Mixtures of compounds are very common in Organic Chemistry. Most reactions produce more than one product. Naturally occurring materials are only rarely 100% pure. It is therefore desirable to have a simple, fast and efficient way to determine the purity of Organic mixtures. The separation of a mixture by passing it, in solution, over an adsorbent (such as Alumina or Silica Gel) is the basic idea of Chromatography. Chromatography is a very general phenomenon. It involves the passage of a mobile phase across a stationary phase in a column. Usually a mixture of compounds is present in the mobile phase

1. Paper Chromatography
by Dr. Robert D.Craig, Ph.D.
.

2. Chromatography
• Chromatography is usually introduced as
a technique for separating and/or
identifying the components in a mixture.

3. The theory
• The basic principle is that components in
a
• mixture have different tendencies to
adsorb onto a surface or dissolve in a
solvent.
• It is a powerful method in industry, where
it is used on a large scale to separate and
• purify the intermediates and products in
various syntheses

4. The theory
• There are several different types of
chromatography currently in use – ie paper
• chromatography; thin layer chromatography
(TLC); gas chromatography (GC); liquid
• chromatography (LC); high performance liquid
chromatography (HPLC); ion
• exchange chromatography; and gel permeation
or gel filtration chromatography.

5. Basic principles
• All chromatographic methods require one
static part (the stationary phase) and one
• moving part (the mobile phase). The
techniques rely on one of the following
• phenomena: adsorption; partition; ion
exchange; or molecular exclusion.

6. Adsorption
• Adsorption chromatography was
developed first. It has a solid stationary
phase and a liquid or gaseous mobile
phase. (Plant pigments were separated at
the turn of the 20th century by using a
calcium carbonate stationary phase and a
liquid hydrocarbon mobile phase.

7. Adsorption
• The different solutes travelled different
distances through the solid, carried along
by the solvent.) Each solute has its own
equilibrium between adsorption onto the
surface of the solid and solubility in the
solvent, the least soluble or best adsorbed
ones travel more slowly.
• The result is a separation into bands
containing different solutes.

8. Adsorption
• Liquid chromatography using a column
containing silica gel or alumina is an
example of adsorption chromatography
(Fig. 1).
• The solvent that is put into a column is
called the eluent, and the liquid that flows
out of the end of the column is called the
eluate.

9. Paper chromotography
• This is probably the first, and the simplest, type of
chromatography that people meet.
• A drop of a solution of a mixture of dyes or inks is placed
on a piece of chromatography paper and allowed to dry.
The mixture separates as the solvent front
• advances past the mixture. Filter paper and blotting
paper are frequently substituted for chromatography
paper if precision is not required. Separation is most
efficient if the atmosphere is saturated in the solvent
vapour

10. Today . . . .
• Some simple materials that can be
separated by using this method are inks
from fountain and fibre-tipped pens, food
colourings and dyes. The components can
be regenerated by dissolving them out of
the cut up paper.

11. Stationary and mobile phases
• The efficiency of the separation can be
optimised by trying different solvents, and
• this remains the way that the best solvents for
industrial separations are discovered
• (some experience and knowledge of different
solvent systems is advantageous).
• Paper chromatography works by the partition of
solutes between water in the
• paper fibres (stationary phase) and the solvent
(mobile phase).

12. Stationary and mobile phases
• Common solvents that are used include
pentane, propanone and ethanol. Mixtures
of solvents are also used, including
aqueous solutions, and solvent systems
with a range of polarities can be made

13. Stationary and mobile phases
A mixture useful for separating the dyes on
Similarities is a 3:1:1 mixture (by
• volume) of butan-1-ol:ethanol ammonia
solution.

14. You goal today . . . Rf factor
determination
• As each solute distributes itself
(equilibrates) between the stationary and
the mobile phase, the distance a solute
moves is always the same fraction of the
distance moved by the solvent. This
fraction is variously called the retardation
factor or the retention ratio, and is given
the symbol R or Rf:

15. 2D chromotography
• It is possible that two solutes have the
same Rf values using one solvent, but
• different values using another solvent (eg
this occurs with some amino acids). This
• means that if a multi component system is
not efficiently separated by one solvent
the
• chromatogram can be dried, turned
through 90 degrees, and run again using a
second solvent.

16. The Rf factor

17. Rise over run of solvent front

18. Procedures for finishing Lab #2
• Find your (2) Chromatography strips
(Known & unknown)
• Compare your strips with other classes
• Note where the amino acid moved
**If it is speared approximate its location using the other classes.
• Measure the distances moved by the solvent and the amino
acid
• Calculate the Rf value and enter your value in the computer
• Identify your unkown using all of the Rf values
• Write up your lab report

19. Introduction/Background
• Purpose of the lab and its importance toward
our study of Chemistry and Biology.
• What are the various ways that compounds
can be separated (use your research)
• Why is paper chromatography being used for
our setting?
• How does this method work?
• What are the goals for this lab?

20. Hypothesis & Preparations for Lab
• Must be a clear statement of what you
predict will happen.
• Follow your prediction with reasons why
you have made this statement
• Identify the independent and dependent
variables
• Provide a list of materials needed to do
this lab. Be sure the list is complete
including size and number of items
needed.

21. Method & Procedures
• Write a step by step procedure indicating
everything you did in this lab.
• A bulleted list can be helpful
• Use the handout I gave you as a starting point and
make all of the necessary modifications to be as
accurate as possible.
• Someone reading this procedure should be able to
follow these instructions and do exactly what you
did. This would include:
– How the data was obtained
– How the data will be analyzed

22. Data- How to Organize it
-show where the solvent and solvent front moved to
with the measured distances (cm)
• Show the Rf value you calculated next to these
strips
• Be sure to explain how all of the data was obtained
in your table

23. Lab # 2: Guidelines for Write Up
• Title
• Introduction/Background
• Hypothesis
• Independent & Dependent Variables
• Materials (modify the list w/specifics)
• Procedure (modify from what I gave you)
• Data (both chromatography strips cut in half)
• Data Table
• Analysis w/ graph
• Conclusion & Evaluation

24. Analysis
• Graph your data
(molecular weight vs. Rf or distance moved)
• Review your hypothesis - were you right?
• Explain what happened vs. what you predicted
• Why were you wrong or right?
• Explain how you interpreted your results
(procedure & methods)
• Look for any patterns, correlations, or relationships
that might be significant
• Were you able to identify the unknown? Explain

25. Sources of Error & Evaluation
• What were some of the problems you
encountered during the lab?
• Why were your results different from
others?
• How can this lab be improved?

26. Lab # 2: Guidelines for Write Up
• Title
• Introduction/Background
• Hypothesis
• Independent & Dependent Variables
• Materials (modify the list w/specifics)
• Procedure (modify from what I gave you)
• Data (both chromatography strips cut in half)
• Data Tables
• Analysis w/ graph
• Conclusion & Evaluation