Araniador, Glaicyl Dame Ann B.

March 3, 2016

Date Performed:

Bronzal, Lucile A.

February 29 and

Date Submitted: March 10, 2016

Mameloco, Neri May G.

EXPERIMENT NO. 6
CHROMATOGRAPHY
I. INTRODUCTION
Chromatography is one of the several techniques, where a chemical mixture is
carried by a liquid or gas, which serves as the mobile phase, is separated into
components as a result of differential distribution of the solutes as they flow around
or over a stationary phase, which may be a liquid or solid.
It has numerous applications in biological and chemical fields. It has also a
number of advantages compared to other techniques like crystallization, solvent
extraction and distillation. It is capable of separating all the components of a
multicomponent chemical mixture without requiring a knowledge of the identity of
the substance present. This technique also helps in analyzing, identifying, purifying
and quantifying unknown separable mixtures.
In this experiment, one major type of chromatography known as paper
chromatography will be performed for separating gumamela (Hibiscus) leaf
components. Paper chromatography works mostly on capillary attractions. This
attraction which depends on adhesive and cohesive forces allows the mobile phase
to move up the stationary phase because of the created surface tension interaction
from the forces.
Paper chromatography has a wide application in the analysis of biologically
important compounds, such as amino acids, steroids, carbohydrates and bile
pigments.
Another type of chromatography will be performed in the experiment, which is
the thin layer chromatography. It is a form of solid-liquid absorption
chromatography. It uses a thin layer of absorbent, usually silica gel, which is
supported on flat surface. This type is advantageous in monitoring processes of
reactions, analyzing crude products of unknown mixtures, determining the number
of components of a mixture, and evaluating the efficiency of purification process.
II.

RESULTS AND DISCUSSION

A. Separation of Plant Pigments by Paper Chromatography
Sample: Gumamela (Hibiscus)
Solvent
System
Spot No.
1
2
3
4
5

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

X2
0.5
1.2
2.0
2.9
3.7

Y2
4.5
4.5
4.5
4.5
4.5

Rf
0.11
0.27
0.44
0.64
0.82

Color
Yellow-green
Yellow-green
Yellow-green
Yellow-green
Yellow-green

9:1:1 (v/v/v)
acetone
X2
Y2
0.6
4.2
1.3
4.2
2.2
4.2
2.8
4.2
3.5
4.2

pet-ether-diethyl etherRf
0.14
0.31
0.52
0.67
0.83

Color
Yellow-green
Yellow-green
Yellow-green
Yellow-green
Yellow-green

The result clearly shows that the Rf of the ether diethyl ether acetone is much
higher compared with the ether acetone. This is due to the polarity of the solvents used
which is the ether diethyl ether acetone and ether acetone. Ether acetone is more polar
compared to ether diethyl ether acetone thus it travels at slower and covers a shorter

distance. The reason why the spots travel at a short pace is because it is more attracted
to the stationary phase.

B. Analysis of the Component Dyes of Black Ink by TLC

Solvent System: 30% ethyl acetate in hexane
Sample: Gumamela (Hibiscus)
Color: White
Comparing the results with the 100% hexane, 5% ethyl acetate in hexane, 10%
ethyl acetate in hexane, 20% ethyl acetate in hexane and crude oil, as the
concentration of ethyl acetate increases the color of the pigment becomes lighter.
Among the five solvents used in column chromatography, it clearly shows that 20%
ethyl acetate in hexane is equal to the color in crude oil. However, 30% ethyl acetate
in hexane used did not produce any visible colors due to the absorption of its color by
the silica gel.
C. Identification of Amino Acids by Paper Chromatography
Solvent System:
Visualization method:
Phenylalanine
Tyrosine (T)
Aspartic
(P)
Trial 1
Trial 2
Trial 1
Trial 2
Trial 1
X2
4.50
4.20
4.20
0
1.70
Y2
6.30
6.30
6.30
6.30
6.30
Rf
0.69
0.67
0.67
0
0.28
Color
Violet
violet
violet
violet
violet
Averag
0.680
0.335
0.265
e Rf

