Chromatography

Chromatography

- Chromatography is a physical method of separation in which the components to be

separated (solute) are distributed between two phases, one of which is the stationary
phase (St. Ph) while the other moves in a definite direction and called the mobile
phase (M. Ph).

 In all chromatographic separations:

a- The sample is transported with the mobile phase (may be liquid or gas) which is
then forced through an immobile and immiscible stationary phase. Components of the
sample distribute themselves between the mobile and the stationary phases to varying
degrees, where those are strongly retained by the stationary phase move slowly with
the flow of the mobile phase and vise versa.
b- As a consequence of these difference in mobility, sample components are
separated into discrete bands or zones that can be analyzed qualitatively or
quantitatively.

 Some chromatographic definitions

Eluate: It is the mobile phase leaving the column.
Eluent: It is the mobile phase leaving the column carrying the analyte.

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 Chromatography

Elution: Moving the mobile phase carrying the analyte through the stationary phase.
 Classification of chromatography
- Chromatographic methods may be classified according to:
I- According to the technique used
a- Columnar chromatography: at which the stationary phase is held in a column
through which the mobile phase is forced.
b- Planar chromatography: at which the stationary phase is spread as a thin layer
on a glass, plastic or aluminum plates ( in thin layer chromatography) or it is held
in network structure of the paper (in paper chromatography).

II- According to the nature of the mobile phase

a- Liquid chromatography
At which the mobile phase is liquid. It is classified according to the nature of the
stationary phase into:
i- Liquid solid chromatography ii- Liquid liquid chromatography
i- Liquid solid chromatography (LSC)
1- The stationary phase is solid. e.g. silica or alumina which may be packed on a
column (columnar) or spread as a thin layer (planar).
2- Separation of the sample components depends on difference in their adsorption
coefficient which is a surface phenomenon, where components that are weakly
adsorbed eluted first.
ii- Liquid liquid chromatography (LLC)
1- The stationary phase is liquid (solid particles surrounded by a thin layer of thick
liquid).
2- This liquid may be:
- Mechanically held on the solid support on the form thin coat (planar) or
packed in a column (columnar).
- Chemically and permanently held to the solid support and in this case the
stationary phase is called bonded phase.

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 Chromatography

3- Separation of the sample components depends on difference in their partition

coefficients (solubility of the components in the liquid stationary and mobile
phases), where components of lower solubility in the stationary phase eluted
first.

b- Gas chromatography
At which the mobile phase is gas. It is classified according to the nature of the
stationary phase into:
i- Gas solid chromatography ii- Gas liquid chromatography
i- Gas solid chromatography (GSC): at which the stationary phase is solid.
ii- Gas liquid chromatography (GLC): at which the stationary phase is liquid which
may be:
- Mechanically held on a solid support in the form of a thin coat and packed in
a column.
- Coats the inner wall of the capillary column.

III- According to the mechanism of sorption (attachment) to the stationary

phase
a- Adsorption chromatography
1- Adsorption chromatography can be applied in LSC and GSC using columnar and
planar techniques.
2- It depends on using active solid stationary phase (e.g. alumina or silica gel) that
contains high surface energy active sites which interact with the functional groups
in the separated components by weak forces (Vander wal), charge transfer or
hydrogen bonding.
3- The chromatographic separation is based on difference in adsorption coefficients
of the separated components.

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 Chromatography

4- Separation occurs due to competition of the mobile phase and the solute
molecules to the active sites of the stationary phase where the weakly adsorbed
component travels faster than which is highly adsorbed.

b- Partition chromatography
1- Partition chromatography can be applied in LLC and GLC using columnar and
planar (paper chromatography) techniques.
2- It depends on using liquid stationary phase which may be carried by a solid support
(e.g. kieselguhr) in column chromatography or cellulose fibers in paper
chromatography.
3- The chromatographic separation depends on difference in partition coefficient of
the separated components.
4- Separation occurs due to difference in relative solubility of the components
between the liquid stationary and mobile phases where the component which is
weakly soluble in the stationary phase travels faster than that of higher solubility.

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 Chromatography

c- Ion exchange chromatography (IEC)

1- Ion exchange chromatography can be applied for separation of ionic compounds.
2- It depends on using stationary phase which is polymer contains large number of
ionizable functional groups. It may be either cationic (e.g. SO 3H+) or anionic (e.g.
R3N+Cl-) exchanger according to the replaceable ion.
3- Chromatographic separation depends on the exchange of cations (H+) or anions
(Cl-) of the stationary phase with cations or anions of the mixture components in
the mobile phase (i.e. separation is due to ionic distribution coefficients of the
sample components) where the component of a charge opposite to that of the
exchanger travels faster.
pH2

- + +
SO3 Na H3N
COOH

Ion-exchange Resin

- +
SO3 H3 N
-
COO pH4.5
+
Na

d- Size exclusion chromatography (SEC) = Gel filtration chromatography

1- It is used for separation of components of different molecular weights (size).

