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‫الكروماتوغر‬ ‫و‬ ‫الكيميائية‬ ‫الفصل‬ ‫طرق‬
‫افية‬
458
‫كيم‬
Chemical Separation and
Chromatographic Methods
Chem 458

High Performance Liquid
Chromatography (HPLC)

Liquid chromatography
It is the first ever described chromatographic method
(by Tswett in 1903)
Unlike gas chromatography, the sample in liquid
chromatography must not be vaporized so, almost all kinds
of compounds can be analysed by liquid chromatography
The development of instrumental liquid chromatography
was later than for gas chromatography because of the
higher pressure needed for the former
HPLC is considered to be the major chromatographic
technique available today for non-volatile or heat-sensitive
substances.

A little history:
The official date of birth of
chromatography is the 21 March 1903
in Warsaw when Mikhail Semenovitch
TSWETT has presented at the
Congress of the Polish Natural
Sciences Society a communication
entitled: « A new class of adsorption
phenomena and their applications in
biochemical analysis » about the
separation and purification of vegetal
pigments (a mixture of chlorophylls
and xantophylls) on a chalk column
plant extract
in solvent

A little history:
1938 : REICHSTEIN proposes a theory for the elution and
separation of solutes on a column
1952 : application of gradient elution
1967 : beginning of HPLC after the works of HUBER and
HUZSMAN, this technique was first named « High Speed
Liquid Chromatography » then « High Pressure Liquid
Chromatography » and finally « High Performance Liquid
Chromatography »
1969 : after the 5th International Symposium
International « Advances in Chromatography » the
development of HPLC was very fast
The term HPLC is appropriate for separations of any size
(from micro-analytical to preparative) if the particles of the
stationary phase are not larger than about 10µm

Fundamental definitions (to IUPAC nomenclature)
Chromatography: a physical method of separation in which the
components to be separated are distributed between two phases, one of which
is stationary (stationary phase) while the other (the mobile phase) moves in a
definite direction
Chromatogram: a graphical or other presentation of detector response,
concentration of analyte in the effluent or other quantity used as a measure of
effluent concentration versus effluent volume or time
Stationary Phase: one of the two phases forming a chromatographic
system. It may be a solid, a gel or a liquid. If a liquid, it may be distributed on
a solid. This solid may or may not contribute to the separation process. The
liquid may also be chemically bonded to the solid (bonded phase: covalently
bonded to the support particles or to the inside wall of the column tubing) or
immobilized onto it (immmobilized phase)
Mobile Phase: a fluid which percolates through or along the stationary
bed, in a definite direction. It may be a liquid (liquid chromatography) or a
gas (gas chromatography) or a supercritical fluid (supercritical-fluid
chromatography)

Principle of liquid chromatography
A liquid used as mobile phase moves along a tube used as column.
This column is packed with a solid support which plays the role of
stationary phase
If the stationary phase and the mobile phase were correctly selected,
the constituents of mixture are unequally retained along the column
This phenomenon called retention means that the injected solutes
move slower than the mobile phase with different speeds. They are thus
eluted successively from the column and separated
The detector connected at the column outlet gives a signal
corresponding to each solute which is recorded as the chromatogram
In the working conditions, the retention time spent by each solute in
the column is characteristic and can be used for qualitative purpose
The peak amplitude corresponding to its area can be used to
measure the concentration of the solute in the injected mixture

Simplified scheme of the chromatographic
separation process
t = 5mn
t = 10mn
most least
interactions with
stationary phase
injector detector
flow of mobile phase
t = 0 column

Main modes in liquid chromatography
There are several modes in high performance liquid
chromatography
They are classified according to the mechanism of separation
Each mode corresponds to a given kind of interaction:
surface adsorption
solvent partitioning
ion exchange
size exclusion

Adsorption chromatography
the stationary phase is a solid adsorbant
retention is due to a series of adsorption / desorption
steps
separation is based mainly on differences between the
adsorption affinities of the sample components for the
surface of the active solid (liquid solid chromatography)

Adsorption chromatography
silica and alumina are the most
used stationary phases
both solute and solvent can be
attracted by the active sites at the
surface of the stationary phase
the molecules are retained by the
interaction of their polar functional
groups with the surface functional
groups such as silanols of silica
if solutes have different interactions
with the adsorbing sites the separation
can occur
Si
O
Si
O
Si
O
O
O
H
H
H
Silanol groups
Si-OH at the
surface of
silica

Partition chromatography
the stationary phase is a liquid coated or linked to a
solid support
retention is due to the partitioning of the solute
between the two liquid phases (relative solubility)
separation is based mainly on differences between the
solubilities of the components in the mobile and
stationary phases (liquid liquid chromatography)

