Name: John Christopher L.

Luces

Date performed: January 11, 2011

Co-worker: Thresdale Andrada

Date submitted: January 21, 2011

Femm Dominique Jaranilla

EXPERIMENT N0. 4
CHROMATOGRAPHY
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.

THEORETICAL BACKGROUND

Chromatography is a widely used method that allows the separation, identification and determination of
the chemical components in complex mixtures. It is a technique in which the components of a mixture are
separated based on differences in the rates at which they are carried through a fixed or stationary phase by a
gaseous or liquid mobile phase.
The stationary phase is fixed in place either in a column or on a planar surface. The mobile phase
moves in a definite direction over or through the stationary phase carrying with it the analyte mixture,
where the sample interacts with the stationary phase and is separated. The mobile phase may be a gas,
liquid, or a supercritical fluid.
High-performance liquid chromatography (HPLC) is the most versatile and widely used type of elution
chromatography. In liquid chromatography, the mobile phase is a liquid solvent containing the sample as a
mixture of solutes. The types of HPLC are often classified by separation mechanism or by the type of
stationary phase. These include (1) partition chromatography, (2) adsorption chromatography, (3) ionexchange, (4) size-exclusion, (5) affinity, and (6) chiral chromatography.
Partition chromatography is also called liquid-liquid chromatography in which the stationary phase is a
second liquid that is immiscible with the liquid mobile phase. Its separations may be attributed to the
differences in the solubility of the sample in the stationary and mobile phases.
Paper chromatography is an example of liquid-liquid or partition chromatography. It is a useful
technique in the separation and identification of different plant pigments. Since the filter paper is made of
highly purified cellulose, it absorbs and retains water molecules strongly. The filter paper with cellulose
and the bound water compose the stationary phase. The solvent is then the mobile phase. The separations
become flavorable due to different affinities of the components. After development, the spots
corresponding to different compounds may be located by their color, ultraviolet light, ninhydrin or by

treatment with iodine vapors. The paper remaining after the experiment is known as the chromatogram.
The components which have been separated differ in the retention factor.
Retention factor may be defined as the ratio of the distance travelled by the substance to the distance
travelled by the solvent.
In adsorption chromatography, the mobile phase is usually an organic solvent and the stationary phase
is finely divided particles of silica or alumina. In adsorption chromatography, the only variable that affects
the distribution coefficient of analytes is the composition of the mobile phase, in contrast with partition
chromatography where the polarity of the stationary phase can also be varied.
Thin layer chromatography is a form of solidpliquid adsorption chromatography. It involves a
stationary phase consisting of a thin layer of adsorbent material, usually silica gel, aluminium oxide, or a
cellulose immobilized onto a flat, inert carrier sheet. The mobile phase constitutes the solvent.
III.
DATA AND RESULTS
A. Separation of Plant Pigments by Paper Chromatography
Sample: San Fransisco (Codiaeum variegatum)
Solvent
System
Spot
no.
1
2

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

X
1. 9 cm
2.8 cm

Rf

Color

6.20 cm
6.20 cm

0.5
0.45

Green
Yellow

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

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

X
Y
Rf
Color
1.3

6.5 cm

0.2

Green

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:
G-TECH
Spot No.
1
2
3

X
2.4
2.9
3.8

Y
4.9
4.9
4.9

Rf
0.49
0.59
0.78

Color
Black
Pink
Yellow

X
3.2
3.6
4.9

Y
4.9
4.9
4.9

R
0.65
0.73
1

Color
Purple
Yellow
Blue

FABERCASTEL
Spot No.
1
2
3
G-TECH

Fabercastel

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
Visualization method: Spraying 2% ninhydrin solution in acetone to the paper (chromatogram)
Phenylalanine (P)

Tyrosine (T)

Aspartic acid (A)

Unknown

Trial1

Trial2

Trial1

Trial2

Trial1

Trial2

Trial1

Trial2

X (cm)

6.8

6.8

6.3

6.5

5.1

5.2

6.9

6.7

Y (cm)

7.2

7.2

7.2

7.2

7.2

7.2

7.2

7.2

Rf

0.95

0.95

0.88

0.90

0.71

0.72

0.96

0.93

Color

Dark
violet

Dark
Violet

Light
Violet

Light
Violet

Light
Violet

Light
Violet

Dark
violet

Dark
Violet

Average
Rf

0.95

0.89

Unknown is: Phenylalanine

IV.

