Chromatographic techniques
TejasviBhatia
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119 slides
May 03, 2020
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About This Presentation
TECHNIQUES
Slide Content
Principle and Applications of
Chromatographic Techniques
TEJASVI BHATIA
Chromatographic Techniques
TEJASVI BHATIA
ORIGIN
Chromatography(fromGreekword)“Chroma”(colour)and
“Grafein"(towrite)collectiveterm-:laboratorytechniquesforthe
separationofmixtures.
RussianbotanistMikhailSemyonoyichTsvetinventedthefirst
chromatographytechniquein1900duringhisresearchon
chlorophyll.
ThemethodwasdescribedonDecember30,1901atthe11th
CongressofNaturalistsandDoctorsinSt.Petersburg.
Chromatographybasicallyinvolvestheseparationofmixtures
duetodifferencesinthedistributioncoefficientofsample
componentsbetween2differentphases.
Chromatography(fromGreekword)“Chroma”(colour)and
“Grafein"(towrite)collectiveterm-:laboratorytechniquesforthe
separationofmixtures.
RussianbotanistMikhailSemyonoyichTsvetinventedthefirst
chromatographytechniquein1900duringhisresearchon
chlorophyll.
ThemethodwasdescribedonDecember30,1901atthe11th
CongressofNaturalistsandDoctorsinSt.Petersburg.
Chromatographybasicallyinvolvestheseparationofmixtures
duetodifferencesinthedistributioncoefficientofsample
componentsbetween2differentphases.
Principle of Chromatography
Whenagas/vapourcomesincontactwithanabsorbent,
certainamountofthegasgetsabsorbedonthesolidsurface.
Thephenomenontakesplaceaccordingtothelawsof
Freaundlich(x/m=kC1/n)orLongmuir(x/m=k1C+k2C)
where x is the mass of the gas or vapour sorbent in mass of the sorbent and
C is the vapour concentration in the gas phase and k, k1 & k2 are constants.
Similarlyifthevapour,gasandacompoundcomesincontact
withaliquid,afixedamountofcompoundgetsdissolvedinthe
liquid.ThisphenomenonisknownasHenry’sLawofpartition
x/m=kC.
Whenagas/vapourcomesincontactwithanabsorbent,
certainamountofthegasgetsabsorbedonthesolidsurface.
Thephenomenontakesplaceaccordingtothelawsof
Freaundlich(x/m=kC1/n)orLongmuir(x/m=k1C+k2C)
where x is the mass of the gas or vapour sorbent in mass of the sorbent and
C is the vapour concentration in the gas phase and k, k1 & k2 are constants.
Similarlyifthevapour,gasandacompoundcomesincontact
withaliquid,afixedamountofcompoundgetsdissolvedinthe
liquid.ThisphenomenonisknownasHenry’sLawofpartition
x/m=kC.
Chromatography terms
Chromatogram:Thevisualoutputofthe
chromatograph.Inthecaseofanoptimal
separation,differentpeaksorpatternsonthe
chromatogramcorrespondtodifferentcomponents
oftheseparatedmixture.
Achromatograph-Equipmentthatenablesa
sophisticatedseparation(Gaschromatographicor
liquidchromatographicseparation)
Chromatogram:Thevisualoutputofthe
chromatograph.Inthecaseofanoptimal
separation,differentpeaksorpatternsonthe
chromatogramcorrespondtodifferentcomponents
oftheseparatedmixture.
Achromatograph-Equipmentthatenablesa
sophisticatedseparation(Gaschromatographicor
liquidchromatographicseparation)
Mobilephase-Themobilephaseconsistsofthesample
beingseparated/analyzedandthesolventthatmovesthe
samplethroughthecolumn.
IncaseofHPLCthesolventismobilephasewhichcarrythe
analytes.Themobilephasemovesthroughthe
chromatographiccolumn(thestationaryphase)wherethe
sampleinteractswiththestationaryphaseandisseparated.
Phasewhichmovesinadefinitedirection.
IncaseofGLCthemobilephaseisgas.
Stationaryphase-Thesubstancewhichiscovalentlybondedto
thesupportparticlesortotheinsidewallofthecolumntubing
inchromatographyprocedure.e.g.silicalayerinthinlayer
chromatography.
Theeffluent-Themobilephaseleavingthecolumn.
beingseparated/analyzedandthesolventthatmovesthe
samplethroughthecolumn.
IncaseofHPLCthesolventismobilephasewhichcarrythe
analytes.Themobilephasemovesthroughthe
chromatographiccolumn(thestationaryphase)wherethe
sampleinteractswiththestationaryphaseandisseparated.
Phasewhichmovesinadefinitedirection.
IncaseofGLCthemobilephaseisgas.
Stationaryphase-Thesubstancewhichiscovalentlybondedto
thesupportparticlesortotheinsidewallofthecolumntubing
inchromatographyprocedure.e.g.silicalayerinthinlayer
chromatography.
Theeffluent-Themobilephaseleavingthecolumn.
Sample-Thematteranalysedinchromatography.Itmayconsistof
a singlecomponentormixtureofcomponents.
Solute-Referstotheanalytespresentinsampleduringpartition
chromatography.
Analyte- Substancethatistobeseparatedduring
chromatography.
Solvent-Referstoanysubstancecapableofsolubilizingother
substance.
a singlecomponentormixtureofcomponents.
Solute-Referstotheanalytespresentinsampleduringpartition
chromatography.
Analyte- Substancethatistobeseparatedduring
chromatography.
Solvent-Referstoanysubstancecapableofsolubilizingother
substance.
Analyticalchromatography-Todeterminetheexistence/
presenceandtheconcentrationofanalyte(s)inasample.
Preparativechromatography-Preparativechromatographyis
usedtoisolateandpurifythecompoundsandcollectthe
sufficientquantitiesofasubstanceforfurtheruse,ratherthan
analysis.
PolarSolvents
Water > Methanol > Acetonitrile > Ethanol > Oxydipropionitrile.
Non-polar Solvents
N-Decane > N-Hexane > N-Pentane >Cyclohexane.
presenceandtheconcentrationofanalyte(s)inasample.
Preparativechromatography-Preparativechromatographyis
usedtoisolateandpurifythecompoundsandcollectthe
sufficientquantitiesofasubstanceforfurtheruse,ratherthan
analysis.
PolarSolvents
Water > Methanol > Acetonitrile > Ethanol > Oxydipropionitrile.
Non-polar Solvents
N-Decane > N-Hexane > N-Pentane >Cyclohexane.
Itisacharacteristicofparticularcompound.Timetakenfora
particularanalytetopassthroughthesystem(fromthecolumn
inlettothedetector)undersetconditions.
Retention time(RT)
particularanalytetopassthroughthesystem(fromthecolumn
inlettothedetector)undersetconditions.
Retention time(RT)
Itisaquantitativeindicationofhowfara
particularcompoundtravelsinaparticular
solvent.
The retention factor( Rf) = distance the solute (D1)
moves / the distance traveled by the solvent front
(D2)
Rf= D1 / D2
where
D1=distancethatcolortraveled,measuredfrom
centerofthebandofcolortothepointwherethe
colorwasapplied.
D2=totaldistancethatsolventtraveled.
Retention Factor(Rf)
particularcompoundtravelsinaparticular
solvent.
The retention factor( Rf) = distance the solute (D1)
moves / the distance traveled by the solvent front
(D2)
Rf= D1 / D2
where
D1=distancethatcolortraveled,measuredfrom
centerofthebandofcolortothepointwherethe
colorwasapplied.
D2=totaldistancethatsolventtraveled.
Retention Factor(Rf)
Important Parameters
Capacity factor (k')
It is a measurement of the retention time of a sample
molecule, relative to column dead volume (V
0)
k'=
V
1
-V
o
Vo
Where:
k' = Capacity Factor of the column
Vo = Void volume ( or dead volume) of the column
(volume at which an unretained component elutes)
V1 = Retention volume of peak 1
Capacity factor (k')
It is a measurement of the retention time of a sample
molecule, relative to column dead volume (V
0)
k'=
V
1
-V
o
Vo
Where:
k' = Capacity Factor of the column
Vo = Void volume ( or dead volume) of the column
(volume at which an unretained component elutes)
V1 = Retention volume of peak 1
Capacity factor (k') depends upon
Solvent polarity
Composition
Purity
Temperature
Column Chemistry
Sample
Solvent polarity
Composition
Purity
Temperature
Column Chemistry
Sample
Column Efficiency (N)
TheColumnEfficiency(N)(alsocalledtheoreticalplatecount)isameasureofthe
bandspreadingofapeak.
Smallerthebandspreadhigherthenumberoftheoreticalplateswhich
indicatesgoodcolumnandcolumnefficiency
Or
LargerthevalueofNisforacolumn,betterseparationoftwocompounds.
-N is independent of solute retention
-N is dependent on the length of the column
Poor column efficiency due to:
Age and history of the column.
Extra column band broadening (such as due to malfunctioning injector or
improper tubing ID)
Inappropriate detector setting .
Change in flow rate and solvent viscosity.
TheColumnEfficiency(N)(alsocalledtheoreticalplatecount)isameasureofthe
bandspreadingofapeak.
Smallerthebandspreadhigherthenumberoftheoreticalplateswhich
indicatesgoodcolumnandcolumnefficiency
Or
LargerthevalueofNisforacolumn,betterseparationoftwocompounds.
-N is independent of solute retention
-N is dependent on the length of the column
Poor column efficiency due to:
Age and history of the column.
Extra column band broadening (such as due to malfunctioning injector or
improper tubing ID)
Inappropriate detector setting .
Change in flow rate and solvent viscosity.
Methods of measuring column efficiency (N)
5Sigma 4Sigma
Tangent 3Sigma
½Height 2Sigma(inflection)
N
= Vr
W
()
2
W Method
W1 4inflection(2)
W
h 5.54 ½ Peak height
W3 9 3
W4 16 4
W5 25 5
Wtan 16 Tangent
Wasymmetry 10 Asymmetry-based
= constant dependent on high
where peak width measured
Vr= retention time
W= peak width
5Sigma 4Sigma
Tangent 3Sigma
½Height 2Sigma(inflection)
N
= Vr
W
()
2
W Method
W1 4inflection(2)
W
h 5.54 ½ Peak height
W3 9 3
W4 16 4
W5 25 5
Wtan 16 Tangent
Wasymmetry 10 Asymmetry-based
= constant dependent on high
where peak width measured
Vr= retention time
W= peak width
Resolution is distance between the peak centre of the
two components peaks divided by the average base
width of the peaks.
Resolution (Rs)
Rs=
V2 -V1
½(W
1
+W
2
)
two components peaks divided by the average base
width of the peaks.
