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Presented By -

 
Faculty
Nirmala College of Pharmacy
Muvattupuzha, Kerala
India
Email ± jacobshaise@gmail.com
 ] iquid 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.
]
he 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.

]
he 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:
he modern form of liquid chromatography
is now referred to as ³flash chromatography´
 Four types of high performance liquid
chromatography (HP C):
] partition
] adsorption (liquid-solid)
] ion exchange
] size exclusion or gel
„ improved performance
„ high pressure
c
 Separation is accomplished by
partitioning b/w a M.P & Stationary
column material.

 

small, uniform particle
gives high column efficiencies
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to achieved desired flow rates
 R c


Based on Modes of chromatography
  
S.P is polar
M.P is non polar
!" 
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
3. Ion pair ³
4. Gel permeation / Size exclusion ³
5. Affinity ³
6. Chiral phase ³
Based on elution technique
Isocratic separation
Gradient separation
 Based on scale of operation
Analytical HP C
Preparative HP C
Based on the type of Analysis
Qualitative analysis
Quantitative analysis
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 "  
Ƅ Capable of handling ³macromolecules´
Ƅ Suitable for pharmaceutical compounds
Ƅ Efficient analysis of liable natural products
Ƅ Reliable handling of inorganic & ionic species
Ƅ Dependable analysis of biochemical's
PRINCIP E
m

Particle size of the S.P material plays a
crucial role in HP C
Micro particulate column packing :
Silica particles ĺ uniform, porous, with
spherical or irregular shape
Diameter ĺ 3.5 to 10 µm
 HP C instrumentation comprises:
1. M.P reservoirs
2. Eluent degas module
3. Solvent delivery pumps
4. Manual / Auto injector
5. Analytical column
6. Detector
7. Data processor
 Mobile phase reservoir
stores M.P (HP C grade solvents)
Ƅ 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
Ƅ 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.)
SO VEN
DE IVERY PUMPS
Reciprocating pumps:
» widely used
» maintain accurate flow rate

 
  

# 

 

 Solvent delivery systems
two types: 1. Isocratic system
2. ow pressure gradient
3. High pressure gradient
Injection system
a. Syringe system:- results best
b. Injection valve :- [Rheodyne injector]
» oading 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
c     
 
» 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


5-10 µm in d.m
 
 
Porous & 40 µm in d.m
$% 
S.P bonded onto an inert support
 DE
EC
ORS
1. UV DE
EC
OR : Based on UV light ab.
> fixed W detector (254nm)
> variable W detector (190-600nm)
2. REFRAC
IVE INDEX DE
EC
OR :
Non specific / Universal detector
Ļ sensitivity & specificity
3. PHO
ODIODE ARRAY DE
EC
OR (PDA)
similar to UV detector, non destructive
190-600 nm for quantization & identification
Spectra is 3D, Response vs time vs W

m &
 Flourimetric detector
Excitation & emission W can be selected
Ĺ sensitive than UV
Disadvantage: Some comp. are not fluorescent
Conductivity detector
based on electrical conductivity
Amperometric detector
Reduction / oxidation
 RECORDERS & IN
EGRA
ORS
] 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 HP C is water
Millipore Milli-Q apparatus produce water
 
  
Non polar & moderately polar comp. ĺ
ADSORP
ION CHRO.
Highly polar molecules by ĺ R.P Chro.
Acids & Bases by ĺ Ion exchange Chro.
 m 'mR'(()c

± Pharmaceutical field
± Chemical & Petrochemical industry
± Forensic
± Biochemical separations
± Food analysis
Qualitative analysis
Checking the purity of a compound
 Quantitative Analysis
&
 

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