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CHROMATOGRAPHY  CHROMATOGRAPHY  DEFINITION OF

DEFINITION OF CHROMACHROMATOGRAPHY TOGRAPHY 

• Science on the study of separation ofScience on the study of separation of

molecules based on dierences in their molecules based on dierences in their structur

structure and/ e and/ or composition.or composition.

• It originated from the Greek wordsIt originated from the Greek words

“chroma”

“chroma” meaning meaning “color”“color”, and, and “graphein”

“graphein” meaningmeaning “to rite”“to rite”..

• It was coined by the Russian botanistIt was coined by the Russian botanist

“Mi!hail "em#ono$ich T%$et &T%ett'” “Mi!hail "em#ono$ich T%$et &T%ett'” during his research about chlorophyll

during his research about chlorophyll where he separated the dierent plant where he separated the dierent plant pigments.

pigments.

• Used for Used for separation, identicaseparation, identication, andtion, and

determina

determination of tion of the chemicalthe chemical components in comple! mi!ture. components in comple! mi!ture.

• It in"ol"es passing a mi!ture dissol"ed in aIt in"ol"es passing a mi!ture dissol"ed in a

#mobile phase$ through a

#mobile phase$ through a #stationary#stationary phase$ which separates the analyte to be phase$ which separates the analyte to be measured from other molecules in the measured from other molecules in the mi!ture and allows being

mi!ture and allows being isolated.isolated.

• %US&' (ened as a procedure by which%US&' (ened as a procedure by which

solutes are separated by a dierential solutes are separated by a dierential migration process in a system consisting migration process in a system consisting of two phases, one of

of two phases, one of which mo"eswhich mo"es continuously in a gi"en direction and in continuously in a gi"en direction and in which the indi"idual substances e!hibit which the indi"idual substances e!hibit dierent mobility by reason of dierences dierent mobility by reason of dierences in adsorption, partition, solubility, "apour in adsorption, partition, solubility, "apour pressure, molecular si)e or ionic charge pressure, molecular si)e or ionic charge density.

density.

• It may beIt may be preparati$epreparati$e oror anal#ticalanal#tical

 Preparati$e Chromatograph#Preparati$e Chromatograph#

 It seeks to separate theIt seeks to separate the components of a mi!ture for components of a mi!ture for further use *and thus is a form of further use *and thus is a form of purication+.

purication+.

 Anal#tical Chromatograph#Anal#tical Chromatograph#

 It normally operates with smallerIt normally operates with smaller amounts of materials and seeks to amounts of materials and seeks to measure the relati"e proportions of  measure the relati"e proportions of  analytes in mi!tures.

analytes in mi!tures. PRINCIP(E" OF

PRINCIP(E" OF CHROMACHROMATOGRAPHY TOGRAPHY 

• It in"ol"es mo"ing a It in"ol"es mo"ing a preparpreparation of aation of a

materials to be separated %test materials to be separated %test

preparation' o"er a stationary support. preparation' o"er a stationary support.

•  he molecules  he molecules in the test prin the test preparations eparations willwill

ha"e dierent reactions with the ha"e dierent reactions with the stationary support.

stationary support.

•  his will  his will lead to sepalead to separation of #siration of #similar$milar$

molecules. molecules.

• (ierent types of molecules can be(ierent types of molecules can be

separated from each other, as they mo"e separated from each other, as they mo"e o"er the support material.

o"er the support material. ) PHA"E" IN CHROMATOGRAPHY* ) PHA"E" IN CHROMATOGRAPHY*

"tationar# Pha%e "tationar# Pha%e

 -i!ed phase %may be porous or-i!ed phase %may be porous or nely di"ided solid or a liuid that nely di"ided solid or a liuid that

has been coated in a thin layer on has been coated in a thin layer on an inert supporting

an inert supporting material.material. Mo+ile Pha%e

Mo+ile Pha%e

&ure liuid or gas or mi!ture of &ure liuid or gas or mi!ture of solutions that mo"es through or solutions that mo"es through or o"er the !ed phase.

o"er the !ed phase.

TERMINO(OGIE" TERMINO(OGIE" Anal#te Anal#te

 he substance

 he substance to be separto be separatedated during

during chromatogrchromatographyaphy Chromatogram

Chromatogram  he "isual outp

 he "isual output/ resulut/ result int in chromatography

chromatography E,-ent

E,-ent

It is the mobile phase lea"ing the It is the mobile phase lea"ing the column

column

It s the phase which mo"es in a It s the phase which mo"es in a denite direction

denite direction

It may be a liuid, a gas, or a It may be a liuid, a gas, or a supercritical uid

supercritical uid

Is a characteristic time it takes for Is a characteristic time it takes for a particular analyte to pass through a particular analyte to pass through the system *from the column inlet the system *from the column inlet to detector+ under set conditions to detector+ under set conditions Stationary phase is solid

Stationary phase is solid 0obile phase is liuid or gas 0obile phase is liuid or gas

Retention and separation depends Retention and separation depends on the ability of the atoms on the on the ability of the atoms on the surface to remo"e analytes from surface to remo"e analytes from the mobile phase and adsorb them the mobile phase and adsorb them temporarily by means of

temporarily by means of electrostatic forces electrostatic forces 1lso known as

1lso known as “%i0e e/cl-%ion”“%i0e e/cl-%ion”,, “gel permeation”

“gel permeation”, or, or “gel“gel 1ltration”

1ltration”

Stationary phase is a polymeric Stationary phase is a polymeric substance containing numerous substance containing numerous pores of molecular dimensions pores of molecular dimensions 0obile phase is a liuid or gas 0obile phase is a liuid or gas Retention and separation depends Retention and separation depends on the

on the dierentdierential migration ofial migration of solute molecules based on solute molecules based on molecular si)e

molecular si)e

Stationary phase is liuid Stationary phase is liuid 0obile phase is liuid or gas 0obile phase is liuid or gas

Retention and separation occur due Retention and separation occur due to the relati"e solubility of the

to the relati"e solubility of the analytes in the two uids as analytes in the two uids as determined by their partition determined by their partition coe2cients coe2cients PRINCIP(E" OF "EPARATION PRINCIP(E" OF "EPARATION Mo+ile Pha%e Mo+ile Pha%e Retention Time Retention Time A.%orption A.%orption Partition Partition Molec-lar E/cl-%ion Molec-lar E/cl-%ion Ion2E/change Ion2E/change

(2)

