CHEM 1451 Lab 2 Paper Chromatography Background and Procedure

BACKGROUND INFORMATION

1. Classification of Matter: Pure Substances and Mixtures

In the beginning of studying chemistry, students learn how to write chemical formulas, determine names, and learn
properties of molecular and ionic compounds. These chemicals are called (pure) substances: a type of matter that
has a fixed composition. Pure substances are further classified as either elements or compounds. Elements are
composed of one type of atom. Compounds are composed of more than one type of atom.

Example: Each of the following matter is a pure substance since it has a fixed composition.

a. Helium gas (He) is an atomic element composed only of helium atoms.

b. Oxygen gas (O 2 ) is a molecular element because the molecules are composed only of oxygen atoms.

c. Water (H 2 O) is a molecular compound composed of two hydrogen atoms and an oxygen atom.

d. Table salt (NaCl) is an ionic compound that has a fixed ratio of sodium ions and chloride ions.

In everyday life, many chemicals we encounter are not in the form of a pure substance. Instead they are mixtures of
more than one substance. Familiar examples are tap water, air and gasoline. They are mixtures that have varying
compositions of more than one pure substance. Mixtures are categorized as either homogeneous or
heterogeneous: homogeneous mixtures have a uniform composition throughout (not to be confused with a fixed
composition) while heterogeneous mixtures do not. Figure 1 summarizes the classification of matter.

Figure 1: Any matter can be classified as a type of a pure substance or a mixture according to this flow chart.

1
CHEM 1451 Lab 2 Paper Chromatography Background and Procedure

2. Paper Chromatography

Visual inspection cannot always help you classify if something is a pure substance or a mixture. If we are given a
material, how can we determine if it is a pure substance or a mixture? Chromatography is a technique to separate
and identify the components of a mixture, allowing you to figure out if something is a pure substance or a mixture.
In this experiment, you will use a paper chromatography to determine if the source of color in food dye is
made of one coloring agent or a mixture of coloring agents.

Figure 2 illustrates the general mechanism of separation of components in a mixture for chromatography. Initially
(time = 0) a mixture contains chemical components A and B. As two components are carried by a mobile phase,
they will interact with (or stick to) a stationary phase (time = T 1 ). The two components may have different
interactions with the stationary phase, that is, for example, B may interact longer with the station phase than A (time
= T 2 ). Therefore now A will move in front of B (time = T 3 ). The process is repeated as the mobile phase carries A
and B. As a result, two components of the mixture are separated.

Time 0 T1 T2 T3
Direction of mobile phase flow
A
A B A
B B A B

Stationary phase
Figure 2: Separation process used in chromatography is illustrated schematically. See the main text for explanation.

In paper chromatography, a very small amount of a sample (a mixture to be separated) is applied near the end of the
chromatography paper (see Figure 3 (a)). The point of sample application on the paper is called the origin. Then
the paper is set upright in a solvent called eluent. As shown in Figure 3 (a), the origin should always be above the
surface of solvent. As the eluent moves upward on the paper by capillary action (Figure 3 (b)), components of the
mixture are carried by the eluent with different speeds: In this example, the mixture is composed of two substances.
When the eluent moves closer to the top of the paper (Figure 3 (c)), two components are clearly separated. The
eluent should not move all the way to the top of the paper, and the paper should be removed from the eluent
beforehand. The top of the eluent (the line made by the boundary between wet and dry part of the paper)
is called the eluent front.

chromatography paper

eluent front

origin
components
eluent of a mixture

(a) (b) (c)

Figure 3: Schematic pictures of separation processes by paper chromatography are shown in chronological order
(from left to right). Two spots (dark gray) in (b) and (c) represent different components in the mixture applied at the
origin (Black point in (a)).

2
CHEM 1451 Lab 2 Paper Chromatography Background and Procedure

Figure 4 shows an example of a finished chromatography paper (called chromatogram). In this chromatogram,
three samples are applied simultaneously at different origins on the same chromatography paper. For the first
sample (left in Figure 4) shows two spots representing separated components of a mixture. For the second (center
in Figure 4) and the third (right in Figure 4) samples, a pure substance X and a pure substance Y were used
respectively.

5.10 cm

4.35 cm
2.80 cm
origin origin origin
for a mixture for X for Y

Figure 4: An example of a chromatogram shows the application of three samples. In this example, two known pure
substances (X and Y) are applied on the right side. The first two spots on the left side are separated components of a
mixture.

According to this chromatogram, you can conclude that X moves slower than Y for this particular eluent used.
The difference in their movements can be characterized by calculating a retention factor (R f ) for each spot:

distance traveled by the spot from the origin

(Eq. 1) Rf =
distance traveled by the eluent front from the origin

It is important to specify the nature of eluent used in an experiment since the values of retention factors and
the order of elution are dependent on a type of eluent. For example, X might moves faster than Y for another type
of eluent.

