Chromatography

Basic introduction and

instrumentation
 A SEMINAR
ON
Chromatography Introduction and
Instrumentation

GUIDED BY: BY: PATEL AKSHAY

J.D.Patel M.PHARM- 1(ph’ceutics)

(head of department) ROLL NO-05

Department of Pharmaceutics
NOOTAN PHARMACY COLLEGE,VISNAGAR
 Chromatography terms
• The analyte is the molecule which is to be purified or isolated during
chromatography
• Analytical chromatography is used to determine the identity and
concentration of molecules in a mixture
• A chromatogram is the visual output of the chromatograph. Different peaks
or patterns on the chromatograph correspond to different components of the
separated mixture
• A chromatograph takes a chemical mixture carried by liquid or gas and
separates it into its component parts as a result of differential distributions of
the solutes as they flow around or over the stationary phase
• The mobile phase is the analyte and solvent mixture which travels through
the stationary phase
• Preparative chromatography is used to purify larger quantities of a
substance
• The retention time is the characteristic time it takes for a particular
molecule to pass through the system
• The stationary phase is the substance which is fixed in place for the
chromatography procedure and is the the phase to which solvents and the
analyte travels through or binds to. Examples include the silica plate in
thin layer chromatography
 Retention
• The retention is a measure of the speed at which a substance
moves in a chromatographic system. In continuous
development systems like HPLC or GC, where the compounds
are eluted with the eluent, the retention is usually measured
as the retention time Rt or tR, the time between injection and
detection. In interrupted development systems like TLC the
retention is measured as the retention factor Rf, the run
length of the compound divided by the run length of the
eluent front:

• The retention of a compound often differs considerably

between experiments and laboratories due to variations of
the eluent, the stationary phase, temperature, and the setup.
It is therefore important to compare the retention of the test
compound to that of one or more standard compounds under
absolutely identical conditions.
 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.
 The Rate Theory of Chromatography
• A more realistic description of the processes at
work inside a column takes account of the time
taken for the solute to equilibrate between the
stationary and mobile phase (unlike the plate
model, which assumes that equilibration is
infinitely fast). The resulting band shape of a
chromatographic peak is therefore affected by the
rate of elution. It is also affected by the different
paths available to solute molecules as they travel
between particles of stationary phase. If we
consider the various mechanisms which contribute
to band broadening, we arrive at the Van Deemter
equation for plate height;
• HETP = A + B / u + C u
• where u is the average velocity of the mobile
phase. A, B, and C are factors which contribute to
band broadening.
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;

where w1/2 is the peak width at half-height.

As can be seen from this equation, columns behave as if they have

different numbers of plates for different solutes in a mixture.
A-Eddy diffusion
The mobile phase moves through the column which is packed
with stationary phase. Solute molecules will take different
paths through the stationary phase at random. This will cause
broadening of the solute band, because different paths are of
different lengths.
B - Longitudinal diffusion
The concentration of analyte is less at the edges of the band
than at the center. Analyte diffuses out from the center to the
edges. This causes band broadening. If the velocity of the
mobile phase is high then the analyte spends less time on the
column, which decreases the effects of longitudinal diffusion.
C - Resistance to mass transfer
The analyte takes a certain amount of time to equilibrate
between the stationary and mobile phase. If the velocity of the
mobile phase is high, and the analyte has a strong affinity for
the stationary phase, then the analyte in the mobile phase will
move ahead of the analyte in the stationary phase. The band of
analyte is broadened. The higher the velocity of mobile phase,
the worse the broadening becomes.
 Van Deemter plots
•A plot of plate height vs. average linear velocity of mobile phase

Such plots are of considerable use in determining the optimum mobile phase flow rate
 Resolution

• Although the selectivity factor, a, describes the separation of

band centres, it does not take into account peak widths.
Another measure of how well species have been separated is
provided by measurement of the resolution. The resolution of
two species, A and B, is defined as

•Baseline resolution is achieved when R = 1.5

• Adsorption Chromatography: Adsorption chromatography is
probably one of the oldest types of chromatography around.
It utilizes a mobile liquid or gaseous phase that is adsorbed
onto the surface of a stationary solid phase. The
equilibriation between the mobile and stationary phase
.
accounts for the separation of different solutes
• Partition Chromatography: This form of chromatography is
based on a thin film formed on the surface of a solid
support by a liquid stationary phase. Solute equilibriates
between the mobile phase and the stationary liquid .
HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
Limitations of GC :
• Only useful for volatile and thermally stable compounds.
• Can’t be used for solid.

Difficulties encountered in LC :

• Non availability of sensitive detectors.