"tationar# pha%e

"tationar# pha%e is a polymeric is a polymeric matri! bonded with ionic

matri! bonded with ionic functionalfunctional groups *usually

groups *usually

%t#rene.i$in#l+en0ene pol#mer %t#rene.i$in#l+en0ene pol#mer++ Mo+ile pha%e

Mo+ile pha%e is always a liuid is always a liuid

Retention and separation is mainly due Retention and separation is mainly due to the

to the electroselectrostatic bonds with thetatic bonds with the functional groups

functional groups PRINCIP

PRINCIPA( O34ECTI5E" A( O34ECTI5E" OFOF CHROMATOGRAPHY 

CHROMATOGRAPHY 

Resolution of mi!tures into constituent Resolution of mi!tures into constituent parts

parts (etermina

(etermination of tion of homogeneityhomogeneity

3omparison of substances suspected of 3omparison of substances suspected of being identical

being identical &urication &urication

3oncentration of substances from dilute 3oncentration of substances from dilute solutions

solutions products products

4uantitati"e separation from comple! 4uantitati"e separation from comple! mi!tures

mi!tures

Indication of molecular structure Indication of molecular structure DIFFERENT TECHNI67E" OF DIFFERENT TECHNI67E" OF CHROMATOGRAPHY  CHROMATOGRAPHY  PRINCIP(E" OF "EPARATION PRINCIP(E" OF "EPARATION  

 echniues by echniues by 3hromatogra3hromatographic 5edphic 5ed Shape

Shape

3olumn

3olumn 3hromatogra3hromatographyphy &lanar 3hromatography &lanar 3hromatography

&aper 3hromatography &aper 3hromatography  hin 6ayer 3

 hin 6ayer 3hromatograhromatographyphy  

 echniues by echniues by &hysical St&hysical State of 0obileate of 0obile &hase

&hase 6iuid

6iuid chromatograchromatographyphy 7&63

7&63 "7PPORT" IN

"7PPORT" IN CHROMACHROMATOGRAPHICTOGRAPHIC PREPARATION" PREPARATION" Immo+ili0e. "ilica on Immo+ili0e. "ilica on Gla%% Plate% Gla%% Plate% Thin (a#er Thin (a#er Chromatograph# Chromatograph# 8T(C9 8T(C9 5

5oollaattiille e GGaa%%ee%% GGaa% % CChhrroommaattooggrraapphh## 8GC9 8GC9 P Paappeerr PPaappeerr Chromatograph# Chromatograph# (i:-i.%; Containing (i:-i.%; Containing H#.rophilic; H#.rophilic; In%ol-+le Molec-le% In%ol-+le Molec-le% (i:-i. (i:-i. Chromatograph# Chromatograph# THEORY OF CHROMATOGRAPHY  THEORY OF CHROMATOGRAPHY  •

• 8!ploits the dierences in8!ploits the dierences in partitioningpartitioning

+eha$ior

+eha$ior between9between9

 0obile &hase0obile &hase

 Stationary &haseStationary &hase

PHA"E" OF

PHA"E" OF CHROMACHROMATOGRAPHY TOGRAPHY  Mo+ile Pha%e

Mo+ile Pha%e

1 gas or liuid that passes through 1 gas or liuid that passes through the column

the column "tationar# Pha%e "tationar# Pha%e

1 solid or liuid that does not 1 solid or liuid that does not mo"e. It refers to the

mo"e. It refers to the chromatograp

chromatographic hic support.support. C(A""IFICA

C(A""IFICATION TION OF OF CHROMACHROMATOGRAPHICTOGRAPHIC METHOD" METHOD"   1dsorption1dsorption   63 63  7&637&63

 3olumn 3olumn 3hromatogra3hromatographyphy

 Ion:8!changeIon:8!change

 7&637&63

 12nity12nity

 (;1 12nity 3hromatography(;1 12nity 3hromatography

 8lectrophoresis8lectrophoresis

 Si)e 8!clusionSi)e 8!clusion

 Gel &ermeationGel &ermeation

 &artition&artition

 7&637&63

 &aper 3hromatography&aper 3hromatography 3A"I" OF INTERACTION"

3A"I" OF INTERACTION" OF AN ANA(OF AN ANA(YTE TOYTE TO THE "TATIONARY PHA"E

THE "TATIONARY PHA"E 3harge

3harge

Relati"e Solubility Relati"e Solubility

 he dynamics

 he dynamics of the solute of the solute particleparticle passes through the "oid spaces passes through the "oid spaces

between the stationary phase particles between the stationary phase particles in a system as well as its kinetics. in a system as well as its kinetics. %Giddings'

%Giddings'

HO< RATE THEORY AND P(ATE THEORY HO< RATE THEORY AND P(ATE THEORY <OR="

<OR="

 he mobile pha

 he mobile phase mo"es thrse mo"es through theough the stationary phase, picking up the stationary phase, picking up the compounds to be tested.

compounds to be tested. 1s the mobile

1s the mobile phase tra"els through thephase tra"els through the stationary phase, it takes the compounds stationary phase, it takes the compounds with it.

with it.

1t dierent points in the stationary phase, 1t dierent points in the stationary phase, the dierent components of the compound the dierent components of the compound are going to be absorbed, and are going to are going to be absorbed, and are going to stop %at a

stop %at a specic distance' mo"ingspecic distance' mo"ing through the mobile phase.

through the mobile phase. Identication and control of

Identication and control of technicaltechnical

Gas 3hromatography Gas 3hromatography

Surface 1dsorption Surface 1dsorption THEORIE" OF

THEORIE" OF CHROMACHROMATOGRAPHY TOGRAPHY  Plate Theor#

Plate Theor#  he chroma

 he chromatographic systographic system is atem is a series of discrete layers of theoretical series of discrete layers of theoretical plates. %0artin and Synge'

plates. %0artin and Synge' Rate Theor#

(3)

RETENTION RETENTION

DEFINITION OF RETENTION DEFINITION OF RETENTION

0easure of speed at which a substance 0easure of speed at which a substance mo"es in

mo"es in a chromatographic system.a chromatographic system.

In continuous de"elopment systems %7&63 In continuous de"elopment systems %7&63 < G3', where the compounds are eluted < G3', where the compounds are eluted with an eluent, it is e!pressed as

with an eluent, it is e!pressed as Retention Time 8Rt9

Retention Time 8Rt9..

In interrupted de"elopment systems %63 In interrupted de"elopment systems %63 < &3', it is e!pressed as

< &3', it is e!pressed as RetentionRetention Factor 8R>9

Factor 8R>9..

Retention >actor

Retention >actor is the run length of theis the run length of the compound di"ided by the run length of compound di"ided by the run length of thethe eluent front.

eluent front.

FORM7(A OF RETENTION FORM7(A OF RETENTION

 Rf 

 Rf 

==

 Distance

 Distancemoved by

moved by the

the compou

compound

nd

 Distance

 Distancemoved by

moved by the

the solv

solv ent 

ent 

 Rf 

 Rf 

==

 D

 D

11

 D

 D

22

"IGNIFICANCE ?

"IGNIFICANCE ? IMPORTIMPORTANCE OFANCE OF RETENTION

RETENTION

1 uantitati"e indication of how far a 1 uantitati"e indication of how far a

particular compound tra"els in a particular particular compound tra"els in a particular sol"ent.

sol"ent.

