The document describes an experiment using paper chromatography to separate mixtures. Students used paper chromatography to separate mixtures of dyes and amino acids. Different compounds traveled different distances up the paper based on their solubility in the solvent and ability to bind to the paper. Rf values were calculated to quantify the mobility of each compound, and could be used to identify unknown samples. Sources of error included inconsistent manual injection amounts for the small volume samples.

1. HBC 1019 Biochemistry 1 Trimester 1, 2012/2013
Osama Barayan - 1091105869
Faculty of Information Science & Technology
HCB 1019 – BIOCHEMISTRY 1
LECTURE: AMELIA BINTI KASSIM
Date: 27–6-2012
PRACTICAL 2
Separation and Identification
Techniques using Paper
Chromatography
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2. HBC 1019 Biochemistry 1 Trimester 1, 2012/2013
Introduction:
Chromatography is a process which can be used to isolate the various components of a
mixture. There are a number of different types of chromatography in use, including gas,
liquid, paper, and gel permeation chromatography, and this process can get quite
involved, especially with complex mixtures. It is also an extremely useful addition to a
variety of fields, including pure and applied sciences, forensics, and athletics, among
many others.
The process relies on the fact that different molecules will behave in different ways when
they are dissolved in a solvent and moved across an absorbent medium. In a very simple
example, one could take ink and make a mark on a piece of paper. The paper could be
dipped into water, and the capillary action of the water would pull the ink through the
paper. As the ink moved, its ingredients would separate out, revealing a distinctive
pattern which could be used to determine the components of the ink.
Chromatography, a group of methods for separating very small quantities of
complex mixtures, with very high resolution, is one of the most important techniques in
environmental analysis. The ability of the modern analytical chemist to detect specific
compounds at ng/g or lower levels in such complex matrices as natural waters or animal
tissues is due in large part to the development of chromatographic methods.
The science of chromatography began early in the twentieth century, with the
Russian botanist Mikhail Tswett, who used a column packed with calcium carbonate to
separate plant pigments. The method was developed rapidly in the years after World War
II, and began to be applied to environmental problems with the invention of the electron
capture detector (ECD) in 1960 by James Lovelock. This detector, with its specificity and
very high sensitivity toward halogenated organic compounds, was just what was needed
to determine traces of pesticides in soils, food and water and halocarbon gases in the
atmosphere. This happened at exactly the time when the effect of anthropogenic
chemicals on many environmental systems was becoming an issue of public concern.
Within a year, it was being applied to pesticide analysis. The pernicious effects of long
lived, bioaccumulating pesticides, such as DDT, would have been very difficult to detect
without the use of the ECD. The effect of this information on public policy has been far-
reaching.
The basis of all types of chromatography is the partition of the sample compounds
between a stationary phase and a mobile phase which flows over or through the stationary
phase. Different combinations of gaseous or liquid phases give rise to the types of
chromatography used in analysis, namely gas chromatography (GC), liquid
chromatography (LC), thin layer chromatography (TLC), and supercritical fluid
chromatography (SFC).
Chromatography has increased the utility of several types of spectroscopy, by
delivering separate components of a complex sample, one at a time, to the spectrometer.
This combination of the separating power of chromatography with the identification and
quantitation of spectroscopy has been most important in environmental analysis. It has
enabled analysts to cope with tremendously complex and extremely dilute samples
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3. HBC 1019 Biochemistry 1 Trimester 1, 2012/2013
Materials, reagents and equipments:
Trypsin
Light Green
Congo Red
Orange G
Buffer pH7
Buffer pH3
Aspartic Acid
Valine
Solvent A (80% Methanol)
Solvent B (0.05% Ninhydrin in 80% Methanol)
Beakers
Chromatography tank containing glass bead
Sample applicators
Chromatography paper strips
Methods:
Experiment I Separation of dyes
Three dyes are supplied: Orange G, Congo Red and Light Green
1. Place 1-2 drops of Orange G in one beaker, Congo Red in another beaker and
similarly for Light Green in the third.
2. Get a TLC sheet (avoid touching the coated surface since fingerprints can leave
significant quantities of protein and will affect the experiment). Draw a light
pencil line about 3/4cm or 0.75cm from the bottom on both side of the sheet and
fold it into half.
