EXPLORATION 7.
Column Chromatography: Is There Vitamin A in Spinach?
The easy answer to the title question is, “No, there is no vitamin A, as such, in spinach (or any other vegetable).”
However, many plants contain pigments (colored substances) called carotenoids, from which animals, such as man,
can make vitamin A in their bodies. (The carotenoids are yellow-orange compounds that get their name from the
Latin word for carrot.) If spinach contains a carotenoid pigment (or more than one), then your answer to the title
question could be, “Spinach contains a carotenoid pigment that can be made into vitamin A.”
The structures of two common carotenoids, β-carotene and lycopene, as well as the structure of vitamin A are
shown here. You can see how similar the two carotenoids are and can imagine how the vitamin A molecule can be
derived from them.
You can use column chromatography to explore the separation of the carotenoids (if any) from the other
substances in several plant products and you can use absorption spectrophotometry (see the “Analysis” section) to
identify which one(s) you have obtained. The carotenoids are not very polar and dissolve well in nonpolar
solvents. They are not strongly attracted to polar solids like aluminum oxide (alumina), so they are easily
separated from more polar components by chromatography on such solids. Most plant products contain a good
deal of water, which interferes with extraction of the carotenoids. You can get around this difficulty by first
extracting the plant products with methanol to get rid of a lot of the water and then with nonpolar solvent to
extract the carotenoids (and possibly other components).
EQUIPMENT AND REAGENTS NEEDED
Thin-stem glass pipets; regular glass pipets; 25 Erlenmeyer flasks; 50-mL beakers; 400-mL waste beaker; glass
stirring rods; ring stand and small clamp to hold a glass pipet; 13 x 100-mm culture tubes; plastic teaspoons;
wax; cotton; scissors; food blender; rubber spatula; Spectronic 20 spectrophotometer (or equivalent).
Methanol; petroleum ether (essentially pentane); 10% (v:v) acetone in petroleum ether; anhydrous sodium
sulfate (granular solid); alumina (acid-washed for chromatography); chopped spinach (thawed frozen package);
tomato paste; baby food: carrots, squash, peas, green beans, etc..
PROCEDURE SAFETY GLASSES OR GOGGLES ARE REQUIRED FOR ALL LABORATORY WORK.
Work in a group of three on this exploration. You should explore spinach, and two other foods (tomatoes, carrots,
squash, peas, green beans, etc.) for the presence or absence of carotenoid pigments. Each group should explore a
spinach sample and divide up the other foods and tasks to be done among the members of the group. When you
have finished, exchange your results, so each member of the group has all the results. Your instructor may also
have you share data with other groups in the class.
PREPARING SPINACH PUREE – ALREADY COMPLETED PRIOR TO THE DAY OF THE LAB EXPLORATION
This is the procedure to prepare enough spinach for the entire laboratory section. Please the thawed contents of a
package of frozen spinach in a food blender. Put the cover on the blender and blend until the contents are very
finely divided (pureed). If necessary, stop occasionally and use a rubber spatula to scrape solid from the wall of
the container back to the bottom for better blending. Loosely cover the mouth of a 600-mL beaker with several
layers of cheesecloth. Pour the entire contents of the blender onto the cheesecloth, allowing the liquid to pass
through into the beaker. Bring the corners of the cheesecloth together to form a bag for the finely chopped
spinach and wring the cheesecloth tightly to squeeze as much water as possible from the solid. Use this solid as
the starting material for the carotenoid extraction in the next section.
EXTRACTING CAROTENOIDS FROM PUREED VEGETABLES
WARNING: Methanol and petroleum ether are very volatile and flammable.
Be sure there are no flames anywhere in the laboratory while they are in use.
Add about 10 mL (2 teaspoons) of the pureed vegetable to a 50-mL beaker. Obtain some methanol in a small
clean, dry flask. Add about enough methanol to cover the food sample and stir thoroughly for about two minutes.
Use a glass thin-stemmed pipet to remove as much of the liquid as possible from the mixture in the beaker (DO
NOT REMOVE FOOD MATERIAL IF POSSIBLE). Discard this liquid in a waste beaker. Repeat with parts of methanol
until the food sample seems dry.
