1. HPLC
Prof. P.Ravisankar
Vignan Pharmacy college
Valdlamudi
Guntur Dist.
Andhra Pradesh
India.
banuman35@gmail.com
00919059994000

2. H
P
igh
ressure
L iquid
C hromatography
Since high pressure is used when compared to classical chromatography
Separation of the components from a mixture is achieved by pumping
mobile phase at High pressure using appropriate displacement pumps
or gas pressure. (Due to the small particle size (3-5 um).

3. H
P
igh
erformance
L iquid
Chromatography
More ever it is improved performance when compared to other
conventional column Chromatographic techniques.

4. H
P
igh
riced
L iquid
C hromatography

5. • Michael Tswett (1906) -separation of plant pigments
• Martin and Synge (1941) liquid-liquid partition chromatography
• 1952 Nobel Prize
• Other chromatography Techniques
• Thin-layer chromatography (TLC)
• Paper chromatography
• Preparative column chromatography
• Medium pressure chromatography
• Gas chromatography
• Ion-exchange chromatography
• Size-exclusion chromatography (Gel Permeation Chromatography)
• HPLC
• VLC
• Flash Chromatography
• Affinity Chromatography
• Chiral Chromatography
• Super Critical Fluid Chromatography
5

6. What means chromatography ?
AABC
AC
ABBC
C C
B
C
B
A
C
B
A
B
C
CC
C C
C
C
BB
B
B
B
B
A
A
A
A
A
A
Sample separated components
Chromatography is the chemical / physical separation of components for qualitative and
quantitative analysis
PrinciplePrinciple
6

7. Different chromatography methods
7
Name Mechanism Stat. Phase Mobile phase
Paper- partition liquid liquid
Thin layer- adsorption solid liquid
Gas- adsorption /
partition
solid or liquid gas
Column- adsorption /
partition
Solid/liquid liquid

8. Liquid Chromatography (LC)
• Two different phases are used to separate
components of the mixture: stationary and
mobile phase
• The solute separates on the column via
interactions based on physical mechanisms like
retention
8

9. Partitioning
• Separation is based on the analyte’s
relative solubility between two liquid
phases
9
Stationary PhaseMobile Phase
Solvent Bonded Phase

10. 10

11. What is HPLC?
• The most widely used analytical separations technique
• Utilizes a liquid mobile phase & packed column to
separate components of mixture
• uses high pressure to push solvent through the column
• Popularity:
– sensitivity
– ready adaptability to accurate quantitative determination
– suitability for separating nonvolatile species or thermally
fragile ones
11

12. • Popularity:
– widespread applicability to substances that are of prime
interest to industry, to many fields of science, and to the
public
• Ideally suited for separation and identification of amino
acids, proteins, nucleic acids, hydrocarbons,
carbohydrates, pharmaceuticals, pesticides, pigments,
antibiotics, steroids, and a variety of other inorganic
substances
12

13. History
• Early LC carried out in glass columns
– diameters: 1-5 cm
– lengths: 50-500 cm
• Size of solid stationary phase
– diameters: 150-200 µm
• Flow rates still low ! Separation times long!
• Decrease particle size of packing causes increase in column
efficiency!
– diameters 3-10 µm
• This technology required sophisticated instruments
– new method called HPLC
13

14. Advantages of HPLC
• Higher resolution and speed of analysis
• Greater reproducibility due to close control of
the parameters affecting the efficiency of
separation
• Easy automation of instrument operation and
data analysis
• Adaptability to large-scale, preparative
procedures
14

15. Advantages to HPLC
• Advantages of HPLC are result of 2 major advances:
– stationary supports with very small particle sizes and large
surface areas
– appliance of high pressure to solvent flow
15

16. Chromatographic terms
16
Baseline
Injection
monitoring
Eluent peak;
dead volume
Peak wide
Peak width
at half-
height
Baseline
noise
Peak
fronting Tailing
poor
resolution
Baseline
drift

