BASICS OF CHROMATOGRAPHY
Prepared by
DR: Sherif Abdel-Naby Abdel-Gawad
PhD Analytical Chemistry-Faculty of Pharmacy Cairo University
History of
Chromatography
Mikhail Tswett is credited with the
invention
of
chromatography.
Developed a technique that separated
various
plant
pigments
(e.g.,chlorophylls and xanthophylls) by
passing solutions through glass columns
filled with finely ground CaCO3.
The separated species appeared as
colored bands on the column.
Hence Greek chroma = color and
graphein = writing
M. S. Tswett
Russian Scientist
(1872-1919)
Tswett’s experiment
CHROMATOGRAPHY
Basic Principles
Two phases considered:
1) Mobile Phase: solvent moving
through the column.
2) Stationary Phase: stays in place
inside of the column.
“Eluent”
COLUMN
“Eluate”
Process is called “elution”
Simple Column
Fresh eluent
Initial band
of A and B
Column
Packing
Porous Disk
•Two phases: mobile and stationary
•Mobile phase is solvent moving through
column
–liquid (Methanol, water, buffer)
–gas (He, H2, N2)
•Stationary phase fixed inside column
–viscous liquid coated on inert support inside of
column
–solid particles packed inside column
•Solutes have different affinities for mobile
phase and stationary phase
Classification of chromatographic methods
I- Classification according to the nature of mobile
phase
A- Liquid chromatography (LC)
i-Liquid-solid chromatography (LSC)
In this type, stationary phase is solid
ii- Liquid-liquid chromatography (LLC)
In this type the stationary phase is liquid, which may be
mechanically held on a solid support in the form of thin coat. It may
be packed in a tubular column or spreaded as thin layer on a plate.
Bonded phase chromatography (BPC)
The liquid stationary phase may be chemically and permanently
held to a solid support.
B- Gas Chromatography (GC)
The mobile phase used in this type is a gas. This type can be
classified according to the nature of stationary phase into:
i- Gas-Liquid chromatography (GLC)
In this type the stationary phase is a liquid, which may be
mechanically held on a solid support in the form of thin coat
and packed in a column or it coats the inner wall of capillary
column.
ii- Gas-Solid Chromatography (GSC)
In this type the stationary phase is a solid.
II- Classification according to the mechanism of
attachment to the stationary phase.
A- Adsorption
Chromatography
Stationary phase : Solid
Mobile phase : Liquid or
Gas
How it separates
–solute adsorbs on to
stationary phase surface
B- Partition Chromatography
Stationary phase: Liquid on
solid support
Mobile phase: Liquid or Gas
How it separates
–solute dissolves into
liquid coating
C- Ion-Exchange
Chromatography
Stationary phase: Anions or cations
covalently bound to solid
stationary phase
Mobile phase :liquid
How it separates
solute ions of opposite charge
attracted to stationary phase
D- Molecular Exclusion
Chromatography
Stationary phase: Porous gel
Mobile phase: Liquid
How it separates
–small molecules
trapped in pores of
stationary phase
Also called
gel filtration,
gel permeation,
or
size-exclusion
chromatography
E- Affinity Chromatography
Stationary phase :
Immobilized molecules on
liquid or solid stationary
phase
Mobile phase: Liquid
How it separates
–Molecules with specific
shape dock with ligands
III- Classification according to the used technique
A- Columnar:
In this type the stationary phase is packed in a
column, e.g. GC, LC and conventional column.
B- Planar:
In this type the stationary phase is spreaded as a thin
layer on glass, plastic or aluminium plates; or it is
held in the network structure of paper (paper
chromatography).
Summary of types of chromatography
Mobile
Phase
Liquid
Gas
Liquid chromatography (LC)
Gas chromatography (GC)
Chromatographic
method
Stationary phase
Solid
liquid
Solid
liquid
method
LSC
LLC
GSC
GLC
Mechanism of
Adsorption
chromatography
Partition
chromatography
Adsorption
chromatography
Partition
chromatography
Columnar &
Planar
Columnar & Planar
Columnar
Columnar
Chromatographic
sorption
Technique
Theory of chromatography
The plate theory
•
The plate theory assumes
that, a column of stationary
phase is regarded as a large
number of horizontal, narrow,
discrete, consecutive theoretical
layers known as theoretical
plates (N).
• At each plate of the stationary
phase continuous partitioning
and equilibration of solutes
(components molecules) occurs
as the mobile phase moves
down the column.