1 good indicator of whether an unknown < 1 good indicator of whether an unknown < a known compound are identical or similar. a known compound are identical or similar. APP(ICA

APP(ICATION" TION" OF OF CHROMACHROMATOGRAPHY TOGRAPHY 

• (i:-i. Chromatograph#(i:-i. Chromatograph#

   est water est water samples for samples for pollutionpollution

• Ga% Chromatograph#Ga% Chromatograph#

 5omb detection in 5omb detection in airportsairports

 Identify/ 4uantify (rugs < 1lcoholIdentify/ 4uantify (rugs < 1lcohol

 In -orensics, to compare bers foundIn -orensics, to compare bers found on a "ictim

on a "ictim

• Thin (a#er Chromatograph#Thin (a#er Chromatograph#

 (etection of (etection of &estic&esticides < ides < InsecticidesInsecticides in food

in food

 In -orensics, to analy)e dyeIn -orensics, to analy)e dye composition of bers

composition of bers

• Paper Chromatograph#Paper Chromatograph#

 Separation of 1mino 1cids and 1nionsSeparation of 1mino 1cids and 1nions

 R;1 -ingerprintingR;1 -ingerprinting

 Separation and testing of histamines <Separation and testing of histamines < antibiotics

antibiotics AD"ORPTION

AD"ORPTION CHROMACHROMATOGRAPHY TOGRAPHY  "tationar# Pha%e

"tationar# Pha%e Solid on which

Solid on which sample components aresample components are adsorbed

adsorbed

0ay be a liuid %liuid:solid' or a gas 0ay be a liuid %liuid:solid' or a gas %gas:solid'

%gas:solid'

e.g.

e.g. Col-mn Chromatograph# 8CC9Col-mn Chromatograph# 8CC9 << Thin (a#er Chromatograph# 8T(C9 Thin (a#er Chromatograph# 8T(C9 PARTITION CHROMATOGRAPHY 

PARTITION CHROMATOGRAPHY  "tationar# Pha%e

"tationar# Pha%e

6iuid supported on inert solid 6iuid supported on inert solid Mo+ile Pha%e

Mo+ile Pha%e Is a liuid

Is a liuid %liuid:liu%liuid:liuid partition' or aid partition' or a gas %gas:liuid'

gas %gas:liuid' e.g.

e.g. Paper Chromatograph#Paper Chromatograph#  

 ype of partitiype of partition chromatogron chromatography inaphy in which the stationary phase is a layer of  which the stationary phase is a layer of  water adsorbed on a sheet of paper water adsorbed on a sheet of paper Normal2Pha%e Chromatograph# Normal2Pha%e Chromatograph#

o

o &olar Stationary &hase %e.g.&olar Stationary &hase %e.g. <ater<ater

or

or MethanolMethanol''

o

o ;on:&olar 0obile &hase %e.g.;on:&olar 0obile &hase %e.g.

He/ane He/ane''

o

o  his fa"ors r his fa"ors retention of polaretention of polar

compounds and elution of non: compounds and elution of non: polar compounds

polar compounds

Re$er%e.2Pha%e Chromatograph# Re$er%e.2Pha%e Chromatograph#

o

o ;on:&olar Stationary &hase;on:&olar Stationary &hase o

o &olar 0obile &hase&olar 0obile &hase

CHROMA

CHROMATOGRAPHY TOGRAPHY  Ion E/change Re%in

Ion E/change Re%in

o

o &olymeric matri! with the surface&olymeric matri! with the surface

of which ionic

of which ionic functional groupsfunctional groups %e.g.

%e.g. Car+o/#lic Aci.%Car+o/#lic Aci.% andand Amine%

Amine%' ha"e been chemically' ha"e been chemically bonded.

bonded. 6iuid

6iuid

Pol#meric %-+%tance containing Pol#meric %-+%tance containing n-mero-% pore% o> molec-lar n-mero-% pore% o> molec-lar .imen%ion%

.imen%ion%

3ontain analytes that are sol"ated 3ontain analytes that are sol"ated molecules separated accordi

molecules separated according to ng to theirtheir si)e, by their ability to penetrate a si)e, by their ability to penetrate a sie"e:like structure

sie"e:like structure

De/tran 8"epha.e/9; Agaro%e De/tran 8"epha.e/9; Agaro%e 8"epharo%e9;

8"epharo%e9; oror Pol#acrilami.ePol#acrilami.e 83io2Gel9 B

83io2Gel9 B G86 -IG86 -IR1R1I=;I=; Mo+ile Pha%e Mo+ile Pha%e (i:-i.2(i:-i. Partition (i:-i.2(i:-i. Partition ION2E@CHANGE ION2E@CHANGE "tationar# Pha%e "tationar# Pha%e Mo+ile Pha%e Mo+ile Pha%e

0ethod of choice for inorganic ions and 0ethod of choice for inorganic ions and attempts of re"ersed phase method attempts of re"ersed phase method unsuccessful

unsuccessful

"IE E@C(7"ION CHROMATOGRAPHY 8"EC9 "IE E@C(7"ION CHROMATOGRAPHY 8"EC9

1lso called

1lso called Gel FiltrationGel Filtration oror Molec-lar2Molec-lar2 "ie$e Chromatograph#

"ie$e Chromatograph#

Mo+ile Pha%e Mo+ile Pha%e

e.g.

e.g. "o>t Gel%"o>t Gel%

"emi2Rigi. or Rigi. Gel% "emi2Rigi. or Rigi. Gel% "tationar# Pha%e

(4)

Pol#%t#rene; Gla%% 3ea.%; Pol#%t#rene; Gla%% 3ea.%; oror Al!#late. De/tran Al!#late. De/tran > G86> G86 &8R081I=; &8R081I=; Mo+ile Pha%e Mo+ile Pha%e 6iuid or Gas 6iuid or Gas

Used for the separation of solutes with Used for the separation of solutes with dierent molecular si)e

dierent molecular si)e

Used e!tensi"ely for the separation of Used e!tensi"ely for the separation of macromolec

macromolecules or ules or biological origin andbiological origin and for purication of

for purication of synthetic:organsynthetic:organicic polymers

polymers AFFINITY

AFFINITY CHROMACHROMATOGRAPHY TOGRAPHY 

Utili)es high specic interactions between Utili)es high specic interactions between one kind of solute molecule and a

one kind of solute molecule and a secondsecond molecule co"alently attached

molecule co"alently attached

%immobili)ed' to the stationary phase %immobili)ed' to the stationary phase Immobili)ed molecule can be an antibody Immobili)ed molecule can be an antibody to a p

to a particulaarticular proteinr protein TECHNI67E" I

TECHNI67E" IN PARN PARTITIONTITION CHROMATOGRAPHY 

CHROMATOGRAPHY  COMMON TYPE"

COMMON TYPE" OF PAROF PARTITIONTITION CHROMATOGRAPHY  CHROMATOGRAPHY  &aper 3hromatography &aper 3hromatography Gas:6iuid 3hromatography Gas:6iuid 3hromatography Gel 3hromatography Gel 3hromatography "PECIA( TYPE"

