Biochomical Techniqu

O Pape ehomatognapy
Tuin loyea chaormalogaphy
HPLC

ELLSA

Chemu umineiceuce ummoaky

365
 CHROMATOGRAPHY
Colour SAQ
wrtng
By Mikhail Tsvet
-

on
seperdt-ton of
plant pyman
Moblle phose -Chlorophyll
Butanol
Solvent Whatmann
filter
paper
Statono
phase
(inert medium)

Water adsorbed
on cellulose of Solvent system
lter poper ,B : A : D peclche
spo
by weak Butono Cdertdhed by
in teractbns 4:1 S *Dstiled Calculahm
water
CH-bond. Acetc
Acid RF-value
Vcnderwabl doras)
overt Mobile
Liquid liguid Cfoper)
phose) Chromatography
Somple (gets seperated on the bass of
their aHnity with
their Alumina &
olsbiliky with solvent system)
Alunina |Stationany phase 7
Ah03)
ord)
Compounds move soluble
in solvent system will euuke
Out dint.

Ltuid- Solid (Column)

romato g0phy
366
* Des
-

Tt i6 a technique or
procadune dor Sepertuhion g
Similar Compounds by i& Continuous e-distribution
between two phases i.e. takionay phose
-
& Mobile phase.
Compound has move ebenbion power (ahrit toward
ess solubil Stahonay
phose

Retomed
TfCompound ha less elenbron power
moe solubiüty

moves in (solvent)
upuard direchion
Classihcation
ChvomatogTapny
Based on Mobile
phase
iquid Based on
Gas
Stahono
hase
Solid iquid Solid Liquid
iquid-solid Liqud Gas.Solid Gos-Lquid
chivomatogophy Chvty.
chromahogrophy
liqud
exColum Chromatogrophy
chromatography
with Alwina
apey
chromaography
later absoTbed on
Inert medtum actr
as Statfonay phose

367
*Baat Princpleg
-

Mechanisms
involved n
o
Physico-cheucal ompounda
seperation o compound ih
CrromatoOphy
ADSORPTZON
T is a
Surtace phenomenon n which
onme
molecule sadovbed on another
- Compouo
Based on Adsorption pouer Compoundg ne
5eperated.
Alumina (adsovbent) in Columo cromatoaraphy
Compounds which Qve Less adsorbed, ae eluted
out.
Mainy n olid- Liquid Chomatography.

2 PARTITION:
- depends on solubitty towords mobile phase
Move solilble in kie mobil phase dcster movement
Less soluble in mobile phase Retamed
Mainly iquid-Gqusid Chomasogrophy.
in
-

e aper Chromcography
Tnin- layer Chromatography (Stkta qol)
HPLC (High fevdormance
quid Comcitogaphy)
MOLECULAR SIEWING
Columns packed wih Gel
LHas beads with pores
- Depends on she shope ot parhical
-Large porrcles will elute out fast.
Smaller partcles will b e reained in beads
-
also known as Gel
Gel-Rlbrahion Chromatogrophy Beada
 ToN-ExcHANGE:-CHROMAToGRAPY:"
Column s Paced with Rassins.
Exchange fmakerral occu bet Racsn
Compounds on the bosts o chavge
LT is dependønt on Tonization.
LTuwo types o Raisns
Cation-exchange Anion-exchange
Raisins Rastns
Retain posibvely Rebain negatively
Charoed Charged
ons ons

AFeINITY CHROMATOGRAPHY:
Columns are packed wi h specahc ligonds
or SubstroaBe (in cose ot
enyme) or
Anbgen&
Specichic to Antibody
Substrabe

Applicattons
& Thin - layer Chromatography
Ohper Chramatography
-

cuhron
seperaron o-TAmino
o acds
LReductng suga
-Rurichrcotion o Caubohydraes Also,
Scr-eening, dor
rotein To-borne-emo
lipids omotobolrm.
Nucleic adds
Enumes
- Drugs
ett
method dor measurement o
HPLC- Gold tandard Method)
analytes. (rimay Raserenca
of Drugs. Vitamns, Homone. 369
Measuring, conc.
eneymes, ekt
 Paper Chromatography
*
Types
Ascendin Descendan Radial
- The solvent
- The solvent
m
2-Dimensional
w
System will he soluet - The solvert

move n. System usil System wil System un

move n ove n allowed & the
upward
downword Radol it otated
divection. dneetion dhecbion by 90.