3. Gently spot a tip or small drop of each color onto the pencil mark with applicator
(the spot should be as small as possible) side by side for 10 times and allow the
sheet to dry.
4. At another side of the sheet, place three larger spots side by side for about 20
times of the drop. (Figure 1)
5. Fold the paper in half and stand for about 5 mins to allow the spots to dry.
6. Add sufficient amount of solvent A to the bottom of the beaker until the solvent
just cover the glass bead.
7. Place the paper strip in the beaker, sample side down, cover it with lid. Allow the
solvent to travel up the sheet in 5 to 10 mins or until the solvent has reach almost
the line of folded paper.
8. Remove the TLC sheet carefully by forceps and allow it to dry. Note the positions
of the solvent immediately that define how far the solvent has traveled and let it
dry. Allow the chromatogram to dry completely and note the position of each spot
later.
9. Take another paper strip and spot the amount of dyes which you now think will
give good spots. Mix the three dyes together in one beaker and spot this mixture
at the other end, but use three times the amount that necessary for any dye, since
they are now diluted with each other. Allow the spots to dry and develop the
chromatogram as before.
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4. HBC 1019 Biochemistry 1 Trimester 1, 2012/2013
10. Calculate Rf values for each amino acid according to the following equation:
Rf = Distance traveled by spot
Distance traveled by solvent
Record Rf values for each sample you tested in the table shown below.
11. Elaborate on your observations for both sides of the TLC sheets.
12. Keep the chromatograms for reference and label fully.
3 large spots
3 small spots about 3/4cm
Figure 1
Experiment 2 Separation of amino acids
1. Place 1-2 drops of amino acid Valine into a beaker and Aspartic acid in another
beaker.
2. Get a TLC sheet (avoid touching the coated surface since fingerprints can leave
significant quantities of protein and will affect the experiment). Draw a light
pencil line about 3/4cm or 0.75cm from the bottom on both side of the sheet.
3. Gently spot a tip or small drop of each amino acid onto the pencil mark with
applicator (the spot should be as small as possible) side by side for 10 times and
allow the sheet to dry.
4. At another side of the sheet, place two larger spots side by side for about 20 times
of the drop. (as in Figure 1)
5. Fold the paper in half and stand for about 5 mins to allow the spots to dry.
6. Develop this in solvent B. Add sufficient amount of solvent B to the bottom of the
beaker until the solvent just cover the glass bead.
7. Place the paper strip in the beaker, sample side down, cover it with lid. Allow the
solvent to travel up the sheet in 5 to 10 mins or until the solvent has reach the line
of the folded paper.
8. Remove the TLC sheet carefully by forceps and allow it to dry. Note the positions
of the solvent immediately that define how far the solvent has traveled and let it
dry. Note the position of each spot later. Allow the chromatogram to dry
completely.
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5. HBC 1019 Biochemistry 1 Trimester 1, 2012/2013
9. After development, take the paper strip from the tank and heat it, either in an oven
at 70 – 110 ºC for 2 to 3 mins or over a hot plate. Amino acids will give purple or
yellow colors when they react with ninhydrin and the spots of Valine and Aspartic
acid will show up dense colorations of the paper. Note the positions of the spots
and note any trailing of the spots; extensive trailing means that the chromatogram
is overloaded that means there is too much application of the sample.
10. Take another paper strip and spot the amount of amino acids which you now think
will give good spots. Mix the two amino acids together and spot onto another
paper strip but use two times the amount that necessary for any dye. Develop
again in solvent B, heat and note the separation achieved.
11. Determine the Rf values for these two amino acids on all the chromatograms.
12. Elaborate on your observations on both sides of the TLC sheets. Keep the
chromatograms for reference and label fully.