Example: Calculate a retention factor (R f ) for X and Y in Figure 4.

2.80 cm
For X:
= Rf = 0.549
5.10 cm

4.35cm
For Y:
= Rf = 0.853
5.10 cm

A retention factor can be used to determine the identification of substances in a mixture. In Figure 3, the values of
retention factors for two spots from the mixture (Left in Figure 3) can be compared to those of X and Y. If their
values are close enough such as in Figure 3, you could conclude the identities of substances in the mixture as X and
Y.

3
CHEM 1451 Lab 2 Paper Chromatography Background and Procedure

PROCEDURE

Materials You Need for Lab 2 Paper Chromatography

You should arrange the following material items for this experiment. Equipment in blue letters are supplied in the
lab kit. You must assemble all chemicals and other equipment prior to performing this experiment.

Chemicals

Distilled water (Do not use tap water or purified water.)

Sodium chloride (table salt; Use the non-iodized kind and DO NOT use “iodized” salt.)
Food dye (Green, Yellow, Red, and Blue)
• You will need one box of McCormick® Assorted Food Colors & Egg Dye
(https://www.mccormick.com/spices-and-flavors/extracts-and-food-colors/food-colors/assorted-food-
colors-and-egg-dye: accessed on 01/06/2019). If you do not use a correct food dye, then your experiment
result may not come out correctly and lose credits.

Equipment

Balance
600-mL beaker
250-mL beaker
One chromatography paper (cut in 7.5 cm by 14 cm)
Ruler
Wash bottle
Glass stirring rod
Spatula
Plastic spoons
Cupcake papers
Plastic cup
Wooden toothpicks
Pencil (Do not use the marker.)
Four small plates (Do not use a paper plate.)
One small plate that can cover the 600-mL beaker
Paper towel
Small stapler and staples
Hair dryer
Cell Phone or Digital Camera (or anything that can take a picture of a chromatogram and save it as a PDF file.)
Scissors to cut a chromatography paper

For this experiment, you must complete all the procedures in one sitting. The experiment itself takes
approximately one hour.

You will measure a distance by using the ruler included in the lab kit. I encourage you to re-read the page 1 and
the page 2 of the Lab 1 Background and Procedure file so that you can record a measurement with the
correct number of significant figures.

4
CHEM 1451 Lab 2 Paper Chromatography Background and Procedure

(A) Preparation of chromatography papers

Before you start, cut a chromatography paper to the size of 7.5 cm by 14 cm. You should be able to make two
chromatography papers of 7.5 cm by 14 cm from one chromatography paper in the kit. You need one
chromatography paper of 7.5 cm by 14 cm in this experiment. The kit includes extra papers in case you make
mistakes.

1. Take one chromatography paper (the size: 7.5 cm by 14 cm), draw a straight line 1.2 cm from the bottom of the
14-cm side of the paper WITH PENCIL (Figure 5). Mark FOUR origins (×) on the line as shown in Figure 5.
Under each origin (× symbol), write “R” for red food dye, “Y” for yellow, “G” for green, and “B” for blue. MAKE
SURE TO USE A PENCIL. DO NOT USE A PEN.

2. On the top margin of the chromatography paper, write “Food Dye 0.1 % NaCl” followed by your name and the
date of the experiment.

Figure 5: The layout of chromatography paper is shown. The origins are indicated by the cross mark (×).

3. Place 1 or 2 drops of food dye on a small plate. Each plate should hold only one color of food dye.

4. Get a toothpick and blunt the end by tapping it on the table top to loosen the wood fibers. Dip the toothpick in the
red food dye. Touch the origin “R” on a chromatography paper briefly with the toothpick. Do not press too hard
and break through the paper. The diameter of the spot should not exceed 3 mm. Repeat this process for all the food
dyes (yellow, green and blue), on the origin with a new toothpick for each color. After the first application of dyes,
allow the paper to dry (about 30 seconds). Then repeat this process two more times allowing the paper to dry in
between. Your goal is to apply sufficient amount of dyes in a spot as small as possible (no larger than 3 mm in
diameter). If a diameter of a spot is too large, the shape of the spot will become too distorted later.

5. Roll the chromatography paper into a cylinder and staple the ends of the paper as shown in Figure 6 (the color
spots should face outside). Make sure to leave a small gap between the ends of paper when you staple them. Now
the chromatography paper should stand by itself.

Figure 6: A chromatography paper is curled up into a cylinder shape maintained by two staples.

