Separation of Plant Pigments by Column Chromatography (CC)

Objectives: Chromatographic Method, Plant Pigments

Peter Keusch

German version

Chemicals and other materials: silica gel 60  (Merck)

petroleum ether

acetone

NaCl

CaCO3

Na2SO4

fresh leaves

Apparatus and glass wares: glass chromatography column fitted

with a fritted disk at the bottom and a stop cock at the outlet

seperation funnel 500 mL

separatory funnel 100 mL

powder funnel

5 measuring cylinders 25 mL

beaker 100 mL

beaker 600 mL

9 Erlenmeyer flask 100 mL

volumetric pipette 20 mL

pipette bulb

mortar & pestler

glass rod

cork ring

swan-neck lamp

Hazards and safety precautions:

Petroleum ether is volatile and very flammable. Petroleum ether presents a high fire risk. The toxicity of petroleum ether varies according to its composition. Many of the components are of quite low toxicity, but some formulations may contain chemicals that are suspected carcinogens. Avoid ingestion and inhalation.

Acetone is highly flammable. Irritating to eyes.

Safety glasses and protective gloves required. The experiment should be performed under a portable fume cupboard giving all-round visibility!

Preparation

Extraction of the leaf pigments: Using a pestle, fresh leaves are grinded in a mortar containing 22 mL of acetone, 3 mL of petrol ether and a spatula tip-ful of CaCO3. The pigment extract is filtered. The filtrate is poured into a separation funnel and is mixed with 20 mL of petrol ether and 20 mL of 10% aqueous NaCl solution. The separating funnel is shaken carefully. When the layers have separated the lower layer is allowed to drain into a beaker. This phase is thrown away. The upper layer is washed 3-4 times with 5 mL of dest water. Afterwards the extract is placed in an Erlenmeyer flask and is dried with about 4 spatula tips of Na2SO4. The liquid is carefully decanted into an Erlenmeyer flask.

Eluting solvent (mobile phase):

mixture of petroleum ether and acetone (7 : 3)

Silica gel slurry:Using a beaker of an Appropriate size, a slurry of silica gel and eluting solvent is prepared.

Packing of the column: A uniform well-consolidated packing of the column is critical to the success of this chromatographic seperation. A clean, dry column is aligned in a vertical position. A beaker is placed under the column outlet. The column is slowly and evenly filled about two-thirds full with silica gel slurry. The stop cock is opened to allow liquid to drain into the beaker. Pouring the slurry down a glass rod held against the wall of the column will minimize bubbling and turbulence. The side of the chromatographic tube is gently tapped with a cork ring or a piece of vacuum tubing during the packing process, to make the silica gel compact.

Meanwhile the stop cock is opened to allow the excess eluting solvent to run out. Using a powder funnel a small amount of sand is carefully added to the top of the silica gel column to prevent it from being disturbed when fresh solvent eluent is added. The solvent level is allowed to drop until it is just barely above the sand. The bottom outlet of the separation column is closed. It is made sure that the column never runs dry. During the experimental procedure there should be always a small column of liquid above sorbent!

Experimental procedure:

Using a volumetric pipette 20 mL of the leaf extract are added directly (or carefully down the side of the column) to the sand layer. Then the mobile phase is drained continuously to the top of the column by aid of a seperation funnel. The bottom outlet of the column is opened. The eluent flows down through the column. The column, with the adsorbent and the sample, is 'developed': As the eluent passes down the column, the components of the mixture begin to move down the column. The separated zones 'flow out' of the column, where the eluates are collected in Erlenmeyers. The flasks are changed as the eluate changes color.

Using a swan-neck lamp a bright beam of light is directed at the leaf extract and at the samples eluted from chromatography column. Leaf extract and the samples containing chlorophyll or pheophytin produce a reddish glow. This phenomenon is known as fluorescence.

   

   

Video clip (Download RealPlayer .rm file)

Results and discussion:

The mobile phase slowly flows down through the silica gel column by gravity leaving behind zones of color - the chromatogram. The theory of column chromatography is analogous to that of thin-layer chromatography. The different components in the sample mixture pass through the column at different rates due to differences in their partioning behavior between the mobile liquid phase and the stationary phase.

leaf pigments color

carotenes golden

pheophytin olive green

chlorophyll a blue green

chlorophyll b     yellow green    

lutein yellow

xanthophylls yellow

References:   Demonstration Experiments on Video   Separation of Food Dyes by Thin Layer

Chromatography (TLC)   Demonstration Experiments on Video   Separation of a Lipophilic Dye Mixture

by Thin Layer Chromatography (TLC)   Demonstration Experiments on Video   Separation of Plant Pigments by Thin

Layer Chromatography (TLC)

Separation of Food Dyes by Thin Layer Chromatography (TLC)

Objectives: Chromatographic Method, Food Dyes

Peter Keusch

German version

Chemicals: Test solution:  a mixture of 7 dyes dissolved in water: Erythrosin, Brillant Black BN, Fast Red E, Naphthol Red S, Yellow Orange S, Ponceau 4R, Tartrazine.

