Jazer John T.

Lirazan
March 12, 2017

Chromatography
I. Results
Table 1: Separation of Plant pigment through paper chromatography.
Sample: Crotons (Codiaeum variegatum)
Distance travelled by the solvent: 74.5 mm 1st try, 70 mm 2nd try

Solvent 9:1 (v/v) pet-ether-acetone 9:1:1 (v/v/v) pet-diethyl-ether-acetone

system
Spot. No. X (mm) Rf Color X (mm) Rf Color
1 38.5 0.52 Light 1 0.71 Light
yellow yellow
2 22 0.30 Light 2 0.37 Light green
Green
3 15 0.20 Olive green 3 0.2 Olive green
Sketch of chromatogram:
Table 2: Analysis of the component of dyes of black ink TLC
Sample: Pilot G-tech (0.4mm)
Developing Solvent : Petroleum ether-diethylether-acetone
Distance travelled by the solvent (Y), mm: 40 mm

Spot No. X (mm) Color Rf

1 25.9 Yellow orange 0.65
2 22 Light green 0.55
3 19 Dark pink 0.48
4 17 Dark green 0.43
5 7.5 black 0.19

A. Sketch Chromatogram
Table 3: identification of Amino acids by Paper Chromatography
Solvent system: 1:2 (v/v) ammonium hydroxide-isopropyl alcohol
Visualization method: Application of Ninhydrin
Distance Travelled by Solvent (Y), mm: 73 mm

Phenylalanine Tyrosine (T) Aspartic Acid Unknown (U)

(P) (A)

Trial 1 Trial 2 Trial 1 Trial 2 Trial 1 Trial 2 Trial Trial 2 Trial Trial
1 1 2

X 61 62 48 mm 48 mm 26 mm 26 mm 62 62 mm 26 26
mm mm mm mm mm

RF 0.84 0.85 0.66 0.66 0.36 0.36 0.84 0.85 0.36 0.36

Color Dark Dark purple purple Blue Blue Dark Dark Blue Blue
blue blue blue blue

Ave. 0.85 0.85 0.66 0.66 0.36 0.36 0.85 0.85 0.36 0.36
RF

Unknown is: Phenylalanine and Aspartic acid

Sketch:
 II. Discussion
Chromatography is a laboratory technique for the separation of a mixture. The mixture is
dissolved in a fluid called the mobile phase, which carries it through a structure holding another
material called the stationary phase. The various constituents of the mixture travel at different
speeds, causing them to separate. The separation is based on differential partitioning between the
mobile and stationary phases. Subtle differences in a compound's partition coefficient result in
differential retention on the stationary phase and thus changing the separation. Paper
chromatography is an analytical method used to separate colored chemicals or substances. It is
primarily used as a teaching tool, having been replaced by other chromatography methods, such
as thin-layer chromatography.
The first part of the experiment is the separation of plant pigment using paper
chromatography. Students have cut and weighed 2g of Crotons (Codiaeum variegatum) leaves.
The leaves were extracted using 20 ml of acetone, mortar and pestle and was then filter by a soft
cotton. The extract was put inside the vial to avoid the evaporation in air. A capillary tube was
used to spot the bottom of the strip of the chromatographic paper about one centimeter from the
bottom. The process was repeated 5 times, allowing the spot to dry each time. Same procedure
was done in a separate strip of chromatographic paper. Two separate test tubes were filled with 5
ml of 9:1:1 petroleum ether-diethyether-acetone and 5 ml of 9:1 petroleum ether-acetone. The
papers were carefully attached with the spotted side at the far end to the hook mounted on a
stopper. The paper strips were carefully inserted into the test tube, making sure that the spot is
above the level of the solvent. The students observed the set up as the solvent rise with the paper.
the paper was then removed when the solvent is about one inch from the top of the strip. The
solvent front was marked using a pencil and the individual outline was also marked.
The substance visible on paper is called pigments. Chlorophyll a is the main pigment that
makes up about 75% of the pigmentation in plants. Chlorophyll b makes up about 25% of the
pigmentation. The paper will display a spectrum of pigments found in the croton leaves. Using
RF one can determine the relationship between the distance traveled to the distance the pigment
travelled. The higher the RF the more affinity the compound has to the solvent, the lower affinity
the lower RF value. Yellow green has the highest Rf its tells us that it has the it has more affinity
and green has the lowest Rf thus tells us it has the lesser affinity.

Thin Layer Chromatography (TLC) is a solid-liquid technique in which the two phases are a
solid, stationary phase and a liquid, mobile phase. The more polar a molecule, the higher affinity
it will have for the plate and will therefore spend less time in the mobile phase. As a result, it will
move up the plate more slowly. Conversely, a less polar molecule will spend more time in the
mobile phase and will therefore move up the plate more quickly. The speed at which the
molecules will move up the plate thus depends on the relative difference in polarity between the
stationary and mobile phases, and will vary depending on the nature of the stationary and mobile
phases used for separation.

