High Performance Liquid Chromatography (HPLC) is a process used for the seperation of the mixture considered in the Pharmaceutical Analysis subject.

1. High Performance Liquid
Chromatography (HPLC)
Prepared by- Sagar Verma (170823101099)
7th Sem B Pharm
Parul Institute of Pharmacy And Research
Parul University, Vadodara

2.  HPLC is the most widely used separations technique
in analysis.
 It utilizes a liquid mobile phase to separate
components of mixture
 It is currently the popular type of chromatography
and is even replacing GC in its more traditional
applications.
 Uses high pressure to push solvent through the
column.
Introduction

3. It is popular because:
 It is sensitive.
 Ready adaptability to accurate quantitative
determination.
 Suitable for separating nonvolatile species and
thermally fragile substances.
 Applicable to substances that are of prime interest to
industry, to every fields of science, and to the public.

4.  Higher resolution and good 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.
Advantages

5. Components of HPLC-
• Mobile phase reservoir
• HPLC Pump(s)
• Sample injector (manual or auto)
• Column
• Detector
• Data system
• Mixing valves
• Mobile phase waste container
Instrumentation of HPLC

6. Components of HPLC

7. Schematic Diagram of HPLC

8.  A carrier for the sample solution.
 Interactions of substance with the mobile phase and
stationary phase are bases for separation process.
Mobile Phase
Interaction
of substance
with mobile
phase
Interaction
of substance
with
Stationary
phase
Elution Retention
Time
Strong Less Faster Less
Less Strong Slower More

9.  Mobile phase must dissolve the sample
 It should have a strong solvent strength
 Interaction with solute should lead to selectivity for
separation of components
 It should not be hazardous
Properties of the Mobile Phase

10.  Made from Glass/stainless steel
 Capacity: 200 to 1000 mL
 May be equipped with degassers which removes dissolved
gases usually O2 and N2 that interfere by forming bubbles
in the columns and detector systems.
 Bubbles cause -band spreading & interfere with the
performance of the detector.
 Degassers and filters may not be integral part of HPLC
system.
Mobile Phase Reservoir

11.  Tubing carries mobile phase from one to another component of
HPLC.
 They should be inert, have ability to withstand pressure and be
able to carry sufficient volume.
 Generally stainless steel pipes are used.
Disadvantage of stainless steel pipes:
 Bore of the tube get collapsed when
bending is done.
 Easily blocked by small particles.
 Difficult to cut effectively.
Tubing

12. Requirements for an HPLC pumping system:
 Generation of required pressures
 Pulse free output
 Flow rate ranging from 0.1 to 10 ml/min
 Corrosion – resistant components (seals of stainless
steel or Teflon);
 High pressure generated by pumps should not lead to
an explosion hazard
Pumping system

13. Typical types of HPLC pumps:
 Reciprocating pumps (used for 90% HPLC
instrumentation)
 Syringe or Displacement pumps
 Pneumatic pumps
Pumping system

14.  Consist of small chamber in which solvent is pumped
by back and forth motion of motor-driven piston.
Reciprocating Pumps

15. Advantages:
 Small internal volume
 High output pressures (up to 10,000 psi)
 Ready adaptability to gradient elution
 Constant flow rates
 Large solvent capacities
Disadvantage:
 Pulsed flow creates baseline noise
Reciprocating Pumps

16.  Consist of large syringe like chambers equipped with a
plunger activated by a screw-driven mechanism powered
by a stepping motor.
 Displacement pumps also produce a flow that tends to be
independent of viscosity and back pressure
 Output is pulse free.
Disadvantage:
 Limited solvent capacity
 Considerable inconvenience when solvents must be
changed.
 No gradient elution
Displacement Pump (Syringe Type pump)

17.  Mobile phase is present in a collapsible container
housed in a vessel which can be pressurized by a
compressed gas.
 Here the pressure from a gas cylinder delivered
through a large piston.
Advantages:
• Pulse free flow
• Cheap
Pneumatic Pump (constant pressure pumps)

18. Disadvantages:
• Limited capacity
• Pressure output and flow rate depend on solvent
viscosity
• No gradient elution
• Pressure output is < 2000 psi.

19.  For injecting the sample through the column
 Minimize possible flow disturbances
 Limiting factor in precision of liquid chromatographic
measurement
 Volumes must be small: 1-500 L
Sample Injection Systems

20.  Used to remove the particulate matter & contaminants
from the solvent.
 Removes sample components that irreversibly binds to the
stationary phase.
 Packing chemically identical to analytical column.
 Particle size is large, so no pressure drop.
 When become contaminated, it is replaced with new one.
 Sacrificed to protect more expensive analytical column &
improves column life
Pre-column (Guard column)

21. Pre-column

22.  Column is the heart of HPLC separation.
 Columns are constructed of heavy-wall; glass-lined metal tubing
(restricted to pressure <600 psi) or stainless steel tubing.
 Withstand high pressures and the chemical action of the mobile
phase.
 The columns are usually 10-30 cm long, 4-10 mm inside diameter
and particle size is 5 μ.
 Contain 40000 to 60000 plates /meter.
 Recently high speed, high performance column with i.d. range
from 1-4.6 mm and packed with 3 or 5 μ, length is 3 -7.5 cm.
 Contain 100000 plates/meter.
Analytical column

23. Characteristic of the ideal detector-
 Adequate sensitivity.
 Good stability and reproducibility
 Not sensitive to change in temperature and mobile phase composition.
 Should have minimal internal volume in order to reduce zone
broadening.
 Nondestructive of sample.
 Wide linear dynamic range
 Short response time
 More Properties of Detector
 High reliability and ease of use
 Similarity in response toward all analytes
 Selective response toward one or more classes of analytes
Detectors

24.  Types of Detectors-
Two types:
 Bulk property detectors: Respond to a mobile phase bulk
property, which is modulated by presence of solutes.
e.g., refractive index, dielectric constant or density
 Solute property detectors: Respond to some property of solutes
 Like UV absorbance, fluorescence or diffusion current that is not
possessed by the mobile phase.
Detectors

25.  Can be used to isolate and purify compounds for further use.
 Can be used to identify the presence of specific compounds in a sample.
 Can be used to determine the concentration of a specific compound in a sample.
 Can be used to perform chemical separations
 Useful in determination of enantiomers
 Useful in determination of Biomolecules
 Isolation and purification of biologically active natural products
 Control of synthetic reactions - Identification of intermediates and target
compound.
 Biosynthesis study - Detection of biogenetic intermediates and enzymes
involved.
 Control the microbiological process
 Used for separation of antibiotic from broth mixture
HPLC Applications

26. THANK YOU