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 Chromatography

2- It depends on using stationary phase which is a polymer with a network structure

(porous particles).
3- Chromatographic separation of the sample components depends on difference in
their diffusion coefficients where components of the mixture permeate through the
pores of the polymer according to their molecular weights.
4- Molecules that are smaller than the pores size will pass inside the pores and
retained, so they have longer path and longer transition time than larger molecules
which can not enter the pores and eluted first.

e- Electrochromatography (Electrophoresis)
1- It depends on applying electric current for separation of charged species where
separation is based on the charge or the mobility of ions.
2- It is used mainly for separation of protein.

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 Chromatography

 Summary of types of chromatography

Mobile phase Liquid Gas
Chromatographic LC GC
technique
Stationary phase Solid Liquid Ion exchange resin Polymer with Solid Liquid
network
structure
Chromatographic LSC LLC IEC SEC GSC GLC
technique
Mechanism of Difference in Difference in Difference in ionic Difference in Difference in Difference in
separation adsorption partition distribution diffusion adsorption partition
coefficient coefficient coefficient coefficient coefficient coefficient
(ion exchange) (size exclusion)
Technique Columnar and planar Columnar
Application Compound of Compounds Ionic and inorganic Compounds of Gaseous and volatile
different of the same compounds different compounds
chemical chemical molecular
types type weights

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 Chromatography

 Theory of chromatographic separations

- Two theories were developed which together explained the separation mechanism
during the chromatographic separation. These are the plate theory and the rate theory.
1- The plate theory
a- It explains the migration of the separated zones along the column.
b- It supposes that the chromatographic column consists of large number of separate,
horizontal, narrow, discrete and consecutive theoretical layers which called the
theoretical plates (N).

c- At each plate, equilibration of the solute between the stationary and the mobile
phases was assumed to take place. Movement of the solute down the column was due
to stepwise transfer of the equilibrated mobile phase from one plate to the next.
d- These plates serve as a way of measuring the column efficiency, either by stating
the number of theoretical plates in the column (N) or by stating the plate height (height
equivalent to theoretical plates = HETP). Increasing number of theoretical plates and
decreasing the height equivalent to the theoretical plates are preferable.
HETP = L/N where L = the column length

2- The rate theory

a- It explains the distribution of the particles inside the separated zones.
b- The distribution of the molecules of certain compound in the zone is random where
the highest concentration will be in the center while it decreases as it spreads at the
periphery (i.e. follow normal distribution curve).

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 Chromatography

 The chromatogram
a- It is a graph showing the detector response as function of elution time.

b- When a mixture of components (A & B) is injected into a column, they undergo

separation by the movement of the mobile phase through the stationary phase. Once a
component is emerged from the column, the detector gives a signal that indicates the
emergence of that component.

c- Following the previous figure, it represents a typical chromatogram for a sample

containing mixture of two components (A & B). The small peak on the left id for
species that does not retained in the column (i.e. the mobile phase). The other two
peaks are for the two solutes (A & B) in the mixture.

d- The chromatogram is used for qualitative (identification) analysis by using t R value

(position of the peak on the time axis) and for quantitative analysis using the area
under the peak or using the peak height.

 Some terms used in chromatography

1- Retardation factor (R value)
a- It relates the migration of the substances in the mixture relative to that of the
mobile phase.
R= Concentration of the substance in the mobile phase = tm

Total concentration of the substance t m + ts

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 Chromatography

Where: tm = the time spend by the substance in the mobile phase.

ts = the time spend by the substance in the stationary phase.

b- R value for a certain substance varies with the change of both or either the mobile
or stationary phase but once the chromatographic conditions are constant the R value
can be used for qualitative analysis of the substance.

c- As the component is more retained as the smaller will be its R value.

2- Rf value
a- It is used for thin layer and paper chromatography where the sample spots move
up to definite distances.
Rf = The distance traveled by the zone center
The distance traveled by the solvent (the solvent front)
b- As the component is more retained as the smaller will be its Rf value.

3- The retention time (Rt or tR value)

a- It is used in GC and HPLC.
b- tR value: it is the time elapsed from injection of the sample component till
appearance of its peak maximum.
c- The void time (tM = t0 value) = it is the time taken for the mobile phase to pass
through the column. Or/ time elapsed from addition of the mobile phase (non
retained substance) at the top of the column till appearance of its peak maximum.
d- The adjusted retention time (tR') = tR - tM
e- As the component is more retained as the higher will be its tR value.

