Laboratory Experiment 6:

Column Chromatography of Food Dyes/Inks.

Objectives: 
 To be able to purify a mixture to separate and collect the components of the mixture using
column chromatography
 To be able to identify the components collected and describe the collection process

Apparatus:
 Pasteur Pipette
 250 mL Erlenmeyer Flask
 250 mL Beaker
 Iron Stand
 Iron Ring
 Iron clamp
 Glass Rod
 Pippette bulb
 Test Tubes

Materials and Reagents Needed:

 Blue and Red Liquid food dyes
 Silica or Alumina
 Distilled Water
 Fine Sand
 Cotton or Glass Wool
 NH4OH
 1-pentanol
 ethanol
 chromatography paper

Theoretical Background:
Chromatography is a technique used to separate the components of a mixture. It can be
used as an analytical technique to gain information about what is present in a mixture, or as a
purification technique to separate and collect the components of a mixture.
In all chromatographic methods, a sample is first applied onto a stationary material that
either absorbs or adsorbs the sample: adsorption is when molecules or ions in a sample adhere to
a surface, while absorption is when the sample particles penetrate into the interior of another
material.
All forms of chromatography work by exploiting different interactions between the dyes
and the two components of the chromatography setup.
The stationary phase – this does not move and the liquids pass through it. (In ordinary paper
chromatography, the paper is the stationary phase.)
The mobile phase – this is the liquid that moves through the stationary phase (like the water
through filter paper)
The main principle that allows chromatography to separate components of a mixture is
that components will spend different amounts of time interacting with the stationary and mobile
phases. A compound that spends a large amount of time mobile will move quickly away from its
original location, and will separate from a compound that spends a larger amount of time
stationary. The main principle that determines the amount of time spent in the phases is the
strength of intermolecular forces experienced in each phase. If a compound has strong
intermolecular forces with the stationary phase it will remain adsorbed for a longer amount of
time than a compound that has weaker intermolecular forces. This causes compounds with
different strengths of intermolecular forces to move at different rates.
Chromatography Setup

Procedure:
A. Preparation of Sample Mixture

The column pictured in this section shows purification of a drop of dilute purple food dye (made from 1
drop red dye, 1 drop blue dye and 15 drops water). The dye is separated as best as possible into its three
components: blue, red and pink. A 2.5” column of silica gel is used and eluted with a solution made from
a 1:3:1 volume ratio of 6 M NH4OH:1-pentanol:ethanol.
1. Run a Thin-Layer Chromatography (TLC) (picture A) of the sample to be purified to determine the
appropriate solvent for chromatography. The desired component should have an R f around 0.35.

B. Preparation of the dry column

1. Use a metal rod or hanger to wedge a bit of cotton or glass wool into the narrow end of a short-
stemmed Pasteur pipette. The cotton should be moderately tight so liquid can trickle through, but
not solid.
2. Scoop silica or alumina into the wide end of the pipette column then invert and raise the column
so the powder falls to the bottom. Continue to use this scooping method to fill the pipette column
to 2 - 2.5 inches high with silica or alumina (this quantity can be altered depending on the amount
of sample).

Alternatively, scoop adsorbent into the wide end of a fresh pipette and use it as a funnel to deliver
adsorbent through the narrow tip and into the pipette column secured to a ring stand with a
threefingered clamp.

Safety note: As silica and alumina are fine powders and lung irritants, be sure to work in a fume
hood when handling silica or alumina. Also tap the pipette after scooping to dislodge adsorbent
on the outside of the pipette (so it doesn’t spill when vertical).

3. Gently clamp the pipette column to a ring stand or latticework using a three-fingered clamp (note:
they are fragile!) and tap it to make sure the silica / alumina is settled and the top edge is
horizontal.
 4. Add approximately 0.5 cm of sand atop the silica / alumina layer. If using very fine sand, use
another pipette to act as a funnel, as described in step 3. For coarse sand, use a small scooper or
the wide end of another pipette to aid in its delivery.

B. Wetting the column

1. Position a test tube supported in a small beaker beneath the column. Add a squirt-full of the
appropriate eluent (previously determined by TLC in Step 1), gently above the sand layer of the
pipette column.
2. Use a pipette bulb (or dropper bulb) to apply gentle air pressure and push the eluent through the
column , stopping when the liquid level is in the sand layer. Throughout the entire elution
process, keep this white column section wet with eluent.

