HPLC Instrumentation, Pharmaceutical analysis, HPLC detectors

1. Presented by:
Sharath .H.N
M pharmacy
Dept of ph. analysis

2. Index
1.Liquid chromatography introduction
2.Types of hplc techniques
3.Hplc Instrumentation

3. Introduction
• Liquid chromatography is the method, in
which a dilute solution of sample is passed
through a column packed with solid particles.
Thus, liquid is passed through vertical columns
under gravitational flow.
•

4. • The origins of Liquid Chromatography began in
the early 1900’s with the work of the Russian
botanist, Mikhail S. Tswett. His famous studies
focused on separating compounds (leaf
pigments), which were extracted from plants
using a solvent.
• During 1970's, most chemical separations were
carried out using a variety of techniques including
open-column chromatography, paper
chromatography, and thin-layer chromatography
• However, these chromatographic techniques
were inadequate for quantification of compounds
and resolution between similar compounds.
•

5. • During this time, pressure liquid chromatography
began to be used to decrease flow through time,
thus reducing purification times of compounds being
isolated by column chromatography
• By the 1980's HPLC was commonly used for the
separation of chemical compounds.
• New techniques improved separation, identification,
purification and quantification far above the previous
techniques. Computers and automation added to the
convenience of HPLC. Improvements in type of
columns and thus reproducibility were made as such
terms as micro-column, affinity columns, and Fast
HPLC began to immerge.

6. Advantages OF HPLC
• Separations is fast and have a high resolving
power
• Continuous monitoring of the column effluent.
• Can be applied to the separation and analysis of
very complex mixtures.
• Repetitive and reproducible analysis using the
same column.
•
•

7. • Adsorption, partition, ion exchange and exclusion
column separations are applicable.
• Capable of handling macromolecules of high
molecular weight.
• Suitable for pharmaceutical compounds.
• Suitable for separating non volatile substances.
•

8. • When a mixture of components is introduced into
a HPLC column, they travel according to their
relative affinities towards the stationary phase.
• The component which has more affinity towards
the adsorbent travels slowest the component
which has less affinity towards the stationary
phase travels faster.
• Since no two components have the same affinity
towards the stationary phase, the components
are separated
PRINCIPLE OF SEPARATION IN HPLC

9. Types of HPLC techniques
• 1.Based on modes of chromatography
•
• i) Normal phase mode.
• ii) Reverse phase mode.
•
• 2.Based on principle of Chromatography.
•
• i) Adsorption chromatography.
• ii) Partition chromatography
• iii) Ion exchange chromatography.
• iv) Size exclusion/gel permeation chromatography.
• v) Affinity chromatography.
• vi) Chiral chromatography
•
•

10. • 3.Based on elution techniques.
• i) Isocratic separation.
• ii) Gradient separation
•
• 4.Based on the scale of operation
• I) Analytical HPLC.
• II) Preparative HPLC.
•
• 5.Based on the type of analysis.
• I) Qualitative analysis.
• ii) Quantitative analysis.

11. 1.Based on modes of chromatography
•
• Interaction or affinity between:
•
• Polar-polar is more
• Non polar-non polar is more where as the
• Interaction or affinity between the polar- nonpolar is less.
•
• Normal phase chromatography:
• Separation of the polar analytes by partioning onto a polar
stationary phase.
• stationary phase- polar (silica gel, alumina)
• mobile phase- non polar.
•

12. •
• Mechanism-interaction of stationary phase (polar
surface) with polar components of the sample
molecules.
•
• Applications –Separation of non-ionic , non polar
substances (Sio2).
•

13. • Reverse phase chromatography:
• Separation of non polar analytes by partitioning onto a
non polar stationary phase.
•
• Mechanism-interaction of stationary phase non polar
hydro carbon chain with non polar parts of the sample
molecules.
•
• Application – separation of non ionic and ion forming non
polar to medium polar substances. (COOH –hydrocarbon).
•
•