4- The retention volume (Rv value)

a- It is the volume of the mobile phase consumed till appearance of peak maximum
of the component.
b- As the component is more retained as the higher will be its Rv value.

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 Chromatography

c- Rv = tR × U where: tR = the retention time of the substance

U = the velocity of the mobile phase (flow rate).
5- The mobile phase velocity (U)
U = L / tM where: L = the column length
tM = the retention time of the mobile phase.
6- Zone velocity (UA)
UA = L / (tR)A where (tR)A= the retention time of the solute.
7- Capacity factor (K')
a- It describes the migration rates of the analytes on the columns.
b- K'A= (tR)A – tM/ tM = (tR') / tM
c- It must be from 1 -10 to be acceptable.
8- Selectivity factor (α)
a- It describes the separation of two adjacent species (A & B) on the column.
c- α = (tR)B – tM = (tR')B/ (tR')A= K'B/K'A
(tR)A – tM
Where: K'B= the capacity factor of the more retained species.
d- α must be > 1. As the selectivity factor increases, the selectivity of the stationary
phase and the efficiency of the separation increase.
9- The column resolution (Rs)
a- It describes the separation of two adjacent species (A & B) on the columns but
differ from (α) in taking into account the peak width.

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 Chromatography

b- Rs = 2 [ (tR)B – (tR)A]
WA + WB
c- The Rs must be ≥ 1.5 for complete separation. The greater the separation of the
zones from each other and/ or the smaller the zone width, the larger will be the
resolution.
10- The efficiency of the column
a- It is determined by two separate parameters:
1- The migration of the zone (the retention time).
2- The zone broadening (the zone width).
b- It is quantitatively determined by calculating the number of theoretical plates (N)
or the height equivalent to theoretical plates (HETP).
N= 16 (tR)2/ W2 (calculated for each component separately)
H = L/N = LW2/ 16 (tR)2 L = NH = 16 (tR)2H/W2
c- As the retention time of the analyte 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:
1- Eddy diffusion
a- It is resulted from imperfect packing of the column with the stationary phase, i.e.
using of irregular large particles.
b- Presence of air bubbles or cracks due to imperfect packing.
2- Ordinary diffusion (longitudinal diffusion)
a- It is resulted from the low flow rates of the mobile phase.
b- At low flow rates, the particles of the separated components move from the
concentrated zone center to the boarders of the zone leading to band broadening.

3- Ordinary diffusion (longitudinal diffusion)

a- It is resulted from the high flow rates of the mobile phase.

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 Chromatography

b- At high flow rates, there is no enough time for some molecules to equilibrate (i.e.
distribute themselves between the stationary and the mobile phases. Some
molecules move a head with the mobile phase and some will remain on the
stationary phase on the back of the zone leading to band broadening.

 How to minimize zone broadening?

1- Using of small and regular stationary phase particles.
2- Perfect packing of the stationary phase (no cracks or air bubbles).
3- Using the optimum flow rate (by trials).

 Optimization of the column performance

- In order to optimize the column performance, we must correlate the resolution (Rs) to
number of theoretical plates (N), selectivity factor (α) and capacity factor (K').

- To optimize the column performance we should:

a- Increases the number of theoretical plates (N) through reducing the height
equivalent to theoretical plates (HETP) by using a stationary phase of small particle
size (not by increasing the column length).
b- Perfect packing of the stationary phase to decrease band broadening and
maximize (N).
c- Controlling the capacity factor (K') by changing the composition of the mobile
phase (in LC) or changing the temperature (in GC).
d- Optimizing the selectivity factor (α) by :-
- Controlling K' value.
- Changing the mobile phase flow rate.
- Changing the stationary phase composition when necessary.

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 Chromatography

I- Gas chromatography (GC)

- Gas chromatography (GC) involves a sample being vaporized and injected onto the
head of the chromatographic column. The sample is transported through the column
by the flow of inert gaseous mobile phase (carrier gas).

 Types of gas chromatography

a- Gas solid chromatography (GSC)
1- The stationary phase is solid.
2- Separation depends on the difference in adsorption affinity of the separated
components to the solid stationary phase.
b- Gas liquid chromatography (GLC)
1- The stationary phase is liquid supported on an inert solid matrix.
2- Separation depends on difference on distribution affinity (partition coefficient) of
the separated components between the carrier gas and the liquid stationary phase.

 Samples determined by gas chromatography

1- Volatile substances that can be volatilized without decomposition, e.g. low
molecular weight hydrocarbons, aldhydes, ketons and esters.
2- Substances that are not volatile but can be converted to volatile components, e.g.
fatty acids (non volatile) are converted to methyl ester derivatives (more volatile).
3- Samples those are organic and inorganic but not ionic.
4- Substances those are thermo stable.
5- Low molecular weight components (2 – 1000 gm).