"PECIA( TYPE" OF PAROF PARTITIONTITION CHROMATOGRAPHY 

CHROMATOGRAPHY 

6iuid 3hromatography %63' 6iuid 3hromatography %63'

7igh &erformance 6iuid 3hromatography 7igh &erformance 6iuid 3hromatography %7&63'

%7&63'

Si)e 8!clusion 3hromatography %S83' Si)e 8!clusion 3hromatography %S83' 3olumn 3hromatography %33'

3olumn 3hromatography %33' TECHNI67E" I

TECHNI67E" IN PARN PARTITIONTITION CHROMATOGRAPHY 

CHROMATOGRAPHY 

=peration of a column %a tube lled with =peration of a column %a tube lled with adsorbent and sol"ent'

adsorbent and sol"ent' 1 solution containing the

1 solution containing the solute is layeredsolute is layered on top of the sorbent and is allowed to on top of the sorbent and is allowed to enter the sorbent

enter the sorbent  he sol"ent i

 he sol"ent is allowed ts allowed to pass continuao pass continuallylly through the column

through the column P

PARTITION COEFFICIENT ARTITION COEFFICIENT 8=98=9

If two phases are in contact with one If two phases are in contact with one another, and if one or both

another, and if one or both phases containphases contain a solute, the solute

a solute, the solute will distribute itselfwill distribute itself between two phases

between two phases It is the ratio of

It is the ratio of the concentrations of thethe concentrations of the solute in the two phases

solute in the two phases

 K 

 K 

==

Concentrationof solute

Concentrationof solute

∈∈

the

the

stationary phase

stationary phase

Concentrationof solute

Concentrationof solute

∈∈

the

the

mobile phase

mobile phase

MATERIA(" 7"ED IN PARTITION MATERIA(" 7"ED IN PARTITION CHROMATOGRAPHY 

CHROMATOGRAPHY 

3olumns containing a matri! that adsorb 3olumns containing a matri! that adsorb solutes

solutes

(iatomaceous 8arth %3elite' (iatomaceous 8arth %3elite' Silica Gel

Silica Gel

3ellulose &owder 3ellulose &owder

3ross:6inked (e!trans %Sephade! 67 3ross:6inked (e!trans %Sephade! 67 ?@'

?@'

7ydrophobic Sol"ent %5en)ene' 7ydrophobic Sol"ent %5en)ene' 7ydrophil

7ydrophilic ic Sol"ent %1lcohol'Sol"ent %1lcohol' 1lcohols < 1mides > for non:polar 1lcohols < 1mides > for non:polar materials

materials

&uried Aater > for polar materials &uried Aater > for polar materials

layer of solid particles layer of solid particles

P

PAPER CHROMATOGRAPHY APER CHROMATOGRAPHY 

can be can be

T<O2DIMEN"IONA(

T<O2DIMEN"IONA( CHROMACHROMATOGRAPHY TOGRAPHY  (ierent colors/ spots will appear on the (ierent colors/ spots will appear on the paper after ? hours %"ertically'

paper after ? hours %"ertically'  hen after

 hen after ? more hou? more hours and the rs and the paperpaper rotated C@ degrees, more colors/ spots will rotated C@ degrees, more colors/ spots will  hin 6ayer 3

 hin 6ayer 3hromatograhromatographyphy

Stationary phase created by suspending Stationary phase created by suspending the support or washing the column with a the support or washing the column with a proper sorbent

proper sorbent

0obile &hase 0obile &hase

P(ANAR

P(ANAR CHROMACHROMATOGRAPHY TOGRAPHY 

• 1 separation techniue in which the1 separation techniue in which the

stationary phase is present as or on a stationary phase is present as or on a plane

plane

•  he plane ca he plane can be a papn be a paper, ser"ier, ser"ing as suchng as such

or impregnated by a substance as the or impregnated by a substance as the stationary bed or a

stationary bed or a

spread on support such as a glass plate spread on support such as a glass plate TECHNI67E" IN

TECHNI67E" IN P

PAPER APER CHROMACHROMATOGRAPHYDE"CRIPTIONTOGRAPHYDE"CRIPTION  his method in"

 his method in"ol"es spotting thol"es spotting the samplee sample solution onto a

solution onto a strip of chromatographicstrip of chromatographic paper

paper

 he paper i

 he paper is placed ins placed into a Bar, cto a Bar, containing aontaining a shallow layer of sol"ent and then sealed shallow layer of sol"ent and then sealed 1s the sol"ent rises through the paper, it 1s the sol"ent rises through the paper, it meets the sample mi!ture, which starts to meets the sample mi!ture, which starts to tra"el up the paper with the sol"ent

tra"el up the paper with the sol"ent mi!ture tra"el dierent distances, mi!ture tra"el dierent distances,

according to how strongly it interacts with according to how strongly it interacts with the paper

the paper  his allows

 his allows the calculathe calculation of the Rftion of the Rf "alues, and compare with a standard "alues, and compare with a standard 1 method in"ented by the 5ritish 1 method in"ented by the 5ritish biochemists,

biochemists, Archer 4ohn Porter MartinArcher 4ohn Porter Martin and

and Richar. (a-rence MillingtonRichar. (a-rence Millington "#nge

"#nge

1n analytical techniue for

1n analytical techniue for separating andseparating and identifying mi!tur

identifying mi!tures that or es that or colored, especially pigments colored, especially pigments

(ierent compounds in the sample (ierent compounds in the sample

(5)

appear on the paper %either scattered or appear on the paper %either scattered or hori)ontally'

hori)ontally' DETECTION OF "POT"

DETECTION OF "POT" IN PIN PAPERAPER CHROMATOGRAPHY  CHROMATOGRAPHY  5y color 5y color 5y its uorescence 5y its uorescence

5y chemical reaction, after spraying the 5y chemical reaction, after spraying the spots with a "isuali)ing reagent

spots with a "isuali)ing reagent 5y

5y radioacti"iradioacti"ityty

IDENTIFICATION OF "POT"

IDENTIFICATION OF "POT" ON PAPERON PAPER CHROMATOGRAPHY 

CHROMATOGRAPHY 

3omparison with standards of known Rf 3omparison with standards of known Rf "alues

"alues

8lution > by cutting out the spot and 8lution > by cutting out the spot and soaking the paper in an appropriate soaking the paper in an appropriate sol"ent

sol"ent

PAPER CHROMATOGRAPHY  PAPER CHROMATOGRAPHY 

Used in the

Used in the identication of (igo!in, US&identication of (igo!in, US& Used in the separation of

Used in the separation of  

 risulfapyrrisulfapyrimidine (rugsimidine (rugs "tationar# Pha%e "tationar# Pha%e

It consists of a sheet of

It consists of a sheet of paper withpaper with controlled te!ture and thickness controlled te!ture and thickness usually made up of

usually made up of cell-lo%ecell-lo%e *lter*lter paper+ > which is polar

paper+ > which is polar Mo+ile Pha%e

Mo+ile Pha%e  he mobile phas

 he mobile phase used in pe used in paperaper chromatography may include any of chromatography may include any of the following9

the following9

0i!ture of alcohol and water with 0i!ture of alcohol and water with added ammonia or acetic acid added ammonia or acetic acid 3hloroform 3hloroform 5en)ene 5en)ene 3yclohe!ane 3yclohe!ane