Reference solutions:  Yellow Orange S and Brillant Black, each dissolved in water.

Developing solvent:  2.5 % sodium citrate solution, ammonia 25 %, 2-propanol  (20 : 5 : 3)

The developing solvent must be freshly

prepared.

Apparatus and materials: developing chamber  (jam glass with a

screw cover  h = 11 cm, d = 5 cm)

Fertigfolie POLYGRAM® CEL 300 plate  (Macherey Nagel)

glass capillaries (1 µL)

Hazards and safety precautions:

Concentrated ammonia solution is extremely damaging to eyes. Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin.

2-Propanol is highly flammable.

Safety goggles and protective gloves required. The developing solvent should be prepared in a laboratory fume hood!

Experimental procedure:

Using a soft pencil, a line is drawn approximately 1,5 cm from the bottom of the plate. The spotting points are numbered (1,2,3). At the spotting points 1 and 3 the reference solutions are applied onto the plate, at the spotting point 2 the dye mixture. Using capillaries approx 0.25 µL of the dye solutions are applied to the TLC plate. The capillaries fill themselves quickly when dipped into organic sample solutions. Before emptying the submerged end of the capillary is rolled horizontally on filter paper. The clean upper end of the capillary is placed on the layer vertically and carefully, vertically so that the capillary empties itself and carefully to avoid damage to the layer. Easy application of samples is allowed with a spotting guide.

When the solvent is completely evaporated (approx. 10 min) from the plate, the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom.

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line). The TLC chamber is covered. The TLC plate is allowed to remain undisturbed. When the solvent front has reached three quarters of the length of the plate, the plate is removed from the developing chamber and the position of the solvent front is immediately marked. The solvent on the plate is allowed to evaporate.

Results:

The dye mixture is separated into individual components. The progress of their separation may be followed by observing the movement of colored spots. The use of referenve solutions allows to identify Yellow Orange S and Brillant Black in the mixture.

   

Video clip (Download RealPlayer .rm file)

Background:

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration. The retention factor, Rf value, is used to characterize and compare components of various samples.

The Rf value is defined as follows:

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent. The composition of the mobile phase and the temperature must remain constant.

Separation of Plant Pigments by Thin Layer Chromatography (TLC)

Objectives: Chromatographic Method, Plant Pigments

Peter Keusch

German version

Chemicals and other materials: petroleum ether

acetone

isopropanol

NaCl

CaCO3

Na2SO4

fresh leaves

Apparatus and glass wares: rotary evaporator

round bottom flask 100 mL

TLC chamber 22 cm × 22 cm × 10 cm

TLC silica gel plate  (DC-Alufolien, Kieselgel 60 / Kieselgur F254, Art.5567   Merck)

seperating funnel 100 mL

5 measuring cylinders 25 mL

measuring cylinder 100 mL

Erlenmeyer 100 mL

mortar & pestle

paint brush

Hazards and safety precautiona:

Petroleum ether is volatile and very flammable. Petroleum ether presents a high fire risk. The toxicity of petroleum ether varies according to its composition. Many of the components are of quite

low toxicity, but some formulations may contain chemicals that are suspected carcinogens. Avoid ingestion and inhalation.

Acetone and isopropanol are highly flammable.

Safety glasses and gloves must be worn. The experiment should be performed under a portable fume cupboard giving all-round visibility!

Preparation

Developing solvent (mobile phase):100 mL of petrol ether, 11 mL of isopropanol and 5 drops of dist. water

Preparation of the TLC chamber:The developing solvent is placed into a TLC chamber. The solvent should completely cover the bottom of the chamber to a depth of approximately 0.5 cm. The chamber is closed and shaken. It is kept covered so that evaporation doesn't change the composition of the developing solvent mixture. After 15 minutes the chamber will be saturated with the solvent vapor.

