Chemistry 108 Name_______________________________

Lab #5 Prelab:
EXTRACTION AND SEPARATION OF PLANT PIGMENTS
Purpose of the lab:
The purpose of this lab activity is for the student to learn about extraction and chemical separation
technology. Specifically, the student will learn how to do a liquid phase-extraction and Thin Layer
Chromatography in order to separate a mixture of molecules.

Introduction
Chromatography is a useful analytical technique that allows various components of a mixture to be
separated based on their polarity and/or size. There are many different types of chromatographic
separation apparatuses and methods. We will use a technique called thin-layer chromatography (TLC).
The thin-layer chromatographic surfaces that we will use in this experiment are plastic slides (TLC slides)
with a coating of very small porous silica (SiO2) particles. The particles are so small that when they are
not adhered to a surface, they behave like dust.
A small drop of the mixture to be separated is applied (spotted) near one end of the TLC slide. The
spotted end of the TLC slide is then dipped into a developing solvent (see figure 1a), called the mobile
phase¸ which flows up the TLC surface by capillary action. As the developing solvent flows up the TLC
surface, it can carry along the components of the mixture. Each component of the spotted mixture will
move upward at a different rate.

The fact that different molecules move along the chromatographic surface/solvent interface at
different rates is the key feature of chromatographic separation. Think of the component molecules as
constantly moving back-and-forth from being adsorbed to the TLC surface to being carried upward with
the mobile phase solvent. In the case of TLC, the more soluble the component is in the solvent, the faster
it travels up the surface. If it is not very soluble, it will remain adsorbed on the TLC surface (stationary
phase) longer and therefore travel at a slower rate. In addition to the component’s affinity for the solvent,
the component’s affinity for the stationary phase also plays a part in the rate at which the component
Chemistry 108 Plant Pigments

moves. If there is a strong affinity between a component molecule and the stationary phase surface, the
molecule will move upward more slowly. The affinity to the surface is determined by:
(1) non covalent attractive interactions, and
(2) in cases of porous stationary phases, the size of the molecule.
If a molecule has a size such that it is easily “stuck” in a pore (hole), it will move upward more slowly
since it takes more time to become “un-stuck”.

The rates of flow are measure in terms of Rf (retardation factor) values. An Rf is the relative
distance that a sample component has moved relative to the distance moved by the mobile phase solvent.
The following illustration will demonstrate how the Rf is calculated. Rf is measured by dividing the
distance the component traveled by the distance the solvent traveled. Therefore, an Rf value can never be
greater than 1. The Rf value for a particular component is characteristic for that component in that
particular solvent. Therefore, it will always be the same (considering that the mobile and stationary phases
are the same) and can be identified in other mixtures

Rf = Distance component traveled

Distance solvent traveled

Example: Calculation of TLC Rf values for a sample containing 4 components:

Solute Distance Rf value

Traveled
(cm)

A 2.0 cm 2.0 cm = 0.33

6.0 cm

B 3.0 cm 3.0 cm = 0.50

6.0 cm

C
3.5cm 3.5 cm = 0.58
6.0 cm

D
5.5cm 5.5 cm = 0.92
6.0 cm
Chemistry 108 Plant Pigments

Prelab Questions
1) Consider the following thin layer chromatograph. Calculate the Rf values for each of the
spots in the illustration below.

Distance
Spot Traveled Rf value
(cm)

A 2.6 cm

B 3.1 cm

C 4.2 cm

D 5.1 cm

2) In the first step of this lab, you will crush the spinach while it is in methanol (CH3OH).
Crushing breaks (opens) the plant cells and the methanol removes much of the water from
the cells. Draw the line bond structure of methanol and of water. Methanol and water are
attracted to each other by noncovalent interactions. Name the noncovalent attractions that
are occur between water and methanol. Which of these attractive forces are the strongest?
Chemistry 108 Plant Pigment Lab

In the second step of the lab, we will extract the pigment molecules in a technique called
liquid-phase extraction. In this step you will separate the hydrophobic plant pigment
molecules from other hydrophilic component molecules and solids. This is done by placing
the pulverized spinach in a flask that contains an extremely hydrophobic liquid called
“petroleum ether” and a hydrophilic liquid. These two liquids will not mix and form two
layers because one is hydrophobic and the other hydrophilic. When the flask is shaken
vigorously, then left to settle so that the two liquids separate into layers again, the spinach
pigment molecules will be extracted into the hydrophobic petroleum ether layer. These
various pigment molecules that are extracted into the petroleum ether layer will be separated
from each other in a final TLC Chromatography step of the lab described earlier.

3) Petroleum ether contains molecules like CH3CH2CH2CH2CH2CH3 and

CH3C(CH3)2CH2CH(CH3)CH2CH3. (Note: Although the name implies that petroleum ether
is in the ether family, it is not; it is a mixture of non-aromatic hydrocarbons. The name is
historical from when “ether” was the same as “spirits” and had to do with the fact that the
hydrocarbon mixture had a high vapor pressure.)
Look at the structure of β-carotene (one of our plant pigments) below and
thoroughly explain why β-carotene is much more soluble in petroleum ether than in water.
Do not simply say that “like dissolves like” (although it is true), name and explain the
nature of the intermolecular force that is involved here.

β-carotene