how do start hplc method development?
how much sample required for developing the method
development?
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Answer Posted / sushant
1.HPLC method development start with literature servey to
get rough idea abt development
2.then find out functionallity, molecular structure,
Polarity of particular API or molecule. from which we can
decide method development phase RPC or NPC.
3.Find out Solubility, Max UV absorbace so that we be
assure about respective diluent & wavelegh for method
development.
if we will not get UV absorbace then there is dervatisation
method.
4.Mostly many oragnic drug molecules are polar or mid polar
so we usually go for RPC.
5.satrt with mobile phase composition Use water & MEthanol
or ACN according to sollubillity in 50:50 ratio.
6.Select Column -start with C-18 column make HYpersil or
Inertsil(3 microne x 4.6 ID )10-15 cm
7. feed other parameter like wavelength,ambient Temp,
1ml/min Flow rate
8.About sample preparation for assay preparation use conc
in between 100-500 ppm as per peak response.
9.If there are more than 10 molecule in particular API go
for gardient method for better resolution & short run time
while developing any method always follow ICH parameter abt
Tailing factor, Capacity factor, Assymetry, thereotical
plates.Change MP composition ,Column.
Once you get better result then for sharpness or to save
time adjust flowrate & increase Temp.
10.If our desired peak isnot retain on water /ACN/Methanol
composition MP then go for Buffer. Phosphate buffer is
widly use buffer for its PKa value,easily Available, gives
more Hydrophillic effect, stable or non degradable for 2-3
days.
...............Before starting any method development on
HPLC Flush the system with water, check pressure, saturate
column with MP,check callibration status, lamp Hours.
Always keep data about whatever trials you have done during
method development.
Method Development is very challenging task so always be
logical before any changes in any parameter.
Mostly For HPLC method Development 10 gm sample is enough
but for overall development max 100 gm sample is sufficient.
Its a tentative quantity.
After method development method validation must be done.
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