Resolution (Rs)
Rs=
V2 -V1
½(W
1
+W
2
)
Factors affecting the Resolution
Increase column length
Decrease column diameter
Decrease flow-rate
Uniformly packed Column
Use uniform stationary phase (packing material)
Sample size
Select proper stationary phase
Select proper mobile phase
Use proper pressure
Use gradient elution
Increase column length
Decrease column diameter
Decrease flow-rate
Uniformly packed Column
Use uniform stationary phase (packing material)
Sample size
Select proper stationary phase
Select proper mobile phase
Use proper pressure
Use gradient elution
Why Do Bands Spread?
a. Eddy diffusion
b. Mobile phase mass transfer
c. Stagnant mobile phase mass transfer
d. Stationary phase mass transfer
e. Longitudinal diffusion
a. Eddy diffusion
b. Mobile phase mass transfer
c. Stagnant mobile phase mass transfer
d. Stationary phase mass transfer
e. Longitudinal diffusion
a) Eddy diffusion–a process that leads to peak broadening due to
the presence of multiple flow paths through a packed column.
As solute molecules travel through the column, some arrive at
the end sooner then others simply due to the different path
traveled around the support particles in the column that result in
different travel distances.
the presence of multiple flow paths through a packed column.
As solute molecules travel through the column, some arrive at
the end sooner then others simply due to the different path
traveled around the support particles in the column that result in
different travel distances.
Asoluteinthecenterofthechannelmovesmorequicklythan
soluteattheedges,itwilltendtoreachtheendofthechannel
firstleadingtoband-broadening
The degree of band-broadening due to eddy diffusion
and mobile phase mass transfer depends mainly on:
1) Size of the packing material
2) Diffusion rate of the solute
b.) Mobile phase mass transfer–caused by the presence of different
flow profile within channels or between particles of the column.
soluteattheedges,itwilltendtoreachtheendofthechannel
firstleadingtoband-broadening
The degree of band-broadening due to eddy diffusion
and mobile phase mass transfer depends mainly on:
1) Size of the packing material
2) Diffusion rate of the solute
b.) Mobile phase mass transfer–caused by the presence of different
flow profile within channels or between particles of the column.
C.) Stagnant mobile phase mass transfer–band-broadening due to
differences in rate of diffusion of the solute molecules between the mobile
phase outside the pores of the support (flowing mobile phase) to the mobile
phase within the pores of the support (stagnant mobile phase).
Since a solute does not travel down the column
when it is in the stagnant mobile phase, it spends
a longer time in the column than solutethat
remains in the flowing mobile phase.
Stagnant mobile phase mass transfer depends on:
1) Size, shape and pore structure of the packing material
2) Diffusion and retention of the solute
3) Flow-rate of the solute through the column
differences in rate of diffusion of the solute molecules between the mobile
phase outside the pores of the support (flowing mobile phase) to the mobile
phase within the pores of the support (stagnant mobile phase).
Since a solute does not travel down the column
when it is in the stagnant mobile phase, it spends
a longer time in the column than solutethat
remains in the flowing mobile phase.
Stagnant mobile phase mass transfer depends on:
1) Size, shape and pore structure of the packing material
2) Diffusion and retention of the solute
3) Flow-rate of the solute through the column
d.) Stationary phase mass transfer–band-broadening due to the movement of
solute between the stagnant phase and the stationary phase.
Sincedifferentsolutemoleculesspenddifferentlengthsoftimeinthe
stationaryphase,theyalsospenddifferentamountsoftimeonthe
column,givingrisetoband-broadening.
Stationary phase mass transfer depends on:
1) the retention and diffusion of the solute
2) the flow-rate of the solute through the column
3) the kinetics of interaction between the solute and
the stationary phase
solute between the stagnant phase and the stationary phase.
Sincedifferentsolutemoleculesspenddifferentlengthsoftimeinthe
stationaryphase,theyalsospenddifferentamountsoftimeonthe
column,givingrisetoband-broadening.
Stationary phase mass transfer depends on:
1) the retention and diffusion of the solute
2) the flow-rate of the solute through the column
3) the kinetics of interaction between the solute and
the stationary phase
e.) Longitudinal diffusion–band-broadening due to the diffusion of the
solute along the length of the column in the flowing mobile phase.
Longitudinal diffusion depends on:
1) Diffusion of the solute
2) Flow-rate of the solute through
the column
solute along the length of the column in the flowing mobile phase.
Longitudinal diffusion depends on:
1) Diffusion of the solute
2) Flow-rate of the solute through
the column
The baselineis any part of the chromatogram where only mobile phase is
emerging from the column.
The peak maximum is the highest point of the peak.
The injection pointis that point in time/position time when/where the
sample is placed on the column.
The dead pointis the position of the peak-maximum of an unretainedsolute.
The dead time(t
o) is the time elapsed between the injection pointand the
dead point.
The dead volume(V
o) is the volume of mobile phase passed through the
column between the injection pointand the dead point.
Thus, V
o= Qt
owhere Q is the flow rate in ml/min.
Terms of Chromatography
emerging from the column.
The peak maximum is the highest point of the peak.
The injection pointis that point in time/position time when/where the
sample is placed on the column.
The dead pointis the position of the peak-maximum of an unretainedsolute.
The dead time(t
o) is the time elapsed between the injection pointand the
dead point.
The dead volume(V
o) is the volume of mobile phase passed through the
column between the injection pointand the dead point.
Thus, V
o= Qt
owhere Q is the flow rate in ml/min.
Terms of Chromatography
The retention time(t
r) is the time elapsed between the injection pointand the
peak maximum. Each solute has a characteristic retention time.
The retention volume(V
r) is the volume of mobile phase passed through the
column between the injection pointand the peak Maximum.
Thus, V
r= Qt
rwhere Q is the flow rate in ml/min. (Each solute
will also have a characteristic retention volume.)
The correctedretention time(t'
r) is the time elapsed between the dead point
and the peak maximum.
The correctedretention volume(V'
r) is the volume of mobile phase passed
through the column between the dead pointand the peak
maximum. It will also be the retention volumeminus the dead
volume. (Thus, V'
r= V
r-V
o= Q(t
r -t
o) where Q is the flow rate in
ml/min.)
r) is the time elapsed between the injection pointand the
peak maximum. Each solute has a characteristic retention time.
The retention volume(V
r) is the volume of mobile phase passed through the
column between the injection pointand the peak Maximum.
Thus, V
r= Qt
rwhere Q is the flow rate in ml/min. (Each solute
will also have a characteristic retention volume.)
The correctedretention time(t'
r) is the time elapsed between the dead point
and the peak maximum.
The correctedretention volume(V'
r) is the volume of mobile phase passed
through the column between the dead pointand the peak
maximum. It will also be the retention volumeminus the dead
volume. (Thus, V'
r= V
r-V
o= Q(t
r -t
o) where Q is the flow rate in
ml/min.)
The peak height (h) is the distance between the peak maximumand the base
line of the peak.
The peak width(w) is the distance between each side of a peak measure at
0.6065 of the peak height (ca0.607h).
The peak width at half height (w
0.5) is the distance between each side of a
peak measured at half the peak height. The peak width measured at half
height has no significance with respect to chromatography theory.
The peak width at the base (w
B) is the distance between the intersections of
the tangents drawn to the sides of the peak and the peak basegeometrically
produced. The peak width at the base is equivalent to four standard
deviations (4s) of the Gaussian curve and thus also has significance when
dealing with chromatography theory.
line of the peak.
The peak width(w) is the distance between each side of a peak measure at
0.6065 of the peak height (ca0.607h).
The peak width at half height (w
0.5) is the distance between each side of a
peak measured at half the peak height. The peak width measured at half
height has no significance with respect to chromatography theory.
The peak width at the base (w
B) is the distance between the intersections of
the tangents drawn to the sides of the peak and the peak basegeometrically
produced. The peak width at the base is equivalent to four standard
deviations (4s) of the Gaussian curve and thus also has significance when
dealing with chromatography theory.
Types of Chromatography
1.Liquid/SolidChromatography(adsorptionchromatography)
Liquid-solid:adsorptiononsolidwhichisgenerallypolar(cellulose,poly
amides)or(silicagel,alumina,magnesiumsilicates).
2.Liquid/LiquidChromatography(partitionchromatography)
Liquid-liquidpartitionbetween2bulkphases(immisciblesolvents)
3.IonExchangeChromatographySpecificinteractionswithionicspecies
(changerelativestrengthsofacidorbase)
4.GelPermeationChromatography(exclusionchromatography)
5.AffinityChromatography.
Types of Chromatography
Liquid-solid:adsorptiononsolidwhichisgenerallypolar(cellulose,poly
amides)or(silicagel,alumina,magnesiumsilicates).
2.Liquid/LiquidChromatography(partitionchromatography)
Liquid-liquidpartitionbetween2bulkphases(immisciblesolvents)
3.IonExchangeChromatographySpecificinteractionswithionicspecies
(changerelativestrengthsofacidorbase)
4.GelPermeationChromatography(exclusionchromatography)
5.AffinityChromatography.
Types of Chromatography
Liquid Solid Chromatography-The separation mechanism
in LSC is based on the competition of the components of the mixture sample for
the active sites on an absorbent such as Silica Gel. (Paper, TLC)
Liquid-LiquidChromatography-Thestationarysolid
surfaceiscoatedwithaliquid(theStationaryPhase)whichisimmisciblein
thesolvent(Mobile)phase.Partitioningofthesamplebetween2phases
delaysorretainssomecomponentsmorethanotherstoeffectseparation.
in LSC is based on the competition of the components of the mixture sample for
the active sites on an absorbent such as Silica Gel. (Paper, TLC)
Liquid-LiquidChromatography-Thestationarysolid
surfaceiscoatedwithaliquid(theStationaryPhase)whichisimmisciblein
thesolvent(Mobile)phase.Partitioningofthesamplebetween2phases
delaysorretainssomecomponentsmorethanotherstoeffectseparation.
Ion-Exchange Chromatography
In Ion-exchange Chromatography the separationis based on
the competition of different ionic compounds of the sample
for the active sites on the ion-exchange resin (anion or cation
resins) (column-packing).
Low salt High salt
In Ion-exchange Chromatography the separationis based on
the competition of different ionic compounds of the sample
for the active sites on the ion-exchange resin (anion or cation
resins) (column-packing).
Low salt High salt
Ion Exchange Chromatography
Applied to
Biochemical drugs, their metabolites,
amino acid, proteins, and peptide
Food preservatives
Vitamin
Mixtures of sugars
Pharmaceutical preparation
Purification of proteins
Applied to
Biochemical drugs, their metabolites,
amino acid, proteins, and peptide
Food preservatives
Vitamin
Mixtures of sugars
Pharmaceutical preparation
Purification of proteins
Gel-PermeationChromatographyisamechanical
sortingofmoleculesbasedonthesizeofthe
moleculesinsolution.
Smallmoleculesareretainedlongertimethanlarge
molecules.
Gel-Permeation Chromatography
sortingofmoleculesbasedonthesizeofthe
moleculesinsolution.
Smallmoleculesareretainedlongertimethanlarge
molecules.
Gel-Permeation Chromatography
Separationmechanismissievingnotpartitioning.
Techniqueapplicablefortheseparationofhigh-molecularweight
species(proteinsandpolymer).
Soluteandsolventmoleculescandiffuseintopores/trappedand
removedfromtheflowofthemobilephase.