Partition chromatography
the most retained species is that having the highest affinity
(solubility) for the liquid stationary phase, relatively to the mobile
phase (eluent)
separation is based on the differences in relative solubility
There are two modes in liquid chromatography
« normal » mode : polar stationary phase and non-polar mobile
phase (the first mode described). In this procedure, the stationary
phase is more polar than the mobile phase. This term is used in
liquid chromatography to emphasize the contrast to reversed-phase
chromatography
« reversed-phase » mode: non-polar stationary phase and polar
mobile phase (the most used mode). In this procedure the mobile
phase is significantly more polar than the stationary phase, e.g., a
microporous silica-based material with chemically bonded alkyl
chains

Ion exchange chromatography (IEC)
the stationary phase has ionically charged groups at the surface
the retention is due to the attractive interactions between ionic
solutes and the opposite charged stationary phase
separation is based mainly on differences in the ion exchange
affinities of the sample components
this technique is now often referred to as Ion Chromatography (IC)

Size exclusion chromatography (SEC)
the stationary phase is a porous material having controlled
pore size
separation is based mainly on exclusion effects, such as
differences in molecular size and/or shape
the terms Gel Filtration and Gel-Permeation Chromatography
(GPC) were used earlier to describe this process

Size exclusion chromatography (SEC)
In this mode, each column can separate solutes having specific
size range
separation mechanism is sieving
the larger species cannot enter all the pores and will elute first
because they have a shorter path in the column
this mode is very useful for the determination of molecular size
of macromolecules (polymers, proteins,…)
large molecules excluded from pores - not retained, first eluted
intermediate molecules: retained, intermediate elution times
small molecules permeate into pores: strongly retained, last
eluted

Importance of polarity in HPLC
the notion of polarity plays a fundamental role in HPLC
all chemicals have a unique and characteristic behaviour
related to their molecular structure and electron charge
distribution
they can be described as being “ polar ” or “ non-polar ”, with a
range of polarities between the most polar and most non-polar
water is a good example of a very polar liquid, and paraffin
based oil is a good example of a very non-polar liquid
this “ polarity ” characteristic of chemicals allows to explain
the chromatographic “ retention mechanisms ” that are used to
create many HPLC separations
a simple rule describes this behavior for polarity-based retention
mechanisms:
“Like Attracts Like, and Opposites are Not Attracted”

Mobile phase composition:
isocratic analysis: in this procedure the composition
of the mobile phase remains constant during the
elution process
gradient elution: in this procedure the composition
of the mobile phase is changed continuously or
stepwise during the elution process

Elution process:
 During the solute transfer in the column, it shows a
typical broadening due to the diffusion phenomena
(transversal and longitudinal)
 The solute band width increases with the retention time
giving a typical peak broadening

The chromatogram:
characteristic parameters
 tM : dead time (for a « non-retained solute »)
 tR : retention time, characteristic of each solute

Some fundamental equations:
 Corrected retention time: t’R = tR – tM
 average linear velocity (ū) is measured from the retention time of an
unretained substance (tM) which moves at the same velocity as the mobile phase:
ū = L / tM
 Retention factor (or capacity ratio) k : corresponds to a relative retention:
k = t’R / tM = (tR – tM) / tM
 since: tM = L / ū
we can write: tR = (1 + k) . tM = (1 + k) . L / ū
Hence the retention time is directly proportional to the column length L and
inversely proportional to the linear flow rate of the mobile phase ū
 when k is ≤ 1.0, separation is poor
 when k is > 30, separation is slow
 when k is 2-10, separation is optimum

Some fundamental equations:
 average linear velocity (ū) is measured from the retention time of an
unretained substance (tM) which moves at the same velocity as the
mobile phase:
ū = L / tM = F / q
where: F is the mobile phase flowrate
and q the free cross-sectional area of the column
 the column porosity eT is:
eT = q / p . r2 = F . tM / p . r2 . L = F . tM / VR
where: r is the radius of the column
and VR the volume of the empty column

Column efficiency:
The chromatographic peaks being supposed gaussian, the peak broadening can be
related to the separation and the column efficiency which is evaluated by the
number of theoretical plates of the column N (similarly to distillation process)
which is a number indicative of column performance.
For a gaussian peak, N can be calculated by one of the following equations:
N = (tR / s)2 (s: standard deviation of the peak)
N = 16 (tR / w)2 (w: width at baseline)
N = 5,54 (tR / d)2 (d: width at half- height)
In order to compare columns having different lengths, one calculate the plate
height or height equivalent to a theoretical plate HETP:
H = L/N (L : column length)
H may vary from centimeters (packed columns) to several microns (high resolution
capillary columns)

