GC and HPLC

Chromatography techniques

1. GAS CHROMATOGRAPHY
Dr.M. Sonia Angeline
Assistant Professor
Department of Life Sciences
KristuJayanti College

2. INTRODUCTION
Gaschromatographydiffersfromotherformsofchromatographyinwhichthemobilephaseisagasand
thecomponentsareseparatedasvapors.
Itisthususedtoseparateanddetectsmallmolecularweightcompoundsinthegasphase.
Thesampleiseitheragasoraliquidthatisvaporizedintheinjectionport.
Themobilephaseforgaschromatographyisacarriergas,typicallyheliumbecauseofitslowmolecular
weightandbeingchemicallyinert.
Thepressureisappliedandthemobilephasemovestheanalytethroughthecolumn.
Theseparationisaccomplishedusingacolumncoatedwithastationaryphase.

3. PRINCIPLE OF GC
•Theequilibriumforgaschromatographyispartitioning,andthecomponentsofthesamplewillpartition
(i.e.distribute)betweenthetwophases:thestationaryphaseandthemobilephase.
•Compoundsthathaveagreateraffinityforthestationaryphasespendmoretimeinthecolumnandthus
elutelaterandhavealongerretentiontime(Rt)thansamplesthathaveahigheraffinityforthemobile
phase.
•Affinity for the stationary phase is driven mainly by intermolecular interactions and the polarity of the
stationary phase can be chosen to maximize interactions and thus the separation.

4. GAS CHROMATOGRAPHY IS MAINLY
COMPOSED OF THE FOLLOWING PARTS:
1.Carriergasinahigh-pressurecylinderwithattendantpressureregulatorsandflowmeters
Helium,N
2,H,Argonareusedascarriergases.
Heliumispreferredforthermalconductivitydetectorsbecauseofitshighthermalconductivityrelativetothatof
mostorganicvapors.
N
2ispreferablewhenalargeconsumptionofcarriergasisemployed.
Carriergasfromthetankpassesthroughatogglevalve,aflowmeter,(1-1000ml/min),capillaryrestrictors,anda
pressuregauge(1-4atm).
Flowrateisadjustedbymeansofaneedlevalvemountedonthebaseoftheflowmeterandcontrolledbycapillary
restrictors.
Theoperatingefficiencyofthegaschromatographisdirectlydependentonthemaintenanceofconstantgasflow.

5. 2.Sampleinjectionsystem
Liquidsamplesareinjectedbyamicro-syringewithaneedleinsertedthroughaself-scaling,silicon-rubberseptum
intoaheatedmetalblockbyaresistanceheater.
Gaseoussamplesareinjectedbyagas-tightsyringeorthroughaby-passloopandvalves.
Typicalsamplevolumesrangefrom0.1to0.2ml.
3.Theseparationcolumn
TheheartofthegaschromatographyisthecolumnwhichismadeofmetalsbentinUshapeorcoiledintoanopen
spiraloraflatpancakeshape.
Copperisusefulupto250
0
Lockfittingsmakecolumninsertioneasy.
Severalsizesofcolumnsareuseddependingupontherequirements.
GAS CHROMATOGRAPHY IS MAINLY
COMPOSED OF THE FOLLOWING PARTS:

6. 4.Liquidphases
Aninfinitevarietyofliquidphasesareavailablelimitedonlybytheirvolatility,thermalstabilityandabilitytowetthe
support.
Nosinglephasewillserveforallseparationproblemsatalltemperatures.
•Non-Polar–Parafin,squalane,siliconegreases,apiezonL,siliconegumrubber.Thesematerialsseparatethe
componentsinorderoftheirboilingpoints.
•IntermediatePolarity–Thesematerialscontainapolarorpolarizablegrouponalongnon-polarskeletonwhichcan
dissolvebothpolarandnon-polarsolutes.Forexample.diethylhexylphthalateisusedfortheseparationofhigh
boilingalcohols.
•Polar–Carbowaxes–Liquidphaseswithalargeproportionofpolargroups.Separationofpolarandnon-polar
substances.
•Hydrogenbonding–Polarliquidphaseswithhighhydrogenbondinge.g.Glycol.
•Specificpurposephases–Relyingonachemicalreactionwithsolutetoachieveseparations.e.gAgNO3inglycol
separatesunsaturatedhydrocarbons.

