PCG 308 for Year 3 Undergraduate students- 2024.ppt

Separating techniques using electrophoresis, GC, HPLC

1. PCG 308
EXTRACTION AND CHROMATOGRAPHIC TECHNIQUES

2. LIQUID-LIQUID EXTARCTION
Liquid-Liquidextractionisversatileanddependable
separationtechniquewhereinanaqueoussolutionisusually
broughtintocontactwithanotherorganicsolvent,
exclusivelyimmisciblewiththeformer,soastoaffecta
legitimateandactualtransferofeitheroneormoresolutes
intothelater.
Theseparationswhichcanthusbeachievedarefoundtobe
rathereasy,fast,convenientandeffectivereasonably.
Suchseparationareperformedbyshakingthetwoliquids
inaseparatoryfunnelforafewminute.

3. The partitioning of solute(s) between two phases is an
equilibrium process and is characterized by an equilibrium
constant called the partition coefficient:

4. Oneimportantthingtonoteisthatitismoreefficientto
extractwithmultiplesmallvolumesthanoncewithan
equivalentlargevolume.Forexample,itismore
efficienttoextractwithtwo100mLvolumesof
extractionsolventthanone200mLvolumeeventhough
thetotalextractionvolumeisthesame.

5. Inliquid-liquidextractionsthefollowingtwoaspectsare
crucialandimportant,namely:
(a)Errorduetothevolumechange,and
(b)Effectivenessofanextraction.
Basedontheappropriatepartitioncoefficientofanimmiscible
solventpairitispossibletocalculatetheeffectivenessofan
extractione.g
Ifthedistributioncoefficientofasolute(0.5g)betweenwater
andethylacetateis1/12,afterfour
successiveextractionswith100mlusing25mleachtime,what
totalpercentageofthesolutemust
havepassedfrom100mlofitsaqueoussolutionintoethyl
acetate?Comparetheeffectivenessof
extractionwithsingleextractionwith100mlofethylacetate.

6. W
n = W
o
KV
1 + V
2
KV
1
n
Using the formula WhereW
n
=amountofsoluteremaininginthe
aqueousphase(water)afterextractionforn
times
W
o
=initialamountinaqueousphase=0.5gK = coefficient of solute between H
2O and EtOAc =
1
12
V
1
=volumeofwater=100ml
V
2
=volumeofEtOAcusedforextraction(25ml)
n=numberoftimesofextraction=4

7. W
n = 0.5
100
4
1
12
x
100 + 25
1
12
x
W
n = 0.5
8.333
4
8.333 + 25
W
n = 0.5
8.333
4
8.333 + 25
= 0.5
8.333
4
33.333
= 0.5
4
0.25 W=0.5(3.9x10
-3
)=1.95x10
-3
gor0.00195
Amount extracted = 0.5 -0.00195 = 0.498 g

8. 100
0.498
0.5
x% extraction = = 99.61% Forsingleextractionusing100ml(V
2)ofEtOAcW
n = 0.5
100
1
1
12
x
100 + 100
1
12
x W
n = 0.5
8.333
1
8.333 + 100
= 0.5
8.333
1
108.333
= 0.50.076920
1
0.0385 g =
Amount extracted = 0.5 g –0.0385 g = 0.461 g100
0.461
0.5
x% extraction = = 92.3%

9. Comparethisvalue0.461g(92.3%)whenonelarge
extractionwasperformedusingthesametotal
volumeofethylacetate,itwillbeseenthatamore
satisfactoryextractionresultsfromusingthesolvent
inseveralseparateportionswhichmayleadtoalmost
completeextractionwhichis0.498g(99.61%)incase
offoursuccessiveextractionswith100mlusing25ml
eachtime

10. FACTORSINFLUENCINGSOLVENTEXTRACTION
Anumberofcardinalfactorsexertapositiveinfluenceonthe
phenomenonofsolventextraction,namely:
(a)Effectoftemperature(maybeestimatedconvenientlyfromits
effectsonthesolubilitiesofthesubstanceinthetworespective
solvents)
(b)Effectofinertsolutes.
ForexampleCaCl2,MgCl2,andsucrosecanbeemployed
efficaciouslytosolveextractionproblemsbyallowingefficient
extractionsfromthewaterintosuchsolventasacetone,
ethanolandmethanolthatarefoundtobecompletelymiscible
withwaterintheabsenceofsalt.MatkovitchandCristian
foundtheaboveinertsolutestobethebestagentsforsalting
acetoneoutofwater.

11. (c)EffectofpHonextraction
Generallyithasbeenfoundthatorganicacidsandbasesdoexistin
aqueoussolutionasequilibriummixturesoftheirrespectiveneutral
aswellasionicforms.Thustheseneutralandionicformsmaynot
havethesameidenticalpartitioncoefficientinasecondsolvent;
therefore,thequantityofasubstancebeingextractedsolelydepends
uponthepositionofthecid-baseequilibriumandultimatelyupon
thepHftheresultingsolution.
(d)Effectofion-pairformation
Ion-pairsareformedbythevirtueofunionbetweencomparativelylarge
organicanionsandmuchsmallercations.Interestingly,theresulting
ion-pairshavebeenfoundtoshowtheirappreciablesolubilityinpolar
solvent;andhencethesespeciesmaybeextractedconvenientlyunder
suchexperimentalparameterswhereneitherindividualcomponention
could.

