FACULTY OF ENGINEERING TECHNOLOGY

DEPARTMENT OF CHEMICAL ENGINEERING TECHNOLOGY

PRINCIPLE OF BIOCHEMISTRY LABORATORY

LABORATORY INSTRUCTION SHEETS

COURSE CODE BNN30104

EXPERIMENT NO. EXPERIMENT 5

EXPERIMENT TITLE BASIC PAPER CHROMATOGRAPHY

DATE
GROUP NO.
LECTURER/ INSTRUCTOR/ 1) DR. SITY AISHAH MANSUR
TUTOR 2)
DATE OF REPORT
SUBMISSION
ATTENDANCE/PARTICIPATION/DISPLINE /5%
INTRODUCTION: /5%
PROCEDURE: /5%
RESULTS & CALCULATIONS /15%
ANALYSIS /15%
DISTRIBUTION OF MARKS
DISCUSSIONS: /20%
FOR LABORATORY
REPORT: ADDITIONAL QUESTIONS: /15%
CONCLUSION: /10%
SUGGESTION & RECOMENDATIONS /5%
REFERENCES: /5%
TOTAL: /100%

EXAMINER COMMENTS: RECEIVED DATE AND STAMP:

 STUDENT CODE OF ETHICS

DEPARTMENT OF CHEMICAL ENGINEERING

TECHNOLOGY

FACULTY OF ENGINEERING TECHNOLOGY

I hereby declare that I have prepared this report with my own efforts. I also admit

to not accept or provide any assistance in preparing this report and anything that

is in it is true.

1) Group Leader: (Signature)

Name : ABDUSSALAM AL-HAKIMI BIN MOHD TAHIR
Matrix No. : AN140037

2) Group Member 1: (Signature)

Name : NUR FADHILA SHAFIKA BT YUSOP
Matrix No : AN150404

3) Group Member 3: (Signature)

Name : FRANCIS CHANG FOOK ANN CADACIO
Matrix No. : AN150368
 FACULTY : ENGINEERING
EDITION:
TECHNOLOGY
LABORATORY: MOLECULAR
REVISION NO:
BIOLOGY LABORATORY
EXPERIMENT 5: BASIC PAPER
EFFECTIVE DATE:
CHROMATOGRAPHY
AMENDMENT DATE:

1.0 OBJECTIVES

To separate components of mixture by their different polarities.

2.0 LEARNING OUTCOMES

At the end of this laboratory session student will be able to;

2.1 Relate the polarity of molecules to their solubility in different solvents.
2.2 Connect molecular motion in a solvent to polarity of molecules.
2.3 Distinguish between stationary and mobile phases and determine the
Rf values for the different components separated.

3.0 INTRODUCTION

Paper chromatography is probably the first, and the simplest, type of chromatography
that people meet. It was introduced in 1961 by schonbein. In this type of chromatography, a
specialized paper is used for stationary phase, due that it is known as paper chromatography.
Paper chromatography is an analytical method technique for separating and identifying
mixtures that are or can be coloured, especially pigments. This method has been largely
replaced by thin layer chromatography, however it is still a powerful teaching tool.
Paper chromatography is a method that is used to separate out materials from a
mixture such as to separate amino acids, is a form of partition chromatography. Water, a
component of the developing solvent, forms hydrogen bonds with the fibers of the paper and
serves as the stationary phase. The organic liquids that are also present in the developing
solvent serve as the mobile phase. The components of the mixture are drawn up the paper to
different heights, depending on their solubility in the mobile phase. A solvent such as alcohol
or water is used to dissolve the components of a mixture. The solvent travels up the paper by
capillary action. The particles of solute that are dissolved in the solvent are carried up the
paper along with the solvent. The particles of solute will be separated according to solubility
as they are carried up the paper; the soluble particles travel faster and will end up at the top,
while the less soluble will travel more slowly and will be seen at the bottom. The pattern on
the chromatography paper is called a chromatograph.
Ink is a liquid or semi-liquid material used for writing, printing or drawing. Chemists
view it as a colloidal system of fine pigment particles dispersed in a solvent.1 The pigment
may or may not be colored, and the solvent may be aqueous or organic. The earliest black

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EDITION:
TECHNOLOGY
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REVISION NO:
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EFFECTIVE DATE:
CHROMATOGRAPHY
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writing inks, developed before 2500BC, were suspensions of carbon, usually lampblack, in
water stabilized with a natural gum or materials like egg albumen. Modern ink formulations
are rather more complex. In addition to the pigment, they contain many other ingredients in
varying levels. Although all black inks may look the same, they may be quite different in
composition. The compositions are generally proprietary and are well guided secrets for
different companies. We will use this process of chromatography to examine the differences
between several different inks. The special chromatography paper is spotted with each ink
and placed in a container with the solvent. The water moves up the paper by capillary action
carrying the soluble portions of the ink. The more soluble the compound is in the mobile
phase, the further it moves up the paper.