Stationary phaseare porous silica or polymer particles
polystyrene, polyacrylamide) (5-10 mm) is used.
Largemoleculesnotretainedintheporesandelutefirst.
Small molecules permeate into pores strongly retained, eluted last
Gel Permeation Size Exclusion
Techniqueapplicablefortheseparationofhigh-molecularweight
species(proteinsandpolymer).
Soluteandsolventmoleculescandiffuseintopores/trappedand
removedfromtheflowofthemobilephase.
Stationary phaseare porous silica or polymer particles
polystyrene, polyacrylamide) (5-10 mm) is used.
Largemoleculesnotretainedintheporesandelutefirst.
Small molecules permeate into pores strongly retained, eluted last
Gel Permeation Size Exclusion
Advantages
Short & well-defined separation times.
Narrow bands spread & good sensitivity.
Solutes do not interact with the stationary phase.
Disadvantages
Only limited number of compounds can be separated
because the time scale of the chromatogram is short.
Inapplicability to samples of similar size, such as
isomers.
At least 10% difference in molecular weight is required
for reasonable resolution.
Short & well-defined separation times.
Narrow bands spread & good sensitivity.
Solutes do not interact with the stationary phase.
Disadvantages
Only limited number of compounds can be separated
because the time scale of the chromatogram is short.
Inapplicability to samples of similar size, such as
isomers.
At least 10% difference in molecular weight is required
for reasonable resolution.
It is generally a low resolution chromatography
technique and thus it is often reserved for the final,
"polishing" step of a purification.
It is also useful for determining the tertiary structure
and quaternary structure of purified proteins.
Application
technique and thus it is often reserved for the final,
"polishing" step of a purification.
It is also useful for determining the tertiary structure
and quaternary structure of purified proteins.
Application
Affinity chromatography
Interactionofthesolutewithanimmobilizedaffinityligand.
Awashingstep,wherenon-bondedsolutesarerinsedoff.
Inelutionstep,wheretheselectivelyattachedmoleculesare
recoveredbyelutionwithacompetingsubstrate,orbyapplying
conditionswhichaltertheconformationoftheattached
macromolecule.
Interactionofthesolutewithanimmobilizedaffinityligand.
Awashingstep,wherenon-bondedsolutesarerinsedoff.
Inelutionstep,wheretheselectivelyattachedmoleculesare
recoveredbyelutionwithacompetingsubstrate,orbyapplying
conditionswhichaltertheconformationoftheattached
macromolecule.
Applications
Inthefieldofbiochemistryfortheseparationofbio
macromoleculesfrombiologicalcompounds.
Forexample,S.Loukasandcolleaguesstudiedtheexistenceofonetypeofsuspected
opiatereceptorinthebrain(1994).Theyseparatedthe(opioidbindingproteinbyusingan
opioidreceptorantagonistthatwasspecifictothe(-opioidbindingprotein.Thesestudies
mayonedayleadtoabetterunderstandingofhowdrugssuchasopiumaffectourbrains.
Affinitychromatographycanbeusedtodetermine
dissociationconstantsofligandsandmolecules.
Inthefieldofbiochemistryfortheseparationofbio
macromoleculesfrombiologicalcompounds.
Forexample,S.Loukasandcolleaguesstudiedtheexistenceofonetypeofsuspected
opiatereceptorinthebrain(1994).Theyseparatedthe(opioidbindingproteinbyusingan
opioidreceptorantagonistthatwasspecifictothe(-opioidbindingprotein.Thesestudies
mayonedayleadtoabetterunderstandingofhowdrugssuchasopiumaffectourbrains.
Affinitychromatographycanbeusedtodetermine
dissociationconstantsofligandsandmolecules.
Paper chromatography.
Thin layer chromatography.
Gas liquid chromatography.
High performance liquid chromatography.
Gas chromatography-Mass spectroscopy.
Common Chromatography
Thin layer chromatography.
Gas liquid chromatography.
High performance liquid chromatography.
Gas chromatography-Mass spectroscopy.
Common Chromatography
Paper Chromatography
Paperchromatographyisatechniquethat
involvesplacingasmalldotofsamplesolution
ontoastripofchromatographypaper.
Differentcompoundsinthesamplemixturetravel
differentdistancesaccordingtohowstronglythey
interactwiththepaper.
Thepaperisplacedinajarcontainingashallow
layerofsolventandsealed.Asthesolventrises
throughthepaperitmeetsthesamplemixture
whichstartstotravelupthepaperwiththesolvent.
ThisallowsthecalculationofRfvalueandcanbe
comparedtostandardcompoundstoaidinthe
identificationofanunknownsubstance
Paperchromatographyisatechniquethat
involvesplacingasmalldotofsamplesolution
ontoastripofchromatographypaper.
Differentcompoundsinthesamplemixturetravel
differentdistancesaccordingtohowstronglythey
interactwiththepaper.
Thepaperisplacedinajarcontainingashallow
layerofsolventandsealed.Asthesolventrises
throughthepaperitmeetsthesamplemixture
whichstartstotravelupthepaperwiththesolvent.
ThisallowsthecalculationofRfvalueandcanbe
comparedtostandardcompoundstoaidinthe
identificationofanunknownsubstance
Thin layer chromatography
Thinlayerchromatography(TLC)isawidely-employed
laboratorytechniqueandissimilartopaper
chromatography.
However,insteadofusingastationaryphaseofpaper,it
involvesastationaryphaseofathinlayerofadsorbentlike
silicagel,alumina,orcelluloseonaflat,inertsubstrate.
Comparedtopaper,ithastheadvantageoffasterruns,
betterseparations,andthechoicebetweendifferent
adsorbents.
Differentcompoundsinthesamplemixturetravel
differentdistancesaccordingtotheinteractionwiththe
adsorbent.ThisallowsthecalculationofanRfvalueand
canbecomparedtostandardcompoundstoaidinthe
identificationofanunknownsubstance.
Thinlayerchromatography(TLC)isawidely-employed
laboratorytechniqueandissimilartopaper
chromatography.
However,insteadofusingastationaryphaseofpaper,it
involvesastationaryphaseofathinlayerofadsorbentlike
silicagel,alumina,orcelluloseonaflat,inertsubstrate.
Comparedtopaper,ithastheadvantageoffasterruns,
betterseparations,andthechoicebetweendifferent
adsorbents.
Differentcompoundsinthesamplemixturetravel
differentdistancesaccordingtotheinteractionwiththe
adsorbent.ThisallowsthecalculationofanRfvalueand
canbecomparedtostandardcompoundstoaidinthe
identificationofanunknownsubstance.
Gas Liquid Chromatography
Theconceptofgas-liquidwasfirstelucidatedin1941byMartinand
Synge,whowerealsoresponsibleforthedevelopmentofliquid-liquid
partitionchromatography.
VolatileOrganiccompoundscanbeseparatedduetodifferencesintheir
participatingbehaviorbetweenthemobilephase(gas)andthe
stationaryphaseinthecolumn.
Inertgases-Ar,Ne,N
2,Hecanbeusedasmobilephase.
O2 is usually avoided since it will oxidize the solid phase.
The lighter gases He and H2 require faster analysis
Themobilephasedoesnotinteractwithmoleculesoftheanalyte;its
onlyfunctionistotransporttheanalytethroughthecolumn.
Theconceptofgas-liquidwasfirstelucidatedin1941byMartinand
Synge,whowerealsoresponsibleforthedevelopmentofliquid-liquid
partitionchromatography.
VolatileOrganiccompoundscanbeseparatedduetodifferencesintheir
participatingbehaviorbetweenthemobilephase(gas)andthe
stationaryphaseinthecolumn.
Inertgases-Ar,Ne,N
2,Hecanbeusedasmobilephase.
O2 is usually avoided since it will oxidize the solid phase.
The lighter gases He and H2 require faster analysis
Themobilephasedoesnotinteractwithmoleculesoftheanalyte;its
onlyfunctionistotransporttheanalytethroughthecolumn.
Schematic diagram of GLC
Capillarycolumns–fusedsilicaparticlesinthepackedcolumnrequire
chemicalmodification(below).Thestationaryphasesurface(silica)isa
hydroylatedsurface.Thiscausedproblemswithnonpolarstationaryphases.
Whenpolarormildlypolarspeciespartitionintothes.p.theystandagood
chanceofbeingtrapped,causingaexcessivebandbroadeningbeyondwhat
isexpectedfromvanDeemterconsiderations.
Packedcolumns–uniformsilicaparticles(150-250μm)requiredto
ensureuniformpathlengths(the“A”terminthevanDeemtereqn.)
Surfacesarechemicallymodified(seebelow).Thecolumnsthemselves
wereeitherglassorstainlesssteel.
Packed ColumnCapillary Column
Types of columns
chemicalmodification(below).Thestationaryphasesurface(silica)isa
hydroylatedsurface.Thiscausedproblemswithnonpolarstationaryphases.
Whenpolarormildlypolarspeciespartitionintothes.p.theystandagood
chanceofbeingtrapped,causingaexcessivebandbroadeningbeyondwhat
isexpectedfromvanDeemterconsiderations.
Packedcolumns–uniformsilicaparticles(150-250μm)requiredto
ensureuniformpathlengths(the“A”terminthevanDeemtereqn.)
Surfacesarechemicallymodified(seebelow).Thecolumnsthemselves
wereeitherglassorstainlesssteel.
Packed ColumnCapillary Column
Types of columns
GC Injector & Syringe–
It is important to rapidly vaporize the sample. Slow vaporization
increases band broadening, by increasing the sample “plug”.
Injection port temperature is usually held 50
0
C higher than the
BP of the least volatile compound.
InjectorInjector Port
It is important to rapidly vaporize the sample. Slow vaporization
increases band broadening, by increasing the sample “plug”.
Injection port temperature is usually held 50
0
C higher than the
BP of the least volatile compound.
InjectorInjector Port
Detectors
Flame Ionization Detector (FID).
Electron Capture Detector (ECD)
Nitrogen-Phosphorous Detector (NPD)
Flame Photometric Detector (FPD)
Photoionization Detector (PID)
Discharge ionization detector (DID)
Hall electrolytic conductivity detector (ELCD)
Mass selective detector (MSD)
Pulsed discharge ionization detector (PDD)
Thermal energy analyzer (TEA)
Flame Ionization Detector (FID).
Electron Capture Detector (ECD)
Nitrogen-Phosphorous Detector (NPD)
Flame Photometric Detector (FPD)
Photoionization Detector (PID)
Discharge ionization detector (DID)
Hall electrolytic conductivity detector (ELCD)
Mass selective detector (MSD)
Pulsed discharge ionization detector (PDD)
Thermal energy analyzer (TEA)
Flame Ionization Detector
(FID)
Sensitivetowardshydrocarbons-
AnalyteisburnedinH2/air,which
producesCH,CHO+&radicalsofthe
compound
Specificfororganiccarbon,insensitiveto
inorganics,CO2,SO2etc.
Responsetospecificorganicdependson
thenumberoforganiccarbons.
