PAPER CHROMATOGRAPHY
The prefix CHROMOS is Greek for the word, COLOR. So, the process of chromatography (krōw-mah-tah-grah-fē)
will create a graph (or picture) of color(s)!
By definition, chromatography is a technique for separating the compounds comprising a mixture by placing a sample
of the mixture in a mobile phase that is passed over a stationary phase (Visualizing Matter Technology Edition Holt 2002 p477) . In this
case, the stationary phase is the chromatography paper and the mobile phase is the solvent (salt water, alcohol, acetone
etc...).
The resulting chromatogram will have multiple component representations, optimally. Each different component should
represent a different compound, relative to that compound’s interaction with the mobile and stationary phase.
The chromatogram could easily be different when various solvents are used as the mobile phase.
Translation: Mixtures, (like pen inks or food dyes) are made up of two or more different compounds. The compounds
(components) can be separated from each other, using their individual solubility in a specific solvent.
Different compounds dissolve in salt water, alcohol, acetone, depending upon their molecular polarity
As the solvent (mobile phase) moves up the paper, soluble compounds travel with the solvent.
The separation of compounds is based on the competition of the solute and the mobile phase for binding places on the
stationary phase.
Therefore, a balance of intermolecular forces determines the position of equilibrium and thus the ability of the solvent
to move the solute up the “plate”. In other words, would the spot prefer to be stuck on the paper or would it prefer to
move along with solvent? http://www.chem.umass.edu/~samal/269/tlc.pdf
This difference in absorbance is relatively unique to the substance and can be used to
1) separate a mixture of compounds to help identify the compounds AND
2) used to compare the composition of one mixture to another
A solvent (like water or ethanol) will dissolve the parts of a mixture and "carry" different compounds different distances,
as that solvent is absorbed by a piece of filter paper.
The most soluble compounds in the solvent will be carried the greatest distance from the source point.
Compounds that are insoluble or only slightly soluble in the solvent will stay closer to the source point.
Once completed, a qualitative comparison may be made based upon the pattern of this separation AND a mathematical
calculation of the Rf values. These Rf values (retention factor or “relative to front) are unique to each compound. These
values change as you change the solvent, but the values are still unique to each component, relative to that solvent.
These values can be matched to accepted industry standards, to determine the identity of the compound.
When reading a chromatogram, you must take into account, the distance of the solvent front from the starting
point and the distance of the component.
Rf= distance of Y / distance of X
and is always ≤ 1
or rather: Rf = DComponent / DSolvent and is always ≤ 1
Diagram of Set Up:
Effectively, you will pleat
chromatography paper. At the base of each
pleat you will place a dot” of ink to be tested.
This paper will then be placed into a beaker
which holds a small amount of solvent.
Pencil Line
2 cm
dot of mixture
Check out:
Animation/Measurement: http://www.800mainstreet.com/e3/e3.html
Materials: 1 ruler, 1 600 mL or 1,000 mL beaker, 1 piece of chromatography paper, pens/inks of various colors
Procedure: (An adaptation of a lab done in EVERYDAY CHEMISTRY @ WCSU …so thanks to Dr. Russ Selzer)
1) Put on your goggles
2) Draw a straight PENCIL line about 2 cm from the bottom of the chromatography paper.
Now, pleat or fan your chromatography paper as demonstrated in class.
3) At the base of each pleat, (approximately 2 cm. above the base ...at the line) place a dot of ink or a dot of
colored marker, using a toothpick. Be sure that your last pleat has a dot of the UNKNOWN mixture
4) Add saltwater to the beaker ... now this is the tricky part. You need to add enough ethanol so that the ink
mark is ABOVE the liquid’s level ... but you need enough water to be drawn up the height of the pleated
paper.
Might I suggest that you put the paper in the beaker and note where the ink dots are ... THEN take
out the paper and then add your saltwater to the beaker? Now, put back the paper with the ink samples and
start the experiment.
Solvent level below the ink dots
5) Permit the solvent in the beaker to rise up the filter paper until it is about 2 cm from the top.
6) Remove the strip. Let it sit up right for a minute or two. Once you have let it rest …mark in pencil the height
of the solvent with a "dash".
