The document provides a comprehensive introduction to high-performance liquid chromatography (HPLC), covering the principles of chromatography, key instrumentation, and the factors affecting resolution and efficiency of separation. It elaborates on chromatographic terms, band broadening, and the interplay between the mobile and stationary phases. Additionally, it details the components of HPLC systems, including pumps and sample injectors, explaining their functions and importance in the chromatographic process.

1. HPLC
Basic introduction, Theory and
instrumentation
By Priyanka Yadav
Associate Professor, SSSPC,
Zundal

2. • 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
Chromatography terms

3. Detector
Signal
tim
e
tr
tm
Injection
w
Chromatogram
Retention time tr
Retention volume Vr
Baseline width Wb
void time, tm
void volume Vm
• Chromatogram
is a plot of the
detector’s signal as
a function of time
or volume of
eluted mobile
phase.
• It consists of a
peak for each of
the separated
solute bands.

4. Chromatographic Resolution
Resolution: is a
quantitative measure of
the degree of separation
between two
chromatographic peaks

5. Resolution value and degree of
peak overlap
0.13 %

6. How to improve resolution
improved selectivit
narrow band
selectivity is unchang
Poor resolution

7. How to improve resolution
• From later equation, it is clear that resolution may be
improved either by increasing ∆tr or by decreasing wA or
wB.
• We can increase ∆tr by enhancing the interaction of the
solutes with the column or by increasing the column’s
selectivity for one of the solutes.
• Peak width is a kinetic effect associated with the solute’s
movement within and between the mobile phase and
stationary phase.
• The effect is governed by several factors that are
collectively called column efficiency.

8. Fundamental equation of resolution
• The fundamental equation of resolution indicates that
the resolution is affected by three parameters
1. Selectivity (separation) factor (α)
2. Efficiency
3. Retention (capacity factor, k)
Efficiency Selectivity Retention
Factors affecting resolution

9. Column efficiency
• The plate number N (column factor) can be
increased by lengthening the column, decreasing
the packing particle diameter, or optimizing flow
rate.
• However, improving resolution by increasing N is
expensive in time.
• Doubling the column length doubles the elution
time, solvent consumption, and pressure while
only increasing Rs by 1.4.
• Likewise, reducing the particle diameter increases
the resolution but may exceed the maximum
allowable pressure.

10. • How to change the capacity factor: Change the mobile phase
strength (polarity in reversed phase chromatography)
Capacity factor
• It is a measure of how strongly a solute is retained
by the stationary phase (k').
tm is the retention time of
the non retained
compound
t`r Adjusted retention time
.
Retention (capacity) factor

11. Significance of K`
Results
Capacity
factor
a component is unretained
k` = 0
all peaks are packed together too closely at
the beginning of the chromatogram.
k` < 2
good separations are observed
k` is between
2 and about
10
peaks are separated, but they take too
long to elute, have become wider and
lower, and if close to baseline noise in
magnitude, they may become difficult to
k` > 10

12. Column Selectivity
The selectivity (or separation factor) is the ability of the
chromatographic system to distinguish between the sample
components
α = if the solutes elute
with identical retention
times,
α> 1 when t r,A > t r,B

13. Effect of selectivity on resolution
The selectivity α can be increased by
1.changing columns to a different stationary phase or
2.by imposing secondary equilibria through changes in mobile-phase pH
3.or the addition of complexing agents to the mobile phase,.

14. Peak asymmetry
Asymmetry factor (AF) = b/a 0.95 – 1.15

15. Non ideal asymmetrical
chromatographic bands
(a) Sharp peak (b) Broad peak (c) Fronting (d) Tailing
• fronting is most often the result of overloading the
column which will lead to poor trapping of the analyte,
too much ‘dead volume’ in the chromatographic sample
some and sample decomposition.
• tailing, system molecules of the analyte are adsorbed
strongly onto active sites in the stationary phase.

16. 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.