• Speed of separation with conventional liquid
chromatography.
• Difficulty of speed overcome by using high pressure.
 Comparision of GC & LC

• Mobile phase gas • Mobile phase liquid

• (very cheap) • (very costly)
• St. Phase solid/liquid • St.Phase solid/liquid
• Mobile phase inert • Mobile phase interacts
with solute.
• Separation mainly based • St. phase limited in no.
on selection of st.phase separation (desired)
achieved by selecting
(wide choice)
mobile phase.
• Wide applications.
• Limited applications.
Flow diagram for a liquid chromatograph
 Eluent Delivery System :

• Reservoirs (one or more )

• Degassers

• Pumps
Function of Degassers :

• Appreciable amount of gases dissolve

at high pressure.
• When pressure released in column
and detector bubbles may form.
• Degassing by heating distilling,
vacuum pumping or purging inert gas
with low solubility like He/Ar.
Requirements of Pump :

• Pulseless flow upto 10 ml/min.

• High pressure upto 1000 psi or more.
• Suitability for gradient elution;
• Reciprocating pump
• Syringe pump
• Pneumatic pump
A reciprocating pump for HPLC
Reciprocating Pump: Schematics

• Most HPLC pumps are

reciprocating
• A motor driven cam
drives the piston to
deliver solvent through
the outlet check valve
• Gradient are formed by
using 2 or more pumps
(high-pressure mixing)
or solenoid-actuated
proportioning valves
(low-pressure mixing)
 Reciprocating pump :
• In 90% instrument used.
• To minimise pulsing pistons and cylinders operate in
cycle.
• Pressure drop caused by slowing of one is compensated
by others.
• Advantages :
• Small volume
• Pressure upto 600 atm(10,000 psi) can be applied.
• Variable flow rate upto 10 ml/min.
• Suitable for gradient elution.
• Disadvantages :
• Not totally pulseless flow.
• Damping device required to have regular flow.
Valve injection systems for liquid sampling : (a) rotary
A sampling loop for liquid chromatography
 Sample Inlet System :
• Syringe injection : Problem of leaking and
blow back of plunger.
• Stop-flow Injection : During injection, flow is
stopped by a valve kept before injection port.
After injection over flow is started again.
• Most widely used method is sampling
valve method :
• Rheodyne injector : In one position sample
fills loop (variable size) when mobile phase goes
directly to column. Then lever is moved, when
eluent carries sample from the loop along with
it to the column.
• Loops of different size 0.5µl to 500µL.
 Columns :
• Stainless steel.
• Length 30 cm.
• Internal diameter 5 mm.
• Particle size 5 µm.
• 40000 to 60000 plates/m.
• Smaller columns available.
• Require less volume of eluent.
• This is important as mobile phase liquids costly.
• Such columns have limited sample capacity.
• Temperature control not important.
• Many separations at room temperature.
• Sometimes temp. 30 – 1500C used accuracy ± 0.20C.
 Guard column :
• Short column containing similar st. phase as analyte
column, but particle of bigger size filled so that no
pressure
drop.

Two functions :
(i) Remove impurity to protect analyte column which is
very costly.
(ii) Presaturates mobile phase with st.phase liq. so that
in analyte column st.phase liq. Is not carried away
with mobile phase liq.
 Detectors :

• No detector is as sensitive, versatile as

detectors of GC like FID and TCD.
• Very few work on bulk property.
• Most of them respond to some physical
property of solute that is different from
eluent.
UV-Absorption Detector
 UV – Absorption detector :
• (i) Single wavelength (ii) Variable wavelength
• Principle :
• Most of organic compound absorb UV radiation.
• If eluent is not absorbing then as soon as solute is
eluted of Column and reach detector a signal obtained.
• For single wavelength source is Hg-vapour lamp that
emits 254 nm wavelength.
• Radiation divided in two beams one passing through
pure eluent other through column effluent.
• If solute eluted it will absorb radiation and detector will
observe difference in intensity of two beams.
 Volume of cell 1 to 10 µL To have pathlength 2 to 10
mm. Diameter of tube very narrow.

Advantages :

(iii) Sensitivity 10-4µg/ml.

(iv) Selective
(v) In sensitive to change in flowrate, temp. & comp. of
mobile phase
(vi) Suitable for gradient elution.
Limitation :

The eluent should not absorb UV radiation.

Refractive Index Detector
 Refractive Index Detector :
Two types : (a) Reflection type (b) Deflection type.

A beam of light is reflected at eluent prism interface.

Other is refracted and that beam after passing through
collimating lens is falling on photocell, which measures its
intensity.
When solute enters the eluent, the RI of eluent
changes. As a result the intensity of beam reflected at
eluent prism interface changes. This leads to change in
intensity of beam refracted which is measured by photocell.

