Column Part 2 Flashcards
Chromatography separates compounds based on (1)
(1) polarity
Column chromatography is used to
separate macroscopic amounts of compounds, like running a “larger TLC”
How do we choose the appropriate solvent to be sued in column chromatography?
Use TLC to determine the appropriate solvent for
separation.
* You want a solvent such that the desired
compound will move off the baseline and
separate well from other compounds.
What was the biggest thing to keep in mind during this experiment?
DO NOT let solvent level fall below the top of the silica
The solvent can be reused as long as
it’s not colored
When preparing the sample of your annatto extract, what are the two very important things that need to be ensured?
- The sample needs to be COMPLETELY DISSOLVED -
YOU WILL NEED TO USE YOUR PIPETTE TO DISSOLVE
AS MUCH SAMPLE AS POSSIBLE - VERY IMPORTANT to load the sample in a narrow
band onto the column.
How will the bands appear in the column?
- TOP MOST
Most polar compound: Norbixin (dark red) - Bixin (bright orange-red)
- BOTTOM(near stopcock)
Least polar compound
Methyl bixin(yellow color)
What is another example of a column chromatography experiment?
Separation of plant pigments
What should the Rf value of your desired compound be to get a good separation in a column chromatography experiment?
0.25-0.5
Why is it important for the solvent level in the column to never fall below the top of the silica gel?
We need to ensure that there is always solvent above the silica gel to ensure the column does not run dry.
Why is a cracked column not desirable?
If the column runs dry, cracks develop inside silica gel in the column, thus
affecting the separation.
cracked column will
allow the compounds to flow directly through the cracks and will impact the separation
of compounds on the silica gel.
What would happen if you left your DCM/Ethanol solvent mixtures uncovered?
they would evaporate
What are the safety hazards associated with silica gel and DCM?
Silica gel: inhalation hazard
DCM: carcinogenic compound
How do you push the solute into the column?
Adding 3-4 mL increments of solvent to push the bixin(orange band) down the column.
Repeat (using 3-4 mL of solvent) until the solvent above the silica layer is no longer colored. Once the sample is loaded (i.e. the solution above the silica gel layer is colorless), gently fill
the column all the way to the top with solvent
When collecting fractions, the ______(1) factions will come off the column first and will be collected in a 125 mL erlenmeyer flask. The intense ____(2) band, which is _____(3) will be next. Once this band has finished eluting, you should stop collecting fractions
(1) yellow
(2) orange-red
(3) bixin
It is important that the annatto extract is loaded on the column in as ______ of a band as possible, which means that _____
small
it is as concentrated as possible
In general, samples are loaded on the column in the same solvent that will
run through the column. What is the exception in our case?
We will be using a more polar solvent to load
the sample so that we are able to load it in a narrow band. While we want as concentrated a
solution as possible, we need everything dissolved. If the material is not completely
dissolved, it will slowly dissolve and contaminate the column.
once the entire sample is adhered to the silica column, as indicated by ____, the
rest of the glass column can be filled with the solvent. Make sure the solvent level never falls
below the top of the silica gel.
the solvent being colorless
One of the advantages of performing column chromatography on colored compounds is that
you are able to see the compounds as they separate. Because all the compounds have
similar colors, the entire column will become orange. However, you should be able to
observe a band that is red-orange moving down the column. This is the Bixin band.
smaller fractions allow you to collect
as much pure bixin as possible.
How will the purity of the fractions be determined?
TLC
What is the general procedure in this experiment
- Prepare your silica gel slurry by mixing silica gel and adding 3% EtOH in DCM. Add to the column
- Rinse with solvent
- Concentrate the annatto extract from last week with 10% EtOH in DCM
- Drain the solvent
- Push the solvent down into the column using 3% EtOH in DCM. Repeat UNTIL there is very little color in
the solution above the column when you add fresh solvent - Fill the column all the way to the top using a powder funnel
- Collect fractions
- Determine the fractions that contain pure bixin
- Run a TLC plate to compare mixture from last week, mixture from this week(concentrated and unconcentrated), bixin standard
- rotary evap solvent
Waste disposal
all test tubes solvent goes into C,H,O-HALOGENATED & Acetone
After all of the solvent has drained from your column, immediately invert your column upside
down over 4 empty weigh boats, leaving a one inch gap between the column and the weigh boats
inse your 50 mL and 100 mL RB flask with Acetone and pour the contents into the “C,H,O
–HALOGENATED & Acetone” waste container.
What observations on the TLC plate indicate pure bixin?
- Anything that has a tail of methyl bixin is note pure. You should look for TLC plates that have one bright orange spot for bixin
