Chapter 6 Laboratory: Separating Mixtures 109solvent and that particular substrate is 0.40 (2 cm/5 cm). The Rf
of a particular compound is a dimensionless number that is fixed
for any particular combination of solvent and substrate, but may
vary dramatically for other combinations of solvent and substrate.
(Many texts incorrectly define Rf as retention factor (k), which is a
related but separate concept.)In this lab, we’ll examine many aspects of paper chromatography,
including ionic and molecular analytes, polar and nonpolar
solvents, and two-dimensional chromatography.dR. pAUL joNES CommENTS:
You can generally correct mid-experiment if you started at
too low of a polarity; it is difficult to make the correction
the other direction on the fly.large (sometimes huge) amounts of analyte, and is used to purify
compounds on a commercial scale.
In paper chromatography, it’s important to choose an
appropriate solvent. Polar solvents are more efficient carriers
for polar analytes, such as ionic compounds. Nonpolar solvents
are more efficient carriers for molecular (covalent) compounds.
Some examples of common solvents in decreasing order of
polarity are:
- water
- amides (e.g., N,N-dimethylformamide)
- alcohols (e.g., ethanol, isopropanol)
- ketones (e.g., acetone, methyl ethyl ketone)
- esters (e.g., ethyl acetate, amyl acetate)
- chlorocarbons (e.g., dichloromethane, carbon
tetrachloride, chloroform) - ethers (diethyl ether)
- aromatics (benzene, toluene)
- alkanes (hexanes, heptanes, petroleum ether)
Mixed solvents are used in paper chromatography to separate
mixtures that contain both polar and nonpolar compounds, or
to increase separation of mixtures of components that have
similar behavior with a single solvent. The rule of thumb for mixed
solvents is that a small amount of a polar solvent mixed with a
large amount of a nonpolar solvent tends to behave as a
polar solvent.
The key metric for paper chromatography is called the
retardation factor (Rf), which is the ratio of distance the analyte
moves from the initial point to the distance the solvent front
moves from the initial point. In other words, Rf = (migration
distance of the analyte)/(migration distance of the solvent).
For example, if a spot of analyte migrates 2 cm from the initial
point during the time it take the solvent front to migrate 5 cm
from the initial point, the Rf for that compound with that particular
SBSTITUTIU oNS ANd modIfICATIoNS (continued)- A coat hanger trimmed to length works well for the
stiff wire. You’ll need three segments, one for each
of the chromatography jars, each longer than the
diameter of the jar. These wires will be used with the
paper clips to suspend the chromatography strips and
squares inside the jars. - You may substitute methyl ethyl ketone (MEK)
for acetone. - You may substitute any other nonpolar solvent, such
as lighter fluid or toluene, for petroleum ether.
CUTIOA nS
Most of the solvents used in this laboratory are flammable.
Use extreme care, avoid open flame, and have a fire
extinguisher handy. Wear splash goggles, gloves, and
protective clothing.z
POCEDURER
This lab has two parts. In Part I, we’ll use basic paper
chromatography to examine the effect of polar and nonpolar
solvents on the dyes contained in permanent and water-
soluble marking pens. In Part II, we’ll use two-dimensional
chromatography to achieve better separation of these dyes.pART I: BASIC CHRomAToGRApHy- If you have not already done so, put on your splash
goggles, gloves, and protective clothing. - Label each chromatography jar with the solvent that
it will contain. Transfer enough solvent into each chro-
matography jar to provide about 2.5 cm of liquid in the
bottom of the jar. Cover the jars. Ideally, we want the air
in each jar to become saturated with the vapor from the
solvent contained in that jar. - Prepare 12 chromatography strips, each about 15 mm to
25 mm wide and about as long as your chromatography
jars are tall. Draw a fine pencil line across each strip about
4 cm from one end. At the opposite end, label the strips
A through L. - Prepare the 12 labeled chromatography strips by spotting
each of them with the water-soluble and permanent
black and brown marking pens in the center of the pencil
line. The goal is to create spots that are as small and as