LAB 1: CHROMATOGRAPHY OF SPINACH
Thin Layer and Column Chromatography
PURPOSE: To separate the pigments of spinach by Column Chromatography.
To analyze Column Chromatography fractions by Thin Layer Chromatography.
SAFETY CONCERNS:
Always wear safety goggles.
Handle and dispose of broken glass safely.
Avoid inhalation of solvent fumes. Acetone and Hexane may be harmful for pregnant women.
Acetone and Hexane are flammable so do not use them near open flames.
READING:
Mohrig, et al; Techiniques in Organic Chemistry; W.H. Freeman and Co.;
 TLC; introduction, preparation & development, visualization techniques, Choosing a Developing
Solvent
 Liquid Chromatography (introduction, preparation & development; Sources of Confusion)
CHROMATOGRAPHY:
Introduction:
Most samples of matter are impure mixtures of two or more substances. Chromatography is a widely used
experimental technique for the separation of a mixture of compounds into its individual components. The word
chromatography means "separation of colors" but today chromato-graphy is used for both colored and colorless
substances.
The separation process is based on the fact that porous solids adsorbs different substances to different extremes
depending upon their polarity. The term “Adsorption” refers to the adhesion or stickyness of a substance to the
surface of another substance, as opposed to the term “absorption” which refers to a substance penetrating into the
inner structure of another substance.
A mixture to be separated is first applied to an immovable porous solid (like paper, or alumina, or fine silica sand)
called the stationary phase. The components of the mixture then get “washed” along the porous solid by the flow
of a solvent called the mobile phase. The mobile phase can be liquid (as in column, paper, or thin-layer
chromatography) or it can be a gas (as in gas chromatography).
Each component of a mixture to be separated will be attracted differently to the porous stationary phase depending
on its polarity and the polarity of the stationary phase chosen. Remember that “Like attracts Like”. If the stationary
phase is polar then polar components will be attracted or stick more to it but non-polar components will move
across the surface easily. If the stationary phase is nonpolar then nonpolar components will be more attracted to it
and the polar compounds will move along more quickly.
Likewise, if the mobile phase or solvent that is washing over the components of a mixture is polar then it will attract
polar components of the mixture and carry them along easily, leaving the nonpolar components behind or moving
slow. A non-polar solvent will attract and carry along the non-polar components of a mixture but leave the polar
substances behind or moving slow. As the mobile phase (solvent) moves through the porous stationary phase by
capillary action, it "pulls" along the molecules of the mixture to be separated at different rates. Because of the
different polarities of the molecules the components have different attractions to the mobile and to the stationary
phases, and therefore do not travel at the same speed through the stationary phase. This leads to a separation of the
various molecules.
The simplest types of chromatography, paper and thin-layer, will be used in this experiment. Other
chromatographic methods, including column chromatography, gas chromatography (GC), and high performance
liquid chromatography (HPLC) are used extensively in chemistry and related fields such as medicine.
In medicine, chromatography is used to separate and identify amino acids and proteins in mixtures. Chromatograms
of blood samples will sometimes reveal the presence of foreign proteins associated with certain diseases. Law
enforcement agencies sometimes require chromatographic analysis of urine specimens from suspected drug addicts.
CH242 Lab 1: Chromatography of Spinach (W15)
1
Thin-Layer Chromatography:
In Thin-layer chromatography the stationary phase is a thin coating of adsorbent material, called the sorbent, on a
sheet of glass, plastic, or metal. The TLC sheet is suspended vertically in an eluent and the eluent travels up the
sheet.
Common sorbents include
 silica (SiO2, very pure, finely ground sand),
 alumina (Al2O3, also used in abrasives, ceramic materials, and dental cement),
 cellulose (similar to very pure, finely ground wood fibers).
In order to separate the substances of a mixture, the substances must have different R f values. By carefully choosing
an eluent and a sorbent, it is usually possible to find a combination that will separate the mixture.
