Chromatographic Techniques of Anaylsis

7/24/2019 Chromatographic Techniques of Anaylsis

1/39

Chromatographic

Techniques of Anaylsis

Dr. Anant R. Kapdi


2/39


3/39

Chromatography

,t is the partitioning of components of a sample according to theirdistri(ution coe)cient (et"een a mo(ile phase and a stationary phase.

Stationary phase is al"ays a solid surface such as silica&o(ile phase represents the sol+ents.

Chromatography "as rst disco+ered (y &i#hail Ts"ett.Calcium car(onate used as the stationary phase for separatingcoloured pigment from plant e!tract using pet. ether as mo(ile phase.

The "ord comes from -ree# "ords Chromos "hich means colour andgraphy "hich means to "rite.


4/39

Classication of chromatographic separations

a/hysical state of mo(ile and stationary phase

&o(ile phase0

i liquid Column chromatography* Thin layer chromatography*

High performance liquid chromatography

ii -as -as chromatography

Stationary phase0

i solid Column* TLC

ii liquid lm coated on solid surface -C* H/LC

iii /orous gel Si1e e!clusion chromatography

2&ethod of contact (et"een the mo(ile and stationary phase

2Chemical or physical mechanism for separation

i Adsorption chromatography0 Column* -C

ii /artition0 H/LC


5/39

Thin Layer Chromatography 3Adsorption chromatography

&o(ile /hase0 liquid 4 Stationary phase0 solid adsor(ent 3silica


6/39

So+ents used0

D&$5&e6H5DC&57t6Ac57t865/et

ether5He!ane5pentane

Sol+ent system0

polar 0 nonpolar sol+ent

7t6Ac He!ane or /et. 7ther

7t86 /entane

&e6H He!ane

-radient elution0 ,ncrease concentration of one sol+ent to elute +ery

closely places solutes.


7/39

Relation (et"een distance tra+elled (y sol+ent and thesu(stance is gi+en (y

e.g.


8/39

TLC preparation process

/reparing the Cham(er

/reparing /lates for De+elopment

De+eloping the /lates

,dentifying the Spots

,nterpreting Data

http0%%""".chemilp.net%la(Techniques%TLC9ideo.htm


9/39

Stain de+elopment of TLC plates

:9light0 for most organic compounds

,odine0 for unsaturated compounds

/hosphomoly(dic acid

Dinitrophenyl hydra1ine0 aldehydes and #etones9anillin0 general organic compounds

K&n6;0 sensiti+e groups to o!idationCerric ammonium moly(date


10/39

6n a TLC plate Rf +alues o(tained "ere *


11/39

Column Chromatography

Column chromatography can (e used on (oth a large and small scale. Theapplications of this technique are "ide reaching and cross many disciplines

including (iology* (iochemistry* micro(iology and medicine.


12/39

Column chromatography is technique "hich "or#s on the same principleas TLC. Ho"e+er* unli#e TLC "here dierent compounds are separatedon a TLC plate* it is possi(le to collect these compounds separately

using column chromatography.

,n TLC the direction of sol+ent Eo" isdo"n to up "hile re+erse is the casefor Column. Ho"e+er* the theoryremains the same as TLC. onpolarcomponent "ill elute rst from the

column follo"ed (y the polar.


13/39

/rocess in+ol+ed in the separation of components using Columnchromatography

Stationary phase0 Silica or Alumina

8 Choice of sol+ent0 The sol+ent system de+eloped during TLC "hichallo"ed (est

separation of the components of the mi!ture

@ Apparatus0 -lass column* (ea#ers

; /ac#ing column0 a Dry /ac#ing ( Slurrymethod


14/39

? Sample application ' -radient elution


15/39

-eneral theory of Chromatographicanalysis

The retention time* tr, is the elapsed timefrom the introduction of the solute to thepea# ma!imum.

The retention time also can (e measuredindirectly as the +olume of mo(ile phase

eluting (et"een the soluteFs introductionand the appearance of the soluteFs pea#ma!imum. This is #no"n as the retentionvolume, Vr.Void time, tm


16/39

Chromatographic resolution

The goal of chromatography is to separate a sample into a series ofchromatographic pea#s* each representing a single component of the sample.Resolution is a quantitati+e measure of the degree of separation (et"een t"ochromatographic pea#s* A and G*


17/39

,n a chromatographic analysis of lemon oil a pea# for limonene has aretention time of =.@' min "ith a (aseline "idth of


18/39

Capacity factor

A soluteFs capacity factor can (e determined from achromatogram (y measuring the columnFs +oid time* tm, andthe solutes retention time, tr

adjusted retention time: The dierence (et"een a

soluteFs retention time and columnFs +oid time 3tr).

,n a chromatographic analysis of lo"molecular"eight acids*(utyric acid elutes "ith a retention time of >.'@ min. The columnFs+oid time is


19/39

The relati+e selecti+ity of a chromatographic column fora pair of solutes is gi+en (y the selectivity factor, a,

which is dened as

Column Selectivity

,n a chromatographic analysis of lo"molecular"eight acids*(utyric acid elutes "ith a retention time of >.'@ min. The columnFs+oid time is


20/39

Column e)ciency

Band broadening0 ith time the "idth of the solute pea# "hilepassing through the column starts (roadening. This aects thee)ciency of the column. Groader the pea#s e)ciency of the columnto seprate the solute reduces.