5
CHEM 1451 Lab 2 Paper Chromatography Background and Procedure

(B) Preparation of 0.1 % NaCl solvent

1. Measure 0.10 g of NaCl in a cupcake paper using the balance. Remember to use the tare function.

2. Transfer NaCl to a 250-mL beaker. Then add distilled water to the 100-mL mark on the beaker. By using a glass
stirring rod, stir the solution to dissolve NaCl.

(C) Chromatography of Food Dyes

1. Transfer a small amount of 0.1% NaCl solution into a 600-mL beaker. Do not transfer a lot of the solution.
The solution in the beaker should be around 0.5-cm deep.

2. Slowly place the chromatography paper into the 600-mL beaker containing 0.1% NaCl solution. The origin line
on the paper should be above the surface of the solution. Carefully center the paper in the beaker so that it does not
touch the side of the beaker.

3. Carefully cover the beaker with a small dish to avoid the evaporation of the solution.

4. Do not disturb the beaker as the eluent moves up the chromatography paper.

5. When the eluent front is about 1 cm from the top edge of the paper, remove the paper from the beaker. It
should not take more than 20 minutes (typically about 10 – 15 minutes). So if it takes more than 20 minutes for the
eluent to climb up, something went wrong. Contact the instructor. Otherwise, move to the next step.

6. This step must be done quickly in a concerted manner. Remove the staples from the paper and lay the
chromatogram flat on a piece of paper towel. Mark the position of the eluent front with a pencil. Lightly mark the
position so that you do not break through the paper with the pencil. Because the eluent front is not always straight,
you need to mark the position of eluent front for each food dye. Use a hair dryer to dry the chromatogram. The
purpose of using a hair dryer is to evaporate some water on the chromatogram. You don’t have to make it
completely dry. Stop using the dryer when it is dry enough that the eluent front does not move. Do not use the
dryer too close to the paper.

7. Measure the distance (in cm) of the eluent front for each food dye from the origin.

8. Mark the position of spots on the paper. Most likely, each color spot will have the shape of an elongated oval or a
truncated oval. Some of the color spots might slightly overlap. In Figure 7, the position of a color spot is marked
by the symbol “×”.

Figure 7: A color spot will have either (a) the shape of an elongated oval or (b) the shape of a truncated oval. The
position of spots are marked by the symbol “×”.

9. Measure the distances (in cm) of every spot marked by “×” on the chromatogram from the origin. Record your
measurements on the data section.

10. Take a picture of the chromatogram using the digital camera. Make sure that a picture clearly shows the
chromatogram. Adjust the size and the focus. Save the picture in PDF format and name the file as “Food Dye
Chromatogram.pdf”.

6
CHEM 1451 Lab 2 Paper Chromatography Background and Procedure

“How can I make a picture in the pdf format?”

If you have a smart phone, you can find a free scanner app that allows you to take a photo and save it in PDF format.
Send it to your computer via email and save the file to your computer. For example, I use the free version of the
“CamScanner” app that takes a picture of a document and save it in PDF format. Then I can email it to my email
account.

The picture of the chromatogram that you create is graded by the following grade rubric (Proficient, Competent,
Acceptable, Novice, and Unacceptable).

Please note that you must make sure to prepare a picture in color. If not, the picture is not accepted, and you will
receive a zero grade.

Proficient (20 points)

The layout of the chromatogram is prepared according to Figure 5 of this file.
The eluent fronts are marked correctly.
The position of each color spot is marked correctly by the cross symbol (×).
For each food dye, two color spots are sufficiently separated.
The eluent front stops about 1 cm from the top edge of the paper.

Competent (16 points)

For each food dye, two color spots are sufficiently separated.
The eluent front stops about 1 cm from the top edge of the paper.
However, one or more of the following are observed.
The layout of the chromatogram is not prepared according to Figure 5 of this file.
The eluent fronts are not marked correctly.
The position of each color spot is not marked correctly by the cross symbol (×).

Acceptable (10 points)

One of the following are observed.
Color spots are not sufficiently separated.
The eluent fronts travels too far and stops close to the tope edge of the paper.
The eluent fronts do not travel enough and stops more than 1 cm from the top edge of the paper.

Novice (5 points)
Two or more of the following are observed.
Color spots are not sufficiently separated.
The eluent fronts travels too far and stops close to the tope edge of the paper.
The eluent fronts do not travel enough and stops more than 1 cm from the top edge of the paper.

Unacceptable (0 point)
One or more of the following are observed.
1) No picture of the chromatogram is presented.
2) The picture of the chromatogram is not in the PDF format. The picture cannot be opened in the Canvas Course.
3) The chromatogram is not created by you. This is considered as academic dishonesty and is prosecuted as such.