Column Column Chromatography Chromatography Separating a mixture of Separating a mixture of food coloring food coloring.
Jan 11, 2016
Column ChromatographyColumn Chromatography
Separating a mixture of food coloringSeparating a mixture of food coloring.
You will be providedYou will be provided with a with a two-foottwo-foot length of glass tubing. length of glass tubing.
From this you can prepare From this you can prepare TWO TWO chromatographic columns.chromatographic columns.
You need a good hot flame!!You need a good hot flame!!Rotate evenly with Rotate evenly with BOTH hands,BOTH hands, while while pushing slightly toward the center to pushing slightly toward the center to
“gather“gather wall thickness”.wall thickness”.
If you hold with one hand and rotate with the If you hold with one hand and rotate with the other, you will tie the glass into an other, you will tie the glass into an UGLY UGLY
KNOTKNOT as it softens. as it softens.
Remove from the flame and pull Remove from the flame and pull steadily in opposite directions as steadily in opposite directions as
the center constricts.the center constricts.
Hold the glass for a few seconds and Hold the glass for a few seconds and allow the glass to cool and re-set.allow the glass to cool and re-set.
When the glass has re-set, burn When the glass has re-set, burn it in two at the constriction.it in two at the constriction.
As the glass separates, allow the “beads” to As the glass separates, allow the “beads” to form at the ends, closing off the columns. Now form at the ends, closing off the columns. Now
you have made TWO columns.you have made TWO columns.
Don’t put the hot glass down on a cold bench top.Don’t put the hot glass down on a cold bench top.Don’t pick it up again until you are Don’t pick it up again until you are SURESURE it has it has
cooled.cooled.
Select a suitable-sized cork to support Select a suitable-sized cork to support the column;the column; or use some other method or use some other method
as suggested in Zubrick’s earlier editionas suggested in Zubrick’s earlier edition . . .. . .
The 7 mm O.D. glass tubing should The 7 mm O.D. glass tubing should fit through the hole in your fit through the hole in your
thermometer holder, -- but DON’T thermometer holder, -- but DON’T FORCE IT!FORCE IT!
With a With a piece of piece of tubing, tubing,
fit a fit a small small funnel funnel to the to the top of top of
the the column.column.
This will This will make it make it easier to easier to pack the pack the columncolumn..
Clamp Clamp the the
column column vertically vertically on a ring on a ring
stand.stand.
Place a Place a small small
receiver receiver beneath beneath
the the column.column.
With the With the small small
diameter diameter glass rod, glass rod,
push a small push a small pellet of pellet of
cotton into cotton into the narrow the narrow end of the end of the column.column.
DO NOTDO NOT pack it pack it
tightly, or tightly, or the the
column column will drain will drain much too much too
slowlyslowly..
Next, Next, add a bit add a bit of clean of clean
sand.sand. No need No need to overdo to overdo
it !it !
Next, obtain Next, obtain a few grams a few grams
of of chromato-chromato-
graphic graphic alumina alumina
and prepare and prepare to pack the to pack the
column.column.
Now, add Now, add the the
alumina to alumina to within within
about 10 about 10 cm (3-4 cm (3-4 inches) inches)
from the from the top!top!
Add in small Add in small portions, portions,
tapping the tapping the column so the column so the vibration will vibration will
cause the cause the alumina to alumina to pack tightly pack tightly and evenly.and evenly.
Now, add Now, add a bit a bit more more
sand to sand to the top of the top of
the the alumina.alumina.
Remove Remove the funnel, the funnel,
and and prepare prepare the 70 the 70
percent percent methanol methanol
eluent.eluent.
Liquids do not mix well in small diameter Liquids do not mix well in small diameter vessels; vessels; ((pousse café )pousse café )??
So, -- pour the mixture into a beaker andSo, -- pour the mixture into a beaker and
MIX IT THOROUGHLYMIX IT THOROUGHLY..
When all When all is ready, is ready,
add add several several
drops of drops of the dye the dye
mixture to mixture to the top of the top of
the the column.column.
