isocratic rp-hplc.. using n-propyl alcohol - principle for elution?? (Aug/18/2007 )
i m trying to understand the principle for an isocratic elution for reverse phase hplc.. using a phosphate-propyl alcohol buffer. could u tel me the basis of binding and elution in such cases.
reverse phase works by hydrophobic interactions with the sample. isocratic elution is performed in a medium which is near strong enough to release the sample from the matrix. as you pass the medium through the matrix the sample will bind loosely and will be knocked off the matrix and rebind and repeat. the more hydrophobic the sample, the stronger it will bind and the later it will elute.
i hope i made it clear.. its like i have got this method which is rp yet isocratic.. i dont understand how that can work.
the method is on a c4 rp column.. with an isocratic elution with phospate-IPA buffer. so wud the protein bind by rp principles? or not bind.. as the binding n elution conditions r same.. as it is isocratic? or does it depend on the size of the protien rather than its hydrophobiciyt here?? i m trying to understand whats happening in the method.
the reason i feel that such a method is there.. cos my protien has low recovery on only rp-acetonitrile gradient methods. cud that b a reason??
lemme know quickkk.. i have losst my sleep over it now!! and there isnt much literature available too on this.
isocratic reversed phase is a binding method. the binding is weak and can be released by the force of the flow of the medium. in fact, you can change selectivity by changing flow rate.
there are some books and pamphlets available from various sources.