(FID)
Sensitivetowardshydrocarbons-
AnalyteisburnedinH2/air,which
producesCH,CHO+&radicalsofthe
compound
Specificfororganiccarbon,insensitiveto
inorganics,CO2,SO2etc.
Responsetospecificorganicdependson
thenumberoforganiccarbons.
Sensitive to electron withdrawing groups
especially towards organics containing –F, -Cl, -
Br, -I & -CN, NO
2 ,
Nickel-63source emits energetic electrons collides
with N2 (introduced as make-up gas or can be
used as carrier gas) producing more electrons:
Ni-63=>ee-
+N2=>2e-+N2
Theresultisaconstantcurrentthatisdetectedby
theelectroncollector(anode).
Electron Capture Detector (ECD)
especially towards organics containing –F, -Cl, -
Br, -I & -CN, NO
2 ,
Nickel-63source emits energetic electrons collides
with N2 (introduced as make-up gas or can be
used as carrier gas) producing more electrons:
Ni-63=>ee-
+N2=>2e-+N2
Theresultisaconstantcurrentthatisdetectedby
theelectroncollector(anode).
Electron Capture Detector (ECD)
The nitrogen phosphorous detector
also called the thermo ionic detector
Verysensitivetonitrogenand
phosphorouscompounds.
Itisbasedontheflameionization
detectorbutdiffersinthatitcontainsa
rubidiumorcesiumsilicate(glass)bead
situatedinaheatercoil,alittle
distancefromthehydrogenflame.
Thesensitivityofthedetectorto
phosphorousisabout10-12ppmand
fornitrogenabout10-11ppm
NPD
Nitrogen-Phosphorous Detector
also called the thermo ionic detector
Verysensitivetonitrogenand
phosphorouscompounds.
Itisbasedontheflameionization
detectorbutdiffersinthatitcontainsa
rubidiumorcesiumsilicate(glass)bead
situatedinaheatercoil,alittle
distancefromthehydrogenflame.
Thesensitivityofthedetectorto
phosphorousisabout10-12ppmand
fornitrogenabout10-11ppm
NPD
Nitrogen-Phosphorous Detector
Nitrogen-Phosphorus Detector (NPD)
Advantages:
Useful for environmental testing
Detection of organophosphate pesticides
Useful for drug analysis determination of amine-containing or basic drugs
Like FID, does not detect common mobile phase impurities or carrier gases
Limit of detection: NPD is 500x better than FID in detecting nitrogen-and
Phosphorus-containing compounds
NPD more sensitive to other heterocompounds, such as sulfur-, halogen-
,and arsenic-containing molecules
Disadvantages:
Destructive detector
Advantages:
Useful for environmental testing
Detection of organophosphate pesticides
Useful for drug analysis determination of amine-containing or basic drugs
Like FID, does not detect common mobile phase impurities or carrier gases
Limit of detection: NPD is 500x better than FID in detecting nitrogen-and
Phosphorus-containing compounds
NPD more sensitive to other heterocompounds, such as sulfur-, halogen-
,and arsenic-containing molecules
Disadvantages:
Destructive detector
Analyteionized by UV radiation.
Electrodes collect ion current.
Compound must absorb UV
radiation (usually 254 nm) to be
detected.
Application
•BTX compounds
•Styrene
•Trichloroethane
•Isobutane
•Cyclohexane
•Polyvinyl
Photo ionization Detector (PID)
Electrodes collect ion current.
Compound must absorb UV
radiation (usually 254 nm) to be
detected.
Application
•BTX compounds
•Styrene
•Trichloroethane
•Isobutane
•Cyclohexane
•Polyvinyl
Photo ionization Detector (PID)
Flame Photometric Detector
Highly specific for S and P containing
compounds.
Sensitive detector with mass flow
dependent response behavior.
Well suited for capillary GC.
Quadratic dependence of response for S.
Highly specific for S and P containing
compounds.
Sensitive detector with mass flow
dependent response behavior.
Well suited for capillary GC.
Quadratic dependence of response for S.
1 HCH
2 HCH
3 βHCH
4 HCH
5 Aldrin
6 -Endo
7 pp-DDE
8 op-DDT
9 pp-DDD
10 pp-DDT
Spiked water containing
120ppbOC mixture
Spiked water containing
60ppbOC mixture
Spiked water containing
30 ppb OC mixture
Spiked water containing
15ppbOC mixture
GC Chromatogr. of pesticides in Water
1 2 3 4 5 6 7 8 9 10
1 2 3 4 5 6 7 8 9 10
2 HCH
3 βHCH
4 HCH
5 Aldrin
6 -Endo
7 pp-DDE
8 op-DDT
9 pp-DDD
10 pp-DDT
Spiked water containing
120ppbOC mixture
Spiked water containing
60ppbOC mixture
Spiked water containing
30 ppb OC mixture
Spiked water containing
15ppbOC mixture
GC Chromatogr. of pesticides in Water
1 2 3 4 5 6 7 8 9 10
1 2 3 4 5 6 7 8 9 10
Uses of Gas Chromatography
Determination of volatile compounds
(gases & liquids).
Determination of partition coefficients
and absorption isotherms.
Isolating pure components from complex
mixtures.
Determination of volatile compounds
(gases & liquids).
Determination of partition coefficients
and absorption isotherms.
Isolating pure components from complex
mixtures.
HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
Once called High Pressure Liquid Chromatography
CHROMATOGRAPHY
Once called High Pressure Liquid Chromatography
HPLC
InHPLCpressure(wellabovetheatmosphere)arerequiredto
operatehighefficiencycolumnswhichispackedwithsmalldiameter
particlesataflowrateoffewml/min.Asolventorsolventmixtureis
pressurizedthroughapumpanddeliveredapulsefreeflowtothe
columntoseparatethecompounds.
Mostwidelyusedanalyticalseparationstechnique.
Givesthesensitivityuptopartspertrillion(ppt)level.
Usedforaccuratequantitativedetermination.
Suitablefornonvolatilespeciesorthermallyfragileones.
InHPLCpressure(wellabovetheatmosphere)arerequiredto
operatehighefficiencycolumnswhichispackedwithsmalldiameter
particlesataflowrateoffewml/min.Asolventorsolventmixtureis
pressurizedthroughapumpanddeliveredapulsefreeflowtothe
columntoseparatethecompounds.
Mostwidelyusedanalyticalseparationstechnique.
Givesthesensitivityuptopartspertrillion(ppt)level.
Usedforaccuratequantitativedetermination.
Suitablefornonvolatilespeciesorthermallyfragileones.
Types of HPLC
APartition chromatography(liquid mobile phase/liquid stationary phase)
solid support: silica or silica-based
treated similarly to GLC supports
1.Normal phase chromatography
nonpolar mobile/polar stationary:nonpolar solutes elute first
2.Reversed phase chromatography
polar mobile/nonpolar stationary:polar solutes elute first
e.g.,water mobile/hydrocarbon stationary
3.Chiral chromatography
stationary phase is chiral
can separate enantiomers
APartition chromatography(liquid mobile phase/liquid stationary phase)
solid support: silica or silica-based
treated similarly to GLC supports
1.Normal phase chromatography
nonpolar mobile/polar stationary:nonpolar solutes elute first
2.Reversed phase chromatography
polar mobile/nonpolar stationary:polar solutes elute first
e.g.,water mobile/hydrocarbon stationary
3.Chiral chromatography
stationary phase is chiral
can separate enantiomers
B.Adsorption chromatography(liquid mobile phase/solid stationary phase)
oldest type stationary phase: silica, alumina
C.Ion chromatography
separate and determine different ions using ion-exchange resins
resins contain “loosely” held ions these ions will exchange with solute
ions that bind more tightly to resin
D.Size exclusion chromatography
packing:approx. 10 m silica or polymer particles forming a network of
pores of uniform size “very small” molecules get trapped in pores and
are last to be eluted “very large” “molecules” are too big to fit in pores
and get eluted first “intermediate sized” molecules undergo fractionation
very useful technique for polymers
oldest type stationary phase: silica, alumina
C.Ion chromatography
separate and determine different ions using ion-exchange resins
resins contain “loosely” held ions these ions will exchange with solute
ions that bind more tightly to resin
D.Size exclusion chromatography
packing:approx. 10 m silica or polymer particles forming a network of
pores of uniform size “very small” molecules get trapped in pores and
are last to be eluted “very large” “molecules” are too big to fit in pores
and get eluted first “intermediate sized” molecules undergo fractionation
very useful technique for polymers
Mobile phase
Mobile phase are liquids which carries analytes
to the column for separation
Types of elution
a) Isocratic-A solvent or premixed two or more solvents are
delivered through pump through whole analysis.
b) Gradient-Two or more solvents are steadily changed
during the analysis.
Mobile phase are liquids which carries analytes
to the column for separation
Types of elution
a) Isocratic-A solvent or premixed two or more solvents are
delivered through pump through whole analysis.
b) Gradient-Two or more solvents are steadily changed
during the analysis.
Normal and Reverse phase
Normalphase(NP)-Thestationaryphaseisstronglypolarin
nature(e.g.silicagel)andthemobilephaseisnonpolar(such
asn-hexaneortetrahydrofuran)
Reversephase(RP)-Thestationaryphaseisnonpolar
(hydrophobic)innaturewhilethemobilephaseispolarliquid
(suchasmixtureofwaterandmethanoloracetonitrile.
Normalphase(NP)-Thestationaryphaseisstronglypolarin
nature(e.g.silicagel)andthemobilephaseisnonpolar(such
asn-hexaneortetrahydrofuran)
Reversephase(RP)-Thestationaryphaseisnonpolar
(hydrophobic)innaturewhilethemobilephaseispolarliquid
(suchasmixtureofwaterandmethanoloracetonitrile.
Stainless steel
2-30 cm length
4 -10 mm internal diameter
0.45-10 µparticle size
High Speed Isocratic / Gradient separation
Variation in solvent changes
Elution at different pH
Analytical Column
HPLC Columns
2-30 cm length
4 -10 mm internal diameter
0.45-10 µparticle size
High Speed Isocratic / Gradient separation
Variation in solvent changes
Elution at different pH
Analytical Column
HPLC Columns
Types of column
Types of Detector
UV/Visible
Photo diode array
Fluorescence
Conductivity
Refractive index
Electrochemical
Light scattering
UV/Visible
Photo diode array
Fluorescence
Conductivity
Refractive index
Electrochemical
Light scattering
UV / Visible
DetectUV&visibleregion
MeasureaccordingtoBearLambertslaw
Photocellmeasuresabsorbanceoflight
ModernUVdetectorhasfilterfor
repetitiveandquantitativeanalysis
Advantages
•highsensitivity
•smallsamplevolumerequired
•linearityoverwideconcentration
ranges
•canbeusedwithgradientdetection
Disadvantage
Notworkforthecompoundsthatdonot
absorblightatthiswavelengthregion.
DetectUV&visibleregion
MeasureaccordingtoBearLambertslaw
Photocellmeasuresabsorbanceoflight
ModernUVdetectorhasfilterfor
repetitiveandquantitativeanalysis
Advantages
•highsensitivity
•smallsamplevolumerequired
•linearityoverwideconcentration
ranges
•canbeusedwithgradientdetection
Disadvantage
Notworkforthecompoundsthatdonot
absorblightatthiswavelengthregion.