WARNING: Petroleum Ether is very dangerous, keep covered at all times, use
only in well ventilated areas (FUMEHOOD BEST OPTION)
Add enough petroleum ether to the food sample to cover it and stir for about two minutes. Allow the solids to
settle. Use a clean thin-stemmed glass pipet to remove as much liquid as possible; save this liquid in small glass
bottle available that can be sealed. Repeat this process with more petroleum ether and continue to collect the
transparent color liquid in the sealed glass bottle.
Add about 3 mL of distilled water to the petroleum ether extract. Mix gently with the lid on or with the pipet
(drawing the liquid from the tube into the pipet, expel it, and draw it up again several times) to mix the petroleum
ether and water as thoroughly as possible. Allow the water and petroleum ether layers to separate and then
carefully remove the water by drawing it up with the thin-stem pipet. Discard this water into the waste beaker.
Add a small amount (a few crystals) of solid anhydrous (“without water”) sodium sulfate to the petroleum ether
extract in the sealed glass bottle. The solid will absorb any water that is left in the petroleum ether and give you a
“dry” sample. (Remember that “dry”, in this case, means “no water”. The sample will still be liquid, since most of
it is the liquid solvent, petroleum ether.) This colored petroleum ether solution that has been washed and dried is
your sample for chromatography and/or spectrophotometric analysis.
CHROMATOGRAPHING YOUR CAROTENOID EXTRACTS – Only for spinach sample
If the sample is yellow or yellow-orange it does not have to be chromatographed to separate the carotenoids.
Yellow samples may be directly analyzed spectrophotometrically without chromatographing them. A group may
want to chromatograph one yellow sample to compare the behavior yellow sample to the behavior of non-yellow
samples. Keep careful records of the appearance of the column, the effluents from it, and so on, as you do any
chromatographic separations.
If the sample is any other color (Green for the Spinach) it must be chromatographed
[Importance of Time – this procedure must be started and completed without stopping]
You have to carry out the chromatographic procedure without stopping, so you should have all the reagents and
equipment ready before you start.
Obtain the regular glass pipet, pack a piece of cotton inside at the base of the tip of the pipet. Heat some wax
(candle) and seal the tip of the pipet. Go to the fume hood and fill the pipet with petroleum ether (the wax must
prevent it from flowing out of the pipet. Add the Alumina (Aluminum oxide) slowly so it flows down and is stopped
by the cotton plug. The pipet should now have Alumina packed in the pipet surrounded by the ether. This should
be clamped as shown in the diagram below.
Have the colored sample ready, the 10% v/v acetone/ether ready, have a glass bottle with fitted lid, and a waste
beaker.
The column of alumina in the stem is your
chromatographic column.
Unseal the tip of the pipet and allow the petroleum ether to drip
out of the column into a waste beaker. When the liquid level of
ether has dropped enough, add the colored sample slowly to the
column pipet. Do the addition gently as to not disturb the column.
Permit the liquid to continue to drip through the column and
continue adding the colored sample until all the liquid has been
added. You may fill the pipet to the top.
Allow the colored sample to drip out of the pipet into the waste
beaker, until there is room to add the 10% v/v acetone/ether.
Begin adding the acetone/petroleum either eluting solvent. Again,
be gentle, so as not to disturb the column and fill the pipet to the
top. As the eluting solvent flows through the column, you should
observe and record what the column looks like; a yellow or
yellow-orange band forming in the column and moving down (if
carotenoids are present in the sample). What is(are) the color(s)
of any other slower (or faster) moving bands?
Watch this band carefully on all sides of the volume. (It doesn’t
always travel at the same rate on all sides of the column.) As soon
as you see the yellow band near the bottom of the alumina
column, collect the yellow pigment solution in a clean, dry, labeled
glass container that can be sealed. Only collect the yellow
solution, once it appears a different color remove the glass
container, seal the glass container, and replace with the with the waste beaker.
