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By: Prof / Dr. Tarek Fayed 
Represented by : Mahmoud Galal Zidan 
E-Mail : M7moud.zidan@yahoo.com 
E-Mail : m7moud.zidan@aol.com 
E-Mail : M7moud.zidan@Gmail.com 
Facebook : www.fb.com/m7moud.zidan 
Twitter : www.twitter.com/m7moudzidane 
Tel : +201272122218 /+201273937378
BBaassiiss ooff CChhrroommaattooggrraapphhyy 
What is the chromatography? 
Chromatography is a physical method of 
separation in which the components to be 
separated are distributed between two 
phases (one is the stationary phase and the 
other is the mobile phase). 
The stationary phase may be a solid or a 
liquid supported on a solid, or a gel and 
can’t be gas . 
The mobile phase may be either a liquid 
or a gas, and can’t be solid
We can only control the stationary and 
mobile Phase, while mixtures to be separated 
are the problem we have to deal with. 
Chromatography is a dynamic process 
where the mobile phase moves in definite 
direction by a controlled rate.
R e s e r v o i r 
m o b i l e p h a s e 
s t a t i o n a r y p h a s e 
o r R e s i n 
F l o w - t h r o u g h 
+ S a m p l e 
F r i t 
The analyte interacting most strongly with the stationary 
phase will take longer time (has high retention and eluted 
later) to pass through the system than those with weaker 
interactions.
Theoretical Concept: DDiissttrriibbuuttiioonn RRaattiioo 
The distribution ratio, KS (also called a partition ratio or 
partition coefficient), for component A, is therefore 
KS = [CS 
A] / [CM 
A] 
Each separated components will have different values of K, 
reflecting their relative affinities for the stationary phase.
Russian scientist Tswett in 1906 
used a glass columns packed 
with finely divided CaCO3 to separate 
the green plant pigments extracted 
by hexane. The pigments after 
separation appeared as colour 
bands that can come out of the 
column one by one. 
Tswett was the first to use the term "chromatography" 
derived from two Greek words "Chroma" meaning 
color and "graphein" meaning to write.
Classification of Chromatographic methods 
Mobile phase: a solvent that flows through the 
supporting medium to elute the mixture components. 
Stationary phase: a layer of solid or liquid film coating 
on the supporting medium that interacts with the 
analytes to be separated. 
Chromatographic methods are classified according to: 
A – Mechanism of separation: 
The mechanism of separation depends mainly on the 
nature of the stationary phase. Based on separation 
mechanisms chromatography can be classified into:
1- Adsorption Chromatography: 
It is the oldest technique. 
Separation is due to 
difference in the 
adsorption power 
of mixture components. 
The stationary phase is 
a solid with adsorption 
characters. 
Silica gel and alumina are the 
most Common stationary 
phase in adsorption 
chromatography. 
DIAGRAM OF SIMPLE LIQUID COLUMN CHROMATOGRAPHY 
A + B + C 
OOOOOOOOOOO 
OOOOOOOOOOO 
OOOOOOOOOO 
OOOOOOOOOOO 
OOOOOOOOOOO 
OOOOOOOOOO 
OOOOOOOOOOO 
OOOOOOOOOOO 
OOOOOOOOOO 
OOOOOOOOOOO 
OOOOOOOOOOO 
OOOOOOOOOO 
OOOOOOOOOOO 
OOOOOOOOOOO 
OOOOOOOOOO 
OOOOOOOOOOO 
OOOOOOOOOOO 
OOOOOOOOOO 
OOOOOOOOOOO 
OOOOOOOOOOO 
OOOOOOOOOO 
OOOOOOOOOOO 
OOOOOOOOOOO 
OOOOOOOOOO 
OOOOOOOOOOO 
OOOOOOOOOOO 
OOOOOOOOOOO 
OOOOO OOOO 
OOOOOOOOOOO 
OOOOOOOOOO 
OOOOOOOOOOO 
OOOOOOOOOOO 
OOOOOOOOOO 
OOOOO OOOO 
OOOOOOOOOOO 
OOOOOOOOOO 
OOOOOOOOOOO 
OOOOOOOOOOO 
OOOOOOOOOO 
OOOOOOOOOOO 
OOOOOOOOOOO 
OOOOOOOOOO 
OOOOO OOOO 
OOOOOOOOOOO 
OOOOOOOOOO 
OOOOOOOOOOO 
OOOOOOOOOOO 
OOOOOOOOOO 
OOOOOOOOOOO 
OOOOOOOOOOO 
Sample 
(A+B+C) 
Column 
Solid Particles 
(packing material-stationary 
phase) 
Eluant (eluate) 
A 
B 
C 
Solvent(mobile or 
moving phase)
2- Partition Chromatography: 
Separation is due to difference in solubility of components 
in two immiscible liquids. 
