http://orgchem.colorado.edu/hndbksupport/chrom.html
Because of toxicity, cost, and flammability concerns, the common solvents are hexanes (or
petroleum ethers, ligroin) and ethyl acetate (an ester). Diethyl ether can be used, but it is very
flammable and volatile. Alcohols (methanol, ethanol) can be used. Acetic acid (a carboxylic
acid) can be used, usually as a small percentage component of the system, since it is corrosive,
non-volatile, very polar, and has irritating vapors. Acetone (a ketone) can be used. Methylene
chloride (halogenated hydrocarbon) is a good solvent, but it is toxic and should be avoided
whenever possible.
If two solvents are equal in performance and toxicity, the more volatile solvent is
preferred in column chromatography because it will be easier to remove from the desired
compound after isolation from a column chromatography procedure.
Ask the lab instructor what solvents are available and advisable. Then, mix a nonpolar solvent (hexanes) with a polar solvent (ethyl acetate or acetone) in varying percent
combinations to make solvent systems of greater and lesser polarity. The charts below should
help you in your solvent selection. Download the pdf file (linked below the charts) for a
printable version to keep for ready reference.
http://www.sciencebuddies.org/science-fairprojects/project_ideas/Chem_Chromatography_resources.shtml
Solvent
Polarity (arbitrary scale of 15)
Suitability
Comments
Water
1 – Most polar
Good
Rubbing alcohol (ethyl type)
or denatured alcohol
2 – High polarity
Good
POISONOUS!
Primary ingredient is ethyl
alcohol (ethanol), but it is
mixed with other ingredients
to make it poisonous.
Rubbing alcohol (isopropyl
type)
3 – Medium polarity
Good
POISONOUS!
Primary ingredient is isopropyl
alcohol (isopropanol).
Vinegar
3 – Medium polarity
Good
Nail polish remover (acetone)
4 – Low polarity
Good
POISONOUS!
USE OUTSIDE or with good
ventilation. You might be able
to find pure acetone at a
hardware store.
Turpentine
5 – Least polar
Good
POISONOUS!
USE OUTSIDE or with good
ventilation-it really smells
bad.
Vegetable oil
5 – Least polar
Poor
Too thick / viscous
Mineral oil
5 – Least polar
Poor
Too thick / viscous
1
http://books.google.com/books?id=HCZgcHbU0sC&pg=PA116&dq=paper+chromatography+solvent&lr=&cd=11#v=onepage&q=paper%20
chromatography%20solvent&f=false
2
Design Experiment No32
2.0 hours
D1=1,D2=2,D3=2
Investigate the factors affecting retention in Paper
Chromatography
Beijing World Youth Academy
Subject: Chemistry
Student name: YeiYoung Choo
Candidate number: 000791 011
January 28, 2009
Teacher Helen Xu
3
Investigating the Effect of Polarity of Solvent on Retention Time
in Paper Chromatography
(Evaluated for Design)
Research Question
How does the polarity of solvent affect an amino acid’s retention time in paper
chromatography?
Introduction
This experiment aims to investigate the effect of polarity of solvent on retention time
in paper chromatography. For investigation, this experiment will carry out paper
chromatography with four different amino acids in four trials. Good solvents for carrying out
paper chromatography with peptides very often appear to be those that are highly polar; nonpolar solvent is rarely mixed together. 1 Based on a Geoffrey Allen’s 2 idea that 1butanol/acetic acid/water (3:1:1, by volume) is one of the especially good solvents in paper
chromatography of peptides, 3 different amounts of hexane would be added for different
solvents to see how the changes made to polarity of solvent can affect retention rate.
In addition, to allow a more agreeable conclusion, four amino acids—two non polar
and to polar—would be used in the experiment, so that a general effect on retention rate can
be discussed without making the result solely focused on either polar or non-polar amino
acids.
Variables
Independent variable
Type of chromatography solvent (Polarity
of solvent)
Dependent variables
Retention time in paper chromatography
Controlled variables
Type of chromatography paper
Manipulating the proportion of hexane
in the chromatography solvent
How to control
Whatman chromatography paper; Grade
1 Chr 3001-9314
Substance
tested
for
paper serine, threonine, tryptophan, glycine for
chromatography
each trial of paper chromatography
System in which paper chromatography is Glass jar
carried out
Substance used to make travelled spots of Ninhydrin
amino acids appear in colour
Amount of amino acid dropped on the One drop of amino acid using a capillary
chromatography paper before initiating tube
paper chromatography
1
Geoffrey Allen, Sequencing of Proteins and Peptides (New York: Elsevier, 1981), 117-118.
Allen is a British chemist who has also took the position of Vice-President of the Royal Society and is
renowned for physics and chemistry of polymers.
3
Geoffrey Allen, 117-118. The solvent is from R. P. Ambler (Ambler quoted in Allen, 117).
4
The world standard chromatography paper. A smooth surface, 0.18 mm thick with a linear flow rate (water) of
130 mm/30 min. Good resolution for general analytical separations. (Whatman Ltd, 2007-2009).
2
4
Materials required
Chemical/Substance
1-butanol
Acetic acid
(De-ionised water)
Hexane
Cadmium acetate5
Ninhydrin
Serine
Threonine
Tryptophan
Glycine
Material
Quantity
Whatman chromatography paper: Grade 1
Chr 3001-9316
Capillary tube
Pencil
1
Ruler
1
Scissors
1
Measuring cylinder
Safety material
Safety glasses7
Safety gloves8
Mask9
Uncertainty
±1mm
10ml; 50ml; ±1ml
Quantity
1
1
Method
5
Although ninhydrin can stain the travelled spots of amino acids, it can destroy the nature of amino acid.