17. • At the beginning of a chromatographic separation the
solute occupies a narrow band of definite width.
• As the solute passes through the column, the width of its
band continually increases in a process called band
broadening.
• Column efficiency provides a quantitative measure of the
extent of band broadening.
• Column efficiency can be explained by two theories:-
1. PLATE THEORY OF CHROMATOGRAPHY
2. RATE THEORY OF CHROMATOGRAPHY
COLUMN EFFICIENCY

18. 1. The 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.

19. • Resolution is a measure of the efficiency of the column.
• There are another parameter which defines the
efficiency as the number of theoretical plates, N.
• Consider a column that is divided into N segments of
equal length, and that each segment is just long
enough to allow complete equilibration of solute
partitioning between the stationary phase and the
mobile phase, according to its partition coefficient.
Each of these segments is called a theoretical plate. .
• It is important to remember that a theoretical plate is
an artificial construct and that no such plates exist in a
chromatographic column.

20. • The relationship between column length (L), number of
theoretical plates, N, and the height of a theoretical
plate, H; are give by the following equation;
• The greater the number of plates for a given column
length, the shorter the height equivalent to a
theoretical plate, H, and the more efficiently the
column.
• A column’s efficiency improves with an increase in the
number of theoretical plates or a decrease in the height
of a theoretical plate.

21. Calculation of number of theoretical
plates
• The number of theoretical plates in a chromatographic
column is obtained by using the following equations:
• An advantage of N as a measure of efficiency is that
– it may be calculated from measurements on a single
peak. Unlike Rs, it does not require a pair of peaks
and is independent of their relative selectivity .
– It may be calculated using either the peak width at
base, Wb, or peak width at half the peak height, Wh.
– The latter (Wh) is sometimes better, when for
broadened peak, fronting or the effects of adsorption
(tailing).

22. 2. 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.

23. 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.
As can be seen from this equation, columns behave as if they have
different numbers of plates for different solutes in a mixture.

24. • Solute molecules passing through a chromatographic column travel
separate paths that may differ in length.
• Because of these differences in path length, solute molecules injected
simultaneously elute at different times.
• The principal factor contributing to this variation in path length is
– a non-homogeneous packing of the stationary phase in the column,
– differences in particle size and
– packing consistency.
– average diameter of the
particulate packing material
• A smaller range of particle sizes and a more consistent packing produce
a smaller value for A.
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.

25. • If a band of molecules or atoms are placed in a container such as a tube at
the center of its length, the molecules will diffuse in the direction of lower
concentration.
• Clearly this occurs in all directions but in a tube the walls are a limit and
so our concern is along the axis of movement caused by the mobile phase
and this is referred to as longitudinal molecular diffusion.
• Even if the mobile phase velocity is 0, solute molecules are constantly in
motion, diffusing through the mobile phase.
• Since the concentration of solute is greatest at the center of a
chromatographic band, more solute diffuses toward the band’s forward
and rear edges than diffuses toward the band’s center.
• The net result is an increase in the band’s width and band broadening
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.

26. 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.
• A chromatographic separation occurs because solutes move between the
stationary and mobile phases.
• For a solute to move from one phase to the other, it must first diffuse to
the interface between the two phases in a process called mass transfer.
• A contribution to band broadening occurs whenever the solute’s
movement to the interface is not fast enough to maintain a true
equilibrium distribution of solute between the two phases.
• Thus, solute molecules in the mobile phase move farther through the
column than expected before passing into the stationary phase.
• Solute molecules in the stationary phase, on the other hand, take longer
time than expected to cross into the mobile phase.

27. 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

28. Plot of the height of a theoretical plate
as a function of mobile-phase velocity
using the van Deemter equation

29. • 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.

30. • 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.

31. HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY

32. Instrumentation of HPLC
The System consist of :
• Mobile Phase Reservoir (Solvent storage bottle)
• De-gasser
• Gradient controller and mixing unit
• High Pressure Pump
• Pressure gauge
• Pre-column/ Guard Column
• Sample inlet system
• Column
• Detector
• Recorder

33. Flow diagram for a liquid chromatograph

34. Eluent Delivery System :
• Mobile phase Reservoirs (one or more )
• Degassers
• Mixing Unit
• Pumps

35. MOBILE PHASE RESERVOIRS
• Glass or stainless-steel containers capable of
holding up to 1 liter mobile phase (pure organic
solvents or aqueous solutions of salts and buffers)
• Inert to a variety of aqueous and non aqueous
mobile phases.
• Stainless steel should be avoided for use with
solvents containing halide ions.

36. DEGASSERS
• In many cases, aqueous solvents & some
organic solvents are degassed prior to use
• Degassing is done to prevent formation of gas
bubbles in the pump or detector ( Mobile phases
are degassed by stirring of the mobile phase
under vacuum, sonication or sparing with helium
gas)
• The mobile phase are filtered to remove
particulate matter that may clog the system

37. Function of Degassers :
• Appreciable amount of gases dissolve at high
pressure.
• So, 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
gas.

38. PUMP
• The solvents or mobile phase must be passed
through a column at high pressures at up to 6000
psi(lb/in²) or 414 bar.
• As the particle size of stationary phase is smaller
(5 to 10μ) the resistance to the flow of solvent will
be high.
• That is, smaller the particle size of the stationary
phase the greater is the resistance to the flow of
solvents.
• Hence high pressure is recommended.

39. Requirements of Pump :
• Pulseless flow upto 10 ml/min.
• High pressure upto 6000 psi.
• Suitability for gradient elution.
• Pumps should be capable of taking the solvent
from a single reservoir or more than one
reservoir containing different solvents
simultaneously.

40. Types of Pumps used in HPLC:
• 1. Displacement Pumps
• 2. Reciprocating Pumps
• 3. Pneumatic Pumps

41. 1. Displacement Pumps
• It consists of large, syringe like chambers equipped
with a plunger activated by a screw driven
mechanism powered by a stepping motor.
• So it is also called as Screw Driven Syringe Type
Pump.
• Advantages:- It produces a flow that tends to be
independent of viscosity & back pressure.
• Disadvantages:- It has a limited solvent
capacity(~250) & considerably inconvenient when
solvents must be changed.

42. Displacement Pump

43. 2. Reciprocating Pumps
• This pump transmits alternative pressure to the
solvent via a flexible diaphragm ,which in turn is
hydraulically pumped by a reciprocating pump.
Disadvantages
• Produces a pulsed flow which is damped because
pulses appear as baseline noise on the
chromatograph.
• This can be overcome by use of dual pump heads
or elliptical cams to minimize such pulsations.

44. A reciprocating pump for HPLC

45. 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)

46. 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.

47. 3. Pneumatic pumps
• In this pumps, the mobile phase is driven
through the column with the use of pressure
produced from a gas cylinder.
• It has limited capacity of solvent.
• Due to solvent viscosity back pressure may
develop.

48. A pneumatic pump for HPLC

49. SAMPLE INJECTOR SYSTEM
• Several injector devices are available
either for manual or auto injection of the
sample.
(i) Septum Injector
(ii) Stop Flow Injector
(iii) Rheodyne Injector

50. i. Septum injector
• These are used for injecting the sample through a
rubber septum.
• This kind of injectors cannot be commonly used ,
since the septum has to withstand high pressures.
ii. Stop Flow injector
• In this type the flow of mobile phase is stopped
for a while & the sample is injected through a
valve.
• During injection, flow is stopped by a valve kept
before injection port. After injection over flow is
started again.

51. III. Rheodyne injector
• It is the most popular injector and is widely
used.
• This has a fixed volume of loop, for holding
sample until its injected into the column, like
20μL, 50μL or more.
• Through an injector the sample is introduced
into the column.
• The injector is positioned just before the inlet of
the column.
• 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.

