HPLC

1. MAHENDRA G S
High Performance Liquid
Chromatography

2. H
P
igh
erformance
Liquid
Chromatography

3. H
P
igh
ressure
Liquid
Chromatography

4. H
P
igh
riced
Liquid
Chromatography

5. Why HPLC?

6. World wide market volumeWorld wide market volume
of liquid phase separation analytical instrumentsof liquid phase separation analytical instruments
Total market volume : US$ 3,454 B

7. The segments HPLC is used in the world marketThe segments HPLC is used in the world market

8. The fields HPLC is used in the world marketThe fields HPLC is used in the world market

10. High Performance Liquid Chromatography
• Mobile phase
– Liquid
• Stationary phase
– Small diameter particles
packed into column.
• Pressure is required to
push liquid through
column.
• Advantages
– Better resolving power
– Faster

11. High Performance Liquid Chromatography
http://academic.sun.ac.za/saf/units/aaa/images/image4_l.jpg

12. History lesson
• 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. HPLC
SAMPLE
STATIONARY PHASE MOBILE PHASE

14. What is HPLC?
• HPLC is a process of separation of mixture
containing 2 or more components under high
pressure by passing the sample through column
containing a stationary phase by means of
pressurized flow of liquid mobile phase.

15. Advantages to HPLC
• Higher resolution and speed of analysis
• HPLC columns can be reused without repacking or
regeneration
• 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

16. Types of HPLC
• Liquid-solid (adsorption) chromatography
• Liquid-liquid (partition) chromatography
• Ion-exchange chromatography
• Size exclusion chromatography

17. Liquid-solid (adsorption) chromatography
• It involves large surface area with
less particle size.
• Separation of components of
mixture is due to affinity of the
components for surface of
stationary phase.
• High affinity compounds comes at
the end.
• Intermediate components come
in middle and low affinity
components comes first.

18. Liquid-liquid (partition) chromatography
• Separation is based on the analyte’s relative
solubility between two liquid phases.
• Normal phase & Reverse phase chromatography.

19. Ion-exchange chromatography
• Reversible exchange of counter
ion takes place between
stationary phase ion & mobile
phase ion.
• The separation was achieved
due to difference in strength of
electrostatic interaction of the
solute with the stationary phase.
• Cation exchange resins
– sulfonic acid group, carboxylic acid
group
• Anion exchange resins
– quaternary amine group, primary
amine group

20. Size Exclusion Chromatography(SEC)
• Gel permeation(GPC), gel filtration(GFC)
chromatography
• Based on separation according to the size of the
molecules, the materials used as stationary
phase contains pores of certain sizes.
• Molecules which are too large, are excluded from
the pores.
• Smaller molecules enter into the pores and
eluted later, while the larger molecule remains in
the flowing mobile phase and are eluted first.

21. • Specific pore sizes, average residence time in the
pores depends on the effective size of the analyte
molecules
– larger molecules
– smaller molecules
– intermediate size molecules
SEC(continued)

22. LC Separation Mechanisms
Source: Rubinson and Rubinson, Contemporary Instrumental Analysis, Prentice Hall Publishing.

23. Instrumentation
• System controller
• Solvent reservoir
• Tubing
• Pump
• Injection device
• Column
• Detector
• Recorder

24. System controller
• Mainly designed for the control of sample introduction
function to those which intelligently communicate with
all HPLC system modules and completely, control
their operation.
• They are PC based devices with sophisticated
software for data processing in addition to provide
control function and also communicate directly with
other system, modules permitting setting up of
analysis parameter from single control panel for
storing for analytical method for future use.

25. Solvent Reservoir
• Made up of stainless steel.
• Ideal characters like
– Composition of these reservoirs should be inhert.
– Should not be reactive with solvents.
• Steel should be avoided for the use of solvent containing
Halide ion.
• If the reservoir is to be pressurized, glass should be
avoided.
• Capacity of the reservoir should be greater than 500ml.
• In many cases the aqueous solvent and some organic
solvents are degassed prior to use. These are done to
prevent formation of gas bubbles in detector.

26. Solvent Reservoir
• Types of degassing solvents:
– Sparging with less soluble gas
– Heating
– Reducing pressure by applying vaccum
– Sonication
• Advantages of degassing are:
– Stability in the baseline and enhanced sensitivity in some
types of chromatographic detectors
– Reproducible retention time for eluting peaks
– Reproducible injection volume for quantitation
– Stable pump operation

27. Tubing
• It is used to connect all parts of systems.
• Inside diameter of the tubing prior to injection device is not
critical but tubing should have ability to withstand pressure
and able to carry sufficient volume of solvents.
• Usually tubing of very small diameter (less than 0.5 mm
inner dia) are used.

28. Pumps
• The function of pumps in HPLC is to pass mobile phase
through the column at high pressure and at controlled flow
rate.
• Pumps must be constructed from material that are inhert to
mobile phase.
• Materials commonly used are glass, stainless steel, Teflon
and sapphire.
• HPLC is high pressure chromatography the pump must be
capable of generating pressure of upto 5000 psi at a flow
rate upto 3ml/min for analytical conditions.

