course materials

1. Column and Thin Layer Chromatography Questions
Column and Thin Layer Chromatography QuestionsColumn and Thin Layer
Chromatography QuestionsRunning head: TLC AND COLUMN CHROMATOGRAPHY LAB
REPORT TLC and Column Chromatography Lab Report Roberto Reinoso Florida National
University 1 TLC AND COLUMN CHROMATOGRAPHY LAB REPORT 2 Introduction Thin
layer chromatography (TLC) is a method used to analyze a mixture of compounds,
especially pigments, by separating the compounds on a sheet of inert substance such as
glass, silica gel, or aluminum foil. Compounds will be separated according to the overall
hydrophobicity of each individual compound: attraction to the stationary phase and
differential solubility in the mobile phase. Column chromatography is also another method
used to separate substances in a solid immobilized stationary phase embedded in a
cylindrical glass or plastic tube. In this method, substances are separated based on
differential adsorption to the adsorbent. In this experiment, we are going to use TLC and
column chromatography to separate, visualize, and characterize various pigments present
in spinach leaves. Method A. Extraction of Pigments 1. Put some frozen spinach leaves on a
paper towel and use it to squeeze water from the leaves as much as possible. 2. Weigh out
around 4 grams of dried spinach leaves and put them in a mortar. 3. To assist the grounding
process, put around 2 grams of sand into the mortar alongside the leaves sample. 4. Weigh
out around 6 grams of anhydrous magnesium sulfate and add into the mortar. 5. Grind the
leaves until become homogenized. Add more magnesium sulfate, if necessary, and continue
grinding until the consistency becomes like a fine powder. 6. Put the homogenized sample
into an Erlenmeyer flask. Add 15 mL of acetone inside the Erlenmeyer flask and pour the
extracted pigment solution into a new beaker. Carefully pour it to prevent the solid
precipitate from getting into the beaker. 7. Inside a fume hood, use a gentle stream of
compressed air to remove the solvent from the pigment solution. B. Column
Chromatography 1. Shove a tiny piece of cotton wool into a glass pipette until it gets down
to the tip. Column and Thin Layer Chromatography QuestionsORDER NOW FOR
CUSTOMIZED, PLAGIARISM-FREE PAPERSClamp the glass pipette and put a funnel on top of
it. 2. Add 2 – 3 mL of sand into the pipette to the stationary phase. Subsequently add silica
gel and fill in the glass pipette by 6 cm in height and use sand to cover the surface of the
silica gel. TLC AND COLUMN CHROMATOGRAPHY LAB REPORT 3 3. Pipette hexane into the
column and keep adding it until the hexane flows out from the column. 4. Add 0.5 mL of
hexane into the crude pigment solution and swirl the mixture until it gets homogenized. 5.
Remove the excess hexane using compressed air and subsequently pipette the pigment

2. solution into the column. Use compressed air to gently push the pigment solution into the
column. 6. Start eluting the column with 90/10 solution of hexane and acetone. Notice the
separation of pigments. 7. Use different test tubes to capture the separated pigments. 8.
Finally, use pure acetone to elute the remaining pigments inside the column. C. Thin Layer
Chromatography 1. To make a micro spotter, put the center of a capillary tube on the hot
part of a Bunsen’s flame, remove the capillary from the flame before the capillary is pulling
apart, and break the capillary tube. 2. Prepare a TLC plate. On the side of TLC plate with
silica gel, apply the hexane-pigment solution on the baseline using the micro spotter. Spot
the pigment two or three times on the plate to make sure that the spot is enough for
visualization of the separated pigments. 3. Fold a filter paper and stick it into a beaker glass
and then saturate the paper with hexane. Keep adding hexane into this developing chamber
until a shallow level of hexane is achieved. 4. Prepare another developing chamber with
different solvents including 70:30 hexane to acetone and 50:50 hexane to acetone solutions.
Cover all developing chambers with a glass lid. 5. Column and Thin Layer Chromatography
QuestionsPut the TLC plates into each of the developing chamber. Wait until the solvent is
running up to the end line. 6. Finally spot the separated pigments from the column
chromatography on to the TLC plate and run it inside the developing chamber. TLC AND
COLUMN CHROMATOGRAPHY LAB REPORT 4 Reference: –
https://www.youtube.com/watch?v=R491QOnkmCs –
https://www.youtube.com/watch?v=8Ff1Xx-On-g – https://youtu.be/EErXChi-fvk –
https://youtu.be/aZnLYGDJiZE – https://youtu.be/wTlT7RifcPE -The documents provided
under the Column and TLC Chromatography Reading material. Principles of
chromatography Let’s first familiarize ourselves with some terms that are commonly used
in the context of chromatography: Term Definition Mobile phase or carrier Stationary phase
or adsorbent Eluent Eluate Elution solvent moving through the column substance that stays
fixed inside the column fluid entering the column fluid exiting the column (that is collected
in flasks) the process of washing out a compound through a column using a suitable solvent
Term Definition Analyte mixture whose individual components have to be separated and
analyzed Now let’s try to understand the principle of chromatography. Let us draw a
pictorial representation of a column chromatographic separation set up. As depicted above,
the analyte is loaded over the silica bed (packed in the column) and allowed to adhere to the
silica. Here, silica acts as the stationary phase. Solvent (mobile phase) is then made to flow
through the silica bed (under gravity or pressure). The different components of the analyte
exhibit varying degrees of adhesion to the silica (see later), and as a result they travel at
different speeds through the stationary phase as the solvent flows through it, indicated by
the separation of the different bands. The components that adhere more strongly to the
stationary phase travel more slowly compared to those with a weaker adhesion. Analytical
chromatography can be used to purify compounds ranging from milligram to gram scale.
Principle of separation of different components: Differential affinities (strength of adhesion)
of the various components of the analyte towards the stationary and mobile phase results in
the differential separation of the components. Affinity, in turn, is dictated by two properties
of the molecule: ‘Adsorption’ and ‘Solubility’. We can define adsorption as the property of
how well a component of the mixture sticks to the stationary phase, while solubility is the