52. Valve injection systems for liquid sampling : (a) rotary

53. A sampling loop for liquid chromatography

54. 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.

55. Precolumn/Guard column :
• It contains a packing chemically identical to that in
analytical column.
• Mainly used to remove the impurities from the solvent and
thus prevents contamination of the analytical column, it
can protect analytical column. It is also called as guard
column or protective column.
• it is having large particle size. It is having short length of 2
to 10 cm, so does not affect separation.
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.

56. Analytical column :
• The success or failure of analysis depends upon
choice of column.
• Actual separation is carried out here.
• Stainless –steel tube
• size – length -25 to 100 cm
• Internal diameter – 2 to 4.6 mm
• Column is filled with small particles 5 – 10 micron.
The solid support can be silica gel, alumina.
• The separation is result of different components
adhering to or diffusion into the packing particles
when the mobile phase is forced through column.

57. A Column for liquid chromatography

58. • C8 and C18 columns are considered as examples
of reversed phase liquid chromatography (RP).
• The stationary phase here is seen as a thin film
of non-polar liquid phase that has been designed
to be chemically similar to an inert material
(Silica gel particles).
• The non-polar layer is chemically linked to the
silica particles surface by reaction with the polar
silanol groups on the stationary phase surface
and so rendering them less polar or non-polar.
• The difference between the two columns will be
in the length of the carbonchain attached to the
silica surface.

59. • Accordingly C8 HPLC columns have
packing material composed of silica
particles attached to C8 carbon units.
• C18 will, of course, have packing materials
coated with C18 hydrophobic units.
• Categorically both are reversed phase but
C18 columns will definitely be more
"hydrophobic rather than the C8 columns.

60. DETECTORS
• Absorbance (UV/Vis and PDA)
• Refractive index (detects the change in turbidity)
• Fluorescence (if the analyte is fluorescent)
• Electrochemical (measures current flowing
through a pair of electrodes, on which a
potential difference is imposed, due to oxidation
or reduction of solute)
• Conductivity (for ions)
• Light scattering
• Mass spectrometry (HPLC-MS)

61. UV – Absorption detector :
• UV visible detector is widely used as it detects
large number of compounds because most drugs
have appropriate structural characteristics for
light absorption.
• These are useful for aromatic compounds and
other type of unsaturated systems.
• These are classified as fixed or variable
wavelength detectors.
• Fixed wavelength detectors employ filter as a
source to provide appropriate wavelength.
• Most common fixed wavelength detectors are
based on 254 nm.

62. • Variable wavelength detectors are employ
a spectrophotometer to provide dispersion
of light and selection of any wavelength in
UV visible regions.
• Diffraction gratings are frequently used for
wavelength dispersion.

63. UV-Absorption Detector

64. Types of 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.

65. Volume of cell 1 to 10 µL To have pathlength 2 to 10
mm. Diameter of tube very narrow.
Advantages :
(i) Sensitivity 10-4µg/ml.
(ii) Selective
(iii) In sensitive to change in flowrate, temp. & comp. of
mobile phase
(iv) Suitable for gradient elution.
Limitation :
The eluent should not absorb UV radiation.

66. PHOTODIODE ARRAY (PDA):
• A photodiode array (PDA) is a linear array of discrete
photodiodes on an integrated circuit (IC) chip.
• Allows a range of wavelengths to be detected
simultaneously. In this regard it can be thought of as
an electronic version of photographic film. Array
detectors are especially useful for recording the full Uv-
vis is a absorption spectra of samples that are rapidly
passing through a sample flow cell, such as in an HPLC
detector.
• PDAs work on the same principle as simple.
Photovoltaic detector
• similar to UV detector, non destructive 190-600 nm for
quantization & identification Spectra is 3D, Response vs
time vs WL.

