Lab K: Isolation of Bixin (column chromatography) Flashcards
column chromatography
a technique used to separate macroscopic amounts (large amounts) of compounds based on their POLARITIES
- like running a “larger TLC”
- most common organic purification techique
how do we choose the appropriate solvent for separation/to use for column chromatography?
use TLC to determine the appropriate/optimal solvent
- want a solvent such that
1. the desired compound will move off the baseline
2. the desired compound will separate well (aka clear separation) from other compounds
what are our choices of solvents and which one was chosen?
DCM
3% EtOH in DCM
10% EtOH in DCM
–> 3% EtOH in DCM was chosen
describe the steps in packing the column
- Make sure the Column is clamped in the middle and it is vertical
- Prepare (in a beaker) a slurry of silica gel and the appropriate solvent
- Add the slurry via POWDER FUNNEL
- Add more solvent to wash slurry down the sides of the column using a pipette
- Open stopcock to collect the solvent in a beaker and reuse solvent (as long as it’s not colored)
- DO NOT let solvent level fall below the top of the silica
how to prepare the sample
the sample needs to be COMPLETELY DISSOLVED –> use pipette to dissolve as much sample as possible
why it is important that the sample is completely dissolved when preparing the sample?
very important to load the sample in a narrow band onto the column
what color are the bands in the column for norbixin, bixin, and methyl bixin
norbixin - dark red
bixin - bright orange-red
methyl bixin - yellow
what does it mean for the column to run dry and what happens if it does?
column running dry means that there is no solvent above the silica gel (aka the solvent level fell below the top of the silica gel)
cracks will develop inside the silica gel in the column and thus will affect the separation
what does it mean for the sample to be LOADED into the column and what can you do afterwards?
the solution above the silica gel layer is colorless
once the sample is loaded, gently fill the column all the way to the top with solvent (3-4mL increments)
how do you avoid broad bands?
- use a concentrated solution of extract as possible but load all your product onto the column
- make sure the extract is fully loaded into the column before adding solvent all the way to the top
(this process usually takes a few rounds of adding 3-4mL of solvent in order to push the colored bands down the column)
what is the order of how compounds/colors come off the column?
yellow fractions (methyl bixin - least polar) come off the column first –> collect in a 125 mL erlenmeyer
intense orange-red bixin (intermediate polarity) comes off next
when the bixin band has finished eluting, stop collecting fractions (the rest would just be norbixin - dark red)
describe the procedure of running TLC of the collected fractions
- Use a TLC plate to determine the purity of fractions containing bixin: begin with one fraction (A) before the intense orange-red band started eluting to the fraction
where it stopped - On the TLC plate, you will spot (A) first – and then spot 1 in every three fractions ONLY
- Develop your TLC plates in a solvent that you have predetermined will provide a good separation between the components of the annatto extract
- once you have identified which test tube fractions contain bixin through the developed TLC plates, combine ONLY all PURE bixin-containing fractions into a 100mL RB flask
Note: At most you may need 2 TLC plates
why do we use TLC after collecting the fractions from the column?
use TLC to determine the purity of ONLY fractions that seem to have bixin in them –> then after determining which fractions contain all PURE bixin (aka bixin only), you collect them into a RB flask and do another TLC plate –> then rotary evaporate them
describe the 4 lanes for the final TLC plate
Lane 1: Initial residue (from 50 mL RB flask – Week 1 of Column lab)
Lane 2: Bixin standard (ask your TA)
Lane 3: a spot from the RB flask containing the combined test tube fractions containing BIXIN
Lane 4: a more concentrated spot from the same RB flask above
why do we rotary evaporate the RB flask containing the bixin-containing fractions?
to remove the solvent from the bixin solid so that we can determine the mass of PURE BIXIN
waste disposal procedure
AFTER you have combined the fractions to be rotovaped –>
AFTER the COLUMN has completely drained –>
TEST TUBES with UNUSED FRACTIONS –>
AFTER you have combined the fractions to be rotovaped –> THE STOPCOCK of the column is opened and left to drain into a waste Erlenmeyer flask
AFTER the COLUMN has completely drained –> The column is immediately turned upside down and 4 weigh boats are placed below the column at a height of 1 inch to contain the Silica gel that will fall out after it dries up (You do not have to clean up/ dispose the Silica gel)
TEST TUBES with UNUSED FRACTIONS –> “CHO Halogenated Waste” Container
TLC is super useful in __ (2 things)
TLC is super useful in
1. monitoring reactions
2. evaluating the purity of samples
while preparing the column, it is important that 1.__ and 2.___
while preparing the column, it is important that…
1. the column is vertical
2. there are no cracks in the silica in the column
why is it important that there are no cracks in the silica gel in the column?
a cracked column will allow the compounds to flow directly THROUGH the cracks –> 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 (aka solvent level is below silica gel) –> cracked column affects separation of compounds
how do we make sure that the initial mixture loaded on the column is in as small of a band as possible? and why do we want a narrow band?
in general samples are loaded on the column in the same solvent that will run through the column BUT in our lab, we use a more polar solvent to load the sample so that we are able to load it into a narrow band
want a narrow band so that the sample is as CONCENTRATED as possible
what will happen if the material/sample is not completely dissolved?
it will slowly dissolve and contaminate the column
what are 2 conditions to be met while loading the sample into the column?
- want as narrow of a band as possible
- need to make sure that the sample/material is COMPLETELY DISSOLVED
why is the extract solution carefully dripped down the side of the column?
to make sure the extract solution does not disturb the bed of silica
what is indicated by the solvent being colorless?
the entire sample of the extract has adhered to the silica column and the column is ready to be filled with the solvent
when you start collecting the fractions from the column, why do you want to collect SMALL fractions?
the smaller fractions allow you to collect as much PURE BIXIN as possible
what is an advantage of performing column chromatography on colored compounds?
you are able to see the compounds as they separate
(since all 3 compounds are similar in color, the entire column will turn orange but you should still see the RED-ORANGE BIXIN BAND move down the column)
how do you spot the first TLC plate(s)? (what increment)
spot the first fraction that contains the compound and then every THIRD fraction
if 2 fractions both contain the same pure compound then…
if 2 fractions both contain the same pure compound then all fractions between the 2 also contain the pure compound
safety hazards:
silica gel:
DCM:
silica gel: inhalation hazard
DCM: carcinogenic compound
–> keep chemicals covered and inside fume hood (keep hood closed when not using them)
–> DOUBLE GLOVE