HPLC Chromatogram of PAHs Std.
and blood sample
1
2
3
4
5
6
7
8
9
10
11
12
13
and blood sample
1
2
3
4
5
6
7
8
9
10
11
12
13
Fluorescence
For compounds having natural
fluorescing capability
Fluorescence observed by
photoelectric detector
Mercury or Xenon source are used
with grating monochromator to
isolate fluorescent radiation
For compounds having natural
fluorescing capability
Fluorescence observed by
photoelectric detector
Mercury or Xenon source are used
with grating monochromator to
isolate fluorescent radiation
Conductivity
Measure conductivity of column effluent
Concentration of the sample
proportional to change in conductivity
Best use in ion-exchange
chromatography
Cell instability
Advantages
•extremely high sensitivity
•high selectivity
Disadvantage
•may not give linear response over
wide range of concentrations.
Measure conductivity of column effluent
Concentration of the sample
proportional to change in conductivity
Best use in ion-exchange
chromatography
Cell instability
Advantages
•extremely high sensitivity
•high selectivity
Disadvantage
•may not give linear response over
wide range of concentrations.
Refractive Index
Measure displacement of beam with respect
to photosensitive surface of detector.
Advantages
•Universal respond to nearly all solutes
•Reliable
•Unaffected by flow rate
•Low sensitive to dirt and air bubbles.
Disadvantages
•Expensive
•Highly temperature sensitive
•Moderate sensitivity
•Cannot be used with gradient elution flow
cell
Measure displacement of beam with respect
to photosensitive surface of detector.
Advantages
•Universal respond to nearly all solutes
•Reliable
•Unaffected by flow rate
•Low sensitive to dirt and air bubbles.
Disadvantages
•Expensive
•Highly temperature sensitive
•Moderate sensitivity
•Cannot be used with gradient elution flow
cell
Electrochemical
Basedonreductionoroxidationoftheeluting
compoundatasuitableelectrodeand
measuretheresultingcurrent
Advantages
•Highsensitivity
•Easytouse
Disadvantages
•Mobilephasemustbemadeconductive
•Mobilephasemustbepurifiedfrom
oxygen,metalcontamination&halides
Basedonreductionoroxidationoftheeluting
compoundatasuitableelectrodeand
measuretheresultingcurrent
Advantages
•Highsensitivity
•Easytouse
Disadvantages
•Mobilephasemustbemadeconductive
•Mobilephasemustbepurifiedfrom
oxygen,metalcontamination&halides
Advantages of HPLC
Higher resolution and speed of analysis can be performed.
HPLC columns can be reused without repacking or regeneration.
Greater reproducibility.
Easy operation of instrument and data analysis.
Adaptability to large-scale for analytical / preparative
procedures.
Stationary supports with very small particle sizes and large
surface areas.
Higher resolution and speed of analysis can be performed.
HPLC columns can be reused without repacking or regeneration.
Greater reproducibility.
Easy operation of instrument and data analysis.
Adaptability to large-scale for analytical / preparative
procedures.
Stationary supports with very small particle sizes and large
surface areas.
Fast Protein Liquid Chromatography
Itisaformofcolumnchromatographyusedtoseparateor
purifyproteinsfromcomplexmixtures.Itisverycommonlyused
inbiochemistryandenzymology.ColumnsusedwithanFPLC
canseparatemacromoleculesbasedonsize,chargedistribution,
hydrophobicity,orbiorecognition(aswithaffinity
chromatography)
Applicationsoffastproteinliquidchromatographyinthe
separationofplasmaproteinsinurineandcerebrospinalfluid.
Chromatographicseparationoftheproteinstakes1hforurine
specimensand45minforCSF.
Itisaformofcolumnchromatographyusedtoseparateor
purifyproteinsfromcomplexmixtures.Itisverycommonlyused
inbiochemistryandenzymology.ColumnsusedwithanFPLC
canseparatemacromoleculesbasedonsize,chargedistribution,
hydrophobicity,orbiorecognition(aswithaffinity
chromatography)
Applicationsoffastproteinliquidchromatographyinthe
separationofplasmaproteinsinurineandcerebrospinalfluid.
Chromatographicseparationoftheproteinstakes1hforurine
specimensand45minforCSF.
Application of HPLC
For the separation, identification and quantification-
Carbohydrates, Nucleosides (purines and pyrimidines)
Amino acids, proteins, Vitamins
Pharmaceutical products
Fatty acids, fats ,Aflatoxins ,Antioxidants
Pollutants like PAHs, PCBs, pesticides, phenols, phthalates
Carotenoids, chlorophylls, cocaine,
Alcohol in blood,
Explosive materials
The Police, F.B.I., and other detectives use chromatography
when trying to solve a crime.
A variety of other organic substances.
For the separation, identification and quantification-
Carbohydrates, Nucleosides (purines and pyrimidines)
Amino acids, proteins, Vitamins
Pharmaceutical products
Fatty acids, fats ,Aflatoxins ,Antioxidants
Pollutants like PAHs, PCBs, pesticides, phenols, phthalates
Carotenoids, chlorophylls, cocaine,
Alcohol in blood,
Explosive materials
The Police, F.B.I., and other detectives use chromatography
when trying to solve a crime.
A variety of other organic substances.
Mass Spectrometry
MSisananalyticaltoolthatisusedtoidentifyunknown
compounds,quantifyknownmaterialandelucidatethestructure
andphysicalpropertiesofions.Itrequirelessthanpicogram
amt.(10-12)ofmaterial
MScanseparatechargedatomsormoleculesaccordingtotheir
mass-to-chargeratio(m/z).
MSisananalyticaltoolthatisusedtoidentifyunknown
compounds,quantifyknownmaterialandelucidatethestructure
andphysicalpropertiesofions.Itrequirelessthanpicogram
amt.(10-12)ofmaterial
MScanseparatechargedatomsormoleculesaccordingtotheir
mass-to-chargeratio(m/z).
Schematic diagram of MS
Working of MS
Sample is introduced under high vacuum to convert into gas
phase.
The compound under investigation is bombarded with a
beam of electron which produce ionic molecule or fragments
of the original species.
Ions (+&-ions) are repelled out of the ion source and
accelerated towards the analyser region.
Analyser can detect either positive or negative ions.
Sample is introduced under high vacuum to convert into gas
phase.
The compound under investigation is bombarded with a
beam of electron which produce ionic molecule or fragments
of the original species.
Ions (+&-ions) are repelled out of the ion source and
accelerated towards the analyser region.
Analyser can detect either positive or negative ions.
Components of MS
Inlet system
Ionization source
Electrostatic accelerating device
Ion separator (magnetic field)
Ion collector (detector)
Vacuum pump
Inlet system
Ionization source
Electrostatic accelerating device
Ion separator (magnetic field)
Ion collector (detector)
Vacuum pump
Ionization Sources
Electron Ionization
Chemical ionization (isobutane, methane,
ammonia)
Fast atomic bombardment
Liquid secondary ionization
MALDI
Electro spray ionization
SELDI
Electron Ionization
Chemical ionization (isobutane, methane,
ammonia)
Fast atomic bombardment
Liquid secondary ionization
MALDI
Electro spray ionization
SELDI
Detector
Detector monitors ion current, amplifies it and then
transmits signal to data system.
Types of detectors used
Field-Electromagnetic field/ Electric field
Quadrupole and ion trap-radio frequency& direct
current
TOF
Photomultiplier tube
Electron multiplier
Micro channel plate
Detector monitors ion current, amplifies it and then
transmits signal to data system.
Types of detectors used
Field-Electromagnetic field/ Electric field
Quadrupole and ion trap-radio frequency& direct
current
TOF
Photomultiplier tube
Electron multiplier
Micro channel plate
5.00 10.00 15.00 20.00 25.00 30.00
Time0
100
%
STDPAHmix261006 Scan EI+
TIC
7.51e4
16.20
15.41
7.31
3.33
3.73
7.44
8.81
11.43
30.92
25.73
21.28
21.43
31.69 1
2
3
4
5
6
7
8
9105.00 10.00 15.00 20.00 25.00 30.00
Time0
100
%
Sam45-261006 Scan EI+
TIC
1.91e5
16.24
8.08
3.04 15.41
13.8512.46
10.33
22.69
18.40
18.05
18.53
21.43
20.38
26.74
25.77
23.15
31.71
30.94
28.78
Time0
100
%
STDPAHmix261006 Scan EI+
TIC
7.51e4
16.20
15.41
7.31
3.33
3.73
7.44
8.81
11.43
30.92
25.73
21.28
21.43
31.69 1
2
3
4
5
6
7
8
9105.00 10.00 15.00 20.00 25.00 30.00
Time0
100
%
Sam45-261006 Scan EI+
TIC
1.91e5
16.24
8.08
3.04 15.41
13.8512.46
10.33
22.69
18.40
18.05
18.53
21.43
20.38
26.74
25.77
23.15
31.71
30.94
28.78
5.00 10.00 15.00 20.00 25.00 30.00
Time0
100
%
Sam45-261006 Scan EI+
TIC
1.91e5
16.24
8.08
3.04 15.41
13.8512.46
10.33
22.69
18.40
18.05
18.53
21.43
20.38
26.74
25.77
23.15
31.71
30.94
28.78 Acenaphthene
Pyrene
Fluoranthene
B(j) fluoranthene
B(ghi)Pyrelene
GC-MS chromatogram after
matching with library (NIST)
Time0
100
%
Sam45-261006 Scan EI+
TIC
1.91e5
16.24
8.08
3.04 15.41
13.8512.46
10.33
22.69
18.40
18.05
18.53
21.43
20.38
26.74
25.77
23.15
31.71
30.94
28.78 Acenaphthene
Pyrene
Fluoranthene
B(j) fluoranthene
B(ghi)Pyrelene
GC-MS chromatogram after
matching with library (NIST)
Reaction monitoring-enzyme activity, chemical modification , protein digestion
Amino acid sequencing
Oligonucleotide sequencing
Protein structure determination
Environmental Monitoring and Cleanup
GC-MSisbecomingthetoolofchoicefortrackingorganicpollutantsintheenvironment.Therearesomecompoundsfor
whichGC-MSisnotsufficientlysensitive,includingcertainpesticidesandherbicides,butformostorganicanalysisof
environmentalsamples,includingmanymajorclassesofpesticides,itisverysensitiveandeffective.
Criminal Forensics
GC-MScananalyzetheparticlesfromahumanbodyinordertohelplinkacriminaltoacrime.Theanalysisoffreedebrisusing
GC-MSiswellestablished,andthereisevenanestablishedAmericanSocietyforTestingMaterials(ASTM)standardforfire
debrisanalysis.
Food,BeverageandPerfumeAnalysis
Fortheanalysisofthesecompoundswhichincludeesters,fattyacids,alcoholsandterpenesetc.Itisalsousedtodetectand
measurecontaminantsfromspoilageoradulterationwhichmaybeharmfulandwhichisoftencontrolledbygovernmental
agencies,forexamplepesticide.
