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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
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
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
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.
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
Ettre, L. S.; Sakodynskii, K. I. (March 1993). "M. S. Tswett and the discovery of
chromatography II: Completion of the development of chromatography (1903–
1910)". Chromatographia. 35 (5-6): 329–338. doi:10.1007/BF02277520.
W. C.; Kahn, M.; Mitra, A. (1978). "Rapid chromatographic technique for preparative
separations with moderate resolution". J. Org. Chem. 43 (14): 2923–
2925. doi:10.1021/jo00408a041.
Anfinsen, Christian B.; Edsall, John Tileston; Richards, Frederic Middlebrook, eds.
(1976). Advances in Protein Chemistry. pp. 6