Chromatographic Techniques of Anaylsis

Chromatographic  Techniques  T echniques of Anaylsis Anaylsis

Dr. Anant R. Kapdi

Historical Signicance Liquidliquid e!tractions "ere #no"n for many years. e.g. $e%&g solution in ' & HCl "here $e forms $eCl! salt is rst treated treate d "ith ethyl acetate. &g "hich acts as an interferent (ecause of higher a)nity for "ater goes into "ater phase* "hile $e is e!tracted into ethyl acetate (y thoroughly thorough ly sha#ing the mi!ture of t"o immisci(le sol+ents.

Historical Signicance Liquidliquid e!tractions "ere #no"n for many years. e.g. $e%&g solution in ' & HCl "here $e forms $eCl! salt is rst treated treate d "ith ethyl acetate. &g "hich acts as an interferent (ecause of higher a)nity for "ater goes into "ater phase* "hile $e is e!tracted into ethyl acetate (y thoroughly thorough ly sha#ing the mi!ture of t"o immisci(le sol+ents.

Chromatography ,t is the partitioning of components of a sample according to their distri(ution coe)cient (et"een a mo(ile phase and a stationary phase. Stationary phase is al"ays a solid surface such as silica &o(ile phase represents the sol+ents. Chromatography "as rst disco+ered (y &i#hail Ts"ett. Calcium car(onate used as the stationary phase for separating coloured pigment from from plant e!tract using pet. ether as mo(ile phase.

 The "ord comes from -ree -ree# # "ords Chromos "hich means colour and graphy "hich means to "rite.

Classication of chromatographic separations

a/hysical state of mo(ile and stationary phase &o(ile phase0 i liquid Column chromatography* Thin layer chromatography* High performance liquid chromatography ii -as -as chromatography Stationary phase0 i solid Column* TLC ii liquid lm coated on solid surface -C* H/LC iii /orous gel Si1e e!clusion chromatography 2ðod of contact (et"een the mo(ile and stationary phase 2Chemical or physical mechanism for separation i Adsorption chromatography0 Column* -C ii /artition0 H/LC

 Thin Layer Chromatography 3Adsorption chromatography &o(ile /hase0 liquid 4 Stationary phase0 solid adsor(ent 3silica

So+ents used0 D&$5&e6H5DC&57t6Ac57t 865/et ether5He!ane5pentane Sol+ent system0 polar 7t6Ac

0

nonpolar sol+ent He!ane or /et. 7ther

7t86  

&e6H

/entane He!ane

-radient elution0 ,ncrease concentration of one sol+ent to elute +ery closely places solutes.

Relation (et"een distance tra+elled (y sol+ent and the su(stance is gi+en (y

e.g.

 TLC preparation process

/reparing the Cham(er /reparing /lates for De+elopment De+eloping the /lates ,dentifying the Spots ,nterpreting Data http0%%""".chem

Stain de+elopment of TLC plates :9light0 for most organic compounds ,odine0 for unsaturated compounds /hosphomoly(dic acid Dinitrophenyl hydra1ine0 aldehydes and #etones 9anillin0 general organic compounds K&n6;0 sensiti+e groups to o!idation Cerric ammonium moly(date

6n a TLC plate Rf +alues o(tained "ere *