Principle Ion chromatography

ion chromatography

ion-exchange chromatography separates molecules based on respective charged groups. ion-exchange chromatography retains analyte molecules on column based on coulombic (ionic) interactions. essentially, molecules undergo electrostatic interactions opposite charges on stationary phase matrix. stationary phase consists of immobile matrix contains charged ionizable functional groups or ligands. stationary phase surface displays ionic functional groups (r-x) interact analyte ions of opposite charge. achieve electroneutrality, these inert charges couple exchangeable counterions in solution. ionizable molecules purified compete these exchangeable counterions binding immobilized charges on stationary phase. these ionizable molecules retained or eluted based on charge. initially, molecules not bind or bind weakly stationary phase first wash away. altered conditions needed elution of molecules bind stationary phase. concentration of exchangeable counterions, competes molecules binding, can increased or ph can changed. change in ph affects charge on particular molecules and, therefore, alters binding. molecules start eluting out based on changes in charges adjustments. further such adjustments can used release protein of interest. additionally, concentration of counterions can gradually varied separate ionized molecules. type of elution called gradient elution. on other hand, step elution can used in concentration of counterions varied in 1 step. type of chromatography further subdivided cation exchange chromatography , anion-exchange chromatography. positively charged molecules bind anion exchange resins while negatively charged molecules bind cation exchange resins. ionic compound consisting of cationic species m+ , anionic species b- can retained stationary phase.

cation exchange chromatography retains positively charged cations because stationary phase displays negatively charged functional group:

r-x


−




c


+



+



m


+





b


−


⇄



r-x


−




m


+



+



c


+



+



b


−




{\displaystyle {\text{r-x}}^{-}{\text{c}}^{+}\,+\,{\text{m}}^{+}\,{\text{b}}^{-}\rightleftarrows \,{\text{r-x}}^{-}{\text{m}}^{+}\,+\,{\text{c}}^{+}\,+\,{\text{b}}^{-}}

anion exchange chromatography retains anions using positively charged functional group:

r-x


+




a


−



+



m


+





b


−


⇄



r-x


+




b


−



+



m


+



+



a


−




{\displaystyle {\text{r-x}}^{+}{\text{a}}^{-}\,+\,{\text{m}}^{+}\,{\text{b}}^{-}\rightleftarrows \,{\text{r-x}}^{+}{\text{b}}^{-}\,+\,{\text{m}}^{+}\,+\,{\text{a}}^{-}}

note ion strength of either c+ or a- in mobile phase can adjusted shift equilibrium position, retention time.

the ion chromatogram shows typical chromatogram obtained anion exchange column.