To apply air pressure using a pipette bulb, create a strong connection between the column and the
bulb, and then squeeze the bulb. It is important when releasing the pressure to first break the seal
while still keeping your hand clenched and THEN release your hand . If you release your hand
while still connected to the column, the bulb will create suction that can violently pull liquid into
the bulb and disrupt the column.
3. Add more eluent if needed, and use bulb pressure until the entire column is saturated with eluent
and the eluent level is in the sand layer.

C. Adding the Sample

1. Use a pipette to add the entire sample to the sand layer. If the sample is a liquid, add it directly. If
it is a solid, dissolve it in the smallest amount of solvent possible, preferably the eluent. If the
solid is not soluble in the eluent, use the minimum amount of dichloromethane. Position the
pipette tip near the sand layer and add the sample carefully, trying not to splash compound onto
the sides.
2. Rinse the original container with a little solvent and add the rinsing to the column using the same
pipette (in order to rinse the pipette as well).
3. Apply pressure with the bulb to force the sample just past the sand layer.
4. Add more eluent (approximately 0.5 cm high) to rinse the sides of the column . Again use bulb
pressure to force the dye onto the adsorbent, and then fill the pipette above the sand layer as high
as possible with eluent.

D. Eluting the Column and Collecting Fractions

1. Apply gentle bulb pressure to begin eluting the sample through the column, refilling the pipette
whenever the solvent level nears the sand layer.
2. Immediately begin collecting the liquid eluting beneath the column into an empty test tube.
Change the test tube for a fresh one periodically, based on your judgment or your instructor’s
guidance (perhaps when a small test tube fills to about 1 cm high).

These different tubes are called “fractions.” The goal of a column is to collect small enough
fractions that most (or some) fractions contain pure material. If the separation of the mixture is
difficult (if the ∆Rf of the components is low), it may be best to collect even smaller fractions
(e.g. 0.5 cm high).
3. Keep the test tubes in order on a test tube rack
Data Collected:

Color Remarks
Tube 1 Transparent Eluent added in the column
First component collected
Tube 2
Pink present in food dye mixture
Second component collected
Tube 3
Red present in food dye mixture
Original mixture collected due
Tube 4 to incomplete separation of red
Purple and blue components
Last component collected
Tube 5
Blue present in food dye mixture

Results and Discussion

In this experiment, we were able to separate the components present in the sample
mixture that was loaded in the chromatography column. Throughout the start of the process until
the end, the separation of colors in the column was easily observable. Although we only added
red and blue dye for the sample mixture, we were able to collect a pink color which was truly
unexpected. This goes to show how efficient column chromatography can be in purifying
mixtures to separate compounds.

During the collection of fractions, we notice that there is an order to which a component
gets collected. The first to be collected was the eluent as it is the mobile phase used in the
process. Next is the pink color followed by the red, the purple (original color of samle mixture),
and lastly the blue color. If we look back at the TLC conducted before proceeding in the column
chromatography, we see that the color pink is ahead of the other two colors which is also
reflected in the column chromatography.

Conclusion 

It is obserable in the TLC conducted before proceeding in the column chromatography

that the pink color goes further ahead than the other colors on the paper. If we recall in the
previous TLC experiment, the color that runs faster has the highest Rf which is why it ends up
further or higher in a TLC plate. With this concept, we can diretly relate the two methods of
chromatography and establish the relationship: A compound with a higher R f runs “faster,” meaning it
will end up higher on a TLC plate, and will be collected first with a column.

From the experiment, it is observable as well that there is a difference on how fast each colors
travel down the stationary phase. From this it can concluded that if this experiment is performed using
two compounds instead of two food dye colors, there would certainly be specific factos that will affect the
order of collection of the components and may also indicate the difference in properties and chemical
compositions between the two compounds that led to the order of their collection.
 Organic Chemistry
CHEORG25P

Laboratory 6:
Column Chromatography of Food Dyes/ Inks

Submitted to:
Ms. Angelica Polana

Submitted by:
Keir Bolero
Mark Romeo Pagayon
Kuvin Guadalupe
Alyssa Samorin
Eunice Anne Sta. Ana

December 17, 2022

References:

Nichols, L. (2017). Organic Chemistry Laboratory Techniques (2nd ed.).

file:///C:/Users/user/AppData/Local/Temp/Rar$DIa11932.36680/OChem_LabTechniques_
2ndEd.pdf

https://www.sserc.org.uk/wp-content/uploads/2016/05/Chromatography.docx

https://www.youtube.com/watch?v=KGTZ3XBEfyc&t=56s