7) From the middle of the original dot of dye, measure the distance to that dash and record it as your Ds value
(distance of the Solvent)
8) From the middle of the original dot of dye, measure the middle of each color that "developed" and
record that distance as a Dc (distance of the Component)
You should measure to the middle of the color band. You may have 1 to 4 different zones of color. Record
these distances as Dc
9) If you wish to you may repeat the experiment using ethanol and/or acetone solvents. Use a new (dry) piece of
chromatography paper for each trial. Use the SAME inks. You can then compare the results with the
1% m/m/ saltwater. You may find that you pull different components out of the dyes.
CHROMATOGRAPHY
Primary Reference: http://www.chemguide.co.uk/analysis/chromatography/paper.html#top
Animation/Measurement: http://www.800mainstreet.com/e3/e3.html
Important: You are required to compare Rf (retention factor) values of various compounds found in common
food dyes, pens, colorants.
However, the Rf value of a compound is not a physical constant. It depends upon (or changes with) the
stationary phase, solvent and temperature at which the experiment was performed. It is important to be able to
refer the experimental results to some reference standard (Rr) ... however, for us, absolute standards are
unavailable. So, you will create your own "standards", by creating chromatograms of known food dyes. You
will then obtain unknown mixtures of those same food dyes, and create a chromatogram of the unknowns. You
will compare the Rf values of the component compounds of the unknown mixtures, to the Rf values of the
known standard dyes (hence, your Rf values for the standard dyes will in essence act as your reference values
(Rr values).
http://www.ljcreate.com/products/product.asp?id=601&program=159&curr=1
http://www.sciencephoto.com/images/download_lo_res.html?id=655000390
http://en.wikipedia.org/wiki/File:TLC-Essential-Oils.jpg
NAME ________________________________________
PAPER CHROMATOGRAPHY RESULTS OF STANDARD DYES USING 0.10 % m/m NaCl(aq) AT ROOM
TEMPERATURE
Mixtures
e.g.) Blue Vis a Vis
#Components
2
Component
Color(s)
a) Blue
b) Pale Teal
Dc1,2,3...
Ds
(cm)
(cm)
8.6
2.2
8.6
Rf
=Dc/Ds
Comments
1.0
0.25
The “teal” is very pale,
but there does seem to be
a component color –
another test is merited.
Question:
1) Using your data, identify two mixtures which seems to contain at least one common substance.
2) Below is a chart describing the results of a chromatography experiment, what mixture(s) is/are found in
the unknown mixture?
Paper Chromatography of Standard Dyes and
an Unknown Mixture, in Acetone at Room Temperature
Mixture
Standard Dye 1
Number of
Components
2
Standard Dye 2
3
Standard Dye 3
2
Standard Dye 4
3
Unknown Mixture of
Dyes
5
Rf Values
A = 0.24
B = 0.71
A = 0.53
B = 0.49
C = 0.99
A = 0.11
B = 0.21
A = 0.81
B = 0.56
C = 0.50
A = 0.55
B = 0.51
C = 0.73
D = 0.80
E = 0.25
The Unknown contains Standard Dyes: _______________________________
3) Use the following table of results of a chromatography experiment. The observations are of a student's paper
chromatography experiment of standard natural dyes and of unknown samples related to those dyes. Which
of the
standard dyes (yellow, green, red, blue, black) does the Unknown Mixture contain?
Paper Chromatography of Standard Dyes and
an Unknown Mixture, in Distilled Water at Room Temperature
Standard Dye
Yellow
Number of
Components
2
Rf Values for
Components
AY = 0.24
BY = 0.71
Green
4
AG = 0.34
BG = 0.25
CG = 0.69
DG = 0.38
Red
3
AR = 0.11
BR = 0.21
CR = 0.39
Blue
1
AB = 0.32
Black
3
ABLK = 0.72
BBLK = 0.56
CBLK = 0.50
Unknown Mixture
of Standard Dyes
3
Component i = 0.33
Component ii = 0.24
Component iii = 0.72
The Unknown contains Standard Dyes: _______________________________