HPLC & UPLC omkar kumar kuwar

1. TOPIC: HIGH PERFORMANCE LIQUID CHROMATOGRAPHY {HPLC} &
ULTRA PRESSURE LIQUID CHROMATOGRAPHY {UPLC}
Presented By:
Omkar kumar Kuwar
Department: pharmacology
M. Pharm, 1st year student
omkar.official.955@gmail.com
Presented To :
Dr. Pooja Chawla
Professor and H.O.D
Department of Pharmaceutical Chemistry
ISF College of Pharmacy, Moga, Punjab
pooja.chawla@isfcp.org

2. Table of content
 Introduction to chromatography
 Types of chromatography
 Introduction to HPLC
 Principle
 Advantages
 Types of HPLC separation
 Instrumentation
 Introduction to UPLC
 Comparison B/W HPLC & UPLC
 Comparison B/W HPTLC & HPLC
 Comparison B/W GC & HPLC
 Applications
 References

3. CHROMATOGRAPHY
 It is analytical method in which separation of active
constituent in complex mixture, and the mixture was
distributed in two phases i.e. stationary and mobile phase, is
known as chromatography.
 It is the technique used for separation, identification,
purification and extraction of compound.
 Basically all chromatography system consist of two phases
 1. MOBILE PHASE – Liquid or gaseous and flows over or
through the stationary phase
 2.STATIONARY PHASE – Solid, liquid or solid/liquid
mixture that is immobilized.
 The basic principle of chromatography is based on
adsorption, partition, Ion exchange, size/molecular
exclusion, affinity chromatography.

4. Types of chromatography
 Based on mode of chromatography
1. Normal phase mode
2.Reverse phase mode
 Based on principle of separation
1.Adsorption chromatography
2.Ion exchange chromatography
3.Partition chromatography
4. Size exclusion
 Based on elution technique
1.Isocratic separation
2.Gradient separation
 Based on scale operation
1.Analytical HPLC 2.Preparative HPLC

5. Principle of liquid chromatography

6. liquidchromatography

7. Introduction to Hplc
 HPLC is high performance liquid chromatography
 It is the modified form of gas chromatography, it is
applicable for both volatile and non-volatile compound
 It is divided into 1. Normal phase HPLC
2. Reversed phase HPLC
 It is having a high resolution and separation capacity
 It is used as qualitative as well as quantitative analysis.
 HPLC is chromatographic technique used to separate
mixture of compounds in analytical chemistry and
biochemistry with the purpose of identifying,
quantifying, purifying the individual components of
mixture

8. HPLC

9. PRINCIPLE
 High performance liquid chromatography’s principle is
based on adsorption as well as partition
chromatography depending on the nature of stationary
phase.
 It is important for the determination of volatile and
non-volatile compounds.
 It is important for qualitative and quantitative analysis.
 It is important for retention time [the time required
after the sample injection maximum angle peak reaches
to the detector].

10. HPLC SYSTEM

11. ADVANTAGES
 It is simple, rapid and reproducible.
 High sensitivity.
 High performance.
 It is having high resolution and separation capacity.
 Accuracy and precision.
 Early recovery of separated component.
 Easily visualization of separated components.
 It is analytical technique which is important for product
validation, product quality and study of product.

12. TYPES OF HPLC SEPARATION
 NORMAL PHASE- Separation of polar analytes by
partitioning onto polar bonded stationary phase.
 REVERSED PHASE- Separation of non polar analytes by
partitioning onto non polar bonded stationary phase.
 ADSORPTION- In between normal and reversed. Separation
of moderately polar analytes by using adsorption onto pure
stationary phase.
 ION CHROMATOGRAPHY- Separation of organic and
inorganic ions by partitioning into ionic stationary phase
bonded to a solid support.
 SIZE EXCLUSION- Separation of large molecules based on
the path they took on the maze of tunnels in stationary
phase.

