Universiti Tunku Abdul Rahman (Kampar Campus)

Faculty of Science, Engineering, and Technology

Bachelor of Science (Hons) Biotechnology

Year 2 Semester 1

UESB 2142 Laboratory 2A

(I) Biochemistry & Cell Metabolism

Lecturer: Mr. James Wee Sheau Yang

Student’s Name: Cheah Hong Leong

Student’s ID: 08AIB03788

Group members: Lim Zhe Choon, Yeah Wei Jin, Ng Ka Swin, Ang Pei Wen

Experiment No. 2

Title: Paper Chromatography – Photosynthetic Pigment Separation.

Date: 15 June 2009

Title: Paper Chromatography – Photosynthetic Pigment Separation

Objectives:
– To separate carotene, xanthophylls, chlorophyll a, and chlorophyll b by paper
chromatography and calculate Rf value for each pigment
– To learn the effect of polarity and molecular size and molecular weight on Rf
value of photosynthetic pigments
 – To learn the effect of different solvents on the results of paper chromatography
and choose the suitable solvent for each type of chromatography

Results:
Table: Distance Moved and Respective Rf Values for Each Photosynthetic Pigment with
Solvent 1 & 2
Pigments’ colors Solvent 1 Solvent 2
Distance, cm Rf Values Distance, cm Rf Values

*Yellow-orange 11.0 0.6548 - -

*Yellow 10.6 0.6310 - -
*Blue-green 4.5 0.2679 9.15 0.4668
*Yellow-green 2.0 0.1190 - -

Notes:
Solvent 1: petroleum ether: acetone = 9: 1
Solvent 2: petroleum ether: acetone = 6: 4
Solvent front for solvent 1 = 16.8 cm
Solvent front for solvent 2 = 19.6 cm
*Yellow-orange pigment is Xanthophylls, yellow pigment is carotenes, blue-green
pigment is chlorophyll a, and yellow-green pigment is chlorophyll b.
Conclusion and Discussion:
Molecular structures for carotene, xanthophylls, chlorophyll a, and chlorophyll b are
shown below:
Chlorophyll a Chlorophyll b

Carotene

Xanthophylls
The chromatography carried out based on solvent 1 and 2 shown different results. Only
chromatography with solvent 1 shown separation of pigments; whereas solvent 2 shown
only one blue-green colored dot ascended the chromatography paper.
One possible cause was the almost equal proportion of petroleum ether and acetone in the
solvent; caused all the pigments to dissolve almost equally in solvent 2. Petroleum ether
was the nonpolar part of solvent while acetone was the polar part of the solvent. Hence,
all the pigments, both polar pigments and nonpolar pigments moved ascend the
chromatography paper at almost equal rate, without any separation from each other.
Only solvent 1 in which the ratio of petroleum ether to the acetone was very high (9:1),
separated those pigments into different distance of movement and Rf values as those
pigments have different solubility in the solvent 1.
Chlorophyll a and b have a larger molecular size and molecular weight than carotene and
xanthophylls; they moved ascended the chromatography paper slower than carotene and
xanthophylls. Hence chlorophyll a and b have significant lower Rf values than carotene
and xanthophylls in solvent 1.
The distance and Rf value of chlorophyll a was higher than that of chlorophyll b. This
was due to different in solubility and polarity of chlorophyll a and b. Solvent 1 was
composed largely of nonpolar petroleum ether. Chlorophyll b was slightly more polar
than chlorophyll a due to presence of –CHO group instead of –CH3 group in chlorophyll
a. Therefore, chlorophyll a was more soluble in solvent 1 than chlorophyll b, and
chlorophyll a was moved by solvent 1 at rate greater than chlorophyll b.

However, carotene is nonpolar pigment whereas xanthophylls are polar pigments due to
presence of additional hydroxyl groups on xanthophylls. Suppose carotene was more
soluble in solvent 1 and moved at greater rate than that of xanthophylls, therefore has a
higher Rf value than that of xanthophylls. But, the result showed that xanthophylls have a
greater distance and Rf value than carotene.
One possible cause of this result was the error occurred in color identification between
yellow and yellow-orange colors. The yellow color of carotene can be easily confused
with the yellow-orange color of xanthophylls. Therefore, the result was reverse from the
theoretical result.
In conclusion, the kinetic rate of pigments ascending the chromatography: carotene >
xanthophylls > chlorophyll a > chlorophyll b.

References:
Campbell, N.A. & Reece, J.B., 2005. Biology. 7th ed. CA: Pearson Benjamin Cummings.
Voet, D.J., Voet, J.G., & Pratt, C.W., 2008. Principles of Biochemistry. 3rd ed. NJ: John
Wiley & Sons, Inc.
Wikipedia 2009, Carotene, online, retrieved 26 June 2009, from
http://en.wikipedia.org/wiki/Carotene
Wikipedia 2009, Xanthophyll, online, retrieved 26 June 2009, from
http://en.wikipedia.org/wiki/Xanthophyll
Wikipedia 2009, Chlorophyll, online, retrieved 26 June 2009, from
http://en.wikipedia.org/wiki/Chlorophyll