Medicine
Incombinationwithisotopiclabelingofmetaboliccompounds,theGC-MSisusedfordetermining.
LawEnforcement
GC-MS is increasingly used for detection of illegal narcotic, and may eventually supplant drug-sniffing dogs.
Security
Explosive detections, many of them based on GC-MS.
Uses of MASS
Amino acid sequencing
Oligonucleotide sequencing
Protein structure determination
Environmental Monitoring and Cleanup
GC-MSisbecomingthetoolofchoicefortrackingorganicpollutantsintheenvironment.Therearesomecompoundsfor
whichGC-MSisnotsufficientlysensitive,includingcertainpesticidesandherbicides,butformostorganicanalysisof
environmentalsamples,includingmanymajorclassesofpesticides,itisverysensitiveandeffective.
Criminal Forensics
GC-MScananalyzetheparticlesfromahumanbodyinordertohelplinkacriminaltoacrime.Theanalysisoffreedebrisusing
GC-MSiswellestablished,andthereisevenanestablishedAmericanSocietyforTestingMaterials(ASTM)standardforfire
debrisanalysis.
Food,BeverageandPerfumeAnalysis
Fortheanalysisofthesecompoundswhichincludeesters,fattyacids,alcoholsandterpenesetc.Itisalsousedtodetectand
measurecontaminantsfromspoilageoradulterationwhichmaybeharmfulandwhichisoftencontrolledbygovernmental
agencies,forexamplepesticide.
Medicine
Incombinationwithisotopiclabelingofmetaboliccompounds,theGC-MSisusedfordetermining.
LawEnforcement
GC-MS is increasingly used for detection of illegal narcotic, and may eventually supplant drug-sniffing dogs.
Security
Explosive detections, many of them based on GC-MS.
Uses of MASS
Chiral chromatography
Counter current chromatography
Lectin chromatography
Hydroxyapatite chromatography
Some specific chromatography
Counter current chromatography
Lectin chromatography
Hydroxyapatite chromatography
Some specific chromatography
Chiral chromatography
Thisinvolvestheseparationofstereoisomers.
Inthecaseofenantiomers,thesehavenochemicalor
physicaldifferencesapartfrombeingthreedimensional
mirrorimages.
Conventionalchromatographyorotherseparationprocesses
areincapableofseparatingthem.
Toenablechiralseparationstotakeplace,eitherthemobile
phaseorthestationaryphasemustthemselvesbemade
chiral,givingdifferingaffinitiesbetweentheanalytes.
ChiralchromatographyHPLCcolumns(withachiral
stationaryphase)inbothnormalandreversedphaseare
commerciallyavailable.
Thisinvolvestheseparationofstereoisomers.
Inthecaseofenantiomers,thesehavenochemicalor
physicaldifferencesapartfrombeingthreedimensional
mirrorimages.
Conventionalchromatographyorotherseparationprocesses
areincapableofseparatingthem.
Toenablechiralseparationstotakeplace,eitherthemobile
phaseorthestationaryphasemustthemselvesbemade
chiral,givingdifferingaffinitiesbetweentheanalytes.
ChiralchromatographyHPLCcolumns(withachiral
stationaryphase)inbothnormalandreversedphaseare
commerciallyavailable.
Countercurrent chromatography
Thisisatypeofliquid-liquidchromatography,
whereboththestationaryandmobilephases
areliquids.Itinvolvesmixingasolutionof
liquids,allowingthemtosettleintolayersand
thenseparatingthelayers.
Thisisatypeofliquid-liquidchromatography,
whereboththestationaryandmobilephases
areliquids.Itinvolvesmixingasolutionof
liquids,allowingthemtosettleintolayersand
thenseparatingthelayers.
Lectin affinity chromatography
Thisisaformofaffinitychromatographywhere
lectinareusedtoseparatecomponentswithinthe
sample.Lectin,suchasconcanavalinA,areproteins
whichcanbindspecificcarbohydrate(sugar)
molecules.Themostcommonapplicationisto
separateproteinbasedontheirglycangroups.
Thisisaformofaffinitychromatographywhere
lectinareusedtoseparatecomponentswithinthe
sample.Lectin,suchasconcanavalinA,areproteins
whichcanbindspecificcarbohydrate(sugar)
molecules.Themostcommonapplicationisto
separateproteinbasedontheirglycangroups.
Future of Chromatography
National safety/ Defense Forensic science
R&D
Health-Drugs
Environmental -Food & beverages and
pollutant chemicals
Material Characterization
National safety/ Defense Forensic science
R&D
Health-Drugs
Environmental -Food & beverages and
pollutant chemicals
Material Characterization
SPECTROSCOPY
Spectral Distribution of Radiant EnergyX-Ray UV Visible IR Microwave
200nm 400nm 800nm
WAVELENGTH(nm)
Wave Number (cycles/cm)
Spectroscopywasoriginallythestudyoftheinteractionbetween
radiationandmatterasafunctionofwavelengthλ.
Spectrophotometercanbeusedtodeterminetheentityof
anunknownsubstance,ortheconcentrationofanumberofknown
substances.Thetypeofsource/filtersusedtypicallydetermines
thetypeofthespectrophotometer.
Spectral Distribution of Radiant EnergyX-Ray UV Visible IR Microwave
200nm 400nm 800nm
WAVELENGTH(nm)
Wave Number (cycles/cm)
Spectroscopywasoriginallythestudyoftheinteractionbetween
radiationandmatterasafunctionofwavelengthλ.
Spectrophotometercanbeusedtodeterminetheentityof
anunknownsubstance,ortheconcentrationofanumberofknown
substances.Thetypeofsource/filtersusedtypicallydetermines
thetypeofthespectrophotometer.
DISPERSION OF POLYCHROMATIC LIGHT WITH A PRISM Polychromatic
Ray
Infrared
Red
Orange
Yellow
Green
Blue
Violet
Ultraviolet
monochromatic
Ray
SLIT
PRISM
Polychromatic Ray Monochromatic Ray
Prism -spray out the spectrum and choose the certain wavelength
(l)that you want by slit.
Ray
Infrared
Red
Orange
Yellow
Green
Blue
Violet
Ultraviolet
monochromatic
Ray
SLIT
PRISM
Polychromatic Ray Monochromatic Ray
Prism -spray out the spectrum and choose the certain wavelength
(l)that you want by slit.
2.Fluorometer -measures the intensity of fluorescent light emitted by a
sample exposed to UV light under specific conditions.Emit fluorescent light
as energy decreases
Ground state
Sample
90C
Detector
UV Light Source
Monochromator
Monochromator
Antibonding
Antibonding
Nonbonding
Bonding
Bonding
Energy
'
'
'
'
'
n->
n
n->'
Electron's molecular energy levels
Fluorometer
sample exposed to UV light under specific conditions.Emit fluorescent light
as energy decreases
Ground state
Sample
90C
Detector
UV Light Source
Monochromator
Monochromator
Antibonding
Antibonding
Nonbonding
Bonding
Bonding
Energy
'
'
'
'
'
n->
n
n->'
Electron's molecular energy levels
Fluorometer
BEER LAMBERT LAW Glass cell filled with
concentration of solution (C)
II
Light
0
As the cell thickness increases, the intensity of I (transmitted intensity of light )
decreases.Light Lens Slit Monochromator
Sample Detector Quantitative Analysis
Slits
concentration of solution (C)
II
Light
0
As the cell thickness increases, the intensity of I (transmitted intensity of light )
decreases.Light Lens Slit Monochromator
Sample Detector Quantitative Analysis
Slits
R-Transmittance
R = I
0-original light intensity
I-transmitted light intensity
% Transmittance = 100 x
Absorbance (A) or optical density (OD) = Log
= Log = 2 -Log%T
Log is proportional to C (concentration of solution) and is
also proportional to L (length of light path
through the solution).
I
I
0
I
I
0
I
0
I
1
T
I
I
0
R = I
0-original light intensity
I-transmitted light intensity
% Transmittance = 100 x
Absorbance (A) or optical density (OD) = Log
= Log = 2 -Log%T
Log is proportional to C (concentration of solution) and is
also proportional to L (length of light path
through the solution).
I
I
0
I
I
0
I
0
I
1
T
I
I
0
A CL = KCL by definition and it is called the Beer
Lambert Law.
A = KCL
K = Specific Extinction Coefficient ----1 g of solute
per liter of solution
A = ECL
E =Molar Extinction Coefficient ----Extinction
Coefficient of a solution containing 1g molecule of
solute per 1 liter of solution
Lambert Law.
A = KCL
K = Specific Extinction Coefficient ----1 g of solute
per liter of solution
A = ECL
E =Molar Extinction Coefficient ----Extinction
Coefficient of a solution containing 1g molecule of
solute per 1 liter of solution
CELLS
UV Spectrophotometer -Quartz (crystalline silica)
Visible Spectrophotometer -Glass
IR Spectrophotometer -NaCl
LIGHT SOURCES
UV Spectrophotometer
1.Hydrogen Gas Lamp
2.Mercury Lamp
Visible Spectrophotometer
1.Tungsten Lamp
IR Spectrophotometer
1.Carborundum (SIC)
UV Spectrophotometer -Quartz (crystalline silica)
Visible Spectrophotometer -Glass
IR Spectrophotometer -NaCl
LIGHT SOURCES
UV Spectrophotometer
1.Hydrogen Gas Lamp
2.Mercury Lamp
Visible Spectrophotometer
1.Tungsten Lamp
IR Spectrophotometer
1.Carborundum (SIC)
CHROMOPHORIC STRUCTURE
Group Structure nm
Carbonyl > C = O 280
Azo -N = N- 262
Nitro -N=O 270
Thioketone -C =S 330
Nitrite -NO2 230
Conjugated Diene -C=C-C=C- 233
Conjugated Triene -C=C-C=C-C=C- 268
Conjugated Tetraene -C=C-C=C-C=C-C=C- 315
Benzene 261
Group Structure nm
Carbonyl > C = O 280
Azo -N = N- 262
Nitro -N=O 270
Thioketone -C =S 330
Nitrite -NO2 230
Conjugated Diene -C=C-C=C- 233
Conjugated Triene -C=C-C=C-C=C- 268
Conjugated Tetraene -C=C-C=C-C=C-C=C- 315
Benzene 261
Type of spectroscopy
Spectroscopy depends on the physical quantity
measured. Normally, the quantity that is measured
is an intensity, either of energy absorbed or
produced.
OpticalSpectroscopyorUltraviolet-visibleSpectroscopy
(involvesinteractionsofmatterwithelectromagnetic
radiationlight).
Electronspectroscopyisananalyticaltechniquetostudythe
electronicstructureanditsdynamicsinatomsandmolecules.
Ingeneralanexcitationsourcesuchasxrays,electrons,will
ejectanelectronfromaninner-shellorbitalofanatom.
Spectroscopy depends on the physical quantity
measured. Normally, the quantity that is measured
is an intensity, either of energy absorbed or
produced.