Samples are applied as a solution in Samples are applied as a solution in "olatile sol"ent *usually in uantities of  "olatile sol"ent *usually in uantities of  @.D to D@@@ mcg+

@.D to D@@@ mcg+

Samples are applied to a strip of Samples are applied to a strip of chromatograp

chromatography paper at hy paper at origins usingorigins using capillary pipettes or microliter syringe capillary pipettes or microliter syringe *for accuracy+

*for accuracy+ -or

-or a%cen.ing chromatograma%cen.ing chromatogram,, the spots are applied at E:F cm the spots are applied at E:F cm from the lower edge

from the lower edge -or

-or .e%cen.ing chromatogram.e%cen.ing chromatogram,, the spots are applied at :C cm the spots are applied at :C cm from the upper edge

from the upper edge -or

-or ra.ial chromatogramra.ial chromatogram, the, the spots are applied on a circle with a spots are applied on a circle with a radius of D:E cm

radius of D:E cm

=ptimum si)e of spots "aries from E:H =ptimum si)e of spots "aries from E:H cm in diameter. 1dBacent spots should cm in diameter. 1dBacent spots should be ?:E cm apart.

be ?:E cm apart.  he paper is

 he paper is then dipped inthen dipped into ato a suitable sol"ent, taking care that the suitable sol"ent, taking care that the

spot is abo"e the surface of the spot is abo"e the surface of the sol"ent and placed in a

sol"ent and placed in a sealedsealed container.

container.  he sol"ent m

 he sol"ent mo"es up the papo"es up the paper byer by capillary action, which occurs as a capillary action, which occurs as a result of the attraction of the sol"ent result of the attraction of the sol"ent molecules to the paper.

molecules to the paper.

1s the sol"ent rises through the

1s the sol"ent rises through the paper,paper, it meets and dissol"es the

it meets and dissol"es the samplesample mi!ture which will then tra"el up the mi!ture which will then tra"el up the paper with the sol"ent solute

paper with the sol"ent solute samplesample (ierent compounds in the sample (ierent compounds in the sample mi!ture tra"el at dierent rates due to mi!ture tra"el at dierent rates due to dierences in solubility in the sol"ent dierences in solubility in the sol"ent and due to dierences in their

and due to dierences in their

attraction to the bers in the paper. attraction to the bers in the paper.  his usually

 his usually takes takes se"eral mse"eral minutes upinutes up to hours.

to hours.

 he components w

 he components which ha"e bhich ha"e beeneen separated dier in their

separated dier in their retentionretention factor or Rf "alues, which are then factor or Rf "alues, which are then computed using9 computed using9

 Rf

 Rf value

value

==

 Distancetraveled

 Distancetraveled

by

by sol

solute

ute

 Distancetraveled

 Distancetraveled

by

by solv

solvent 

ent 

chromatogra chromatographyphy

inert, at substrate %glass inert, at substrate %glass Silica Gel Silica Gel 1lumina 1lumina 3ellulose 3ellulose It consists of a thin

It consists of a thin layer of adsorbentlayer of adsorbent material immobili)ed onto at,

material immobili)ed onto at, inertinert carrier sheet.

carrier sheet.

  63 &lates 63 &lates "ample Preparation an. Application

"ample Preparation an. Application

E$al-ation o> Chromatogram E$al-ation o> Chromatogram

TECHNI67E" IN

TECHNI67E" IN THIN (AYERTHIN (AYER CHROMATOGRAPHY 

CHROMATOGRAPHY  THEORIE"

THEORIE"

Aidely used laboratory techniue, similar Aidely used laboratory techniue, similar to paper

to paper

Stationary phase used is a thin layer of Stationary phase used is a thin layer of adsorbent on an

adsorbent on an plate'9

plate'9

AD5ANTAGE" OF THIN (AYER AD5ANTAGE" OF THIN (AYER CHROMATOGRAPHY  CHROMATOGRAPHY  Rapid Rapid Sensiti"e Sensiti"e Simplicity Simplicity 3heap 3heap

THIN (AYER CHROMATOGRAPHY  THIN (AYER CHROMATOGRAPHY 

• "tationar# Pha%e &T(C Plate%'"tationar# Pha%e &T(C Plate%'



8!cellent Resolution 8!cellent Resolution

(6)

  63 &lates are made  63 &lates are made by mi!ing theby mi!ing the adsorbent, such as

adsorbent, such as %ilica gel%ilica gel with with a small amount of

a small amount of inert binder likeinert binder like calci-m %-l>ate &g#p%-m'

calci-m %-l>ate &g#p%-m' andand water.

water.

  he mi!ture is spr he mi!ture is spread as thicead as thickk slurry on an unreacti"e carrier slurry on an unreacti"e carrier sheet, usually

sheet, usually gla%%gla%%,, thic! thic! al-min-m >oil

al-min-m >oil oror pla%tic pla%tic, and the, and the resultant plate is then dried and resultant plate is then dried and acti"ated by heating in an o"en for acti"ated by heating in an o"en for E@ mins. at DD@3.

E@ mins. at DD@3.

  he thickness of the adsorb he thickness of the adsorbentent layer is typically around

layer is typically around 2)2) mm

mm for for anal#tical p-po%e%anal#tical p-po%e% and and around

around 2) mm2) mm for for preparati$epreparati$e T(C

T(C..

   o ensure o ensure the stationarthe stationary adhery adheres rmles rmlyy on the backing plate and does not on the backing plate and does not ake o during de"elopment,

ake o during de"elopment, +in.er%+in.er% are added to the

are added to the adsorbent.adsorbent.

 3alcium Sulfate %gypsum'3alcium Sulfate %gypsum'

 StarchStarch

 3arbomethylcellulose3arbomethylcellulose

• Mo+ile Pha%eMo+ile Pha%e

  he common sol"en he common sol"ents are tts are thehe following9 following9   7eptane7eptane   7e!ane7e!ane   IsooctaneIsooctane   3yclohe!ane3yclohe!ane   0ethanol0ethanol 

 1cetic 1cid1cetic 1cid

 AaterAater

 8thyl 8ther8thyl 8ther

  3hloroform3hloroform   1cetone1cetone   33l33lJJ     olueneoluene   5en)ene5en)ene   1cetonitrile1cetonitrile   &yridine&yridine 

 8thylene 3hloride8thylene 3hloride

  8thanol8thanol   I:&ropanolI:&ropanol   (io!ane(io!ane 

 8thyl 1cetate8thyl 1cetate

 -or mi!tures of unknown -or mi!tures of unknown composition,composition, +en0ene

+en0ene or or chloro>orm ith chloro>orm ith  ethanol

ethanol is the best sol"ent for is the best sol"ent for e!plorotatory runs.

e!plorotatory runs.