A tiny drop of the mixture to be separated is placed on the plate near the bottom of the paper. A lightly drawn pencil
line marks the location of the spot. This location is called the origin. The paper is suspended vertically in the
mobile phase, a solvent or eluent. The eluent could be water or alcohol, or a solvent solution made form several
reagents whose proportions are chosen to enhance their ability to "pull" along some substances in the mixture being
separated better than others. We want each chemical in our mixture to have different attractions to the solvent so
that they will travel at different speeds and be separated.
The origin must be above the surface of the eluent. The eluent rises up the paper by capillary action. When the
eluent reaches the origin, the components of the mixture rise at different rates. The container must be covered to
prevent evaporation of eluent. The chromatogram must be removed from the eluent before the eluent reaches the
top of the paper.
As the substances in the mixture rise up the paper, they spread out and the spots become larger. For this reason, the
original spot should be as small as possible, less than 5 mm in diameter. If too much material is applied to the small
spot, the spot may develop a long "tail." If too little material is applied to the spot, the color of the spot may be too
faint to see as the spot enlarges while moving up the paper. Trial-and-error and experience help the experimenter
obtain both a small spot and one with the proper amount of material.
Substances can be identified by the heights they reach on the completed chromatogram by calculating Rf (rate of
flow or retention factor) values. The Rf value is a constant for a given substance under the same experimental
conditions. The Rf value may be calculated from the following equation. The Rf value itself is unitless.
Rf =
Distance of the center of the sample spot from the origin
Distance of the solvent front from the origin
Figure 6.1 shows the finished chromatogram of substance A, substance B, and a mixture containing substances A
and B. To determine the distance traveled by each component measure the distance from the origin to the center of
the migrated spot. If the spot is large with a "tail," measure to the "center of gravity" or densest concentration of the
spot.
Rf (substance A) =
3.1 cm = 0.28
11.2 cm
Rf (substance B) = 8.5 cm = 0.76
11.2 cm
Solvent front
11.2
8.5
3.1
Origin
(With original
sample spots)
2
A
B
&
A&B
&
A
B
&
A&B
&
Figure 6.1 Typical finished chromatogram of two substances.
CH242 Lab 1: Chromatography of Spinach (W15)
Once the Rf value is known, the substance can sometimes be identified by comparing its R f value with those
reported in the literature. To check the identity of an unknown substance, it is usually necessary to run a
chromatogram of a known sample simultaneously with the unknown.
Paper Chromatography:
Paper chromatography is almost identical to thin layer chromatography however the stationary phase is a sheet of
absorbent paper, such as filter paper. TLC offers advantages over paper chromatography in that TLC can be
performed with a variety of sorbents and gives better separation of mixtures with less spreading of the spots
Liquid Column Chromatography:
Column chromatography is similar to thin layer chromatography however the stationary phase is a column of
adsorbent rather than a layer stuck to a backing. The advantage of column chromatography over paper or TLC is
that it can be performed on a larger scale for the purpose of purifying samples.
Vegetable Pigments:
Deeply colored vegetables such as spinach contain a mixture of pigments including

Carotenes (1 spot) (yellow-orange)
 Pheophytin a (gray, may be nearly as intense as chlorophyll b)
 Pheophytin b (gray, may not be visible)
 Chlorophyll-a (blue-green, more intense than chlorophyll b)
 Chlorophyll-b (green)
 Xanthophylls (possibly 3 yellow spots)
When exposed to air the chlorophyll pigments are slowly oxidized to form brown-colored products. The
pigments are nonpolar and do not dissolve in water, a highly polar solvent; that's why grass stains are so
difficult to launder from clothing. The pigments do dissolve in acetone, a common solvent found in
fingernail polish remover.
IN THIS EXPERIMENT:
In this experiment we will isolate the pigments of spinach by column chromatography and then analyze
the chromatography fractions by Thin Layer Chromatography.
The eluents for the chromatography of these pigments will be variations of hexane, acetone, and methanol
with increasing polarity.
(Traditionally a solvent called Ligroin was used instead of hexane. Ligroin is a non-polar solvent similar
to gasoline, mineral spirits, or painter's naphtha--it is a mixture of hydrocarbons with a boiling point range
of 60-90°C sometimes called petroleum ether. A 2:1 ligroin-acetone eluent mixture has polarity that gives
a good separation of the spinach pigments and could be used as an alternative.)
