Column Chromatography of Carotenoids%2b(Repaired)

Isolation of Carotenoids

Introduction

Carotenoids are organic, fat-soluble pigments that are naturally occurring in plants and some other photosynthetic organisms, called terpenes. These compounds are responsible for the red, yellow, and orange color of fruits and vegetables. There are over 600 different carotenoids known, they are split into two classes, xanthophylls and carotenes. Animals are incapable of synthesizing carotenoids, and must obtain them through their diet. Structurally carotenoids are tetraterpenes (eight isoprene units) of a 40- carbon atoms ( C4O H56 ) which could be considered the backbone of the molecule. The chain may be terminated by cyclic end-groups (rings) and may be complemented with oxygen-containing functional groups. The hydrocarbon carotenoids with molecules containing oxygen are known as xanthophylls . The unoxygenated (oxygen free) carotenoids are known as carotenes. Carotenes typically contain only carbon and hydrogen. Some familiar examples of carotenoids coloration are the orange of carrots and citrus fruits, the reds of peppers and tomatoes. The two commonest carotenoids are lycopene, a red pigment found in tomato and beta-carotene, a yellow pigment found in carrots. Other pigments also occur in the leaves of plants but they are not obvious because their colors masked by the chlorophylls in live, healthy leaves. These pigments become visible in the fall when the leaf dies and the chlorophyll rapidly decompose. Carotenes act as a precursor of vitamin A. Only alpha-carotene and beta-carotene are converted to significant amounts of vitamin A in the body, and beta-carotene is by far the most plentiful carotenoid found in fruits and vegetables. Beta-carotene cleaves to form two molecules of vitamin A when it is ingested. Also called retinol, vitamin A plays an important role in vision. Beta-carotene is also a powerful antioxidant, and has been shown to help guard against cancer and heart disease. Lycopene, is an antioxidant stop free radical production, and it may lower the risk of prostate cancer. The best food source of lycopene are processed tomato products such as ketchup, tomato paste, and tomato juice.

Organized by: Badahdah, M. S. & Alghazzawi, W. M. ~ 1 ~

Principle

This experiment performed to try and tentatively identify and isolate lycopene and beta-carotene from two foods rich in them, tomato paste and carrots using refluxing, column chromatography and thin layer chromatography. Analyze the fractions by thin-layer chromatography to determine if the fraction contains more than one component. Since both lycopene and β-carotene are colored pigments that absorb light in the UV

and visible range, they can be identified from their maximum wavelength.

PART I: Dehydration and Extraction

In this part you will extract pigments from tomato paste and carrots, using ethanol and chloroform as an organic solvents.

Apparatus & glassware

Condenser Hot plate Beaker Filter paper Separation funnel Glass funnel Round-bottom flask 2 Conical flask

Chemicals & other material:

Tomato paste or carrot (natural or baby food) Ethanol (95 %) Chloroform Saturated sodium chloride solution Anhydrous sodium sulfate


Procedure

1. Place 3 gm of tomato paste and 9 gm carrots (natural or baby food) and 30 ml of 95 % absolute ethanol in a

small beaker. The suspension is then stirred for at least five minutes.

2. Filter to separate ethanol from the plant sample through a small funnel. Press out all the liquid of the semi-solid residue collected in the funnel by pressing it gently with the flat side of a spatula and discard the solvent. All glassware from this point must be dry.

3. Return the solid residue, with or without any adhering filter paper, to a round-bottom flask, then add 15 ml of chloroform. The flask is fitted with a condenser and refluxed at 40°C for 4 minutes. This will extract the β-carotene and lycopene from the plant. The yellow or red extract is separated from the solid residue by decantation, and then the solid residue is returned to the flask.

4. Repeat the extraction procedure two more times with 15 ml chloroform each time. Combine the organic solvent extracts in a flask and discard the plants.

5. Pour the combined organic solvent extracts into a separation funnel, add 20 ml of saturated sodium chloride solution (aid in layer separation), then shake.

6. Allow the layers to separate and then slowly drain the colored lower layer (Chloroform is more dense than water) through a funnel which has a cotton with 4 spatula tips of anhydrous sodium sulfate on top of it. This removes any water from the colored extract.

7. Evaporate the colored extract on a hot plate under the hood until about 1 ml of solvent remains in beaker. Do not allow the solvent to completely evaporate.

8. Warp the flask with foil to avoid light oxidation, label the flask with your name and place it in a beaker. Store it in the freezer until the next lab.


PART II: Column chromatography

In this part you will use the preparative method of column chromatography to separate two major fractions of pigments, the carotenoids from extract.Since the different components are colored differently, the separation is easily followed visually. Many factors can affect the separation. These include: adsorbent, polarity of the column or solvents, size of the column, and the rate of flow.