The stationary phase is a liquid thin film on an inert solid 
support. The stationary liquid is usually more polar than 
the mobile phase. Cellulose powder and wetted silica gel 
are examples of supports in partition chromatography that 
carry film of water act as stationary phase. 
Solid Support Film of the liqiud 
stationary Phase
3- Ion Exchange Chromatography (IEC): 
It is used for separation of charged molecules. The 
stationary phase is an ion exchange resin to which a 
cationic or anionic groups are covalently bonded. Ions of 
opposite charges (counter ions) in the mobile phase will 
be attracted to the resin and compete with the 
components of the mixture for the charged group on the 
resin. The mobile phase must be charged (electrolyte or 
buffer solution). 
H+ 
H+ 
COO- NH4 
+ 
Resin Resin 
COO- NH4 
+ 
Counter counter 
ions ions 
Anionic exchanger Cationic exchanger 
OH- OH-
4- Gel Filtration Chromatography 
Dr Gihan Gawish 
Also called " Gel Permeation, 
Molecular Sieve or Size exclusion“: 
Separation is according to difference 
of size or Molecular weight (MW). 
 Stationary phase (column matrix): 
is beads of hydrated, porous polymer. 
 Mobile phase: is buffer or solvent.
5- Zone Electrophoresis: 
Separation of electrically charged bio-molecules under the 
effect of an electric field of strength E, they will freely 
move towards the electrodes of opposite charge. 
Sample application 
Bands of negativey 
charged compounds 
Bands of postivey 
charged compounds
6- Affinity Chromatography: 
The separation is based on the affinity of proteins to 
specific ligands such as enzymes. The ligand is attached 
to suitable polysaccharide polymer such as cellulose - 
agarose – dextran. 
7- Chiral Chromatography: 
In this type we can separate enantiomers – we used chiral 
stationary phase that react with one enantiomer more 
then the other so separation takes place.
B- According to the nature of the mobile and 
stationary phase: 
In this regard chromatography is classified into: 
1- Liquid Chromatography (LC): 
The mobile phase is liquid. In case of separation by 
adsorption the stationary phase is solid so it is called: 
Liquid-Solid Chromatography (LSC). If separation occurs 
through partition, the stationary phase is liquid so it is 
called: Liquid -Liquid Chromatography (LLC). 
2- Gas Chromatography (GC) 
The mobile phase is an inert gas nitrogen or helium. 
Again if the stationary phase is solid it is called: Gas– 
Solid Chromatography (GSC). When stationary phase is 
liquid it is called: Gas-Liquid Chromatography (GLC).
C- According to the technique (methods of holding the 
Stationary Phase): 
1- Planar or Plane Chromatography: 
In this type the stationary phase is used in the form of 
layer. Plane chromatography is further classified into: 
a- Thin Layer Chromatography (TLC): 
The stationary phase is spread on glass or plastic or 
aluminum sheets. 
b- Paper Chromatography (PC): 
A specific type of papers is used as stationary phase. 
2- Columnar or Column Chromatography (CC): 
The stationary phase is held in to a tube made of glass or 
metal (gel – ion exchange – adsorption).
CCllaassssiiffiiccaattiioonn ooff CChhrroommaattooggrraapphhyy
Chromatography Nomenclature 
Typical response obtained by chromatography is called 
a chromatogram: chromatogram- concentration versus elution time 
Wh 
Wb 
Inject 
Where: tR = retention time tM = void time 
Wb = baseline width of the peak in time units 
Wh = half-height width of the peak in time units
Forces Responsible for Separation 
The affinity differences of the components for the 
stationary or the mobile phases can be due to several 
different chemical or physical properties including: 
 Ionization state 
 Polarity and polarizability 
 Hydrogen bonding / van der Waals’ forces 
 Hydrophobicity 
 Hydrophilicity 
The rate at which a sample moves is determined by how 
much time it spends in the mobile phase.
