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Paper Chromatography Formal Report ORG chem

Paper Chromatography Formal Report ORG chem

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Published by Rachel Anne Barlao

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Published by: Rachel Anne Barlao on Sep 17, 2011
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12/15/2012

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COLUMN AND THIN LAYER CHROMATOGRAPHY
Michael Dominique B. Allag, Eryll Joy H. Agojo, Camille A.Baetiong, Greanne P. Danica Ballesta,
Rachel
 
Anne S. Barlao
Group 1 2C Medical Technology Organic Chemistry Laboratory
Abstract
Chromatography is a technique for separating mixtures into their components in order to analyze,identify, purify, and quantify the mixture or components. There are different types of chromatographyand each has its own advantages and disadvantages. In this experiment, DCM-hexane was used toextract the different pigments of the siling labuyo.
 
Extract was introduced into the column and eluatewas collected, this process is the column chromatography (CC) method. The purity of the componentswas determined by using thin later chromatography (TLC). UV lamp was used to visualize thedeveloped TLC plate and the Retention or Retardation Factor was measured.
I.Introduction
Chromatography can be defined as alaboratory technique that separatescomponents within a mixture by using thedifferential affinities of the components for amobile medium and for a stationary mediumthrough which they pass. The underlying principleof chromatography is that different substanceshave different partition coefficients between thestationary and mobile phases. A compound thatinteracts weakly with the stationary phase willspend most of its time in the mobile phase andmove rapidly through the chromatographicsystem. Compounds that interact strongly withthe stationary phase will move slowly. All formsof chromatography work on the same principle.Diverse types of Chromatography arepossible, depending on the physical states of the phases. Employing a gas the mobilephaseis termed gas chromatography (gc) or vaporphase chromatography (vpc). Separations usinggas chromatography involve vapor phase versusadsorption and/or equilibria. LiquidChromatography(lc)referstoanychromatographic process that employs a mobileliquid phase. Chromatographic separations canalso be carried out using thin layerchromatography (tlc) and column chromatographywhich a variety of supports, including immobilizedsilica on glass plates.Chromatography separates a substanceinto its component parts, which is very useful, assubstances are often unique in their composition.It can identify a substance and show how itdiffers from others that may look alike on thesurface. All types of chromatography areuseful for analytical purposes. Underappropriate
conditions
, all types
 
of chromatography can beused forpreparative scale separations. In everytype of chromatography there are threeelements to be considered: the size of thesample (Load), relative separation of components (Resolution), and the Speed.It would be
 
ideal ifall three elements could bemaximized so that complete separation of samples of any desired size could be quicklyachieved. In practice, generally two of theseelements can be maximized at the expense of the third. For routine analytical work,resolution and speed are maximized at theexpense of the load. In preparative scaleseparations,load, and
 
speed can bemaximized but then separations are usuallyincomplete. Complete separations of largesamples can be achieved but the overalloperation is likely to be slow and tedious, andmay involve the use of large quantities of solvent that must be distilled for reuse, ordiscarded.
 
Intheexperiment,ColumnChromatographyandThinChromatography were used.Column chromatography isadvantageous over most otherchromatographic techniques because it can beused in both analytical and preparativeapplications. Not only can columnchromatography be used to determine thenumber of components of a mixture, but it canalso be used to separate and purify substantialquantities of those components for subsequentanalysis. This is in contrast to paperchromatography, which is solely an analyticalmethod. The disadvantage of a columnchromatography is that it is time-consumingand tedious, especially for large samples. If itis unnecessary to preparative separate largequantities of sample, analytical methods suchas paper chromatography may be moresuitable and easier to perform.Thin-LayerChromatography (TLC)involves the same principles as columnchromatography; it is also a form of solid liquidadsorption chromatography. In this case,however, the solid adsorbent is spread as athin layer on a plate of glass or rigid plastic.The solvent travels up by plate throughcapillary action. A drop of the solution to beseparated is placed near one edge of the plate,and the plate is placed in a container, called adeveloping chamber, with enough of theeluting solvent to come to a level just belowthe point of origin. The solvent migrates up theplate, carrying with it the components of themixture at different rates. The result then, is aseries of spots on the plate, falling on a lineperpendicular to the solvent level in thecontainer.TLC has a number of advantages: It issimple, fast, efficient to use and it requiresonly small amounts of sample. TLC is generallyused a qualitative analytic technique, such aschecking the purity of a compound ordetermining the number of components in amixture or column chromatographic function.In addition, TLC is useful for determining thebest solvents for a column chromatographicseparation. It can be used for an initial checkon the identity of an unknown sample.Preparative plates can be carried out withspecial thick-layered TLC plates.DCM hexane or Dichloromethanehexane is the solvent system used to elutethroughachromatographycolumn. This means that the mobile phase(solvent system) consists of 1:1 (ratio of volume)mixtureof dichloromethane(DCM; CH2Cl2), and hexane (C6H14).The solid phase (silica gel) is eluted withthis solvent system until fully solvated, thecompound to be purifiedisthenloadedontothe solvated solid phase,and the column is eluted with the samesolvent system until your desired compoundhas come off the columnThe Retention or Retardation Factor(Rƒ value) is the ratio of the distance that thespot travelled relative to the distance movedby the solvent which in this case is the DCM-hexane.
 
The objectives of the experimentare the following: separate the coloredcomponents of red siling labuyo using columnchromatography, to predict the purity of components using column and thin layerchromatography (TLC) and lastly, to measurethe Retention/Retardation Factor (Rƒ values) of colored components in TLC.
II.Experimental
Pigments of the siling labuyo were extractedby cutting it to pieces and by pouring DCM-hexane and eventually triturating it by using amortar and pestle with the ratio of 1:1. Theextracted pigments were set aside for awhile.Silica Gel Column was prepared by pluggingthe column with cotton followed by thesilica gel which was uniformly packed andcontained no holes or air bubbles until itreached the indented part of the Pasteurpipette.0.5 ml of the extract was placed on top othe column using Pasteur pipette. Thepigment mixture was eluted using 10mlDCM-hexane. The system solvent wasintroduced in portions. The column was notallowed to run dry and the colorless eluatecollected was discarded. Test tubes werechanged each time the color of the eluatevaries. The number of drops for each colorwas noted.After collecting the eluates from thecolumn, Thin Layer Chromatography wasperformed.The color of the eluate varies. The numberof drops for each color was noted.After collecting the eluates from thecolumn, Thin Layer Chromatography wasperformed.The eluates were applied on the5cm X 8cm pre-coated TLC plate byequidistantly spotting each spot 10 times. Thespot was allowed to dry first beforeapplyingthe succeeding spots. It was ensured that thespots made were small as possible so thatwhen the plate develops,the colors wouldnot be disarray.Developing Chamber was prepared byplacing the approximate amount of DCM hexane. The inner wall of the chamberwas lined with filter paper to allow theTLC plate to stand. The developing chamberwas covered with watch glass and was allowed
to
equilibrate.The TLC plate was carefully introducedin the developing chamber. The solventsystem was allowed to rise up until itreaches just 1cm from the upper end. TheTLC plate was then removed carefully from thechamber. The solvent front was immediatelymarked and the plate was allowed to dry.
 
Thecomponents were visualizedusing the ultraviolet lamp after the plate hasdeveloped after elution process and this causessubstances to appear as colored spots.The values were measured andchromatographic plates were documented.
III.Results andDiscussionPlant used:
Siling Labuyo
Solvent System used
: DCM-Hexane

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