Migration Parameter
Common solvents that are used include pentane, propanone and ethanol. Mixtures of
solvents are also used, including aqueous solutions, and solvent systems with a range of
polarities can be made. A mixture useful for separating the dyes on Smarties is a 3:1:1
mixture (by volume) of butan-1-ol: ethanol:0.880 ammonia solution. As each solute
distributes itself equilibrates) between the stationary and the mobile phase, the distance a
solute move is always the same fraction of the distance moved by the solvent. This fraction
is variously called the retardation factor. Also known as resolution factor or retention ratio.

Figure 1: retention factor paper chromatography

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 FACULTY : ENGINEERING
EDITION:
TECHNOLOGY
LABORATORY: MOLECULAR
REVISION NO:
BIOLOGY LABORATORY
EXPERIMENT 5: BASIC PAPER
EFFECTIVE DATE:
CHROMATOGRAPHY
AMENDMENT DATE:

4.0 INSTRUMENTS /APPARATUS / CHEMICAL / REAGENTS

a. Equipment
i. Chromatography paper
ii. Filter paper
iii. Glass rod or pencils
iv. Tape
v. Capillary tubes for dyes or toothpick
vi. Two beakers or jars
vii. Clear plastic wrap or watch glasses to cover beakers during activity or
aluminium foil can be used
viii. Food dyes (3 colours) / possible liquids
ix. Pestle and mortar
b. Reagent
i. Deionized water for mixing and mobile phase element
ii. Isopropyl alcohol / 50% water ; acetone
iii. Butanol : acetic acid : water in ratio 4:1:5

5.0 PROCEDURE
For one piece of paper into one beaker of one solvent.

1. Chromatography paper was cut into 10.0 cm and was handled by the edges.
2. Line was marked by pencil 2 cm from the bottom, a spot was labelled for each sample
tested.
3. The different samples/dyes were applied to the labelled spots on the pencil line by
using capillary tube. Assigned spots was recorded.
4. The paper was taped to the glass rod such that the line with the sample spots is not
submerged in the solvent.
5. 25 ml solvent was added to the beaker approximately half from the marked line, then
glass rod was placed with tape.
6. The solvent was run at least 10 minutes up the paper.

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 FACULTY : ENGINEERING
EDITION:
TECHNOLOGY
LABORATORY: MOLECULAR
REVISION NO:
BIOLOGY LABORATORY
EXPERIMENT 5: BASIC PAPER
EFFECTIVE DATE:
CHROMATOGRAPHY
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7. The solvent front was marked by pencil.

8. The distance was measure for the solvent moved and the distance each component of
the dyes moved.

6.0 RESULTS

Table 6.1: Rf value for each dye.

Solvent Colours Distance of Distance of Rf
Solvent (cm) Compound
(cm)
acetone Red 11.0 10.2 0.927
Yellow 10.6 9.7 0.915
Green 9.8 8.8 0.898
butanol : Red 6.6 5.8 0.879
acetic acid : Yellow 7.3 6.4 0.877
water green 6.6 5.7 0.864

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EDITION:
TECHNOLOGY
LABORATORY: MOLECULAR
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Acetone Butanol : acetic acid : water

10.2 5.8
Rf of red dye = 11.0 = 0.927 Rf of red dye = = 0.879
6.6
9.7 6.4
Rf of yellow dye = 10.6 = 0.915 Rf of yellow dye = 7.3 = 0.877
8.8 5.7
Rf of green dye = = 0.898 Rf of green dye = 6.6 = 0.864
9.8

7.0 ANALYSIS

The collected data includes the solvent distance and the compound distance. The data
was then used to calculate the presented Rf results. For the first sample, which is the red colour
dye, it managed to travel up to 11.0 cm above the starting point for acetone solvent while for
butanol it only managed to travel up to 6.6 cm. Second dye, for acetone the yellow colour
climbs up to 10.6 cm whereas 9.8 cm for the green colour dye while for butanol 7.3 and 6.6
respectively. We can see that in yellow dye, there are a little bit of blue colour started from the
starting point. This means that in yellow colour dye, it contains some concentration of blue dye
in it. We also measured the distance travelled for the compound, so that we could calculate the
Rf value. From calculation, red dye has the highest Rf amount for both in acetone and butanol
solvent compared to the others. Meanwhile, the green has the lowest Rf amount among the
other dye.

