High Performance Liquid
Chromatography
(HPLC)
PHC 213
Classification according to the technique used.
1.Columnar:
In this type the stationary phase is packed in a column,
e.g. GC, LC and conventional column.
2.Planar:
In this type the stationary phase is
spreaded as a thin layer on glass,
plastic or aluminum plates; it is held
in the network structure of paper
(paper chromatography)
HPLC Separation Modes:
Classified according to:
 Polarity
 Electrical charge
 Molecular size
Separations based on
polarity:

To design a chromatographic separation system,
we create competition for the various
compounds contained in the sample by choosing
a mobile phase and a stationary phase with
different polarities.

Compounds in the sample that are similar in
polarity to the stationary phase will be
delayed because they are more strongly
attracted to it.

Compounds whose polarity is similar to that
of the mobile phase will be attracted to it and
move faster.

Based upon differences in the relative
attraction of each compound for each phase,
a separation is created by changing the
speeds of the analytes.

Normal Phase HPLC.
This is the same as in thin layer chromatography or column chromatography
Although it is described as "normal", it isn't the most commonly used in HPLC.
-Polar stationary phase and non-polar solvent .The column is filled
with tiny silica particles, and the solvent is non-polar e.g. hexane.
Polar compounds in the mixture being passed through the column will stick
longer to the polar silica than non-polar compounds will.
The non-polar ones will therefore pass more quickly through the column.
• Reverse Phase.
Non-polar stationary phase and a polar mobile phase
The same column size , but the silica is modified to be non-polar by attaching
long hydrocarbon chains to its surface - typically with either C8 or C18 atoms .
A polar solvent is used e.g. a mixture of water and methanol or acetonitril.
Polar molecules in the mixture having strong attraction with the polar
solvent (mobile phase) than hydrocarbon chains attached to the silica
(the stationary phase) therefore pass more quickly through the column.
Non-polar compounds in the mixture will tend to form attractions with the
hydrocarbon groups (stationary phase) and be less soluble in the solvent
(mobile phase) therefore pass slow down through the column.
Reversed phase HPLC is the most commonly used form of HPLC.
Reversed phase HPLC is the most commonly used form of
HPLC.
- “Like dissolves like”. In Normal Phase the analyte will be
partitioned preferentially in the mobile phase and provide little
interaction with the stationary phase. This is not desirable
since selective retention on the column will be very hard to
control. It can be controlled by modifying the stationary phase,
a very time consuming and expensive proposition even if
feasible.


In Reverse Phase the opposite is true. The analyte will be
partitioned preferentially in the stationary phase (“Like
dissolves like”). and by simply modifying the mobile phase, by
adjusting the polarity, ionic strength or pH, selectivity can be
virtually fully controlled.
Separations based on charge:
Ion-exchange chromatography:

In ion-exchange chromatography, Likes repel,
while opposites attract each other.

Stationary phases for ion-exchange separations
are characterized by the nature and strength of
the acidic or basic functions on their surfaces
and the types of ions that they attract and
retain.
 Cation
exchange is used to retain
and separate positively charged
ions on a negative surface.
 anion
exchange is used to retain
and separate negatively charged
ions on a positive surface.
Types of Ion-Exchanger
MECHANISM OF ION-EXCHANGE
CHROMATOGRAPHY OF AMINO ACIDS
pH2
SO3
-
+
Na
H3N
+
COOH
Ion-exchange Resin
SO3
-
H3 N
Na
+
+
-
COO
pH4.5
Selecting an operating mode
Sample type
LC Mode
•
Moderate polarity molecules
LSC or LLC
•
Compounds with similar functionality
LSC or LLC
•
Ionizable species
IEC
•
Compounds with differing solubility
LLC
•
Mixture of varying sized molecules
GPC
Retention parameters

1. Retention volume (VR)
The retention volume is the volume of
mobile phase passed through the
column between the injection point and
the peak maximum
2- Retention time (tR) = the distance from
the injection point to the peak maxima in
time units.
It is used as an identifier for a given
analyte.
It depends on mobile phase flow rate.
VR = F x tR
3. Void volume (V0)
The volume of liquid phase in the column.
V0 = F x t0
Also called retention volume of unretained
component.
4. Retention factor (k\)
Sometimes called capacity factor
k\ = VR – V0 / V0
= tR – t0 / t0
Independent on mobile phase flow rate and
column dimensions.
K\

The optimum values for k\ are in the range 1-10;
very little can be gained by increasing k\ further.

High values of k\ also mean long analysis times.

The capacity factor is controlled largely by
adjustment of the mobile phase composition.

Increasing k\ improves resolution by causing the
solutes to spend more time in the stationary phase(
increase anal. Time), increasing selectivity moves
one or both peaks relative to the other.
5.
Selectivity ()
The ability of the chromatographic system to discriminate
different analytes (distance between the peak maxima).
= k2 /k1 = tR2 – t0 / tR1 – t0
It is always greater than unity.
It depends primarily on the nature of the analyte and the
difference in its interaction with the stationary phase.
It is not affected by the mobile phase composition unless
this parameter modifies the analyte nature (ionization).

for a desired degree of resolution, three
conditions have to be met:
a- The peak must be separated from
each other (α > 1 )
b- The peak must be retained on the
column (k\ > 0 )
c- The column must develop some
minimum number of plates.
6. Efficiency
 It is a property of the column.