• "ample Preparation an. Application"ample Preparation an. Application

 (ierent compounds in the sample(ierent compounds in the sample mi!ture tra"el at dierent rates due to mi!ture tra"el at dierent rates due to the dierences in their attraction to the dierences in their attraction to the stationary phase and because of the stationary phase and because of dierenc

dierences in solubility in es in solubility in the sol"ent.the sol"ent.

 Separation of compounds is based onSeparation of compounds is based on the competition of the solute and the the competition of the solute and the

mobile phase for binding places on the mobile phase for binding places on the stationary phase.

stationary phase.

• "pecial Detecting Metho.%"pecial Detecting Metho.%

 CharringCharring

 In"ol"es the spraying ofIn"ol"es the spraying of

concentrated sulfuric acid and concentrated sulfuric acid and heating the plate

heating the plate

  he resu he result is seen lt is seen by the charby the charringring of the spots

of the spots

 7%e o> Io.ine 7%e o> Io.ine 5apor5apor

 In this method, In this method, the chromatogramthe chromatogram is placed in a closed container is placed in a closed container holding a few

holding a few iodine crystalsiodine crystals

  he sample s he sample spots reapots react with thect with the iodine "apor and form brown spots iodine "apor and form brown spots

  he reac he reaction is rtion is re"ersiblee"ersible

 E/amination -n.er 75 Ra.iationE/amination -n.er 75 Ra.iation

 Useful for compounds thatUseful for compounds that uoresce

uoresce

   wo UK light swo UK light sources arources are useful ane useful andd commercially a"ailable9 commercially a"ailable9 75 %hort2a$e &)nm' 75 %hort2a$e &)nm' 75 long2a$e &Jnm' 75 long2a$e &Jnm'

Preparation o> Deri$ati$e% Preparation o> Deri$ati$e% F7NCTIONA F7NCTIONA ( GRO7P" ( GRO7P" R REEAAGGEENNTT"" CCOO((OORR PROD7CED PROD7CED A

Accii..%% 33rroommccrree%%ooll Green Green  Y  Yelloello Al.eh#.e% Al.eh#.e% an. an. =etone% =etone% );2 );2 .initrophen#lh#. .initrophen#lh#. ra0ine ra0ine  Y  Yelloello Amine% an. Amine% an. Amino Amino Aci.% Aci.% N

Niinnhh##..rriinn FFll--oorree%%cceenn tt

A

All!!aallooii..%% MMeerrcc--rriic Nc Niittrraattee YYeelllloo t too 3ron 3ron 3ar+it-rate 3ar+it-rate % % Diphen#lcar+a0o Diphen#lcar+a0o ne ne P-rple P-rple Car+oh#.ra Car+oh#.ra te% te% Aniline Aniline Phthalate Phthalate Gra#23lac!  Gra#23lac!  (

(iippii..%% 33rroommtthh##mmooll 3l-e 3l-e (ight (ight Green Green "

"tteerrooii..%% AAnnttiimmoonn## Trichlori.e Trichlori.e

5ario-% 5ario-%

DETECTION OF "POT"

DETECTION OF "POT" ON THIN (AYERON THIN (AYER CHROMATOGRAPHY 

CHROMATOGRAPHY  5y its natural color 5y its natural color

(7)

5y uorescence 5y uorescence

5y spraying with "isuali)ation reagents 5y spraying with "isuali)ation reagents "PECIFIC "PECIFIC 5I"7A(IING 5I"7A(IING AGENT" AGENT" "PEICIFC "PEICIFC COMPO7ND" COMPO7ND" IDENTIFIED IDENTIFIED N

Niinnhh##..rriinn AAmmiinno o AAccii..%% R

Rhhoo..aammiinne e 33 ((iippii..%% An

Antitimomon# Cn# Chhloloriri.e.e "ter"teroioi.%.%; T; Tererpipinnoioi.%.% "-l>-ric aci. K "-l>-ric aci. K Heating Heating 7ni$er%al 5i%-ali0ing 7ni$er%al 5i%-ali0ing

Agent >or Mo%t Agent >or Mo%t Organic "-+%tance% Organic "-+%tance% Pota%%i-m Pota%%i-m Permanganate in Permanganate in "-l>-ric Aci. "-l>-ric Aci. H#.rocar+on% H#.rocar+on% Ani%al.eh#.e in Ani%al.eh#.e in "-l>-ric Aci. "-l>-ric Aci. Car+oh#.rate% Car+oh#.rate% 3

3rroommiinne e 55aappoorr OOllee11nn%% AD5ANTAGE" OF THIN (AYER

AD5ANTAGE" OF THIN (AYER CHROMATOGRAPHY 

CHROMATOGRAPHY 

• Great resol"ing power because spots areGreat resol"ing power because spots are

smaller smaller

• Greater speed of separationL 7igherGreater speed of separationL 7igher

resolution resolution

• Aider choice of materials as sorbentsAider choice of materials as sorbents •

• 8asy detection of spots8asy detection of spots •

• 8asy isolation of substances from the8asy isolation of substances from the

chromatogram chromatogram

DR7G" ANA(YED IN THIN (AYER DR7G" ANA(YED IN THIN (AYER CHROMATOGRAPHY  CHROMATOGRAPHY  • • 1nalgesics1nalgesics • • 1ntipyretics1ntipyretics •

• 1spirin, &henacetin, 1cetaminophen1spirin, &henacetin, 1cetaminophen •

• 1nti:Inamma1nti:Inammatory tory (rugs(rugs •

• Uricosuric (rugsUricosuric (rugs •

• 3aeine and 3aeine:3ontaining (rugs3aeine and 3aeine:3ontaining (rugs

GA"2(I67ID CHROMATOGRAPHY PRINCIP(E" GA"2(I67ID CHROMATOGRAPHY PRINCIP(E" GA" CHROMATOGRAPHY 

GA" CHROMATOGRAPHY  1lso known as #

1lso known as #ga%2li:-i.ga%2li:-i. chromatograph#

chromatograph#$ or #$ or #ga%2li:-i.ga%2li:-i. partition chromatograph#

partition chromatograph#$$ 1 separation techniue in which the 1 separation techniue in which the mobile phase is gas

mobile phase is gas It is used in the

It is used in the analysis of gaseous andanalysis of gaseous and "olatile substances

"olatile substances Mo+ile Pha%e Mo+ile Pha%e  his is the c

 his is the carrier arrier gasgas

3onsiderations in the choice of

3onsiderations in the choice of carriecarrierr gas9 gas9 Safety Safety &urity &urity Inertness Inertness 1"ailability 1"ailability 3ost 3ost (etector to be used (etector to be used SampleMs matri! SampleMs matri! 7elium 7elium ;itrogen ;itrogen 7ydrogen 7ydrogen 1rgon 1rgon 1ir 1ir

Small diameter glass tube Small diameter glass tube %%Capillar# Col-mnCapillar# Col-mn''