A summary of chromatographic terms are defined as 
follows: 
The baseline is any part of the chromatogram where 
only mobile phase is emerging from the column. 
The peak maximum is the highest point of the peak. 
The injection point is that point in time/position 
time when/ where the sample is placed on the column. 
The dead point is the position of the peak-maximum 
of an unretained solute. 
The dead time (to) is the time elapsed between the 
injection point and the dead point.
The dead volume (Vo) is the volume of mobile 
phase passed through the column between the 
injection point and the dead point. 
The retention time (tr) is the time elapsed 
between the injection point and the peak 
maximum. Each solute has a characteristic 
retention time. 
The retention volume (Vr) is the volume of mobile 
phase passed through the column between the 
injection point and the peak maximum. 
The peak height (h) is the distance between the 
peak maximum and the base line geometrically 
produced beneath the peak.
Chromatography Lec 1
The Development Procedures 
It is the process, by which the mixture components 
(substances) are moved through the chromatographic 
system (through a column or along a plate). The solute 
progresses through the chromatographic system, only 
while it is in the mobile phase. There are three types of 
chromatographic development: 
Elution development, 
Displacement development 
Frontal analysis. 
Elution development is the only development technique 
employed in both GC and LC
Displacement Development 
Displacement development is only effective with a solid 
stationary phase where the solutes are adsorbed on its 
surface. 
The components array themselves along the distribution 
system in order of their decreasing adsorption strength. 
The sample components are usually held on the 
stationary phase so strongly that they are eluted very 
slowly or even not at all. 
The solute must be displaced by a substance more 
strongly held than any of the solutes (called displacer 
which is the mobile phase). 
In displacement development the solutes are never 
actually separated from one another. 
The solutes leave the system sequentially and somewhat 
mixed with its neighbor.
Frontal Analysis 
It can be effectively employed in a column chromatography. 
The sample is fed continuously onto the column as a dilute 
solution in the mobile phase. 
Frontal analysis can only separate part of the first component 
in a relatively pure state, each subsequent component being 
mixed with those previously eluted. 
For a three components mixture, containing solutes (A), (B) 
and (C) as a dilute solution is fed continuously onto a column: 
The first component to elute, (A), will have less affinity to the 
stationary phase. Then the second solute, (B), will elute but it 
will be mixed with the first solute. Finally, the third solute (C), 
will elute in conjunction with (A) and (B). 
It is clear that only solute (A) is eluted in a pure form. 
Thus, frontal analysis is not suitable for for most practical 
analytical applications.
Elution Development 
Elution development is best described as a series of 
adsorption-extraction processes which are 
continuous from the time the sample is injected. 
As the elution proceeds, the moving phase will 
continuously displace the concentration profile of the 
solute in the mobile phase forward, relative to that in 
the stationary phase.
Chromatography Lec 1
Types of elution techniques 
Simple elution: the column is eluted with the same 
solvent all the time. This is suitable when the 
components have similar affinities for the stationary 
phase and are therefore eluted rapidly, one after 
another. 
Stepwise elution: the eluent (solvent) is changed 
after a predetermined period of time. The eluents are 
chosen to have increasing eluting power, that is, 
increasing affinity to the remaining components, and 
there for releasing them from the stationary phase.
Gradient elution: the composition of the eluting 
solvent is changed gradually to achieve separation of 
widely varying affinities for the stationary phase. The 
ratio of two solvents is gradually changed to increase 
slowly the eluting power of the mobile phase. Thus 
enhancing the resolution (narrows the zones and 
reduces tailing). The solvent composition gradient 
may be a concentration, pH, polarity or ionic strength 
gradient. 