17. Parameters used in HPLC
• Retention parameters
• Column efficiency parameters
• Retention : When a component in a sample interacts
with the stationary phase in the column and a delay
in elution occurs
• Column efficiency : Goodness of a column
17

18. Column dead time, retention time
18
time
signal
t
t t
t
0
R 1 R 2
R 3
0
tR 1' tR 2' tR 3'
t´R = tR - t0
t0 = column dead time = time an unretarded compound needs to pass the
column
tR = retention time

19. Capacity and Selectivity
19

20. Capacity Factor
• Capacity factor
20
k´ =
tR - t0
t
0
t´R
t
0
=
•If the substance is not retained by the stationary phase,
the capacity factor is k' = 0.
•Small k' (k < 1) values show that the components are
only retained slightly by the separation column. Their
peaks are located close to the non-retained peak (k' = 0).
•the optimum separation range to be k' values between 1
and 15. Values for k' > 5 mean long retention times with
associated band broadening.

21. Resolution
21
If the peaks are separated almost down to the baseline, R » 1.5.
Higher resolutions than R = 1.5 are not desirable because they
significantly extend the analysis time but do not result in additional
information.
Generally the values of R » 1.0 are sufficient to achieve qualitative or
quantitative results.Even values of R » 0.5 are sufficient to determine the
number of components present. For quantitative analysis, however, the
peak areas overlap too much

22. Condition for Good separation
22

23. Plate NumberPlate Number
23
The theoretical plate number or N is a quantitative measure
for the column efficiency
In formulas (1) or (2) the peak base width WB or the half width
WH are compared with the retention time tR

24. Plate Number and Plate Height
 Since an efficient separation column delivers sharp peaks with narrow base
widths, a better column has relatively high value of N
 The concept of the theoretical plates is a useful tool to describe the efficiency of a
separation column
 The number of theoretical plates is proportional to the column length L. The
longer a column, the more theoretical plates it has; however, the column back
pressure increases
 To be able to compare separation columns of various lengths, the theoretical plate
height, H is used
24
H = L/N

25. 25

26. Five modes in HPLC
LC mode Packing materials Mobile phase Interaction
Normal phase chromatography Silica gel n-Hexane/IPE Adsorption
Reversed phase chromatography Silica-C18(ODS) MeOH/Water Hydrophobic
Size exclusion chromatography Porous polymer THF Gel permeation
Ion exchange chromatography Ion exchange gel Buffer sol. Ion exchange
Affinity chromatography Packings with ligand Buffer sol. Affinity
26

27. 27
HPLC Basic Instrumentation
Mobile
phase
Pump
Solvent Delivery
Injector
Sample Injection
Column
Separation
Detector
Data Processor
Waste

28. 28

29. Composition of HPLC System
• Solvent
• Solvent Delivery System (Pump)
• Injector
• Sample
• Column
• Detectors
• Waste Collector
• Recorder (Data Collection)
29

30. Mobile Phase Degassing
• Dissolved gases in the mobile phase can come out of
solution and form bubbles as the pressure changes
from the column entrance to the exit
– May block flow through the system
• Sparging is used to remove any dissolved gas from
the mobile phase
– An inert and virtually insoluble gas, such as helium, is
forced into the mobile phase solution and drives out any
dissolved gas.
• Degassing may also be achieved by filtering the
mobile phase under a vacuum
30

31. Are used to store Mobile-Phase. The solvent reservoir must be
made of inert material such as glass and must be smooth so as to
avoid growth of microorganisms on its walls. It can be transparent
or can be amber colored. A graduated bottle gives a rough
estimate of mobile-phase volume in the bottle. Solvent reservoirs
are placed above HPLC system (at higher level) in a tray. They
should never be kept directly above the system as any spillage of
solvent on the system may damage electronic parts of HPLC.
31
Solvent Reservoir

32. Eluotropic series
32

33. Mobile Phase Mixing
• Solvent proportioning valve(3.) can be
programmed to mix specific
amounts of solvent
from the various
reservoirs to
produce the
desired mobile
phase composition
33
3.