The most common solvents are arranged in order of their elution powers as the following:

Water > Methanol > Ethanol > Propanol > Acetone > Ethyl Acetate > Diethyl Ether > Chloroform > Dichloromethane > Benzene > Carbon Tetrachloride > Cyclohexane > Hexane > Petroleum ether.

In this experiment the carotenoids separating according to the differences in polarity (Beta carotene is nonpolar while lycopene is more polar).

Principle

Column chromatography (LSC) is one of the most useful methods in biochemical work in separation and purification of both solids and liquids. The theory of column chromatography is analogous to that of thin-layer chromatography.

In TLC, the stationary phase (adsorbent) is a thin layer of the most common stationary phase, silica gel (SiO2) or alumina (Al2O3) on a glass, metal or plastic plate. The cellulose powder has often been used in the past.

In column chromatography the same materials (adsorbent) is packed into a vertical glass column. A fine powder of the adsorbent in a tube (called a column) serves as the stationary phase, and mixtures of substances in appropriate solvents are

applied to the top of the column and passed through the column, more or less like filtration. The less-polar components of the mixture emerge from the bottom of the column first, followed by the more-polar components.

The solvent (eluent), instead of rising by capillary action up a TLC, flows down through the column filled with the adsorbent by gravity action or by the application of air pressure.


Just as in TLC, there is an equilibrium established between the solute adsorbed on the silica gel or alumina and the eluting solvent flowing down through the column.The major advantages of column chromatography are its ability to handle large amounts of material and the ability to change the eluting solvent throughout the course of the separation. This allows one to remove one component while a desired product remains essentially unmoved. Then a solvent change moves the desired product through the column. Solvents changes may include such things as changes in polarity, canges pH, or changes in ionic strength. The last two are used largely in biological separations. Thus, by varying the stationary phase and by changing solvent or solvent systems,an efficient separation may be achieved. In this assignment, alumina serves as the adsorbent stationary phase. The more polar components are held to the alumina more tightly and, therefore, move through the column more slowly. Increasing the polarity of the solvent moves all components faster but has largest effect on polar compounds.

The column is a piece of cylindrical glass (polyethylene) tubing with a stopcock attached at the bottom of the column to control the flow of the solvent through the column, or a piece of flexible tubing is often attached at the bottom of the column , and a screw clamp would be placed on the Flexible tubing at the bottom to stop or regulate the flow of the solvent. The tubing must not be dissolved by the chromatographic solvents used to achieve the separation.

Column chromatography can be small or big, according to the amount of material which needs to be loaded onto the column. Various sizes of chromatography columns are used; it is often convenient to use an ordinary burette as a chromatography column. A pasteur pipette is also used as a chromatography column.


The stationary phase (column packing) in the column is polar.The polar compound interacts more strongly with the stationary phase and moves more slowly. The non-polar compound moves more rapidly.

The stationary phase is non-polar. Non-polar compounds will move more slowly because they are attracted to the column packing. The more polar compound moves more quickly down the column.Components of the sample separate from each other by partitioning between the stationary phase (silica or alumina) and the mobile phase (eluent). Molecules with different polarity partition to different extents, and therefore move through the column at different rates. The eluent is collected in fractions.

Apparatus & glassware

1. Column chromatography 2. 2 Beakers3. Glass rod4. Conical flask5. Glass funnel6. Pasteur pipette7. Small vial

Chemicals & other material

1. Crude caroteniods (Dried pigment)2. Petroleum ether (60-80°C)

Care: Avoid ingestion and inhalation of petroleum ether because it is volatile and very flammable. The toxicity of petroleum ether varies according to its composition but some formulations may contain chemicals that are suspected carcinogens. The experiment should be performed under a portable fume cupboard giving all-round visibility.

3. Safety glasses and protective gloves 4. Alumina, aluminum oxide (Al2O3). 5. Hexane


Procedure

Preparation of the column

1. Wear gloves and goggles at all times.2. Place a very small glass wool plug in the bottom of the column with a long glass rod, until all entrapped air is forced out. The plug is just to keep the solid alumina from entering the stopcock. The plug must not interfere with the flow of the solvent through the stopcock.3. Clamp the column to a ring stand in vertical position.4. Close the stopcock, and fill the column with 6 ml of the eluent.5. Place a small beaker under the stopcock of the column to catch any solvent that may accidentally drip out of the tip of your column.

Preparation of the slurry

Prepare slurry (Mixture of a solvent and an un-dissolved solid) by pouring dry alumina (approximately 30 ml) slowly into a beaker of double volume of petroleum ether (60 ml), a little at a time while swirling to form thick but flowing slurry.

The addition adsorbent to solvent should be followed strictly because the adsorbent will solvate and liberate heat. If the solvent is added to the adsorbent, it may boil away almost as far as it is added due to heat evolved, especially if ether or another low boiling solvent is used.

