BASIC ANALYTICAL CHEMISTRY

CHM 256
LABORATORY CASE STUDY REPORT
(CHROMATOGRAPHY)

TITLE OF CASE STUDY (VIDEO):

SEPARATION OF PIGMENTS FROM THE EXTRACT OF SPINACH LEAVES

BY PAPER CHROMATOGRAPHY

NO NAME OF MEMBERS STUDENT ID

1
2
3
4
5

GROUP :

LECTURER’S NAME :

DATE OF SUBMISSION :

1
3.0 ANALYSIS

Under analysis subtopic, the procedure to separate the pigments from the the extract of
spinach leaves using paper chromatography will be discussed. Below are listed the apparatus
and chemicals needed for this separation.

3.1 Apparatus

1. Chromatographic Chamber with Lid

2. Whatman Filter Paper
3. Fine Capillary Tube
4. Thread
5. Ruler with Scale
6. Pencil

3.2 Chemicals

1. Extract of Spinach Leaves

2. Isopropyl Alcohol-Water mixture

3.3 Procedure

1. Whatman filter paper strip was taken.

2. A line of 2cm above the filter paper end was drawn, using a pencil.
3. A lengthwise line was drawn from the centre of the paper towards the previous
line.
4. Point of intersection between these two lines was named as P.
5. The capillary tube was used to taking the spinach leaves extract.
6. Point P was dropped with a spinach leaves extract and left to dry in opened air.
7. Another drop of spinach leaves extract was placed on the same spot ensured it to
be rich with a mixture of spinach leaves.
8. Leave point P to dry again.
9. The filter paper was tied with a piece of thread.
10. The chromatographic chamber that contained solvent mixtures of isopropyl
alcohol and distilled water was used to suspend the filter paper.
11. The solvent level was ensured to remain about 1cm below the pencil lines, as
shown in Figure 1.

Figure 1. Chromatographic Chamber with a Filter Paper.

12. The chamber was left undisturbed for a period of time.
13. The solvent that rose along the leaf extract was noticed.
14. The pigments of carotene, xanthophyll, chlorophyll a and chlorophyll b obtained
from spinach leaf extract were on different spots of the filter paper.
15. The filter paper was taken out of the chamber.
16. The distance of the solvent has risen on filter paper was marked using a pencil.
17. The solvent front was labelled at the highest solvent, reached the straight line.
18. The filter paper was dry, and the centre of the pigment spots was marked with a
pencil.
19. The distance of solvent from the original line to solvent front and distance of spots
from original line was measured.
20. The formula of Reference Front (RF) =

Distance Pigment Migrated ( X )

Front Migrated (Y )¿was used for calculating the
Distance Spots ¿
value of the pigments as shown in Figure 2.
 Figure 2. The formula of Reference Front.

21. Observation and values were recorded in Table 1

3.3 Procedure

A Whatman filter paper strip was taken. With a pencil, a line of 2cm above the filter paper
was drawn. A lengthwise line was drawn from the centre of the paper towards the previous
line made. Point of intersection between these two lines was labelled as P. A capillary tube
was used to taking the spinach leaves extract. The point P was dropped with a spinach leaves
extract and was left to dry in the opened air. Another drop of spinach leaves extract was
placed on the same spot ensured it to be rich with a mixture of spinach leaves. The point P
was left to dry again. The filter paper was tied with a piece of thread. A chromatographic
chamber contained solvent mixtures of isopropyl alcohol and distilled water was used to
suspend the filter paper. The solvent level was ensured to remain about 1cm below the pencil
lines, as shown in Figure 1.

Figure 1. Chromatographic Chamber with a Filter Paper.

The chamber was left undisturbed for a certain period of time. The solvent that rose along the
leaf extract was noticed. The pigments of carotene, xanthophyll, chlorophyll a and
chlorophyll b obtained from spinach leaf extract were on different spots of the filter paper.
The filter paper was taken out of the chamber. The distance of the solvent had risen on the
filter was marked using a pencil. The solvent front was labelled at the highest solvent,
reached the straight line. The filter paper was dry, and the centre of the pigment spots was
marked with a pencil. The distance of solvent from the original line to solvent front and
distance of spots from the original line was measured. The formula of Reference Front (R F) =

Distance Pigment Migrated ( X )

Front Migrated (Y )¿ was used for calculating the values of
Distance Spots¿
the pigments as shown in Figure 2.

Figure 2. The formula of Reference Front.

Observation and values were recorded in Table 1.