34. • Isocratic elution:
Use of a constant-composition mobile phase in liquid chromatography
• Gradient elution:
– Vary the mobile phase composition with time
– If there is a wide polarity range of components to be eluted.
– Allows for faster runs.
– Ex: mobile phase composition can be programmed to vary from
75% water: 25% acetonitrile at time zero to 25% water: 75%
acetonitrile at the end of the run.
• More polar components will tend to elute first.
• More non-polar components will elute later in the gradient
34

35. Common Reverse Phase Solvents
• Methanol
35
CH3OH
• Acetonitrile CH3CN
• Tetrahydrofuran
• Water H2O

36. Pumping system
36

37. In order to reduce separation time and allow the use of smaller particle size
packings (10 microns and below), we must force the liquid mobile phase
through the column under pressure. This is the function of the pump (also
called the "solvent delivery system") to maintain a constant flow of mobile
phase through the HPLC regardless of the pressure (back pressure) caused by
the flow resistance of the packed column.
There are several types of pumps are available,
Reciprocating Piston Pumps
Syringe Type Pumps
 Constant Pressure Pumps
.
37

38. 38
Reciprocating Piston Pumps
Consist of a small motor driven piston which moves rapidly
back and forth in a hydraulic chamber that may vary from 35-
400 µL in volume.
On the back stroke, the separation column valve is closed, and
the piston pulls in solvent from the mobile phase reservoir. On
the forward stroke, the pump pushes solvent out to the column
from the reservoir.
A wide range of flow rates can be attained by altering the
piston stroke volume during each cycle, or by altering the stroke
frequency. Dual and triple head pumps consist of identical
piston-chamber units which operate at 180 or 120 degrees out
of phase

39. 39
Reciprocating Piston Pumps

40. Syringe Type Pumps
The syringe pump is a large, electrically operated simulation of a
hypodermic syringe.
Are most suitable for small bore columns because this pump delivers
only a finite volume of mobile phase before it has to be refilled.
These pumps have a volume between 250 to 500 mL. The pump
operates by a motorized lead screw that delivers mobile phase to the
column at a constant rate. The rate of solvent delivery is controlled
by changing the voltage on the motor.
40

41. Constant Pressure Pumps
The mobile phase is driven through the column
with the use of pressure from a gas cylinder
 A low-pressure gas source is needed to
generate high liquid pressures.
The valving arrangement allows the rapid refill
of the solvent chamber whose capacity is about
70 mL. This provides continuous mobile phase
flow rates.
41

42. Tubing
• Very small inner
diameter
• Consistent i.d.
• Very strong
• Easy to cut
• Fittings available
42

43. Auto samplers
Are fully automatic injection systems enabling greater productivity
and the highest level of precision.
The HPLC Autosampler incorporates an elegant swivel head
that allows a manual injection and a special Rheodyne injection
valve to give accurate full or partial loop fill injections.
43

44. Injection Port
The sample introduction device such as injector to introduce the sample in a
flow of mobile phase at high pressure.
 It is not possible to use direct syringe injection on column like GC as the
inlet pressure in LC is too high.
The valve injection through fixed or variable loop is a common way of
introducing the sample.
The Rheodyne valve is the mostly used devise. The loop can be partially or
fully filled. There are both the types of injectors available.
The advantage of partial filling is the possibility of using small amount of
sample, when there is scarcity of sample.
The precision of the injection is 1% RSD
44

45. Samples are injected into the HPLC via an injection port. The injection port of an HPLC commonly
consists of an injection valve and the sample loop. The sample is typically dissolved in the mobile
phase before injection into the sample loop. The sample is then drawn into a syringe and injected into
the loop via the injection valve. A rotation of the valve rotor closes the valve and opens the loop in
order to inject the sample into the stream of the mobile phase. Loop volumes can range between 10
µl to over 500 µl. In modern HPLC systems, the sample injection is typically automated
45

46. Column
HPLC Column Hardware
Stationary Phase
46

47. Columns
• Solid Support - Backbone for bonded phases.
– Usually 10µ, 5µ or 3µ silica or polymeric particles.
• Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support.
– Extremely stable
– Reproducible
• Guard column - Protects the analytical column:
– Prolongs the life of the analytical column
47
• Analytical column - Performs the separation.

