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Orgo Lab > Lab K: Isolation of Bixin (column chromatography) > Flashcards

Lab K: Isolation of Bixin (column chromatography) Flashcards

1
Q

column chromatography

A

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

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2
Q

how do we choose the appropriate solvent for separation/to use for column chromatography?

A

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

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3
Q

what are our choices of solvents and which one was chosen?

A

DCM
3% EtOH in DCM
10% EtOH in DCM

–> 3% EtOH in DCM was chosen

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4
Q

describe the steps in packing the column

A
  1. Make sure the Column is clamped in the middle and it is vertical
  2. Prepare (in a beaker) a slurry of silica gel and the appropriate solvent
  3. Add the slurry via POWDER FUNNEL
  4. Add more solvent to wash slurry down the sides of the column using a pipette
  5. Open stopcock to collect the solvent in a beaker and reuse solvent (as long as it’s not colored)
  6. DO NOT let solvent level fall below the top of the silica
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5
Q

how to prepare the sample

A

the sample needs to be COMPLETELY DISSOLVED –> use pipette to dissolve as much sample as possible

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6
Q

why it is important that the sample is completely dissolved when preparing the sample?

A

very important to load the sample in a narrow band onto the column

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7
Q

what color are the bands in the column for norbixin, bixin, and methyl bixin

A

norbixin - dark red
bixin - bright orange-red
methyl bixin - yellow

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8
Q

what does it mean for the column to run dry and what happens if it does?

A

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

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9
Q

what does it mean for the sample to be LOADED into the column and what can you do afterwards?

A

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)

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10
Q

how do you avoid broad bands?

A
  • 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)
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11
Q

what is the order of how compounds/colors come off the column?

A

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)

  1. methyl bixin (least polar) - yellow
  2. bixin (moderately polar) - orange red
  3. norbixin (most polar) - dark red
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12
Q

describe the procedure of running TLC of the collected fractions

A
  1. 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
  2. On the TLC plate, you will spot (A) first – and then spot 1 in every three fractions ONLY
  3. Develop your TLC plates in a solvent that you have predetermined will provide a good separation between the components of the annatto extract
  4. 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

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12
Q

why do we use TLC after collecting the fractions from the column?

A

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

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13
Q

describe the 4 lanes for the final TLC plate

A

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

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14
Q

why do we rotary evaporate the RB flask containing the bixin-containing fractions?

A

to remove the 3% EtOH in DCM solvent from the bixin solid so that we can determine the mass of PURE BIXIN

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15
Q

waste disposal procedure
AFTER you have combined the fractions to be rotovaped –>
AFTER the COLUMN has completely drained –>
TEST TUBES with UNUSED FRACTIONS –>

A

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

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16
Q

TLC is super useful in __ (2 things)

A

TLC is super useful in
1. monitoring reactions
2. evaluating the purity of samples

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17
Q

while preparing the column, it is important that 1.__ and 2.___

A

while preparing the column, it is important that…
1. the column is vertical
2. there are no cracks in the silica in the column

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18
Q

why is it important that there are no cracks in the silica gel in the column?

A

a cracked column will allow the compounds to flow directly THROUGH the cracks –> impact the separation of compounds on the silica gel

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19
Q

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

A

columns tend to crack if they are allowed to dry out (aka solvent level is below silica gel) –> cracked column affects separation of compounds

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20
Q

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?

A

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 (10% EtOH in DCM to dissolve the annatto extract) and 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

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21
Q

what will happen if the material/sample is not completely dissolved?

A

it will slowly dissolve and contaminate the column

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22
Q

what are 2 conditions to be met while loading the sample into the column?

A
  1. want as narrow of a band as possible
  2. need to make sure that the sample/material is COMPLETELY DISSOLVED
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23
Q

why is the extract solution carefully dripped down the side of the column?