 Advantages of gas chromatography

1- Highly sensitive (10-9 – 10-12 gm).
2- Highly selective and accurate.
3- Short analysis time (high speed) because the mobile phase is gas which is faster
than the liquid mobile phase (in LC).

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 Chromatography

 Disadvantages (limitations) of gas chromatography

1- Can not be used for substances that are not volatile and can not be derivatized to
volatile components. Also it can not be used for thermo labile components.
2- Can not be used for high molecular weight polymers.
3- Can not be used for ionic substances (Salts).
4- Can not be used for samples in aqueous solvents because aqueous solvents are less
volatile than organic solvents.
5- Can not be used biological samples (blood and tissue) unless clean up procedure is
carried out because the impurities in the biological samples block the column.
6- Gas chromatography must be connected to (coupled with) mass spectrometer for
accurate identification of the compounds.

 Instrument used for gas chromatography

- Gas chromatographic instrument is formed of five components:
a- The gas supply system with its regulator, purifier and flow rate.
b- Sample injection system.
c- Column and thermo stating system.
d- Detector.
e- Integrator and computer data station.

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 Chromatography

a - Gas supply system

- This includes: 1- The carrier gas 2- The gas used for the detector
 The carrier gas
a-The mobile phase in GC in contrast to most other types of chromatography does not
interact with molecules of the analyte (i.e. inert). Its only function is to transport the
analyte through column and so it is called the carrier gas.
b- The most commonly used carrier gases are nitrogen, helium, hydrogen and argon.
c- The gas supply is associated with pressure regulator and flow meter which control
the gas flow rate. Also it is connected to gas purifier to remove water and other
impurities present in the carrier gas.
d- The carrier gas must be:-
i- Highly pure because the impure gas may damage the stationary phase, cause
noise and give unidentified peaks.
ii- Inert and does not interact with the components of the mixture.
iii- Dry because the presence of water along with the high temperature used during
the analysis may hydrolyze the sample components and the stationary phase.
iii- The choice of the carrier gas depends on the detector used:-

The detector The carrier gas The detector gas

Flame ionization detector Nitrogen, helium, argon or Mixture of air and
hydrogen. hydrogen
Electron capture detector Nitrogen No gases needed
Thermal conductivity The carrier and the detector gases are the same. Helium
detector and hydrogen are used for both.

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 Chromatography

b – Column and thermo stating system

1- Columns
- It is where the separation process occurs, there are two types of columns used in GC
technique, packed and capillary columns (open tubular columns).

 Comparison between different types of columns used in GC technique.

Item Packed column Capillary column
(open tubular column)
Fabrication Made from glass, metal or Made from stainless steel, glass or fused
material teflon. silica.
Length of column 2 – 3 m length 15 – 100 m length
Diameter 2 – 4 mm internal diameter 0.2 – 0.5 mm internal diameter
Packing material May be uniform and finely - The packing material is only a very thin
divided packing material (GSC) layer of liquid stationary phase coated or
or inert solid support coated with bonded to the internal walls of the column.
a very thin layer of liquid (i.e. LGC only).
stationary phase (GLC) - Two types of capillary columns are found:
a- WCOT (Wall coated open tubular): it is a
capillary tube coated with a thin layer of
liquid stationary phase (i.e. no support).
b- SCOT (Support coated open tubular): it
is a capillary tube at which its inner surface
is lined with a thin film of a support
material coated with the mobile stationary
phase.
The used flow The used flow rate is 30 – 50 The used flow rate is 1 mL/min as there is
rate mL/min no resistance for the gas flow as the column
is in the form of open tube.
Advantages 1- It has higher sample capacity 1- It has higher resolution (Rs) value and
higher number of theoretical plates (N).
2- Shorter time of analysis

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 Chromatography

 Requirements of liquid stationary (immobilized) phase used in gas

chromatographic technique.
1- It must be of low volatility; ideally the boiling point of the liquid stationary phase
should be at least 100°C higher than the maximum column operating temperature.
2- It must be thermally stable and chemically inert.
- The most common stationary phases in gas-chromatography are polysiloxanes,
which contain various substituent groups to change the polarity of the phase.

 Order of separation of components in gas chromatography depends on:

1- The boiling points of the separated components where components of higher
boiling point are more retained.
2- Different in solubility or adsorption of the analytes in the stationary phase: one of
the main reasons of the separation of different components in GC is their
interaction with the stationary phase. According to "like dissolve like", the
component of stronger interaction with the stationary phase is more retained.