48. HPLC Columns
Particle size Column ID Sample Load
Analytical 3 – 5 µ 0.3 − 4.6 mm ng – µg
Semi-prep 10 µ 8 – 10 mm 1 – 100 mg
Preparative 10 – 30 µ 5 – 200 mm gram
scale
48
•An HPLC column consists of a stainless steel tube which is sealed with fittings
on both ends. Steel frits in the end fittings keep the packing material in the
column.
•Analytical columns have inner diameters of 1 - 10 mm and lengths of 25 - 250
mm. They are operated at flow rates of 60 µl - 5.0 ml/min.
•To protect the actual separation column from chemical contamination, a guard
column with the same packing material as the separation column is installed.

49. Column Packing
– Usually spherical silica particles of uniform
diameter (2-10µm)
• The smaller particles yield higher separation
efficiencies.
– The silica particles are very porous
• Allows for greater surface area for interactions
between the stationary phase and the analytes.
– Other packing materials may also be used:
• Zirconia (ZrO2) 49
http://hplc.chem.shu.edu/NEW/HP
LC_Book/Adsorbents/ads_part.html

50. Particle Diameter
• Has a greater effect on resolution than
column length
• Short columns with small particles ideal
• 5µm is standard size
• 3µm better, but restricted range of packings
available
• Downside is high back pressure and issues
with retention of small particles inside the
column, blockages
50

51. Cap nut with
compression
Guard
column
Split collets
Housing with
outer thread
cartridge
51

52. Refers to the solid support contained within the column over which the
mobile phase continuously flows.
 The sample solution is injected into the mobile phase through the injector
port. As the sample solution flows with the mobile phase through the
stationary phase, the components of that solution will migrate according to
the non-covalent interactions of the compounds with the stationary phase.
The chemical interactions of the stationary phase and the sample with the
mobile phase, determines the degree of migration and separation of the
components contained in the sample.
 For example, those samples which have stronger interactions with the
stationary phase than with the mobile phase will elute from the column less
quickly, and thus have a longer retention time, while the reverse is also true.
Columns containing various types of stationary phases are commercially
available.
Stationary Phases
52

53. Monofunctional surface modification of SiO2
OH
Si
O
Si
O
Si
O
SiO
OH
OH
OH
OH
X Si
CH3
CH3
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
C6H13
OH
Si
O
Si
O
Si
O
SiO
O
OH
O
OH
Si
CH3
C18H37
CH3
Si
CH3
C18H37
CH3
Si
CH3
CH3
CH3
OH
Si
O
Si
O
Si
O
SiO
O
O
O
O
Si
CH3
CH3
Si
CH3
CH3
Si
CH3
CH3
CH3
Endcapping
monofunctional
modification
Endcapping
Stationary Phases
53

54. Reversed Phase Chromatography
• Bonded phases made by covalently
bonding a molecule onto a solid
stationary phase like silica
• Typical stationary phases are
nonpolar hydrocarbons, waxy liquids
or bonded hydrocarbons (such as
C18, C8, C4, etc.)
• pH range 2.5 to 7.5
• 60-90% of all analytical LC
separations are done on bonded
phases in the reverse phase mode.
54