Isocratic elution: in isocratic elution mixed solvent 
with a fixed composition is used along the elution 
process (as an example 30% EtOH – 70 Ether)
PPeeaakk RReessoolluuttiioonn oorr sseeppaarraattiioonn
Peak Poor Preesaokl u RRtieeossnoolluuttiioonn oorr sseeppaarraattiioonn ((RR)) 
More separation 
Less band spread 
V 2 
- V 
1/2(W + W ) 
2 
1 
1 
R = 
V is the retention volume, and W is the peak width
SSeelleeccttiivviittyy FFaaccttoorr ((a)):: parameter used to describe how well two 
components are separated by a chromatographic system 
For two component mixture where B 
is more retained than A, then; a >1 
B 
A 
K 
K 
B 
k 
= 
a 
a 
Where; k = (tR –tM)/tM 
k t t 
= ( ) - = ( ) 
- 
R A M 
t t 
= ( ) 
- 
R B M 
R A M 
and k t t 
R B M 
M 
B 
M 
A 
A 
t t 
t 
t 
k 
- 
= 
( ) 
a 
K = Distribution Constant 
Retention factor 
t = Retention time
Separation (column) Efficiency (s): 
The efficiency of separation is related experimentally to a component’s 
peak width. 
- an efficient system will produce narrow peaks, 
- narrow peaks  smaller difference in interactions 
It is determined by the processes that are involved in solute retention 
and transport in the column 
Wh 
Estimate s from peak widths, assuming 
Gaussian shaped peak: 
Wb = 4 s 
Wh = 2.354 s 
Dependent on the amount of time that a solute spends in the column (k’ or tR)
Factors affecting separation effeciency (Peak Broadening) 
Number of theoretical plates (N): compare efficiencies of a system for 
solutes that have different retention times 
N = (tR/s)2or for a Gaussian shaped peak 
N = 16 (tR/Wb)2 or N = 5.54 (tR/Wh)2 
The larger the value of N is for a column, the better the column will be 
able to separate two compounds.
Plate height (H): compare efficiencies of columns with different 
lengths 
H = L/N 
where: L = column length N = number of theoretical plates for the 
column 
H can be also used to relate various chromatographic parameters 
(e.g., 
flow rate, particle size, etc.) to the kinetic processes that give rise to 
peak broadening. 
Why Do Bands Spread? 
a. Eddy diffusion 
b. Mobile phase mass transfer 
c. Longitudinal diffusion
a.) Eddy diffusion : a process that leads to peak (band) broadening 
due to the presence of multiple flow paths through a packed column. 
As solute molecules travel through the column, 
some arrive at the end sooner then others simply 
due to the different path traveled around the 
support particles in the column that result in 
different travel distances. 
Longer path arrives at end of column after (1).
b.) non equilibrium mass transfer: a process of peak broadening 
caused by non equilibrium attainment due to the transfer of olute 
from the higher concentration region to the low concentration 
one 
The degree of band-broadening due to eddy diffusion and mobile 
phase mass transfer depends mainly on: 
1) the size of the packing material 
2) the diffusion rate of the solute
c.) Longitudinal diffusion: band-broadening due to the diffusion of the 
solute along the length of the column in the flowing mobile phase. 
The degree of band-broadening 
due to 
longitudinal diffusion 
depends on: 
1) the diffusion of the solute 
2) the flow-rate of the solute 
through the column 
A solute in the center of the channel 
moves more quickly than solute at 
the edges, it will tend to reach the 
end of the channel first leading to 
band-broadening
Van Deemter equation: relates flow-rate or linear velocity to H: 
H = A + B/m + Cm 
where: 
m = linear velocity (flow-rate x Vm/L) 
H = total plate height of the column 
A = constant representing eddy 
diffusion & mobile phase mass transfer 
B = constant representing 
longitudinal diffusion 
C = constant representing stagnant 
mobile phase & stationary phase 
mass transfer 
This relationship is used to predict what the resulting effect would be of varying 
this parameter on the overall efficiency of the chromatographic system.
Factors increasing peak resolution 
1. Increase column length 
2. Decrease column diameter 
3. Decrease flow-rate 
4. Pack column uniformly 
5. Use uniform stationary phase (packing material) 
6. Decrease sample size 
7. Select proper stationary phase 
8. Select proper mobile phase 
9. Use proper pressure 
10. Use gradient elution

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Chromatography Lec 1

  • 1. By: Prof / Dr. Tarek Fayed Represented by : Mahmoud Galal Zidan E-Mail : M7moud.zidan@yahoo.com E-Mail : m7moud.zidan@aol.com E-Mail : M7moud.zidan@Gmail.com Facebook : www.fb.com/m7moud.zidan Twitter : www.twitter.com/m7moudzidane Tel : +201272122218 /+201273937378
  • 2. BBaassiiss ooff CChhrroommaattooggrraapphhyy What is the chromatography? Chromatography is a physical method of separation in which the components to be separated are distributed between two phases (one is the stationary phase and the other is the mobile phase). The stationary phase may be a solid or a liquid supported on a solid, or a gel and can’t be gas . The mobile phase may be either a liquid or a gas, and can’t be solid
  • 3. We can only control the stationary and mobile Phase, while mixtures to be separated are the problem we have to deal with. Chromatography is a dynamic process where the mobile phase moves in definite direction by a controlled rate.