13. SEPARATION MODES

14. HPLC CONTAIN
 STATIONARY PHASE- It is non polar and the solid
surface is coated with a second liquid which is
immiscible in mobile phase
 MOBILE PHASE-A polar mobile phase[ACN, MeOH,
Water and Buffer]
 BONDED PHASE
1.C-2 Ethyl Silyl
2.C-8 Octyl Silyl
3.C-18 Octadecyl Silyl

15. BLOCK DIAGRAM OF HPLC

16. WHY TO USE HPLC
 Simultaneous analysis
 High resolution
 High sensitivity
 Good repeatability
 Moderate analysis condition
 Easy to fractionate and purify
 Not destructive

17. INSTRUMENTATION OF HPLC
 SOLVENT STORAGE BOTTLE.
 GRADIENT CONTROLLER AND MIXING UNIT.
 DE-GLASSING OF SOLVENTS.
 PUMP.
 PRESSURE GAUGE.
 PRE-COLUMN.
 SAMPLE INRODUCTION SYSTEM.
 DETECTOR.
 RECORDER.

18. INSTRUMENTATION

19. SOLVENT MOBILE PHASE RESEVOIRS
 Glass or steel containers capable of holding
up to 1 l of mobile phase
 Inert to a variety of aqueous and non
aqueous mobile phase
 Stainless steel should be avoided for use with
the solvents containing halide ions

20. DEGASSION OF MOBILE PHASE
 In many case aqueous solvents and some organic
solvents are degassed prior to use
 Degassing is done to prevent formation of bubbles in
the pump or detector
 The mobile phase are filtered to remove particulate
matter that mat clog the system
TUBIING
Should be inert
Have ability to withstand pressure
Able to carry sufficient volume

21. Pump
 The solvents or mobile phase must pass
through high pressure column
 As the particle size of stationary phase is
smaller, the resistance to the floe of solvent is
high
 That is smaller the size of stationary phase ,
greater is the resistance to the flow of solvent
 Hence high pressure is recommended

22. REQUIREMENT FOR PUMPS
 Generation of pressure of about 6000 psi
 Pulse free output and all material in the pump
should be chemically resistant to the solvent
 Flow rate ranging from 0.1 to 10ml/min
 Pump should be capable of taking the solvent from
the single reservoir or more than one reservoir
containing different solvents simultaneously

23. TYPES OF PUMP USED IN HPLC
I. DISPLACEMENT PUMPS
II. RECIPROCATING PUMP
III. PNEUMATIC PUMP

24. HPLC PUMPS

25. DISPLACEMENTS PUMP
 It consist of large syringe like chambers
equipped with plunger activated by screw
driven mechanism powered by stepping
motor
 So it is called screw driven displacement
pump
 It produce a flow that tends to be
independent of viscosity and back pressure
 It has limited solvent capacity and
considerably inconvenient when solvent
must be changed

26. DISPLACEMENT PUMP

27. RECIPROCATING PUMP
 This pump transmits as an alternative pressure to
the solvent via flexible diaphragm which in turn is
hydraulically pumped by a reciprocating pump
DISADVANTAGE
Produces a pulsed flow which is damped because
pulses appear as baseline noise on the
chromatograph
This can be overcome by use of dual pump heads or
elliptical camps to minimize such pulsation

28. RECIPROCATING PUMP

29. SCHEMATICREPRESENTATION

30. ADVANTAGES
 Have small internal volume
 Higher output pressure up to 10000
psi
 Adaptability to gradient elution
 Large solvent capacities and constant
flow rates
 Largely independent of back pressure
and solvent viscosity

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

32. PNEUMATIC PUMPS

33. SAMPLE INJECTOR SYSTEM
 Several injectors system are available
either for manual or automatic
injection of samples
I. Septum injector
II. Stop flow injector
III. Rheodyne injector