The eluent mixture must be free of water--one drop of water would considerably change the polarity of the mixture.
Resources: Pavia; Introduction to Organic Laboratory Techniques
CH242 Lab 1: Chromatography of Spinach (W15)
3
NOTES:
PROCEDURES:
ACTIONS:
1
I. ISOLATION OF SPINACH PIGMENTS:
A. Extraction of pigments
The crushing helps break
cell walls and free the
pigments from the cells.
1. Use a mortar and pestle to grind about 0.5 grams of cut or torn
fresh1 (or 0.25g frozen)2 spinach leaves (avoid stems or thick
veins) with 1 mL acetone3 until the leaves have broken into
particles too small to be clearly seen. 4
2
2. With a Pasteur pipet, transfer the mixture to a centrifuge tube.
3
3. Rinse the mortar and pestle with 1.0 mL cold acetone and add it
to the remaining mixture in the centrifuge tube.
4. Centrifuge the mixture (be sure to balance the centrifuge) to allow
the sediment to fall to the bottom. Using a Pasteur pipet,
transfer the liquid extract to another centrifuge tube with a tight
fitting cap.
5. Add 2.0 mL of hexane to the extract. Cap and shake to mix.
6. Add 2.0 mL of water and shake thoroughly with occasional
venting.
7. Centrifuge the mixture to break the emulsion.5 Remove the
bottom aqueous layer with a Pasteur pipet and discard.
8. Dry the remaining hexane layer (which contains the dissolved
pigments) by filtering it through a column6 containing anhydrous
sodium sulfate7 into a dry test tube labeled “E” for extract.
Fresh leaves are preferred
but if frozen spinach must be
used then thoroughly dry
thawed leaves by pressing
between paper towels.
CAUTION: The solvents,
Acetone and Hexane, used in
this
experiment
are
flammable.
No flames
should be present!
4
If too much acetone has
evaporated add an additional
0.5-1.0 mLs.
5
The
emulsion
usually
appears as a cloudy green
layer in the middle of the
mixture.
6
Into a 5 ¾ inch Pasteur
pipet put a plug of cotton and
tamp it into position using a
glass rod. Add about 0.5 g
of powdered or granular
anhydrous sodium sulfate,
and tap the column with your
finger to pack the material.
Clamp the column vertical.
Column for drying extract.
9. Flush the drying column with an additional 0.5 ml hexane to
extract all the pigments from the drying agent.
0.5g Anhydrous sodium sulfate
Cotton
10. Evaporate the solvent by placing the test tube (E) in a warm
water bath (40-60oC) in a hood. Redissove the residue in 0.5
mL hexane.
11. Stopper test tube (E) and set aside in the dark until you are
ready to run the alumina chromatography column.
4
CH242 Lab 1: Chromatography of Spinach (W15)
7
Water will attract to the
anhydrous sodium sulfate and
be pulled away from the
organic solvent.
B. Column Chromatography
1. In preparation have ready:
 4 tubes of chromatography solvents of increasing polarity:
A. 10.0 mL hexane
B. 6.0 mL 70% hexane–30% acetone solution (by volume)
C. 6.0 mL acetone
D. 6.0 mL 80% acetone-20% methanol solution (by

Increasing polarity of solvents
8
volume)
5 dry test tubes for sample collection numbered 1-5.
2. Prepare a chromatography column8 packed with about 1.25 g of
alumina.9
Read and understand the following procedures before continuing. The
procedure takes about 15 minutes. If using a column without a stopcock
then once started it cannot be stopped until all the material is eluted from
the column.
If preparing a column from a
Pasteur pipet push a loose plug
of cotton into the pipet with a
glass rod. Add the measured
amount of alumina while
tapping the column gently with
your finger to reduce air
pockets. Continue tapping to
ensure that the alumina is
tightly packed.
9
10
3. Using a Pasteur pipet, slowly add about 3.0 mL of hexane to
the column and let drain to completely moisten the alumina.