The second part of the experiment is the analysis of the component dyes of black ink by
TLC. First students have filled a capillary tube with ink from a ballpoint pen (G-tech) and spot
on the TLC one inch from the bottom. Students have poured about 20 ml of the solvent system
6:2:2 n-butanol-ethanol-ammonia in a developing chamber. A filter paper was lined at the side
and soaked in a solution for about 15 minutes to allow the system to equilibrate. The TLC plates
were place inside the developing chamber and the student made sure that the spot is above the
level of the solvent system. The camber was covered using a watch glass and the students allow
the setup to equilibrate for about 20 minutes. The students measured the distance travelled by the
solvent and the spot then they analyze all results. RF values has a corresponding color,
Adsorptivity of compounds increases with the increased polarity. The eluting power of
solvents increases with the polarity. Low polarity compounds can be eluted with the low polarity
solvents, while higher polarity compounds require solvents of higher polarity. The stronger the
compound is bound to the adsorbent, the slower it moves up the TLC plate. Non-polar
compounds move up the plate most rapidly thus having higher RF value, whereas polar
substances travel up the TLC plate slowly or not at all means lower RF value. In the experiment
Yellow green has the highest Rf value which is 0.65 this mean it is least polar the the color black
is at the near bottom end of the TLC plate with an Rf value of 0.19 this indicates that it is the
most polar among compounds composing the inc of Gtech pen.
A paper chromatography variant, two-dimensional chromatography involves using two
solvents and rotating the paper 90° in between. This is useful for separating complex mixtures of
compounds having similar polarity, for example, amino acids. The third part of the experiment is
the identification of amino acids by paper chromatography. The students obtain a clean sheet of
Whatman no. 1 filter paper then they have drawn a thin line parallel to the side and also drawn a
eight along the line with equal intervals. The X’s were mark with their corresponding amino
acids and was spot. The paper was then rolled cylindrically in such a way that the labels are
facing and staple the ends. The developing chamber was filled with the solvent system 1:2
ammonium hydroxide-isopropyl alcohol until it is 1 cm deep. The paper was place and observed
until it is developed. When the paper was fully developed it was put in a board and the distance
travelled by the solvent was mark using a pencil. The paper was sprayed with 2 percent
ninhydrin the allows it to evaporate. The results were analyzed and then gathered. The collected
data includes the solvent distance, and spot distance. The data was then used to calculate the
presented R f results. The results clearly show the relationship of the unknown mixtures to the
known amino acids. Analyzing the color of one of the mixture compared to the known amino
acids. The findings of this experiment in terms of spots color and RF results shows that the
unknown contains two types of amino acids which is Phenylalanine and Aspartic acid.

Answers to question:
1.
A. If the solvent level in the developing chamber is higher than the spotted sample, the
spotted sample will no longer run and separate in the chromatographic paper, but
rather it will dissolve the solvent. This will not show any chromatograms.

B. If too much sample is applied to the paper, the paper will derive scattered
chromatograms- making it more difficult to identify the components of Rf values.

C. If the paper is allowed to remain in the chamber after the solvent front has reached the
top of the plate, the components will bunch on the top, and in some cases, the
 components disappear because of the components being crowded on top: the Rf
values will also not be computed.

2. It is necessary to cover the developing chamber tightly during the development of a

chromatogram because if solvent will left opened, the solvent will evaporate along the
whole length of the sample. Due to this situation, Rf values will be inaccurate. Covering
the chamber will isolate the solvent inside.
3. Both TLC and paper chromatography are not methods to separate and identify very
volatile substances. It is because of the fact that volatile substance contains volatile
components that will volatilize into the air. Separating these components that volatilize
into the air. Separating these components require high performance liquid or gas
chromatography.
4. We are required to handle the chromatographic paper only at its conrners in part C
because the hands might contain other components that will react with the solvent and
disturb the chromatographic expected outcome.
5. The unknowns are Phenylalanine and Aspartic acid. It is possible to identify through
clear observation on the Rf values. The unknowns Rf values had a close resemblance to
the standard amino acids.

III. Conclusion
The students have successfully learned the techniques of paper chromatography and thin
layer chromatography. They have learned that proper solvents and materials used. They have
learned that the solvent level in the developing chamber should not be higher than the spotted
sample. Students should not apply too much sample. The paper should not be allowed to remain
in the chamber after the solvent front has reached the top of the plate. It is also necessary to
cover the developing chamber during the development of chromatogram.
The students also apply chromatographic methods in the separation of the components in the
mixture. Students have read the MSDS and the lab procedure carefully and they are able to apply
these methods and precautions during the experiment.
By computing Rf values of the amino acids and comparing it with the unknown the students
were able to identify the unknown. Colors also have contributed with the identification of the
unknown with the standard.
 IV. References

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