• The efficiency of the column is increased as
the number of theoretical plates (N) is
increased, i.e the efficiency of column is
measured by N.
• Also, the column efficiency is measured by
Height Equivalent of Theoretical plates (HETP)
which is denoted by (H)
• N = L/H or H = L/N
Where:
N = number of theoretical plates.
L = Column length.
Chromatogram:
•
It is a plot of solute
concentration as given by
detector response versus
elution time.
• The position of peak on
the time axis can be used
for identification of the
sample
• The area under the peak
provides a quantitative
measure of the amount of
each species.
A typical chromatogram for a sample
containing two components
Some terms used in chromatography
1- R Value (retardation factor):
R=
tm
_________
tm + ts
where tm is the time that the substance spends in the mobile
phase and ts is the time that it spends in the stationary
phase.
•R value for a certain substance varies with change of both or
either of the mobile and stationary phases , but once we
state the conditions of a particular chromatographic method
R value will be characteristic for the substance and used for
its identification .
•The differences in migration rates cause the components in
a mixture to separate into bands located along the column.
2-Rf Value in thin layer chromatography, paper
chromatography and in column chromatography
where the sample band moves down the column to a
definite distance:
Rf = the distance traveled by the zone center
The distance traveled by solvent (solvent front)
As the solute is more retained as the smaller will be the
Rf value.
3- Rt or tR Vaue The Retention Time : (GC and HPLC)
it is the time from the injection to the emergence of the
peak maximum, (notice that as the solute is more
retained the larger will be the Rt value).
4- Rv The Retention Volume :
It is the total volume in ml of eluent (mobile phase or
solvent ) required to elute the center of the zone of a
particular substance. It is used in column, HPLC, and GC,
(notice that as the solute is more retained the larger will
be the Rv value).
5-Capacity Factor K\:
It is widely used to describe the migration rates of solutes
on columns. K\ = K Vs / Vm also
K\ = t R - t m
tm
6-Column resolution (Rs)
A- Selectivity factor () : for a column separating two
components A and B
K\B
(tR)B – tm
 = ________ = ______________
K\A
(tR)A – tm
K’B is the capacity factor of the more retained substance
and K'A is that for the less retained one.
• The larger the  the more selective the stationary
phase and the more effective the separation, 
should be more than one.
B- Resolution factor
Rs = 2z / WB + WA
= 2 [tR(B) – tR(A)]/WB + WA
It is clear from the equation that, the greater
separation of the zones from each other and/or
smaller the zone width the larger will be
resolution. Rs should be at least 1 and 1.5
complete separation
the
the
the
for
7- The Efficiency of the Column:
It is determined by two parameters , the migration
of the zone and the zone broadening i.e. the
retention time and the zone width
• Column efficiency is quantitatively determined by
calculating the number of theoretical plates N or H
(HETP)
N =16 tR2 / W2
while H = LW2 / 16 tR2
So as the retention time increases and / or the zone
width decreases the more efficient will be the
column .
Factors affecting zone broadening.
Zone broadening is due to three factors:
Eddy diffusion (Multiple path effect)
• This effect occur in packed column not in open
tubular columns.
• Eddy diffusion is caused by imperfect packing of the
column with the stationary phase. Imperfect packing
causes the presence of air bubbles or cracks.
Imperfect packing is also, due to the use of irregular
large particles stationary phase.
Ordinary diffusion.
It results from low flow rate of the mobile phase,
where molecules of the separated component move
from the concentrated zone center to the borders of
the zone.
Non equilibrium mass transfer
It results from high flow rate of the mobile phase. At
high flow rate of the mobile phase there is no
enough time for molecules of the separated
component to distribute themselves between the
mobile phase and the stationary phase.
• Some of the molecules move ahead with the
mobile phase at the front of the zone and
some will remain on the stationary phase on
the back of the zone.
• Thus to minimize zone broadening we have to
use small regular stationary phase particles,
perfect packing (no cracks or air bubbles) and
choose optimum flow rate.
Van Deemter Equation
• Putting the three factors together yields the Van
Deemter equation that helps predict how the
column flow rate will affect the theoretical plate
height
H= A + B/Ux + CUx
• A (Eddy diffusion) H independent on flow rate.
• B/Ux (Ordinary diffusion) H inversely proportional to
flow rate.
• CUx (Non equilibrium mass transfer) H proportional
to flow rate.