However, cadmium ions can prevent amino acids from destruction due to ninhydrin sprayed later by entering the
alcohol layer of the chromatography solvent. Also, using cadmium ions can develop the colours emerged on the
chromatogram. A. D. Semenov, I.N. Ivleva and V.G. Datsko, “The determination of microgram quantities of
amino acids in natural waters” in Russian Chemical Bulletin (1961) vol. 10 published online (Springer New
York, 2004). Online available at: http://www.springerlink.com/content/r64u330hwx325652/. Access date:
January 28, 2010.
6
The world standard chromatography paper. A smooth surface, 0.18 mm thick with a linear flow rate (water) of
130 mm/30 min. Good resolution for general analytical separations. (Whatman Ltd, 2007-2009).
7
Wear safety glasses because almost all of the chemical used in the experiment are harmful. Oxford University.
“Chemical Safety Data: 1-Butanol.” Dr Hugh Cartwright - Hands-on Science.
http://cartwright.chem.ox.ac.uk/hsci/chemicals/1-butanol.html (accessed January 28, 2010); Oxford University.
“Chemical Safety Data: Acetic Acid.” Dr Hugh Cartwright - Hands-on Science.
http://cartwright.chem.ox.ac.uk/hsci/chemicals/ethanoic_acid.html (accessed January 28, 2010); Oxford
University. “Chemical Safety Data: Hexane.” Dr Hugh Cartwright - Hands-on Science.
http://cartwright.chem.ox.ac.uk/hsci/chemicals/hexane.html (accessed January 28, 2010); Oxford University.
“Safety Data for Cadmium Acetate Dihydrate.” Dr Hugh Cartwright - Hands-on Science.
http://msds.chem.ox.ac.uk/CA/cadmium_acetate_dihydrate.html (accessed January 28, 2010).
8
Same reason as that in footnote 7.
9
Wear a mask especially for cadmium acetate which is very harmful. Oxford University. “Safety Data for
Cadmium Acetate Dihydrate.” Dr Hugh Cartwright - Hands-on Science.
http://msds.chem.ox.ac.uk/CA/cadmium_acetate_dihydrate.html (accessed January 28, 2010).
5
1. Measure 1-butanol, acetic acid, de-ionsied water and hexane as instructed on the table
below to make four different solvents for paper chromatography.
Table 1. Table showing how solvents A to D should be prepared with the given volumes of
1-butanol, acetic acid, de-ionised water and hexane to make them different in terms of
polarity
Volume [ml]
1-butanol
Acetic acid
De-ionised water Hexane
Solvent A
15
5
5
0
Solvent B
15
5
5
5
Solvent C
15
5
5
10
Solvent D
15
5
5
20
2. Add 3mL of cadmium acetate to each solvent.
3. Measure 14.5cm (width) X 30cm (length) from the provided cellulose chromatography
paper and cut it with a pair of scissors.
4. Measure 5cm of from one end of the paper and draw a line across with a pencil, so that the
length of the paper is divided into 5cm and 25cm by the pencil line.
5. Measure, now in width, 5cm from one side of the chromatography paper and mark the spot
gently with a pencil. (A small, sharp, vertical line recommended for a pencil mark).
6. Do the same from the opposite end.
7. Make the same pencil marks between the two spots marked in steps 3) and 4) but at 1.5centimetre-interval, so that there are three more spots marked with a pencil.
8. Use a capillary tube to spot the five types of amino acids along the pencil line at five
distinct spots.
9. After spotting one time for each amino acid, leave the chromatography paper until all five
spots are dry and absorbed by the paper.
10. Label the spots by writing down the names of the amino acids with a pencil below each
spot.
11. Fill the glass jar with solvent A and put the chromatography paper into the jar.
12. Make sure the solvent does not reach the pencil line or touch the amino acid spots on the
line.
13. Wait until the chromatogram has developed.
14. Meanwhile, repeat steps 3. to 13. with same amino acids and other remaining three
solvents.
15. Make sure to take out the chromatography papers before they are entirely wet by the
solvent.
16. Draw a line along the line up to which the solvent has traveled on each chromatography
paper.
17. Measure the distance between the two pencil lines on the chromatography paper and
record the measurement on the top of the paper. Do this for every chromatography paper.
18. Spray ninhydrin on all four chromatography papers to stain the spots of amino acids.
19. Note that the amino acid spots have traveled upwards. Draw “x” to indicate the center of
each spot on the chromatogram.
6
20. Measure the distance between the line from which the spots have begun traveling upward
and the center of each spot.
21. Record the measurement right next to each spot.
Reference
Allen, Geoffrey. Sequencing of Proteins and Peptides. New York: Elsevier, 1981.
Whatman Ltd 2007-2009, http://www.whatman.com/CelluloseChromatographyPapers.aspx
Semenov, A. D., Ivleva and V.G. Datsko. “The determination of microgram quantities of
amino acids in natural waters” in Russian Chemical Bulletin (1961) vol. 10 published online
(Springer
New
York,
2004).
Online
available
at:
http://www.springerlink.com/content/r64u330hwx325652/. Access date: January 28, 2010
7