7. 5.Supports
Thestructureandsurfacecharacteristicsofthesupportmaterialsareimportantparameters,which
determinetheefficiencyofthesupportandthedegreeofseparationrespectively.
Thesupportshouldbeinertbutcapableofimmobilizingalargevolumeofliquidphaseasathinfilmover
itssurface.
Thesurfaceareashouldbelargetoensuretherapidattainmentofequilibriumbetweenstationaryand
mobilephases.
Supportshouldbestrongenoughtoresistbreakdowninhandlingandbecapableofpackedintoauniform
bed.
Diatomaceousearth,kieselguhrtreatedwithNa
2CO
3for900
0
Ccausestheparticlefusionintocoarser
aggregates.
Glassbeadswithalowsurfaceareaandlowporositycanbeusedtocoatupto3%stationaryphases.
Porouspolymerbeadsdifferinginthedegreeofcross-linkingofstyrenewithalkyl-vinylbenzenearealso
usedwhicharestableupto250
0

8. GAS CHROMATOGRAPHY IS MAINLY
COMPOSED OF THE FOLLOWING PARTS:
6.Detector
Detectorssensethearrivaloftheseparatedcomponentsandprovideasignal.
Theseareeitherconcentration-dependentormassdependent.
Thedetectorshouldbeclosetothecolumnexitandthecorrecttemperaturetopreventdecomposition.
7.Recorder
Therecordershouldbegenerally10mv(fullscale)fittedwithafastresponsepen(1secorless).Therecorder
shouldbeconnectedwithaseriesofgoodqualityresistancesconnectedacrosstheinputtoattenuatethelarge
signals.
Anintegratormaybeagoodaddition.

9. PROCEDURE OF GC
•Step1:SampleInjectionandVaporization
1.Asmallamountofliquidsampletobeanalyzedisdrawnupintoasyringe.
2.Thesyringeneedleispositionedinthehotinjectionportofthegaschromatographandthesampleisinjected
quickly.
3.Theinjectionofthesampleisconsideredtobea“point”intime,thatis,itisassumedthattheentiresampleentersthe
gaschromatographatthesametime,sothesamplemustbeinjectedquickly.
4.Thetemperatureissettobehigherthantheboilingpointsofthecomponentsofthemixturesothatthecomponents
willvaporize.
5.Thevaporizedcomponentsthenmixwiththeinertgasmobilephasetobecarriedtothegaschromatographycolumn
tobeseparated.

10. PROCEDURE OF GC
•Step2:SeparationintheColumn
Componentsinthemixtureareseparatedbasedontheirabilitiestoadsorbonorbindto,thestationaryphase.
Acomponentthatadsorbsmoststronglytothestationaryphasewillspendthemosttimeinthecolumn(willbe
retainedinthecolumnforthelongesttime)andwill,therefore,havethelongestretentiontime(Rt).Itwillemerge
fromthegaschromatographlast.
Acomponentthatadsorbstheleaststronglytothestationaryphasewillspendtheleasttimeinthecolumn(willbe
retainedinthecolumnfortheshortesttime)andwill,therefore,havetheshortestretentiontime(Rt).Itwillemerge
fromthegaschromatographfirst.
Ifweconsidera2componentmixtureinwhichcomponentAismorepolarthancomponentBthen:
1.componentAwillhavealongerretentiontimeinapolarcolumnthancomponentB
2.componentAwillhaveashorterretentiontimeinanon-polarcolumnthancomponentB

11. PROCEDURE OF GC
•Step3:DetectingandRecordingResults
1.Thecomponentsofthemixturereachthedetectoratdifferenttimesduetodifferencesinthetimetheyareretainedin
thecolumn.
2.Thecomponentthatisretainedtheshortesttimeinthecolumnisdetectedfirst.Thecomponentthatisretainedthe
longesttimeinthecolumnisdetectedlast.
3.Thedetectorsendsasignaltothechartrecorderwhichresultsinapeakonthechartpaper.Thecomponentthatis
detectedfirstisrecordedfirst.Thecomponentthatisdetectedlastisrecordedlast.

