CHROMATOGRAPHY

BY
Prof. Dr.
Mohamed wafaa nassar
Chromatography:
Technique used for separation ,
isolation and identification of
mixture
when mixture applied on stationary
phase ,its component move by mobile
phase in different ratios and
separation occurs
Chromatography is a physical
separation technique which resolves
the individual components of a
mixture based on their distribution
between the stationary phase and
mobile phase
The stationary phase:
In chromatography is fixed in
place either in a column or on a
planar surface.
The mobile phase:
In chromatography moves over or
through the stationary phase
carrying with the analyte mixture.
The mobile phase may be a gas or a
liquid.
CHROMATOGRAPHY
The most widely used means of
performing analytical
separations is
chromatography, a method
that finds application to all
branches of science
HPLC Applications
Bioscience
Chemical proteins
peptides
polystyrenes nucleotides
dyes
phthalates

tetracyclines
Pharmaceuticals corticosteroids
antidepressants
Consumer Products
barbiturates
lipids
antioxidants
sugars
Environmental
polyaromatic hydrocarbons
Clinical
Inorganic ions amino acids
herbicides vitamins
homocysteine
5
The name
"chromatography" is
derived from two
Greek words
"chroma" meaning
"color" and
"graphein" meaning
"to write"
Classification of chromatographic methods
Chromatographic separations
Chromatography:
Is a technique in which components of
mixture are separated based on difference
in the rate at which thy are carried
through a fixed or stationary phase by a
gaseous or liquid mobile phase.
The stationary phase:
In chromatography is fixed in place either
in a column or on a plannar surface.
The mobile phase:
In chromatography moves over or through
the stationary phase carrying with the
analyte mixture.
The mobile phase may be a gas or a liquid.
Parameters of chromatography
Eluent : Is a solvent used to carry
the components of a mixture
through a stationary phase.
Elution : Is the process in which
solute, are washed through
stationary phase by the
movement of mobile phase.
eluate. The mobile phase that exits
from the column is termed eluate
Distribution constant:
is equal to the ratio of solute
molar concentration in the
stationary phase to its molar
concentration in mobile phase.
Retention time (tR): Is the time
between injection of a sample and
appearance of a solute peak at
the detector of a chromatographic
column.
The retention factor (kA): For solute A
is related to the rate at which A
migrates through a column. It's the
amount of time a solute spends in the
stationary phase relative to time it
spends in mobile phase.
The selectivity factor (): For solute
A and B is defined as the ratio of
distribution constant of the more
strongly retained solute (B) to the
distribute constant for the less
strongly held solute (A).
The Dead time (Void time )tm:
time takes for an
unretained species to
pass through a
chromatographic
column.
Resolution:
• The more efficient a column the
greater degree of resolution it will
produce between closely eluting
peaks. The resolution between two
peaks (A) and (B) .
• Complete separation is considered
to be Rs = 1.2
Efficiency of Column :
More efficient of column when
retention time increase and zone
width decrease
The efficiency of Column is
determined by calculating the
number of theoretical plates (N)
•Plate count or number of
theoretical plates (N)
Chromatographic column efficiency
Depend upon
1- Plate hight (H)
2- Plate count or number of
theoretical plates (N)
The efficiency of chromatography
increase as the plate count (N)
become greater
and as the plate height (H) becomes
smaller.
Brodening of peak factors
Zone (peak) brooding is due to three factors
1- Eddy diffusion
Caused by
a) Bad backing of column,
b) Presence of air baubles,
c) The use of irregular large particles
stationary phase
d) the presence cracks in the column.
2- Ordinary diffusion (Longitudinal diffusion)
Due to low mobile phase flow. That causes
migration of solute from the zone center to
the borders of zone.
3- Non equilibrium mass transfere:
Results from high mobile phase flow rate,
e.g. no enough time for distribution of
To minimize broadening
1- Chose optimum flow rate.
2- Use small, regular statonary phas Particles with
perfect packing, and absence of cracks and
air bubbles.
To optimize the column performance.
1- Optimize the composition of the mobile phase (
in LC) or temp program in (GC).
2- Change flow rate of mobile phase
3- Column must be perfectly packed with small
regular particles without cracks.
4- If resolution < 1.5 stationary phase must be
changed
• To relate the resolution with the column
efficiency, capacity factor and selectivity
factor, some equations are used.
Theory of chromatography
When sample in mobile phase applied
at the head of column as a layer, the
components of sample distribute
itself between 2 phases when mobile
phase forced to the column , the layer
will moved to another fresh portion of
stationary phase. By continuous
addition of mobile phase, it will carry
components to the down part of
column.
• The difference in affinity of
components to stationary phase
causes their separation from
each other.

