Vanessa Olga J.

Dagondon

Date performed: March 23, 2015

Diandra Dawn J. Sanchez

Date submitted: March 31, 2015

CHROMATOGRAPHY
Experiment 4
I.

Objectives
To learn the techniques of paper chromatography and thin layer chromatography
To apply chromatographic methods in the separation of the compounds of a mixture
To identify an unknown by comparing Rf value and other characteristics with those
of a standard.
II. Data and Discussion
A. Separation of Plant Pigments by Paper Chromatography
Sample: San Fransisco (Codiaeum variegatum)
Solven
9:1 (v/v) pet-ether-acetone
t
System
Spot
X
Y
Rf
Color
no.
1. 1
3.40
0.32
1
Yellowcm
cm
Green
1.8
3.40
0.53
2
Redcm
cm
orange

9:1:1 (v/v/v) pet-ether-diethyl etheracetone

X

Rf

0.85
cm
-

2.8
Cm

0.30

Color
YellowGreen
-

Chromatography is widely used technique in separating mixtures of substance into

their components. There are many types of chromatography. Examples are Paper
chromatography and thin layer chromatography. Even having some differences, all types of
chromatography use the same principle. All have a stationary phase (a solid, or a liquid
supported on a solid) and mobile phase (liquid or gas). The mobile phase runs through the
stationary phase carrying the components of the mixture with it and thus, separating them
into components.
Paper chromatography was used in separating and identifying pigments in the leaf
sample, San Fransisco (Codiaeum variegatum). There were two set-ups prepared, each using
a different solvent. The first and second set-up made use of 9:1 (v/v) pet-ether-acetone
which is polar and 9:1:1 (v/v/v) pet-ether-diethyl ether- acetone which is nonpolar,
respectively.

The first set-up separated two pigments with the colours of yellow-green and redorange. With the use of a polar solvent, the red-orange pigment obtained a higher rf value
(the red-orange pigment rose higher than the yellow-green pigment). This signifies that the
red-orange pigment is more polar than the yellow-green pigment. This is because the solvent
used is polar and therefore by the rule of thumb, like dissolves like, polar substances will
be soluble in polar solvents and thus, would be eluted last (placed at the top of the strip).
The red-orange pigment has lower affinity to stationary phase and high affinity to mobile
phase. The second set-up, only the yellow-green pigment was identified. This indicates that
the yellow-green pigment is less polar. This is because, the solvent used in this set-up was
non-polar and therefore the red-orange pigment didnt dissolve in it. To generalize, when
using a polar solvent, a polar substance will migrate higher in the paper and the same goes
for non-polar solvents.
In conclusion, the solvent system 9:1 (v/v) pet-ether-acetone is more beneficial and
more appropriate to be used as solvent in separating the pigments of the San Francisco
leaves (Codiaeum variegatum) than 9:1:1 (v/v/v) pet-ether-diethyl ether- acetone.
B. Analysis of the Component Dyes of Black Ink by TLC
Solvent System: 6:2:2 (v/v/v) n-butanol-ethanol-NH3
Sample: Pilot (black)
Spot
X
Y
No.
2
2.2 cm
3.4 cm
3
2.7 cm
3.4 cm

Rf

Color

0.65
0.79

Violet
Yellow

Thin layer chromatography is another type of chromatography where the mobile

phase is a liquid and the stationary phase is a solid adsorbent (silica) coated on a stationary
support such as glass or plastic. In the analysis of dyes in the ink of a back Pilot ballpoint
pen, such chromatography was used. The TLC plate given by the instructor was spotted. The
chromatogram is then developed by placing the TC plate into a TLC chamber containing the
mobile phase or the solvent. The solvent will then travel up carrying the sample with it
forming distinct spots on the TLC plate.Two spots were observed in the plate, a yellow and a
violet spot. The yellow spot took the higher position than the violet spot. This distinction is
due to the relative polarity of the sample (the ink) and the solvent. The sample will be either
adsorbed to the stationary phase or may be eluted by the solvent. If a polar solvent is used,
the polar compound in the sample will adhere more to the adsorbent and thus would yield a
lower rf value.
The analysis made use of a polar solvent (mobile phase) and a polar stationary phase
(TLC plate with silica gel). Since the violet dye took the lowest position, it is more polar
than the yellow dye. Furthermore, the violet dye had a lower rf than the yellow dye.

