DEFINATIONThe technique of separatingthe components of a mixture isachieved by the differentialmovement of individualcomponents through astationary phase under theinfluence of a mobile phase.Chromatography is widelyused for separation ,purification , identificationand characterisation of thecomponents of a mixturewether coloured or colourless.
Principles of chromatography Chromatography is a physical process. Any chromatography system is composed of mainlythree things:1) Stationary phase2) Mobile phase3) Mixture to be separated
Chromatography is an dynamic process in whichmobile phase moves in definite direction. In chromatography process we can only controlstationary &mobile phase as mixture is the problem wehave to deal with.
TERMINOLOGIES Differential: showing a difference. Affinity: natural force of attraction between thesubstances. Adsorption :phenomenon of higher concentration ofmolecular species(liquid or gas) on the surface of solid inbulk. Adsorbent: the substance’s surface on which adsorptiontook place. Adsorbate : substance which get adsorbed on adsorbent. Stationary phase : part of apparatus that does not movewith the sample. Mobile phase : gas or liquid that carries the sample withit.
WORKING OF CHROMATOGRAPHY The substance which has to be separated has manycomponents, some of them has affinity to mobilephase & some has affinity to stationary phase. The substance having affinity to mobile phase getdissolved in it and travels large distance it. While substance having affinity to stationary phase getadsorbed on it and travels very short distance.
Uses of chromatography Analyze: examine of mixture, it’s components & thererelation with one another. Identify : determine the identy of mixture orcomponents based on known components. Purify: separates components in order to isolatecomponents for further studies. Quantify : dertimens the amount of substancespresent in the sample.
STATIONARY PHASEThe stationary phase or adsorbent in columnchromatography is a solid. The most common stationary phase for columnchromatography is silica gel, followedby alumina. Cellulose powder has often been used in thepast. The stationary phases are usually finely ground powdersor gels and/or are micro porous for an increased surface.
MOBILE PHASE•The mobile phase or eluent is either apure solvent or a mixture of differentsolvents.• The eluent has also been chosen so thatthe different compounds can be separatedeffectively
Column chromatography Column chromatography in chemistry is a method usedto purify individual chemical compounds from mixtures ofcompounds. The main advantage of column chromatography is therelatively low cost and disposability of the stationaryphase used in the process. The latter prevents cross-contamination and stationary phase degradation due torecycling We can’t separate different amino acid’s &sugars. Wecan’t also do quantitative analyses accurately.
6 beakers or jars 6 covers or lids Distilled H2O Isopropanol Graduated cylinder 6 strips of filter paper Different colors of Sharpiepens Pencil Ruler Scissors TapeMaterials List
STATIONARY PHASE•The stationary phase or adsorbent in paperchromatography is a liquidPaper is made of cellulose fibres, and cellulose is apolymer of the simple sugar, glucose.The key point about cellulose is that the polymerchains have –OH groups sticking out all aroundthem. To that extent, it presents the same sort ofsurface as silica gel or alumina in thin layerchromatographycellulose fibres attract water vapour fromthe atmosphere as well as any water thatwas present when the paper was made.You can therefore think of paper as beingcellulose fibres with a very thin layer ofwater molecules bound to the surface.
MOBILE PHASE The mobile phase used in paper chromatography is liquid . non-polar solvent such as hexane, Acetone,etc, Polarsolvents water, alcohols,etc as the solvent The solvent has also been chosen so that the differentcompounds can be separated effectively
PRINCIPLE OF PAPERCHROMATOGRAPHYCapillary Action – the movement of liquid within the spaces of aporous material due to the forces of adhesion, cohesion, and surfacetension. The liquid is able to move up the filter paper because itsattraction to itself is stronger than the force of gravity.Solubility – the degree to which a material (solute) dissolves into asolvent. Solutes dissolve into solvents that have similar properties.(Like dissolves like) This allows different solutes to be separated bydifferent combinations of solvents.It can be used to separate differentamino acids & sugars but quantitativeanalyses is not accurately.
THIN LAYER CHROMATOGRAPHYHere the mobile phase is a liquid, flowing pasta thin layer of powder on a solid support byCAPILLARY ACTION; a variation of columnchromatography.Substances that are less attracted tothe solid or are more soluble in theliquid move faster. And so movefurther up the plate by the time thatthe process has been stopped bytaking the plate out of the liqiud. -larger Rf
. Detection If the spots are not colored and can’t be seen by theeye, use:• UV lamp for UV-active compounds; most aromaticsare UV-active• If compounds are not UV-active, use an iodine .
Rf value(retention factor) The ratio of distance travelled by the component (fromorigin) compared with the distance travelled by thesolvent front (from origin) is called the Rf value.Rf = Migration distance of a substanceMigration distance of solvent frontOrigin LineSolvent Front LineDistance traveledby solventDistance traveled by spot
28••The ratio of distance travelled by the component (from origin) comparedwith the distance travelled by the solvent front (from origin) is called the Rfvalue.Solvent frontxabcRf of = a/xRf of = b/xRf of = c/x
Rf Value•The Rf value is notinformative•What affects the Rf value?TemperatureSolventThickness and amountof spot
?GAS CHROMATOGRAPNY1) In gas chromatography the movingphase is a gas and the stationary phaseis either liquid or solid.2) The technique is suitable forseparation of materials which arevolatile without decomposition
It can be used for qualitative as wellas quantitative analyses ofsubstances.It can’t be used to separate non-volatile substances.
HIGH PRESSURE LIQUIDCHROMATOGRAPHY Different analytes have different equilibria between themobile phase and stationary phase Equilibrium is dynamic; thus we can view it as a givenanalyte molecule spending a fraction of time dissolved inthe mobile phase Since different solutes gave different fractions, a separationof the analytes occur as they are pushed through thecolumn by the mobile phase
Uses for ChromatographyReal-life examples of uses for chromatography:• Pharmaceutical Company – determine amount ofeach chemical found in new product• Hospital – detect blood or alcohol levels in apatient’s blood stream• Law Enforcement – to compare a sample found ata crime scene to samples from suspects, LikeExplosion residue, Arson cases Poisons in viscera.• Environmental Agency – determine the level ofpollutants in the water supply• Manufacturing Plant – to purify a chemicalneeded to make a product• Dyes –Identifying coloring dyes used in differentfood products