14. 2.Based on principle of
chromatography
•
• Adsorption chromatography
• Usually polar stationary phase and non-
polar mobile phase are employed.
•
• Polar s.p – silica gel, alumina, porous glass bead.
• Non polar m.p – heptane, benzene, acetone
•

15. • Partition chromatography
• In this case liquid stationary phase is used.
•
• Polar s.p - alcohol, methanol, water.
• Non polar s.p – benzene, CHCl3,acetone.
•

16. • Ion exchange chromatography:
•
• The principle involves reversible exchange of the functional
groups.
• Exchange of similar charged ion.
•
• Stationary phases :
• 1) Cation exchangers.
• 2) Anion exchangers.
• Mobile phase: Aqueous buffer system.
• Applications: organic acids, organic bases, proteins, nucleic acid .
•

17. • Size exclusion chromatography:
•
• Mixture of components with different molecular size
are separated by gels.
•
• Gel acts as a sieves which has crossed polymer linkage
and hence a mixture of substances with different
molecular sizes is separated.
•
• Mechanism by its diffusion effects

18. • Soft gels like dextran, agarose or
polyacrylamide are used.
•
• Semi rigid gels like polystyrene, alkyl dextran
in non aqueous medium are also used.
•
•

19. • Affinity chromatography:
• Affinity of the sample with specific stationary
phases.
• Technique widely used in field of Bio technology,
Microbiology, Biochemistry.
•
• 3.Based on elution technique:
• Isocratic elution.
• Isocratic Elutions – Constant solvent composition,
mobile phase
• polarity stays constant throughout elution process.
This is equivalent to isothermal separations in GC.
•
•

20. 2) Gradient Elutions – Mobile phase composition (and
thus polarityvaries throughout elution. This is
equivalent to temperatureprogramming in GC.
•
• 4.Based on the scale of operation
•
• Analytical HPLC - For analytical purpose.
• -No recovery of the sample.
• -sample quantity very small (micro grams)

21. • Preparative HPLC - For preparative samples.
• -individual fractions of the sample collected
using fraction collector.
•
• -Collected samples can be re-used.
• Ex: Separations of few grams of a mixture

22. 5.Based on
the type of analysis
• Qualitative Analysis : To identify the compound
and to detect the presence of any impurities.
•
• Quantitative Analysis : To detect the amount of
individual or several components in the mixture.
•
•

23. • Principle of separation in HPLC:
•
• -Mixture of components introduced into the
column.
•
• -These travel according to their relative affinities
towards the stationary phase.
•
• - Component with more affinity towards
stationary phase travel slower and Component
with less affinity towards stationary phase travel
faster.

24. • STATIONARY PHASES
• (PACKING MATERIALS)
•
• Stationary phases are the material consisting particles
which are about 1 to 10 microns in average (often
irregularly shaped).
•
• Polar : Normal phase .
• -Silica ,Alumina
• -Cyano, amino or diol terminations on the bonded
phase.
•
• Non polar : Reversed Phase.
• -C18 to about C8 terminals on the bonded phase.
• -Also Phenyl and Cyano terminals.

25. • Mobile Phases in the HPLC
•
•
• Must do the following:
• - solublize the analyte molecules and the solvent they are in.
•
• - be suitable for the analyte to transfer back and forth between
during the separation process.
•
• It must :
• - be pure .
• - be compatible with instrument.
• - not interfere during detection.
• - not dissolve the stationary phase.
• - be readily available.
• - Not be too compressible (causes pump /flow problems)

26. INSTUMENTATION OF HPLC
1) Degassing system
2) Pump solvent delivery system
3) Check valves
4) Pulse damper
5) Pre-columns
6) Guard column
7) Flow splitter
8) Auto sampler
9) Sample injection port
10) Column

27. HPLC Instrumentation

28. DEGASSING SYSTEM
Sparging/Bubbling
Vacuum filtration
Ultra sonication

29. PUMP-SOLVENT DELIVERY SYSTEM
 The pumps are used to pass mobile phase through the column at high
pressure because the particles that are used pack HPLC columns are
small enough ie: <50μm and also particle size of packing material is 5-
10 μm
 To prevent solvent flow from gravity pumps that develop pressure up
to 5000psi are needed to force the mobile phase through column so
that solvent stream enters the instrument at constant flow
rate/pressure. In addition to this the pumps used in HPLC should have
the following features