55. spherical
irregular
Structure of Packing Material
monolithic
55

56. Synthesis of RP Packings
56

57. Common RP Packings
57
column
Whatman
Zorbax
Partisil
Inertsil
Styragel
Ultrasphere
kromasikl

58. Reversed Phase ChromatographyReversed Phase Chromatography
modification: RP-8, RP-select B, RP-18, -CN, -Diol, -NH2
base material: LiChrosorb®
, LiChrospher®
, Superspher®
, Purospher®
,
Purospher®
STAR, Chromolith®
, Aluspher®
, Polyspher®
Normal Phase ChromatographyNormal Phase Chromatography
modification: Si, -Diol, -CN, -NH2
base material: LiChrosorb®
, LiChrospher®
, Superspher®
, Aluspher®
,Purospher®
STAR ,
Chromolith®
Ion Exchange ChromatographyIon Exchange Chromatography
modification: -NR+
3, -N+
HR2, NH2 for cations
-SO3
-
, -COO-
for anions
Base material: Polyspher®
, LiChrosil®
Size Exclusion ChromatographySize Exclusion Chromatography
Sorbents: LiChrogel®
PS for SEC in organic solvents
Fractogel®
for aqueous SEC
58

59. Detector
59
•Detect various compounds as they elute out from column. The
detector gives response in terms of a milivolt signal that is then
processed by the computer (integrator) to give a
chromatogram.
•Basically detector consists of a flow-cell through which the
mobile phase and resolved sample moves optics shine through
the detector cell and variation in optical properties are
detected.
•A Ultra violet or UV detector detects absorbance of UV light by
chromophores in the analyte compound.
•A refractive index detector will sense variation in refractive
index of mobile phase stream passing through flow-cell
• Similarly Fluorescence Detectors checks for Florescence.

60. Various Detetectors are Listed below
• Ultraviolet (UV)
Fixed wavelength detector
Variable wavelength detector
Diode Array
• Fluorescence
• Electrical Conductivity
• Refractive Index
• Electrochemical
• Light scattering
• IR Absorbance
• Mass-Spectrometric
60

61. Fraction collector
61http://www.lplc.com/old/instruments/img/cf1big.jpg

62. Automated Waste Collection
62

63. Recorder
63
http://www2.okbu.edu/academics/natsci/chemistry/people/mrjordan/webtutorials/hplc/images/recorder.JPG

64. Picture of a Typical HPLC System
64

65. 65

66. HPLC Data
66
http://www.mac-mod.com/tr/plumbing-tr.html

67. 67

68. • 2004: Further advances in column technology
and chromatography instrumentation
– Utilized even smaller packing particle sizes (1.7µm)
– Higher pressures (15000psi)
– Allowed for significant increases in LC speed,
reproducibility, and sensitivity.
• New research utilizing particle sizes as small as
1µm and pressures up to 100,000psi!
68
What is Ultra Performance
Liquid Chromatography?

69. Chromatograms of simvastatin
69

70. Why UPLC more efficient
• Peak capacity (P) is the number of peaks that
can be resolved in a specific amount of time
• P is proportional to the inverse of the square
root of the Number of theoretical plates (N):
N = L/H
• Plate heights are correlated through the Van
Deemter equation
70

71. Contrasting HPLC and UPLC
• UPLC gives faster results with better
resolution
• UPLC uses less of valuable solvents like
acetonitrile which lowers cost
• The reduction of solvent use is more
environmentally friendly
• Increased productivity can increase you
revenue in an industrial setting
71

72. 72
http://caffeineawareness.org/index.php

73. Waters (USA)
Agilent (USA)
Thermo (USA)
Varian (USA)
Dionex (USA)
Jasco (Germany)
Knauer (Germany)
Shimadzu (Japan)
Manufacturers
73
Ningbo Yujie Optical Instruments Co., Ltd.

74. HPLCHPLC
DETECTORSDETECTORS
74

75. DETECTORS
 UV-Vis/PDA
 Refractive index
 Electrochemical
 Conductivity
 Fluorescence
 Radioactivity
 Evaporative light scattering
 Corona CAD
 PDA-MS
 PDA-NMR
 FTIR
75

76. OVERVIEW
Detector Analyte /attributes sensitivity Nature
UV-Vis
Works with all molecules containing
chromophores absorbing UV-Vis
ng
Specific
PDA Works for all wavelengths in UV-Vis ng
Fluorescence
Compound with native fluorescence or
fluorescence tag
fg-pg
Conductivity
Anions, cations, organic acids,
surfactants
ng
Radio activity Radioactive labeled compounds ng-pg
76