Solid matri! inside a larger metal Solid matri! inside a larger metal tube %

tube %Pac!e. Col-mnPac!e. Col-mn'' 3hemically stable

3hemically stable 7igh boiling point 7igh boiling point 6ow "iscosity 6ow "iscosity

Specic sol"ent properties towards Specic sol"ent properties towards the components to be separated the components to be separated 7ydrocarbons

7ydrocarbons

Silicone oils and gums Silicone oils and gums &olyesters

&olyesters

&olyalcohols *carbowa!+ &olyalcohols *carbowa!+

or

or gla%%gla%% and contains and contains a a

@.?:@.F mm and @.?:@.F mm and

o

o <COT<COT *column walls are*column walls are

coated with the acti"e coated with the acti"e materials+

materials+

o

o P(OTP(OT *columns are uasi:solid*columns are uasi:solid

lled with many parallel lled with many parallel micropores+

micropores+

3arrier gases employed in G39 3arrier gases employed in G39

"tationar# Pha%e "tationar# Pha%e

It is adhered to the inside of a9 It is adhered to the inside of a9

3haracteristics9 3haracteristics9 8!amples9 8!amples9 TYPE" OF GC CO(7MN TYPE" OF GC CO(7MN Pac!e. Col-mn% Pac!e. Col-mn%

6arge core columns with a length of 6arge core columns with a length of D.F:D@ meters and an internal

D.F:D@ meters and an internal diameter of ?:J mm

diameter of ?:J mm  he tubing is

 he tubing is usually mausually made ofde of %tainle%% %teel

%tainle%% %teel a packing of

a packing of nely di"ided, inert, solidnely di"ided, inert, solid support material that is coated with support material that is coated with stationary phase

stationary phase Capillar# Col-mn% Capillar# Col-mn%

-le!ible columns with a "ery small -le!ible columns with a "ery small internal diamete

internal diameter of r of a length of ?F:N@ meters a length of ?F:N@ meters

ith a pol#imi.e o-ter coating ith a pol#imi.e o-ter coating  

 ypes9ypes9

COMPONENT" AND F7NCTION" OF GC COMPONENT" AND F7NCTION" OF GC "Y"TEM

"Y"TEM

Carrier Ga% Tan!  Carrier Ga% Tan! 

 he tubing is

(8)

1 chamber made of

1 chamber made of stainless steel thatstainless steel that supplies the carrier gas needed in the supplies the carrier gas needed in the analysis

analysis

Pre%%-re Reg-lator Pre%%-re Reg-lator

1 suitable two:stage

1 suitable two:stage diaphragmdiaphragm controlled pressure regulator that controlled pressure regulator that reduces the pressure le"el compatible reduces the pressure le"el compatible with the reuirement of the instrument with the reuirement of the instrument Flo Controller

Flo Controller

It is contained within

It is contained within a thermostatica thermostatic chamber capable of maintaining a chamber capable of maintaining a constant temperatur

constant temperature as high e as high as J@@as J@@ degrees centigrade

degrees centigrade

It is important to control gas ow rate It is important to control gas ow rate because it aects the analysis of the because it aects the analysis of the samples

samples InLector Port InLector Port

1 small chamber where the sample is 1 small chamber where the sample is introduced into the system

introduced into the system  he primary

 he primary reuirreuirement of theement of the

inBection system is that the sample be inBection system is that the sample be "apori)ed instantaneou

"apori)ed instantaneously so tsly so that ahat a narrow band of "apor is introduced into narrow band of "apor is introduced into the beginning of the column

the beginning of the column Col-mn or Col-mn O$en Col-mn or Col-mn O$en

 he #heart$

 he #heart$ of the G3 Syof the G3 System. It isstem. It is contained in an o"en,

contained in an o"en, the temperaturethe temperature of which is precisely controlled

of which is precisely controlled  he rate at

 he rate at which a sawhich a sample passesmple passes through the column is

through the column is directlydirectly

proportional to the temperature of the proportional to the temperature of the column

column

 he higher the

 he higher the column temperatcolumn temperature,ure, the faster the sample mo"es

the faster the sample mo"es throughthrough the column

the column

7owe"er, the faster a sample mo"es 7owe"er, the faster a sample mo"es through the column, the less it

through the column, the less it

interacts with the stationary phase and interacts with the stationary phase and the less analytes are separated

the less analytes are separated Detector

Detector

1 number of detectors are used in G3 1 number of detectors are used in G3  he most common det

 he most common detectors arectors are thee the following9 following9 Thermal Con.-cti$it# Thermal Con.-cti$it# Detector Detector o

o 1 uni"ersal detector1 uni"ersal detector o

o Simple, ine!pensi"e, andSimple, ine!pensi"e, and

non:destructible to the non:destructible to the sample

sample

Flame Ioni0ation Detector Flame Ioni0ation Detector &FID'

&FID'

o

o 7ighly sensiti"e detector7ighly sensiti"e detector o

o 1lmost a uni"ersal detector1lmost a uni"ersal detector

=ther detectors that are sensiti"e only =ther detectors that are sensiti"e only to specic types of substances or work to specic types of substances or work well only in narrower ranges of

well only in narrower ranges of concentrations concentrations Di%charge Ioni0ation Di%charge Ioni0ation Detector &DID' Detector &DID'

Electron Capt-re Detector Electron Capt-re Detector &ECD'

&ECD'

Flame Photometric Detector Flame Photometric Detector &FPD' &FPD' Hall Electrol#tic Hall Electrol#tic Con.-cti$it# Detector Con.-cti$it# Detector &EICD' &EICD' Nitrogen Pho%phor-% Nitrogen Pho%phor-% Detector &NPD' Detector &NPD'

Ma%% "electi$e Detector Ma%% "electi$e Detector &M"D'

&M"D'

Photo Ioni0ation Detector Photo Ioni0ation Detector &PID'

&PID'

P-l%e. Di%charge Ioni0ation P-l%e. Di%charge Ioni0ation Detector &PDD'

Detector &PDD'

Thermal Energ# Anal#0er Thermal Energ# Anal#0er &TEA'

&TEA'

the the

the column at a dierent time %Rt' the column at a dierent time %Rt' Recor.er Integrator

Recor.er Integrator

Used to graphically reproduce the Used to graphically reproduce the output of the detector and record the output of the detector and record the result #chromatogram$

result #chromatogram$ Retention Time 8tR9 Retention Time 8tR9

o

o  he time r he time reuired euired by an a"erby an a"erageage

molecule of component to pass molecule of component to pass from the inBector port through the from the inBector port through the column to the detector

column to the detector Retention 5ol-me 8$R9 Retention 5ol-me 8$R9

o

o  he "olume of th he "olume of the carrie carrier gaser gas

necessary to carry an a"erage necessary to carry an a"erage molecule of the component from molecule of the component from the point of inBection to the

the point of inBection to the detector detector APP(ICATION OF GA"2(I67ID APP(ICATION OF GA"2(I67ID CHROMATOGRAPHY  CHROMATOGRAPHY 