• (component of highest affinity,

the last one eluted from column
and vice versa).
Chromatographic separation of
mixture A and B
High performance (pressure) liquid
chromatography (HPLC) was,
developed in the mid1970's and
quickly improved with the
development of column packing
materials and the additional
convenience of on-line detectors.
In the late 1970's, new methods
including reverse phase liquid
chromatography allowed for
improved separation between
very similar compounds.
High-performance (Pressure) liquid
Chromatography (HPLC)
High pressure liquid chromatography
(HPLC) is a technique used to separate
the components of a chemical mixture.
These components (or solutes) are
first dissolved in a liquid solvent, and
then forced to flow through a
chromatographic column under high
pressure using pumping system. In this
column, the mixture is separated into
its components after interaction
between the stationary phase mobile
phase.
• The stationary phase defined as the
fixed packing material in the column.
• The moving part of the system is the
mobile phase, which is a liquid.
• The interaction of the solute with
mobile and stationary phases can be
practiced through different choices of
solvents and columns.
• HPLC has the ability to easily
separate a wide variety of chemical
mixtures.
Advantage of HPLC

1-High speed of analysis (take only few

minutes)
2- High resolution and determination of
complex mixture .
3- High accuracy (relative error less
than 1% for quantitative analysis)
4- High sensitivity according to the
nature of samples and detector
5- Automatic system, the whole operation
staring from injection of the sample to
identification of the peak and then repeat
the cycle with next sample
6- Used for determination of intact drug in
presence of its degradation product.
7-High resolution for isomers.
Disadvantage ( Limitation ) of HPLC
1- Expensive instrumentation
2- The requirement of practical
experience need at least 6 – 12 months to
become a professionally operator
3- Expensive supplies ( e.g. columns ,
special high grade solvents )
Classification of HPLC according to
separation mechanism:
(By the type of stationary phase)
1- Liquid-liquid chromatography :
This type is called (Partition) e.g.
normal phase chromatography
(NPC) and reversed phase
chromatography ( RPC)
2- Liquid solid chromatography(LSC)
: This type is called (Adsorption)
3- Ion exchange
chromatography(IEC): Separation
of mixture depend on cation and
anion exchange
4- Size exclusion
chromatography (SEC)
Separation of mixture depend
on size &shape by partition
and sieving
5- Affinity chromatography (AC)
Separation of mixture depend
on function group
6- Chiral chromatography :
Separation of chiral
compounds (enantiomers).
The main components of an HPLC system
The essential components parts
in HPLC instrument include
- Mobile phase Reservoirs and
solvent mobile systems:
- Pump,
- Injector,
- Column,
- Detector
- Recorder.
• Mobile phase Reservoirs and solvent
mobile systems:
• A modern HPLC instrument is
connected with one or more glass
reservoirs. Dissolved gases and dust
must be removed from the liquid.
Remove dissolved gases and dust
from liquid which interfere with the
performance of most detectors.
Degasses may consist of a vacuum
pumping system, distillation system,
a device for heating and stirring or
by inert gas which the dissolved gas
are swept out.
• Choice of mobile phase
• The order of polarities of common mobile
phase solvents are
water>acetonitrile>methanol>ethanol>tetra
hydrofuran>propanol>cyclohexane>hexane
• There are two types of elution:
• Isocratic elution :
Single solvent of constant composition is
used through the analysis method.
• Gradient elution :
is one in which composition of the solvent is
changed continuously or in series of steps.
• Column for high-performance liquid
chromatography
• Column constructed from stainless
steel tubing. Most column range in
length from 10 to 30 cm and inside
diameter 2-5 mm which packed with
5-10 μm. Column of this type provides
40,000 to 60,000 plate /m.
• Recently length of 3-7.5 cm, inside
diameter 1-4.6 mm packed with 3-5μm
particles; contain as many as 100,000
plates /m.
The most packing for liquid
chromatography (L.C) is prepared
from silica particles. Other
packing materials include alumina
particles, porous polymer particles
and ion exchange resins.
• Detectors:
Detector is the eye of liquid
chromatography LC system
and measures the compounds
after their separation on the
column.
• Detectors for HPLC should be
small and compatible with
liquid flow. The detector used
depends on the nature of the
sample.
• Some of common detectors
1) Absorbance 2) Flourescence
3) Refractive index 4) Electrochemical
5) Conductivity 6) Mass spectrometry
• Criteria for detectors
• High sensitivity
• Higher liner dynamic range
• Applicable to most of the solutes
• Does not contribute to band broadening
• Non-destructive
• Faster response
• UV detectors