Column efficiency:
 Column selectivity:
a = t’R2 / t’R1 = k2 / k1 ( separation occurs only if a > 1 )
 Resolution between two neighboring peaks:
RS = 2 (tR2 – tR1) / (w2 + w1) = 1,18 (tR2 – tR1) / (d2 + d1)
 For two neighboring peaks, a resolution RS higher than 1 means
a complete separation (for RS = 1, the overlapping peak surface is
2%)
 When RS is less than 0.8, the separation between the two
peaks is considered to be incomplete

Optimization of column efficiency and resolution
Resolution (and zone broadening) depends on:
 ū (linear flow rate): low flow
favors increased resolution
 H (plate height) (or N number
of plates): use smaller particles,
lengthen column, viscosity of
mobile phase (diffusion)
 a (selectivity factor): vary
temperature, composition of
column/mobile phase
 k (capacity factor): vary
temperature, composition of
column/mobile phase
time
detector
signal
A B
A B
A
B
tM tRA tRB
RS = 0.75
RS = 1.0
RS = 1.5

Optimization of chromatographic separation
then
From the equations of:
 resolution: R = 2 (tr,B – tr,A) / (wB + wA)
 and column efficiency: NB = 16 (tr,B / wB)2
 if we assume that: wB  wA
To obtain the desired resolution, we can control:
 N number of plates (or plate height H): use smaller particles, lengthen
column, change viscosity of mobile phase (diffusion)
 a (selectivity factor): vary temperature, composition of column/mobile
phase
 k (capacity factor): vary temperature, composition of
column/mobile phase
we can deduce that
the resolution is
given by:

Main parts of HPLC equipment
a
b
d
c e
f
g
h
a- solvent reservoirs e- precolumn and column
b- gradient elution controller f- detector
c- pumping system g- data processing
d- sample injection h- solvent waste

Agilent HPLC equipment
mobile phase storage
filtration and degassing
preparation of eluent mixtures
HPLC pump
autosampler
column box and oven
control unit
injection valve
detection unit with
UV-visible lamp

High performance liquid chromatography (HPLC) animation
https://www.youtube.com/watch?v=ZN7euA1fS4Y

The mobile phase
In HPLC the quality of the mobile phase is crucial
The solvent is either water (preferred) or organic
All solvents should be of ‘HPLC grade’
This means:
a high purity
a prior filtration on a 0.2µm filter
The ‘HPLC grade’ solvent can be either purchased or
produced using a convenient filtration device
Using filtered and pure solvents avoids many problems with
pumps and columns and also improves the quality of analyses
All solvents should be degassed prior to use in HPLC

Reciprocating piston pump
It is the most widespread pumping system used in HPLC:
a- motor
b- transmission
c- tightness seal
d- piston
e- solvent inlet
f- inlet and outlet
checkvalves
g- solvent outlet
a
g
f
e
d
c
b

Injection system in HPLC
The best injection device uses a switching six-port valve with
a sampling loop
This system allows an easy and reproducible injection of
liquid samples
It operates in two steps: load and inject
« load »
position
sample sample
mobile phase mobile phase
column column
outlet outlet
Sample
loop
« inject »
position

The HPLC column
Most stationary phases are chemically bonded to
the solid support which means a greater stability
The stationary phase parameters are accurately
controlled to assure constant and reproducible
performances
Packing techniques have also improved in order to
obtain smaller but more efficient columns
A typical HPLC column End fittings

Detection in HPLC
HPLC detectors are commonly classified as:
general (or bulk property detector): it measures the
difference in some physical property between the
chromatographic effluent and the pure mobile phase. This kind
of detector, such as refractive index detector, is considered as
universal and has a general use but a low sensitivity and a
limited dynamic range
specific (or solute property detector): responds to a
physical or chemical property characteristic of the solute. It
provides high sensitivity with a wide linear range such as in
spectrophotometric detectors. Although this kind of detectors is
specific it is more widely used than bulk property ones

Spectrophotometric UV/visible detector
It is a simple and cheap detector
and the most widely used detector in HPLC
It is a solute property detector
It is specific to solutes which exhibit an absorption in the
UV/visible range (generally unsaturated compounds)
The mobile phase must not significantly absorb at the
measured wavelength
With favourable solutes, the sensitivity (minimum detectable
concentration) is about 5.10-8g.mL-1

UV detector (with photodiode array )
photodiode array
sample cell
deuterium lamp
holographic
grating
quartz
lens

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