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.