29. Pumps
• Pumps should posses some of following characters:
a) Interior of pump should not be corroded by any of
solvent used.
b) A range of flow rates should be available and it should
be easy to change the flow rate.
c) The solvent flow should be non-pulsing.
d) It should be easy to change from one mobile phase to
another.
e) Pump should be easy to dismantle and repair.
Classification of pumps:
1. Constant pressure pumps
2. Constant flow pumps

30. Constant pressure pumps
Pneumatic amplifier pump:
 Air from the cylinder at pressure upto 150 psi is applied to
gas piston that has relatively large surface area.
 The gas piston is attached to hydraulic piston that has small
surface area.
 One drive stroke, the outlet valve on the pump head is open
to the column and inlet valve closed to the mobile phase
reservoir.
 At the end of stroke, the air in the chamber is vented and air
enters on other side of piston to start the return stroke.
 On return stroke the outlet valve closes, inlet valve opens
and pump head refills with mobile phase.

31. Pneumatic amplifier pump

32. Constant pressure pumps
 The pump can be started and stopped by open of a valve
fitted between the cylinder regulator and pump.
Advantages:
 Pump is very economic
 Used when column need very high pressure
Disadvantages:
 Difficult to disconnect and maintain (repair)
 Does not provide constant flow of mobile phase

33. Constant flow rate pumps
 There are 2 principle types of pumps in this category:
1. Syringe drive pump
2. Reciprocating piston pump

34. Syringe drive pump
 In this pump mobile phase is
displaced from a chamber by
using variable speed stepper
motor to turn a screw which
drive a piston.
 The chamber has a volume of
200 – 500 cm3
.
 The pump produces pulse less
flow and requires no check
valves.
 The flow must be interrupted
periodically to refill the
chamber.

35. Syringe drive pump

36. Syringe drive pump
Advantages:
 Output is pulse free
Disadvantages:
 Limited solvent capacity (~20 mL) and inconvenience when
solvents need to be changed

37. Reciprocating piston pump
 This pump is most widely used in modern HPLC.
 There are 2 principle operations that is filling and pumping
cycles.
 During filling cycles, piston is withdrawn from a syringe type
chamber and check valves are connected to this chamber.
 They are fixed such that during the piston withdrawal,
solvent flow from solvent reservoir, but not into the pump
outlet.
 When piston direction is reversed, check valve operates to
allow solvent to flow from the outlet valve only.
 By altering the distance that piston travels we can adjust the
volume of solvent discharged from pump per unit time.

38. Reciprocating piston pump

39. Reciprocating piston pump

40. Typical HPLC Pump (runs to 4,000+ psi)

41. Reciprocating piston pump
Advantages:
 Small internal volume (35-400 µL)
 High output pressures (up to 10,000 psi)
 Ready adaptability to gradient elution
 Constant flow rates
Disadvantages:
 Pulsed flow creates noise

42. Sample injection systems
 HPLC involves very high pressure. introduction/injection of
sample to system presents some problems.
 Injection system should have certain qualities, which are
important:
 Sample should be injected in a narrow plug. In order to
achieve maximum efficient separation.
 Size of the samples should be variable in order to
accommodate wide variety of samples.
 The injection technique should be reproducible for
maximum accuracy in quantitative techniques
 most importantly, system must be able to inject against
high pressure without sample loss.

43. Sample injection systems
 Direct sample introduction via syringe is the simplest form of
injection.
 Syringe must be specifically constructed to withstand
pressures upto 1000-1500 psi without leakage.
 Another problem is caused by coring of the septum and
subsequent build up of septum particles on the front end of
the column.
 This may cause excessive pressure in the column.
 Since septum comes in direct contact with the mobile
phase, it must be constructed with inert material.
 Commonly used injectors are:
 Septum injectors
 Loop injectors

44. Sample injection systems
 The most widely used sample injection system is loop
injection valve.
 The valve provides easy injection of sample against high
back pressure.
 The sample loop can be any size from 10ml upto 1-2ml for
preparative scale HPLC.

45. LOAD (the sample loop)
Inject (move the sample
loop into the mobile
phase flow)

47. LC column
LC injector

48. Stop flow injection device
• Syringe injection techniques can be used at much higher
pressure if stop flow techniques are used.
• The sample can be injected/introduced in to one chamber of
piston, while mobile phase is flowing.
• The piston is moved rapidly to align the sample chamber
with flowing mobile phase in order to introduce the sample
to column.
• This may lead to disturbance in solvent flow, which may
sometime cause baseline disturbance.

