NTRODOCDAN

Chonnalogaapy ihcatty depenol

soluuhon on tw availahih
Hh Tuis gi
Qcoloumnpadangs nmall ueage paxhclo bge
to #he
a wwmum peak boaolimuna on hu Colovmn ouwng
aint Relahousep betuoeen tea heorehical plat hegat pasanetes,H
Onmo pachil s2e. Petbrmans auud Csomagvaply qralu
bnnall
Cplot dolourmn paskmgs wit awesoge dllammels as as

vaita
0 um. ttouwunta , u L Kuauine uiaplesmae bystene o
OMgac 6oluenk auo a e gnerall inste to loiua lovo sanple

Loaongs. To prouide a biocwmpakiba Luglr Ausolub' sepuakio

matu

biopdsymers, ucluolhing prokeiy, Fast Pota auuid Clonaoaph,

uoas atecd. FPLC pouides u pull Aauge q Caloakogaapl
mools Buch s ton xchoamge , luuomabfpuwng. al Peaahou
vphopik mtaachons auA ANeRse phase bageo ou pashces otp
aNcaage oomta sgu w asimnla rauge as kose used ph tPL

8pecottpus

PRINCIPLE
Baseol u e 7ropeub ne tosgel to 6epesalo pvcteins, FPLO
works on 6annt pncipes as 6i2eCrclusco toa - xebhange

an anty Chtoatoghaplhy Duwg ge exclusin, a colomn

2ge beads poRous glass gnanles altowek o eguelibriats
uwt a &olveb (soue) Butable fpr a moleules to be sepezated

Sutl au eapui wbratad Coloomn, ha lasgee moleuules pass aoug

d unaRBtal épacu betioau tha beads. Tuu us betause Hhe
Pokes q he gel haue bmalea diantee h a n Lokab w nakcl

h a lasge. roleuilu to eutr.

otans a aluo be 6eptaled b iou-Ccage hute
protns
excamg MAVeRsib oth os elutstukicall bouol to inek
6uppoae mecium h govetmung ockos iu fon-exchauge itachous
t e etcto stake fos 9 atthackiw, uouie tun olepeds main
ou the Rulakiua. charge , he adiue q he uydiatiol ious auud the
dege non-bouong intetachons. Smu poteis ae Capobl
pobwng Dof pos+Ena amd ntgae Clargee they Can bind b bot
anien amo Caton excamges bouoevea thiy cam be maol to btaa
MMe neqakie Casgu by wnciasi he pt, o4 beaa posku
Catge b dutwg t pH. Foa exauple, the olsied qotins
maybe made o behae as a cahon by louoeuig t pH a d
uohe
Chromaographuo ow a caho extuonget voîuemoe ma
the amionie proteuw speues

Prvteks ,speuiallg egms bind. to ucepbas épuuipeally anmd tis

ropea a pe fity s wcd to 8epeaato potuns KOn

a xke pooteis Contamang he ngme b be pudhid alloved

fo percolake haoug &uch a column n y t h e duud uum,

uoniet ús Capabe g buoda 8 p c a y to tu immobiigec a a c ,
otu he etasdrd by the matrixAU othe moleuules , oue hau
no sptdflha foa + gaue l pas douou aud out the
Coloumn. 4te at tu undushel Componuuk hae ben usked out
n the uoasl bolutow altuas
4h coloomn , the ConolHns ase

6o Hhakr Ha u g a otissockats from th lzannd and ubs fom

h e Colovnm

NSTRDMENTAT OA
to a HPLG, Ha base iubrumunkatn pr FPLC is
Stni lar
t H &me The majoa companeula neudd to peafm PPLC
) SAToNARY PHAs Tuoo mai forme mabi/stahiouay
phase mateual a cuailoke , based m a ad &old
stuehse
Eoth foms mvolve opproxmalay sphurial paahiels
7a umomm ige
to minimuse space or oliuoa amd hca bomd
bioadutg lo
u. e an
mad witolly nmaolifieo sdlia o tysane
hesun Composed o- ked
agoto beads uo a a ustae
gauds.
a)MOBLE PHASE - Mosty bufeas, ongame 8olventk aue used
as he mobile phase. s csubial thak tu mobile pBhase is

olgassed because gassung h eluunk Cam altea tw colovmn

Agolukion and wmtesfre oth Couaous mondhoa2 t eluab.

8)POMP- Constauk Conboled dlouo by petsbaltic pumps. Th

ouo 1alt vasie based on sal a pAparahoa e aalyheal
ot pAupatabiue kromagnaphy

) MIXGR St poLoeRed aud Conbrolltd by Hhe Pump

espeuallyPe vey importout uoheu toamuna anadieuti betuoem
bufa80uug Tu m Xea onsus hat h bufes sed axe i tu
CoAieu propoabiou Relahiue to eal othe duung Cowe the FPL
Ruum

5)UNSECTLON VALUG The most Common metuod sampe

mbrodiuit u by the ue aLoop weute Ts Coa a mba
toop o émall volume that cam be leo uoit tta kane.T
eluewb from h
pump tn Canneled haoug hu oop y
meams a ola Buottelung &ysann and the sampe shuol o n

h Coovmn uoithoub teanuphiou loo q elueul

 COLOMNL - Th materals, ulated to FPLC, wsd foa

seperati ae ticatiy nmade of wwest ptashc uinert Puuid

Sufaess k e Teplon , titnniwrm mnd glass. Coluns are pauked

w t bmall posticls0 gel beads uo a ollanater 3-78pp.

aud haut
hey a duignnd t operake at not mose thau S8opi
a Lasge t n a l oliamulie. Te cdumns ouls be shnk o4f
hauol 1 H20 ohn n o m
use

RACToN COLLELTDR - A Fed vouume g he elual s colleetad

frachous and s amayed o r th pusena g t h pnahd

6ample

FLon RESTRICToR Genaak

back psAL o a
&ady
pAavet he oAnakiow n a-bubles, as use buhses hindea the
amalysit amd Chuales brood peaks.

DETeCTOR Vasable uoautluugli olteutass a bassd upou

vLvavcolut - visibe specbophotbrmebngihey haue a aleteuho 6uitiut
o k d e 6 xe m mo a ineas Komge Usually t e

pe deteuton method ued dpends ou the amakyto n inteees

Oto detehas dommonly used ae Conoluthius oletewn and
Rerac Suder Detecto

PPLIC AloAG

aPokw amaly
b) poprotuw 8cperato
PuAcaton amwal venonm
) Seperaton nplasma pmbens Daina anol Cuubrospinal u d
PIAGRAMoFTHEEPLC2NSTRLYMENIATIO

ButerA Buter B

MIxER

Sample
loop insechin volve

Colunn
waste

Men tDy

Fvachon
colecor

Ssoloktou 9 pophobilnopn olanminaus po human utiiocy

Diago n B+alasaumia
Raps pualfcakon PNA
Analysus 9 ntrguous Conshituenla n beo

uot loo pmuns and th auailahil a oide nange tolumn

betauw n t h u e Lovo p i u u s h dustem o seperal protens uoit

aAnak souhon s Fasb Protem/Perfpmana Liauwd

Chsomabaqrp