View
235
Download
1
Category
Preview:
Citation preview
7/24/2019 Chromatographic Techniques of Anaylsis
1/39
Chromatographic
Techniques of Anaylsis
Dr. Anant R. Kapdi
7/24/2019 Chromatographic Techniques of Anaylsis
2/39
7/24/2019 Chromatographic Techniques of Anaylsis
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.
7/24/2019 Chromatographic Techniques of Anaylsis
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
2ðod 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
7/24/2019 Chromatographic Techniques of Anaylsis
5/39
Thin Layer Chromatography 3Adsorption chromatography
&o(ile /hase0 liquid 4 Stationary phase0 solid adsor(ent 3silica
7/24/2019 Chromatographic Techniques of Anaylsis
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.
7/24/2019 Chromatographic Techniques of Anaylsis
7/39
Relation (et"een distance tra+elled (y sol+ent and thesu(stance is gi+en (y
e.g.
7/24/2019 Chromatographic Techniques of Anaylsis
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
7/24/2019 Chromatographic Techniques of Anaylsis
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
7/24/2019 Chromatographic Techniques of Anaylsis
10/39
6n a TLC plate Rf +alues o(tained "ere *
7/24/2019 Chromatographic Techniques of Anaylsis
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.
7/24/2019 Chromatographic Techniques of Anaylsis
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.
7/24/2019 Chromatographic Techniques of Anaylsis
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
7/24/2019 Chromatographic Techniques of Anaylsis
14/39
? Sample application ' -radient elution
7/24/2019 Chromatographic Techniques of Anaylsis
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
7/24/2019 Chromatographic Techniques of Anaylsis
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*
7/24/2019 Chromatographic Techniques of Anaylsis
17/39
,n a chromatographic analysis of lemon oil a pea# for limonene has aretention time of =.@' min "ith a (aseline "idth of
7/24/2019 Chromatographic Techniques of Anaylsis
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
7/24/2019 Chromatographic Techniques of Anaylsis
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
7/24/2019 Chromatographic Techniques of Anaylsis
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.
7/24/2019 Chromatographic Techniques of Anaylsis
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
7/24/2019 Chromatographic Techniques of Anaylsis
22/39
KGJ Capacity factor* J column selecti+ity* R J resolution
7/24/2019 Chromatographic Techniques of Anaylsis
23/39
-AS CHR6&AT6-RA/HM
&o(ile /hase0 -as 3carrier gas
Stationary /hase0 solid or liquid coated onsolid support
-assolid chromatography -asliquidchromatography
7/24/2019 Chromatographic Techniques of Anaylsis
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
7/24/2019 Chromatographic Techniques of Anaylsis
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
7/24/2019 Chromatographic Techniques of Anaylsis
26/39
( Capillary columns0 also #no"n as open tu(ular columns&ade0 fused silica coated on polymer supportLength0
7/24/2019 Chromatographic Techniques of Anaylsis
27/39
7/24/2019 Chromatographic Techniques of Anaylsis
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 @
7/24/2019 Chromatographic Techniques of Anaylsis
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;ðo!y aniline
7/24/2019 Chromatographic Techniques of Anaylsis
30/39
Hyphenated Techniques0 -as Chromatography&assSpectrometry
http0%%""".shsu.edu%PchmQtgc%sound
s%Eashles%-C&S.s"f
7/24/2019 Chromatographic Techniques of Anaylsis
31/39
7/24/2019 Chromatographic Techniques of Anaylsis
32/39
7/24/2019 Chromatographic Techniques of Anaylsis
33/39
High /erformance LiquidChromatography
7/24/2019 Chromatographic Techniques of Anaylsis
34/39
H/LC Columns
Analytical Columns,nternal diameters0 8. mm to ;.' mmLength0 @
7/24/2019 Chromatographic Techniques of Anaylsis
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
7/24/2019 Chromatographic Techniques of Anaylsis
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
7/24/2019 Chromatographic Techniques of Anaylsis
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
7/24/2019 Chromatographic Techniques of Anaylsis
38/39
Hyphenated Technique0 Liquid Chromatography &ass Spectrometry
7/24/2019 Chromatographic Techniques of Anaylsis
39/39
Recommended