34. SEPTUM AND STOP FLOW INJECTOR
 SEPTUM
These are used for injecting the samples
through rubber septum. This kind of septum
cannot be generally used as they need to
withstand high pressures
 STOP FLOW
In this type, the flow of mobile phase is
stopped for a while and the sample is injected
through a valve

35. RHEODYNE INJECTOR
This is most widely
used injector and has
capacity of high
volume for loop, for
holding samples
until it is injected to
the columns.
Through an injector
the sample is
introduced in the
column. The injector
is positioned just
before the inlet of the
column

36. COLUMN
 PRE COLUMN
 It contains a packing
chemically identical to that
in analytical column
 Mainly used to remove the
impurities from solvent
and thus prevent the
contamination of
analytical column
 It is also called as the
guard column of or
protective column
 It is having large particle
size
 It is having short length of
2 to 10 cm so does not
affect the separation
 ANALYTICAL COLUMN
 The success or failure
depends on the choice of
the column.
 Actual separation is
carried out here
 Stainless steel tube
 Size length is up to 25 to
100cm
 Column is fixed with size
particle of 5 to 10 micron.
The solid support can be
silica gas or alumina
 The separation is result of
different components
adhering or diffusion into
packaging particle

37. detectors
 Absorbance [UV/Vis and PDA]
 Refractive index[ detects the change in
turbidity]
 Fluorescence[if the analyte is fluorescent]
 Conductivity for ions
 Light Scattering
 Mass spectrometry [ HPLC-MS]

38. DETECTORS

39. DETECTORS

40. IDEAL DETECTOR PROPERTIES
High sensitivity
Universality or predictable specificity
Large linear response range
Low dead volume
Non destructive
Insensitive to temperature and mobile phase
Reliable to use
Continuous operations

41. RECORDERS AND INTEGRATERS
 Recorders are used to record and response
obtained from the detectors after
amplification
 They record the baseline
 Retention time can be find out from this
recordings
 The integrators are improved version of
recorders
 Integrators provide information on peaks
than recorders

42. Introduction to UPLC
 UPLC refers to ultra performance liquid
chromatography.
 Which enhance mainly in three areas: “speed,
resolution and sensitivity.
 The principles of UPLC are same principle as
HPLC.
 The basic difference is in designer of the column
material particle size which less than 2-μm.
 The separation and quantification in UPLC is done
under very high pressure (up to 100M Pa).
 Under high pressure it is observed that not any
negative influence on analytical column.
 Time and solvent consumption is less in UPLC.

43. HPLC AND UPLC

44. HPLC AND UPLC

45. HPTLC HPLC
COMPARISION B/WHPTLC ANDHPLC
 High performance thin
layer chromatography
 Planar
chromatography
 Simultaneous method
for test as well as
reference material
 High performance
liquid chromatography
 Column
chromatography
 Not simultaneous
method for test as well
as reference material

46. GC HPLC
 Gas chromatography
 Less resolution
 Limited flexibility
 Determination of
volatile components
 High performance
liquid chromatography
 High resolution
 Extreme flexibility
 Determination of
volatile and non
volatile compounds
COMPARISION B/WGC AND HPLC

47. APPLICATIONS
 Drug discovery.
 Clinical analysis.
 Proteomics.
 Forensic chemistry.
 Drug metabolism study.
 Environmental chemistry.
 Diagnostics studies.
 Pharmaceutical applications.
 Qualitative and quantitative analysis.
 Therapeutic Drug monitoring.

48. REFERENCES
 Introduction to high performance liquid chromatography
by Lab-Training.com Auriga Research Ltd. Page no: 1-34.
 Handbook of HPLC HPTLC 2021 edition by Rajesh kumar
Nema, G yuvaraj, CBS publishers. Page no: 2-24.
 Chatwal, G.R. and Anand, S.K.J. (2003) instrumental
methods of chemical Analysis, Himalaya publishing House,
Mumbai, page no 2.108-2.109,

49. Thank you