In analytical chemistry < biochemistry In analytical chemistry < biochemistry &etrochemical en"ironmental monitoring &etrochemical en"ironmental monitoring In chemistry research %e!tensi"ely'

In chemistry research %e!tensi"ely'

0easurement of to!ic substances in water, 0easurement of to!ic substances in water, soil, or air

soil, or air

In drug uality control, to assure the In drug uality control, to assure the uantitati"e/ ualitati"e features of uantitati"e/ ualitati"e features of pharmaceuticals

pharmaceuticals

PRINCIP(E AND THEORY OF G(C PRINCIP(E AND THEORY OF G(C

G63 uses a ow:through narrow tube G63 uses a ow:through narrow tube %column', through which dierent %column', through which dierent

chemical constituents of the sample pass chemical constituents of the sample pass in a gas stream %mobile phase' at dierent in a gas stream %mobile phase' at dierent rates, with the stationary phase

rates, with the stationary phase 1s the chemical e!it at the end of 1s the chemical e!it at the end of column, they are detected and are column, they are detected and are identied electronically

identied electronically  he function of the

 he function of the stationary phstationary phase in thease in the column is to separate the dierent

column is to separate the dierent components

components  his causes

 his causes each of the each of the component to e!component to e!itit 1ssay of "olatile oils content

(9)

=ther parameters that can alter the Rt =ther parameters that can alter the Rt are9

are9

Carrier Ga% Flo Rate Carrier Ga% Flo Rate Temperat-re

Temperat-re D.

D. 1 kno1 known "olwn "olume oume of gas f gas or lor liuid iuid analanalyte iyte iss inBected into the entrance/ head of the inBected into the entrance/ head of the column using9

column using9

 Micro%#ringeMicro%#ringe

 Ga% %o-rce %itching %#%temGa% %o-rce %itching %#%tem ?.

?. he he carcarrier rier gas gas #sw#sweepseeps$ th$ the ane analytealyte molecules through the column

molecules through the column E.

E. he he sweesweeping ping motimotion is on is inhiinhibited bited by tby thehe adsorption of the analyte molecules onto adsorption of the analyte molecules onto the9

the9

 Col-mn all%Col-mn all%

 Pac!ing material% in the col-mnPac!ing material% in the col-mn J.

J. SincSince eae each typch type of me of molecuolecule hale has dis diereerentnt progr

progression, the "arious ession, the "arious components ofcomponents of the analyte mi!ture are separated as they the analyte mi!ture are separated as they progress through the column, at dierent progress through the column, at dierent times %Rt'

times %Rt' F

F.. 11 .etector.etector is used to monitor the outlet is used to monitor the outlet stream from the column

stream from the column

G(C DETECTOR" IN4ECTOR" G(C DETECTOR" IN4ECTOR" -lame Ioni)ation (etector -lame Ioni)ation (etector Split/ Splitless InBector Split/ Splitless InBector AD5A

AD5ANTAGNTAGE" E" OF OF GA"2(I67IDGA"2(I67ID CHROMATOGRAPHY 

CHROMATOGRAPHY 

8!cellent separation, usually less than N@ 8!cellent separation, usually less than N@ seconds

seconds

Sensiti"ity < speed are e!traordinary, with Sensiti"ity < speed are e!traordinary, with D@:D? gram sample being detectable for D@:D? gram sample being detectable for many substances

many substances

G3 can be run D@@@ times

G3 can be run D@@@ times faster than 63faster than 63 purication of samples

purication of samples MO(EC7(AR "IE5E

MO(EC7(AR "IE5E GE( CHROMATOGRGE( CHROMATOGRAPHYAPHY PRINCIP(E"

PRINCIP(E"

DEFINITION OF M" GC DEFINITION OF M" GC 1 common type of

1 common type of partitionpartition chromatogr

chromatography in aphy in which separation ofwhich separation of components is based on

components is based on molecular si)emolecular si)e THEORY OF M" GC

THEORY OF M" GC

• 1 column is prepared of tiny particles of an1 column is prepared of tiny particles of an

inert substance that contain small pores inert substance that contain small pores

• If a If a sample solution %containing moleculessample solution %containing molecules

of "arious dimensions' is passed through of "arious dimensions' is passed through

the column, the

the column, the following mo"ementsfollowing mo"ements happen9

happen9

 0olecules larger than the pores mo"e0olecules larger than the pores mo"e only in the

only in the space between the particlesspace between the particles and are not retarded by the column and are not retarded by the column material

material

 0olecules smaller than the pores0olecules smaller than the pores diuse in and out of

diuse in and out of particles %withparticles %with probability that it increases with probability that it increases with

decreasing molecular si)e' > this slows decreasing molecular si)e' > this slows down their mo"ement through the down their mo"ement through the column

column

• 0olecules are eluted from the column in0olecules are eluted from the column in

the order of9 the order of9

 (ecreasing si)e(ecreasing si)e

 (ecreasing molecular weight %for(ecreasing molecular weight %for relati"ely constant shapes >

relati"ely constant shapes > Glo+-larGlo+-lar or

or Ro.Ro.'' MA

MATERIA(" 7"ED IN TERIA(" 7"ED IN M" GCM" GC Gel%

Gel%

1 E( network with a "ery random 1 E( network with a "ery random structure. 3onsist of cross:linked structure. 3onsist of cross:linked polymers that are9

polymers that are9 Inert Inert ;on:Reacti"e ;on:Reacti"e Uncharged Uncharged

Used for aueous solution Used for aueous solution

De/tran De/tran

o

o &olysaccharide composed of&olysaccharide composed of

glucose residues. &roduced by glucose residues. &roduced by fermentati

fermentation of on of glucose byglucose by

Leuconostoc mesenteroides Leuconostoc mesenteroides

o

o &repared by "arious degrees of&repared by "arious degrees of

cross:linking to control pore si)e cross:linking to control pore si)e

o

o "epha.e/"epha.e/ : tradename : tradename

Agaro%e Agaro%e

o

o =btained from seaweeds=btained from seaweeds o

o Its pore si)e is larger thanIts pore si)e is larger than

Sephade! Sephade!

o

o Useful for the analysis <Useful for the analysis <

separation of (;1 separation of (;1

o

o "epharo%e 3iogel"epharo%e 3iogel > >

tradenamesL supplies as wet tradenamesL supplies as wet beads

beads

Pol#acr#lami.e Pol#acr#lami.e

o

o Its pore si)e is determined byIts pore si)e is determined by

the degree of cross:linking the degree of cross:linking

o

o &repared by cross:linking&repared by cross:linking

1crylamide with

1crylamide with N;N2N;N2

Meth#lene23i%2Acer#lami.e Meth#lene23i%2Acer#lami.e

o

o 3iogel P3iogel P : tradename: tradename

6arger preparati"e G63Ms can be used for 6arger preparati"e G63Ms can be used for

References

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