Basics of Chromatography.ppt

Basics of Chromatography

1. Basics of Chromatography
JonyMallik

2. Introductory Principles
Chromatographyisacombinationoftwowords;
*Chromo–Meaningcolor
*Graphy–representationofsomethingonpaper

3. History of Chromatography
Chromatography,literally"colorwriting",wasfirstemployedby
RussianscientistMikhailTswettin1903/1906.Hecontinuedtowork
withchromatographyinthefirstdecadeofthe20thcentury,primarily
fortheseparationofplantpigmentssuchaschlorophyll,carotenes,
andxanthophylls.Sincethesecomponentshavedifferentcolors
(green,orange,andyellow,respectively)theygavethetechniqueits
name.

4. Itisaphysicalseparationmethodofseparationin
whichthecomponentsofamixtureareseparatedby
differencesintheirdistributionbetweentwophases,
oneofwhichisstationary(stationaryphase)while
theother(mobilephase)movesthroughitina
definitedirection.Thesubstancesmustinteractwith
thestationaryphasetoberetainedandseparatedby
it.

5. Definition of chromatography
IUPACdefinition(InternationalUnionofpureandapplied
Chemistry)(1993):
Chromatographyisaphysicalmethodofseparationinwhich
thecomponentstobeseparatedaredistributedbetweentwo
phases,oneofwhichisstationarywhiletheothermovesina
definitedirection.
Thestationaryphasemaybeasolid,oraliquidsupportedon
asolidorgel,themobilephasemaybeeitheragasora
liquid.

6. Mixture
Separate
Analyze
•Identify
•Purify
•Quantify
Components
CHROMATOGRAPHY

7. Chromatograph: Instrument employed for a chromatography.
Eluent: Fluid entering a column.
Eluate: Fluid exiting the column.
Elution: The process of passing the mobile phase through the
column.
Flow rate: How much mobile phase passed / minute (ml/min).
Linear velocity: Distance passed by mobile phase per 1 min in
the column (cm/min).

8. MobilePhase–gasorliquidthatcarriesthe
mixtureofcomponentsthroughthestationary
phase.
StationaryPhase–thepartoftheapparatusthat
holdsthecomponentsastheymovethroughit,
separatingthem.

9. Uses for Chromatography
Chromatography is used by scientists to:
•Analyze–examine a mixture, its components, and
their relations to one another
•Identify–determine the identity of a mixture or
components based on known components
•Purify–separate components in order to isolate one of
interest for further study
•Quantify–determine the amount of the a mixture
and/or the components present in the sample

10. Real-life examples of uses for chromatography:
Pharmaceutical Company
Hospital
Law Enforcement
Environmental Agency
Manufacturing Plant

11. Chromatogram:
Itisthevisualoutputofthechromatograph.
Chromatograph:
ItisequipmentthatenablesasophisticatedSeparation.
Stationaryphase(boundedphase):
Itisaphasethatiscovalentlybondedtothesupportparticlesorto
theinsidewallofthecolumntubing.

12. Mobile phase:
Itis the phase which moves in a definite direction.
Analyte (Sample):
Itis the substance to be separated during chromatography.
Eluate:
Itis the mobile phase leaving the column.

13. Retentiontime:
Itisthecharacteristictimeittakesforaparticularanalyteto
passthroughthesystem(fromthecolumninlettothedetector)
undersetconditions.
Eluent:
Itisthesolventthatwillcarrytheanalyte.

14. Retardation factor(R):
Fraction of an analyte in the mobile phase of a
chromatographic system.

15. •LiquidChromatography–separatesliquidsampleswithaliquid
solvent(mobilephase)andacolumncomposedofsolidbeads
(stationaryphase)
•GasChromatography–separatesvaporizedsampleswitha
carriergas(mobilephase)andacolumncomposedofaliquidorof
solidbeads(stationaryphase)
•PaperChromatography–separatesdriedliquidsampleswitha
liquidsolvent(mobilephase)andapaperstrip(stationaryphase)
•Thin-LayerChromatography–separatesdriedliquidsamples
withaliquidsolvent(mobilephase)andaglassplatecoveredwitha
thinlayerofaluminaorsilicagel(stationaryphase)

17. GEL FILTRATION
Gelfiltrationseparatesmoleculesaccordingtothe
differencesinsizeastheypassthroughthefiltration
mediumpackedinthecolumn.
Itiswellsuitedforbiomoleculesthataresensitiveto
pH,concentrationandharshenvironment.
Parametersthataffectsgelfiltrationare,particlesize,
flowrate,packagingdensity,porosityoftheparticle
andviscosityofthemobilephase.

20. MATERIALS REQUIRED
Cross linked dextrans (sephadex)
Agarose (sepharose)
Polyacrylamide
Porous glass gel.
APPLICATIONS
Fractionation(purificationofthedesiredproteinusingsuitable
gel)
Molecularweightdetermination

21. ION EXCHANGE
Ion exchange
chromatography is used
to remove ions of one type
from a mixture and
replace them by ions of
another type.
The basic principle is
reversible competitive
binding

22. ION EXCHANGERS
•Cation exchangers (negative ions –stationary)
•Anion exchangers (positive ions -stationary)
Four types of polymers are commonly used. They are,
•Synthetic hydrophobic polymer resins crosslinked
with divinylbenzene.
•Naturally occuring as well as synthetic
polymers(cellulose)
•Synthetic hydrophilic polymers
•Silica gel

23. AFFINITY CHROMATOGRAPHY
•Affinitychromatography
includesbioaffinity,dye-
ligand affinityand
immobilizedmetalion
afffinitytechniques.
•Itisbasedontheformationof
thespecificandreversible
complexesbetweenapairof
biomolecules.

24. HPLC
HPLCisaphysicalseparationtechniqueinwhicha
sampledissolvedinaliquidisinjectedintoacolumn
packedwithsmallparticlesanditisseparatedintoits
constituentcomponents
HPLCisprobablythemostimportantandwidelyused
analyticaltechniqueforquantitativeanalysisof
organicsandbiomolecules
HPLCisapplicabletomanykindofsamples:
Mostusefulforpharmaceuticals,biomolecules,and
labileorganics

25. HPLC Instrumentation Overview
25
Principle Pattern An Example
Detector
Thermostatted
Column Compartment
Autosampler
Binary Pump
Vacuum DegasserSolvent Cabinet
Solvent Reservoirs
Controller

27. HPTLC
HPTLCisasophisticatedformofTLC.
Fastestofallchromatographictechniques.
Anycombinationsofstationaryandmobilephases
canbeused.
AnalyticalHPTLCisusedformicropreparative
analysis(ie.,separationofmilligramscalefor
analysisoffraction)
Givesmoresharperandcompactbandswith
minimumdistanceofmigration.
Usedforbothqualitativeandquantitativeanalysis.

28. HPLC Vs. HPTLC

29. THANK YOU