An introduction to High Performance Liquid Chromatography (HPLC)

1. HIGH PERFORMANCE (PRESSURE) LIQUID
CHROMATOGRAPHY
PRESENTED BY:
STUTI SRIVASTAVA
M.PHARM
Ist YEAR

2. HPLC
High-performance liquid chromatography (i.e. HPLC; formerly
referred to as high-pressure liquid chromatography)
Is a technique in analytical chemistry used to:
 Separate
 Identify, and
 Quantify
each component in a mixture.

3.  High performance liquid chromatography is basically a
highly improved form of column chromatography.
 Instead of a solvent being allowed to drip through a
column under gravity, it is forced through under high
pressures of up to 400 atmospheres. This makes it much
faster.
 It also allows us to use a very much smaller particle size
for the column packing material which gives a much
greater surface area for interactions between the
stationary phase and the molecules flowing past it.
 This allows a much better separation of the components
of the mixture.

4.  The other major improvement over column
chromatography concerns the detection methods
which can be used.
 These methods are highly automated and extremely
sensitive.
 HPLC has been used for many purposes, such as:
 Manufacturing
 Legal
 Research
 Medical purposes,etc.

5. PRINCIPLE
 The main principle is ADSORPTION.
 The process involves the interaction of the
compounds in the analyte (which travels
along with a mobile phase) across an
immobile surface ( stationary phase).
 The compounds bind at a specific region of
stationary phase based on certain physical
and chemical properties.

6.  These bound molecules are then eluted with a
suitable buffer and the same are collected with
time.
 The analyte particles are separated on the basis
of:
 Polarity
 Charge
 Molecular weight
 Presence of functional group

7. TYPES OF HPLC TECHNIQUES
1.) Based on modes of chromatography:
a) Normal phase mode:
Stationary Phase is polar.
Mobile Phase is non polar.
b) Reverse phase mode:
Stationary Phase is non polar.
Mobile Phase is polar.

8. 2.)Based on elution technique:
a)Isocratic separation: In this mode of analysis,
the composition of the mobile phase does not
change during an analysis (i.e. the composition is
constant), the method is said to be isocratic.
b)Gradient separation: In this mode of
analysis, the mobile phase composition is altered
during the analysis – normally by increasing the
amount of organic modifier.

9. INSTRUMENTATION
1. MOBILE PHASE (SOLVENT) RESERVOIRS
2. ELUENT DEGAS MODULE
3. SOLVENT DELIVERY PUMPS
4. MANUAL / AUTO INJECTOR
5. ANALYTICAL COLUMN
6. DETECTOR
7. DATA PROCESSOR

12. APPARATUS AND MATERIALS
COLUMNS
Made up of stainless steel.
Withstand pressure 5.5*107 Pa.
Dimension:-
20-50 cm in length and 1-4 mm in width.
Porous plug of stainless steel or teflon used in ends
to retain the packing material.

13. COLUMN PACKING
• Particle size of the Stationary Phase material plays
a crucial role in HPLC.
• Silica, Sepadex, alumina, Zipax ANH, Zipax PAM
may be used for packing the columns.
• Particles must be uniform and porous, along with a
spherical shape.
• Diameter → 3.5 to 10 µm.

14. MOBILE PHASE RESERVOIR
• Stores Mobile Phase (HPLC grade solvents).
• Determines the resolution & Speed of analysis .
• Choice of solvent is based on the Flow rate, polarity
& pH of Mobile Phase.
• Isocratic separation made by using single solvent.
• Gradient separation may be done by use of various
suitable solvents.
• We may use diethyl ether, methylene chloride, and
chloroform individually or together with hexane.

15. ELUENT DEGAS MODULE
Gassing is the presence of air bubble in the solvent.
Dissolved gases in Mobile Phase pose a number of
problems such as alteration of column resolution,
interference with continuous monitoring, excessive detector
noise, result fluctuations, etc.
Degassing can be carried out by:
 Warming the solvent.
 Using magnetic stirrer to stir the solvent.
 Subjecting the solvent to vacuum.
 Ultrasonic vibrations (converts ultra high frequency
to mechanical vibrations).
 By bubbling helium gas through the solvent
reservoir.

16. SOLVENT DELIVERY SYSTEMS
2 types of solvent systems may be used:
1. Isocratic system
2. Gradient system:
a) Low pressure gradient
b)High pressure gradient
A pulseless flow through the column is produced, pumps
operate by the introduction of high pressure gas in the
pump chamber, gas in turn forces the solvent from pump
chamber into the column.

17. INJECTION SYSTEM
o Syringe system:- Results best.
Loading through the sample loop (20-
50µl)

18. DETECTORS
Desired Detector Characteristics:
 High sensitivity and reproducibility.
 Respond to all solutes.
 Response unaffected by changes in
temperature and mobile phase flow .
 Respond independently of the mobile phase.
 Reliable and convenient to use .
 Nondestructive to the solute.
 Fast response.

19. Some common detectors used are:
 UV-Visible spectrophotometer.
 Fluorescence detectors.
 Electrochemical detector.
 Radioactive detectors.

20.  UV-VISIBLE DETECTORS: The UV-visible
absorbance detector is the most common HPLC
detector in use such as PMT, PDA, etc.
 FLUORESCENCE DETECTORS:
Fluorescence detectors (FL) measure the optical
emission of light by solute molecules after they
have been excited at a higher energy wavelength
and can be very sensitive for compounds that have
native fluorescence or that can be made to
fluoresce through derivatization.

21.  ELECTROCHEMICAL DETECTOR:For
compounds that can be oxidized or reduced, the
electrochemical (EC) detector is one of the most
sensitive and selective HPLC detectors available.
 RADIOACTIVITY DETECTORS:
Radioactivity detectors (sometimes referred to as
radiometric or radio-flow detectors) are used to
measure radioactive analytes as they elute from
the HPLC column.

24. COLUMN EFFICIENCY AND SELECTIVITY
The column efficiency depends on partition coefficient,
K, where K is given by the relation :
K = conc. Of solute in stationary phase/conc.
Of solute in the mobile phase
Efficiency of the column is:
 Directly proportional to: Column length,
Resolving power, No. of theoretical plates per unit
area and Surface area of adsorbent material.
 Inversely proportional to : Particle size

25. HPLC OFFERS NUMEROUS ADVANTAGES
a) Capable of handling “micromolecules”.
b) Suitable for pharmaceutical compounds study.
c) Efficient analysis of liable natural products.
d) Reliable for handling of inorganic & ionic species.
e) Dependable analysis of biochemicals.
f) Time saving method.
g) Extremely sensitive.

26. APPLICATIONS
 LEGAL: This technique is also used for detection
of illicit drugs in urine.
 MANUFACTURING: It is a common technique
used in pharmaceutical development, as it is a
dependable way to obtain and ensure product purity.
 FOOD: For the estimation of food sample to
contain Vitamins, antioxidants, sugars, amino acids,
cholesterol, dyes, antioxidants, residual pesticides,
etc.

27.  MEDICAL: Medical use of HPLC can include drug
analysis, while urine is the most common medium for
analysing drug concentrations, blood serum is the
sample collected for most medical analyses with
HPLC.
 RESEARCH: Similar assays can be performed for
research purposes, detecting concentrations of
potential clinical candidates like anti-fungal and
asthma drugs.
 Assay.
 Analytical method validation.
 Stability studies.
 Compound identification.