Extraction of the leaf pigments: Using a pestle fresh leaves are grinded in a mortar containing 22 mL of acetone, 3 mL of petrol ether and a spatula tip-ful of CaCO3. The pigment extract is filtered. The filtrate is put into a separating funnel and is mixed with 20 mL of petrol ether und 20 mL of 10% aqueous NaCl solution. The separating funnel is shaken carefully. When the layers have separated the lower layer is allowed to drain into a beaker. This phase is thrown away. The upper layer is washed 3-4 times with 5 mL of dest water. Afterwards the extract is placed in an Erlenmeyer flask and is dried with about 4 spatula tips of Na2SO4. The liquid is carefully decanted into a round bottom flask. Using a rotary evaporator the leaf extract is concentrated to a final volume of about 3 mL.

Application of the extract to the TLC plate: With a pencil a line is drawn approximately 1,5 cm from the bottom of the plate. The coating of the plate should not be scraped! Using a paint brush or a Pasteur

pipet the leaf extract is applied as a line to the TLC plate. The procedure is repeated until the line is very dark green. The transferred extract is allowed to dry thoroughly after each addition. The line is kept as thin and straight as possible.

Experimental procedure:

The loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom. The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the developing solvent (solvent surface must be below the extract line). The TLC chamber is covered. The TLC plate is allowed to remain undisturbed. When the solvent front has reached three quarters of the length of the plate, the plate is removed from the developing chamber and the position of the solvent front is immediately marked.

   

Video clip (Download RealPlayer .rm file)

Results and discussion:

As the solvent rises by capillary action up through the TLC plate, the components of the pigment mixture are partitioned between the mobile phase (solvent) and the stationary phase (silica gel) 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 weakly a component is adsorbed the faster it will migrate up the TLC plate. On the other hand, the running distance depends on the solubility of the pigment in the solvent. Since the experiment employs a high non-polar solvent (petroleum ether), the pigments that are least polar (carotenes) will be best solved in the non-polar solvent ("similia similibus solvuntus") and will thus have the largest running distance.

  

leaf pigments color

carotenes golden

pheophytin olive green

chlorophyll a blue green

chlorophyll b   yellow

green

lutein yellow

violaxanthin yellow

neoxanthin yellow

Separation of a Lipophilic Dye Mixture by Thin Layer Chromatography (TLC)

Objectives: Chromatographic Method, Lipohilic Dyes

Peter Keusch

German version

Chemicals: Test solution:   Dye mixture (Butter Yellow, Sudan Blue II, Sudan Red G and Indophenol in toluene.

Reference solutions:  Sudan Red G and Sudan Blue II each dissolved in toluene.

Developing solvent:  toluene

Glass wares and materials: developing chamber  (jam glass with a

screw cover h = 11 cm, d = 5 cm )

DC plate POLYGRAM® ALOX N/UV254  (Macherey Nagel)

glass capillaries (1 µL)

Hazards and safety precautions:

Toluene is toxic by inhalation, ingestion or by absorption through skin. Serious irritant. Experimental teratogen.

Safety goggles and gloves required. The experiment should be performed in a portable laboratory fume hood giving all-round visibility!!

Experimental procedure:

Using a soft pencil, a line is drawn approximately 1 cm from the bottom of the plate. The spotting points are numbered (1,2,3). At the spotting points 1 and 3 the reference solutions are applied onto the plate, at the spotting point 2 the dye mixture. Using capillaries approx 1 µL of the dye solutions are applied to the TLC plate. The capillaries fill themselves quickly when dipped into organic sample solutions. Before emptying the submerged end of the capillary is rolled horizontally on filter paper. The clean upper end of the capillary is placed on the layer vertically and carefully, vertically so that the capillary empties itself and carefully to avoid damage to the layer. Easy application of samples is allowed with a spotting guide.

When the solvent is completely evaporated (approx. 10 min) from the plate, the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom. The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the developing solvent (solvent surface must be below the extract line). The TLC chamber is covered. The TLC plate is allowed to remain undisturbed. When the solvent front has reached three quarters of the length of the plate, the plate is removed from the developing chamber and the position of the solvent front is immediately marked. The solvent on the plate is allowed to evaporate.

Result:

   

Videoclip (Download RealPlayer .rm-Datei)

The dye mixture is separated into individual components. The progress of their separation may be followed by observing the movement of colored spots. The use of referenve solutions allows to identify Sudan Red G and Sudan Blue II in the mixture.

Background:

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration. The retention factor, Rf value, is used to characterize and compare components of various samples.

The Rf value is defined as follows:

In order to get reproducible Rf values the atmosphere in the developing chamber must be saturated with the solvent. The composition of the mobile phase and the temperature must remain constant,