Packing of column

1. Quickly and carefully pour the slurry (10-12 cm) into the top of a clean, dry column using a glass funnel. Stir and pour immediately to maximize the amount of alumina that goes into the column instead of remaining behind in the beaker. You may find a clean spatula or glass rod helpful in transferring the alumina. The column is tapped constantly and gently by tubing or rubber stopper on the side during the pouring of slurry to help the alumina settle uniformly, compact, and to remove air bubbles inside the column.


2. Place a beaker under the column outlet, open the screw clamp, and allow the solvent to drain into it. Add more solvent as necessary. The solvent collected prior to the sample can be re-used.

3. Use a Pasteur pipette to rinse any alumina that is sticking to the sides of the column. Allow the alumina to settle while eluent continues to drip into the beaker. Once the alumina has settled, carefully add about 1cm of sand to the top of the column to prevent it from being disturbed when fresh solvent is added.

4. When finish packing, drain the excess solvent until it just reaches the top level of alumina (about 0.5 ml). Close the screw clamp. Your column is now “packed”.

5. Never let the solvent drop below the top of the adsorbent to prevent the column from going dry out as the column progresses. Cracks will form within the alumina column if it dries, and compounds can fall down the cracks instead of partitioning between mobile and stationary phases.

6. The success of your separation will be dependent on how well you pack and load the column.

Adding the sample (pigment)

1. The sample is first dissolved in a very small amount of the solvent. It is important to use a minimum amount (1-1.5ml) of solvent to dissolve the pigments. A thin horizontal band of sample is best for an optimal separation. If the extract has dried out, re-dissolve it in 1ml of the solvent.

2. Place a beaker under the column then open the clamp at the tip of the column allowing the solvent to drain just to the top of the column 1 mm above the surface of the column.

3. Add about half (and save the other half for the thin layer chromatography later) of the crude plant pigment extract drop wise to the top of the sand using a long Pasteur pipette being careful not to disturb the top of the column. Allow the sample to adsorb onto the top of the alumina before adding more eluting solvent.

4. Carefully add solvent to the top of the column. To avoid disturbing the top of the column, it’s a good idea to carefully pipette an inch or two of solvent onto the column instead of pouring solvent directly onto the column. If the sample is colored, and the fresh layer of solvent acquires this color, add small amount of eluting solvent until the solvent above the top of the column is colorless.


1212

5. Fill the column to the top with solvent. The column is now ready to run.

6. The components of the sample (pigment) begin to move down the column and separate according to their polarity. The yellow beta-carotene moves rapidly through the column, while the red lycopene moves slowly. Increasingly more polar solvents will be used to elute the various components from the column.

Sample collection

1. Continue adding solvent at the top. Begin collecting solvent until you see the band of the yellow β-carotene is just above the stopcock of the column. Change the collecting beaker and begin collecting the yellow fraction in vial. 2. Switch collection vial and allow the solvent to run down to just the top of the column and then add a solvent mixture of 5:45 petroleum ether/chloroform to elute the orange/red lycopene band.3.Switch beaker again when there appears to be a color change and begin to collect the orange band in another vial when it reaches the middle to the lower half of the column to minimizes contamination of the lycopene with β-carotene. The process is discontinued when the band(s) desired is (are) off the column. Save the collected fractions for TLC analysis and UV-Vis spectrum.4. If the compounds to be separated from column chromatography are colorless, small equal sizeFractions of the eluent are collected sequentially in labeled vials and the composition of

each fractions are analyzed by thin layer chromatography.


Petrolum ether

Alumina

Stopcock

Tomato sample

Glass wool

Clamp

PART III: TLC analysis of sample fractions

In this part you will use the analytical method of thin layer chromatography to estimate the number of pigments in the extract.

Preparation of the Slurry

One gram of silica gel to 3 ml of chloroform is placed in a wide-mouth screw cap jar. The jar is shaken vigorously to obtain smooth, homogeneous slurry. The slurry may be stored in the tightly capped screw cap jar until it is to be used.

Preparing (Coating) the Slides

Microscope slides or Silica gel G plates are used as TLC plates.1. Wash microscope slides with soap and water, then rinse with 50% aqueous methanol

and dry thoroughly on paper towels. Handle by the edge.2. Stirring the slurry with a glass rod, then dip the slides two by two (as sandwich) in it. 3. Dry the plates at 110°C in an oven for about 1 hour.

Applying the sample

1. Using a capillary tubes, spot the solutions to be analyzed on the silica gel side of the TLC plate (or microscope coated slide) along the pencil line. One spot should be from the yellow band (i.e., β-carotene) from the column. One spot should be from the red band (i.e., lycopene) from the column. One spot should be from the colored crude extract that was not ever put on the column. The spots should be very small in diameter, but clearly visible.