The degree of band broadening (width of the
chromatographic peak).

Tow terms are used as quantitative
measures of column efficiency:
Plate height(H) and Plate number(N).
N=L/H
The Theoretical Plate Model of Chromatography
The plate model supposes that the chromatographic column is
contains a large number of separate layers, called theoretical
plates. Separate equilibrations of the sample between the
stationary and mobile phase occur in these "plates". The analyte
moves down the column by transfer of equilibrated mobile phase
from one plate to the next.
It is important to remember that the plates do not really exist; they
are a figment of the imagination that helps us understand the processes at
work in the column. They also serve as a way of measuring column efficiency,
either by stating the number of theoretical plates in a column, N (the more
plates the better), or by stating the plate height; the Height Equivalent to a
Theoretical Plate (the smaller the better).
If the length of the column is L, then the HETP is
HETP = L / N
The number of theoretical plates that a real column possesses can be
found by examining a chromatographic peak after elution;
N = 16 (tR / W )2
where w is the peak width
tR is the retention time
N is the plate hight
As can be seen from this equation, columns behave as if they
have different numbers of plates for different solutes in a
mixture
At each plate of the stationary phase , continuous partitioning
and equilibration of solutes (components molecules) occurs as
the mobile phase moves down the column.
Thus the movement of solutes and solvent is regarded as
a series of stepwise transfer from one plate to the next.
The efficiency of the column is increased as the number of
theoretical plates (N) is increased,
Efficiency and selectivity are complementary
chromatographic descriptors.
6.
Resolution (R)
The resolution of a column provides a quantitative measure of
its ability to separate two analytes.
The ratio of the distance between two peaks to the
average width of these peaks (at baseline).
R = 2 (tR2 –tR1) /w2 +w1
1.5 is sufficient for the baseline separation of closely eluted
peaks.
2. Parameters used in HPLC
Condition for good separation
A larger Rs value means a better separation.
Rs =
1
4
k’2
1 + k’2
- 1
N
- 1
k’2
1 + k’2
N
: Capacity term
increases the retention time
: Selectivity term
increases the time interval between peaks
: Column efficiency term
produce narrow peaks
Comparison of the variation of selectivity and
efficiency necessary to increase resolution from 1
to 1.5
Resolution
1
Efficiency
10000
Selectivity
1.04
1.5
22500
1.04
1.5
10000
1.06
Optimization of column performance.
To optimize the column performance we have to change α
and َK' to obtain Rs value of 1.5.
This can be achieved by:
1-Change the composition of mobile phase and /or its flow rate
2-The column must be perfectly packed with small regular
spherical particles, without any cracks, gabs or spaces to
minimize zone broadening and to maximize N.
3-If resolution is still less than 1, the stationary phase must be
changed

In regular gravity column the particle size of the
stationary phase ranges from 150-250 μm.
Decreasing the particle size leads to increase in
surface area and consequently better separation is
achieved. In HPLC the particle size used is ranging
from 3-20 μm. Particle of ≤ 5 μm would yield more
than 10000 theoretical plates / meter.

The capacity of HPLC column are usually low less
than 20 μ g of sample so detector must be very
sensitive.

Pump is necessary to push mobile phase through
the finely divided stationary phase.
High Performance Liquid Chromatography
(HPLC)

Instrumentation:
123456-
Solvent reservoir (S).
High pressure pump.
Injection.
Column.
Detector.
Recorder.
1- Solvent reservoirs:
May be one container in which the required solvent system is
added or may be two or more each containing one solvent and
mixing is done in a solvent mixing unite.
The HPLC solvents must be of:
-High purity as HPLC grade solvents
-Low viscosity
-limited flammability and toxicity
-low reactivity to avoid chemical interaction with solutes
-Compatibility with the detector
The solvents must be filtered and degassed.
Degassing is very important for the following reasons:
a- To get ride of dissolved gases especially oxygen that may
react with stationary or mobile phase.
b- Bubbles formation will disturb the detectors.
Degassing can be done by:
1- Sonicator.
2- Passing helium in the solvents
The mobile phase may be, organic solvents-water-buffers.
2- Pumps:
They are necessary to enable the flow of mobile phase through
the finely divided stationary phase.
Pumps should be able to
-deliver up to 7000 psi (Pound/square inch).
-Have flow rates ranging from 0.1 to 10ml/min
-have high resistance to corrosion by a variety of solvents.
-Stepwise elution means the use of several eluting agents of
gradually increasing strength for successive desorption of the
separated components.
-Continuous change of the desorption power of the mobile phase
is achieved by adding stronger eluting agent (more polar solvent)
gradually to less polar one which is currently used
i.e. we start with the less polar solvent then the more polar is
added and mixed gradually according to a definite program.
3- Injection.
Injectors mostly are 6-port rotary valves
In order to introduce a sample onto
the column for analysis, a special
valve called the injector must be
used to transfer the sample into the
pressurized system..
Guard (or pre-) Column :
They are placed between injector and
analytical columnIt
-The job of the guard column is to to remove or retain sample and
solvent impurities that could be irreversibly adsorbed onto the analytical
column ,thus prevent contamination of the analytical column which
decrease its performance.
-A precolumn is used to protect the column against high-pH mobile phases
-They are very short columns about 3 cm , packed with material similar
to that contained in the analytical column, for example C18.
4- Analytical Columns:
Analytical column are typically 10-25 cm long and 5-10 mm
internal diameter (id).
Columns are made of stainless steel lined with glass to prevent
metal interaction with solvent or solutes.
Particle size from 3-20 μm. Particles ≤ 5 μm yield >10,000
theoretical plates/ meter.
Recently high speed and high performance microcolumns with 1-4mm(id) and length
of 3 to 7.5 cm having the advantages of speed and minimal solvent consumption.
Column Packing Materials:
Porous micro particles have diameter ranging from 3 to 10m, they
are composed of silica, alumina, porous polymer or ion exchange
resin.
Silica particles may be coated with thin organic film, which is
physically or chemically bonded to the surface.
Preparation of bonded phase
Si---oH + RoH → Si---oR
 Thermaly un stable and easy hydrolysis.
 Si---oH + Cl-Si-(CH3)2 R →