8.0 DISCUSSIONS
Please discuss on the experimental result and make a conclusion

9.0 ADDITIONAL QUESTIONS

1. Justify the reason(s) of covering the chamber with aluminium foil during
experiment.

To prevent the evaporation of the mobile phase. The mobile phase is a

mixture of organic solvents and water. Typically, the solvent used for the
chromatography is rather volatile If paper chromatography was performed
in an open container, the organic part of the mobile phase evaporates
much faster than water, and the composition of the mobile phase will
change. Because of that, the chromatogram result will surely change too.

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EDITION:
TECHNOLOGY
LABORATORY: MOLECULAR
REVISION NO:
BIOLOGY LABORATORY
EXPERIMENT 5: BASIC PAPER
EFFECTIVE DATE:
CHROMATOGRAPHY
AMENDMENT DATE:

2. Can you relate the principle of thin layer chromatography (TLC) in food
industry? Extensively explain

Thin-layer chromatography (TLC) is widely used in laboratories throughout

thhe world for food analysis and quality control. In food, TLC method is
used for separation and identification of colours, preservatives, sweetening
agent, and various food products. The principle of thin-layer
chromatography (TLC) will be applied to investigate water soluble food
dyes. The TLC plates to be used consist of a thin layer of solid silica gel
coated onto a flexible plastic material. Numerous applications of TLC have
been reported in the areas of food composition, intentional additives,
adulterants, contaminants, and decomposition involving determinations of
compound classes such as amino acids, lipids and fatty acids, sugars,
vitamins, and organic acids.

Based on the principle of separation. The separation depends on the

relative affinity of compounds towards stationary and mobile phase. The
compounds under the influence of mobile phase travel over the surface of
stationary phase. During this, movement the compounds with higher
affinity to stationary phase travel slowly while the others travel faster. Thus
separation of components in the mixture is achieved. Thin-layer
chromatography is an improvement on paper chromatography because the
more uniform particles used generally make the method more
reproducible. It is a very fast and convenient method of performing
separations for quality control and for screening unknown mixtures for
their composition.

3. Compare and contrast between paper and column chromatography

Definition Paper chromatography is the is a group of chromatographic

type of chromatography where
it take place upon the
stationary phase of cellulose
filter paper when the mobile
phase of solvent along with the
mixture components run
across and separate out due to
difference in affinity with
water. Paper chromatography
that using a sheet of blotting
paper, usually filter paper, for
the adsorption column
Basic The stationary phase is
principles applied to a flat surface. A
sample is applied at one end
and the mobile phase is
allowed to flow and move the
sample across the thin layer
techniques which use columns
to house the stationary phase.
The column can be small or
large, but typically, it contains
larger amount of stationary
phase Column
chromatography that in which
the various solutes of a
solution are allowed to travel
down an absorptive column,
the individual components
being absorbed by the
stationary phase.
In column chromatography
the sample is applied to the
top of the column and the
liquid mobile phase is allowed
to flow through the column

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 FACULTY : ENGINEERING
EDITION:
TECHNOLOGY
LABORATORY: MOLECULAR
REVISION NO:
BIOLOGY LABORATORY
EXPERIMENT 5: BASIC PAPER
EFFECTIVE DATE:
CHROMATOGRAPHY
AMENDMENT DATE:

effecting separation of the

Advantage identifying the affinity of
molecules especially when a
mixture of polar and non-polar
components are taken for
separation.
applied sample.
Column chromatography is
useful for preparative
separations. They are
relatively inexpensive, but
their chromatographic
separation power are much
lower than HPLC

10.0 CONCLUSION

Conclusion is merely a summary, presented in a logical order, of the important findings

already reported in the discussion section. It also relates to the objectives stated earlier.

11.0 REFERENCES

1. Sherma, J. (2000). Thin-layer chromatography in food and agricultural analysis. Journal of

Chromatography A, 880(1), 129-147.
2. Lorenzo, R. A., Pena, M. T., Fernández, P., González, P., & Carro, A. M. (2015). Artificial
sweeteners in beverages by ultra performance liquid chromatography with photodiode array
and liquid chromatography tandem mass spectrometry. Food control, 47, 43-52.
3. Poonia, N. S. (1967). Qualitative analysis with paper chromatography. J. Chem. Educ, 44(8),
477.
4. Toennies, G., & Kolb, J. J. (1951). Techniques and reagents for paper
chromatography. Analytical Chemistry, 23(6), 823-826.
5. vlab.amrita.edu,. (2011). Separation of Compounds Using Column Chromatography.
Retrieved 25 December 2017, from vlab.amrita.edu/?sub=2&brch=191&sim=341&cnt=1

Prepared by/Disediakan oleh : Approved by/Disahkan oleh :

Signature/Tandatangan : Signature/Tandatangan :
Name/Nama : PN. SITY AISHAH Name/Nama : PROF. MADYA DR. ISHAK
MANSUR BABA
Date/Tarikh : Date/Tarikh :