67. PDA Detector

68. REFRACTIVE INDEX (RI)
DETECTOR
• Detection occurs when the light is bent due to
samples eluting from the columns, and this is
read as a disparity b/w the two channels.
• It is not much used for analytical applications
because of low sensitivity & specificity.
• When a solute is in the sample compartment,
refractive index changes will shift the light beam
from the detector.

69. Refractive Index Detector

70. Types of 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  concentration of solute
of refracted beam

71. 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.

72. 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.

73. • It is based on the fluorescent radiation emitted by some
compounds.
• The excitation source passes through the flow cell to a
photo detector while a monochromatic measures the
emission wavelengths.
• More sensitive and specific.
• The disadvantage is that most compounds are not
fluorescent in nature.
• Fluorescence is a type of luminescence in which the
light energy is released in the form of a photon in
nanoseconds to microseconds.

74. RECORDERS AND INTEGRATORS
• Recorders are used to record responses obtained
from the detectors after amplification, if necessary.
• They record the baseline & all the peaks obtained
with respect tot time.
• Retention time can be found out from this
recordings, but area under curve cannot be
determined.
• The Integrators are improved versions of
recorders with some data processing capabilities.
• They can record the individual peaks with
retention time, height, width of peaks, peak area,
percentage area, etc.

75. • Integrators provides more information on peaks
than recorders.
• In recent days computers and printers are used
for recording and processing the obtained data &
for controlling several operations.

76. 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.

77. Silica is heated in dilute acid for a day or two to generate
silonal group. As follows:
Si O Si O Si
OH OH OH
Silica particles
This is then treated with an organochlorosilane.
Si OH Si R
CH3
CH3
Cl Si O Si R
CH3
CH3
ClH
+ +
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.

78. 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.

79. 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.

80. Points of comparision is given below :
• St. phase : Polar
• Mobile phase : Non polar
• On increasing polarity of
mobile phase retention
time decreases.
• Non polar
• Polar
• On increasing polarity of
mobile phase retention
time increases.

81. Mobile phase liquids :
Criteria to select :
1. Low viscosity
2. High polarity to avoid contamination with sample.
3. High stability, should not react with solute/st.phase liq.
4. Low volatility so that bubbles not formed.
5. Immiscible with st. phase liq. If used in adsorbed form.
6. Suitable for separation to be done.
7. Compatibility with detector.

82. 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

83. 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.

84. With polar mobile phase
Derivatisation :
1. To prepare UV absorbing deri.of alcohol comp. treated
with 3-5 dinitrobenzoyal chloride.
2. To prepare fluorescent deri.of carboxylic acids treated
with 4-bromomethyl 7-methoxy coumarin.
It must be quantitative.
Detector
response
Time
Detector
response
Time

85. Varian HPLC System
9010 Solvent
Delivery System
9050 Variable
UV/Vis Detector
HPLC Solvent
Reservoirs
HPLC
Column
Rheodyne
Injector
9060 Polychrom
(Diode Array) Detector
Computer
Workstation
Keep an eye on
these 4 screens!

86. Varian Solvent Delivery System

88. Varian 9010 Solvent Delivery System
Rheodyne
Injector
%A %B %C Flow Rate Pressure
{H2O} {MeOH} (mL/min) (atmos.)
Ready
Ternary Pump
A
C
B
from solvent
reservoir
Column
to
detector
to column
through
pulse
dampener
to injector
through pump
load
inject

89. Variable UV/Vis
Detector
ABS AUFS l RunTime EndTime
0.001 2.000 238 0.00 min 10.0 min
Ready

90. APPLICATION
Drug Discovery
Clinical Analysis
Proteomics
Forensic Chemistry
Drug Metabolism study
Environmental chemistry
Diagnostic studies
Cosmetic analysis
Determination of Green Florescent Protein
Structural Determination
Pharmaceutical Applications
Identification of Bile Acid Metabolite
Clinical Applications
Biochemical Genetics
qualitative and quantitative analysis
Therapeutic Drug Monitoring

91. THANK YOU