77. Detector Analyte /attributes sensitivity Nature
Refractive Index
Temperature sensitive; polymers,
sugars, triglycerides, incompatible
with gradients
0.1-10 µg
Universal
Evaporative light
Scattering(ELSD)
Uniform response; nonvolatile to semi
volatile compounds; compatible to
gradients
ng
Electrochemical Redox reactions pg
Corona CAD
Can detect non UV absorbing
chromophores
ng
Mass Spectrometer Definite analyte identification fg-pg
Both universal and
specific
IR Works with all molecules mg
77

78. Uv- visible detector
Senstive
Wide linear range
Unaffected by changes in temp.
And mobile phase composition
Principle :
• Lambert-Beer’s law:
A= ε b c
At constant cell thickness and constant wavelength
a linear connection between the Absorbance A and
the Concentration C is achieved.78

79. Types of uv-visible detectors
A. Fixed wavelength detectors
B. Variable wavelength detectors
C. Photodiode array detectors
79

80. Fixed wavelength detectors
 Operates only at 1 wavelength (254 nm)
 Best overall precision is noted for peak
area measurements as compared to
variable wavelength detectors.
 Uses a discrete source:
– Low pressure mercury lamp (253.6 nm)
– Silicone photodiode light detector
 It can also be used at other wavelengths by
filtering the emission source to give other lines or
even using phosphor screens to give lines not
available from mercury.
80

81. Variable wavelength detectors
 Uses continuous source of light
• Deuterium or xenon lamp or tugston lamp
• Desired wavelength is isolated by monochromator
 Improved sensitivity from operating at λmax of solute of
interest
 Drawback: limited lamp lifetime as compared to
mercury lamp (Deuterum lamp: 1000-2000 hrs)
 Dual wavelength detection feature:
– Useful for simultaneous monitoring of two
substances
– Baseline noise is higher because this feature is
achieved by toggling the monochromator between 2
wavelengths 81

82. 82

83. Photodiode array detectors
Uses charge coupled DA with 512 to 1024 diodes capable
of spectral resolution of 1 nm
Earlier, they suffered from sensitivity problems, which has
been solved by advanced flow cell design using fiber
optics technology to extend path length without
increasing noise or chromatographic band dispersion
Performs a simultaneous measurement of absorption as a
function of analysis time and over a chosen wavelength
range, so we get UV spectrum for each eluted peak
Used for method development where λmax of impurities in
drug are unknown
Every compound can be quantitated at its λmax , so useful
for trace analysis
Compounds not resolved chromatographically can
sometimes be resolved spectrally
Molecular absorption spectrum can be used for peak
identification and peak tracking
83

84. Measures
light
intensity at
each λ
Measures
light
intensity at
each λ
84

85. Indirect photometric detection
Used when solute of interest does not possess
chromophore
Here, mobile phase possess a chromophore and
absorbs light
When analyte without a significant
chromophore passes through the detector cell,
the absorption of mobile phase is decreased and
is recorded as negative peak
85

86. 86

87. 87

88. Features of modern uv-vis
detectors
Dual or multiple wavelength detection and stop
flow scanning features
Front panel access to self alligned sources and flow
cells for easy maintanance
Self validation features such as:
– Power up diagnostics
– Leak sensors
– Time logs for lamps
– Built in holmium oxide filters for wavelength calibration
– Filter wheels for linearity verification
88

89. Refractive index detectors
Measurement response is a function of the refraction
index difference between pure mobile phase and the
mobile phase with the dissolved separated
components
Due to the strong temperature dependence of the
refraction index, good temperature control of the
measurement cell must be ensured
The detection limit is in the range of 10-6
- 10-8
g/ml
To achieve a high sensitivity, in practice solvents are selected that have
a very high or very low refraction index
This ensures that the difference between the refractive indices of
89

90. 90

91.  One half of the measurement cell is purged with the flowing mobile phase, the second
half of the measurement cell (reference cell) is filled with mobile phase. The refraction
index n is at first identical in both cells.
 If a sample component is added to the eluent, the refraction index n in the
measurement cells changes. The light beam experiences a deflection on the path
Operating principle
91

