How we can analysed unknown sample by hplc....
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First take ir of that sample and judge the functional
goroups attached to that compound and also structure of
that compound.
then check solubility.
then UV scan with known concentration and find lymda max.
then selection of column.
first try with regular solvent like water, acn,
methanol.and then go to the buffers of different pH.near to
its pka value.
also different coloumn.
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first check the structure of the compound.identify the
chromophore groups(for UV Detector),refractive index status
(for RI Detector),reactive status with other chromophore
group compounds(Derivitation method for UV.then check the
solubility status of the compound.prepare a known
concentration solution.
In HPLC select a C18 Column which can serve better for the
polar and not polar compounds.select a buffer pH Oof +/-1
unit of pka value of the compound prepare a buffer mix with
some 20% of organic solvent to chnge the elution early
(based on polarity)
inject the prepared known concentration sample solution in
to above system by observing the chromatogram procced
further by altering buffer
pH,COLUMN,FLOW,TEMPERATURE,ORGANIC RATIO Sample size etc.
but to quantify the unknown concentration we need a known
concentration of standard.
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