C

H
R
O
M
A
T COLUMN CHROMATOGRAPHY
O
G
and THIN LAYER
R CHROMATOGRAPY
A PRELAB
P
EXPERIMENT NO. 5
H
Y
 VISUALIZATION
GENERAL PROCEDURE

COLUMN and Rf Values

CHROMATOGRAPHY
COMPUTATION

EXTRACTION
THIN LAYER
OF PLANT CHROMATOGRAPHY
PIGMENTS
 1 2
PREPARATION OF PLANT EXTRACTS

3
4
Extracting Solvent (10ML)
Siling Labuyo DCM-Hexane (1:1)
Malunggay Hexane-Acetone
(7:3)
COLUMN CHROMATOGRAPHY

1 2 3 SOLVENT SYSTEM:
Introduce successively 3
mL of each of the ffg.
S.S.

A. Siling Labuyo:
1. DCM-Hex (1:1)
2 INCHES

2. DCM
3. DCM : Methanol (1:1)

B. Malunggay:
1. Hexane: Acetone
(7:3)
PUT 0.5 ML OF THE 2. Acetone
RUN THE COLUMN
PLANT EXTRACT INTO 3. Acetone Methanol
SUCCESIVELY WITH
THE COLUMN (1:1)
DIFFERENT SOLVENT
SYSTEMS
COLUMN CHROMATOGRAPHY
 0.25 cm
CHROMATOGRAPHY
THIN LAYER

Developing Chamber
0.75 cm Developing Solvents:

Sili: Hex-DCM (1:1)

Crude Extract
Malunggay: Hex-Acetone
Colored Eluates (7:3)
CHROMATOGRAPHY

Sample/Standard Application - small spots of std/sample are

applied to avoid overlapping and tailing during
THIN LAYER

development
Development - equilibration (saturation of chamber with
mobile phase) to hasten development
Visualization – chemical visualizing agent
Evaluation –comparing Rf values of sample and standards
Documentation – chromatogram
 • The distance a spot travels on a plate is referred
to as the Rf (Rf = b/a).
• In lane 1 an optimal spot (shape and Rf) is
CHROMATOGRAPHY

shown.
• In lane 2 the Rf is good, but the spot is tailing; in
this case a combination with a hydrogen
THIN LAYER

bonding solvent component (MeOH, AcOH, or

Optimal Rf range
NH4OH) could be useful.
a

0.3 to 0.6
• In lane 3 a more polar combination is required.
• In lane 4 the MP is too polar; a less polar
combination is required.
• In lane 5 the Rf is good, but the compound was
b spotted too heavily; spot more lightly.
• In lane 6 the Rf is good, but the compound was
spotted too lightly; spot more heavily.
• In the cases of lanes 2-6 the TLC should be
1 2 3 4 5 6 repeated with the suggested adjustments.
VISUALIZATION and Rf Values

TLC Identify the visible spots by Visualization of UV Active Spots,

encircling them Identify also the UV Active spots by
encircling them
!"#$%&'( $)%*(++(, -. $/( #0+1$( (#30$)
Rf= !"#$%&'( 5)%*(++(, -. $/( #0+*(&$
 THANK YOU!

CHROMATOGRAPHY