Thus change in intensity α concentration of

solute
of refracted beam
 Characteristics :

• Non selective
• Sensitivity 10-3µg/ml (much less than UV)
• Not sensitive to change in flow rate
• Very sensitive to change in temp. and change in
composition of eluent
• Not suitable for gradient elution.

Mainly used for carbohydrates, aminoacid etc.

 Fluorescence Detector :

Can only be used for sub. emitting fluorescence like plant

pigments, vitamins, alkaloids, pharmaceuticals, flavoring
agents etc.

 Highly sensitive 10-5µg/ml.

 Suitable for gradient elution.
 Non fluorescent compounds can be converted to
fluorescent derivatives.
 This can be done on column.
 Derivatisation column kept before or after analyte
column.
 Stationary phases :

They can be either liquid or solid.

If liquid is used it is coated on inert solid, but there are

problems in it.

Hence, bonded phase supports prepared.

 Silica is heated in dilute acid for a day or two to generate
silonal group. As follows:
OH OH OH

Si O Si O Si

Silica particles

This is then treated with an organochlorosilane.

CH3 CH3

Si OH + Cl Si R Si O Si R + ClH

CH3 CH3

R = long alkyl chain of 8 or 18 carbon then Nonpolar

(Reversed phase)
R = -(CH2)n-CN, then polar (Normal phase)
= -(CH2)n-NH2
Stable upto pH 2 & 9 and upto 800C. How they retain
solute molecules is not certain.
Solid stationary phases :

Commonly used are (1) Silica (2) Alumina (3) Polyamides.

Silica is preferred as it can be obtained in different
forms.

 Porous microparticles
 Size 3 to 10 µm
 Surface area 100-900 m2/gm
 Small particles (large area) are suitable to separate
solutes having narrow range of particles.
 Particles with bigger size and small surface area are
effective in separating solutes with wide range of
polarities.
Normal phase and Reversed phase
Chromatography :

Initially the stationary phase used to be polar and

mobile phase used to be non-polar. This combination
became popular as normal phase chromatography. Later
the use of non-polar stationary phase and polar stationary
phase started. This was reverse to the established
combination and hence it is called reversed phase
chromatography.
Points of comparision is given below :

• St. phase : Polar • Non polar

• Mobile phase : Non polar • Polar
• On increasing polarity of • On increasing polarity of
mobile phase retention mobile phase retention
time decreases. time increases.
Mobile phase liquids :

Criteria to select :

3. Low viscosity
4. High polarity to avoid contamination with sample.
5. High stability, should not react with solute/st.phase liq.
6. Low volatility so that bubbles not formed.
7. Immiscible with st. phase liq. If used in adsorbed form.
8. Suitable for separation to be done.
9. Compatibility with detector.
Solvents Polarity Index B. P.
Cyclohexane 0.04 81
n-hexane 0.1 69
Toluene 2.4 110
THF 4.0 66
Ethanol 4.3 78
Ethyl acetate 4.4 77
Methanol 5.1 65
Acetonitrile 5.8 82
Nitromethane 6.0 101
Water 10.2 100
Isocratic & Gradient elution :
If comp. of mobile phase does not change during
elution, it is isocratic.
If comp. changes then Gradient.

Need for Gradient elution :

If we want to separate mix. of 10 solutes, 5 N.P. and
5 highly polar and NP mobile phase used then NP solutes
eluted in reasonable time but polar solutes take long time to
come out. If mobile phase is polar then NP solutes eluted so
quickly that no resolution but now polar solutes come out in
reasonable time. If we make mobile phase gradually from
NP to polar then the problem is solved and that is called
gradient elution.
 Detector
response

Time

With polar mobile phase

Detector
response

Time
Derivatisation :
2. To prepare UV absorbing deri.of alcohol comp. treated
with 3-5 dinitrobenzoyal chloride.
3. To prepare fluorescent deri.of carboxylic acids treated
with 4-bromomethyl 7-methoxy coumarin.
It must be quantitative.
 Varian HPLC System

9060 Polychrom Computer

(Diode Array) Detector Workstation

9050 Variable
9010 Solvent UV/Vis Detector
HPLC Solvent Delivery System
Reservoirs

Rheodyne
Injector

Keep an eye on
HPLC these 4 screens!
Column
Varian Solvent Delivery System
 %A %B %C Flow Rate Pressure to column
{H2O} {MeOH} (mL/min) (atmos.)
load

Ready
inject

Rheodyne
Injector
Varian 9010 Solvent Delivery System to injector

through pump

Column
through C
pulse
dampener
A B

from solvent to
Ternary Pump reservoir detector
Variable UV/Vis
Detector
ABS AUFS λ RunTime EndTime
0.001 2.000 238 0.00 min 10.0 min

Ready
THANK YOU