SPECTROPHOTOMETRIC ANALYSIS [All Samples]
The spectrophotometer (Spec – 20) must be turned on and warm up for 20+ minutes. You will need the
appropriate blank for the analysis (Petroleum ether (non-columned chromatographic or 10% acetone/ether for
columned chromatographic). You will also need your colored samples.
The blank is used to calibrate the Spec – 20. Place the appropriate blank solution into a glass curvet and place the
curvet into the Spec – 20 . Be sure to label near the top of the tube and to wipe any finger prints with a Kim-wipe,
so as not to interfere with light passing through near the bottom of the tube. Set the wavelength for 450, and set
the 100% transmittance, now select absorbance (should be zero). This process is telling the machine to read the
glass cuvette and the solution as allowing 100% of the light at the wavelength of 450 nm to pass zero being
absorbed.
Now place the colored sample to be analyzed in a clean, dry cuvette. Be sure to label near the top of the tube and
to wipe any finger prints with a Kim-wipe, so as not to interfere with light passing through near the bottom of the
tube. Measure the Absorbance and record the value.
Return the appropriate blank, but change the wavelength to 510 nm, and calibrate/blank with 100%
transmittance, now select absorbance (should be zero). Place colored solution back into Spec – 20 and measure
the Absorbance and record the value.
If any colored samples have absorbance values that are very high (over 1.00) at either wavelength, you should
dilute them to get readings that are more in the middle of the scale. Dilute using the appropriate solvent (ether or
10% v/v acetone/ether. If any of your samples have very low absorbance values (less than 0.05), consult your
instructor about how to proceed.
Title/Date(s)
Purpose
Drawn Procedure
Data Table(s) Qualitative (draw colors, bands, etc.) and Quantitative (numbers)
Vegetable
Extract Color
Chromatographed? Describe the colors from the process
spinach
Vegetable
spinach
tomato
A510
A450
A510/A450
carotenoid
Class Data: Share on Board
ANALYSIS
You may assume that a yellow or yellow-orange extract contains a
carotenoid. Your spectrophotometric results should help you
Determine which one predominates, assuming that it is
either β-carotene or lycopene. The absorption spectra of β-carotene
and lycopene are shown in figure to the side.
What is the approximate ratio of the absorbancies at 510 and 450 nm for
β-carotene?
What is the approximate ratio of the absorbancies at 510 and 450 nm for
lycopene?
Even if the β-carotene (or lycopene) concentration in a sample is different from that which gave the spectrum in
figure, the ratio of absorbance at these two wavelengths will still be essentially the same as that in the figure. If
the absorbance ratios for the two carotenoids are quite different, you can use the measured ratio for an unknown
sample as a way to identify which carotenoid you have.
Conclusion Statement
Discussion of Theory (can include a discussion of this process, polarity, transmittance vs. absorbance, vitamin A
(how it is synthesized in the body, benefits, dangers, why is it one of the few fat soluble vitamins), etc.)
Sources of Error: Please discuss two possible procedural errors and the effect the error would have on the results,
please be specific.
NO Questions:
EXPLORING FURTHER
Compounds that stick to an alumina column are generally more polar. To remove them requires a more polar
solvent. After the carotenoids have been separated and collected from one of your samples, change the
developing solvent to pure acetone to explore what, if anything, happens to other colored compounds still on the
column. What color(s) are these compounds? What might they be?
There are many green and yellow vegetables you could explore to see whether they contain carotenoids (vitamin A
precursors). Or, you might focus on one type of vegetable, such as squash or tomatoes, to see if all the kinds
available contain carotenoids or whether they seem to be more prevalent in one than another. If you obtain
yellow extracts (or can separate yellow components by chromatograph), you probably should record the entire
absorption spectrum, in the 360 to 580 nm range, to find out whether the spectrum corresponds to β-carotene,
lycopene, or whether it seems to be a different compound altogether. Write a brief description of the exploration
you propose, check it with your instructor for safety and feasibility, and, if approved, carry it out.
“Vitamin A supplements” are available at drug stores and health food stores. You might explore these products to
see whether they might really contain carotenoids (which the body converts to vitamin A). Write up, check, and
carry out your approved exploration.