49. Column & Packing

50. Column & Packing

51. Structure of Silica Gel
 Amorphous, porous matrix of silicon atoms joined
together with oxygen atoms to form “siloxane bonds” =
(Si – O – Si)
Silicon
Oxygen

52. What is a Silanol Group?
Si – O – H
O
Si
O
Si
O
Si
O
O
H
O
H
O
H
O O O
Comes from the silica gel particle (substrate)
used to make reversed-phase packing materials

53. What are Silanols?
 Residual unreacted surface hydroxyl groups left over from
polymerization
 Reactive sites for use in bonding ligands (C18) to the silica gel
surface
Silanol
(Si-O-
H)
= H
= O
= Si

54. Column & Packing
• Column forms the heart of any HPLC system.
• The quality of its packing materials are essential for good
separation and minimum dispersion.
• The stainless steel column of internal diameter 4.0 to 4.6
mm and length 10-30cm are used.
Types of columns:
1. Analytical columns
2. Preparative columns
3. Microbore columns

55. Guard column
 Since HPLC columns are expensive devices, these guard
columns are used to increase the lifetime of HPLC column.
 The guard column is placed between the injector &
analytical column.
 This can be packed with material similar to main column or
with pellicular material (coated porous glass beads) of the
same type.
 Guard column acts like a filter and absorb impurities which
are either combined in sample or in the elute or formed by
contact of sample with packing. Thereby protect the HPLC
column.

56. Analytical columns
• These are employed to obtain qualitative or quantitative
information about the sample.
• This involves conventional liquid chromatography where
very less amount of solute is used that is upto 10mg.
• They are packed with the stationary phase with the particle
size of 3 – 20 µ.
• Small volume of injection that is <10ml usually 50 – 150 µl.
• Internal diameter <8mm.
• Pore size will be <300 A0
.
• The column load should be between 10-10
to 10-3
gm of
sample/gm of packing.
• These have good column efficiency & more separation
power.

57. Preparative columns
• This is improved/modified analytical column.
• It is used to isolate or purify the sample quantities in the
range of 10 – 1000mg from complex mixture.
• There are of 3 types based on sample size employed:
– Micropreparative/Semipreparative: To purify the sample
of about <100mg. The column tend to be slightly larger
version of analytical column use the same packing.
– Preparative: Sample size is 0.1 to 100 gms the column
are 2-5cm in diameter and 25cm length, packing of 15-
100 µm.
– Macro preparative: Sample size is >0.1kg, the columns
are 20 – 30cm and inner diameter of 60cm with long flow
rates upto 1000ml/minute.

58. Preparative columns (cont.)
• All the preparative columns are having internal diameter of
>8mm.
• The packing is economical as it involves larger particles (20-
100µ) column load is about 0.001 to 0.1 gm of sample/gm of
packing load capacity.
• Packing load capacity is defined as maximum amount of
sample that can be applied without impairing the separation
efficiency.

59. Micro bore columns/small bore
columns
• The size is too small & used for high performance.
• Diameter 0.5 – 2 mm.
• More advantage because of
– its high speed due to narrow/small diameter.
– Less solvent consumption, better performance and
economic.
– Small peak width.
– Sensitivity is more, which allows to detect smaller
quantities of material.
 The problem is the system should have low dispersion.
 Volume of solute injected causes some dispersion,
therefore volume should be <1µl.

60. Column packing methods
Dry packing method:
 Used for packing with particle size of >20mm.
 The dry packing is filled into vertically clamped column in
small amounts and helped to deposit by vibration or tapping.
Wet or slurry packing method:
 Used for packing with particle size of <20mm.
 Small particles with dia 3,5,7 and 10mm can only be placed.
 They are first made into suspension.
 This should not sediment.
 Stable suspension can be prepared by:
 By using ultrasonic treatment
 Balanced density

61. Packings for HPLC
• Particles of 3–10µm diameter are preferred for packing.
• Generally used ones are:
– Silica Gel: Spherical totally porous ‘NUCLEOSIL’.
– Silica Gel: Irregularly shaped, totally porous
‘POLYGLSIL’
– VYDAC packing: Eg. vydac guard columns – glass beads
with porous silica gel surface.
– Aluminium oxide: Irregularly shaped, totally porous
‘ALOX’.
– Cellulose acetylated, irregularly shaped.
– Polyamide, irregularly shaped particles.

62. Bonded Phases
• C-2 Ethyl Silyl -Si-CH2-CH3
•CN Cyanopropyl Silyl -Si-(CH2)3-CN
• C-18 Octadecyl Silyl -Si-(CH2)17-CH3
•C-8 Octyl Silyl -Si-(CH2)7-CH3

63. Neutral Compounds: C18 versus C4
(Same Brand - Different Ligands)
Minutes
0 4 8 12 16 20 24
YMC-Pack™ Pro C18™
YMC-Pack™ Pro C4™
Acenaphthene
Naphthalene
Butylparaben Note: Similar selectivity due
to same silica gel particle.

64. MOBILE PHASE IN HPLC
Polarity is a term that is used in chromatography as an index of
ability of compounds to interact with one another, in these
various ways.
 The eluents are arranged according to polarity ,the strong ones
being polar and weak ones nonpolar.
 The series of weak, medium and strong solvents thus derived
is referred to as the Eluotropic series.