NB. When the polarity of the stationary phase matches that of the sample
components, the order of elution is affected only by difference in their boiling points.

2- Columns thermo stating system

a- It is the place where the chromatographic column is situated and the sample is
separated into its components.
b- The temperature of the column usually equal or slightly higher than the sample
boiling point.
c- The optimum column temperature depends on:
i- The boiling points of the sample components.
ii- Degree of separation required.

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 Chromatography

c- When the sample components are varying greatly in their boiling points,
temperature programming is used.
 Temperature programming:
- It is a technique used in GC used when the sample components have variable boiling
ranges. It is used to improve the resolution and decrease the analysis time where by
the column temperature is increased gradually according to a program.

c- Detectors
1- Its function is to detect the presence of chemical components of in the flow gas.
2- All the detectors measure a relative value (a sample component in the carrier gas
compared to the pure carrier gas).
3- There are three types of detectors used in GC, thermal conductivity detector, flame
ionization detector and electron capture detector.

 Characteristic of good detectors

1- Sensitive to very low concentration of the sample (10-9 10-12 gm).
2- Accurate, reproducible and reliable.
3- Rapid response to low concentrations of the sample.

1- Thermal conductivity detector (TCD) = Katharometer

a- The principle:
- It is based on changing in the thermal conductivity of the gas stream brought about
by the presence of the analyte molecules.
b- Structure of the detector:
- It consists of two channels system, the pure gas flow through one channel and the
carrier gas coming from the column flows through the other.
- The two channels contain electrically heated filaments of platinum or gold held in
a ceramic block. The two filaments form the two arms of wheatstone bridge.

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 Chromatography

c- Mechanism of the detector:

- When the pure carrier gas passes through the channels, the heated filaments are
cooled due to thermal conductivity of the gas and so the resistance of the filaments
(which depend on their temperatures) changes and hence the balancing of the bridge
changes.
- The bridge is in equilibrium when the composition of the gases passing through the
channels is the same (i.e. no sample). When the sample is eluted, the composition of
the gases changes and hence filaments are cooled with different degrees which lead
to change in their resistance and bridge imbalance. Then current flows giving a
signal.
d- The carrier and the detector gases:
- The carrier and the detector gases are the same. They must have high thermal
conductivity to maximize the temperature difference of the two filaments and hence
their resistance which enhances the sensitivity of the detector. Hydrogen and helium
are the most suitable gases.
e- Sample determined by the detector: it is a universal detector and so can be used for
organic and inorganic samples.

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 Chromatography

f- Advantages of the detector:

- Universal detector which can be used for even in organic samples (e.g. NO2 & SO2).
- Non destructive detector.
g- Disadvantages of the detector:
- Non selective.
- Has low sensitivity.

2- Flame ionization detector (FID)

a- The principle:
- Most organic compounds when pyrolyzed at the temperature of hydrogen/air
produce ions and electrons that can conduct electricity which depends on the number
of carbon atoms in the flame.

b- Structure of the detector:

-It consists of cathode (the flame itself) and anode (collecting electrode) situated
above it.

c- Mechanism of the detector:

- When the pure carrier gas and the detector gas pass to the flame, ionization of gases
occurs and electrons are attracted to the anode producing a very small current.

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 Chromatography

- When the organic sample passes with the carrier gas to the flame, the number of the
produced electrons increases and so the produced current increases giving a signal.
d- The carrier gases: hydrogen, helium, argon or nitrogen.
e- The detector gases: mixture of hydrogen and air.
f- Samples determined by the detector: organic samples only.
g- Advantages of the detector:
- It has high sensitivity.
- It has wide linear range.
h- Disadvantages of the detector: it causes destruction of the sample.

3- Electron capture detector (ECD)

a- Structure of the detector: it consists of a cavity that contains two electrodes and a
radiation source that emits β- radiation (e.g. Ni63, H3).

b- Mechanism of the detector:

- When the pure carrier gas is passed over a β-emitter, electrons from the emitter
cause ionization of the carrier gas producing a burst of electrons.
- In absence of the analyte, constant current is produced between the two electrodes
resulted from ionization of the pure carrier gas.

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 Chromatography

- When the electronegative species is carried with the carrier gas, electrons are
captured and therefore the resulted current is decreased.
c- The carrier gases: Nitrogen is used as the carrier gas for ECD.
d- The detector gases: No gases are needed for ECD.
e- Samples determined by the detector: molecules that contain electronegative
functional group, e.g. halogens, peroxides and nitrogen containing compounds.
f- Advantages of the detector: it is highly selective for compounds containing
electronegative functional group.
g- Disadvantages: it has small linear range.

II- High performance (pressure) liquid

chromatography (HPLC)

-High pressure liquid chromatography is a type of LLC and is an improved form of

column chromatography.

