HIGH PERFORMANCE(PRESSURE)
LIQUID CHROMATOGRAPHY
HPLC
Presented By Mr. Shaise Jacob
Faculty
Nirmala College of Pharmacy
Muvattupuzha, Kerala
India
Email – jacobshaise@gmail.com
• Liquid chromatography is a separation
technique that involves:
• the placement (injection) of a small volume of
liquid sample into a tube packed with porous
particles (stationary phase)
• where individual components of the sample are
transported along the packed tube (column) by
a liquid moved by gravity.
• The components of the sample are separated from
one another by the column packing that involves
various chemical and/or physical interactions
between their molecules and the packing particles.
• The separated components are collected at the
exit of this column and identified by an external
measurement technique, such as a
spectrophotometer that measures the intensity of
the color, or by another device that can measure
their amount
• Note:The modern form of liquid chromatography
is now referred to as “flash chromatography”
Four types of high performance liquid
chromatography (HPLC):
•
•
•
•
partition
adsorption (liquid-solid)
ion exchange
size exclusion or gel
◊ improved performance
◊ high pressure
HPLC- Separation is accomplished by
partitioning b/w a M.P & Stationary
column material.
Packing material
small, uniform particle
gives high column efficiencies
High pressure
to achieved desired flow rates
Types of HPLC techniques
Based on Modes of chromatography
1. Normal phase mode:
S.P is polar
M.P is non polar
2. Reverse phase mode:
S.P is non polar
M.P is polar
Different columns used: ODS,C18,C8,C4…
Based on principle of separation
1. Adsorption chromatography
2. Ion exchange “
3. Ion pair
“
4. Gel permeation / Size exclusion “
5. Affinity
“
6. Chiral phase “
Based on elution technique
Isocratic separation
Gradient separation
Based on scale of operation
Analytical HPLC
Preparative HPLC
Based on the type of Analysis
Qualitative analysis
Quantitative analysis
HPLC offers numerous advantages
♠ Capable of handling “macromolecules”
♠ Suitable for pharmaceutical compounds
♠ Efficient analysis of liable natural products
♠ Reliable handling of inorganic & ionic species
♠ Dependable analysis of biochemical's
PRINCIPLE
Adsorption
Particle size of the S.P material plays a
crucial role in HPLC
Micro particulate column packing :
Silica particles → uniform, porous, with
spherical or irregular shape
Diameter → 3.5 to 10 µm
HPLC instrumentation comprises:
1. M.P reservoirs
2.
3.
4.
5.
6.
7.
Eluent degas module
Solvent delivery pumps
Manual / Auto injector
Analytical column
Detector
Data processor
Mobile phase reservoir
stores M.P (HPLC grade solvents)
♠ Resolution & Speed of analysis }
Flow rate, polarity & pH of M.P
Can't use metallic reservoir
Eluent degas module
Dissolved gases in M.P pose a number of problems
∆ flow
∆ excessive detector noise
∆ Rt fluctuation
♠ Bubbling the pump & detector,
Degas module with reservoir of inert gases
He or N2
1. Vacuum filtration
2. Helium purging
3. Ultrasonication (converts ultra high
frequency to mechanical vibrations.)
SOLVENT DELIVERY PUMPS
Reciprocating pumps:
» widely used
» maintain accurate flow rate
Cross-sectional diagram of a simple single – piston reciprocating pump
Solvent delivery systems
two types: 1. Isocratic system
2. Low pressure gradient
3. High pressure gradient
Injection system
a. Syringe system:- results best
b. Injection valve :- [Rheodyne injector]
» Loading through the sample loop (20-50µl)
u
c. Automated injection device :commercially available, automatically
inject 100samples
Guard column
Pre-filter :- useful for industry
Analytical column
Heart of any chromatographic system
» Actual separation of components takes place
Several S.P available
 depending upon tech. or mode of separation
Column material
S.S, glass, polyethylene, PEEK
Column length Column diameter Particle size
5-30 cm
2mm-50mm
1µ-20µ
Particle nature:
Spherical, uniform sized porous material
Surface area
1g S.P provides surface area 100-860 sq.m
Functional group
Depends on the type of chromatographic
separations.
Normal phase mode: hydroxy group
Reverse phase mode: C18 (octa decyl silane)
Bondapak ( waters)
C8 octyl column, C4 butyl column, CN Nitrile column
NH2 Amino column
b
Column packing
three forms
1.Microporous support
5-10 µm in d.m
2. Pellicular
Porous & 40 µm in d.m
3.Bonded phase
S.P bonded onto an inert support
DETECTORS
1. UV DETECTOR : Based on UV light ab.
> fixed WL detector (254nm)
> variable WL detector (190-600nm)
2. REFRACTIVE INDEX DETECTOR :
Non specific / Universal detector
↓ sensitivity & specificity
3. PHOTODIODE ARRAY DETECTOR (PDA)
similar to UV detector, non destructive
190-600 nm for quantization & identification
Spectra is 3D, Response vs time vs WL
Photodiode Array Detector
Flourimetric detector
Excitation & emission WL can be selected
↑ sensitive than UV
Disadvantage: Some comp. are not fluorescent
Conductivity detector
based on electrical conductivity
Amperometric detector
Reduction / oxidation
RECORDERS & INTEGRATORS
• Recorders – to record the responses
• Integrators - data processing capabilities
• ◊ record individual peaks with Rt, height, width
of peaks, peak area, % of area..
Selection of solvent systems
Solvent compatibility with the detector
e.g.. Hexane, chloroform, ACN , Methanol…
Most widely used solvent in HPLC is water
Millipore Milli-Q apparatus produce water
Selection of Column
Non polar & moderately polar comp. →
ADSORPTION CHRO.
Highly polar molecules by → R.P Chro.
Acids & Bases by → Ion exchange Chro.
APPICATIONS OF HPLC
♥ Pharmaceutical field
♥ Chemical & Petrochemical industry
♥ Forensic
♥ Biochemical separations
♥ Food analysis
Qualitative analysis
Checking the purity of a compound
Quantitative Analysis
Direct comparison method
injecting the sample & std. separately &
comparing their peak areas.
Area of the peak = peak height x width of peak at
half height
Calibration curve method
Multi component analysis
Isolation & identification of drugs
Stability studies