  • 4. R e s e r v o i r m o b i l e p h a s e s t a t i o n a r y p h a s e o r R e s i n F l o w - t h r o u g h + S a m p l e F r i t The analyte interacting most strongly with the stationary phase will take longer time (has high retention and eluted later) to pass through the system than those with weaker interactions.
  • 5. Theoretical Concept: DDiissttrriibbuuttiioonn RRaattiioo The distribution ratio, KS (also called a partition ratio or partition coefficient), for component A, is therefore KS = [CS A] / [CM A] Each separated components will have different values of K, reflecting their relative affinities for the stationary phase.
  • 6. Russian scientist Tswett in 1906 used a glass columns packed with finely divided CaCO3 to separate the green plant pigments extracted by hexane. The pigments after separation appeared as colour bands that can come out of the column one by one. Tswett was the first to use the term "chromatography" derived from two Greek words "Chroma" meaning color and "graphein" meaning to write.
  • 7. Classification of Chromatographic methods Mobile phase: a solvent that flows through the supporting medium to elute the mixture components. Stationary phase: a layer of solid or liquid film coating on the supporting medium that interacts with the analytes to be separated. Chromatographic methods are classified according to: A – Mechanism of separation: The mechanism of separation depends mainly on the nature of the stationary phase. Based on separation mechanisms chromatography can be classified into:
  • 8. 1- Adsorption Chromatography: It is the oldest technique. Separation is due to difference in the adsorption power of mixture components. The stationary phase is a solid with adsorption characters. Silica gel and alumina are the most Common stationary phase in adsorption chromatography. DIAGRAM OF SIMPLE LIQUID COLUMN CHROMATOGRAPHY A + B + C OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOO OOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOO OOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOO OOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO Sample (A+B+C) Column Solid Particles (packing material-stationary phase) Eluant (eluate) A B C Solvent(mobile or moving phase)
  • 9. 2- Partition Chromatography: Separation is due to difference in solubility of components in two immiscible liquids. The stationary phase is a liquid thin film on an inert solid support. The stationary liquid is usually more polar than the mobile phase. Cellulose powder and wetted silica gel are examples of supports in partition chromatography that carry film of water act as stationary phase. Solid Support Film of the liqiud stationary Phase
  • 10. 3- Ion Exchange Chromatography (IEC): It is used for separation of charged molecules. The stationary phase is an ion exchange resin to which a cationic or anionic groups are covalently bonded. Ions of opposite charges (counter ions) in the mobile phase will be attracted to the resin and compete with the components of the mixture for the charged group on the resin. The mobile phase must be charged (electrolyte or buffer solution). H+ H+ COO- NH4 + Resin Resin COO- NH4 + Counter counter ions ions Anionic exchanger Cationic exchanger OH- OH-
  • 11. 4- Gel Filtration Chromatography Dr Gihan Gawish Also called " Gel Permeation, Molecular Sieve or Size exclusion“: Separation is according to difference of size or Molecular weight (MW).  Stationary phase (column matrix): is beads of hydrated, porous polymer.  Mobile phase: is buffer or solvent.
  • 12. 5- Zone Electrophoresis: Separation of electrically charged bio-molecules under the effect of an electric field of strength E, they will freely move towards the electrodes of opposite charge. Sample application Bands of negativey charged compounds Bands of postivey charged compounds
  • 13. 6- Affinity Chromatography: The separation is based on the affinity of proteins to specific ligands such as enzymes. The ligand is attached to suitable polysaccharide polymer such as cellulose - agarose – dextran. 7- Chiral Chromatography: In this type we can separate enantiomers – we used chiral stationary phase that react with one enantiomer more then the other so separation takes place.