DISCUSSION

0.49

0.95

Paper chromatography can be used to identify and separate plant pigments in leaves. The plant sample
that was used is Codiaeum variegatum. The solvents that were used are 9:1 (v/v) pet-ether-acetone (polar)
and 9:1:1 (v/v/v) pet-ether-diethyl ether- acetone (nonpolar).
In the first set-up where in PEA was used as a solvent, 2 pigments were identified while in the other
solvent (PEDA), only one color was determined (green). Based on the Rf values obtained in setup A, the
yellow pigment is more polar than the green pigment. This is because the yellow pigment rose higher in the
polar solvent (PEA) while it didnt appear in the nonpolar solvent (PEDA). Based on the principle like
dissolves like since yellow pigment is more polar than the green pigment it is more soluble to the polar
solvent that is why it rose higher on it. Also, the yellow pigment has lower affinity to stationary phase and
high affinity to mobile phase. Thus the solvent system 9:1 (v/v) pet-ether-acetone is more advantageous
and more appropriate to be used as solvent in the Codiaeum variegatum leaves than any other solvent.
In Setup B, only the green pigment is visible. This indicates that the green pigment is less polar because
it appeared in the nonpolar solvent. Yellow pigment was not visible because it didnt dissolve in the
nonpolar solvent.
In the analysis of dyes in G-tech ink, the yellow dye rose the highest then the pink and the black dye in
the lowest position. Since the solvent used is polar, and the stationary phase (TLC plate with silica gel) is
polar, the dyes can be arranged according to increasing polarity. Yellow > Pink > Black. And also, the dyes
can be arranged in increasing solubility, that is according to their rise and Rf value as in Black< Pink<
Yellow. So the most polar is the most soluble in the solvent while the least polar is the least soluble.
On the other hand, in the Faber castelle ink, also three colors were determined blue, yellow and purple.
This shows that the ink component in Faber castelle is different in that of G-tech. In the ink of Faber
castelle, the blue dye rose the highest then the yellow and lastly the purple lie in the lowest position. Since
the solvent used is polar, and also the stationary phase is polar, just like in G-tech the dyes can be arranged
according to increasing polarity. Blue>Yellow> Purple
There were three known amino acids and one unknown amino acid tested under paper chromatography.
The development and placement of spots are according to the solubility, polarity and affinity of the amino
acids to the solvent 1:2) (v/v) 2 % ammonium hydroxide-isopropyl alcohol. Tyrosine has a light violet
spots measuring 6.3-6.5 cm above the original sample. The Rf is therefore 0.88 and 0.9 respectively.
Aspartic acid, on the other hand, also has light violet color but is darker than that of tyrosine and has the
lowest rise with the Rf of 0.49. Of the four amino acids, phenylalanine and the unknown are the most
soluble to the given solvent because they have travelled/ risen the highest. As well, they are amino acids
giving the highest Rf values. Also, they have higher affinity to the mobile phase (solvent) as they travelled
easily and farther with it. On the other hand, they have lower affinity towards the stationary phase
(chromatographic paper) because they dont interact strongly with the paper but rather proceeds faster with
the solvent. Aspartic acid has the lowest rise. So it is the least soluble to the solvent. Also it has the highest
affinity to the stationary phase. But has the lowest affinity to the mobile phase. Based on the color and
solubility of the amino acids, it can be identified that the unknown is phenylalanine. Both the phenylalanine

and the unknown have dark violet spots and have Rf value approximately 0.95. The same Rf values
indicates that the amino acids have the same solubility to the solvent system used.
V.

CALCULATIONS
distance travelled by compound
distance travelled by solvent
Rf =
the origin(x) origin( y)

A. Separation of Plant Pigments

1.) 9:1 (v/v) pet-ether-acetone
2.) 9:1:1 (v/v/v) pet-ether-diethyl-acetone
Spot No.1
Spot No 2.
x = 1 90 cm
X= 2.8
x= 1.3 cm
y= 6.20 cm
Y= 6.20
y= 6.5 cm
Rf = 1.9cm / 6.20cm
Rf= 2.8/6.20 cm
Rf = 1.3cm / 6.5cm
Rf = 0.5 cm
Rf= 0.45
Rf = 0.2
B.

G-TECH
Black
X= 2.4
Y= 4.9
Rf= 2.4/4.9
Rf= 0.49
FABER CASTELL
Purple
X= 3.2
Y= 4.9
Rf= 3.2/4.9
Rf= 0.65

Pink
X= 2.9
Y= 4.9
Rf= 2.9/4.9
Rf= 0.59
Yellow
X= 3.6
Y= 4.9
Rf= 3.6/4.9
Rf= 0.73

Yellow
X= 3.8
Y= 4.9
Rf= 3.8/4.9
Rf= 0.78
Blue
X= 4.9
Y= 4.9
Rf= 4.9/4.9
Rf= 1

C. The same method was used in calculating Rf in C (Identification of Amino Acids by Paper
Chromatography)
VI.
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
A: There will be no result because the spot will smear out and will be dissolved by the solvent
b. Too much sample is applied to the paper
A: If too much sample is applied to the paper, a large spot on the developed chromatogram will appear
and combine with others and may run together making color identification difficult.
c. The paper is allowed to remain in the chamber after the solvent front has reached the top of the plate.

A: If this would happen, the solvent front will reach the maximum point , and the value of R f will vary,
so the real value will be slightly unreliable, since the Y continues to goes up and X remains.
2. Why is it necessary to cover the developing chamber tightly during the development of the
chromatogram?
A: To avoid rapid evaporation of the volatile solvents in the developing chamber, the chamber must be
covered tightly. This is because most of the solvents used are volatile and volatile solvents tend to
evaporate faster and easier than non-volatile ones Also, if the chamber is not covered tightly, there is a
tendency that the chromatographic paper will become dry and it would be difficult to locate the solvent
front since the solvent has gone to the gas phase.
3. Identify your unknown. Explain clearly how you made this identification.
A: The unknown is the Amino Acid- phenylalanine. It was identified through comparing the height of
the development of the unknown sample with the development of that of other amino acid. Its height of
the development of the sample is the same with that of Phenylalanine. Basically, the unknown was
identified with respect to the Rf of known Amino Acids.
4. Can TLC or paper chromatography be used to separate and identify very volatile substances? Explain

your answer.
A: Thin-Layer Chromatography is not for the separation and identification of very volatile substances.
It is because volatile substances evaporate quickly, thus the solvent used easily becomes dry. Therefore,
TLC cant be used to separate and identify very volatile substances.
5. Why were you required to handle the chromatographic paper only at its corners in part C?
A: The chromatographic paper should only be handled to its sides. So as not to absorb unnecessary
moistures and thus to reduce contamination. Also, not touching the chromatographic paper would
prevent the contact to our skin which has also amino acids present.