65. Normal Phase Reverse Phase
Solvent Solvent
strength δ
Solvent
strength (p’)
Solvent Sol strength
(S-18)
n-Hexane 0.00 0.0 Water 0.0
Chloroform 0.40 4.1 Methanol 2.6
DCM 0.42 3.4 Acetonitrile 3.2
THF 0.45 4.0 Acetone 3.4
Acetone 0.56 5.1 Ethanol 3.6
DMSO 0.62 7.2 2-proponal 4.2
Acetonitrile 0.65 5.8 THF 4.5
Ethanol 0.88 4.3
Methanol 0.95 5.1
water 1.00 10.0
Eluotropic series for some common solvents

66. Just as in your understanding of solubility -
where “like dissolves like”:
•Polar compounds are attracted to other Polar
compounds
•Non-polar compounds are attracted to other Non-polar
compounds
•Polar compounds are not attracted to Non-polar
compounds
Polarity - Compound Behavior

67. POLAR NON-POLAR
Mobile Phases:
Water - Acetonitrile - THF - Hexane
O
H
H C H3
H2
C
C
H2
H2
C
C
H2
CH3
Polarity Scale

68. Stationary Phases:
Polarity Scale
POLAR NON-POLAR
Silica - CN - C8 - C18

69. Schematic of liquid chromatograph

70. Isocratic elution
• Isocratic elution has a constant mobile phase composition
– Can often use one pump!
– Mix solvents together ahead of time!
– Simpler, no mixing chamber required
– Limited flexibility, not used much in research
• mostly process chemistry or routine analysis.

71. Gradient Elution
• Gradient elution has a varying mobile phase composition
– Uses multiple pumps whose output is mixed together
• often 2-4 pumps (binary to quarternary systems)
– Changing mobile phase components changes the polarity
index
• can be used to subsequently elute compounds that
were previously (intentionally) “stuck” on the column
• Some additional wear on the stationary phase
• Column has to re-equiluibrate to original conditions
after each run (takes additional time).

73. Detectors
• HPLC detectors can be classified as:
– Solute property detectors -respond to physical or
chemical properties of the solute that is generally not
exhibited by the mobile phase.
– Bulk property detectors -respond to an overall change
in the physical property of mobile phase with and
without solute.
There is no single detector that can be employed for allThere is no single detector that can be employed for all
HPLC separations “the magic box”!HPLC separations “the magic box”!

74. Classification of different Detector
types
Solute PropertySolute Property
FluorescenceFluorescence
ElectrochemicalElectrochemical
Fixed WavelengthFixed Wavelength
Variable WavelengthVariable Wavelength
Photodiode arrayPhotodiode array
UV/VisUV/Vis
Bulk PropertyBulk Property
ConductivityConductivity
Refractive IndexRefractive Index

75. Criteria for HPLC Detectors
• High sensitivity
• Negligible baseline noise
• Large linear dynamic range
• Response independent of variations in operating
parameters (pressures, temperature, flow-rate…etc.)
• Response independent of mobile phase
• Low dead volume
• Non-destructive
• Stable over long periods of operations
• Selective

76. Types of Detector
• Refractive index
– Deflection Refractometer
– Fresnel Refractometer
• UV/Visible
– Fixed wavelength UV detector
– Variable wavelength UV detector
– Diode array detectors
• Fluorescence
• Conductivity
• Evaporative light scattering
• Electrochemical

77. Refractive Index Detectors
• First HPLC detector developed
• Typically referred to as Universal detectors.
– Detects all dissolved solutes- “non-specific”
• RI response depends on the difference in RI between
mobile phase and solute(s).
• Sensitivity reaches maximum when RI differences are
greatest.
∀µg sensitivity but only for isocratic runs
• Commonly used for: Sugars, Polymers and Fatty
Acids

78. Refractive Indices of Some Common
Solvents
Solvents RI
Air 1.0
Methanol 1.329
Water 1.330
Acetonitrile 1.344
Acetic Acid 1.372
Tetrahydrofuran 1.408
Methylene Chloride 1.424
Toluene 1.496

79. Deflection Refractometer
 It measures the deflection of a beam of monochromatic light
by a double prism in which the reference and sample cells
are separated by a diagonal glass divide.
 When both cells contain solvent of same composition, no
deflection of light beam occurs.
 If the composition of the column mobile phase is changed
because of the presence of a solute, then the altered RI
causes the beam to be deflected.
 The magnitude of this deflection is dependent on solute
concentration in the mobile phase.