13. APPLICATIONS OF GC
GCanalysisisusedtocalculatethecontentofachemicalproduct,forexampleinassuringthequalityof
productsinthechemicalindustry;ormeasuringtoxicsubstancesinsoil,airorwater.
Gas chromatography is used in the analysis of:
(a) air-borne pollutants
(b) performance-enhancing drugs in athlete’s urine samples
(c) oil spills
(d) essential oils in perfume preparation
https://www.youtube.com/watch?v=UycPljfrnWo

14. HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY

15. INTRODUCTION
•Highperformanceliquidchromatography(HPLC)hasbecomeaveryversatileand
powerfulseparationandanalyticalmethodovertheyears.
•Itisanadvancedformofliquidchromatography(LC).
•Insteadofintroducingthesolventintothecolumnandallowingittodripdownunderthe
influenceofgravity,inHPLCthesampleisforcedthroughthecolumnunderhigh
pressuresofnearly400atm,resultinginfasterandmoreefficientseparation.
•Thistechniqueisalsocalledhighpressureliquidchromatography.
•HPLC makes it possible to perform structural, and functional analysis, and purification of
many molecules within a short time.
•This technique yields perfect results in the separation, and identification of amino acids,
carbohydrates, lipids, nucleic acids, proteins, steroids, and other biologically active
molecules.

16. PRINCIPLE OF HPLC
•HPLC follows the same basic principle as chromatography.
•Different components in the sample have varying affinities to the adsorbent material.
•This causes a difference in the flow rate for each component which leads to their separation as they come
out of the column.
•The only difference is that the speed and sensitivity of HPLC is much higher than that of LC due to the
application of a high pressure.
•The magnitude of pressure applied depends on several factors such as the length and diameter of the
column, flow rate, size of particles in the stationary phase, and mobile phase composition.

17. PRINCIPLE OF HPLC
•Thepurificationtakesplaceinaseparationcolumnbetweenastationaryandamobilephase.
•Thestationaryphaseisagranularmaterialwithverysmallporousparticlesinaseparationcolumn.
•Themobilephase,ontheotherhand,isasolventorsolventmixturewhichisforcedathighpressurethrough
theseparationcolumn.
•Viaavalvewithaconnectedsampleloop,i.e.asmalltubeoracapillarymadeofstainlesssteel,thesample
isinjectedintothemobilephaseflowfromthepumptotheseparationcolumnusingasyringe.
•Subsequently,theindividualcomponentsofthesamplemigratethroughthecolumnatdifferentratesbecause
theyareretainedtoavaryingdegreebyinteractionswiththestationaryphase.
•Afterleavingthecolumn,theindividualsubstancesaredetectedbyasuitabledetectorandpassedonasa
signaltotheHPLCsoftwareonthecomputer.
•Attheendofthisoperation/run,achromatogramintheHPLCsoftwareonthecomputerisobtained.
•Thechromatogramallowstheidentificationandquantificationofthedifferentsubstances.

18. THE COMPONENTS OF HPLC
•Columns:HPLCcolumnsarenormallymadeofstainlesssteelandare50-300mmlongwithaninternaldiameterof2-
5mm.Theyarefilledwiththeadsorbents(stationaryphase)ofparticlesize3–10µm.
•SampleInjector:Thesampleisinjectedintothecolumnbyaninjectorwhichiscapableofhandlingsamplevolumesin
therangeof0.1-100mLunderhighpressuresofupto4000psi.
•Reservoir:Thesolventorthemobilephaseisplacedinaglassreservoir.Itisusuallyablendofpolarandnon-polar
liquidswhoseconcentrationsdependonthesamplecomposition.
•Pump:ThesolventinthemobilephaseisaspiratedbyapumpfromthereservoirandforcedthroughtheHPLCcolumn
andthenthedetector.
•Detector:ThedetectorinaHPLCsystemislocatedattheendofthecolumnanditdetectsthecomponentsofthe
samplethatelutefromthecolumn.Differenttypesofdetectorssuchasfluorescence,mass-spectrometric,UV-
spectroscopic,andelectrochemicaldetectorsareused.