Results:
Experiment 1
Distance from origin to the centre of
x10 spot x20
Rf value
Solvent front 4.0 cm 8.2 cm -
Orange G 7.7 cm 8.2 cm
Congo Red 5.1 cm 4 cm
Light Green 3.5 cm 8.2 cm
Experiment 2
Distance from origin to the centre of
x10 spot x20
Rf value
Solvent front 8.2 cm 8.2 cm -
Valine 5.7 cm 7.0 cm
Aspartic acid 8.1 cm 8.10 cm
The pic shows when adding 20 drop it become very obvious
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6. HBC 1019 Biochemistry 1 Trimester 1, 2012/2013
Questions:
1. Why do different compounds travel different distances on the piece of paper?
Two reasons:
1. They have different solubilities in the solvent that's being used.
2. They bind to the paper to differing degrees. This is more true with silica
platesrather than paper, but it is a possibility.
2. In this experiment, name the stationary phase and the mobile phase.
With paper chromatography the stationary phase is the paper while the
mobile phase is the solvent in which the sample is dissolved.
3. How is an Rf value useful? What will the range of a Rf value? You are given Rf
values for particle A and particle B, 0.2 and 0.8, what is your conclusion on the
mobility of these particles?
RF value is the distance travelled by a component of a mixture relative to the
solvent. That is, it is the distance travelled by the component divided by the
distance travelled by the solvent.
RF value for any component has a fixed value less than unity. By calculating
RF value for a component and comparing it with known values, a given
component can be identified. The lower the Rf value the less soluble the
pigment is in the solvent.
Rf = Distance traveled by spot
Distance traveled by solvent
Rf = 0.2 = 0.25
0.8
4. Base on your experiment results, which has the highest mobility and which
particle is the least mobile?
The highest mobility with 20 times of the drop and the lowest mobility with
10 times of the drop.
5. What is chromatography used for?
Chromatography is a process which can be used to isolate the various
components of a mixture. There are a number of different types of
chromatography in use, including gas, liquid, paper, and gel permeation
chromatography, and this process can get quite involved, especially with
complex mixtures. It is also an extremely useful addition to a variety of fields,
including pure and applied sciences, forensics, and athletics, among many
others.
6. List the major sources of errors you can observe in this experiment.
A major source of error in this method is the reproducibility of the injection,
especially if manual injections are being made using a syringe. Since most
chromatographic samples are only a microliter or less, accurate
measurement is difficult. Automatic injectors and sampling valves can
reduce this error to a few percent.
Page 6 of 6

7. HBC 1019 Biochemistry 1 Trimester 1, 2012/2013
Questions:
1. Why do different compounds travel different distances on the piece of paper?
Two reasons:
1. They have different solubilities in the solvent that's being used.
2. They bind to the paper to differing degrees. This is more true with silica
platesrather than paper, but it is a possibility.
2. In this experiment, name the stationary phase and the mobile phase.
With paper chromatography the stationary phase is the paper while the
mobile phase is the solvent in which the sample is dissolved.
3. How is an Rf value useful? What will the range of a Rf value? You are given Rf
values for particle A and particle B, 0.2 and 0.8, what is your conclusion on the
mobility of these particles?
RF value is the distance travelled by a component of a mixture relative to the
solvent. That is, it is the distance travelled by the component divided by the
distance travelled by the solvent.
RF value for any component has a fixed value less than unity. By calculating
RF value for a component and comparing it with known values, a given
component can be identified. The lower the Rf value the less soluble the
pigment is in the solvent.
Rf = Distance traveled by spot
Distance traveled by solvent
Rf = 0.2 = 0.25
0.8
4. Base on your experiment results, which has the highest mobility and which
particle is the least mobile?
The highest mobility with 20 times of the drop and the lowest mobility with
10 times of the drop.
5. What is chromatography used for?
Chromatography is a process which can be used to isolate the various
components of a mixture. There are a number of different types of
chromatography in use, including gas, liquid, paper, and gel permeation
chromatography, and this process can get quite involved, especially with
complex mixtures. It is also an extremely useful addition to a variety of fields,
including pure and applied sciences, forensics, and athletics, among many
others.
6. List the major sources of errors you can observe in this experiment.
A major source of error in this method is the reproducibility of the injection,
especially if manual injections are being made using a syringe. Since most
chromatographic samples are only a microliter or less, accurate
measurement is difficult. Automatic injectors and sampling valves can
reduce this error to a few percent.
Page 6 of 6