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 R f f value for each pigment
– – To learn the effect of polarity and molecular size and molecular weight on R f f 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:
TTable: Distance Moved and Respective R f f Values for Each Photosynthetic Pigment with Solvent 1 & 2
PPiiggmmeennttss’cc oolloorrss SSoollvveennt11 SSoollvveennt22
Distance, cm R R f f VVaalluueess DDiissttaannccee,cc mm Rf f Values
**YYeellllooww--oorraannggee 1111..00 00..66554488 -- -- **YYeellllooww 1100..66 00..66331100 -- -- **BBlluuee--ggrreeeenn 44..55 00..22667799 99..1155 00..44666688 **YYeellllooww--ggrreeeenn 22..00 00..11119900 -- --
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
*Yellellow-orange pigment is Xanthophylylls, yellow pigment is carotenes, blue-greenn 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 bb
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 ththe pipigments, both popolalar pipigmenents anand nonpopolalar pipigmenents moved asascend ththee 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 R R f f 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 R f f values than carotenene and xanthophylls in solvent 1.
The distance and R R f f 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 –CH33 group in chlorophyll a. 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 R f f value than that of xanthophylls. But, the result showed that xanthophylls have a
greater distance and R f f 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. 77. thth ed. CA: Pearson Benjamin Cummings.
Voet, D.J., Voet, J.G., & Pratt, C.W., 2008. Principles of Biochemistry. 33rdrd ed. NJ: John Wiley & Sons, Inc.
WWiikkiippeeddiia 22000099, CCaarrootteennee, oonnlliinnee, rreettrriieevveed 226 JJuunne 22000099, ffrroomm 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