HPLC is a chromatographic technique used to separate components of a mixture. The key components of an HPLC instrument are the pump, injector, column, and detectors. The pump forces the mobile phase through the column while the injector introduces the sample. Various detectors can then analyze the separated components as they elute from the column. HPLC is widely used in fields like biochemistry and pharmaceutical analysis for applications such as quantifying drug purity and identifying unknown compounds.

1. INSTRUMENTATION OF HPLC
Presented by:
SK.SAMIYA,
Dept. of Pharmacology.

2. CONTENTS
 Introduction
 Types
 Instrumentation
 Advantages
 Disadvantages
 Applications

3. INTRODUCTION
 HPLC- High Performance Liquid Chromatography
Or
High pressure liquid chromatography
Definition:
 It is a chromatographic technique used to separate components of
mixture for the purpose to identify, quantify or purify the individual
components of the mixture.
 This is widely used in field of biochemistry and analytical chemistry.

4. Types of HPLC techniques
 Based on mode of chromatography
 Based on principle of separation
 Based on elution technique
 Based on scale of operation
 Based on type of analysis

5. Based on modes of chromatography
Normal phase mode:
 The stationary phase is polar in nature & the mobile phase is non-
polar.
 This is not advantageous in pharmaceutical application since most of
the drug molecules are polar in nature and takes longer time to be
eluted and detected.
Reverse phase:
 The stationary phase is non-polar & the mobile phase is polar in
nature.
 Since most of the drugs and pharmaceuticals are polar in nature, they
are not retained for a longer time and eluted faster.

6. Based on principle of separation
 Adsorption chromatography: Separation of components takes place
because of the difference in affinity of compounds towards stationary
phase.
 Ion exchange chromatography: An ion is used to separate a mixture
of similar charged ions.
 Size exclusion or gel permeation chromatography: A mixture of
components with different molecular sizes are separated by using gels
which acts as sieve.

7. Based on elution technique
 Isocratic separation: In this technique, the same mobile phase
combination is used throughout the process of separation. The same
polarity or elution strength is maintained throughout the process.
 Gradient separation: In this technique, a mobile phase combination
of lower polarity or elution strength is used followed by gradually
increasing the polarity or elution strength.

8. Based on the scale of operation
 Analytical HPLC: Where only analysis of the samples are done.
Recovery of the samples is not done
 Preparative HPLC: Where the individual fractions of pure
compound can be collected using fraction collector. The collector
samples are reused.

9. Based on the type of analysis
 Qualitative analysis: Which is used to identify the compound, detect
the impurities, to find the number of components, etc
 Quantitative analysis: Which is done to determine the quantity of
the individual or several components in a mixture. This can be done
by comparing peak area of the standard and sample

10. INSTRUMENTATION
HPLC instrument consists of following components:
 Pump
 Mixing unit
 Solvent degassing
 Injector
 Column
 Detectors
 Application

13. PUMP
 The role of the pump is to force a liquid ( called the mobile phase)
through the liquid chromatography at a specific flow rate, expressed
in milliliters per min (mL/min)
 Normal flow rates in HPLC are in the 1-2mL/min range.
 During the chromatographic experiment, a pump can deliver a
constant mobile phase composition (isocratic) or an increasing mobile
phase composition (gradient).
 Best for the analysis of complex samples.

14. Types of pumps:
Mainly three types:
• Constant flow reciprocating pump
• Syringe type pump
• Pneumatic pump

15. Constant flow reciprocating pump
 The term reciprocating describes
any continuously repeated
backwards and forwards motion.
 Widely used type of pump.
 Solvent is sucked during back
stroke and gets deliver to the
column in forward stroke.

16. Syringe or displacement type pump
 Consists of large syringe like chamber.
 Suitable for small bore column.

17. Pneumatic pump
 Gas is used to pressurize the mobile phase present in a collapsible
solvent container.

18. MIXING UNIT
 Mixing unit is used to mix solvents in different proportions and pass
through the column.
 There are two types of mixing units.
- they are low pressure mixing chamber which uses helium for
degassing solvents.
- high pressure mixing chamber does not require helium for degassing
solvents
 Mixing of solvent is done either with a static mixer which is packed
with beads or a dynamic mixer which uses magnetic stirrer and
operates under high pressure.

19. SOLVENT DEGASSING
 Several gases are soluble in organic solvents.
 When solvents are pumped under high pressure, gas bubbles are
formed which will interfere with the separation process, steady
baseline and the shape of the peak.
 Hence degassing is necessary.

20. This can be done by using following techniques:
 Vacuum filtration:- which can remove air bubbles, but it is not always
reliable and complete.
 Helium purging:- By passing helium through the solvent. This is very
effective but expensive.
 Ultrasonification:- By using Ultrasonicator, which converts ultra high
frequency to mechanical vibrations. This causes the removal of air
bubbles.

21. INJECTOR
 The injector serves to introduce the liquid sample into the flow stear
the mobile phase.
 Typical sample volumes are 5-20microliters
 The injector must also be able to withstand the high pressure of the
liquid system.
 An auto sampler is the automatic version for when the user has many
samples to analyze or when manual injection is not practical.

22. Types of injectors:
1) Septum injectors: For injecting the sample through a rubber septum.
This is not common, since the septum has to withstand high pressure.
2) Stop flow: In which the flow of mobile phase is stopped for a while and
the sample is injected through a valve device.

23. 3) Rheodyne injector: It is the most popular injector. This has a fixed
volume loop like 20µL or 50µL or more. Injector has 2 modes, i.e., load
position when the sample is loaded in the loop and inject mode when
the sample is injected.

24. COLUMN
 It is the heart of the chromatograph
Column length: varies from 5cm to 30cm
Column diameter: ranges from 2mm to 50mm
Particle size: from 1µ to 20µ
Particle nature: spherical, uniform sized, porous materials are used.

26. Materials of construction for the tubing:
 Stainless steel ( the most popular, gives high pressure
capabilities)
 Glass (mostly for biomolecules)
 PEEK (poly ether ether ketone) polymer (biocompatibility and
chemically inert to most solvents).
Packing material:
 The packing material is prepared from SILICA particle,
ALUMINA particle and ion exchange RESIN.
 Porous plug of stainless steel or Teflon are used in the end of the
columns to retain the packing material.

27. DETECTORS
 UV detectors
 Refractive index detector
 Flourimetric detector
 Conductivity detector
 Amperometric detector

28. ADVANTAGES
 Separation of volatile and non-volatile components
 Quick analysis
 High resolution
 More reproducibility
DISADVANTAGES
 High cost
 Complex to operate

29. APPLICATIONS OF HPLC
 Qualitative analysis
 Checking the impurity of a compound
 Presence of impurities
 Quantitative analysis
 Isolation and identification of drugs
 Isolation and identification of mixture of components
 Biopharmaceutical and pharmacokinetic studies
 Stability studies
 purification.