Optimization of column performance
To optimize the column performance we have to
change  and KB to obtain Rs value of 1.5. This can be
achieved by:
1. Change the composition of mobile phase in LC .
2. Change the flow rate of the mobile phase.
3. The column must be perfectly packed with small
regular spherical particles, without any cracks,
gabs or spaces to minimize zone broadening and
to maximize N.
4. If resolution is still less than 1, the stationary
phase must be changed.
Types of elution
A- Isocratic elution (Simple elution)
In this type of elution a mobile phase is either
single solvent or mixture of solvents without
change of its composition.
B- Gradient elution
In this type of elution the composition of the mobile
phase is changed during chromatographic separation
either stepwise or continuously.
Requirements of solvents used as mobile phase
1. It must dissolve the components of the mixture to
be separated.
2. It must be of different eluting power.
3. Solvents of weaker elution power can be tried
successively by addition of solvent of higher elution
power (as in case of gradient elution).
The elution power of a solvent is measured by its
adsorbability on the stationary phase. Solvents are
arranged according to their elution power in what is
called the elutropic series.
Example
Eluting power of a series of solvents for
substances adsorbed on silica gel, arranged in
descending series.
H2O > methanol > ethanol > propanol > acetone >
ethyl acetate > ether > chloroform >
dichloromethane > benzene > toluene >
trichloroethylene > carbon tetrachioride >
cyclohexane > hexane.
• This order is the order of decreasing dielectric
constant of the solvents.
• The purity of solvents should be as high as
possible
Choice of chromatographic separation methods.
• In case of substances of similar chemical types,
partition chromatography is preferable.
• In case of substances of different chemical types,
adsorption
chromatography
gives
better
separation.
• Gaseous and volatile substances need separation
adopting gas chromatographic separation.
• Ionic and inorganic substances are better
separated on ion exchange column and zone
electrophoresis.
HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY (HPLC)
• For conventional column chromatography where the
mobile phase is liquid (Liquid Chromatography LC) and
the stationary phase is either a solid or a liquid which is
coated on solid support.
• In trials to improve chromatographic separation by
increase the stationary phase surface area either we
may decrease the particle size in this case the mobile
phase flow rate will decrease and stops after few ml or
we may increase the column length in this case the
elution will take a very long time may be several hours.
• However, it was not until the late 1960s, that the
technology of producing and using small size
columns packed with stationary phase of 10m
particle diameter.
• To allow the flow of mobile phase within
reasonable time it is introduced under Pressure
for this reason the method was first referred as
High Pressure Liquid Chromatography.
• In HPLC system, a pressure is applied to the
column, forcing the mobile phase through at a
much higher rate. The pressure is applied using a
pumping system.
Types of Samples For HPLC:
• The sample can be organic, inorganic (including
ionic) compounds, volatile, nonvolatile thermally
stable or labile.
• Molecular weight ranges from ~80-~6000.
Advantages of HPLC
• Speed the analysis (takes only few minutes).
• High resolution; very complex mixture can be
resolved to its components.
• High accuracy; Relative error less than 1% for
quantitative analysis.
• High sensitivity; according to the nature of sample
and detector it is possible to measure a
concentration of 10-6 - 10-9 g/ml.
• Automatic systems; the whole operation starting
from injection of the sample to identification of the
peaks and determination of the concentration, and
then repeat the cycle with next sample.
Limitation of HPLC
• Expensive instrumentation and supplies.
• The requirement of experience at least 6-12 months
of practical experience in methods developments are
generally required to become a professionally
operator and obtain the maximum benefit, from
HPLC.
HPLC - Instrument
Schematic diagram showing liquid flow in a typical liquid
chromatographic instrument.
Mobile Phases:
A good solvent must possess the following
requirements:
• High purity and readily available.
• Low viscosity, limited flammability and toxicity
and low reactivity to avoid chemical
interaction with solutes or stationary phase.
• For LLC, immiscibility with stationary phase.
• Compatibility with the detector.
PUMPS:
The requirements for pumping system are
mainly the following
• generate a pressure up to 500 atmosphere
• produce a pulse free output.
• The flow rates ranging from 0.1 to 10ml/min
• To have high resistance to corrosion by a
variety of solvents.
There are THREE types of pumps:
1. Reciprocating pumps
• This is the most commonly used.
• It consists of a small cylindrical chamber that
is filled and then emptied by the back and
forth motion of a piston.
• The pumping motion produces a pulsed flow
that must be damped.
2-Syringe type: single stroke type it has the
advantage of introducing the solvent at pulse
free constant flow rate, but it suffers from
impossibility of solvent gradient and the
limited solvent volume.