acid (A)
Trial 2
1.60
6.30
0.25
violet

The collected data includes the solvent distance and spot distance. The data was
then used to calculate the presented Rf results. The table shows that phenylalanine has
the highest Rf, therefore, it is the most polar solvent among the three. Tyrosines Rf is in
between the Rfs of phenylalanine and aspartic acid. But as viewed in the
chromatography paper it location is near the solvent line meaning it is much attracted
to the mobile phase. Aspartic acid on the other hand, has a lower Rf value therefore it is
a polar solvent and is also attracted to stationary phase.
III. CONCLUSION
The paper chromatography did show that the plant pigments traveled at different
distances after being saturated at two different solvents. The two solvents, ether
acetone and diethyl ether acetone are both polar solvents. However, diethyl ether
acetone is less polar than ether acetone. The adsorptivity of compounds increases with
increased polarity, the more polar the compound then the stronger it binds to the
adsorbent. The eluting power of solvents increases with polarity. Therefore, low polarity
compounds can be eluted with low polarity solvents, while higher polarity compounds
require solvents of higher polarity. The stronger a compound is bound to the adsorbent,
the slower it moves up the TLC plate. Non-polar compounds move up the plate most
rapidly (higher Rf value), whereas polar substances travel up the TLC plate slowly or not
at all (lower Rf value). In our case pigments saturated under diethyl ether acetone has
recorded higher Rf than those of the ether acetone. In summary, low polarity
compounds have higher Rf values than higher polarity compounds.
Phenylalanine and Tyrosine has higher Rf compared to Aspartic Acid meaning it is
more attracted to the mobile phase. In conclusion, Phenylalanine and Tyrosine are less
polar and travel faster going to the mobile phase. On the other hand, aspartic acid

travels slower compared to others and is more polar and more attracted to the
stationary phase.
Errors committed in this experiment may be due to uneven spotting too much
application of sample on the paper.
IV. REFERENCES/BIBLIOGRAPHY
Chemistry 33.1 Laboratory Manual
Paper Chromatography Experiment Report. Retrieved March 5, 2016 from Chem
%2033.1/5/Paper%20Chromatography%20Experiment%20Report%20_%20Examples
%20and%20Samples.html
Vlab.Amarita. Edu. (2011). Separation of Amino Acids by Thin Layer
Chromatography. Retrieved March 6, 2016 from http://vlab.amarita.edu/?
sub=3&brch=63&sim=154&cnt=1
Volstate.Edu. (2016). Amino Acids. Retrieved March 6, 2016 from
www.volstate.edu/CHEM/1030/labs/Amino11.htm
V.

ANSWERS TO QUESTION/CALCULATIONS
1. What would be the effect of the following errors in chromatographic work?
a. The solvent level in the developing chamber is higher than the spotted sample.
If the solvent level in the developing is higher than the spotted sample
chromatography, solvent becomes more polar enough which causes the spots to move
some distance.
b. Too much sample is applied to the paper.
Applying too much sample causes the spots to overlap each other which results
into poor separation or no separation will be obtained at all. It also makes the
interpretation of the results more difficult and decreases the accuracy of the separation
of the compound of interest from everything else. It is easier to determine the Rf of
smaller spots than it is in larger spots.
c. The paper is allowed to remain in the chamber after the solvent front has reached
the top of the plate.
Allowing the paper to remain in the chamber after the solvent reaches the top of
the plate increases the possibility of the components to disperse all over the paper.
Since the solvent has no more space to move, the paper remains saturated and the
compounds continue to diffuse and spread out which results into cluttered
chromatograph. Moreover, it will be difficult to measure the distance traveled by the
solvent because there's no more fixed point to move, but the sample compound may
continue moving.
2. Why is it necessary to cover the developing chamber tightly during the development
of the chromatogram?
It is necessary to cover the developing chamber tightly in order for the
chromatogram to develop properly and for the components to show the correct Rf
values for the solvent. It is also essential that the chamber is saturated with solvent
vapour. Otherwise instead of moving up, the base material solvent will evaporate. If the
developing chamber is left open, much of the solvent would escape because of the air
in the surroundings. This event would change the composition of the developing solvent
which will give inaccurate Rf values.

3. Can TLC or paper chromatography be used to separate and identify very volatile
substances? Explain your answer.
Paper chromatography or thin layer chromatography (TLC) is not a method used
for separating very volatile substances. Instead, high performance liquid (HPLC) or gas
chromatography (GC) was used. The reason for not utilizing the method of TLC or paper
chromatography in identifying very volatile substances was due to the removal of the
volatile components during the process. For example, removing a thin layer plate from
a developing tank, requires evaporation of the developing solvent, which is sometimes
done by heating the plate or moving it through a stream of air. During the process, any
volatile component on the plate is removed at the same time.
4. Why were you required to handle the chromatographic paper only at it corners in part
C?
Chromatographic paper is only allowed to handle on the edges in order to
prevent getting oils from the fingers, to avoid fingerprints and to prevent
contamination. It is also required in order to leave the chromatographic paper
uncovered for as little time as possible to sustain a saturated environment.