Lab K: Isolation of Bixin from Annatto Seeds Extract using Column Chromatography

Why is it important that the column is vertical and that there are no cracks in the silica in the column?

a cracked column will allow the compounds to flow directly through the cracks and will impact the separation of compounds on the silica gel

Why is it important that the solvent level in the column never falls below the top of the silica gel?

columns tend to crack if they are allowed to dry out

Since columns tend to crack if they are allowed to dry out

its important that the solvent level in the column never falls below the top of the silica gel and it is important that the initial mixture is loaded on the column in as small of a band as possible

Make sure that the solvent level never falls.........

below the top of the silica gel

What's an advantage of performing column chromatography on colored compounds?

you are able to see the compounds as they separate

- you should be able to observe a band that is red-orange moving down the column (bixin band)

when you begin to collect the material compounds, you want to start collecting _________ fractions

smaller

Why do we start collecting smaller fractions?

it allows you to collect as much pure bixin as possible

Chromatography helps with the separation of compounds based on

polarity

column chromatography is used to

separate macroscopic amounts of compounds; like running a "larger TLC"

its important to load the sample in a ________ band onto the column

narrow

the sample needs to be completely _______

dissolved

do not let the column run dry and make sure there is always solvent above the ______ ___

silica gel

if the column runs dry, cracks develop inside silica gel in the column, thus affecting the

separation

what color is norbixin?

dark red

what color is bixin?

bright orange-red

what color is methyl bixin?

yellow

What should the Rf value of your desired compound be to get a good separation in a column chromatography experiment?

the Rf value of your desired compound should be such that the desired compound is off the baseline and there is good separation from the other compounds. You want the Rf value to be large enough that the compound elutes off the column but also small enough that it doesn't move off the column too quickly. Want between 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?

because that would mean drying and columns tend to crack if they are allowed to dry out, so its important that the solvent level in the column never falls below the top of the silica gell

Why is a cracked column not desirable?

a 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 dichloromethane/ethanol solvent mixtures uncovered?

the solvent mixtures would evaporate due to their volatility

safety hazard of the silica gel

inhalation hazard

safety hazard of dichloromethane

carcinogenic compound

what is the solvent of this lab?

3% ethanol in DCM (best separation for bixin)

why do we stir and transfer the slurry immediately?

don't want to trap air bubbles and don't want the silica gel to be left inside the beaker

Before we load the bixin extract onto the column, we must make sure to drain the solvent level in the column to about

half a centimeter above the silica gel bed

to concentrate the bixin extract, we add 3 ml of the more polar 10% EtOH in DCM to redissolve the solid extract and put it back into solution. This step is always done with the minimum volume of solvent necessary because....

we don't want to dilute the solution too much

- we want to keep the solution as concentrated to be so that when its added to the column, it packs into a very narrow band

What is important when adding extract onto the column?

- you want to go as close as possible to the top of the column

- add extract solution in circular motion

why do you add the extract solution in a circular motion when adding it to the column?

you want to create a circular band of loaded extract into the column over the silica gel bed

- if you don't do this, you will end up seeing the extract material streaking right through the silica gel band in one direction which will affect the separation of bixin

When loaded in a circular band, the material stays

well packed and moves in a narrow circular band all through the column

- make sure to save two drops of the extra solution to develop a final TLC plate

What are the three bands?

(1) norbixin - deep red at the top

(2) bixin - orange in middle

(3) methyl bixin - yellow at the bottom

every time we add solvent, we must open the _______ to drain

stopcock

adding more solvent and draining the solvent helps

to push the colored bands further down into the column

When will you reach a point when the extract sample is completely loaded into the column

this happens when the solvent above the silica gel layer in colorless. when you reach this point, you can fill the column with solvent all the way to the top

never let your column run dry to......

to prevent the column from drying and developing cracks

- if it dries out and develops cracks, it will impact separation and will not have even separation of bixin

when the solvent above the silica is colorless, add 3-4 pipetefulls of solvent to

create a buffer in order to top off the column with solvent all the way to the top

we create a solvent buffer because if we don't do so and if we added solvent directly from a graduated cylinder directly from the top of the column, it would impact the bands.....

gush of solvent would impact the colored bands at the top of the silica bed which may lead to contaminating bixin with the other two components so its important to create a buffer

trying to collect pure fractions of bixin

start with collecting larger fractions of yellow until you reach orange, which is when you start collecting even smaller fractions (will see a sharp transition in color)