19. INSTRUMENTATION

20. The Pump
•ThedevelopmentofHPLCledtothedevelopmentofthepumpsystem.
•Thepumpispositionedinthemostupperstreamoftheliquidchromatographysystemandgeneratesaflowof
eluentfromthesolventreservoirintothesystem.
•High-pressuregenerationisa“standard”requirementofpumpsbesideswhich,itshouldalsotobeableto
provideaconsistentpressureatanyconditionandacontrollableandreproducibleflowrate.
•MostpumpsusedincurrentLCsystemsgeneratetheflowbyback-and-forthmotionofamotor-drivenpiston
(reciprocatingpumps).Becauseofthispistonmotion,itproduces“pulses”.
Injector
•Aninjectorisplacednexttothepump.
•Thesimplestmethodistouseasyringe,andthesampleisintroducedtotheflowofeluent.
•Themostwidelyusedinjectionmethodisbasedonsamplingloops.
•Theuseoftheautosampler(auto-injector)systemisalsowidelyusedthatallowsrepeatedinjectionsinaset
scheduled-timing.
INSTRUMENTATION

21. INSTRUMENTATION
•Column
•The separation is performed inside the column.
•The recent columns are often prepared in a stainless steel housing, instead of glass columns.
•The packing material generally used is silica or polymer gels compared to calcium carbonate.
The eluent used for LC varies from acidic to basic solvents.
•Most column housing is made of stainless steel since stainless is tolerant towards a large variety of solvents.
•Detector
•Separation of analytes is performed inside the column, whereas a detector is used to observe the obtained
separation.
•The composition of the eluent is consistent when no analyte is present. While the presence of analyte changes
the composition of the eluent. What detector does is to measure these differences.
•This difference is monitored as a form of an electronic signal. There are different types of detectors available.

22. INSTRUMENTATION
Recorder
•The change in eluent detected by a detector is in the form of an electronic signal, and thus it is still not visible to our eyes.
•In older days, the pen (paper)-chart recorder was popularly used. Nowadays, a computer-based data processor (integrator) is
more common.
•There are various types of data processors; from a simple system consisting of the in-built printer and word processor while
those with software that are specifically designed for an LC system which not only data acquisition but features like peak-
fitting, baseline correction, automatic concentration calculation, molecular weight determination, etc.
Degasser
The eluent used for LC analysis may contain gases such as oxygen that are non-visible to our eyes.
•When gas is present in the eluent, this is detected as noise and causes an unstable baseline.
•Degasser uses special polymer membrane tubing to remove gases.
•The numerous very small pores on the surface of the polymer tube allow the air to go through while preventing any liquid
to go through the pore.
Column Heater
The LC separation is often largely influenced by the column temperature.
•In order to obtain repeatable results, it is important to keep consistent temperature conditions.
•Also for some analysis, such as sugar and organic acid, better resolutions can be obtained at elevated temperatures (50 to
80°C).
•Thus columns are generally kept inside the column oven (column heater).

23. COLUMN
•Stainless steel tubing to withstand high pressure
•Heavy-wall glass or PEEK tubing for low pressure (< 600 psi)
•Analytical column: Straight column with length (5 ~ 25 cm)
•Microcolumn: Length of 3 ~ 7.5 cm, high speed and minimum solvent consumption
•Guard column: Remove particulate matter and contamination protect analytical column. Have
similar packing as analytical column
•Température control: < 150 °C±0.1 °C