  • 14. B- According to the nature of the mobile and stationary phase: In this regard chromatography is classified into: 1- Liquid Chromatography (LC): The mobile phase is liquid. In case of separation by adsorption the stationary phase is solid so it is called: Liquid-Solid Chromatography (LSC). If separation occurs through partition, the stationary phase is liquid so it is called: Liquid -Liquid Chromatography (LLC). 2- Gas Chromatography (GC) The mobile phase is an inert gas nitrogen or helium. Again if the stationary phase is solid it is called: Gas– Solid Chromatography (GSC). When stationary phase is liquid it is called: Gas-Liquid Chromatography (GLC).
  • 15. C- According to the technique (methods of holding the Stationary Phase): 1- Planar or Plane Chromatography: In this type the stationary phase is used in the form of layer. Plane chromatography is further classified into: a- Thin Layer Chromatography (TLC): The stationary phase is spread on glass or plastic or aluminum sheets. b- Paper Chromatography (PC): A specific type of papers is used as stationary phase. 2- Columnar or Column Chromatography (CC): The stationary phase is held in to a tube made of glass or metal (gel – ion exchange – adsorption).
  • 17. Chromatography Nomenclature Typical response obtained by chromatography is called a chromatogram: chromatogram- concentration versus elution time Wh Wb Inject Where: tR = retention time tM = void time Wb = baseline width of the peak in time units Wh = half-height width of the peak in time units
  • 18. Forces Responsible for Separation The affinity differences of the components for the stationary or the mobile phases can be due to several different chemical or physical properties including:  Ionization state  Polarity and polarizability  Hydrogen bonding / van der Waals’ forces  Hydrophobicity  Hydrophilicity The rate at which a sample moves is determined by how much time it spends in the mobile phase.
  • 19. A summary of chromatographic terms are defined as follows: The baseline is any part of the chromatogram where only mobile phase is emerging from the column. The peak maximum is the highest point of the peak. The injection point is that point in time/position time when/ where the sample is placed on the column. The dead point is the position of the peak-maximum of an unretained solute. The dead time (to) is the time elapsed between the injection point and the dead point.
  • 20. The dead volume (Vo) is the volume of mobile phase passed through the column between the injection point and the dead point. The retention time (tr) is the time elapsed between the injection point and the peak maximum. Each solute has a characteristic retention time. The retention volume (Vr) is the volume of mobile phase passed through the column between the injection point and the peak maximum. The peak height (h) is the distance between the peak maximum and the base line geometrically produced beneath the peak.
  • 22. The Development Procedures It is the process, by which the mixture components (substances) are moved through the chromatographic system (through a column or along a plate). The solute progresses through the chromatographic system, only while it is in the mobile phase. There are three types of chromatographic development: Elution development, Displacement development Frontal analysis. Elution development is the only development technique employed in both GC and LC
  • 23. Displacement Development Displacement development is only effective with a solid stationary phase where the solutes are adsorbed on its surface. The components array themselves along the distribution system in order of their decreasing adsorption strength. The sample components are usually held on the stationary phase so strongly that they are eluted very slowly or even not at all. The solute must be displaced by a substance more strongly held than any of the solutes (called displacer which is the mobile phase). In displacement development the solutes are never actually separated from one another. The solutes leave the system sequentially and somewhat mixed with its neighbor.
  • 24. Frontal Analysis It can be effectively employed in a column chromatography. The sample is fed continuously onto the column as a dilute solution in the mobile phase. Frontal analysis can only separate part of the first component in a relatively pure state, each subsequent component being mixed with those previously eluted. For a three components mixture, containing solutes (A), (B) and (C) as a dilute solution is fed continuously onto a column: The first component to elute, (A), will have less affinity to the stationary phase. Then the second solute, (B), will elute but it will be mixed with the first solute. Finally, the third solute (C), will elute in conjunction with (A) and (B). It is clear that only solute (A) is eluted in a pure form. Thus, frontal analysis is not suitable for for most practical analytical applications.
  • 25. Elution Development Elution development is best described as a series of adsorption-extraction processes which are continuous from the time the sample is injected. As the elution proceeds, the moving phase will continuously displace the concentration profile of the solute in the mobile phase forward, relative to that in the stationary phase.
  • 27. Types of elution techniques Simple elution: the column is eluted with the same solvent all the time. This is suitable when the components have similar affinities for the stationary phase and are therefore eluted rapidly, one after another. Stepwise elution: the eluent (solvent) is changed after a predetermined period of time. The eluents are chosen to have increasing eluting power, that is, increasing affinity to the remaining components, and there for releasing them from the stationary phase.
