3. HPLC> Is the powerful tool in the development and production of
pharmaceuticals. High performance Liquid chromatography is an
incredible useful analytical technique with a broad range of
applications . HPLC involves a column of adsorbent material (Such as
silica beads ) through which a sample is passed under pressure .
.Each component of the sample Mixture will interact differently with
material in the column and as such have a different flow rate . As a
result , the various components will leave the Column at different time
and can be collected separately .
4. PRINCIPAL OF HPLC
.The principal of HPLC is to force the sample through the Column of the
stationary phase by pumping the mobile phase at high pressure . The to
be analyzed is introduced small volume to the stream of mobile phase
and the sample molecules are retained by specific chemical and
physical interaction with the material of the stationary phase as it
travels the length of the column . The amount of retention depends on
a) The nature of the sample
b) The nature of the composition of the stationary phase and
c) The nature of the composition of the mobile phase
5. DETAILS
>Mobile phase : commonly used solvent are acetonitrile ,methanol and
water .
.Acidifier , buffer solution are used for better separation.
>Stationary phase:
.particle size range is 3.5= 10Um
1)two type of Stationary phase :
Normal phase : silica gel
Reversed phase :ODS silica gel
6. >Columns:
>Two types of columns are used:
i) Normal phase columns
ii) Reversed phase columns
Columns is made up of stainless steel
Internal diameter is 4-5mm(usually 4.6)
Detectors : Bulb property detector, Mass property detector ,
Multipurpose detector.
7. INSTUMENTATION OF HPLC
>Degassing System
>Pump solvent delivery system
>check valves
>Pulse damper
>Pre – column
>Guard column
>Sample injection part
> Column
8. DESAGGING SYSTEM
Sparging / Bubbling
>vacuum filtration
>Ultra sonication
2) Pump solvent delivery system:
>The pump are used to pass mobile phase through the Column at high
pressure because the particles that are used pack HPLC column are small
enough ie : <50um and also particle size of packing material is 5-10um
9. PUMPS
>Generation of pressure upto 5000psi
>flow rate ranging from 0.1 to 100ml/min
>It should be composition resistant and give a pulse free Out put
2) Pumps are thus categories into
i) Mechanical pumps
.Displacement pump
.Reciprocating pumps
ii) Pneumatic pumps
10. MECHANICAL PUMPS
Displacement pump:
>working: I ) Work on the principal of Positive solvent pressure
Ii ) Consist of screw or plunger Which revolve continuously driven by
motor .
Iii ) Rotatory motion provides continuous movement of the mobile
phase which is propelled by the revolving screw at greater speed and
pushes solvent through small needle like outlet .
iii) Consist of large syringe like chamber of capacity 250-500 ml.
11. RECIPROCATING PUMPS
>Working:
I) Pressure from gas cylinder delivered through a large piston drivers the
mobile phase .
II) Pressure on the solvent is proportional to the ratio of piston usually
50:1.
III ) Check Valves :
.they are used to control the flow of solvent and back pressure.
IV ) pulse damper :
Pulse are used to dampen or reduce the reduce observed from the way
baseline caused by pumps.
12. APPLICATION OF HPLC
I) Quality control testing of drugs
II) In qualitative and quantitave analysis
III) Separation and control of impurities
IV) In analysis of biological fluids
V) Stability studies
VI) Industrial application
VII) Stability studies ex : Acyclovir
VIII) Determination of synthetic intermediate Ex : atenolol
