Spinach Lab
Objective
To determine whether "fresh" packaged spinach, frozen spinach, or canned spinach has higher
levels of pheophytin, indicating that it has been dead longer and the leaves have degraded.
Introduction
In modern America, people are far removed from the food they eat. Fruits and vegetables may
travel hundreds of miles before appearing on supermarket shelves. And the preserving
techniques used on these "fresh" foods are not always good for the buyer. However, preserving
the food by freezing or canning is not necessarily more effective. This procedure examines
spinach (Spinacia oleracea) to determine whether fresh, frozen, or canned spinach has
undergone more degregation of the chlorophyll of the leaves.
The amount of degregation will be determined by chromatography, which divides a mixture
based on the molecular structure of the componants. In this case, the spinach leaves are adhered
to a polar coating on a plate. A non-polar solvent then passes along the plate, carrying with it the
molecules that make up spinach leaves. Those that are non-polar travel the farthest, while polar
molecules are more attracted to the plate and move more slowly. The resulting bands of pigment
identify the molecules that make up the spinach leaves.
The major componant of spinach leaves is chlorophyll, the green pigment that acts in
photosynthesis. It comes in two forms, chlorophyll a (chl a) and chlorophyll b (chl b). When
spinach leaves are removed from the plant and allowed to sit, the chlorophyll loses the
magnesium ions it normally contains and becomes pheophytin a and b (phe a and phe b). The
magnesium ions can also be removed in the lab. In this experiment, each type of spinach is
analyzed through chromatography as it is and with magnesium ions removed. If the two patterns
are the same, that indicates that most of the magnesium ions were already removed and the
spinach degraded before it was purchased. However, if they differ, this may indicate that the
spinach is less degraded. It is hypothesized that fresh spinach leaves will be less degraded than
those which have been frozen or canned and thus that they will have more chlorophyll and less
pheophytin.
Methods
First the fresh spinach was analyzed. Half a gram of spinach was mixed with half a gram of
anhydrous magnesium sulfate (to remove the water from the leaves) and one gram of sand. This
was ground to a fine powder and mixed in a vial with two mililiters of acetone by vigorous
shaking to extract the pigments from the leaves. The mixture was allowed to separate and the
solvent was transfered to another vial, being filtered through a Kimwipe. This was the leaf
extract (e). Three hundred microliters of the leaf extract was transferred to another vial and
mixed with a tenth of a gram of Dowex 50WX8 H+ resin. This was allowed to stand, mixing
occasionally, to remove the magnesium ions from the chlorophyll. Then the solvent was
transferred to another vial, and labelled demetallated leaf extract (d).
The two samples were placed on a thin layer chromatography (TLC) plate, each on a labelled
dot about a centimeter from the bottom of the plate. A third dot was added with beta carotene to
provide a baseline. The dots were allowed to dry and reapplied to provide a dark pigment. Then
the plate was suspended in a TLC chamber which contained approximately half a centimeter of
solvent in the bottom. The plate stood in the solvent for ten minutes, allowing the solvent to
move upwards and separate the pigment. The results were observed in natural light and under
UV light. This procedure was repeated with canned and frozen spinach.
Results
The following table shows the distance each major pigment travelled and the rate at which the
pigment moved up the plate is reported as Rf, which was found by dividing the distance the
pigment moved by the distance the solute front moved.
Fres
he
Fres
hd
Solut Xanthop
e
hyll
Front (cm)
(cm)
Rf
Xanthop
hyll
Chl b Rf
Chl a Rf
Phe
(cm) Chl b (cm) Chl a b
(cm)
Rf
Phe
b
Phe a Rf
(cm) Phe a
7.3
1.5
0.21
2.5
7.3
1.6
0.22
n/a
0.34
3.4
0.47
5
0.68
5.7
0.78
3.4
0.47
5.8
0.79
6.5
0.89
Cann
ed e
Cann
ed d
Froz
en e
Froz
en d
6
0.4
0.07
1
0.17
2.3
0.38
4.4
0.73
5.3
0.88
6
0.7
0.12
1
0.17
2.3
0.38
4.4
0.73
5.3
0.88
6
1.7
0.28
2
0.33
2.8
0.47
3.5
0.58
4.4
0.73
6
1.7
0.28
2.1
0.35
3
0.5
4.1
0.68
4.7
0.78
Discussion
Canned spinach looked exactly the same before and after demetallation. This implies that it
went through a great deal of degredation before being purchased and had entirely demetallated
on its own. The frozen spinach had more variation - the original extract had more bands than the
demetallated one, implying more variation in the molecular compounds of the leaf and possibly
less degradation. The fresh spinach also had variation in the rates of movement of pheophytin a
and b between the original extract and the demetallated extract, so it too was not entirely
degraded before purchasing. The poor results on the fresh spinach plate are probably due to not
using enough dye and make it difficult to compare the fresh and frozen spinach. However, these
preliminary results support the hypothesis to some extent by showing that fresh and frozen
spinach are both less degraded than canned spinach.
The data for the plates under UV light is not pictured above, but the pheophytin fluoresced
while the chlorophyll did not. Perhaps the metal ions prevent the chlorophyll from fluorescing.
The Rf values for beta-carotene are also not shown, because they were all virtually one. It is the
most nonpolar molecule in the spinach and moved the farthest, practically at the rate of the
solute. Pheophytin was next most polar, and chlorophyll was more non-polar, with b being more
polar than a in both types. Xanthophyll was the most polar molecule of all and barely moved off
the initial dot (its Rf values are not shown either). This is clear because the film on the plate is
polar and the solvent is non-polar. The polar molecules are attracted to the film and don't move
much with the solvent; the nonpolar molecules are attracted to the solvent and move far because
the plate does not hold them back. To make the polar molecules travel the farthest, all that
would be necessary would be to make the solvent polar and the film non-polar. This is also why
TLC plates cannot be marked with ink, because ink includes pigment molecules that would be
attracted by the solute and move up the plate, contaminating the sample.