9
AscendinX
Paper
chromathogTaphy Descending&
Pape
Adv Chvomatograpy
Better esol -Ady
becauee two Tt equres
doce act Less hme
(GraviyT+
Japilloy acion)

Principle: Liquid Liquid Chomatogrophy

Stohono
phase Mobile
phase
Woer-adsovped
on Colludose
olvent
y weak inkevachon system

370
Steps
Satrate the chamber with solvent ssten
For amno aotla,
8 A: D
4: 1:S
kanp the chamber
k paeked avemight
Cut Wlattman dtlter paper
n appropridbe sze

Wash it 0l NHCa & allou it

dy in Hot oven.
D
(t will
alv
clear all împuvites preret
on &lte poper

Mork Son above lower edge o

dil ter paper with a pencil.
(Dont use pen because ink of pen will
move wih solvent system)

Apply Sampla(2-S ul)

with help of Mitropippette S om
OY Capilloy tube

Dip the louwer edge nto solvent

& hong the papev in
Chomotogaphy
hambe
Allow it to Tun dor l0-t2 hm tll it eaches upper
mark with pe»ol bovder

Dy& Spray with Nnhydnm

371
 HPLC- SAQ
HPL.C
High eormanee igud nomaogophy
(Pessu).
- Mobile phase Liquid

HPLC
Sdvent honaht
Reserv
diagrom) M DIsplay
Ressue ample HPLC Detector
Qauz yechon Coumn
&Rump ysten wth
Cinlet) Adsorbet
materfaM
mall diamekey
acked with smalue
Poricle
- HPLC ollous

Ihe
pvincple of -

Adsorption
- Partitio
- Gel-ilteration depending
pending
on mateial
Lon- exchange used
Afnity
x Ady
-Very tost ( High pessure applied)
- Beter Resoluhdn

4Applicatrun:
LGold stondod/Primay Referenc Method
dor measure ment df al analyles.
Vitamins
Drug3
Hormones
Enymes
Amine 372
ea.
 0S. Ninhydm X-amino auds

Hydrindantm
+Ninhydin
Ruhemann
Purple

pobr visualred
Cabculate RF value

Ratio o Fronk
Distance travelled by solukt
by Solvet

RF value is Specichc
CRoO)fronh)
Relatve Hou)
Recognite Amino aid

Retovdahion daco)

Appicobiong
O Seperahion o Amino acids & Neducing suGors
Rrihicahion o ditlevent compounds
Metabolism
To dekeck Tn-borne-Errorr of

s obsoleke.
*Now, pe Comatogrophy

373
*TLC
Thin-Loyey Chromatoqrephy
Similar to that of, Paper Chonaogropy
excapt in place o popeY,
J
Theve s Glass plate ushtth has
Ihin-Layeed ahrorbent mabeia.

Thin-layevad
od aiorbent

AdyFast (vequire 1-2 hm)

Bebter Resoubion.
Jses
Same 0s Paper
Chromatography.

374
 CHEMILUMINESCENCE
IMMUNOASSAY Spontaneou
emission
Chemical

Agncio D
detechion
- Ag- Homones

Proteins
Peptides
Gytokiles
Thterleukin
Ab Vival-speciche
Backenad-specikc
-

Tt is a Ultra-sesitive & Ultra-speciic

technique for meosurement
eshimation)
ot onalytea

With use
o
Ag-Ab We defermine concenbahon
ert various Anticens Antibodieg

ypesDhect
-Indhect
L'Sandwit"
Advantogg
(deteck upto l0 moles)
UltrasenSitive
pecfic as Ag & Ab involved)
Eosy dov cutomahon
No vdiahon hozords
Longer halt-Like/Sheltlike of Rangon

*ELISA chaQper than IA.

375
Substrode
- Various suhstrakes[ Chomicals oe:
Chomical Ttself cauges Gmisoon of Lght-

Acidinium Hho
eSters
Excited Ground
State state

lighb
Delecked by
wminométer

Though vaous enymes Catalyst:

Substrades: Enyme Oxidoa
- Lwmnol -
-Ha02
HRP

Isoluminol
- AMPPD
-ALP
- Mitro-oxidase
Hypachlonite
- Oxygen

- Metol

HhOL Excted Ground

wmino HRP stale stnke

ght

detecked by
Lwminométer.
Electrochemluminesconce
- Emission photon by Ung elecodas
-ubstrae Rutheniuwm tribipymdne
=

