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Experiment No. 4 Chromatographic Separation

This document describes an experiment using paper chromatography to separate components of food dyes. Paper chromatography works by using a mobile phase to carry solutes through a stationary phase at different rates based on their interactions with each phase. In this experiment, filter paper is used as the stationary phase and either a 5% NaCl solution or isopropyl alcohol are used as mobile phases to separate yellow, green, and red food dyes. The experiment measures the distance traveled by each component and calculates their retention factor (Rf) values. Yellow and red dyes each separate into one component of their color, while green separates into yellow and blue components.

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Hans Bugarin
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189 views

Experiment No. 4 Chromatographic Separation

This document describes an experiment using paper chromatography to separate components of food dyes. Paper chromatography works by using a mobile phase to carry solutes through a stationary phase at different rates based on their interactions with each phase. In this experiment, filter paper is used as the stationary phase and either a 5% NaCl solution or isopropyl alcohol are used as mobile phases to separate yellow, green, and red food dyes. The experiment measures the distance traveled by each component and calculates their retention factor (Rf) values. Yellow and red dyes each separate into one component of their color, while green separates into yellow and blue components.

Uploaded by

Hans Bugarin
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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EXPERIMENT NO.

4
CHROMATOGRAPHIC SEPARATION

Chromatography is a method of separation employed for mixtures of closely related substances.


The word chromatography, coined by a Russian botanist named M.S. Tswett in 1906, comes from two
Greek words khroma, meaning color, and graphein, meaning to draw a graph or to write.
Several chromatographic methods have been developed over the years by different scientists
ranging from the crude paper chromatography to the highly sophisticated techniques like Gas
Chromatography (GC) and High Pressure Liquid Chromatography (HPLC). However, all forms of
chromatography employ the same general principle: A mixture of solutes in a mobile phase passes over a
selectively absorbing stationary phase. Separation occurs because the components of the mixture have
different affinities for the stationary and mobile phases. In paper chromatography, the stationary phase is
the layer of water, hydrogen-bonded to the cellulose structure of the paper; the mobile phase is the solvent
which moves through paper fibers by capillary action.
In this experiment, paper chromatography will be employed to separate the components of food
dyes. The Rf value (the retention factor), for each component, will be calculated.

Rf = Distance traveled by the solute___


Distance traveled by solvent front

The Rf value mathematically expresses the movement of the solute on the paper.

MATERIALS: 5% (g/L) aqueous NaCl, liquid food colors (yellow, green, red)

APPARATUS: 1000mL beaker, filter paper, ruler, pencil, toothpick, plastic food wrap, stapler, rubber
band

PROCEDURE:
1. Cut an 11.5- x 19-cm piece of filter paper.
2. With a pencil, draw a line parallel to the long dimension about 2 cm from the edge. Then put 4 small
x’s on the line, beginning 5 cm from the edge of the paper and spacing the marks about 3 cm apart.
3. With the use of a toothpick, apply a spot (about 1-2 mm) of food color in the first X. (For best
results, make at least 2 – 3 applications, allowing the spot to dry before the next application).
4. Continue spotting the food colors (blue, green, red, orange, yellow, etc.), putting a different color in
each x. Label each spot to identify the color of the dye spotted there.
5. Allow the spots to dry. Then, form a paper into a cylinder and staple the edges together leaving a gap
so that the edges do not quite meet.
6. Put 15 mL of the developing solvent (5% NaCl solution) in the bottom of a clean, dry 1000-mL
beaker.
7. Put the paper cylinder in the beaker containing the developing solvent, placing the spots at the
bottom. Make sure that the paper does not touch the wall of the beaker and that the spots are above
surface of the solvent in the beaker.
8. Immediately cover the beaker with plastic wrap secured by a rubber a rubber band. Allow the
solvent to rise to within 1 cm of the top of the paper (about 30-40 min).
9. When development is finished, remove the paper from the beaker and immediately mark the solvent
front with a pencil line before the solvent evaporates.
10. Dry the paper thoroughly
11. After the paper has dried, locate the densest part of the band for each color. Measure the distance
(millimeters) from the point of application to the densest part of the band color, and the distance
from the point of application to the solvent front. Record the values.
12. Calculate and record the Rf values for every colored spot on the paper.
13. Repeat using isopropyl or ethyl alcohol as the solvent.

