exp10 chromatography

8/20/2019 EXP10 Chromatography

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EXPERIMENT 1C H R O M A T O G R A P H Y

A L F O N S O , G O T E R A , L A U S A


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PAPER CHROMATOGRAPHY

• Paper chromatography is used as an analytical chemistry technique for

and separating colored mixtures like pigments.• Paper chromatography has several components:

– Stationary phase

• To separate chemicals, we have to have a platform on which to separatcase, the platform is paper. The paper does not move in the process ofchromatography, therefore, we call it the stationary phase.

– !o"ile phase

• is the solvent that carries the chemicals through the stationary phase. #move on paper alone, "ut if we add a solvent, it can draw the chemicalthe paper.

– Point of origin

• the spot where you put your chemicals on the stationary phase.


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– %eveloping

• after putting chemicals on the point of origin, the chromatogram can "that is, the mo"ile phase can "e pulled through the stationary phase.

– (etention factor )(f*

• The distance our chemicals move during chromatography is typicalldistance the mo"ile phase moves. +e can measure the dierence in dithe (f value. This is the distance a chemical moves on our paper )staduring our separation divided "y the distance the mo"ile phase moves

– #hromatograph

• the -nished result from chromatography that shows the separated sstationary phase after the mo"ile phase has dried.


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SIGNIFICANCE OF PAPER

CHROMATOGRAPHY • mong all the chromatography methods paper chromatography

inexpensive and rapid method that provides graphic and clear re

• /sed as a qualitative method for identifying the components in

• The separated spots on the -nished and dried chromatogram ca

and re0dissolved to o"tain a pure sample of component of the sa

mixtures.

• /sed in several scienti-c studies in identi-cation of unknown org

inorganic compounds from a mixture.


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FACTORS THAT AFFECT RF VALUE

• Solvent concentration

• Temperature

• The quality of the paper

• Solvent purity


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OBJECTIVE

• To "e a"le to separate compound components 1owing across a s

medium• To "e a"le to identify the three colors staining the -lter paper

• To determine the (f )rate of 1ow*value of each color found on th

paper.

• To know what is chromatography.

• To know the procedure of chromatography.

• To determine the most eective solvent for chromatography.


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MATERIALS

• %eveloper solution

• 234 ml "eaker• #lear cellophane

• (u""er "and

• 5 g of leaf sample

• 54 ml acetone

•!ortar and pestle

• 6ilter paper strip


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PROCEDURES• Prepare the developer solution

•

7our instructor will assign the ratio of the solvent system to each g• Pour 84 ml of the developer solution into a 234 ml "eaker

• #over the "eaker with a clear cellophane with a ru""er "and tied a

saturate the space a"ove the liquid with vapor*

• +eigh 5 g of leaves sample

• #ut the leaves into small si9es

• dd 54 ml of acetone

• #rush the leaves using a clean mortar and pestle


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PROCEDURES

• Pour the mixture into a vial

•Place the vial into a water "ath and heat until the volume of the leaves mreduced to a"out 5 ml

• "tain a -lter paper strip

• %raw a pencil line across the paper a"out 2 cm from the "ottom

• long this line, make sample x$s a"out every ; cm

• Spot the chromatography paper at each x mark with the previously prep

mixture• Place the -lter paper in the "eaker with the developer

• llow the developer to rise up through the paper


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PROCEDURES• +hen the developer has almost advanced to the top, remove the paper and mar

point

• llow the paper to dry

• There should now "e three colors staining the paper

– The yellow color is a pigment called carotene

– The darker green color is a pigment called chlorophyll a

– The lighter green color is a pigment called chlorophyll b

– The pinkish


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• +hat is the signi-cance of the (f values>

– This value represents the ratio of the istance a pi!"ent tthe chro"ato!ra" relative to the istance the solvent fro

Scientists use the Rf value of a sa"ple to ientify the "olecu

• n (f value that is close to 5 indicates that the pigment is verythe solvent.

• n (f value that is very small indicates that the pigment is not tin the solvent.


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PRE LAB ASSIGNMENT

• +rite the structural formula for carotene, chlorophyll , chloroph

$arotene % $&'()* +

-$arotene is a strongly colored red0orange pigment a"undant plants and fruits. t is an organic compound and chemically classi-ed as a hydrocar"on and speci-cally as a terpenoid, re1ectin

its derivation from isoprene units.