A

to make sure the extract solution does not disturb the bed of silica

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24
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
25
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
26
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)
27
how do you spot the first TLC plate(s)? (what increment)
spot the first fraction that contains the compound and then every THIRD fraction
28
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
29
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
30
what is the frit filter and where is it located?
frit filter - a porous filter that allows liquids to pass through but prevents the silica gel from flowing out of the column - located at the bottom of the vertical column
31
why do you place an erlenmeyer flask at the stem of the column (aka at the bottom of the setup)?
erlenmeyer flask placed to trap any leaks from the column
32
why do we double glove in this lab?
because we are working with large volumes of DCM which is a carcinogenic compound
33
what is the first step of the procedure?
checking the flow of solvent in the column --> done by using 3% EtOH in DCM solvent and letting the solvent drain by opening the stopcock of the column and making sure that there are NO AIR BUBBLES
34
how do you make the silica gel slurry?
12g silica gel + 30mL of solvent (3% EtOH in DCM) --> stir with a glass rod in a beaker to make the silica gel slurry
35
why must the silica gel slurry be made inside the fume hood?
because silica gel is an inhalation hazard
36
why do we need to add the silica gel slurry quickly into the column after mixing and forming it?
you quickly pour the slurry into the column ALL at once and quickly after stirring to prevent any silica gel from being left behind inside the beaker - quick stirring quick pouring
37
after we add the silica gel slurry into the column, why do we use a pasteur pipette to add a few more pipette fulls of solvent in a circular motion in the column?
you add the solvent in a circular motion to rinse the silica gel that is stuck to the WALLS of the column --> add solvent until it looks like there is no more silica gel left on the column
38
what do we need to do in between preparing the column with silica gel and loading it with the extract?
need to drain the solvent level to about 0.5cm above the silica gel layer
39
how do we concentrate the bixin extract in the RB flask from column lab 1? how much solvent do we use?
we add the more polar solvent (10% EtOH in DCM) to redissolve the solid extract and put back into solution (solid --> liquid) we use the most MINIMUM amount of solvent necessary to dissolve it (in this case 3mL) because we do NOT want to dilute the solution, we want to keep it as concentrated as possible so that when it is loaded into the column, the extract packs into a very narrow band
40
what is the technique to use when loading the extract into the column?
use a pipette and bring the extract RB flask and pipette as close as possible to the top of the column --> draw up the extract into the pipette and add in a CIRCULAR MOTION to the column (hugging the walls of the column)
41
why must the extract be loaded in a circular motion? what happens if it is not added in a circular motion?
the extract is loaded in a circular motion to form the narrow circular band of extract over the silica gel bed that will flow evenly through the entire column - if the extract was NOT added in a circular motion, the extract material will STREAK right through the silica gel bed in one direction --> affects the separation of bixin
42
why do we save a bit of extract while loading the extract into the column?
save a drop or two of the extract to develop the final TLC plate
43
what do we do after loading the extract?
open the stopcock and drain the extract into the silica gel bed --> remember only do so until the solvent level reaches half a cm above the silica gel bed layer (just eyeballing)
44
how do we rinse the bixin extract off the wall so the column?
adding the 3% EtOH in DCM solvent in a circular motion down the walls of the column
45
what does the process of adding more solvent and draining the solvent do and what is it called?
it helps push the 3 colored bands of methyl bixin, bixin, and norbixin further down into the column RUNNING the column
46
what does it mean to RUN the column?
adding 2-3mL of solvent into the column and then opening the stopcock to drain to push the bands down into the column --> can reuse the solvent being drained as long as the solvent is NOT colored
47
what indicates that the extract is FULLY LOADED into the column?
this happens when the solvent above the silica gel bed layer is COLORLESS --> then can fill the solvent all the way to the top
48
what happens to the separation of bixin if the column dries out and develops cracks?
bixin can get trapped in those cracks, wont have an even separation for bixin, the order of which methyl bixin, bixin, and norbixin come out of the column gets messed up as well
49
after the extract is fully loaded into the column, what is the technique to add the solvent all the way to the top of the column?
first add 3-4 pipettefulls of solvent to create a BUFFER between the solvent being directly poured down and the silica gel bed --> the buffer helps prevent the gush of solvent from impacting the colored bands at the top of the silica gel bed -> contaminating bixin with the other 2 components then add solvent directly from the graduated cylinder and then pour into column using a powder funnel at the top of the column
50
why do we don't have to collect the methyl bixin that first comes out of the column in test tubes and instead collect them in a separate erlenmeyer flask?
because we aren't interested in methyl bixin and analyzing it through TLC later
51
how much do you fill the test tubes up for bixin?
about 1/3 of the test tube --> want to collect smaller fractions to try to collect PURER fractions of bixin --> when there is a sharp color change, collect even SMALLER fractions of bixin
52
why do we use different TLC capillary tubes for EACH test tube?
to avoid cross contamination of the test tube solutions
53
why do we need to spot each test tube solution 3 times?
because the solutions in the test tubes are very dilute
54
what is the general rule of how to choose which test tubes to spot and run TLC in?
in general, spot 1 in every 3 test tubes
55
what are the developing solvents for TLC plates after collecting test tubes?
the same solvent as the column; 3% EtOH in DCM
56
when do you remove the TLC plate?
after the solvent front has crossed half way on the TLC plate --> then mark solvent front with a pencil
57
what does a yellow "tail" or "streak" indicate on the TLC plates for the test tube fractions?
if the TLC spots have a yellow tail, then it indicates methyl bixin is also in the solution if the TLC spots have NO tail/streaking at all and there is only a singular spot, then that fraction contains pure bixin
58
how do we develop the final TLC plate?
preweigh the RB flask and then combine all of the fractions with pure bixin into the RB
59
describe how the final TLC plate looks like
lane 1 - initial bixin extract from column lab 1 (has a leading methyl bixin tail) lane 2 - bixin standard (has a leading methyl bixin tail) lane 3 - one spot of combined bixin fractions (NO TAIL) lane 4 - more concentrated spot of bixin fractions (NO TAIL) --> no methyl bixin leading tail for lanes 3 and 4 ==> those two only contain bixin (pure bixin), successful isolation
60
how do we remove the 3% EtOH in DCM solvent from the pure bixin in the RB flask?
rotary evaporation --> the pure bixin solid will be a dark orange/red spots around the walls of the RB flask --> weigh the RB flask again to get the MASS OF PURE BIXIN
61
how do you clean the column?
1. open the stopcock of the column and drain out the remaining solvent into an erlenmeyer flask --> dispose the solvent collected into the CHO halogenated waste container 2. turn the column upside down over 4 weigh boats and leave a one-inch gap between the mouth of the gap and the weigh boats to trap the dried silica gel from the column --> dispose the silica gel into the waste silica gel container
62
why do we dispose the solvent used into the CHO halogenated waste container?
because the solvent contains DCM (dichloromethane) which contains chlorine which is a halogen
63

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