- With HPLC a pump (rather than gravity) provides the higher pressure required to
propel the mobile phase and the analyte through the densely packed column. The
increased density arises from smaller particle size of the column packing material
which gives a much greater surface area for interactions between the stationary phase
and the molecules flowing past it. This allows better separation on column of shorter
length when compared to ordinary column chromatography and so it is called high
performance liquid chromatography.

 Samples determined by HPLC

1- Volatile and non volatile substances.
2- Thermally labile and thermally stable substances.
3- Organic and inorganic substances (including ionic samples).
4- Low and high molecular weight substances (800 – 6000).

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 Chromatography

 Advantages of HPLC
1- High analysis speed.
2- High resolution for complex mixtures.
3- High accuracy with small relative error.
4- High sensitivity (10-6 – 10-9 gm).
5- Automatic system: the whole system starting from injection till detection of the
sample is carried out automatically.

 Disadvantages of HPLC
1- Expensive instrument and supplies (e.g. columns, high grade solvents and
syringes).
2- It needs long time experience to obtain maximum benefit from HPLC.

 Instrument used in HPLC

1- The mobile phase

 Requirements of good solvent used in HPLC
1- It must be highly pure and available.
2- Inert to avoid chemical interaction with the solutes or with the stationary phase.
3- Has low viscosity and immiscible with the stationary phase.
4- It must be compatible with the detector.

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 Chromatography

 Preparation of the mobile phase

1- It must be filtered to remove any impurities that may damage the pump and the
injection system or cause clogging to the column.
2- Any dissolved gases, usually oxygen and nitrogen, must be removed by passing
the mobile phase through degasser. These gases form bubbles in the column which
leads to band spreading, also they may affect the detector performance.
3- Filtration and degassing may be carried out by filtration of the mobile phase
through Millipore filter under vacuum.

 Type of mobile phase elution

- There are two types of elution, isocratic and gradient elution.
a- Isocratic elution:
- In this type only one and the same mobile phase, either single solvent or mixture of
solvents is used till the analyzed mixture is completely separated to its components
(i.e. using a mobile phase of constant composition).
b- Gradient elution:
- In this type the composition of the mobile phase is changed during the
chromatographic separation in a programmed way and so called the solvent
programming.
- It is used to enhance the separation efficiency when there is great difference in the
retention times of the sample components.

2- The pumping system

- It is the system which pushes the mobile phase under certain flow rate to the column.
 Requirements for pumping system
1- Generates pressure up to 6000 psi.
2- Produces pulse free output.
3- Gives flow rates 0.1 – 10 mL/min.
4- Has flow control and flow reproducibility.
5- Has high resistance to corrosion by a variety of solvents.

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 Chromatography

3- The pre-column
1- It contains the same packing material identical to that in the analytical column.
2- Its function is to:
- Remove the impurities from the used solvent (mobile phase) and so prevent
contamination of the analytical column.
- Saturate the mobile phase with the stationary phase and thus prevent the stripping
of the stationary phase of the analytical column during elution with the mobile
phase.

4- The sample injection system

1- It is used to introduce the sample to the analytical column. It consists of sampling
loop inserted in a sliding or rotator valve.
2- Its mechanism of action:
- When the loop is in the position out of the stream of the solvent, it can be filled
with a syringe till overflow occurs.
- Changing the position of the loop by rotating or sliding the valve, it allows passing
of the sample with the solvent to the column.

5- The analytical column

1- It is manufactured from heavy walled glass tube or stainless steel.
2- It is 15 – 25 cm length with inside diameter of 5 – 10 mm.
3- The column is packed with small particles (5 – 10 μm diameter).
 Type of column packing material
1- Pellicular particles
- It consists of spherical, non porous glass or polymer beads (30 – 40 μm diameter).
- On the surface of the beads, there is a thin porous layer or crust of silica, alumina,
porous polymer or ion exchange resin.
- An additional liquid stationary phase may be physically or chemically bonded to the
surface of the porous layer.

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 Chromatography

2- Porous micro particles

- It consists of porous micro particles of silica, alumina, porous polymer or ion
exchange resin (3 – 10 μm diameter).
- An additional liquid stationary phase may be physically or chemically bonded to
the surface of the particles.
 Classification of HPLC technique
- According to the polarity of the stationary phase, HPLC technique is classified to
normal phase HPLC (N-HPLC) and reversed phase HPLC (R-HPLC).
N-HPLC R-HPLC
1- The used stationary phase is polar, e.g. 1- the stationary phase used is non polar, by
silica or alumina. attaching long chain hydrocarbons to the
surface of silica. e.g. C8 or C18.
NB. In case of attaching C18 hydrocarbon,
the stationary phase is called ODS.
2- The used mobile phase is usually non 2- The used mobile phase is usually polar.
polar.
3- Mechanism of separation is by adsorption. 3- Mechanism of separation is by partition.
4- Non polar substance is eluted before the 4- Polar substance is eluted before the non
polar one (which is more retained to the polar one (which is more retained to the
stationary phase according to "like stationary phase according to "like dissolve
dissolve like"). like").
5- Using of more polar solvents (e.g. water), 5- Using of more non polar solvents (e.g.
decreases the retention time of the analyte. hexan), decreases the retention time of the
analyte.