92. 92

93. Fluorescence detectors
During fluorescence detection,
the sample is irradiated with
UV light of suitable wavelength
and excited for emission of long
wave light.
The Xenon lamp continuously sends light of 325 -
410 nm.
Provides LOD values 100 times lower than
absorption detectors
Highly selective
93

94. 94

95. 95

96. 96

97. 97

98. Electrochemical detectors
Electron transfer processes offer highly selective and
sensitive method
Easily adaptable for use with microcolumns
As background noise is dependent on mobile phase
conditions, it is difficult to utilize these detectors
with gradient elution separations
2 types:
1. Amperometric detection: fixed potential is applied to
the electrode (glassy carbon) and a solute which will
oxidize at that potential yields an output current
2. Coulometric detection: 100% of the solute species is
converted, which offers advantages of no mobile phase
flow dependence on signal and absolute quantitation
through Faraday’s law
98

99. `
99

100. Conductivity detectors
 Measurement of electrical conductance is a subset of
ECD, although it is generally considered separately
since it is non-Faradaic electrochemistry; i.e. no
electron transfer reaction takes place
 The electrical conductivity of the mobile phase is used
as characteristic for detection.
 Universal technique for ionic solutes and are used
mainly for the separation of ions in water or polar
eluents
 Since the conductivity can change approx. 2 % per °C,
conductivity detector are often equipped with
automatic temperature compensation
100

101. With the removal of electrolytes contained in the eluent system
when using ion exchangers, a significant increase in the sensitivity
can be achieved (otherwise it creates difficulty in measuring low
concentration of an ionic solute in the presence of highly
conducting mobile phase
The resulting extremely low basic conductivity makes it possible, to
generate good measurement signals from the smallest sample
volumes.
During conductivity detection the specific conductivity is measured
continuously.
The individual eluted components of the material sample are each
displayed through a change in conductivity.
101
Conductivity detectors

102. Principle of 4 electrode method
The two outer electrodes are used to transmit an
electronically stable alternating current through the cell
The electro-motor force (E), which is measured
deenergized on the two inner electrodes, is a measure for
the conductivity of the cell content according to the
equation.
χ = (K/E)*I
χ = electric conductivity
K = cell constant between the 2nd and 3rd electrode
E = electro-motor force
I = current strength
The deenergized measurement of the electro-motor force
at the two inner electrodes caused no passivity, hence the
instrument operates stably for a long period of time.
102

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105.  ELSD can outperform traditional detectors when analysing non-
chromophoric samples by HPLC
 Traditional HPLC detectors such as UV and RI have limited capabilities
 UV and RI are not compatible with a wide range of solvents
 RI detection is not gradient compatible
 Different analytes produce different UV responses depending on their
extinction co-efficient
 ELSDs can detect anything that is less volatile than the mobile phase
 ELSD is universal and compatible with a wide range of solvents
Evaporative Light Scattering Detector
105

106. Unique Method of Detection
Three steps:
• Nebulization
• Mobile Phase Evaporation
• Detection
106

107. Step 1: Nebulization
 Column effluent passes through nebulizer needle
 Mixes with nitrogen gas
 Forms dispersion of droplet
107

108. Step 2: Mobile Phase Evaporation
 Droplets pass through a heated zone
 Mobile phase evaporates from the sample particle
 Dried sample particles remain
108

109. Step 3: Detection
 Sample particles pass through an optical cell
 Sample particles interrupt laser beam and
scatter light
 Photodiode detects the scattered light
109

110. ELSD – A Powerful Detector for HPLC
Universal
Sensitive
Gradient Compatible
AN ELSD IS AN EFFECTIVE REPLACEMENT OR A PERFECT COMPLEMENT TO EXISTING
LC DETECTORS
 RI
 UV
 MS
 Fluorescence
110