Some of Some of the the
mixture mixture may cling may cling
to the to the glass glass
walls of walls of the the
column.column.
FIRST,FIRST, break break off the off the tip at tip at the the
bottom bottom of the of the
column.column.
Let the Let the mixture be mixture be absorbed absorbed into the into the sand; sand;
THEN,THEN, rinse rinse the sides of the sides of the column the column with a few with a few drops of drops of eluent.eluent.
As the As the column column drains, drains,
continue continue to add to add
eluent to eluent to the top of the top of
the the column.column.
When the When the leading color is leading color is about half way about half way down, down, fill the fill the
column.column. Label Label the column the column with with your your name,name, and and leave it to leave it to
drain until the drain until the next lab.next lab.
Place your Place your column into column into the beaker the beaker
provided for provided for your section. your section. They will be They will be drained and drained and dry by your dry by your
next lab next lab period.period.
Thin – Layer ChromatographyThin – Layer Chromatography
Determining the components Determining the components in “proprietary aspirinsin “proprietary aspirins”
Place 5 – 6 mLs of TLC solvent Place 5 – 6 mLs of TLC solvent into a 100 – 150 mL beakerinto a 100 – 150 mL beaker.
Cover the beaker with a watch glass Cover the beaker with a watch glass or a piece of Parafilm.or a piece of Parafilm.
Heat a melting point capillary Heat a melting point capillary over a over a very small flamevery small flame..
Rotate the capillary evenly with BOTH hands until Rotate the capillary evenly with BOTH hands until the center softens and begins to sag.the center softens and begins to sag.
BUT DON’T PULL YET!!BUT DON’T PULL YET!!
Remove the capillary from the Remove the capillary from the flame and flame and PULL . . .PULL . . .
Hold it for a few seconds until the glass Hold it for a few seconds until the glass cools and re-sets.cools and re-sets.
Snap the capillary in two at the Snap the capillary in two at the center.center.
Now you have TWO Now you have TWO “spotters”“spotters” . . . . . .
Assume an Assume an origin lineorigin line on the TLC plate is on the TLC plate is JUSTJUST ABOVEABOVE the solvent level in the beaker; the solvent level in the beaker;
or or make a reference “notch” (Zubrick).make a reference “notch” (Zubrick).
Place Place four evenly spaced dotsfour evenly spaced dots on the origin on the origin line, and identify the proposed spots at the line, and identify the proposed spots at the
top of the plate.top of the plate.
Place samples of the Place samples of the unknownunknown and and 3 references3 references into spotting plates into spotting plates
Dip a Dip a “spotter”“spotter” into a sample; a bit of into a sample; a bit of liquid is drawn into the capillary.liquid is drawn into the capillary.
Touch the Touch the “spotter”“spotter” to the plate; a bit to the plate; a bit of sample will wet the dot.of sample will wet the dot.
Blow on the spot to hasten drying; and re-Blow on the spot to hasten drying; and re-spot if necessary. Repeat with other spot if necessary. Repeat with other “spotters”“spotters” at their respective spots. at their respective spots.
Place the plate into the developing Place the plate into the developing beaker.beaker.
It may take 10 – 15 minutes for the solvent front to It may take 10 – 15 minutes for the solvent front to approach the top. Mark the solvent front with a approach the top. Mark the solvent front with a
pencil, and allow the plate to air-dry.pencil, and allow the plate to air-dry.
When it is dry, examine the plate When it is dry, examine the plate while it is in the UV light box.while it is in the UV light box.
Compare the height to which the spots Compare the height to which the spots have risen . . .have risen . . .
Identify the components by comparing Identify the components by comparing their positions with the references.their positions with the references.
Sketch the results on your lab report.Sketch the results on your lab report.
Identify the components you found.Identify the components you found.
Turn in your Lab Reports from the Turn in your Lab Reports from the Extraction Experiment along with Extraction Experiment along with
the benzoic acid AND the the benzoic acid AND the p-dichlorobenzene.p-dichlorobenzene.
You may be asked to KEEP the You may be asked to KEEP the aniline to be used in a later aniline to be used in a later
experiment.experiment.