Download THIN-LAYER CHROMATOGRAPHY MINI LAB

Professor Amorde
Organic Chemistry I
Thursday, February 5, 2009
THIN-LAYER CHROMATOGRAPHY MINI LAB
Abstract
The separation of pigments in spinach leaves using Thin-Layer Chromatography (TLC).
Procedure
In a mortar, several fresh spinach leaves were mixed with approximately 8 mL of a mixture
of petroleum ether and ethanol mixed in a 2:1 ratio. The mixture was ground until the spinach
leaves released a dark green liquid. Using a pipette, the extracted dark green liquid was
transferred into a separation funnel (Fig. 1). Water was added into the separation funnel in a
volume approximately equal to the dark green liquid previously placed in the separation funnel.
The mixture was swirled, vented, allowed to separate for 1 min, and the bottom aqueous layer
was removed, and the process repeated. The remaining dark green liquid was transferred into a
small beaker. A small scoop of anhydrous sodium sulfate was added to remove any water left in
the dark green pigment solution. A line was drawn at 1 cm on a 10 cm by 2 cm strip of silica gel
thin layer chromatography plate (Fig. 2). Using a capillary tube, a spot of green pigment solution
was placed on the center line. The spot was allowed to dry, and the process was repeated until a
dark green spot of about 2 mm in diameter was achieved. The chromatogram was developed in a
70:30 solution of petroleum ethanol and acetone. After development, the TLC was read under
regular fluorescent light and ultra-violet (UV) light. Visible spots and colors were noted and
marked for each on the chromatogram.
Dark green layer of petroleum ether and organics.
Light green aqueous layer of leaves and ethanol.
Fig. 1. Separation funnel set-up.
Data
Under the regular fluorescent light, six spots in yellow, light green, and green were visible
(Fig. 2). Six spots in the same location as those visible in normal light (Fig. 2) were also visible
under UV light. The UV spots ranged in color from red to brown and had a diameter slightly
larger than the spots visible in normal light.
UV
RL
5.6 cm
4.3 cm
3.6 cm
3.1 cm
2.2 cm
1.5 cm
1.0 cm
Figure 2.
The TLC plate
as viewed in regular fluorescent light (right) and
UV light (left).
Results
The yellow spots on the chromatogram indicate the presence of xanthophyll in the
spinach leaves. The green spots indicate the presence of chlorophyll b.
Conclusion
The TLC process worked, but did not reveal the presence of all possible organics
identifiable in spinach. The missing indicators on the TLC plate were likely the result of
insufficient grinding or overdevelopment of the chromatogram. It is also possible that the results
were not viewed quickly enough as some of the color indicators vanish over time.
Works Consulted
Lehman, J. W. (1999). Operational organic chemistry: A problem-solving approach to the
laboratory course. 3rd Ed. Upper Saddle River, NJ: Prentice Hall.
McMurry, J. (2008). Organic Chemistry. 7th Ed. Belmont, CA: Brooks/Cole CENGAGE
Learning.