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ORGO LAB > Isolation of B-Carotene (Column Chromatography) > Flashcards

Isolation of B-Carotene (Column Chromatography) Flashcards

1
Q

Chromatography is used to…

A

Separate molecules on the basis of their interactions with other molecules

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2
Q

TLC is used to…

A

Take a “snapshot” of what’s happening in a solution of molecules

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3
Q

Column chromatography is used to…

A

SEPARATE and PURIFY compounds!

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4
Q

Main difference between TLC and column chromatography:

A

TLC = Analyze the components of a mixture
(NO collection of the components)

Column = SEPARATE (and analyze) components of a mixture
(COMPONENTS ARE COLLECTED!!!!)

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5
Q

Analytes that interact better with the mobile phase will…
(in both column + TLC)

A

Move farther up the TLC plate

Move faster through the column = Come out of the column (elute) first!

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6
Q

In column chromatography, what is the mobile phase called?

A

ELUENT

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7
Q

Stationary phase in column chromatography

A

Silica (SiO2)

–> Has OH bonds though due to interaction with environmental H2O = makes it highly polar!

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8
Q

Preparative technique

A

Method used to isolate or purify a specific component from a mixture

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9
Q

Polarity Index (Pi)

What is it used for?

A

An indicator of the ability of a solvent to dissolve polar molecules

–> Trend of Pi is analogous to trend of the dielectric constant

–> Used to help with the selection of solvents for chromatography

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10
Q

TLC vs Column Migration

A

Obey the same principles (extent of movement is determined by the same factors)

BUT the direction of migration is different!

TLC –> Bottom to top migration

Column –> TOP to BOTTOM migration!

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11
Q

Which of the following will interact most strongly with silica stationary phase?

  1. Octane
  2. Octanoic Acid
  3. Octanol
A
  1. Octane = hydrocarbon (NON-polar)
    –> Will interact LEAST with silica

2. Octanoic Acid = Carboxylic Acid (MOST polar)
–> Will interact MOST STRONGLY with silica!

  1. Octanol = Alcohol (slightly Polar)
    –> Will interact a bit with silica but not the MOST strongly
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12
Q

Which analytes would reach the bottom of the column first?

A

The least polar analytes! (Stronger affinity for the solvent than the stationary phase)

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13
Q

Analyte molecule migrate faster down column in polar solvents due to TWO factors:

A

1) Analyte and solvent compete for binding sites on stationary phase

2) Solvent better solvates the analyte
(analyte has a higher affinity for the solvent than for adsorption to the stationary phase!)

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14
Q

Factors to consider when choosing an eluent (2):

A
  1. Analyte MUST be soluble in the eluent!
  2. The stationary phase CANNOT be soluble in the eluent!
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15
Q

Loading Capacity

A

The maximum amount of sample that can be loaded onto the column while still achieving satisfactory separation

–> There needs to be a proper balance between amount of analyte and stationary phase

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16
Q

If too little analyte is added to too much silica…

A

The analyte gets diluted over the entire column = will collect only a minimal amount back

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17
Q

If too much analyte is added to too little silica…

A

The separated analyte components will overlap = won’t be able to purify the compound you are seeking to isolate

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18
Q

The faster the solvent can travel through the stationary phase…

A

The less chance analyte has to adsorb = decrease in amount of separation achieved

= decreased purity of collected compound

(Insufficient time was allowed for proper separation)

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19
Q

The slower a solvent travels through the stationary phase…

A

The components of the analyte can begin to diffuse independently down the column = not separating based off of properties anymore

= impure collection

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20
Q

Factors impacting how column chromatography runs:

A
  1. Chosen eluent
  2. Loading capacity
  3. Solvent mobility
21
Q

Column Volume (CV)

A

Volume of solvent required to saturate the amount of silica in the column

22
Q

1 CV =

A

The volume of solvent needed to entirely move a NON-ADSORBING compound (does NOT interact with stationary phase) through a column

–> Measured by how much volume you collect while moving the non-adsorbing compound all the way to the bottom (the displaced volume due to its movement!)

23
Q

How is column volume (CV) related to retention factor (Rf)?

A

CV is INVERSELY proportional to the Rf!

Rf = 1/CV
CV = 1/Rf

24
Q

To get good separation of components in an analyte, you want to use an eluent that will…

A
  1. Elute (release) the desired compound in 3-5 CVs (corresponds to Rf = 0.2-0.3)
  2. Has a delta-Rf value >= 0.2 (higher delta-Rf = greater separation between separated solutes)
25
Q

To get good separation of components in an analyte, you want to use a developer that will…

A

1) “Deposit” wanted compound at around Rf = 0.2-0.3 (will form spot at this Rf)

2) Maximize the distance between the wanted compound and the next closest separated component! (Delta Rf >= 0.2)

26
Q

Ideal solvent produces the following TLC results:

A
  1. Placement of desired compound at Rf = 0.2-0.3
  2. Delta Rf from desired compound to next closest compound is >= 0.2!
27
Q

What value do we want delta Rf to be?