3. property of how well a component of the mixture dissolves in the mobile • phase. • Column
and Thin Layer Chromatography Questionswill move through the column. Higher the
adsorption to the stationary phase, the slower the molecule Higher the solubility in the
mobile phase, the faster the molecule will move through the column. So, the interplay
between the above two factors determines the differential rates at which the different
components of the analyte will move through the column. Adsorption and solubility of a
molecule can be manipulated by choosing the appropriate stationary phase and mobile
phase. Now, the question arises why do different compounds possess different affinities
towards the stationary and mobile phases? “Polarity” of the compounds dictates their
affinities towards the stationary and mobile phases. Let’s understand this through an
example. Suppose we have a mixture of two molecules A and B, where ‘A’ is a protein and ‘B’
is a lipid. Our column is packed with silica, which is polar in nature; our mobile phase is
hexane, which is non-polar in nature. What do you think will happen when we load this
mixture of A and B onto this column? ‘A’, being polar in nature, will adsorb on to the polar
stationary phase (silica). ‘B’ being non-polar in nature, will readily dissolve in the nonpolar
mobile phase (hexane) without adhering to silica, and will thus elute out of the column with
hexane. Once B is eluted out, the mobile phase will be changed to something polar like
acetonitrile. By doing so we will now force A to detach from the silica and dissolve in the
polar solvent, acetonitrile, and get eluted out of the column with acetonitrile. This is
illustrated in the diagram below. Dyes in Spinach The green in spinach is mainly due to
chlorophyll a and chlorophyll b. Absorption Spectra of Chlorophylls a and b chlorophyll a, R
= CH3 chlorophyll b, R = CHO N Absorbance R N Mg N N O O O O O Wavelength / nm
Pheophytin a and pheophytin b are decomposition products in which chlorophyll a and b
have lost their magnesium ions. The yellow dyes in spinach include beta carotene and
xanthophylls. beta carotene Xanthophylls are derivatives of beta carotene that contain
oxygen. The xanthophyll in Spinach is about 96% lutein and 4% zeaxanthin. OH HO lutein
OH HO zeaxanthin (Differs from lutein in the position of a double bond in the ring on the
right.) A typical separation of dyes in spinach looks like this: sf Solvent front ß-c ß-carotene
chl a pheophytin a pheophytin b chlorophyll a chlorophyll b lutein chl b lut fresh spinach
frozen spinach Troy University – Column and Thin Layer Chromatography
QuestionsDepartment of Chemistry Separation of Plant Pigments by Thin Layer
Chromatography From: Quach, H. T.; Steeper, R L.; Griffin, G. W. J. Chem. Educ. 2004, 81,
385-7. Introduction The principal pigment in higher plants is chlorophyll a. Chlorophyll b,
carotenes and xanthophylls play a secondary role by transferring the energy they absorb to
chlorophyll a for use in photosynthesis. In the following experiment, the pigments found in
the leaves of spinach (Spinacia oleracea) or kale (Brassica oleracea acephala) are separated
by means of thin layer chromatography. Chromatography is an analytical method
permitting the separation of a mixture into its molecular components. The basic principle
upon which it works is that a mixture first adheres to the dry chromatography plate. A
developer or solvent is then passed through the coating on the plate in a fixed direction
moving the pigment molecules of the mixture along at different rates. The greater the
attraction between the molecules and the absorbing medium (coated on the plate), the
slower the molecules ascend the coating. The greater the solubility of the components in the