 The eluotropic series

It is the arrangement of solvents according to their elution power.
e.g. water, methanol, acetonitrile, hexan.
Increasing the strength in N-HPLC
Increasing the strength in R-HPLC

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 Chromatography

6- Detectors
- They must have the same characters as in GC, in addition the detectors used in HPLC
should have minimum internal volume to reduce the zone broadening.
1- UV absorbance detector
a- It is the most widely used detector in HPLC, used for determination of most
substances except aliphatic components (which do not absorb the UV radiation).
b- It depends on measuring the difference in the absorbance of UV light by the pure
mobile phase and the mobile phase carrying the sample.
c- It consists of:
- A source of UV radiation. The beam which is split and passes through two flow
cells, one through which the pure solvent flows and the other through which the
solvent containing the sample flows (test cell).
- The light from both cells will pass through a wavelength selector then through two
phototubes for detection of the absorbed light.

d- When the same solvents flow through the two cells, no signal is obtained. When
the sample passes through one cell, difference in the absorbed light occurs which
converted to a current that gives a signal.

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 Chromatography

2- Fluorescence detector
a- It is used for detection of fluorescent compounds, e.g. vitamins and polycyclic
compounds.
b- The detector is a type of fluorimeter, the source of radiation is xenon lamp using
grating monochromator and photomultiplier tube is the detector.
c- It is characterized by its high sensitivity and selectivity.

3- Refractive index detector (RI)

a- It is a universal detector that can be used determination of all components including
those with no absorbance in near UV or VIS region such as aliphatic amines.
b- It depends on the difference in the refractive index of pure mobile phase and mobile
phase carrying the sample.
c- RI detector has low sensitivity and selectivity.

Qualitative analysis in HPLC and GC techniques

a- It includes separation of component of interest from other components in the
mixture and then identification of the separated components.
b- Identification is carried out by comparing the retention time (t R) of the unknown
component with that of a reference one chromatographed under the same conditions.

Quantitative analysis in HPLC and GC techniques

- It depends on measuring the peak height or area which is directly proportional to the
concentration of the analyte.
1- Peak height measurement
a- It is preferred to the peak area method because of its simplicity.
b- For accurate analysis, the chromatographic conditions must be kept constant and the
peak width does not vary during the determination.
2- Peak area measurement
a- It is used when peak broadening occurs due to over loading of the sample, long
retention time and variation in operating conditions.

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 Chromatography

III- Paper chromatography (PC)

- Paper chromatography is a typical partition system (i.e. LLC system) where the
stationary phase is a solvent held on cellulose molecules which in turn are kept in a
fixed position by the fibrous structure of the paper. This method is used to separate
components of similar chemical structure.
 Principle of paper chromatographic technique
1- A sample is spotted onto a strip of filter paper with a micropipette where the
chromatogram is developed by using a suitable solvent (developing system) present in
a closed chamber.

2- The solvent drawn up the paper by the capillary action. Separation of the sample
components depends on their solubility and their degree of retention by the paper (i.e.
difference in their partition coefficient).
3- The separated spots are detected by treatment with a reagent that forms colored
derivatives.
4- The position of each spot is characterized by its retardation factor (Rf value).

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 Chromatography

 The retardation factor (Rf value):

a- It is the ratio of the distance to which the substance is reached compared with the
distance reached by the solvent front, both measured from the point of application of
the sample.
Rf = Distance moved by the substance
Distance moved by the mobile phase

b- It is affected by temperature, composition of the solvent and time of equilibrium

before the actual development.

c- The Rf value is characteristic for each component (under constant condition) and so
is used for identification (qualitative) purposes.

 Type of stationary phases used in paper chromatographic technique

1- Aqueous stationary phase
a- In this type, water which is held on the cellulose molecules of the paper is the
stationary phase.
b- Solvents which are water immiscible are used as mobile phases but they must
dissolve the tested substances.

2- Hydrophilic stationary phase

- Polar hydrophilic solvents (e.g. methanol and formamide) are used as stationary
phases by saturation of the paper with vapors of these solvents.