3-The pneumatic or constant pressure type, it
is cheap but it has the same disadvantages as
the syringe type.
PRECOLUMN:
• It contains a packing chemically identical to
that in the analytical column.
• The purpose of the precolumn is to remove
impurities from the solvent, and thus prevent
contamination of the analytical column.
• In addition the precolumn saturates the
mobile phase with the stationary phase thus
eliminate stripping (washing) of the
stationary phase from the analytical column
during elution with the solvent.
SAMPLE INJECTION SYSTEM
• It is used for introduction of the
sample.
• By changing the position of the
valve, the loop can be switched in or
out the stream of solvent as it passes
from the pump to the column.
• While the loop in the position out of
the stream of the solvent they can be
filled with syringe till overflow
occurs, this insures that the loop is
completely filled, then the position
of the loop is changed by rotating or
sliding the valve, and allow it to pass
with the solvent to the column.
• Loops of different volumes are used
(10-50l).
COLUMNS USED FOR HPLC
• Columns for HPLC are manufactured from heavy
walled glass tubing or stainless steel.
• The typical range for column length is from 1525cm with an inside diameter from 5-10mm.
• Column packings typically have particle sizes of 5
to 10m diameter.
• Recently high speed and high performance
microcolumns with an inside diameter of 1 to 4
mm and length of 3 to 7.5 cm are also available.
• They have the advantages of speed and minimal
solvent consumption.
Column Packing Materials:
There are TWO main types of column packing:
1- Pellicular Particles:
• This consists of spherical, nonporous glass or polymer
beads 30-40 m diameter, deposited on its surface a
thin porous layer or crust of silica, alumina, porous
polymer or an ion exchange resin.
• Also liquid stationary phase or chemically bonded
phase can be attached to the surface.
2- Porous Micro Particles:
• This particles have diameter ranging from 3 to 10m,
they are composed of silica, alumina, porous polymer
or ion exchange resin.
• Silica particles may be coated with thin organic film,
which is physically or chemically bonded to the surface.
CH3
Si
OH + Cl
Si
CH3
R
Si
O
Si
CH3
Giass Surface
CH3
organochlorosilane
(R: C4H17 or C18H37)
surface of a silica
particles
R
Si
OH
HO - R
Hydroxyl
Si
OH
HOOC - R
Carboxyllc acid
Si
OH
HN-R
1o and 2o Amino
Si
OH
HS-R
Thiols
Silanol Group
Detectors
Questions to ask when evaluating an LC detector:
–Is the detection universal?
–Is the detector response linear?
–What is the limit of detection (LOD)?
–Is the detector useful with gradient elution?
UV Absorbance Detector:
• The detector consists of source of UV
radiation, the beam of which is split and pass
through two flow cells 10 to 14l.
•
•
•
•
•
•
One through which pure solvent flows and
the other through which solvent containing
the sample flows.
Light from both cells will then pass to
wavelength selector, then to two phototubes
for detection of absorbed radiation.
When the same solvent flows through the
cell no signal is obtained, while if sample
passes through one cell difference in current
occurs and a signal is obtained.
LOD ~ 0.1 ng
Good detection method with gradient elution
Use solvents that do not absorb UV radiation
Fluorescence Detector
•
•
•
•
•
•
•
•
•
Similar idea to UV detector
Few molecules fluoresce
Derivatize solutes with fluorescent tag
Derivatize solutes as they elute from column
before detection(Post-column Derivatization)
Essentially linear
LOD ~ 0.001 ng
Fine with gradient elution
Use solvents that do not fluoresce
Refractive Index Detector
• Compare refractive indices of eluate &
reference
• Cannot use with gradient elution
• Essentially universal
• LOD ~ 100 ng
• Essentially non-linear (Linear range very small)
• Disaster with gradient elution
Comparison of LC Detectors
Type
Sample
Advantage
Comments
UV-VIS
For light absorbing
compounds
Detect wide range of samples.
Common detector.
Of intermediate
sensitivity.
Fluorescence
For Fluorescent
compounds
Highly sensitive detector.
Most samples need
derivatization (except
native fluorescent
molecules)
MS
Universal detector
Can be used for both separation
and structure elucidation.
Of intermediate
sensitivity.
Electrochemical
conductometric
Specific detector; for
all ions
Specific for ions
Electrochemical
amperometric
Specific detector;
Electroactive compounds
Specific for electroactive
elements.