Thereotical lates: &artin and Synge ha+e proposed hypotehticalplates that are nothing (ut crosssections of the column "here thedistri(ution of the solute (et"een the stationary and mo(ile phaseta#es place.


21/39

Thereotical plate could (e calculatedas0

J num(er of thereotical plates* HJ height of each thereotical plate*L J length of the column

A chromatographic column ha+ing a length of 8 m has 8


22/39

KGJ Capacity factor* J column selecti+ity* R J resolution


23/39

-AS CHR6&AT6-RA/HM

&o(ile /hase0 -as 3carrier gas

Stationary /hase0 solid or liquid coated onsolid support

-assolid chromatography -asliquidchromatography


24/39

Schematic of -C

a &o(ile /hase0 also called as Carrier -as as it helps in carrying the

solutes from the inNector to the detector. ,nertness "ith the solute isan important criteria.

ArgonHeliumitrogen


25/39

Chromatographic columnsa /ac#ed columns

$or -SC/artitioning of solute (et"een mo(ile phase and solid stationaryphase

&ade0 stainless steel* aluminium* glass* copperLength0 8> mStationary phase0 Diatomeceous earth-LC/artitioning of solute (et"een mo(ile phase and liquid stationaryphase coated on a solid support

&ade0 Stainless steel* glass etc.Solid support0 To a+oid the adsorption of solute molecules one!posed pac#ing material* "hich degrades the quality of theseparation* surface silanols are deacti+ated (y silani1ing "ithdimethyldichlorosilane and "ashing "ith an alcohol 3typicallymethanol (efore coating "ith stationary phase


26/39

( Capillary columns0 also #no"n as open tu(ular columns&ade0 fused silica coated on polymer supportLength0


27/39


28/39

Detectors in -C0aThermal conducti+ity detector0 it is (ased on the thermal conducti+ityof the mo(ile phase. Higher the capacity of the carrier gas to conductheat* (etter is the detection capactiy. Helium is used as carrier gas due

to its high thermal conducti+ity.. :ni+ersally applica(le8. nondestructi(le@. poor detection limit

( $lame ,oni1ation detector0 Com(ustion of organic compounds in H8%air

leads to electrons and ions. 9oltage of @


29/39

!" #cetaldehyde $%& C'%" (thyl #cetate $)) C'*" +ethanol $- C'

." (thanol $)/ C'-" n01roanol $/2 C'" 3sobutanol $!&2 C')" #myl #lcohol43soamyl #lcohol $!*2C'2" #cetic #cid $!%* C'

#cetaldehyde and (thylacetate haveless stronger 50bonding interactionwith the stationary hase so theycome out rst, followed by +e65,(t65 etc" #myl alcohol in comarison

to #cetic acid has higher boiling ointbut acetic acid has a stronger 50bonding"

Composition of Scotch his#ey

Arrange the follo"ing compounds in the order of elutiona&e6H(;itro (en1oic acidcGen1aldehyde

d;&etho!y aniline


30/39

Hyphenated Techniques0 -as Chromatography&assSpectrometry

http0%%""".shsu.edu%PchmQtgc%sound

s%Eashles%-C&S.s"f


31/39


32/39


33/39

High /erformance LiquidChromatography


34/39

H/LC Columns

Analytical Columns,nternal diameters0 8. mm to ;.' mmLength0 @


35/39

Stationary phases0 -enerally the same as -C (ut depending on thepolarity the column could (e classied as ormal or Re+erse phasecolumn.

ormal /hase Chromatography0,f R a(o+e is a polar group then the column stationary phase is polarin nature and in that case use of a normal polar or slightly polarsol+ent. -eneral organic compounds can (e separated using this type ofchromatography.Less polar solute comes out rst follo"ed (y the more polar 3sameprinciple as TLC* Column

Re+ersed phase0 R group in most cases is a nonpolar group such as octyl oroctyldecyl hydrocar(on chain. Highly polar sol+ent such as &e6H or (uered solution could (eused. Ho"e+er* the pH cannot (e more than >.? due to the possi(ility


36/39

&o(ile phase0

/reparati+e H/LC0 ,n normal H/LC the solutes can (e detectedand quantied. Ho"e+er* physical separation is not possi(le.

/reparati+e H/LC allo"s physical separation to gi+e theproducts as analytically pure samples.

Detectors0


37/39

Detectors0aSpectroscopic detectors0 :99is or $luorescence detectors

. A(sor(ance is detected as function of time8. +olumes are lo" around < L@. ,mportant criteria is the mo(ile phase should strongly a(sor( in

the gi+en range;. 9ery good detection limit upto ng

( 7lectrochemical detectors0

. Change in potential "rt reference electrode is used to detectsamples

8. 9ery good detection limit upto < pg@. 9olumes are high


38/39

Hyphenated Technique0 Liquid Chromatography &ass Spectrometry


39/39

Chromatographic Techniques of Anaylsis

Documents