Kera Pezzuti

Chromatography is used widely throughout the world of science from the field of
forensics to molecular biology. Many different types of chromatography exist such as size
exclusion, ion exchange, affinity, thin layer, and paper. Each kind of chromatography is
utilized for different purposes and to discover dissimilar characteristics of a substance.
Affinity, for example, sorts particles based on their attraction to a certain molecule usually
with the use of antibodies, while ion exchange uses charges, plus or minus, to separate
specific proteins from one another. In our labs, we performed a few types such as size
exclusion chromatography (SEC), paper chromatography, and thin layer chromatography
(TLC). During the size exclusion, we separated hemoglobin and vitamin B12 from a
solution. In both paper chromatography and TLC, different components of various plant
pigments like chlorophyll and carotenoid were divided.
In size exclusion chromatography, an aqueous solution is applied to a
chromatography column which is coated with an assortment of porous spheres. These
beads are able to filter the small molecules and larger molecules to alter the speed in which
they exit the column. In our lab, the molecules that would pass through the beads had to
have a molecular weight under 60,000 daltons. The small molecule in this lab was vitamin
B12 with a molecular weight of 1,350 daltons which hemoglobin, the large molecule, has a
molecular weight of 65,000 daltons. Due to the extremely miniscule size of a molecule of
B12, it is forced to pass through the beads while the molecules of hemoglobin are able to
slide past the beads and exit the column easily. The drops of solution were captured in
various test tubes so the color and intensity could be observed as the main component of
the exiting solution changed.
Although SEC has strictly qualitative data, paper chromatography has an Rf value
that is used to determine an unknown substance. In our lab, the paper was placed in a
solution of plant pigment and petroleum ether, the solvent. As the acetone solution traveled
up the paper, it took the plant pigment with it which dispersed and climbed at different
rates. When the solvent finished moving, blatant separations in the colors of the pigment
were seen and then could be calculated. By dividing the distance one component traveled
by the distance the solvent traveled, we could obtain the Rf value for the pigment. These
values are constant for any and every solution so this type of chromatography is useful in
concluding the composition of an unidentified substance. The Rf value for olive green was
0.391, green was 0.641, yellow was 0.953.
TLC operates along the same lines of paper chromatography, but uses a different
solvent and unlike coating on the strip. The solvent used for this TLC lab was iso-octane-
acetone to separate the components of two plant pigments, chlorophyll and carotenoid.
When chlorophyll separates you see greens and yellows, but carotenoid produces reds and
oranges. For chlorophyll, the Rf values obtained were 0.211 for yellow-green, 0.263 for
yellow, 0.329 for light green, 0.368 for green, 0.408 for gray, 0.461 for dark gray, and 0.526
for light gray. For carotenoid, we got 0.448 for peach, 0.552 for light yellow, 0.598 for
yellow, 0.672 for pink, and 0.821 for red-orange.
When the Rf values of different substances are compared, they will be similar, if not
exact, to those of other groups. While the intensity of the colors produced by the SEC may
differ, they will show similar results in the separation of hemoglobin and vitamin B12.
Because chromatography always produces immensely comparable results for the same
substance, it is very useful for the scientific community when substances need to be
identified.
Sources
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/A/AffinityChrom.html
http://www4.gelifesciences.com/aptrix/upp00919.nsf/Content/LabSep_EduC~LC_tech~AC
http://www.proteinchemist.com/tutorial/iec.html