CH3
Si
OH + Cl
Si
R
CH3
surface of a silica
particles
organochlorosilane
(R: C4H17 or C18H37)
CH3
Si
O
Si
R
CH3
endcapping

In chromatography, the replacement of
accessible silanol groups in a bonded stationary
phase by trimethylsilyl groups is referred to as
endcapping
CH3
Si
OH
Si
O
Si
CH3
CH3
Silanol
"Active"
Doactlvated
Silanol "inactive"
Example of deactivated Silanol
a tnimethylsily (TMS)

Endcapping technology prevents the tailing of a polar
compound's peak adsorp polar comp.by silinol gp
Bonded phase must be used within
 pH 2-8
 PH <2 bonded moity will be removed.
 Ph > 8 silica will dissolve

5- Detectors:
They are placed at the column exit and used to detect a
specific property of solute materials in the column
effluent.
Examples of the commonly used detectors:
1- UV Detectors:
They detect solutes that can absorb UV
light due to the presence of conjugated system.
These are the most commonly used detectors.
2- Fluorescence Detectors:
Limited number of compounds can be
detected by such detectors.
3- Infrared Detectors:
It is more general. The solvent use must
not absorb IR at the chosen wavelength for
solute detection.
4- Radioactivity Detectors:
Very specific for radioactive materials.
5- Refractive Index Detectors:
Measure the change in the Refractive Index of
the mobile phase.
6. Mass Spectroscopy Detectors.
For a particular compound, the retention time
will vary depending on:
-the pressure used (because that affects the
flow rate of the solvent)
-the nature of the stationary phase (not only
what material it is made of, but also particle
size)
-the exact composition of the solvent
-the temperature of the column
That means that conditions have to be carefully controlled
HPLC Column & efficiency
*If a column bed is stable and uniformly packed, its
mechanical separation power is determined by the column
length and the particle size.
*Mechanical separation power, also called efficiency, is often
measured and compared by a plate number [symbol = N].
*Smaller-particle chromatographic beds have higher efficiency
and higher backpressure. More mechanical separation power
is gained by increasing column length.
*However, the trade-offs are longer chromatographic run
times, greater solvent consumption, and higher backpressure
Shorter column lengths minimize all these variables but also
reduce mechanical separation power.
Effect of column length
Effect of particle size
Effect of Increasing Efficiency (N)
The efficiency N can be doubled by halving the particle size (i.e.
10µm to 5µm). In the above example, resolution, Rs increased
from 2.0 to 2.8. Column length can then be halved to decrease
analysis time
Importance of Peak Asymmetry (As)
These chromatograms illustrate
the negative effect of increased
peak asymmetry, As, on
resolution, Rs.
When investigating the effect of
bonded phase chain length on
retention, it is important to
maintain the use of a high purity
silica so that peak asymmetry is
minimized
GENERAL FACTORS INCREASING RESOLUTION
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Increase column length
Decrease column diameter
Decrease flow-rate
Pack column uniformly
Use uniform stationary phase (packing material)
Decrease sample size
Select proper stationary phase
Select proper mobile phase
Use proper pressure
Use gradient elution
Quiz
Atest mixture consisting of phenylmethyl ketone , nitrobenzene,benzene &
hexane is separated on a C18 column with a mobile phase of CH3OH: H2O
(60:40) . With these conditions, the ketone is eluted first.The order of the
elution of the other solutes is
•Nitrobenzene, benzene & hexane
•Nitrobenzene, hexane & benzene.
•Benzene , hexane & nitrobenzene.
• Hexane, benzene& nitrobenzene.
In the above question, If the mobile phase used is CH3OH:
H2O (40:60) ,
the retention time of the other solutes will:
•Increase
•Decrease.
•Not changed.