EXPERIMENT NO. 4
CHROMATOGRAPHIC SEPARATION

I. AIM:
-To observe and determine the components of each dye in the filter paper.
-To determine the Retention Factor of each component of the dye.
-Learn what paper chromatography is all about including the process, setup, and etc.
II.
III. 1

II. DATA AND RESULTS

USING 5% NaCl

FOOD COLORS YELLOW GREEN RED


Component 1
Yellow Yellow Red
Color of the spot
Distance travelled, in mm
31mm 23mm 26mm
By the spot
By solvent front 104mm 104mm 104mm
Rf value 0.30mm 0.22mm 0.25mm
Component 2
Blue
Color of the spot
Distance travelled, in mm
82mm
By the spot
By solvent front 104mm
Rf value 0.79mm

USING isopropyl alcohol

FOOD COLORS GREEN RED YELLOW


Component 1
Yellow Red Yellow
Color of the spot
Distance travelled, in mm
22mm 41mm 40mm
By the spot
By solvent front 41mm 41mm 41mm
Rf value 0.54mm 1.00mm 0.98mm
Component 2
Blue
Color of the spot
Distance travelled, in mm
42mm
By the spot
By solvent front 41mm
Rf value 1.02mm

QUESTIONS:

1. In paper chromatography, why must the spots of food color (on the paper) be kept above the surface
of the solvent?

It is important to keep the spots of dye above the level of solvent, because we need to prevent the
dyes from dissolving into the mobile phase. If ever the baseline started at the level of the amount of
solvent, then most likely that the dyes will cease to separate by its components. Thus, failing the aim
of the experiment which is to determine and separate the components of the dyes.

2. For each original spot of food color, describe the number of components you found and their colors.
How would you account for the results?

For both the yellow and red food dye in either isopropyl alcohol solvent or 5% sodium chloride
solution, only one component is observable. Yellow and red color respectively. It is unclear if there
is a yellow component in the red food dye, because it is not actually observed in person. We can also
consider the concentration of the two solvents. Wherein the red food dye is more soluble in a higher
concentration of isopropyl alcohol while the possible yellow component is soluble in lower
concentrations of isopropyl alcohol. In the case of the green dye, it separated into two components:
Yellow and Blue. The blue appeared on top of the yellow due to the difference in solubility of the
components in different concentration of solvents.

3. Is there a green dye in the green food color? Explain.

There is no green dye in the green food color. Because if you will observe the color wheel,
combining the primary colors will produce the three secondary colors. The green color contain both
the yellow and blue colors. A green segment appeared in the paper chromatography of the green dye
because of the decrease in brightness and density. It could also be taken into account that light plays
a role in what color we see on the paper chromatography.
4. Discuss any possible sources of error in this laboratory investigation.

In an experiment, it is inevitable to not have sources of error. In these experiment, paper


chromatography, the possible sources of error are the following:
 The values were observed in a video, thus it makes observing quite difficult. These may
result to inaccurate measurements and component identification.
 The Rf value may not be as accurate as the solvent front is jagged. These makes a confusing
basis of measurement.
 The solvent’s concentration may vary the colors or components that the paper
chromatography will display. Also, the concentration of isopropyl alcohol is not stated.
 The quality of filter paper dictates the distance of the solvent front, since it is dependent on
capillary action, cohesion, adhesion, and surface tension, fibers of the filter paper may play a
factor.
 The temperature of the room were the experiment is conducted can affect the results of the
paper chromatography.
 Some possible component might not be seen because it is possible that the brightness of the
components will fade over a short period of time.

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