30. PUMPS
1. Generation of pressure upto 5000psi
2. Flow rate ranging from 0.1 to 100ml/min
3. Flow control & flow reproducibility of plus
or minus 0.5%
4. It should be composition resistant and give
a pulse free out put
Pumps are thus categorized into
1.Mechanical pumps
• Displacement pumps
• Reciprocating pumps
2.Pneumatic pumps

31. MECHANICAL PUMPS
1. DISPLACEMENT PUMPS

32. WORKING
• Works on the principle of positive solvent pressure.
• Consist of screw or plunger which revolves continuously
driven by motor.
• Rotatory motion provides continuous movement of the
mobile phase which is propelled by the revolving screw at
greater speed and pushes solvent through small needle like
outlet.
• Consist of large syringe like chamber of capacity 250 – 500
ml.

33. ADVANTAGES
• Flow is pulse free.
• Provide high pressure upto 200 – 475 atm.
• Independent of column back pressure and viscosity of
solvent.
• Simple operation.
DISADVENTAGE
• Limited solvent capacity
• Gradient elution is not easy.

34. 2. RECIPROCATING PUMPS
WORKING
• Pressure from a gas cylinder delivered
through a large piston drivers the
mobile phase.
• Pressure on the solvent is proportional to
the ratio of piston usually 50: 1.

35. 3. CHECK VALVES
They are used to control the flow of solvent &
back pressure
4. PULSE DAMPER
Pulse are used to dampen or reduce the pulses
observed from the wavy baseline caused
by pumps

36. PULSE DAMPER
Important damping methods include
• A triple headed pump: - two heads in different stages of
filling as the third is pumping.
• A tube with an air space or a flexible bellows or tube: Here
a gas (air space) or a flexible metal vessel takes up some of
the solution energy. When pump refills, this energy is
released and a smooth pressure pulsation result.
• A restrictor: - In this method, a 25 cm length of 4 mm bore
.stainless steel tubing. Packed with 20µm glass beds, is
placed between the pump and the column.

37. 5. PRE-COLUMNS
 A pre-column is packed with 37-53μm silica particles
(saturator column).
 It is being fitted between pump & the injector valve
ensures that the mobile phase is fully saturated with
the silicate ions prior to the sample injection.
 Thus its use reduces the adverse effects of low or high
pH mobile phases which in turn substantially extend
the life of the column. Their use is recommended in
ion-exchange chromatography using buffered aqueous
phase.

38. 6. GUARD COLUMN
• A short column placed between the sample injector and
the inlet of the main ("analytical") column
• The guard column is packed with the same kind of
packing as the main analytical column, and is intended
to absorb or pick up impurities in the sample or mobile
phase that might damage the main column & increase
the life time of main analytical column.

39. 7. FLOW SPLITTER
• When a differential type of detector is used
the flow of solvent is split just before it enters
the sample injection port
• so that one portion directly goes to the
reference side of the detector & a portion to
the analytical column housed in a constant
temperature chamber

40. 8. AUTOSAMPLER
• Here in this type of instruments their will be a piston metering
syringe type pump to suck the prestabilised sample volume
into a line & than transfer it to the relatively large loop
(approx 100ml)
• In a standard six port valve. The simplest Autosampler utilizes
the special vial & displace the sample through the needle in to
the valve loop.
• Most of the Autosamplers are microprocessor controlled &
can serve as a master collector for the whole instrument

41. 9. SAMPLE INJECTOR PORT
a. Septum injector
b. Stop flow septum less injection
c. Rheodyne injectors

42. SEPTUM INJECTION PORT.
• Syringe is used to inject the sample through a self sealing
inert septum directly into the mobile phase.
• Drawback: - leaching effect of the mobile phase with the
septum resulting in the formation of ghost peaks.
STOP FLOW SEPTUMLESS INJECTION.
• Flow of mobile phase through the column is stopped for a
while.
• Syringe is used to inject the sample.
• Drawback: formation of ghost peak.