81. LAMPLAMP
LED orLED or
IncandescentIncandescent
RR
SS
To AmplifierTo Amplifier
No sampleNo sample
With sampleWith sample
RR
SS
Deflection Refractometer

83. Deflection Refractometer

84. Reflection (Fresnel) Refractometer
• This type of detector is based on the measurement of
change in the fractions of reflected and transmitted light at a
glass liquid interface as a result of change in the refractive
index of the liquid.
• In the optical path two collimated beams from the projector
illuminate the reference and sample cells.
• The cells are made with a Teflon gasket, which is clamped
between the cell prism and a stainless steel reflecting back
plate.

85. Reflection (Fresnel) Refractometer
• As the light beam is transmitted through the cell interfaces, it
passes through the flowing liquid and impinges on the
surface of reflecting back plate. This diffuse reflected light
appears as two spots of light that are imaged by lens onto
dual photo detectors.
• As the ratio of reflected light to transmitted light is function of
refractive index of two liquids, the illumination of the cell back
plate is direct measure of the refractive index of the liquid in
each chamber.

86. Fresnel refractometer optical pathway

87. Reflection (Fresnel) Refractometer

88. Refractive Index Detectors
• Advantages
– universal respond to nearly all solutes
– reliable
– unaffected by flow rate
– low sensitive to dirt and air bubbles in the flow cell
• Disadvantages
– expensive
– highly temperature sensitive
– moderate sensitivity
– cannot be used with gradient elution

89.  In this detectors the measurement of the resistance of an ionic
solution, using an alternative voltage to eliminate spurious effects
(because of electrode polarization) can be used as a detection
principle for ionic species.
 One of the most important advantages of such a detector is the
very small dead volume.
 When used with buffer solutions detection limit is very small,
being in the low ppm range.
with non-ionic mobile phases of low electrical conductivity the
detection limit can be decreased by several orders of magnitude.
Conductivity detectors

90. The primary usefulness of the conductometric detector is for
small anions or cations such as inorganic ions (e.g. , Clo4
-
,No3
-
,
Po4
3-
, Na+
, NH4
+
,etc) or small organic anions ( e.g., acetate ,
oxalate,citrate,etc) or cations (e.g., di- , tri- and tetra-alkyl
ammonium ions).
 The combination of the electrical conductivity detector with
ion exchange chromatographic columns has led to the technique ,
known as ion chromatography.
 In spite of the excellent selectivity of ion exchange resins, the
analysis of non-UV absorbing ions has been hampered essentially
due to the lack of suitable universal detector.

91.  Inorder to suppress the overwhelming conductivity of the
anion exchange suppressor column for cation separation and a
cation exchange suppressor column, for cell.
 In this way, the eluent is neutralised and the conductivity of
the elute ions can be conveniently measured.
 The suppressor column increases the analysis time and the
band broadening and may give some undesired effects such as
ion exclusion, reaction with some ions etc.

92. Conductivity detector optical pathway

93. Conductivity detectors

94. UV Detectors
• UV absorption detectors respond to those substances that
absorb light in the range 180 to 350 nm.
• Most frequently used detector in HPLC analysis.
• Compounds must contain a UV absorbing chromophore.
• Must work in the linear range of Beer’s Law.

95. Fixed wavelength UV detector
• The fixed wavelength UV detector uses light of a single
wavelength (or nearly so) which is produced by a specific
type of discharge lamp.
• The most popular lamp is the low pressure mercury vapor
lamp, which generates most of its light at a wavelength of
254 nm.
• Other lamps that could be used are the low-pressure
cadmium lamp which generates the majority of its light at
225 nm and the low pressure zinc lamp that emits largely at
214 nm.

96. Fixed wavelength UV detector (cont..)
• Light from the UV source is collimated by a suitable lens and
passed through both the sample cell and the reference cell
and then on to two photo cell.
• The cells are cylindrical with quartz windows at either end.
The reference cell compensates for any absorption that
mobile phase might have at the sensing wavelength.
• The outputs from the two photo cells are passed to a signal
modifying amplifier so that output is linearly related to the
concentration of solute being detected.

97. Fixed wavelength UV detector

98. Variable wavelength UV detector
• Light from a deuterium lamp is focused a two curved mirrors
onto a diffraction grating and the selected wavelength is
reflected back onto the second curved mirror.
• The light then passes to a third curved mirror and is focused
by a plane mirror and a quartz lens through the absorption
cell.
• After leaving the absorption cell, the transmitter light is
focused by a second quartz lens onto the photocell.
• Rotating the grating allows the wavelength of the light
passing through the cell to be selected to provide the
maximum sensitivity.

99. Variable wavelength UV detector (cont..)
• Light of the selected wavelength is then focused by means of
a lens through the flow cell and, consequently, through the
column eluent.
• The exit beam from the cell is then focused by another lens
onto a photo cell which gives a response that is some
function of the intensity of the transmitted light.
• The detector is usually fitted with a scanning facility that
allows the spectrum of the solute contained in the cell to be
obtained.