Collect any eluent in test tube #1. Drain the excess hexane until
the level of hexane just reaches the top of the alumina.11 If
necessary, add more hexane.
Use activated alumina from
EM science, No. AX0612-1);
Particle sizes 80-120 mesh;
material Type F-20. Dry
overnight at 110oC and store in
a tightly sealed bottle.
10
4. When the hexane has drained to the top of the column add about
half (0.25 mL) of the dissolved pigments from tube “E”.12
Add the solvent slowly to
avoid disrupting the level
surface of the alumina.
11
5. Continue collecting the eluent in tube 1. Just as the pigment
solution penetrates the top of the column, add 1 mL of hexane
and drain until the surface of the liquid has reached the alumina.
6. Add about 4 mL of hexane. If the yellow band (carotenoid
pigments) begins to separate from the green band (chlorophyll
pigments), continue to add hexane until the yellow band passes
through the column. If the yellow band does not separate from
the green band, change to the next more polar solvent (70%
hexane-30% acetone)13 Collect the yellow band in test tube #2.
The top of the column must
not be allowed to run dry. It
is essential that the liquid
level not be allowed to drain
below the surface of the
alumina at any point during
the procedure.
12
Stopper and save the
remainder of the pigment in a
dark place for use with TLC
later.
13
7. When the yellow band is through and the eluent becomes
colorless again start collecting in tube #3.
8. Add several mLs of the next more polar solvent.13 If the green
band moves down the column continue to add this solvent to
elute the green band.14 Collect the green band in tube #4.
9. When there is little or no green color in the eluent, place test
tube #5 under the column and stop the procedure.
When changing solvents, do
not add the new solvent until
the last solvent has nearly
penetrated the alumina.
14
If the green band does not
move or if a diffuse yellow
band begins to move, change
to the next more polar solvent.
Change solvents again if
necessary.
15
10. Place the carotenoids (tube #2), the chlorophylls (tube #4), and
the remaining extract (tube E) in a warm water bath (40-60oC)
just until15 the solvent has evaporated.
CH242 Lab 1: Chromatography of Spinach (W15)
Do not allow any of the tubes
to continue heating after the
solvent has evaporated.
5
16
Whatman Silica Gel Plates No.
4410 222. Handle carefully by the
edges to avoid flaking. Do not touch
the surface.
C. Thin Layer Chromatography
16
11. Obtain a 10 cm x 3.3 cm silica gel TLC sheet.
12. With a pencil17, draw a faint line (the origin line) lightly &
carefully18 across the bottom of the silica gel sheet about
1-1.5 cm from the bottom edge.
13. Carefully18 keeping at least 0.6 cm from the edges make 3
small vertical pencil marks on the origin line as shown19
to indicate the place where the spinach pigments will be
applied.
12. Redissolve the pigments in tubes 2, 4, & E by adding two
drops of 70% hexane-30% acetone to each of the tubes.
13. Dip a clean capillary tube into each sample tube. Apply
each pigment to the corresponding mark at the origin of
the silica gel TLC sheet by quickly touching the capillary
tube to the sheet. Hold the capillary tube at right angles to
the sheet. Do not scrape off the adsorbent with the
capillary tube. Allow each spot to dry20 for 30 seconds
and repeat the application about 15 times or until dark.21
17
Do not use a pen as the ink may run
in the chromatography solvent.
Pencil “lead” is graphite, a form of
carbon and will not dissolve or run in
the organic solvents
used.
18
Make your pencil
marks very carefully
so as not to flake off
the silica adsorbent.
19
Indicate origin spots
for tubes 2, 4, & E.
20
A drying period is
necessary to ensure
small spots.
2 4 E
The
repeated
&&
application is necessary to ensure
B
sufficient material is applied.
21
&
22
14. Prepare a dry22 chromatography chamber with 70%
hexane-30% acetone eluent mixture.
The hexane-acetone mixture must
stay dry--we don't want water added
to the mixture.
23
15. Lower the TLC sheet into the test tube making sure that
the origin on the TLC sheet stays above the surface of the
hexane-acetone eluent mixture and keeping the plate from
coming in contact with the filter paper liner.