4. developer, the greater the distance the molecules move. The developer used in this
experiment contains a mixture of nonpolar solvents. No open flames solvents are extremely
flammable. The solvent molecules contain non-polar covalent bonds and any net charges
are equally distributed within the molecules. (Water, a common solvent, shares electrons
unequally such that the oxygen end is negative and the hydrogen ends are positive. Such
molecules are called polar molecules.) As the nonpolar solvents move up the
chromatography plate, the pigments move along with it. The more nonpolar the pigment,
the more soluble it is in the nonpolar organic solvents, the faster it will move and the
greater distance it will proceed up the film. Chromatography was discovered by M. Tswett
in 1906. He dissolved a mixture of plant pigments in petroleum ether and passed the
solution through a column of calcium carbonate. Yellow and green zones always formed in
the same relative positions in the column. . Experimental Procedure 1. On a balance weigh
out 0.5 grams of fresh spinach and combine with 0.5 grams of anhydrous magnesium sulfate
and 1.0 grams of sand. Transfer these materials to a mortar and using a pestle grind the
mixture until a fine dry powder is obtained (grind the mixture really well). The anhydrous
magnesium sulfate will remove the water from the leaves. 2. Transfer the powder (2.0
grams total) to a small test tube and combine with 2.0 mL of acetone. Stopper the test tube
and shake vigorously for approximately one minute. You need to make sure that the solid
and solvent are well mixed. 3. Allow this mixture to stand for 10 minutes, then using a pipet
carefully transfer the solvent above the solid into a small microcentrifuge tube. Use care not
to transfer any of the solid material. Column and Thin Layer Chromatography QuestionsThe
solvent extract should be green. Cap the microcentrifuge tube to minimize solvent
evaporation. 4. Repeat the first three steps with a sample of frozen spinach. Repeat yet
again with a sample of canned spinach. 5. Obtain a TLC chamber (a 400 mL glass beaker
covered with parafilm or aluminum foil) and add developing solvent (a mixture of
petroleum ether, acetone, cyclohexane, ethyl acetate and methanol). The solvent should
completely cover the bottom of the chamber to Troy University – Department of Chemistry
a depth of approximately 0.5 cm. Keep the chamber covered so that evaporation doesn’t
change the composition of the solvent. 6. Obtain a TLC plate (a silica gel coated plastic
sheet) which has been precut (5.5 x 9.0 cm) and make four dots equal distance apart with a
pencil (why not an ink pen?) on the coated side approximately 1.0cm from the bottom of the
strip. The dots should be parallel with the bottom of the strip. Label the first dot with the
letter “e” (leaf extract), the second dot “f” (frozen spinach), the third dot “c” (canned
spinach), and the last dot “b” (beta-carotene). 7. Fill a capillary tube (TLC applicator) by
placing it in the leaf extract (it will fill by capillary action) Keep your finger off the end of the
capillary tube. Apply the extract to the center of the first dot (e) on the TLC plate by quickly
touching the end of the TLC applicator to the plate. Allow to dry (you can gently blow on the
strip). Repeat several times to make a concentrated dot of extract. Be sure to let dry
between applications. In a similar manner, place an additional dot on each side of the first
dot to make a short line of extract. Try to make the spots as small as possible but dark
enough to see the color clearly. 8. Repeat steps 5 & 6 with the extracts of frozen spinach,
canned spinach, and betacarotene. Place on appropriate pencil mark. You will be able to
determine the positions of the pure pigments (or, at the beta-carotene) in your leaf

5. separation by comparing them to the positions of the pure extracts. 9. Carefully place the
TLC plate in the TLC chamber. The TLC plate should sit on the bottom of the chamber and be
in contact with the solvent (solvent surface must be below the extract dots). Cover the TLC
chamber. 10. Allow the TLC plate to develop (separation of pigments) for approximately 10
minutes. As the solvent moves up the TLC plate you should see the different colored
pigments separating Column and Thin Layer Chromatography Questions