100. Variable wavelength UV detector

101. Variable wavelength UV detector

102. Diode array detector
• Light from the broad emission source is collimated by an
achromatic lens system so that the total light passes through
the detector cell onto a holographic grating.
• In this way the sample is subjected to light of all wavelengths
generated by the lamp.
• The dispersed light from the grating is allowed to fall onto a
diode array. The array may contain many hundreds of
diodes and the output from each diode is regularly sampled
by a computer and stored on a hard disc.

103. Diode array detector (cont..)
• At the end of the run, the output from any diode can be
selected and a chromatogram produced using the UV
wavelength that was falling on that particular diode.
• The more common tool for research-grade HPLC
instruments.
• Quite versatile.
• DAD scans a range of wavelengths every second or few
seconds. At each point in the chromatogram one gets a
complete UV-VIS spectrum.

104. Diode array detector

105. U.V. Cut-offs for some Common
Solvents
SolventSolvent UV CutoffUV Cutoff SolventSolvent UV CutoffUV Cutoff
WaterWater 180180 N-HeptaneN-Heptane 197197
MethanolMethanol 205205 CyclohexaneCyclohexane 200200
N-PropanolN-Propanol 205205 Carbon tetrachlorideCarbon tetrachloride 265265
AcetonitrileAcetonitrile 190190 ChloroformChloroform 245245
THFTHF 225225 BenzeneBenzene 280280
AcetoneAcetone 330330 TolueneToluene 285285
Methyl acetateMethyl acetate 260260 Methylene chlorideMethylene chloride 232232
Ethyl AcetateEthyl Acetate 260260 TetrachloroethyleneTetrachloroethylene 280280
NitromethaneNitromethane 380380 1,2-Dichloroethane1,2-Dichloroethane 225225
All wavelengths reported in nm.All wavelengths reported in nm.
Remember Solvents chosen can affect detection!!Remember Solvents chosen can affect detection!!

106. UV Detector
• Advantages
– high sensitivity
– small sample volume required
– linearity over wide concentration ranges
– can be used with gradient elution
• Disadvantage
– does not work with compounds that do not absorb
light at this wavelength region

107. Fluorescence Detector
• Increased Selectivity and Sensitivity
• Analytes not only absorbs UV/Vis radiation but also
releases the energy in the form of light of longer
wavelength.
• This property is typically associated with non-ionic
molecules which are strongly conjugated and have rigid
structures.
• Sensitivity is in the pg region (up to 1000x UV)
• Detector is sensitive to the presence of dissolved gasses
and other “quenchers”

108. Fluorescence Detector
• Incident light reaches the cell through a hole in the centre of
the mirror and one half of all emitted light can be collected.
• Light sources are usually xenon discharge lamp, sometimes
augmented by mercury, although deuterium discharge lamps
are sometimes used.
• The excitation light is normally provided by a low pressure
mercury lamp which is comparatively inexpensive and
provides relatively high intensity UV light at 253.7 nm. Many
substances that fluoresce will be excited by light of this
wavelength.
• The excitation light is focused by a quartz lens through the
cell. A second lens, set normal to the incident light, focuses
the fluorescent light onto a photo cell.

109. Fluorescence Detector

110. Fluorescence Detector
• Advantages
– extremely high sensitivity
– high selectivity
• Disadvantage
– may not yield linear response over wide range of
concentrations

111. Choosing a Detector for HPLC
RRII UUVV//VVIISS FFlluuoorr.. EECCDD CCoonndd..
RReessppoonnssee UUnniivveerrssaall SSeelleeccttiivvee
((CChhrroommoopphhoorree))
SSeelleeccttiivvee
((FFlluuoorroopphhoorree))
SSeelleeccttiivvee
((OOxxiiddaattiioonn oorr
RReedduuccttiioonn))
SSeelleeccttiivvee
((IIoonnss))
SSeennssiittiivviittyy µµ ggrraamm nnaannooggrraamm ppiiccooggrraamm ppiiccooggrraamm ppiiccooggrraamm
LLiinneeaarr
RRaannggee
110044
110055
110033
110066
110055
FFllooww
SSeennssiittiivviittyy
YYeess NNoo NNoo YYeess YYeess
TTeemmpp..
SSeennssiittiivvee
YYeess NNoo NNOO YYeess YYeess

112. DERIVATISATION IN HPLC

113. INTRODUCTION
DERIVATISATION:
Derivatisation is a technique of treatment of the
sample to improve the process of separation by the
column or detection by the detector.
 HPLC derivatization plays an important role in the
determination of many pharmaceutical compounds
(aminoacids, antibiotics) in agrochemistry (proteins,
peptides, toxins) and in the environmental pollutants.

114. Reasons for Derivatisation
 Improve peak shape
 Suitable volatility
 Better sensitivity & detectability
 Increased thermostability
 Important for trace analyses of complex biological
samples

115. DERIVATISATION IN HPLC
 The primary purpose for derivatisation in HPLC becomes
that of introducing substituents to increase sensitivity &
selectivity for detection.
 Derivatisation is mostly done in HPLC for the analytes
(aminoacids) which exist as charged ions in solution [anions,
cations or zwitter ions]
 The original complete seperation of complex mixtures of
these analytes require very lengthy runs on ion-exchange
resin columns.

