Isolation of Chlorophyll from Spinach Objectives To extract and separate the chlorophyll from the spinach leaves by thin

n layer chromatography (TLC) To find out the proportion of acetone and hexane solvent for the best resolution in running TLC in this experiment To determine the Rf values of substances in a chromatogram Curriculum Links in NSS curriculum Topic XV Analytical Chemistry Background Chlorophylls are the green pigments that act as the principal photoreceptor molecules of plants They are capable of absorbing certain !avelengths of visible light and then convert them into chemical energy T!o different forms of chlorophyll can be found in plants and they are chlorophyll A and " #n this experiment$ !e are trying to isolate the chlorophylls from the spinach leaves by means of TLC
H O

N N O O O O O H Mg N
2+

N
O O

Mg N

2+

O O O H

Chlorophyll A

Chlorophyll B

aterials and Apparatus %pinach leaves Acetone "rine &exane

Anhydrous sodium sulphate %cissors (ortar and )estle A pair of forceps ,-.mm x ,-mm scre!/cap test tubes 12mL conical flas3s 0ilter funnel )aper to!el

Capillary tubes TLC plates' *lass container !ith lid for TLC +roppers 0ilter paper ,.mL measuring cylinder 2.mL bea3er Cotton !ool

'Cut the TLC plates into small rectangular pieces The length of each plate should be about the same height of the glass container !"perimental #rocedure )art #4 5xtraction $% Clean some spinach leaves !ith !ater and dry it !ith a paper to!el Cut 1g of spinach leaves into small pieces !ith scissors and put into the mortar Add 6mL of acetone into a mortar and grind the leaves !ith the pestle +ecant the solution through a filter funnel !ith a piece of cotton !ool to a scre!/cap test tube and s7uee8e the residue for getting more extract )our 6 mL of acetone into the mortar and grind the residue !ith the pestle again )our the li7uid part into the scre!/cap test tube through the filter funnel Then pour 6 mL of acetone and 2 mL of hexane into the mortar and grind the residue again and then filter the extract into the scre!/cap test tube The residue left over should be very pale green

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Add 6 mL of brine into the scre!/cap test tube )ut on the cap tightly and sha3e the scre!/cap test tube vigorously to !ash the chlorophyll/acetone/hexane solution 9pen the scre! cap of the test tube occasionally to release the gas pressure Then set the tube bac3 on the test/tube rac3 and allo! the mixture to separate The top layer should be a dar3 green and the bottom layer clear

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#f emulsion is hard to separate$ add some brine to facilitate the separation "y using a dropper$ separate and collect the organic layer to a clean scre!/cap test tube (or to discard the bottom a7ueous layer)

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Add enough anhydrous sodium sulphate to the solution containing the chlorophyll inside the scre!/cap test tube to dry the solution Then filter the solution !ith a filter paper into a 12/mL conical flas3

)art ##4 Thin Layer Chromatography $% +ra! a line !ith a pencil , cm from one edge of the plate :se a capillary tube to ta3e out some chlorophyll solution from the conical flas3 and lightly dot the solution onto the starting line of the TLC plate Allo! the solvent to completely evaporate off Then dot more sample onto the same spot Repeat the above procedures until a colored spot is clearly seen )ut a large strip of filter paper around the interior !all of the glass container and a round paper piece at the bottom

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Add a mixture of acetone and hexane in volume ratio 14; to the glass container The depth of the solution in the container should be less than , cm Cover the lid of the container for a fe! minutes to let the solvent vapors saturate the air in the container "y using a pair of forceps$ put the TLC plate vertically into the glass container containing acetone and hexane to let the TLC run <hen the solvent almost reaches the top end of the TLC plate$ ta3e the plate out and mar3 the solvent front

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Try out other mixtures of acetone and hexane (64= and >4-) to see !hich mixture can give the best resolution 0ind the Rf values

*emarks , +uring the extraction process$ the chlorophyll/acetone/hexane solution is !ashed !ith brine and sha3en vigorously in a scre!/cap test tube #t is recommended that the scre! cap should be opened fre7uently to release the gas pressure accumulated from the volatile organic solvent in the test tube #t is recommended that the TLC plate is cut into pieces inside the fumehood !ith the silica side facing the bottom The large strip of filter paper surrounding the interior !all of the glass container is used for solvent saturation inside the container$ !hile the round paper piece placed at the bottom is used to prevent the sliding do!n of the TLC plate positioned vertically in the container The mixture of acetone and hexane in volume ratio 64= should be able to give the best resolution #f lesson time is limited$ students may ?ust !or3 on this designated mixture for the TLC analysis

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The result of the TLC analysis 4 As the solvent rises by capillary action up through the TLC plate$ the components of the pigment mixture are partitioned bet!een the mobile phase (organic solvent) and the stationary phase (silica plate) due to their different adsorption and solubility strength The more strongly a given component is adsorbed to the stationary phase$ the less easily it is removed by mobile phase The more !ea3ly a component is adsorbed the faster it !ill migrate up the TLC plate 9n the other hand$ the running distance depends on the solubility of the pigment in the solvent %ince the experiment employs a highly non/polar solvent (acetone/hexane mixture)$ the pigments that are least polar (carotenes) !ill be best dissolved in the non/polar solvent and !ill thus have the largest running distance As chlorophyll A is more non/polar than chlorophyll " (since chlorophyll " contains polar aldehyde group)$ thus chlorophyll A moves faster !ith mobile phase to an upper position on TLC plate than chlorophyll "

+uestions and suggested ans,ers $% <hy do !e need to cut and grind the leaves@ Ans!er4 #t increases surface area of the leaves for the extraction of chlorophylls &% Can ethoxyethane be used as extraction solvent instead of acetone@ 5xplain Ans!er4 Aes 5thoxyethane is an organic solvent !hich is immiscible !ith !ater "esides$ it has no reaction !ith chlorophyll :pon sha3ing !ith the li7uid mixture$ the organic product !ill dissolve preferentially in the organic solvent '% %tate the stationary phase and the mobile phase for the thin layer chromatography conducted in this experiment Ans!er4 %tationary phase B thin layer of silica gelC mobile phase B li7uid solvent (the acetone/hexane mixture) (% <hy can a mixture of organic components be separated by thin layer chromatography@ Ans!er4 #n TLC$ silica gel used as the stationary phase can be considered polar !hile the organic solvent used as the mobile phase is non/polar %ince the components of the mixture differ in polarity$ the components in a mixture have different tendencies to adsorb onto the silica gel or dissolve in the organic solvent The more polar component has a stronger interaction !ith the silica gel and is therefore adsorbed on the silica gel strongly The more strongly a compound is adsorbed$ the less distance it can travel up the plate #n contrast$ the

less polar compound moves higher up the plate (resulting in a higher Rf value) )% %uggest t!o other types of chromatography )aper chromatographyC high performance li7uid chromatography (&)LC) -% &o! to recognise chlorophyll A and chlorophyll " from the chromatogram@ Ans!er4 Chlorophyll " is more polar than chlorophyll A Therefore$ chlorophyll " should have stronger attraction to silica and conse7uently shorter running distance up the plate compared !ith chlorophyll A$ i e it has lo!er Rf value .% 0or thin layer chromatography$ a capillary tube !as used to ta3e some solution and dotted onto the silica plate &o! the chromatogram !ill be affected by the si8e of the spot@ Ans!er4 #f the spot is too large$ DtailingE may occur (i e the diameter of the spot on the chromatogram is large) and thus lead to poor separation of the mixture of compounds