C. Identification of Amino Acids by Paper Chromatography

Solvent System: 1:2 (v/v) 2% ammonium hydroxide-isopropyl alcohol
Total distance travelled by solvent system: 7.2 cm
Unknown
Phenylalanine
Aspartic acid
Tyrosine (T)
(P)
(A)
Spot 1
Spot 2
Trial1

Trial2

Trial1

Trial2

Trial1

Trial2

Trial1

Trial2

Trial1

Trial2

X
(cm)

6.0

6.1

5.7

5.7

3.1

3.2

6.2

6.0

3.2

3.2

Y
(cm)

7.2

7.2

7.2

7.2

7.2

7.2

7.2

7.2

7.2

7.2

Rf

0.83

0.85

0.79

0.79

0.43

0.44

0.86

0.83

0.44

0.44

Color

Dark
violet

Dark
Viole
t

Light
Violet

Light
Violet

Light
Viole
t

Light
Violet

Dark
violet

Dark
Violet

Light
Viole
t

Light
Viole
t

Ave
Rf

0.84

0.79

0.44

0.85

0.44

Unknown is: Phenylalanine and Aspartic Acid

Three amino acids (Phenylalanine, Tyrosine and Aspartic acid) and one unknown
were tested under paper chromatography. The solvent used was 2 % ammonium hydroxideisopropyl alcohol. The development of the spots is due to the polarity affinity of the amino
acids to its solvent. The above table summarizes the results including the amino acids
respective rf values. The unknown was found out to be a mixture of two amino acids
namely, phenylalanine and aspartic acid yielding almost the rf values of the said amino
acids.
Phenylalanine and also the unknown (since it contained phenylalanine) travelled
the highest. This signifies that they have a higher affinity to the mobile phase (solvent)
since they easily travelled easily and farther from it. Aspartic acid (which was also in the
unknown), on the other hand, has travelled the lowest distance signifying a lower affinity
towards the mobile phase.
III.

QUESTIONS

1. What would be the effect of the following errors in the chromatographic work?
a. The solvent level in the developing chamber is higher than the spotted sample.
The spot will be smudged and will be dissolved by the solvent and thus would yield no
result.
b. Too much sample is applied to the paper
Too much sample applied on the paper would mean a spot with a large diameter on the
chromatogram. In this case, the sample may combine with adjacent samples and hence,
would make color identification difficult.
c. The paper is allowed to remain in the chamber after the solvent front has reached the top
of the plate.
The solvent front will reach the maximum point and therefore would alter the value of R f
since the value of Y (distance travelled by solvent from the origin) will continue to
increase while X (distance travelled by compound from the origin) will remain.
2. Why is it necessary to cover the developing chamber tightly during the development of
the chromatogram?
The chamber was covered tightly to avoid the evaporation of the volatile solvents in the
developing chamber. Another reason is to prevent the chromatogram to dry up since if
this would happen, it will be difficult to locate the solvent front.
3. Identify your unknown. Explain clearly how you made this identification.
The amino acids that were identified in the unknown are phenylalanine and aspartic
acid. These acids were identified through the comparison made from the height of
distance travelled of the amino acids and the unknown with respect to the origin. It was
found out that the distance travelled of the said amino acids coincides with the distance
travelled of the unknown. The Rf values also verified this assumption.
4. Can TLC or paper chromatography be used to separate and identify very volatile
substances? Explain your answer.
No, Thin-Layer Chromatography or paper chromatography cannot be used to separate
and identify very volatile substances. Volatile substances will be dry easily because of it
will evaporate quickly and thus will make identification harder and not feasible.
5. Why were you required to handle the chromatographic paper only at its corners in part C?
The chromatogram was handled only on the sides to prevent it to absorb any unnecessary
moistures and contaminations that may be present in our fingers.

IV. Conclusion

Chromatography is a method of separating compounds into their respective components.

There are lots of types of chromatography. All use the same principle. One type is called the paper
chromatography. In paper chromatography, a paper is marked with an unknown (plant extract)
and is then placed in a developing chamber with a solvent. The solvent, then will carry the
components of the plant extract as it moves up the paper. The pigments will have different rates
because they have different solubility, polarity and affinity to the solvent. A pigment that is the
most soluble will travel the greatest distance and a pigment that is less soluble will move a shorter
distance. Another type of chromatography is the thin layer chromatography. Thin layer
chromatography is somehow similar to paper chromatography. The only difference is that the
stationary phase is a solid adsorbent coated on a stationary support such as a glass or plastic.
In the experiment, Rf values or the retention factor were calculated by dividing the distance
travelled of the compound by the distance travelled by the solvent. Retention factors can be used
to identify and verify the identity of a substance.
V. References
1. McMurry, John. (2010). Foundations of Organic Chemistry Philippine Edition. Quezon
City, Philippines: C&E Publishing, Inc.
2. Solomons, Graham, Fryhle, Craig B., Snyder, Scott A. (2014). Organic Chemistry 11E.
111 River Street, Hoboken: Wiley & Sons, Inc.
3. Brown, Lemay, Bursten, Murphy, Woodward. (2012). Chemistry: The Central Science
12th Edition. Lake Ave., Glenview, IL: Pearson Education, Inc.
4.

5.

Chromatography. Retrieved on March 30, 2015 on

http://www.infoplease.com/encyclopedia/science/chromatography-thin-layer-paperchromatography.html

Theory: Thin Layer Chromatography (TLC). Retrieved on March 30, 2015 on

http://www.chem.ualberta.ca/~orglabs/Techniques%20Extra%20Info/TLC.html

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

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

Black Ink Pilot

Ballpoint Pen

A. Identification of Amino Acids by Paper Chromatography