CHROMATOGRAPHY CHROMATOGRAPHY DEFINITION OF
DEFINITION OF CHROMACHROMATOGRAPHY TOGRAPHY
•
• Science on the study of separation ofScience on the study of separation of
molecules based on dierences in their molecules based on dierences 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 dierent plant where he separated the dierent plant pigments.
pigments.
•
• Used for Used for separation, identicaseparation, identication, 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&' (ened as a procedure by which%US&' (ened as a procedure by which
solutes are separated by a dierential solutes are separated by a dierential 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 dierent mobility by reason of dierences dierent mobility by reason of dierences 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 purication+.
purication+.
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 dierent reactions with the ha"e dierent reactions with the stationary support.
stationary support.
•
• his will his will lead to sepalead to separation of #siration of #similar$milar$
molecules. molecules.
•
• (ierent types of molecules can be(ierent 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 liuid that nely di"ided solid or a liuid 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 liuid or gas or mi!ture of &ure liuid 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 denite direction
denite direction
It may be a liuid, a gas, or a It may be a liuid, 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 liuid or gas 0obile phase is liuid 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 liuid or gas 0obile phase is a liuid or gas Retention and separation depends Retention and separation depends on the
on the dierentdierential migration ofial migration of solute molecules based on solute molecules based on molecular si)e
molecular si)e
Stationary phase is liuid Stationary phase is liuid 0obile phase is liuid or gas 0obile phase is liuid 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
"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 liuid is always a liuid
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 &urication &urication
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
echniues by echniues by 3hromatogra3hromatographic 5edphic 5ed Shape
Shape
3olumn
3olumn 3hromatogra3hromatographyphy &lanar 3hromatography &lanar 3hromatography
&aper 3hromatography &aper 3hromatography hin 6ayer 3
hin 6ayer 3hromatograhromatographyphy
echniues by echniues by &hysical St&hysical State of 0obileate of 0obile &hase
&hase 6iuid
6iuid 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 dierences in8!ploits the dierences 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 liuid that passes through 1 gas or liuid that passes through the column
the column "tationar# Pha%e "tationar# Pha%e
1 solid or liuid that does not 1 solid or liuid 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 dierent points in the stationary phase, 1t dierent points in the stationary phase, the dierent components of the compound the dierent 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 specic distance' mo"ingspecic distance' mo"ing through the mobile phase.
through the mobile phase. Identication and control of
Identication 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#
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
11D
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 liuid %liuid:solid' or a gas 0ay be a liuid %liuid: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
6iuid supported on inert solid 6iuid supported on inert solid Mo+ile Pha%e
Mo+ile Pha%e Is a liuid
Is a liuid %liuid:liu%liuid:liuid partition' or aid partition' or a gas %gas:liuid'
gas %gas:liuid' 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. 6iuid
6iuid
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 -IG86 -IR1R1I=;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
"IE E@C(7"ION CHROMATOGRAPHY 8"EC9 "IE 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
Pol#%t#rene; Gla%% 3ea.%; Pol#%t#rene; Gla%% 3ea.%; oror Al!#late. De/tran Al!#late. De/tran > G86> G86 &8R081I=; &8R081I=; Mo+ile Pha%e Mo+ile Pha%e 6iuid or Gas 6iuid or Gas
Used for the separation of solutes with Used for the separation of solutes with dierent molecular si)e
dierent 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 purication of
for purication of synthetic:organsynthetic:organicic polymers
polymers AFFINITY
AFFINITY CHROMACHROMATOGRAPHY TOGRAPHY
Utili)es high specic interactions between Utili)es high specic 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:6iuid 3hromatography Gas:6iuid 3hromatography Gel 3hromatography Gel 3hromatography "PECIA( TYPE"
"PECIA( TYPE" OF PAROF PARTITIONTITION CHROMATOGRAPHY
CHROMATOGRAPHY
6iuid 3hromatography %63' 6iuid 3hromatography %63'
7igh &erformance 6iuid 3hromatography 7igh &erformance 6iuid 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
&uried Aater > for polar materials &uried 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 (ierent colors/ spots will appear on the (ierent 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 techniue in which the1 separation techniue 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 dierent distances, mi!ture tra"el dierent 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 techniue for
1n analytical techniue for separating andseparating and identifying mi!tur
identifying mi!tures that or es that or colored, especially pigments colored, especially pigments
(ierent compounds in the sample (ierent compounds in the sample
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 identication of (igo!in, US&identication 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 (ierent compounds in the sample (ierent compounds in the sample mi!ture tra"el at dierent rates due to mi!ture tra"el at dierent rates due to dierences in solubility in the sol"ent dierences in solubility in the sol"ent and due to dierences in their
and due to dierences 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 dier in their
separated dier 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 techniue, similar Aidely used laboratory techniue, 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
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) mm2) 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
(ierent compounds in the sample(ierent compounds in the sample mi!ture tra"el at dierent rates due to mi!ture tra"el at dierent rates due to the dierences in their attraction to the dierences in their attraction to the stationary phase and because of the stationary phase and because of dierenc
dierences 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 %hort2a$e &)nm' 75 %hort2a$e &)nm' 75 long2a$e &Jnm' 75 long2a$e &Jnm'
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 Yelloello Al.eh#.e% Al.eh#.e% an. an. =etone% =etone% );2 );2 .initrophen#lh#. .initrophen#lh#. ra0ine ra0ine Y Yelloello 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 3ron 3ron 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
5y uorescence 5y uorescence
5y spraying with "isuali)ation reagents 5y spraying with "isuali)ation reagents "PECIFIC "PECIFIC 5I"7A(IING 5I"7A(IING 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(YED IN THIN (AYER DR7G" ANA(YED IN THIN (AYER CHROMATOGRAPHY CHROMATOGRAPHY • • 1nalgesics1nalgesics • • 1ntipyretics1ntipyretics •
• 1spirin, &henacetin, 1cetaminophen1spirin, &henacetin, 1cetaminophen •
• 1nti:Inamma1nti:Inammatory tory (rugs(rugs •
• Uricosuric (rugsUricosuric (rugs •
• 3aeine and 3aeine:3ontaining (rugs3aeine and 3aeine: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 techniue in which the 1 separation techniue 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
Specic sol"ent properties towards Specic 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, solidnely 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
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 reuirement of the instrument with the reuirement 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 aects the analysis of the because it aects 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 reuirreuirement 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 specic types of substances or work to specic 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 dierent time %Rt' the column at a dierent 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 reuired euired 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 dierent %column', through which dierent
chemical constituents of the sample pass chemical constituents of the sample pass in a gas stream %mobile phase' at dierent in a gas stream %mobile phase' at dierent 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 identied electronically
identied electronically he function of the
he function of the stationary phstationary phase in thease in the column is to separate the dierent
column is to separate the dierent components
components his causes
his causes each of the each of the component to e!component to e!itit 1ssay of "olatile oils content
=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 liuid iuid 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 dis diereerentnt 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 dierent progress through the column, at dierent 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 purication of samples
purication 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 diuse in and out of
diuse 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 aueous solution Used for aueous 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;N2N;N2
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