties of the molecules.
Ion- exchange chromatography retains analyte molecules based on
ionic interactions.
The stationary phase surface displays ionic functional groups (R-X)
that interact with analyte ions of opposite charge.
 Ion-exchange chromatography

Stationary phase

• The stationary phase in IEC consists of beads made of a polystyrene

polymer crosslinked with divinylbenzene.
• The crosslinked polymer (resin) has free phyenyl groups attached to the
chain, which can easily be treated to add ionic functional groups.
 Ion-exchange chromatography

Cross linkage:
• The greater the crosslinkage of the resin, the greater the difference in
selectivity.

• Generally, crosslinkage also increases the rigidity of the resin, reduces

swelling, reduces porosity, and reduces the solubility of the resin.

• In general, medium-porosity materials are used for low-molecular-

weight ionic species and high-porosity materials are used for high-
molecular-weight ionic species.

• The degrees of crosslinkage is expressed by the manufactures as

percent of divinylbenzene. Generally, crosslinkage of 8 to 10% is
used.
 Ion-exchange chromatography

A good stationary phase should have following properties:

• It should be chemically inert.

• It should be inexpensive.

• It should not react with component to be separated.

• It should not react with eluent.

• It should be colorless, uniform in size and shape.

• It should be mechanically stable.

 Ion-exchange chromatography

Two general types of stationary phase can be used in IEC:

1. Cationic exchangers: have fixed negatively charged groups , used to
separate positively charged ions
2. Anion-exchanger: have fixed positively charged groups , used to
separate negatively charged ions
 Ion-exchange chromatography

Reversible exchange of ion between the ion present in the solution and those present in
the ion exchange resin .
Cation exchange:
-Separation of cations
- Solid-𝑯+ + 𝑴+ → Solid- 𝑴+ + 𝑯+
(solution) (solution)
The cations retained by the solid matrix of ion exchange resin can be eluted by using
buffers of diff strength and hence separation of cations can be effected.
 Ion-exchange chromatography

Anion exchange :
- Separation of anions using anion exchange resin
- Solid-OH + A- Solid -A + OH -
(solution) (solution)
-The anions retained by the solid matrix of ion exchange resin can be eluted
by using buffers of diff strength
 Ion-exchange chromatography

Chemical Structure Functional Group Chemical Nature Type of Exchange

-SO-H+ Sulfonic acid Strong acid Cation

-COO-H+ Carboxylic acid Weak acid Cation

-CH2COO-H+ Carboxymethyl Weak acid Cation

-CH2N+(CH3)3Cl- Quaternary ammonium Strong base Anion

Quaternary ammonium Strong base Anion

CH3

CH2N+ CH2CH2CH(Cl-)

CH3
CH3 Tertiary ammonium Weak base Anion

CH2NH+ OH-

CH3
CH2CH3 Diethylaminoethyl (DEAE) Weak base Anion

CH2CH2NH+ OH-

CH2CH3
 Types of ion exchangers

1. Ion exchange resins are used for the separation of small molecules
2.Ion exchange gels are used for the separation of large molecules like
proteins nucleic acid.
3. Inorganic ion exchanger is used in separation involving harsh
chemical conditions (high temperature high radiation , levels, strongly,
basic solution or powerful oxidizing agents ).
 The exchange capacity of a resin

• It is the total number of equivalents of replaceable hydrogen per unit

volume or per unit weight of resin, and it is determined by the number
and strength of fixed ionic groups on the resin.

• The ion exchange capacity affects solute retention, and the exchangers of
high capacity are most often used for separating complex mixtures,
where increased retention improves resolution.
 Regeneration of the ion exchange resin

- The ion exchange resin after separation may not be useful for next separation
as exchangeable functional groups are lost .
- Regeneration: replacement of the exchangeable cations or anions present in the
original resin
- regeneration of the cation exchange resin is done by the charging the column
with strong acid like HCl.

Advantages:

1. this chromatography has a high loading capacity to handle large sample

volume .

2. It is very simple.
 Factors affecting ion exchange separations

1-Nature & properties of ion exchange resins.

2-Nature of exchanging ions:
A) valency of ions:
at low conc & ordinary temp , extent of exchange increases with increase in valency
𝑁𝑎+ < 𝐶𝑎2+ < 𝐴𝑙 3+ < 𝑇ℎ4+
B) Size of ions:
for similar charged ions, exchange increases with decrease in the size of hydrated ion.
Li+ < 𝐻 + < 𝑁𝑎+ < 𝑁ℎ4+ < 𝐾 + < 𝑅𝑏 + < 𝐶𝑠 +
C) Polarizability:
exchange is preferred for greater polarizable ion :
𝐼 − < 𝐵𝑟 − < 𝐶𝑙 − < 𝐹 −
D) concentration of solution:
in dilute solutions, polyvalent anions are generally adsorbed preferentially
 Factors affecting ion exchange separations

E) concentration and charge of ions:

- if resin has higher +ve charge and solution has lower +ve charge ,
exchange is favoured at higher conc.
- If the resin has lower +ve charge and solution has high +ve charge , then
exchange is favoured at low conc.

resins

Ion exchangers Gels

Inorganic
exchangers
 Example

• Amino acids may be positively or negatively charged or neutral (isoelectric point). These three
forms may be separated by a combination of cation and anion exchange resins.
• At specific pH, they can exist in an ionic(-),cationic (+)or zwitterion (no net charge) stage.
• Amino acids are amphoteric (can act as acids or bases).

cationic pH =pI * anionic

pH increase

* isoelectric point (pI): is the pH at which the net charge on the molecule is zero.
 Applications of the ion exchange
chromatography

1- Ion exchange chromatography is used to convert one salt to other.

2- Ion exchange is used to prepare de-ionized water

3- Separation of similar ions:

A mixture of sodium, hydrogen and potassium can be separated using cation
exchanger resin.
4- It can be used for almost any kind of charged molecule including large
proteins, small nucleotides and amino acids. It is often used in protein
purification, water analysis.
 Applications of the ion exchange
chromatography

Ion exchange chromatography for water treatment:

1- Obtaining soft water:
Water is passed through a cation-exchange resin (Na+ form).

What happens to CaSO4 if present in the water?

Ca2+ is replaced with 2 Na+ ,SO42- is not affected.

The resin can be regenerated with NaCl solution.
 Applications of the ion exchange
chromatography
Ion exchange chromatography for water treatment:
2- Obtaining deionized water:

Water is passed through an anion-exchange resin (OH- form) and cation

exchange resin (H+ form) .

What happens to Cu(NO3)2 if present in the water?

Cu2+ is replaced with 2 H+ and NO3- is replaced with OH-

H+ + OH- = H2O is eluted while Cu(NO3)2 is "trapped“.