• Variable wavelength detector:

Grating assembly in positioned
between lamp and flow cell
which shall split the light into its
individual compounds allowing
selection of appropriate
wavelength.
Photodiode array detector (PAD oDAD):
The grating assembly is positioned after the flow

cell so that light of different wavelengths can be

measured simultaneously. PAD detectors allow

simultaneous collection of entire chromatograph

at different wavelengths and absorption spectra

can be displayed at any time in the

chromatogram.
• The most widely used detectors for
liquid chromatography are based on
absorption of ultraviolet or visible
radiation

• Spectrophotometric detectors are

more widely used in HPLC. Modern
instrument use diode-array detector
that can display an entire spectrum as
an analyte exits the column.
• Use of combinations of HPLC and
mass spectrometry detector is
currently becoming quite popular
such as liquid chromatography the
mass spectrometry system can
identify the analytes exiting from the
HPLC column.
• Another detector (e.g.) is based on the
changes in the refractive index (RI) of the
solvent that is caused by analyte
molecules.

• The Disadvantage of this detector (RI) is

its limited sensitivity.

• Several detectors are based on

potentiometric, conductometric and
voltammetric measurements have been
introduced.
Recorder and computer system:
The electrical signal obtained from detector
is recorded on recorder fitted with integrator
and chart paper to give a typical plot of
chromatogram representing the elution of
the sample as shown in the following figure

• Fig. Typical chromatogram

• Vo = Void volume
• tR = Retention time
• w = Width at baseline
I. High Performance Partition
Chromatography:
• The partition mode is the most widely used
mode for routine HPLC. In this mode,
solute components distribute themselves
between the mobile and stationary phases.
This distribution depends on the relative
phase solubility of the components.
Because components differ in their relative
solubilities, they spend varying amounts of
time in the mobile phase and thus elute
from the column separately. The extent of
interaction with the stationary phase
governs the degree of retention (less
interactive components elute from the
column earlier than more interactive
components).
The partition mode is divided into two
types: normal phase and reversed
phase. The conditions for normal
separations are a polar stationary
phase for separation of polar
compounds, where the mobile phase
is nonpolar. The bonded, reversed
phase partitioning conditions mirror
the normal phase separation
conditions. Thus, the stationary
phase is nonpolar and the mobile
phase is polar. Examples of normal
and reversed phase separations are
given in Figures 3 and 4.
• Time (minutes)
• Fig. 3: Normal phase chromatogram
• Fig. 4: Reverse phase chromatography
• MOST COMMONLY USED METHODS IN
CHROMATOGRAPHY
• A) Normal-phase chromatography
• Mechanism: Retention by interaction of
the stationary phase's polar surface with
polar parts of the sample molecules.
• Stationary phase: polar e.g SiO2, Al2O3,
• Mobile phase: non polar e.g Heptane.
hexane, cyclohexane. CH2Cl2.
• Application: Separation of non-ionic, non-
polar to medium polar substances.
Stationary phase of
Normal-phase chromatography

H
O
O Si O
O H
O Si O
O
O CH2
N
O Si O
O H
Separation of solutes :
A) Normal phase chromatography

Based on their polarity,

the more polar is the solute, the
greater is its retention on the
column.

