β-Carotene, or pro-Vitamin A, is a lipid found in carrots and leafy or root vegetables. It is converted into retinal (Vitamin A) then into 11-cis-retinol, bound to the protein opsin and deposited into the rod and cone cells of the eye. Light energy isomerizes the double bond into the trans form and the movement stimulates the optic nerve, producing a sensation of light. Vitamin A or its precursor is an essential nutrient and must be supplied in the diet.
β-Carotene has a characteristic orange color and is a plant pigment. It is a hydrocarbon with many conjugated double bonds. Due to resonance delocalization, there are small energy differences between molecular orbitals. Electrons can absorb light energy and become excited. Since the wavelength of maximum absorption, λmax, is in the blue portion of the spectrum, red and yellow light passes through the sample, giving it the characteristic orange color.
Since the Beer-Lambert Law (Abs. = ε · c · l) shows that absorbance of light at a specific wavelength is proportional to the concentration of the colored species in solution, we can determine the concentration of β-carotene in a solution once the extinction coefficient, ε, has been determined. We will prepare standard β-carotene solutions of different concentrations, measure their absorbance at λmax and draw the standard curve. We will then prepare an extract of β-carotene from a weighed sample of carrot. The absorbance of the extract (or a diluted solution of the extract) will allow us to find the concentration of the extract then the total amount of β-carotene recovered from our carrot sample.
The sample will also be examined by thin layer chromatography. This is a solid-liquid partitioning technique where a solute is applied to an adsorbent material, the stationary phase (here finely ground hydrated SiO2 or silica gel) that is spread out onto a support material like plastic or glass. Then, solvent is allowed to ascend the adsorbent layer. As the solvent and the adsorbent both interact with the solute, the sample is partitioned between the stationary phase and the moving phase. Different solutes in the originally applied material have different equilibria between the two phases and so the mixed components separate. The result of these interactions is summarized in the Rf value, or retardation factor or alternatively the “ratio of fronts”, of each component. This is calculated by dividing the distance traveled by the substance in any one spot from the point of application by the distance traveled by the solvent front from the same original spot.
Objectives: after this experiment, you will be able to
Extract β-carotene from a natural source
Prepare a serial dilution of known β-carotene
Construct a standard absorbance curve and determine the extinction coefficient
Use the standard curve to determine the solute concentration in an experimental sample
Test the purity of the experimental sample by thin-layer chromatography
Use proportions to determine the total amount of β-carotene and its concentration in the source
Carrot, you’ll need to bring one from home. If you are bringing baby carrots, you’ll need enough for 10. g. Note the brand and where purchased.
Solvents: hexane, 95% ethanol
Drying agent, CaCl2(s)
TLC plate with silica gel coating a solid support; microcapillary applicators Chromatography chamber (400-mL beaker with a Parafilm cover) Developing solvent: 70% hexane: 30% acetone
Part A-Extraction of β-Carotene from Carrot
Grate or blend carrots, place 10. g in a 250-mL round-bottom flask and add 100 mL of 95% ethanol. Attach a reflux condenser and heat, refluxing the mixture gently for 30 minutes. Cool. Pour the ethanol extract into a 250 mL Erlenmeyer flask then add an additional 25 mL of ethanol to the carrot pulp to rinse it. Mix the rinse ethanol with the original extract. Discard the carrot pulp.
Place the ethanol extract in a 250-mL separatory funnel. Add 15 mL of water to form an 85% ethanol solution. (This increases the polarity and reduces β-carotene’s solubility in the alcohol solution.) Add 25 mL of hexane to the solution, mix by swirling (but DO NOT shake) then hang the separatory funnel upright and allow the phases to separate. (Hexane is less dense than 85% ethanol.) To remove the hexane layer, which is now more concentrated in β-carotene, drain and save the lower layer, pour the hexane layer out through the top of the separatory funnel and save in a dry 100 mL volumetric flask, then return the ethanol layer to the funnel for further extraction. Extract the ethanol layer similarly with 2 additional 15 mL portions of hexane. Combine the hexane extracts in the 100 mL volumetric flask. Discard the ethanol solution. Bring the volume up to
exactly 100 mL, add a few pellets of drying agent to the flask, stopper and let it stand for a few minutes while the drying agent absorbs any water from the extract.
Part B-Preparation of the Standard Absorbance Curve and Determination of the Extracted β-Carotene Concentration
A solution of β-carotene in hexane of known concentration will be provided. Note the concentration. Take 10 mL and use it to make standard solutions. Determine the λmax of β-carotene and read the absorbance of this primary standard solution. It will probably have a high absorbance and the spectrophotometer is not sensitive enough to small changes in concentration at high absorbance to give a linear relationship. It is necessary to dilute the primary standard to obtain a number of solutions of different concentrations.