A

at LEAST 0.2!

28
Q

What value do we want Rf to be for the desired compound?

A

Rf = 0.2 - 0.3

29
Q

B-Carotene vs Chlorophyll

POLARITY

A

B-Carotene = LESS polar

Chlorophyll = MORE polar

30
Q

What type of molecule / what is the overall structure of beta carotene?

A

B-Carotene is a PURE HYDROCARBON (made up of entirely C + H)

Has two terminal rings (cyclohexane)
–> Separated by a long chain of conjugated double bonds

–> Conjugated DBs = Double bonds separated from one another by a single bond

31
Q

What compound is this?

A

Beta-Carotene

32
Q

Why is B-Carotene less polar than chlorophyll?

A

B-Carotene is a complete hydrocarbon, chlorophylls on the other hand have carbonyl groups within them (increases polarity)

33
Q

Since B-carotene is the least polar pigment in spinach, where will it be found in TLC and column chromatography?

A

Will be found at the TOP of the TLC plate (will move the farthest)

Will be found at the FIRST band of the column (will move the fastest through the column)

34
Q

An analyte compound at the top of a TLC plate corresponds to what band in a column chromatography of the same analyte?

A

TOP TLC spot corresponds to FIRST column band (most bottom band)

35
Q

Based upon the TLCs, which tested developer should be used as the eluent to separate out B-carotene by column chromatography?

A

A –> LIGROIN

–> This is because the TLC shows that using just ligroin positions beta-carotene at Rf ~ 0.2-0.3 and the delta-Rf seems to be of a decent size as well

B is no good because even though the delta Rf is sufficient, the B-carotene is positioned at a HIGH Rf value!

C is no good because the delta Rf is basically zero and the B-carotene is positioned at a HIGH Rf value

36
Q

Based upon the following TLCs, which developer is best to utilize for product purity and identity ANALYSIS?

A

B –> 2:1 (Ligroin : EA)

–> This is because there is good separation of the different components = easy to see what the mixture contains

A is no good because there is not sufficient separation of the other components (can’t tell what else is present in the mixture! = can’t determine purity) –> Not polar enough

C is no good because the components are overlapping too much to be able to really tell what is going on –> TOO polar

37
Q

Purity is shown on a TLC plate by

A

ONE SPOT showing up

38
Q

Identity is determined on a TLC plate by

A

Spot of sample matching Rf with spot of standard

39
Q

How were pigments extracted from spinach?

A

Using ETHYL ACETATE and mashing it around

40
Q

What occurred after pigment extraction?

A

1) Pipette solution of ethyl acetate (organic) and pigments into a separate flask

2) Removal of any visible aqueous layer (will form due to the immiscibility with ethyl acetate)

3) “Drying the organic solvent” –> Removing “invisible water” from the solution with sodium sulfate

41
Q

What do we use sodium sulfate for?

A

To remove “invisible water” (AKA dissolved water)

–> Reacts with water and clumps together

42
Q

What is sodium sulfate?

A

A drying agent

43
Q

Why did we need to make sure the extracted pigment solution was completely devoid of water?

A

Because any water in the solution would make the column POLAR

–> Would make everything (ALL pigments) run faster through the column = B-carotene would not be able to be isolated!!!

44
Q

How do you know when all/most water has been removed using sodium sulfate?

A

There will be no more big clumping, any added sodium sulfate will flow like sand in the solution

45
Q

Answer the following:

A

ONLY fraction 6 can definitively be said to contain only the blue compound

–> We have no idea if what came before it had some other components in it
–> We see that at the next tested fraction, the red compound is also present. Therefore, we have no idea in what fraction that red compound appears after fraction 6; there exists the chance that the red compound appears for the first time in fractions 7-10!

46
Q

Answer the following:

A

C –> Fractions 2-5 AND 7-10

–> Because we know that the components AFTER fraction 11 have the red compound BUT we don’t know exactly which fraction between 7 + 11 the red originated in, we analyze 7-10 to see if there are any other fractions with just pure blue

–> Because we know that fraction 1 has NEITHER red or blue, we’d want to analyze fractions 2-5 to see if blue appears before the already tested fraction 6

NOTE: We would NOT analyze 2-10 BECAUSE we already have the results for fraction 6! Why would we re-run it??

47
Q

Fraction

A

Different portions (“batches”) of eluent collected throughout column chromatography

48
Q

What type of extraction process was utilized to extract the pigments?

A

Solid-liquid extraction

–> The solid components (pigments) were soluble in the solvent utilized and overtime became part of the solution (dissolved) allowing for the extraction of them from the other plant remnants

ORGO LAB (15 decks)

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