43. RHEODYNE INJECTORS
• Operation of sample loop.
– sampling mode
– Injection mode.
• Sample is loaded at atmospheric pressure into an external
loop in the micro volume sampling valve, and subsequently
injected into the mobile phase by suitable rotation
of the valve.
Micro volume sampling valve operation of a Sampling loop.

44. 10. Analytical Column
• Analytical column is most important part of the
instrument dimensions of column are
• Column length: 5cm to 30cm
• Column diameter: 2mm to 50mm
• Particle size: 1μ to 20 μ
• Particle nature: Spherical, uniform sized, porous
materials are used

45. Few Analytical columns

46. CLASSIFACTION OF CLOUMN BASED ON
APPLICATION
Standard column Packed column
Narrow bore column
Short fast column
Preparative column
Micro preparative column
Preparative column
Macro preparative column

47. STANDARD COLUMN
• Internal diameter 4 – 5 mm and length 10 – 30 cm.
• Size of stationary phase is 3 – 5 µm in diameter.
• Used for the estimation of drugs, metabolites,
pharmaceutical preparation and body fluids like plasma.
NARROW BORE COLUMN
• Internal diameter is 2 – 4 mm. ( signal is increased 4 times )
• Require high pressure to propel mobile phase.
• Used for the high resolution analytical work of compounds
with very high Rt

48. SHORT FAST COLUMN
• Length of column is 3 – 6 cm.
• Used for the substances which have good affinity
towards the stationary phase.
• Analysis time is also less (1- 4 min for gradient
elusion & 15 – 120 sec for isocratic elusion).
PREPARATIVE COLUMN
• Used for analytical separation i.e. to isolate or
purify sample in the range of 10-100 mg form
complex mixture.
Length – 25- 100 cm
Internal diameter – 6 mm or more.

49. Preparative column are of three type :
• Micro preparative or semi preparative column
• Modified version of analytical column
• Uses same packaging and meant for purifying sample less then 100 mg.
Preparative column
• Inner diameter – 25 mm .
• Stationary phase diameter – 15- 100 µm
Macro Preparative Column
• Column length – 20 – 30 cm
• Inner diameter – 600 mm

50. PACKING OF COLUMN
• The most widely used method of packing column is by high pressure
slurring technique.
• A suspension of packing is made in a solvent of equal density to the
packing material.
• The slurry is then rapidly pumped at high pressure onto a column with a
porous plug at its outlet.
• The resulting bed of packed material with in the column can then be
prepared for use by running the developing solvent through the column,
hence equilibrating the packing with the developing solvent.
• When hard gels are packed, it is necessary for them to be allowed to swell
first in the solvent to be used in the chromatographic process before
packing under pressure.
• Soft gels cannot be packed under pressure and have to be allowed to
pack from a slurry in the column under gravitational sedimentation only,
in a similar way of packing conventional column

51. METHOD OF PACKING
• Depends on the mechanical strength of stationary phase.
• Particle size of the stationary phase.
– Particles of greater then 20 µm – dry packing
– Particles of lesser then 20 µm – slurry packing / wet
packing.
DRY PACKING
• Particle size greater then 20 µm filled into vertical
clamped column in small quantity.
• Deposition is done by tapping or vibrating the
column.
• Column is unclamped and the tapped on the firm
surface to obtain dense and reproducible packing

52. WET / SLURRY PACKING
• Particle size with diameter less then 20 µm
can only be placed wet as a suspension.
• Suspension should be stable, it should not
sediment, and agglomentation should be
avoided.