Ru (B)3 Ru(8 R(BR"

hotone
lioo-parhicles ane adrovbesl
on suvaca o elecrooles
- Ady dekcked by
wnanoméka.
Moe pluhib 1o molea. 376
Ptnciple step
YYYYY mioobihe
Cweu
Antibodies
immobilized on solid surdace a
mioro-tihe uwell
pecthc &ov Aa to be delecled) lolyvinylechlovide
Blystyrene
Anolyte
Ada (
Analyte

WaghAdd Conjugaked
Biobin-labelled Ab

Wash AdoComplexHoneRad
beptavidine, Peaxido4e
Adol
Suhstoke:
LUMINOL
Anen
Sandwiched
Lwninol excted.
e h v fo Gound stake
bewpen two
Abs

Photons ae veleased Called

Sandwrch
mmunooBroy
Detected bel
By
Lumano metor ( Conc. d
analyte) 377
* LUMINOMETER :
-
Tt is an instrument which dalecb Jaht energ
Tt has 3 pavr
Light Tight Chamber -Holds the solukion
Tnecton system - dor addition of reagent
tube
Detecbor -has photpmuulhpUer
dotech
phohons
* Most common -
andwich CUTA 7.T. TSH
FSh. LM
Applicodions CIA (Rrolactin.et
O Detechon o Antigons lephidss, Homones. ek
Detecbon of Anbbodies Vialin, Backead int., ebt
Meosuement ot
ATP Specidt enymes
o m die Ay
uciderin +AT+0 Luiere, Oxylucikerin
Mg Oryluciderin
+ AMP + CO
Esbimahon o
Hinases Uke Ceahine kinose)
+Photons f
Determinahon o Conc. od co-enymes

NADH
NADPH
Can be uwed dor dolection
ovamoUs analytes in which
there produchon of
NADM& NADPM

378
ELISA-Enzyme Linked Immunosorbent Assay RE

SAQ Immunoa 3say

Depends an
ORL4
Ag-Ab reachbng

Used dor detechon o

H - oteins, peptides,
HoYmones
Bactenal/ Viral Aa
Ab against vaous
-

indehious pavhtles
-t is Quaitahive 0 ued o uanitahve.
Can be used dor
Measuement aff
Analytej
+Type: ELSA RIA

CLIA

*Disadvanta ges o
- Radiotion Hozards
RIA-{Radio-Iwmuno Asay)
-

horter shel-Lile aë reaqenbr

These Disadvantages
ave overcomy by Elz9
*n ELZSA,
-

No Radiohon Hazardr
-Longer Sheld-lile of
eaqen
Ensy ouomabon specickic
seitive 4
qually
*Commonly used ¢or detechon r
-T3.T4.TSH
- HZV 379
SARS COV-2
ELTSA
- Tt is an Tmmuno-chemical method based on
based on
eacnon in whith one oE the
Ag- Ab
immunoeagent is immobiused on a solid-phse
Support rrough Pasive adsorphion

methods ddr
Caphuring A
aciely,adsorbed As
Dhect Tndiect
mphne capu
ELISA:
Enymes used h
- Horseraddish feroxidase (1C)
ALP
B-Glucosidase
-Gucose oxidose
Achylcholine Esease

Linking Substance to antibodies

used 6ov linking enymes
e Glutaraldehyde
Perio doke
Melamine
Sorbent
adsorbed on solud-suppors via pasive
adovphon
blyvinylchbmde
Pblystynena

380
 *
ypeg on 69A
Based on Debecbon technique wed:

Diecb Thdoech Qondwch Compekibive

- Deteckr Ag
Dekeckr B Detacb Aa Debeebr s
Ab Small Aq

aka
*Huhstrates (od inteyt Ab Primoy
TMB (Tebramethy
\8eruicline
A

Ued Thhibition
ELTSA
Ady dor
iy Amino salicylic acd TSH
1OPD (Ortho-phenlare
Dfamin
Amplibtahon deler dor daterminahon
igno4 o
Azino didiammonium Used do T &T4
Salt
datechon
Ab again Sbedobelled Ag

Compebhdn
bet senum Aq
Detected *Avidin-Biotn Chønuhy & labelled A
Spechophomebically AbBioin tAndin
*T$, Puoescent Conc.
Subshak ed Seun Colou
Sub. Roduct
Fuoeswnce maosurtod
Ummuno Asay
FZA)
Delecteod by
Furomeler

381