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PRE LAB ASSIGNMENT


$hlorophyll A %$))(./O)N&0! +

$hlorophyll a a"sor"s most energy from wavelengths of violetorange0red light. t also re1ects green


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PRE LAB ASSIGNMENT


$hlorophyll 1 %$))(.'0!N&O*+

$hlorophyll b helps in photosynthesis "y a"sor"ing light energy. solu"le than chlorophyll a in polar solvents "ecause of its car"onyl gcolor is yellow, and it primarily a"sor"s "lue light.


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WHAT IS THE DIFFERENCE BETWEEN TSTRUCTURAL FORMULA OF CHLOROP

A AND CHLOROPHYLL B?$hlorophyll A• Principal photosynthetic pigment• Present in all phototrophs other than

"acteria• ?lue green in pure state•

@mpirical formula #33ABB3C;!g• The 8rd car"on of the side group is methyl

group• !olecular weight is DB8

$hlorophyll 1

• ccessory photosynthe• Present in all phototrop

diatoms, cyano"acteria"rown algae

• live green in pure stat• @mpirical formula #33A• Side group at the 8rd ca

aldehyde group• !olecular weight is F4B


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PRE LAB ASSIGNMENT

• +hat vitamin is related to carotene>

– ?iologically, "eta0carotene is most importantprecursor of vitamin in the human diet. t alsooxidant properties and may help in preventing cother diseases.

– 2ita"in A has several functions in the "ody. The

known is its role in vision 0 hence carrots Gmake ysee in the darkG. The retinol is oxidi9ed to its retinal, which complexes with a molecule in the eopsin. +hen a photon of light hits the complex, tchanges from the 550cis form to the all0trans forma chain of events which results in the transmiss

impulse up the optic nerve


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DEVELOPING SOLVENTS USED


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DATA AND RESULTS

• (esults:

–Solvent 3: #arotenoids0 !ost solu"leH #hlorophyll a0 Ieast solu"le

– Solvent /: #hlorophyll a0 !ore solu"leH #hlorophyll "0 least solu"le

• Came of Sample: La!uni

• %eveloping solvent: Solvent 3: F hexane, 5 @tc JJ Solvent /: Bhexan

#I( %STC#@ T(K@II@% ?7 SI/T@ SIK@CT

)#I(*

#arotenoid )yellow* E.3cm JJ 000 D.3cm JJ B.;cm 4.BE;

#hlorophyll a )darkgreen*

8.4cm JJ ;.8cm D.3cm JJ B.;cm 4.832

#hlorophyll " )lightgreen*

3.2cm JJ 8.Ecm D.3cm JJ B.;cm 4.E52

Lanthophyll)pink


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POST LAB QUESTIONS

• +hat would "e the consequence of having so much solvent in th

developing cham"er that it would cover the application spot>

– t would dissolve the spot, so you$d have to start over. The cham"econtain Must enough solvent to cover the "ottom


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POST LAB QUESTIONS

• +hat eect would it have on the chromatogram with a "road sa

– The chromatogram could "e messy, overlapping of spots can occhard to determine the distance your sample traveled


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POST LAB QUESTIONS

• Aow is thin layer chromatography technique of separation similachromatographic separation>

– n "oth types of chromatography, samples are placed near the "stationary phase which is then set into a "eaker containing a smthe mo"ile phase, which travels up the stationary phase via capillasamples travel dierent distances up the stationary phase depenpolarity. This is also useful for separating the components of one sa


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CONCLUSION

• Plants dier in the photosynthetic pigments present in them. These pvarying properties which were investigated in this experiment.

• Paper chromatography proved to "e an accurate method of seo"serving the various colors of plant pigments. The pigments disssolvent and migrated upward. The colors were o"served and thdistances measured O recorded. The (f value of each pigment was d

dividing its migration "y the migration of the solvent. t was deterpigments were present in the original spot 000 carotene, chlorochlorophyll ". #arotene was the most solu"le, while chlorophyll " wsolu"le.

• These results were due to the polarity of the pigment chlorophyllorganic compound, in the same types of compounds such as acetone.

exp10 chromatography

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