111. Four Reasons to Replace an RI with an
ELSD:
1. Better sensitivity
2. Gradient compatible
3. Stable baselines
4. No solvent front peaks
111

112. The ELSD Improves Baseline Stability and Detection Sensitivity Compared
to RI
112

113.  Reasons to operate an ELS detector in series with a
UV detector:
1. Obtain a more accurate representation of
sample mass than UV
2. See what may be missing from your UV
chromatogram
113

114. The ELSD responds to all components in this antihistamine
formulation
1. Diphenhydramine
2. PEG, Gelatin
3. Sorbitan
4. Glycerol
5. Sorbitol
114

115. Detect Difficult Samples like Triglycerides without Derivatizing
1. LLO
2. LLP
3. OOL
4. POL
5. PPL
6. OOO
7. OOP
8. PPO
9. OOS
115

116. Although the Mass Spectrometer is a Universal
Detector, ELSDs offer many Benefits over MS:
1. Lower investment and operating costs
2. Less complicated operation
3. Less maintenance
Since chromatographic requirements for ELSD
and MS are similar, methods developed for
ELSD are usually transferable to MS without
Use ELSD in Parallel with MS to Obtain Maximum Structural and Concentration
Information
Use ELSD in Parallel with MS to Obtain Maximum Structural and Concentration
Information
116

117. Replace Fluorescence Detectors with ELSD to Simply Methods by
Eliminating Pre- or Post-column Derivatization
1. Glycine (Gly)
2. Serine / Asparagine (Ser/Asn)
3. Aspartic Acid (Asp)
4. Glutamine (Gln)
5. Alanine / Threonine (Ala/Thr)
6. Glutamic Acid (Glu)
7. Cysteine / Lysine (Cys/Lys)
8. Histidine (His)
9. Proline (Pro)
10. Arginine (Arg)
11. Valine (Val)
12. Methionine (Met)
13. Tyrosine (Tyr)
14. Isoleucine (Ile)
15. Leucine (Leu)
16. Phenylalanine (Phe)
17. Tryptophan (Trp)
117

118. Summary:
Benefits of ELSD as a Replacement or
Complement to Existing Detectors
• See what you might be missing
• More sensitive and stable than RI
• Get a more accurate representation of sample
mass than UV
• Simplify methods by eliminating derivatization
118

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APPLICATIONS OF HPLC :
 Assay of cephalosporins
 Assay of theophylline
 Separation of barbiturates, phenothiazines, benzodiazepine
derivatives, rauwolfia alkaloids etc. by C-18 reversed phases
Quantitative analysis of several analgesics like aspirin, caffeine,
paracetamol, phenacetin, etc.
Analysis of urine and serum samples
Separation of antipyrine and benzocaine in ear drops

128. 128
Separation and purification of nucleic acids. (eg: purines
and pyrimidine compounds by reversed phase technique.)
Determination of preservatives and antioxidants
(eg: sorbic acid)
Analysis of vitamins
Separation of coal and oil products
Analysis of pesticides
Analysis of carcinogens. (eg: aflatoxins, metabolites of
aspergillus flavus.)

129. 129
Separation of steroids like cortisone and cortisol which are
used in the treatment of Rheumatoid arthritis.
Assay of hydro cartisone, fluocinolone acetonide,
triamcinolone acetonide in tablets and other formulations.
Analysis of oestrogen in female urine.
Analysis of lipids.
Separation of tropane akaloids, ergot alkaloids, indole
alkaloids, cinchona alkaloids, etc.
Analysis and separation of amino acids and proteins.
Analysis of carbohydrates.

130. 1. REFERENCES:
2. Instrumental methods of chemical analysis-B.K
Sharma.
3. Pharmaceutical analysis by Ashutosh Kar
4. Fundamentalsof Analyttical
chemistrySkoog,West,Holler
5. Instrumental analysis of chemical analysis-
Gurudeep R Chatal
6. www.ctsholc.com/tools
7. 6.www.wikipedia.org/hplc
8. www.chemguide co.uk/analysis
9. www.hplcindia.com 130

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