116.  By treating these analytes (aminoacids) with
orthophthalaldehyde produces isoindole derivatives whose
polarity is compatible with RP-C 18 columns leading to
much more rapid separations.
 Additionally, these derivatives also display intense
fluorescence at 425nm, which greatly improves the
detection limits when monitoring with a fluorescence
detector.

117. These derivatisation techniques are of two types based upon
the need:
1 Pre-column derivatisation
2 Post-column derivatisation
1 Pre-column derivatisation:
This is done to improve some properties of the sample for
separation of compounds by the column.
2 Post-column derivatisation:
This is done to improve responses shown by the detector

118. PRE-COLUMN DERIVATISATION
 The derivatisation can be carried out in solution as a
last step in sample preparation.
 Because it allows for whatever temperature & reaction
time may be necessary to form the derivative.

119. Examples for pre-column derivatisation
1 Benzoylation of Hydroxysteroids
2 Esterification of Fatty acids
3 Introduction of Flourophores into Aminoacids, Biogenic
amines & alkaloids by treatment with Dansyl chloride (5-
dimethyl aminonapthalene-1-sulphonyl chloride)
4 Epimeric forms of some Vit D3 metabolites have been
resolved as their trimethylsilyl derivatives

120. DERIVATISATION REAGENT ANALYTE
O-Phthalic anhydride Primary amines, Thiols
Flourescamine Primary amines
Dansyl chloride(Dns-cl) Primary & Secondary amines
9-Fluorenyl methyl chloroformate(FMUC) Primary & Secondary amines
Napthyl Iso cyanate Hydroxy compounds
Dansylhydrazine(Dns-H) Aldehydes & Ketones
Anthysldiazomethane(ADAM) Carboxylic acids
N-Acridinyl maleimide Thiols
Reagent for pre-column fluorescence labeling

121. POST-COLUMN DERIVATISATION
 Post-column Derivatization is carried out on the separated
solutes as they emerge from the chromatographic column.
 Consequently, very fast reactions must be used & the
reagents & mobile phase used must be compatible.

122. PROCEDURE FOR POSTCOLUMN
DERIVATISATION
 In this the labeling agent is added to the column effluent
by constant rate in fusion from a separate pump, passes
through a mixing chamber or coil, possibly at elevated
temperature, for a precisely regulated & reproducible
interval & then passes into the detector cell.
 The purpose of this mode is only to convert the analyte to a
better detected form.
 The post-column reaction needs to be fast, as too long a
mixing chamber could lead to extra column band
broadening.

123. ^^^
ELUENT PUMP INJECTOR COLUMN
PUMP
MIXINGCHAMBER
DETECTOR
Block diagram of HPLC post-column derivatisation system

124.  When properly implemented, post-column derivatization
is actually more reproducible in its yield & quantitation
than the pre-column mode.
 Both pre-column or post-column reactions may be
employed to produce electro active compounds
analogues for the formation of fluorescent derivatives.

125. Reagents for post-column derivatization
ANALYTE DERIVATISATION REAGENT
Pantothenic acid in food Orthophthaldialdehyde,
3-mercaptopropionic acid
Carbamate pesticides o-phthaldialdehyde, 2-
mercaptoethanol
(added to mobile phase)
Phenothiazines Peroxyacetic acid
Vitamin C & its derivatives Benzamidine

126. Examples of post column derivatisation reactions for
use with UV detectors include
 Reaction of aminoacids with Ninhydrin and flourescamine.
 Reaction of fatty acids with o-nitrophenol.
 Reaction of ketones with 2,4-DNP.
 Acid/phenol treatment of carbohydrates.

127. Reaction of Ketone with 2,4-DNP

128. Reaction of Aminoacid with Ninhydrin

129. COMPARISON OF TLC & HPLCCOMPARISON OF TLC & HPLC
HPLCHPLC TLCTLC
It is capable of handling macro moleculesIt is capable of handling macro molecules Not possibleNot possible
Pressure is requiredPressure is required Not requiredNot required
Pumps are required to pass mobile phasePumps are required to pass mobile phase Pumps are not requiredPumps are not required
Guard Colum is requiredGuard Colum is required Not requiredNot required
Smaller diameter & smaller surface areaSmaller diameter & smaller surface area Bigger diameter & bigger surface areaBigger diameter & bigger surface area
Solvent reservoir is requiredSolvent reservoir is required Solvent reservoir is not requiredSolvent reservoir is not required
Derivatization is carried out in HPLCDerivatization is carried out in HPLC Not in TLCNot in TLC
Detectors are requiredDetectors are required Detectors are not requiredDetectors are not required
Polar solvent can be identifiedPolar solvent can be identified Not possibleNot possible
More efficiencyMore efficiency Less efficiencyLess efficiency
Auto sampling is possibleAuto sampling is possible Auto sampling is not possibleAuto sampling is not possible
Complicated &expensiveComplicated &expensive Not Complicated &expensiveNot Complicated &expensive