53. DETECTORS
Criteria
• Selectivity
• Sensitivity and detection limit
• Stability
• Reproducibility
• Economically affordable
• It should only record the component of interest

54. UV absorption detectors
• Fixed wavelength detectors
• Multi wavelength detectors
• Photo diode array detectors

55. Fixed wavelength detectors

56. Multiple wavelength detectors

57. Diode array detector
• Here broad emission source like deuterium lamp is
collimated by an achromatic lens
• Sample is subjected to all wavelength generated by
the lamp
• Dispersed light from gratings allowed to fall on to
diode array
• Array contain hundreds of diodes, output from each
diode is sampled by a computer

58. Photo diode array detector

59. FLUORESCENCE DETECTOR
• The single wavelength excitation fluorescence detector is
probably the most sensitive LC detector that is available, but
is achieved by forfeiting versatility. A diagram of a simple
form of the fluorescence detector is shown in figure.
• 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. A fixed wavelength
fluorescence detector will have a sensitivity (minimum
detectable concentration at an excitation wavelength of 254
nm) of about 1 x 10-9 g/ml and a linear dynamic range of
about 500 with a response index of 0.96 < r <1.04.

60. Flourosence detectors

61. ELECTROCHEMICAL DETECTORS
This detector is based on the measurements of the current
resulting from oxidation/reduction reaction of the analyte
at a suitable electrode. Since the level of the current is
directly proportional to the analyte concentration, this
detector could be used for quantification

62. • The eluent should contain electrolyte and be electrically
conductive. Most of the analytes to be successfully
detected require the pH adjustments.
• The areas of application of electrochemical detection are
not large, but the compounds for which it does apply,
represent some of the most important drug such as
phenol, catecholamines, nitrosamines, and organic acids
are in the picomole (nanogram) range
• The specificity, and sensitivity make it very useful for
monitoring these compounds in complex matrices such as
body fluids and natural products.

63. Deflection detectors
• The optical schematic of the deflection detector is shown in
below. This detector based on the deflection principle of
refractometry
• where the deflection of a light beam is changed when the
composition in the sample flow-cell changes in relation to
the reference side (as eluting sample moves through the
system).
• When no sample is present in the cell, the light passing
through both sides is focused on the photodetector (usually
photoresistor).
• As sample elutes through one side, the changing angle of
refraction moves the beam. This results in a change in the
photon current falling on the detector which unbalances it.
The extent of unbalance (which can be related to the sample
concentration) is recorded on a strip chart recorder.

64. The advantages of this type of detector are:
(1) Universal response;
(2) Low sensitivity to dirt and air bubbles in the cells;
and
(3) The ability to cover the entire refractive index
range from 1.000 to 1.750 RI with a single, easily
balanced cell.

65. Refractive index detectors
• It is very sensitive to changes in ambient
temp,pressure,flow rate
• It can not be used for the gradient elution
technique
• Extremely used for the comp that not adsorb
in uv region and not fluorescence

67. WORKING
• It passes the visible light through two compartments
• The differential refractometer monitors the deflection of
a light beam caused by the diff in refractive index
between the contents of the sample and the reference
cell
• The beam of the light from lamp passes through an
optical mask
• That confines the beam to the region of the cell
• The lens collimates the light beam which passes through
both the cells to a mirror
• The mirror reflects the beam back to a lens which focus it
on to a photocell

68. APPLICATIONS
• It is widely used in the separation and
analysis of the polymers
• Used in case of those polymers that contains
more than six monomers
• RI is directly proportional to the
concentration of the polymer and is
practically independent of mol. weight

69. APPLICATIONS OF HPLC1. Quality control testing of drugs
2. In Qualitative & Quantitave analysis
3. Therapeutic monitoring of drug metabolism studies
4. Separation & control of impurities
5. In analysis of biological fluids
6. Stability studies
7. Study of metabolic pathways in basic biochemical pathways
8. Separation of positional isomers, enantiomers, Optica
isomers
9. Industrial applications
a. Determination of synthetic intermediates ex: atenolol
b. In determining traces of impurity ex:Tolnafate
c. Stability studies ex Acyclovir

70. REFRENCES
• Pharmaceutical Analysis - Dr. A. V. Kasture .
• Instrumental Analysis by G .R . Chatwal
• Internet source