OpticalSpectroscopyorUltraviolet-visibleSpectroscopy
(involvesinteractionsofmatterwithelectromagnetic
radiationlight).
Electronspectroscopyisananalyticaltechniquetostudythe
electronicstructureanditsdynamicsinatomsandmolecules.
Ingeneralanexcitationsourcesuchasxrays,electrons,will
ejectanelectronfromaninner-shellorbitalofanatom.
Massspectrometryisananalyticaltechniquethat
measuresthemasstochargeratioofions.Itismost
generallyusedtofindthecompositionofaphysical
samplebygeneratingamassspectrumrepresenting
themassesofsamplecomponents.Themass
spectrumismeasuredbyamassspectrometer.
Dielectricspectroscopy(sometimescalledimpedence
spectroscopy)measuresthedielectricpropertiesofa
mediumasafunctionoffrequency.Itisbasedonthe
interactionofanexternalfieldwiththeelectricdipole
momentsofthesample.
measuresthemasstochargeratioofions.Itismost
generallyusedtofindthecompositionofaphysical
samplebygeneratingamassspectrumrepresenting
themassesofsamplecomponents.Themass
spectrumismeasuredbyamassspectrometer.
Dielectricspectroscopy(sometimescalledimpedence
spectroscopy)measuresthedielectricpropertiesofa
mediumasafunctionoffrequency.Itisbasedonthe
interactionofanexternalfieldwiththeelectricdipole
momentsofthesample.
Most spectroscopic methods are differentiated as either
Atomic or molecular
Absorptionspectroscopy(Usestherangeofthe
electromagneticspectrainwhichasubstanceabsorbs.)
Thisincludes{AtomicAbsorptionspectroscopyandvarious
moleculartechniques(infraredspectroscopy,Nuclear
magneticresonancespectroscopy(NMR)}
Emissionspectroscopy{Usestherangeofelectromagnetic
spectrainwhichasubstanceradiates(emits)}.Techniques
includespectrofluorimeter.
lightscatteringspectroscopy-measurestheamountoflight
thatasubstancescattersatcertainwavelengths,incident
angles,andpolarizationangles.Thescatteringprocessismuch
fasterthantheabsorption/emissionprocess.e.g.(Raman
spectroscopy)
Atomic or molecular
Absorptionspectroscopy(Usestherangeofthe
electromagneticspectrainwhichasubstanceabsorbs.)
Thisincludes{AtomicAbsorptionspectroscopyandvarious
moleculartechniques(infraredspectroscopy,Nuclear
magneticresonancespectroscopy(NMR)}
Emissionspectroscopy{Usestherangeofelectromagnetic
spectrainwhichasubstanceradiates(emits)}.Techniques
includespectrofluorimeter.
lightscatteringspectroscopy-measurestheamountoflight
thatasubstancescattersatcertainwavelengths,incident
angles,andpolarizationangles.Thescatteringprocessismuch
fasterthantheabsorption/emissionprocess.e.g.(Raman
spectroscopy)
Atomic absorption
spectrophotometer
Atomic absorption spectroscopy is a quantitative method of analysis that is
applicable to metals and Metalloids .
An atomic absorption spectrophotometer consists of a light source, a sample
compartment and a detector.
In this method, light from a source is directed through the sample to a
detector. The greater the amount of sample present, the greater the
absorbance produced by the sample.
The source of light is a lamp whose cathode is composed of the element being
measured.
Each element requires a different lamp.
One of the most common means of introducing the sample into the flame is
by preparing a solution of the sample in a suitable solvent, frequently water.
The flame gases flowing into the burner create a suction that pulls the liquid
into the small tube from the sample container. This liquid is transferred to the
flame where the ions are atomized. These atoms absorb light from the
source.
spectrophotometer
Atomic absorption spectroscopy is a quantitative method of analysis that is
applicable to metals and Metalloids .
An atomic absorption spectrophotometer consists of a light source, a sample
compartment and a detector.
In this method, light from a source is directed through the sample to a
detector. The greater the amount of sample present, the greater the
absorbance produced by the sample.
The source of light is a lamp whose cathode is composed of the element being
measured.
Each element requires a different lamp.
One of the most common means of introducing the sample into the flame is
by preparing a solution of the sample in a suitable solvent, frequently water.
The flame gases flowing into the burner create a suction that pulls the liquid
into the small tube from the sample container. This liquid is transferred to the
flame where the ions are atomized. These atoms absorb light from the
source.
Atomic Absorption spectroscopy(AAS)
Basic components-
Nebulizer-create a sample mist
Burner -Slot-shaped which
gives a longer path length
flame
Lamps
Graphite furnace-Solid or
slurry samples, greater
sensitivity
Recorder
Basic components-
Nebulizer-create a sample mist
Burner -Slot-shaped which
gives a longer path length
flame
Lamps
Graphite furnace-Solid or
slurry samples, greater
sensitivity
Recorder
A hollow cathode lamp for Aluminum (Al)
The lamp is housed inside
the lamp compartment of
the instrument.
The sample compartment is really the
flame since it is in the flame that the
atoms absorb radiation from the
source.
The lamp is housed inside
the lamp compartment of
the instrument.
The sample compartment is really the
flame since it is in the flame that the
atoms absorb radiation from the
source.
Quantitativeanalysiscanbe
achievedbymeasuringthe
absorbanceofaseriesof
solutions of known
concentration.
Acalibrationcurveandthe
equationforthelinecanbeused
todetermineanunknown
concentrationbasedonits
absorbance.
achievedbymeasuringthe
absorbanceofaseriesof
solutions of known
concentration.
Acalibrationcurveandthe
equationforthelinecanbeused
todetermineanunknown
concentrationbasedonits
absorbance.
Atomic Fluorescence
spectroscopy
“Atomicfluorescencespectroscopy(AFS)istheoptical
emissionfromgas-phaseatomsthathavebeenexcited
tohigherenergylevelsbyabsorptionofradiation.”
“AFSisusefultostudytheelectronicstructureofatoms
andtomakequantitativemeasurementsofsample
concentrations.”
spectroscopy
“Atomicfluorescencespectroscopy(AFS)istheoptical
emissionfromgas-phaseatomsthathavebeenexcited
tohigherenergylevelsbyabsorptionofradiation.”
“AFSisusefultostudytheelectronicstructureofatoms
andtomakequantitativemeasurementsofsample
concentrations.”
The atomic emission spectrumof an element is the set of frequencies of
the electromagnetic waves emitted by atoms of that element. Each atom's
atomic emission spectrum is unique and can be used to determine if that
element is part of an unknown compound.
The emission spectrum characteristics of some elements are plainly visible
to the naked eye when these elements are heated. For example, when
platinum wire is dipped into a strontium nitrate solution and then inserted
into a flame, the strontium atoms emit a red color. Similarly, when copper
is inserted into a flame, the flame becomes light blue. These definite
characteristics allow elements to be identified by their atomic emission
spectrum. Not all lights emitted by the spectrum are viewable to the naked
eye, it also includes ultra violet rays and infra red lighting.
Thefactthatonlycertaincolorsappearinanelement'satomicemission
spectrummeansthatonlycertainfrequenciesoflightareemitted.Eachof
thesefrequenciesarerelatedtoenergybytheformula:
E=h ν
herein Eis energy, hisPlank’s constant and νis the frequency.
Atomic emission spectroscopy
the electromagnetic waves emitted by atoms of that element. Each atom's
atomic emission spectrum is unique and can be used to determine if that
element is part of an unknown compound.
The emission spectrum characteristics of some elements are plainly visible
to the naked eye when these elements are heated. For example, when
platinum wire is dipped into a strontium nitrate solution and then inserted
into a flame, the strontium atoms emit a red color. Similarly, when copper
is inserted into a flame, the flame becomes light blue. These definite
characteristics allow elements to be identified by their atomic emission
spectrum. Not all lights emitted by the spectrum are viewable to the naked
eye, it also includes ultra violet rays and infra red lighting.
Thefactthatonlycertaincolorsappearinanelement'satomicemission
spectrummeansthatonlycertainfrequenciesoflightareemitted.Eachof
thesefrequenciesarerelatedtoenergybytheformula:
E=h ν
herein Eis energy, hisPlank’s constant and νis the frequency.
Atomic emission spectroscopy
UV SPECTROMETER : Protein,Amino Acids (aromatic),Pantothenic Acid,Glucose
Determination,Enzyme Activity (Hexokinase).
FLUOROMETER : Thiamin (365 nm, 435 nm),Riboflavin,Vitamin A,Vitamin C.
VISIBLE SPECTROPHOTOMETER :Niacin,Pyridoxine,Vitamin B12,Metal
Determination (Fe),Fat-quality Determination (TBA),Enzyme Activity (glucose
oxidase).
APPLICATIONS-
Determination,Enzyme Activity (Hexokinase).
FLUOROMETER : Thiamin (365 nm, 435 nm),Riboflavin,Vitamin A,Vitamin C.
VISIBLE SPECTROPHOTOMETER :Niacin,Pyridoxine,Vitamin B12,Metal
Determination (Fe),Fat-quality Determination (TBA),Enzyme Activity (glucose
oxidase).
APPLICATIONS-
Chromatography
Generic Protocol
1. Prepare Column (±)
2. Apply Sample
3. Wash
4. Elute
5. Analyze Fractions
Equipment
1.HPLC/FPLC
2.GLC
3.TLC
4.Paper Chromatography
Generic Protocol
1. Prepare Column (±)
2. Apply Sample
3. Wash
4. Elute
5. Analyze Fractions
Equipment
1.HPLC/FPLC
2.GLC
3.TLC
4.Paper Chromatography
TheoryofChromatography
1.)Typicalresponseobtainedbychromatography(i.e.,achromatogram):
chromatogram-concentrationversuselutiontime
W
h
W
b
Where:
t
R= retention time
t
M= void time
W
b= baseline width of the peak in time units
W
h= half-height width of the peak in time units
Inject
1.)Typicalresponseobtainedbychromatography(i.e.,achromatogram):
chromatogram-concentrationversuselutiontime
W
h
W
b
Where:
t
R= retention time
t
M= void time
W
b= baseline width of the peak in time units
W
h= half-height width of the peak in time units
Inject
(A)Adifferenceintheretentionofsolutes(i.e.,adifferenceintheirtimeor
volumeofelution)
(B)Asufficientlynarrowwidthofthesolutepeaks(i.e,goodefficiencyforthe
separationsystem)
Asimilarplotcanbemadeintermsofelutionvolumeinsteadofelutiontime.Ifvolumesare
used,thevolumeofthemobilephasethatittakestoeluteapeakoffofthecolumnisreferredtoas
theretentionvolume(V
R)andtheamountofmobilephasethatittakestoeluteanon-retained
componentisreferredtoasthevoidvolume(V
M).
Peak width & peak position
determine separation of peaks
Theseparationofsolutesinchromatographydependsontwofactors:
volumeofelution)
(B)Asufficientlynarrowwidthofthesolutepeaks(i.e,goodefficiencyforthe
separationsystem)
Asimilarplotcanbemadeintermsofelutionvolumeinsteadofelutiontime.Ifvolumesare
used,thevolumeofthemobilephasethatittakestoeluteapeakoffofthecolumnisreferredtoas
theretentionvolume(V
R)andtheamountofmobilephasethatittakestoeluteanon-retained
componentisreferredtoasthevoidvolume(V
M).
