Flash Chromatography.pdf

By Dhanashree R. Kavhale M.Pharm. (Pharmaceutical Chemistry) Sem II.
Flash chromatograpy is the advanced form of column chromatography in which the pressure is aplied for faster elution..

1. Flash Chromatography
Presented by
Dhanashree R. Kavhale
M. Pharm. (Pharmaceutical Chemistry) Sem-II
Department of Pharmaceutical Sciences
Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur -440033

2. Contents
Introduction
Principle of flash chromatography
Instrumentation
Applications
References
2

3. What is Flash chromatography?
ItwaspopularizedseveralyearsagobyClarkStillofColumbiaUniversity.
Flashchromatography,alsoknownasmediumpressurechromatography.
Anairpressuredrivenhybridofmediumandshortcolumnchromatography
optimizedforrapidseparation.
Analternativetoslowandofteninefficientgravity-fedchromatography.
Differsfromtheconventionaltechniquein2ways:
Slightlysmallersilicagelparticles(250-400mesh)areused.
3

4. Continue..
Duetorestrictedflowofsolventcausedbythesmallgelparticles,
pressurizedgas(10-15psi)usedtodrivethesolventthroughthe
columnofstationaryphase
Thenetresultisarapid"overinaflash"andhighresolution
chromatography.
4

5. Principle
Theprincipleisthattheeluentis,undergaspressurerapidlypushedthrougha
shortglasscolumn.
Theglasscolumnispackedwithanadsorbentofdefinedparticlesizewith
largeinnerdiameter.
Themostusedstationaryphaseissilicagel40-63um,butobviouslypacking
withotherparticlesizescanbeusedaswell.
Particlessmallerthan25µmshouldonlybeusedwithverylowviscosity
mobilephases,becauseotherwisetheflowratewouldbeverylow.
5

6. Continue..
Normallygelbedsareabout15cmhighwithworkingpressuresof1.5-
2.0bars.
Inthemeantime,however,andparalleltoHPLC,reversedphase
materialsareusedmorefrequentlyinflashchromatography.
ThecomputerizedsystemcontroltheWorkingofFlashchromatography.
6

7. Instrumentation of Flash chromatography7
Fig: Components of flash chromatography

8. Adsorbents
Silica:Slightlyacidicmedium.Bestforordinarycompounds,good
separationisachieved.
Alumina:Basicorneutralmedium.Canbeeffectiveforeasyseparations,
andpurificationofamines.
Reversephasesilica:Themostpolarcompoundselutefastest,themost
non-polarslowest.
8

9. Selection of stationary phase
Themostimportantstationaryphaseincolumnchromatographyissilica.
Silicagel(SiO2)andalumina(Al2O3)
Adsorbentparticlesizeaffectshowthesolventflowsthroughthecolumn.
Smallerparticles(highermeshvalues{70-230})areusedforflash
chromatography;largerparticles(lowermeshvalues{230-400})areusedfor
gravitychromatography.
TheamountofsilicageldependsontheRfdifferenceofthecompoundstobe
separated,andontheamountofsample.
9

10. Solvent Systems
Flash column chromatography is usually carried out with a
mixture of two solvents, with a polar and a nonpolar component.
1.Hydrocarbons: pentane, petroleum ether, hexanes.
2.Ether and dichloromethane (very similar polarity)
3.Ethyl acetate
The most common two-component solvent system:
1. Ether/Petroleum Ether
2.Ether/Hexane
3.Ether/Pentane Ethyl
4.Acetate/Hexane
5.Methanol/Dichloromethane
10

11. Pump Systems
Apressurerangeuptoeither10baror50bargivesoptimumseparationresults
forabroadrangeofapplications.
Thepumpmodulescanbecontrolledbythreedifferentunits.
PumpControllerC610PumpManagerC615ControlUnitaredesignedfor
isocraticseparations.
Theflowratecanbeeasilyadjustedbyturningaknobandisindicatedbya
largeilluminatedLCD-display.
Deliveredwithaoverpressuresensorformaximumsafety.
11

12. Sample Injection Systems
WayofinjectingsampleInjectionsystemsaredesignedtofacilitatecolumn
loadingwithliquidsandlowsolubilityoilsandsolids.
1.ColumnsGlassColumns:
Awiderangeofcolumnsoffermaximumflexibilityforeverysituation.
Dependingonthenatureandthequantityofthesampleoffersaseriesof
columntypeswhichvaryinform,sizeandperformance.
12

13. 2.ColumnPlastic+GlassColumn:
Plastic+Glass-coatedGlassColumnsareavailableforlargeramountsof
samplesandhigherpressureapplicationsonahighsafetylevel.
Precolumns:Precolumnareminimizingdeadvolumesandenhancethe
lifetimeofthemaincolumnbytrappingcontaminants.
ThesmallPrecolumn,fitstoGlassColumnsofinnerdiameterofID15,26,
36and49mm.
13

14. Continue..
ThelargePrecolumn,fitstoGlassColumnsofID70and100mminner
diameter.
FillingSetsforGlassColumnsDryFillingSet:TheDryFillingSetis
employedforfillingglasscolumnswithsilicagelusingcompressedgas.
Silicagelinthesizerangeof25-200micrometerscanbepackedwith
thismethod.
14

15. Sample loading
Two different methods are used to load the column.
Wet Method
Dry Method
15

16. 1. Wet Loading Method
Inthewetmethod,thesampletobepurified(orseparatedintocomponents)is
dissolvedinasmallamountofsolvent,suchashexanes,acetone,orother
solvent.
Thissolutionisloadedontothecolumn.
Sometimesthesolventchoicetoloadthesampleontothecolumnismore
polarthantheelutingsolvents.
Inthiscase,ifweusethewetmethodofcolumnloading,itiscriticalthatwe
onlyuseafewdropsofsolventtoloadthesample.
Ifweusetoomuchsolvent,theloadingsolventwillinterferewiththeelution
andhencethepurificationorseparationofthemixture.
Insuchcases,thedrymethodofcolumnloadingisrecommended.
16

17. 2. Dry Loading Method
Firstdissolvethesampletobeanalyzedintheminimumamountof
solventandaddabout100mgofsilicagel.
Themixtureuntilthesolventevaporatesandonlyadrypowder
remains.
Placethedrypowderonafoldedpieceofweighingpaperandtransfer
ittothetopofthepreparedcolumn.
Addfreshelutingsolventtothetopnowwearereadytobeginthe
elutionprocess.
17

18. Advantages & Disadvantages
Advantages Disadvantages
1.Lowcost Lowresolution
2.Itispossibletoemploystepped
gradientandisocraticsolvent
elution.
Itisdifficultandtime-consumingto
separatecomplicatedcrudereaction
mixtures.
3.Thechemisttotallyregulatesthe
introductionofstationaryphaseand
packingtechniques.
18

19. Applications
Itisnowoneofthemostwidelyusedmethodsforpurifying
pharmaceuticalintermediatesaswellasfinalproducts.Moreover,itis
commonlyusedinthestudyofnaturalproducts.Forexamples;
•Isolationandseparationofcatechinfromgreenteaextracts.
•Purificationoffattyacids
•Purificationofsteroids
19

20. Thank you..
20