16. Cover the TLC chamber and allow it to sit undisturbed
until the eluent front is 1-2 cm from the top23 of the TLC
sheet.
17. Use forceps to remove the TLC plate and with pencil
immediately mark the position of the solvent front.24
Do not allow the eluent to reach the
top of the sheet.
24
You must draw the line quickly
before the eluent disappears. Hexane
and acetone solvent evaporates very
rapidly and soon you will no longer
be able to see the position of the
eluent front.
25
Outline and observe the spots soon
after the plates have dried as some
pigments may change color when
exposed to air & light.
26
18. As soon as the plate is dry lightly outline all visible spots
with a pencil and make note of the colors. 25
19. Draw a diagram of the chromatogram but tape26
original in your notebook as well.
the
20. Label all spots as A, B, C, D, etc., calculate their Rf
values, and identify the pigments by their colors.27
21. Disposal: Place the excess Hexane-acetone in the "Waste
Organic Solvent" container.
6
Attach the chromatogram to paper
by completely covering it with
transparent tape to prevent the silica
from flaking off.
27
In the crude extract the following
components should be visible (in
order of increasing Rf values).
Carotenes (1 spot) (yellow-orange)
Pheophytin a (gray, may be nearly as
intense as chlorophyll b)
Pheophytin b (gray, may not be visible)
Chlorophyll-a (blue-green, more intense
than chlorophyll b)
Chlorophyll-b (green)
Xanthophylls (possibly 3 yellow spots)
CH242 Lab 1: Chromatography of Spinach (W15)
LAB 1: CHROMATOGRAPHY
NAME_____________
DATE______________
PRE LAB EXERCISES:
1. Rank the following solvents in order of increasing polarity (lease polar to most polar).
____ Ethanol
____75% Ethyl acetate/ 25% hexane
____ Ethyl acetate
____ hexane
____ water
____50% Ethyl acetate/ 50% hexane
____70% acetone/ 30% hexane
____acetone
____30% acetone/ 70% hexane
____Dichloromethane
2. Match the following terms with the phrase that best describes it:
1.___
2.___
3.___
4.___
5.___
6.___
7.___
Sorbent
Eluent
chlorophyll-b
Eluent front
Origin
Adsorption
Absorption
D. line marking the placement of a mixture on a chromatogram.
E. solvent used as a mobile phase
F. the final edge of the mobile phase after development of a chromatogram.
G. adhesion of a substance to the surface of the stationary phase
H. penetration of one substance into the inner structure of another.
I. thin coating of porous material used as a stationary phase.
J. yellow-green
3.___ What safety precautions are necessary when using acetone and hexane (or ligroin)?
A. Avoid flames as these solvents are flammable.
C. Both A and B.
B. Avoid inhaling the solvents as they are hazardous.
4.___ Why is a pencil used to mark the origin line and not a ball-point or ink pen?
A.
B.
C.
D.
E.
5.
6.
Pencils are more dependable since they never run out of ink.
Ink could dissolve in the eluent and rise up chromatogram.
Pencils are less likely to flake off the sorbent.
Ink is harder to erase if you make a mistake in the labeling.
More than one of these.
Point out two errors that would prevent an accurate Rf determination in the TLC setup illustrated below:
Calculate the Rf value for substances A and B on the chromatogram
shown at the right. Show your calculations:
A
B
A&B
&
7.
About 0.5 g of a sample mixture is to be separated on a silica chromatography column. How much silica should
be used to pack the column?
CH242 Lab 1: Chromatography of Spinach (W15)
7
8.
Once the adsorbent is packed in the column, it is important that the level of the elution solvent not drop below
the top of the adsorbent. Why?
9.
What precautions must be taken when you introduce the mixture of compounds to be separated onto the
adsorbent column? Why?
10.
8
What effect will the following factors have on a chromatographic separation:
a. Too strong an adsorbent
b.
Collection of large elution fractions
c.
Solvent level below the top of column adsorbent
CH242 Lab 1: Chromatography of Spinach (W15)