Peak width & peak position
determine separation of peaks
Theseparationofsolutesinchromatographydependsontwofactors:
Retentiontime:
Asolute’sretentiontimeorretentionvolumeinchromatographyisdirectlyrelatedtothe
strengthofthesolute’sinteractionwiththemobileandstationaryphases.
Retentiononagivencolumnpertaintotheparticularsofthatsystem:
-sizeofthecolumn
-flowrateofthemobilephase
Capacityfactor(k’):moreuniversalmeasureofretention,determinedfromt
RorV
R.
k’=(t
R–t
M)/t
M
or
k’=(V
R–V
M)/V
M
capacityfactorisusefulforcomparingresultsobtainedondifferentsystemssinceitis
independentoncolumnlengthandflow-rate.
Asolute’sretentiontimeorretentionvolumeinchromatographyisdirectlyrelatedtothe
strengthofthesolute’sinteractionwiththemobileandstationaryphases.
Retentiononagivencolumnpertaintotheparticularsofthatsystem:
-sizeofthecolumn
-flowrateofthemobilephase
Capacityfactor(k’):moreuniversalmeasureofretention,determinedfromt
RorV
R.
k’=(t
R–t
M)/t
M
or
k’=(V
R–V
M)/V
M
capacityfactorisusefulforcomparingresultsobtainedondifferentsystemssinceitis
independentoncolumnlengthandflow-rate.
Thevalueofthecapacityfactorisusefulinunderstandingtheretentionmechanismsfor
asolute,sincethefundamentaldefinitionofk’is:
k’isdirectlyrelatedtothestrengthoftheinteractionbetweenasolutewiththe
stationaryandmobilephases.
MolesA
stationaryphaseandmolesA
mobilephaserepresentstheamountofsolutepresentin
eachphaseatequilibrium.
Equilibriumisachievedorapproachedatthecenterofachromatographicpeak.
k’ =
moles A
stationary phase
moles A
mobile phase
When k' is # 1.0, separation is poor
When k' is > 30, separation is slow
When k' is = 2-10, separation is optimum
asolute,sincethefundamentaldefinitionofk’is:
k’isdirectlyrelatedtothestrengthoftheinteractionbetweenasolutewiththe
stationaryandmobilephases.
MolesA
stationaryphaseandmolesA
mobilephaserepresentstheamountofsolutepresentin
eachphaseatequilibrium.
Equilibriumisachievedorapproachedatthecenterofachromatographicpeak.
k’ =
moles A
stationary phase
moles A
mobile phase
When k' is # 1.0, separation is poor
When k' is > 30, separation is slow
When k' is = 2-10, separation is optimum
A simple example relating k’ to the interactions of a solute in a column is
illustrated for partition chromatography:
A (mobile phase) A (stationary phase)
K
D
where: K
D= equilibrium constant for the distribution of A between the
mobile phase and stationary phase
Assuming local equilibrium at the center of the chromatographic peak:
k’ =
[A]
stationary phase Volume
stationary phase
[A]
mobile phaseVolume
mobilephase
k’ = K
D
Volume
stationary phase
Volume
mobilephase
As K
Dincreases, interaction of the solute with the stationary phase
becomes more favorable and the solute’s retention (k’) increases
illustrated for partition chromatography:
A (mobile phase) A (stationary phase)
K
D
where: K
D= equilibrium constant for the distribution of A between the
mobile phase and stationary phase
Assuming local equilibrium at the center of the chromatographic peak:
k’ =
[A]
stationary phase Volume
stationary phase
[A]
mobile phaseVolume
mobilephase
k’ = K
D
Volume
stationary phase
Volume
mobilephase
As K
Dincreases, interaction of the solute with the stationary phase
becomes more favorable and the solute’s retention (k’) increases
k’ = K
D
Volume
stationary phase
Volume
mobilephase
Separation between two solutes requires different K
D’s for their
interactions with the mobile and stationary phases
since
peak separation also represents different changes in free energy
DG = -RT ln K
D
D
Volume
stationary phase
Volume
mobilephase
Separation between two solutes requires different K
D’s for their
interactions with the mobile and stationary phases
since
peak separation also represents different changes in free energy
DG = -RT ln K
D
3.)Efficiency:
Efficiencyisrelatedexperimentallytoasolute’speakwidth.
-anefficientsystemwillproducenarrowpeaks
-narrowpeakssmallerdifferenceininteractionsinordertoseparatetwo
solutes
Efficiencyisrelatedtheoreticallytothevariouskineticprocessesthatare
involvedinsoluteretentionandtransportinthecolumn
-determinethewidthorstandarddeviation()ofpeaks
W
h
Estimate from peak widths,
assuming Gaussian shaped peak:
W
b= 4
W
h= 2.354
Dependent on the amount of time that a solute spends in the column (k’ or t
R)
Efficiencyisrelatedexperimentallytoasolute’speakwidth.
-anefficientsystemwillproducenarrowpeaks
-narrowpeakssmallerdifferenceininteractionsinordertoseparatetwo
solutes
Efficiencyisrelatedtheoreticallytothevariouskineticprocessesthatare
involvedinsoluteretentionandtransportinthecolumn
-determinethewidthorstandarddeviation()ofpeaks
W
h
Estimate from peak widths,
assuming Gaussian shaped peak:
W
b= 4
W
h= 2.354
Dependent on the amount of time that a solute spends in the column (k’ or t
R)
Efficienciesofcolumnswithdifferentlengths
H=L/N
where:L=columnlength
N=numberoftheoreticalplatesforthecolumn
Note:Hsimplygivesthelengthofthecolumnthatcorrespondstoonetheoreticalplate
Hcanbealsousedtorelatevariouschromatographicparameters(e.g.,flowrate,particlesize,
etc.)tothekineticprocessesthatgiverisetopeakbroadening:
Plate height (H)
H=L/N
where:L=columnlength
N=numberoftheoreticalplatesforthecolumn
Note:Hsimplygivesthelengthofthecolumnthatcorrespondstoonetheoreticalplate
Hcanbealsousedtorelatevariouschromatographicparameters(e.g.,flowrate,particlesize,
etc.)tothekineticprocessesthatgiverisetopeakbroadening:
Plate height (H)
Numberoftheoreticalplates(N):compareefficienciesofasystemforsolutesthathave
differentretentiontimes
N=(t
R/s)
2
orforaGaussianshapedpeak
N=16(t
R/W
b)
2
N=5.54(t
R/W
h)
2
differentretentiontimes
N=(t
R/s)
2
orforaGaussianshapedpeak
N=16(t
R/W
b)
2
N=5.54(t
R/W
h)
2
Resolution(R
S)–resolutionbetweentwopeaksisasecondmeasureofhowwell
twopeaksareseparated:
R
S=
where:
t
r1,W
b1=retentiontimeandbaselinewidthforthe
firstelutingpeak
t
r2,W
b2=retentiontimeandbaselinewidthforthe
secondelutingpeak
t
r2–t
r1
(W
b2+ W
b1)/2
R
sis preferred over since both
retention (t
r) and column efficiency
(W
b) are considered in defining
peak separation.
R
s$1.5 represents baseline
resolution, or complete separation
of two neighboring solutes ideal
case.
R
s$1.0 considered adequate for
most separations.
S)–resolutionbetweentwopeaksisasecondmeasureofhowwell
twopeaksareseparated:
R
S=
where:
t
r1,W
b1=retentiontimeandbaselinewidthforthe
firstelutingpeak
t
r2,W
b2=retentiontimeandbaselinewidthforthe
secondelutingpeak
t
r2–t
r1
(W
b2+ W
b1)/2
R
sis preferred over since both
retention (t
r) and column efficiency
(W
b) are considered in defining
peak separation.
R
s$1.5 represents baseline
resolution, or complete separation
of two neighboring solutes ideal
case.
R
s$1.0 considered adequate for
most separations.
VanDeemterequation:relatesflow-rateorlinearvelocitytoH:
H=A+B/+C
where:
= linear velocity (flow-rate x V
m/L)
H = total plate height of the column
A = constant representing eddy diffusion &
mobile phase mass transfer
B = constant representing longitudinal diffusion
C = constant representing stagnant mobile phase
& stationary phase mass transfer
Oneuseofplateheight(H)istorelatethesekineticprocesstobandbroadeningtoa
parameterofthechromatographicsystem(e.g.,flow-rate).
Thisrelationshipisusedtopredictwhattheresultingeffectwouldbeofvaryingthis
parameterontheoverallefficiencyofthechromatographicsystem.
Number of theoretical plates(N)(N) = 5.54 (t
R/W
h)
2
peak width (W
h)
H = L/N
H=A+B/+C
where:
= linear velocity (flow-rate x V
m/L)
H = total plate height of the column
A = constant representing eddy diffusion &
mobile phase mass transfer
B = constant representing longitudinal diffusion
C = constant representing stagnant mobile phase
& stationary phase mass transfer
Oneuseofplateheight(H)istorelatethesekineticprocesstobandbroadeningtoa
parameterofthechromatographicsystem(e.g.,flow-rate).
Thisrelationshipisusedtopredictwhattheresultingeffectwouldbeofvaryingthis
parameterontheoverallefficiencyofthechromatographicsystem.
Number of theoretical plates(N)(N) = 5.54 (t
R/W
h)
2
peak width (W
h)
H = L/N
optimum
Plot of van Deemter equation shows how H changes with the linear velocity (flow-rate) of
the mobile phase
Optimum linear velocity (
opt) -where H has a minimum value and the point of maximum
column efficiency:
opt= rB/C
optis easy to achieve for gas chromatography, but is usually too small for liquid
chromatography requiring flow-rates higher than optimal to separate compounds
Plot of van Deemter equation shows how H changes with the linear velocity (flow-rate) of
the mobile phase
Optimum linear velocity (
opt) -where H has a minimum value and the point of maximum
column efficiency:
opt= rB/C
optis easy to achieve for gas chromatography, but is usually too small for liquid
chromatography requiring flow-rates higher than optimal to separate compounds
Separationfactor(a)–parameterusedtodescribehowwelltwosolutesare
separatedbyachromatographicsystem:
=k’
2/k’
1 k’=(t
R–t
M)/t
M
where:
k’
1=thecapacityfactorofthefirstsolute
k’
2=thecapacityfactorofthesecondsolute,
withk’
2$k’
1
Avalueof$1.1isusuallyindicativeofagoodseparation
Does notconsider the effect of column efficiency or peak widths, only retention.
separatedbyachromatographicsystem:
=k’
2/k’
1 k’=(t
R–t
M)/t
M
where:
k’
1=thecapacityfactorofthefirstsolute
k’
2=thecapacityfactorofthesecondsolute,
withk’
2$k’
1
Avalueof$1.1isusuallyindicativeofagoodseparation
Does notconsider the effect of column efficiency or peak widths, only retention.
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