2. If the spots are not visible, check that you have placed enough of each sample on the plate.

3. Follow the steps as in experiment 1 page 12.

Note: Immidiatly circle the visible spots in pencil since the colors may fade over time.

PART IV: UV/Vis spectroscopy

In this part you will explain the colors of pigment fractions by measuring the absorption of electromagnetic spectrum of each, using UV-visible spectrophotometer. Also you will correlate the results of the two types of chromatography with your spectral data.


The result of this kind of measurement is an absorption spectrum, a plot of absorbance (on the y-axis) as a function of wavelength (on the x-axis). Such a plot shows how much radiation is absorbed by the sample at each wavelength of radiation. The shape of the spectrum and the wavelength of maximum absorbance are characteristic of the chemical compound. The absorbance of compounds is also directly related to the concentration of the sample. You will use a spectronic 20 to do UV-Vis spectroscopy on the extract (from tomatoes or carrots) and the fraction that you collected during the column chromatography. You will manually generating absorption spectrum. You will need to determine a λmax for the carotene from this curve. You will need to get a petroleum ether solution of carotene into the glass cuvettes. Do not use an ordinary test tube for your measurement. Care should be taken not to drop the cuvettes.

The following spectrum reveals that the sample absorbs UV radiation strongly at wavelengths below 240 nm, and absorbs visible light near 410 nm. A sample transmits light of the wavelengths that it does not absorb. It should not surprise you that this sample appears orange in color. The sample absorbs light at the blue end of the visible spectrum, allowing light of higher wavelengths (yellows, oranges, reds) to pass through and reach the eye of the observer.

Lycopene and β-carotene pigments separated from tomato paste and carrots may be identified from their absorption spectra. For example, lycopene and β-carotene absorb light in the 400-500 nm region of the visible spectrum. Record the spectra of your column chromatography fractions. Make sure a blank is run first with the appropriate solvent, 100% petroleum ether or 10% petroleum ether in chloroform. Record the spectrum of your sample from 300-800 nm, ( this is actually just visible light spectroscopy). Determine the λmax of any peaks. There are actually several absorption peaks in the electronic spectrum of lycopene, but the peak of maximum intensity corresponds to a


wavelength near 473 nm. β-carotene behaves similarly on irradiation, but the peak of maximum absorption is found at shorter wavelengths, around 448 nm. The exact position of maximum absorption absorption depending somewhat on the solvent.

β-carotene: λ max at 426,448,474 nmLycopene: λ max at 444,473,502 nm

Note

Each student will clean their benches and dispose of the waste chemicals. Each student will submit a lab report the following week to report their findings.

References

1. All pictures are captured by KAU/Biochemistry department.2. http://www.wfu.edu/academics/chemistry/courses/CC/index.htm

To learn more information click on these links:LycopeneCarotenoidsColumn chromatographyColumn Chromatography ProceduresSeparatory Funnel Extraction Procrdure


http://orgchem.colorado.edu/hndbksupport/ext/extprocedure.html

http://orgchem.colorado.edu/hndbksupport/colchrom/colchromproc.html

http://www.chemguide.co.uk/analysis/chromatography/column.html

http://www.whfoods.com/genpage.php?tname=nutrient&dbid=116

http://www.lycopene.org/

Result sheet

Title: …………………………………………………………………………..

Test sample: …………………………………… Group partner: ……………………… ……………………….. ……………………….

Answer the following:

PART I: Dehydration and Extraction

Is the principle components in tomatoes lycopene or β-carotene ? Explain your answer.

………………………………………………………………………………………………

Why we add ethanol?……………………………………………………………………………………………

PART II: Column chromatography

Describe the separation that you saw in the column. ? Was the separation easy to visualize?

………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………

How many fractions (bands) are separated from the column? Are all they colored? What the color of each fraction?

………………………………………………………………………………………………

What fraction elute first? Why?

………………………………………………………………………………………………

Give brief description of your observations?

………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………


PART III: TLC analysis of sample fractions

Fasten your TLC plates here or sketch it, and properly annotate them including all R f

values.

List Rf values for all components off the TLC. (Calculation should be shown).

Distance moved by pigment Distance moved by a solvent

Source of pigment

Number of spots

Color of spot

R f Name of plant pigment

Extract\

Fraction

Fraction

What information did you get from TLC about the purity of the compounds?

………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………


Rf =

PART IV: UV/Vis spectroscopy

Tape a copy of the UV-Vis spectrum. Be sure to label everything properly (title, axes).

What is λ max for the carotene? ……………………….

Explain why these pigments are of different colors?

……………………………………………………………………………………………………………………………………………………………………………………………… ………………………………………………………………………………………………………………………………………………………………………………………………

Discuss how your UV-Vis spectra is.

………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………


Column Chromatography of Carotenoids%2b(Repaired)

Documents

Transcript of Column Chromatography of Carotenoids%2b(Repaired)