12. (d)Effectofsynergisticextraction
EMULSIONPROBLEMENCOUNTERED INEXTRACTIONS
Emulsionmaybedefinedas‘adispersedsystemcontainingatleasttwo
immiscibleliquidphases
Theeffectiveandmeaningfulextractionofananalyteisrenderedalmost
impossiblewhenoneencountersanemulsionformationduringanextraction
processtherebytheseparationofthetwophasesbecomesdifficult
BreakingofanEmulsion
(i)Centrifugation---especiallywhenthedensitiesofthetwosolventsare
appreciablydifferent
(ii)Additionofmonovalentanddivalentions:---Simpleemulsionarebroken
byaddingmonovalentsaltslikeNaClordivalentionssuchasCaCl
2orMgCl
2
incaseofcharge-stabilizedemulsion

13. (iii)Additionofsmallquantitiesofeitherethanolorsometimes
higherhomologousalcoholwillaidcoalescinganemulsion.
(iv)Siliconedefoamingagent:--Afewdropsofthesilicone
defoamingagentssometimeshelpinbreakingemulsion
(v)Thin-Bedofanadsorbent:--Sometimessimplypassingan
emulsionthroughathin-bedofanadsorbentcanremarkably
helpsinachievingemulsionprovidedtheanalyteswillnotbe
absorbedfromeithersolvent.
(vi)Suddencoolingofemulsion(Thermalshock)----Sudden
temperaturedroporfreezing(i.e.givingathermalshock)ofan
emulsionmostlyenhancestheinterfacialtensionbetweenthetwo
immisciblephasestherebycausingcoalescence
(vii)Breakingofemulsioncanalsobeachievedbyalteringthe
ratiooftheprevailingsolvent/dispersedphase.

20. DISCOVERY
M.S. TSWETT, 1903, WARSAW
POLYTECHNIC INSTITUTE
(SEPARATION OF THE
CHLOROPHYLLS OF GREEN LEAVES
EXTRACT)

21. There are four major separation modes that are
used to separate most compounds:
1. Reversed-phase chromatography
2. Normal-phase and adsorption chromatography
3. Ion exchange chromatography
4. Size exclusion chromatography

22. GSC GLC
GAS SFC
NP RP IEC
GPC GFC
SEC
Column
TLC Paper
Planar
LIQUID
CHROMATOGRAPHY

23. Adsorption Chromatography (NP, RP, IEX)
Interactions of the analyte with the
adsorbent surface causing its slower
movement compared to the eluent
molecules
Size-Exclusion Chromatography
Exclusion of the analyte molecules from
the adsorbent pore volume due to their
size
No interactions with the adsorbent
surface

24. NORMAL PHASE
Principle:Adsorption of analytes on the polar,
weakly acidic surface of silica gel.
Stationary Phase.:Silica (pH 2-8), Alumina (pH 2 -
12), Bonded Diol, and NH
2, or CN
Mobile Phase:Non-polar solvents (Hexane, CHCl
3)
Applications: Non-polar and semi-polar samples;
hexane soluble; positional isomers.

25. NP: SEPARATION PRINCIPLE
Polar (specific but nonionic) interactions of
analyte with polar adsorption sites (SiOH, -NH
2, -
CN, Diol) cause its retention
Different sorption affinities between analytes
result in their separation
More polar analytes retained longer
Analytes with larger number of polar
functional group are retained longer
Structural isomers are often separated

26. Principle: Partition of analytes between mobile phase
and stagnant phase inside the pore space +
adsorption on the surface of bonded phase.
Stationary Phase: Hydrophobic surfaces of moieties
bonded on silica (C18, C8, C5, Phenyl, CN)
Mobile phase: Methanol or Acetonitrile and Water.
Applications: ~80% of all separations performed with
RP HPLC.
80%Octadecylsilica (ODS, C18)
10%Octylsilica (C8)
5%Butylsilica (C4)
3% Phenyl
2%Cyano (CN)

27. REVERSED PHASE
SEPARATION PRINCIPLE
Nonpolar (nonspecific) interactions of analyte
with hydrophobic adsorbent surface (-C18, C8,
Phenyl, C4)
Different sorption affinities between analytes
results in their separation
More polar analytes retained less
Analytes with larger hydrophobic part are
retained longer
structural isomers maybe more
challenging in this mode

28. Ion Exchange
Principle: Reversible adsorption of ions on S.P. with
oppositely charged functional groups.
Anionic polymers are known as cation exchange
resins and these resins can be strong or weak
cation exchange resins which are strongly
dependent upon the anionic group that is
bonded to the polymer.
Cationic polymers on the other hand are known as
anion exchangeresins and these resins can also
be weak or strong anion exchange.

29. Stationary Phase:
For cations (cation exchange) -SO
3
-(strong)
,
CO
2
-(weak)
For anions (anion exchange)-NR
4
+ (strong)
,
NH
3
+ (weak)
Mobile Phase: Aqueous buffer with pH and buffer
strength carefully controlled.
Applications: All ionic compounds common anions,
cations, sugars, amines, etc.

30. Size Exclusion
Principle: Internal pores of stationary phase exclude
analytes molecules based on their hydrodynamic
volume. Vr is correlated to M.W. by calibration curve.
Stationary Phase: Porous polymeric particles
(SDVB) with pore diameters of 80, 100, 150, 300, 500
or 1000 Å.
Mobile Phase: Good solvent for polymer. Solvent
must suppress all possible interactions with the
stationary phase surface.
Applications: Organic polymers, biopolymers.