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 Chromatography

3- Hydrophobic stationary phase (Reversed phase)

- Hydrophobic solvents (e.g. paraffin and olive oil) are used as stationary phases after
saturation of the paper with vapors of these solvents. Hydrophilic solvents are used
as mobile phases.

 Advantages of paper chromatography

1- The main advantage of this technique is its high separating power that can be
achieved by using the techniques of multiple chromatography (multiple
developments chromatography and two dimensional chromatography and).
2- It is very useful in biochemistry as it is used for separation of complex samples
with small amounts.
3- It can be used for qualitative (using the Rf value) and for quantitative analysis
(after cutting the spots and extraction of the solutes).

 Multiple chromatography techniques

- They are repeated development chromatographic methods used to obtain better
separation of complex mixtures (i.e. increasing the resolution). They include
multiple developments and two dimensional chromatography.
a- Multiple developments chromatography
- In this technique development is repeated in the same direction either with one
solvent system in several runs or with different systems.
b- Two dimensional chromatography
1- In this technique development is repeated in a direction perpendicular to the flow
of the first system.
2- After development with a given solvent system, the paper is turned 90° and further
development is obtained with a second solvent.

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 Chromatography

Diagram showing two dimensional chromatography

 Detection of spots in paper chromatography

a- If the solutes are colorless and fluoresce, they can be detected under UV light.
b- If the solutes are colorless and none fluoresce, color developing reagents are used.
- e.g. Ninhydrin reagent: gives blue or purple color with amino acids and amines.
- I2 vapors: give brown color with most of solutes.

IV- Thin layer chromatography (TLC)

- It is similar to paper chromatography, however instead of using a stationary phase
of paper; it involves a stationary phase of thin layer of adsorbent supported on a
glass, alumina or plastic strip.
 Advantages of TLC over paper chromatography
1- The development is generally rapid and reproducible.
2- It has better separation and less tendency for tailing.
3- Wide choice between different stationary phases.

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 Chromatography

 Stationary phases used for TLC

- It may be adsorbent, ion exchanger, molecular sieve or liquid film on solid support.
1- Adsorbent
- The most commonly used stationary phases in TLC, e.g. silica gel, alumina and
powdered cellulose.
a- Silica gel
1- It contains OH groups on their surface which form hydrogen bonds with polar
molecules.
2- It must be activated by heating at 110°C for several hours to remove the surface
adsorbed water that prevents polar molecules from reach the surface.
3- It is preferred in separation of polar compounds (e.g. amino acids and sugars).
b- Alumina
a- It contains OH groups or oxygen atoms.
b- It is preferred in separation of weakly polar compounds.

2- Liquid stationary phases

a- Water: when the adsorbents are not activated by heating, so the surface adsorbed
water acts as a stationary phase.
b- Non polar solvents: either silica gel or diatomaceous earth may be silanized to
convert the surface OH groups to non polar methyl groups. The technique in this
case is called reversed phase TLC (R-TLC).
c- Ion exchange resin: the stationary phase is ion exchanger, e.g.:
- Dowex 50 W: strong acid cation exchange resin (in the form of Na+ or H+).
- Dowex 1: strong base anion exchange resin (in the form of Cl-).
d- Size exclusion: such as sephadix which is soaked in water and spread on the plate.
 Mobile phases used for TLC
1- The mobile phase is a suitable liquid solvent or mixture of solvents.
2- Strong solvents push the analytes up the plate, while weak solvents barely move
them.

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 Chromatography

3- The order of strength/ weakness depends on the coating of the TLC plate (i.e. the
stationary phase). e.g. in adsorbent chromatography, the eluting power of solvents
increase in the order of their polarities from hexan, acetonitrile, alcohol to water.
4- Separation of compounds depends on the competition of the solute and the mobile
phase for the binding places on the stationary phase.

 Detection of spots in TLC

1- The stationary phase for TLC often contains substance which fluoresces in UV
light. When it is held under UV light, dark spots appear where the sample spots occur
due to quenching of the plate fluorescence.
2- By using developing reagents, e.g.:
- I2 vapors and ninhydrine.
- Sulfuric acid: after spraying the plate with sulfuric acid and then heating it to char
the organic compounds, black spots are developed.

 Quantitative measurements in TLC

1- By scraping the spot off the plate and then extraction of the analyte for measuring
its concentration.
2- By measuring the optical density of the separated spots by measuring the
transmittance of light or measuring the fluorescence intensity.

Modern TLC
- The power of TLC has been enhanced by:
1- Automatic sample application.
2- Enhancement of detection techniques.
3- Using of very fine particles stationary phase which resulted in faster and more
efficient separation which called high performance thin layer chromatography
(HPTLC).

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