130. Applications
• Organic chemistry
• Inorganic chemistry
• Pharmaceutical industry
• Chemical and petrochemical industry
• Forensic chemistry
• Environmental applications
• Biotechnology
• Food analysis
• Biochemistry field
• Clinical medicine

132. Applications
Organic chemistry
• HPLC techniques are widely used in the field of organic
chemistry from its advent for the separation of different
organic compounds and their analysis.
• E.g.: separation of mixture of aromatic compounds like
benzyl alcohol, phenol, benzoin, ethyl benzoate, toluene,
2,6 di methoxy toluene on hypersil ODS column(c18 on
5µmsilica) where the eluent used is buffer (25mM potassium
di hydrogen phosphate and 1g/L sodium azide adjusted to
p.H-3.5 with hydrochloric acid) followed by acetonitrile.

133. Applications
Inorganic chemistry
E.g.: I) Separation and analysis of anions
– By using the principle of Ion exchange chromatography
Analysis of I-
,IO3
-
,SCN-
,ClO4
-
,S2O3
2-
,Cr2o4
2-
,wo4
2-
,MoO4
2-
,ReO4
2-
etc.. anions using HPLC-AG4 column using a stronger
eluent (6-8Mm CO3
2-
).
II) Separation and analysis of cations
– Separation of cations from Li+
toCs+,NH4
+
using HPLC-AG4
column using stationary phase superficially sulphonated
inert polymer resin using eluent 1mM Hcl.

134. Applications
Pharmaceutical industry
• In product development and product control.
• Isolation of pharmaceuticals.
• In raw material control.
• In pharmacokinetic and stability studies.
• Separation of different pharmaceuticals like antibiotics,
sulphonamides, steroids, psycho active pharmaceuticals.

135. Applications
Chemical and petrochemical industry
 HPLC is used in chemical and petrochemical industry for the
separation and analysis of petroleum products, coal
products, condensed aromatics, surfactants, propellants etc.
Forensic chemistry
• It is the branch of scientific criminology or forensic
toxicology.
• HPLC is primarily used in trace analysis and material
science.
Eg: I) classification of traces of paint after a traffic accident
II) It is used in the investigations of biological human
materials such as blood, urine, gastric contents, etc

136. Applications
Environmental applications
 HPLC is used in monitoring levels and effects of pesticides
and carcinogens in our air, waterand food supplies.
Pesticides include insecticides, herbicides, fungicides,
rodenticides etc.
Biotechnology
• Studies regarding regulatory effects of cyclic nucleotides
• Determination of composition of hydrolysates of nucleic
acids
• Metabolic profiling of normal and diseased subjects
• Separation and purification of nucleic acids

137. Applications
Food analysis
 HPLC is used in food analysis for the analysis of antioxidants,
preservatives, aflatoxins, additives etc. preservatives and
antioxidants are the additives which prevent microbial or
chemical deterioration of food products.
Biochemistry field
• HPLC is used widely in the field of biochemistry for the
separation of amino acids, proteins, carbohydrates and lipids.
Clinical medicine
 In the field of clinical medicine for predicting pharmacokinetic
parameters by Studying concentration of drugs in bile acids,
urine, blood, serum, blood metabolites etc for determining the
dosage of drug, dosing schedules etc

138. References
• Practical HPLC method development by LLOYD R.Synder,
Joseph l.Glajch, I.J.KIRKLAND, 2nd
edition.
• Instrumental methods of analysis, Hobart H.Willard, Lynne
l.MERITT, 7th edition.
• Instrumental methods of chemical analysis, B.K.SHARMA, 26th
edition.
• Instrumental analysis by Douglas A Skoog.
• James w. Robinson et al., Undergraduate instrumental analysis.
• Raymond P.W. Scott, EWING’S Analytical Instrumentation
Handbook, Third Edition.
• Thomas H. Stout, John G. Dorsey, Handbook of Pharmaceutical
Analysis, Drugs and the Pharmaceutical Science Vol. 117.

139. Questions
• Explain the principle and working of detectors used in HPLC.
• What is guard column? Mention its significance.
• Explain the principle and working of solute property detectors
used in HPLC.
• Name the pumps used in HPLC and explain with a neat
diagram.
• Explain the applications and merits of HPLC.
• List the types of detectors used in HPLC.

140. Questions
• Compare GC and HPLC methods.
• What is HPLC and explain the principle involved in HPLC.
• With a neat diagram, explain the different parts and working of
HPLC.
• Classify the types of columns used in HPLC.
• Explain the principle involved in the functioning of a PDA
detector.
• What is the difference between normal and reversed phases in
HPLC?

141. USP Plate Count Calculation

142. Tailing Factor Calculation

143. Resolution Calculation

144. Capacity Factor (K’) Equation

145. Selectivity Equation