24. HPLC
https://www.knauer.net/en/Systems-Solutions/Analytical-HPLC-UHPLC

25. Detector type Measurement principle
UV/Vis detector Absorbance
PDA detector-Photodiode-Array
Detection
Absorbance
Differential refractive index
detector
Refractive index
Fluorescence detector Fluorescence
Electrochemical detector Oxidation / reduction
Electrical conductivity detectorConductivity
Mass spectrometry detectorMolecular size, mass
Optical rotation detector Optical rotation
Circular dichroism detectorCircular dichroism
Evaporative light scattering
detector
Light scattering
•Thereisavarietyofdetectorsthatcan
beuseddependingonthetarget
sample.
•Todetectonlyafewspecific
components,ahighlysensitive
fluorescencedetectororaUV/Vis
detectorcanbeused.
•Todetectawiderangeofcomponents,
thenaPDAdetector,anevaporative
light-scatteringdetector,ora
differentialrefractive-indexdetectoris
moreappropriate.
TYPES OF DETECTORS

26. TYPES OF HPLC
•Normal Phase:
Separation of polar analytes by partitioning onto a polar, bonded stationary phase.
•Reversed Phase:
Separation of non-polar analytes by partitioning onto a non-polar, bonded stationary phase.
•Adsorption: In Between Normal and Reversed.
Separation of moderately polar analytes using adsorption onto a pure stationary phase (e.g.
alumina or silica)
•Ion Chromatography:
Separation of organic and inorganic ions by their partitioning onto ionic stationary phases
bonded to a solid support.
•Size Exclusion Chromatography:
Separation of based in the paths they take through a “maze” of tunnels in the stationary phase.

27. TYPES OF HPLC
1.Normal phase:
•Column packing is polar (e.gsilica) and the mobile phase is non-polar. It is used for water-sensitive
compounds, geometric isomers, cis-trans isomers, and chiral compounds.
2.Reverse phase:
•The column packing is non-polar (e.gC18), the mobile phase is water+miscible solvent (e.g
methanol). It can be used for polar, non-polar, ionizable and ionic samples.

28. THE TECHNIQUE OF HPLC
ThekeystepsintheHPLCseparationtechniqueareasfollows:
Injectionoftheliquidsampleintothecolumncontainingthestationaryphase.
Therearetwotypesofsampleinjectionmethods:manualinjectioninwhichasyringeisinsertedintotheinjectorand
thesampleisinjectedmanually,andautosampling,wherebyalargenumberofsamplescanbeinjectedautomatically.
Individualsamplecomponentsareforceddownthetubebyhighpressurefromthepump.
Componentsareseparatedundertheinfluenceofvariouschemical/physicalinteractionswiththeparticlesinthe
stationaryphase.
Theseparatedanalytesareidentifiedbythedetectorpresentattheendofthecolumn.
Thedetectormeasurestheconcentrationofthecomponents.
Datafromthedetectorisprocessedandachromatogramisproduced.

29. APPLICATIONS OF HPLC
TheHPLChasdevelopedintoauniversallyapplicablemethodsothatitfindsitsuseinalmostallareasofchemistry,biochemistry,and
pharmacy.
•Analysisofdrugs
•Analysisofsyntheticpolymers
•Analysisofpollutantsinenvironmentalanalytics
•Determinationofdrugsinbiologicalmatrices
•Isolationofvaluableproducts
•Productpurityandqualitycontrolofindustrialproductsandfinechemicals
•Separationandpurificationofbiopolymerssuchasenzymesornucleicacids
•Waterpurification
•Pre-concentrationoftracecomponents
•Ligand-exchangechromatography
•Ion-exchangechromatographyofproteins
•High-pHanion-exchangechromatographyofcarbohydratesandoligosaccharides

30. LIMITATIONS
1.Cost:Despite its advantages, HPLC can be costly, requiring large quantities of expensive organics.
2.Complexity
3.HPLC does havelow sensitivityfor certain compounds, and some cannot be detected as they are irreversibly
adsorbed.
4.Volatile substances are better separated by gas chromatography.

31. ADVANTAGES OF HPLC
•Higher resolution and speed of analysis
•HPLC columns can be reused without repacking or regeneration
•Greater reproducibility due to close control of the parameters affecting the
efficiency of separation
•Easy automation of instrument operation and data analysis
•Adaptability to large-scale, preparative procedures
•Two major advances:
•stationary supports with very small particle sizes and large surface areas
•appliance of high pressure to solvent flow

32. https://www.youtube.com/watch?v=ZN7euA1fS4Y-HPLC