  • 28. Gradient elution: the composition of the eluting solvent is changed gradually to achieve separation of widely varying affinities for the stationary phase. The ratio of two solvents is gradually changed to increase slowly the eluting power of the mobile phase. Thus enhancing the resolution (narrows the zones and reduces tailing). The solvent composition gradient may be a concentration, pH, polarity or ionic strength gradient. Isocratic elution: in isocratic elution mixed solvent with a fixed composition is used along the elution process (as an example 30% EtOH – 70 Ether)
  • 29. PPeeaakk RReessoolluuttiioonn oorr sseeppaarraattiioonn
  • 30. Peak Poor Preesaokl u RRtieeossnoolluuttiioonn oorr sseeppaarraattiioonn ((RR)) More separation Less band spread V 2 - V 1/2(W + W ) 2 1 1 R = V is the retention volume, and W is the peak width
  • 31. SSeelleeccttiivviittyy FFaaccttoorr ((a)):: parameter used to describe how well two components are separated by a chromatographic system For two component mixture where B is more retained than A, then; a >1 B A K K B k = a a Where; k = (tR –tM)/tM k t t = ( ) - = ( ) - R A M t t = ( ) - R B M R A M and k t t R B M M B M A A t t t t k - = ( ) a K = Distribution Constant Retention factor t = Retention time
  • 32. Separation (column) Efficiency (s): The efficiency of separation is related experimentally to a component’s peak width. - an efficient system will produce narrow peaks, - narrow peaks  smaller difference in interactions It is determined by the processes that are involved in solute retention and transport in the column Wh Estimate s from peak widths, assuming Gaussian shaped peak: Wb = 4 s Wh = 2.354 s Dependent on the amount of time that a solute spends in the column (k’ or tR)
  • 33. Factors affecting separation effeciency (Peak Broadening) Number of theoretical plates (N): compare efficiencies of a system for solutes that have different retention times N = (tR/s)2or for a Gaussian shaped peak N = 16 (tR/Wb)2 or N = 5.54 (tR/Wh)2 The larger the value of N is for a column, the better the column will be able to separate two compounds.
  • 34. Plate height (H): compare efficiencies of columns with different lengths H = L/N where: L = column length N = number of theoretical plates for the column H can be also used to relate various chromatographic parameters (e.g., flow rate, particle size, etc.) to the kinetic processes that give rise to peak broadening. Why Do Bands Spread? a. Eddy diffusion b. Mobile phase mass transfer c. Longitudinal diffusion
  • 35. a.) Eddy diffusion : a process that leads to peak (band) broadening due to the presence of multiple flow paths through a packed column. As solute molecules travel through the column, some arrive at the end sooner then others simply due to the different path traveled around the support particles in the column that result in different travel distances. Longer path arrives at end of column after (1).
  • 36. b.) non equilibrium mass transfer: a process of peak broadening caused by non equilibrium attainment due to the transfer of olute from the higher concentration region to the low concentration one The degree of band-broadening due to eddy diffusion and mobile phase mass transfer depends mainly on: 1) the size of the packing material 2) the diffusion rate of the solute
  • 37. c.) Longitudinal diffusion: band-broadening due to the diffusion of the solute along the length of the column in the flowing mobile phase. The degree of band-broadening due to longitudinal diffusion depends on: 1) the diffusion of the solute 2) the flow-rate of the solute through the column A solute in the center of the channel moves more quickly than solute at the edges, it will tend to reach the end of the channel first leading to band-broadening
  • 38. Van Deemter equation: relates flow-rate or linear velocity to H: H = A + B/m + Cm where: m = linear velocity (flow-rate x Vm/L) H = total plate height of the column A = constant representing eddy diffusion & mobile phase mass transfer B = constant representing longitudinal diffusion C = constant representing stagnant mobile phase & stationary phase mass transfer This relationship is used to predict what the resulting effect would be of varying this parameter on the overall efficiency of the chromatographic system.
  • 39. Factors increasing peak resolution 1. Increase column length 2. Decrease column diameter 3. Decrease flow-rate 4. Pack column uniformly 5. Use uniform stationary phase (packing material) 6. Decrease sample size 7. Select proper stationary phase 8. Select proper mobile phase 9. Use proper pressure 10. Use gradient elution