Prepare six diluted standard solutions, calculate the new concentration, read their absorbance, and prepare a graph of absorbance as a function of concentration for these standards. The dilutions need to have absorbance in the range of 0.100 to 1.200. A 10-fold dilution does not necessarily mean 1 part concentrated solution plus 9 parts solvent. Instead, place 1 part concentrated solution in volumetric glassware and add additional solvent until the total volume is 10 times greater. Hexane is volatile so stopper all solutions immediately to minimize concentration change due to evaporation. It is usually more accurate to do several serial dilutions rather than one single dilution to achieve a large dilution factor. Rather than a single 1:100 dilution, it would be better to prepare a 1:10 dilution then dilute the new solution again by 1:10.
Take the extracted β-carotene in hexane and record its absorbance at λmax. The extract is probably too concentrated to get an accurate absorbance reading. If absorbance is greater than 1.200, dilute the extract by known dilution factors until its absorbance is within the range of values used to construct the standard curve. Record the overall dilution factor and the absorbance of the final diluted solution.
Part C-TLC of the Extract
If needed, prepare a microcapillary applicator by rolling an open-end capillary tube in a flame until the glass softens. Then gently pull the ends in opposite directions, stretching the tube into a thinner region in the center. Let the glass cool then snap into two parts in the center of the thin section. NOTE: the flame must be kept well away from any flammable solvents and far away from reflux apparatus, extraction procedures and TLC developing chambers. OR, use a commercial microcapillary applicator.
Prepare a thin layer chromatography plate by lightly drawing with a lead pencil (NOT a pen!) a line across the plate about 1 cm from the bottom. Lightly mark 2 dots on the plate at least 0.6 cm in from the edges and 1 cm from each other.
Prepare a developing chamber by placing some 70% hexane: 30% acetone solvent in the bottom of a 400 mL beaker so the bottom is covered but so that the height of the solvent on the beaker bottom is below the marked line on the TLC plate. Place a small filter paper circle in the beaker so the paper edge rests in the solvent and so the paper will wick up solvent. This saturates the air in the sealed container with solvent vapors so that solvent does not evaporate off the plate during development. Cover the beaker with a loosely attached layer of Parafilm and set it aside.
Take a 1 mL sample of the original hexane extract. In a warm (40 – 60°C) water bath, evaporate the pigment solution to dryness. DO NOT allow the dried material to sit in the bath! Using a Pasteur pipet, add 2 drops of the 70%hexane-30% acetone solvent to the dried pigment. Apply this solution to the TLC plate in 1 or 2 quick touches. Keep the spot diameter small and allow the solvent to evaporate completely.
Similarly apply some of the primary standard β-carotene solution to the TLC plate. Place the TLC plate in the development chamber; make sure that the plate does not come in contact with the filter paper liner. Seal the top tightly and set aside undisturbed for development. Remove the plate when the solvent front is 1-2 cm from the top of the plate. Using a lead pencil, mark the position of the solvent front immediately after removing the plate. As soon as the plates have dried, outline the spot(s) with pencil and note the color(s). If the spots are large with much “trailing” of solute, repeat the chromatography on a new plate, applying less material.
Prepare the standard absorbance curve by plotting measured absorbance as a function of the diluted standard solution’s concentration in mg β-carotene per unit volume. Fit the data points, do not connect the dots.
Determine ε, the extinction coefficient at this wavelength. The path length across the sample is 1.oo cm. Determine the concentration of β-carotene in the diluted carrot extract and determine the concentration in the original (undiluted) extract.
How many mg of β-carotene were recovered from the sample of shredded carrot? Determine the % recovery of β-carotene from the original carrot sample. Calculate the Rf value of each spot observed in the β-carotene extract and in the standard solution.
As of 2001 the recommended daily intake of β-carotene is 12μg. Assuming a “standard carrot” weighs 20.g, how many carrots should you eat in a day to satisfy the recommended intake?
- What is the reported λmax for β-carotene? Provide a citation.
- What is the reported extinction coefficient for β-carotene? Provide a citation.
- Draw the structure of β-carotene and circle the system of conjugated double bonds.
- Chlorophyll can be extracted from spinach leaves and appears green. Lycopene can be extracted from tomato and appears red. What are the approximate λmax values for these pigments? Provide citations.
- Carrots are mainly cellulose and water. Why was ethanol used at first to extract the pigment rather than using hexane directly, even though β-carotene is more soluble in hexane?
- Sketch the reflux set-up for the extraction. Label the glassware and label the reagents.
On a new page, prepare data table(s) for the results you’ll collect in this experiment.