Retention mechanism:
Mainly by partition
• Order of elution of compounds:
• Elution is order of increasing polarity
i.e.1. Saturated hydrocarbons.
• 2. Olefins
• 3.Aromatic hydrocarbons & organic
halides
• 4.Sulfides & ethers
• 5.Nitro compounds
• 6.Esters, aldehydes & ketones
• 7.Alcohols & amines
• 8.Carboxylic acids
• Applications:
• Commonly applied to analyses of
samples soluble in non-polar
solvents e.g. fat-soluble vitamins,
hydrocarbons and pesticides
using hexane as the mobile
phase.
• B) Reversed-phase chromatography
Mechanism: Retention by interaction of the
stationary phase's non-polar hydrocarbon
chain with non-polar parts of the sample
molecules.
Stationary phase: non-polar e.g n-octadecyl
(RP- C18) and n-octyl (RP- C8), .
Mobile phase: polar e.g Methanol or
acetonitrile/water or buffer (sometimes
with additives of THF or dioxane).
Application: Separation of non-ionic and ion-
forming non-polar to medium polar
substances (carboxylic acids ~
hydrocarbons). If ion forming substances
(as carboxylic acids) are to be separated.
a pH control by buffers is necessary.
Stationary phase of
Reversed-phase chromatography

CH3 H2O CH3OH H2O

O
O Si O Si
O CH3
O Si O H CH3OH
H2O CH2-OH
O CH3
O Si O Si
O CH3
CH3OH CH3OH H2O
• Choice of mobile phase
• The order of polarities of common
mobile phase solvents are
water>acetonitrile>methanol>ethanol
>tetrahydrofuran>propanol>cyclohex
ane>hexae
• In general, the most chromatographic
preparations are achieved by
matching the polarity of the analyte
to that of the stationary phase, a
mobile phase of considerably
different polarity is then used.
 B. Reversed-phase (RP)
Chromatography (also called bonded-
phase chromatography) (see following
table)
• Separation of solutes:
• Neutral molecules in solution are separated

• based on their hydrophobicity. The more

• polar are eluted first, non-polar

• (hydrophobic) are retained, i.e. elution is in

• order of decreasing polarity.

• Order of elution of compounds:
• Elution is order of decreasing polarity
i.e.
1.Carboxylic acids
2.Alcohols & amines
3.Esters, aldehydes & ketones
4.Nitro compounds
5.Sulfides & ethers
6.Aromatic hydrocarbons & organic
halides
7. Olefins
8. Saturated hydrocarbons.
• Retention mechanism:
• By partition
• Biomedical Applications:
• Widely used, for separating non-polar,
non-ionic compounds and polar (water
soluble)compounds.Pharmaceutical
industry: analysis of steroids, vitamins, β-
blockers, cholesterol, triglycerides &
fatty acids.Isolation & purification of
proteins amino acids, peptides, nucleic
acids& oligosaccharides.Clinical
laboratories: analysis of
catecholamine.Environmental pollution:
analysis of pesticides & herbicides.
• Typical application of high performance parition
Chromatography.
• 1) Pharmaceuticals :
• Antibiotics, Analgesic, sedatives, Steroids
• 2) Biochemical :
• Amino acid, proteins, carbohydrates, lipids
• 3) Food products:
• Artificial sweaters, Additives, Alfatoxins, Anti-
oxidants
• 4) Industrial chemistry:
• Condensed aromatics, surfactants, propellants,
dyes.
• 5) Pollutants:
• Pesticides, Herbicides, phenols
• 6) Forensic chemistry:
• Drugs, Poisons, Blood alcohol, Narcotics
• 7) Clinical medicine:
• Bile acids, Urine extracts, estrogens, Drug
metabolites